LGR5 regulates sequential tooth development: evidence from single-cell transcriptomics and a gene inactivation model

LGR5 regulates sequential tooth development: evidence from single-cell transcriptomics and a gene inactivation model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article LGR5 regulates sequential tooth development: evidence from single-cell transcriptomics and a gene inactivation model Marcela Buchtova, Kristyna Olbertova, Dusan Hrckulak, Petra Kompanikova, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7607583/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Tooth replacement in vertebrates depends on the persistence of the dental lamina, yet the molecular mechanisms that determine species-specific regenerative capacity remain poorly understood. Here we combine single-cell transcriptomics, lineage tracing, and genetic inactivation to define the role of LGR5, a canonical epithelial stem cell marker, in sequential molar development. We identify an unrecognized epithelial Lgr5⁺ population in the dental stalk and rudimentary successional dental lamina, together with a distinct mesenchymal Lgr5⁺ progenitor pool outside the tooth germ. Lineage tracing demonstrates that epithelial Lgr5⁺ cells contribute regionally to second molar formation, implicating them in sequential tooth initiation. Loss of Lgr5 disrupts lamina architecture, leading to shortened stalks, reduced SOX2 expression, impaired basement membrane integrity, and altered Wnt signaling, including downregulation of the LGR5-interacting protein PTK7. Organoid assays further show that Lgr5⁺ epithelial cells function as niche stabilizers rather than classical proliferative progenitors, providing structural and signaling support for replacement tooth development. Comparative analysis in diphyodont minipigs reveals conserved Lgr5 expression in non-regressing lamina domains, linking Lgr5 activity with species capable of multiple tooth generations. Together, these results identify Lgr5 as a key regulator of dental lamina stability and sequential tooth development, uncovering molecular mechanisms that couple Wnt signaling with epithelial integrity and establishing a framework for Lgr5-based regenerative strategies in dentistry. Biological sciences/Developmental biology/Stem-cell niche Biological sciences/Anatomy LGR5 SOX2 PTK7 Wnt signaling stem cells odontogenesis organoids single cell RNA sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION Functional tooth germs in vertebrates originate from the dental lamina (DL), whose longevity influences the number of tooth generations. Monophyodont species (e.g., mice) possess a rudimentary successional dental lamina (RSDL), while polyphyodont species (e.g., snakes) maintain a permanent DL. Diphyodont species (e.g., humans, pigs) initiate a second generation of teeth before the lamina fragments and regresses. However, the mechanisms establishing species-specific tooth generation remain unclear. Moreover, three molars are initiated by a sequential developmental process in mice. Tooth germs of the second (M2) and third (M3) molars are formed as the protrusion of the tooth epithelia of the first molar (M1) 1 . Dental epithelial stem cells (DESCs) can transiently reside in the cervical loop of M1 and contribute to the subsequent tooth germ. Using mouse molars, we can therefore not only assess the presence of stem cells in the DL, but also their contribution to later tooth formation 2. Regulation of tooth renewal also occurs through DESCs that reside in specialized niches. While mammalian tooth regeneration is limited, continuously growing mouse incisors provide a model for stem cell proliferation and differentiation. In these, Wnt, BMP, and FGF signaling regulate stem cell activity 3 . Here, we investigate the role of Wnt signaling in the formation of successional dental laminae (SDLs) in the molar region — an area that has received relatively little attention to date. Wnt signaling is critical for multiple stages of odontogenesis, with Wnt ligands exhibiting distinct expression patterns 4,5 . Canonical Wnt/β-catenin signaling maintains stem/progenitor cell renewal, while non-canonical, i.e. b-catenin-independent, Wnt signaling promotes differentiation 6 . The asymmetric expression of Wnt ligands could regulate dental progenitor cells, like in other tissues such as skin or gut 7 . Wnt inhibition arrests tooth development, while Wnt pathway upregulation induces ectopic teeth 8,9 . As the most of previous studies have focused on incisor development, molar formation has received comparatively little attention yet 13,14,15,16 . To address this gap, we investigated the heterogeneity of mesenchymal and epithelial cell populations in the molar region at embryonic day (E) 18.5—a critical stage corresponding to the maximal projection of the RSDL. By integrating single-cell transcriptomics with histochemistry and in situ RNA hybridization, we provide a detailed characterization of the epithelial–mesenchymal interactions that support the maintenance and function of this transient developmental structure. To investigate the existence of a stem cell niche in the rudimentary successional dental lamina (RSDL) of mouse molars, we combined single-cell transcriptomics and lineage tracing to analyze the localization, identity, and behavior of stem/progenitor cells. We focused on Lgr5 , a Wnt target gene and established marker of epithelial stem cells in various tissues 4,10–12 , whose role in molar development remains unresolved. Using Lgr5-EGFP-IRES-CreERT2 and Rosa26-tdTomato reporter mice, we visualized Lgr5-expressing cells and traced their progeny during the formation of M1 and M2. Single-cell RNA sequencing (scRNA-seq) revealed Lgr5 expression in both epithelial and mesenchymal compartments, highlighting distinct Lgr5 + populations. In Lgr5-deficient mice, we investigated the functional effects of gene loss on molar development and the activity of the Wnt signaling pathway. These approaches enable to follow spatial restriction of Lgr5 expression within the DL and evaluate its role in regulating sequential tooth formation. Taken together, our data reveal distinct epithelial and mesenchymal subpopulations with specialized molecular signatures, suggesting a more complex regulation of RSDL development than previously appreciated. The consistency in marker gene expression across studies underscores the robustness and biological relevance of our dataset. MATERIAL AND METHODS Experimental animals To analyze gene expression of LGR5 during odontogenesis we used Lgr5-EGFP-IRES-CreERT2 mice [B6.129P2-Lgr5tm1(cre/ERT2)Cle/J] 17 . These mice express fluorescent protein EGFP (for full list of abbreviations see Supplementary Table 1) and tamoxifen-regulated variant of Cre recombinase from the endogenous Lgr5 locus. Simultaneously, the “knock-in” allele disrupts Lgr5 gene function. To follow the fate of Lgr5-positive cells, we combined Lgr5-EGFP-IRES-CreERT2 mice with Rosa26-tdTomato mice [Jackson Laboratory, strain: 129S6- Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J mice] 18 . Under the control of ubiquitously active Rosa26 promoter, the Rosa26-tdTomato allele encodes tandem dimer of red fluorescent Tomato (tdTomato) protein following transcriptional stop signal that is flanked by loxP sites and sensitive to Cre-mediated excision. Therefore, Lgr5-positive cells were permanently labeled by tdTomato fluorescence after tamoxifen treatment in compound Lgr5-EGFP-CreERT2 x Rosa26-tdTomato mice. The Cre activity and labeling were induced in embryos by a single dose of tamoxifen in corn oil (2 mg in 100ul) that was gavaged orally into pregnant females at different time points of embryonic development (E12.5; E13.5). Samples were collected at different developmental stages, including E11.5 to 18.5 (E11.5–E18.5) and postnatal day 4 (P4). At selected time points, embryos were euthanized by decapitation and fixed in 4-10% PFA at least overnight depending on developmental stage. Animal care and experimental procedures were approved by the Animal Care Committee of the Institute of Molecular Genetics, Prague, Czech Republic (Approval No. 26/2019). To evaluate diphyodont dentition, we selected the E50 developmental stage of the minipig, when the successional dental lamina is already well developed. Minipig embryos were obtained from the Liběchov animal facility (IAPG, Liběchov, Czech Republic). The day after insemination was established as day 1 of gestation. Staged embryos were obtained by hysterectomy, fixed in 10% neutral formaldehyde. Sections were stained with Haematoxylin-Eosin and alternative slides were used for immunohistochemical labeling or gene expression analyses by RNAScope. All procedures were conducted following a protocol approved by the Laboratory Animal Science Committee of IAPG (Approval No. 97/2011). Processing of embryos for histological analyses Specimens for histological and immunohistochemical analysis were decalcified in 12.5% EDTA in 4% PFA in the fridge and then embedded into paraffin. Paraffin embedded tissues were cut in the transverse plane to get serial histological sections. These sections were stained with Haematoxylin-Eosin and alternative slides were used for immunohistochemical analysis. All photos were taken using the fluorescence microscope Leica DM LB2 (Leica Microsystems, Germany) unless otherwise mentioned. Pictures were processed by Adobe Photoshop (Adobe Systems Incorporated, USA). Immunohistochemical labeling Alternative slides were deparaffinized and rehydrated through a series of ethanol. Water bath (97°C) in Dako solution (pH=6) or citrate buffer for 10 min was used for antigen retrieval. To prevent nonspecific binding of antibodies, blocking serum was applied on the samples for 30 min. Next, slides were incubated with primary antibody (Supplementary Table 2) for 1 hour or overnight, alternatively. The secondary antibody (Supplementary Table 2) was applied for 30 min and Fluoroshied with 4′,6-diamidino-2-phenylindole (DAPI; P36935, Invitrogen, USA) was used for the counterstaining. Analyses of label-retaining cells by 5-Bromo-2'-Deoxyuridine (BrdU ) Pregnant mice (embryos E18.5) were injected peritoneally with 10 nM BrdU (50 mg/kg, Sigma-Aldrich, USA) two hours before collection and then embryos were euthanized by decapitation. Immunohistochemical analyses were performed on alternate slides using 2NHCl and 0.1 % trypsin as a pre-treatment. The sections were incubated at 37 °C for 10 minutes in both solutions. Next, the protocol was processed the same way as was described previously. Analysis of cell fate of LGR5-positive cells Cell fate mapping was performed in Lgr5-EGFP-IRES-CreERT2 mice crossed with Rosa26-tdTomato reporter mice. The combination of the two alleles allowed us to study the relationship between LGR5-expressing cells (which produce EGFP, green fluorescence) and LGR5-descendant (LGR5-D) cells (which produce tdTomato, red fluorescence). Tamoxifen was applied intraperitoneally as described previously 19 at E12.5 or E13.5. Animals were collected at three time points (E15.5, E16.5, E18.5) to cover transition through different developmental stages. Analyses of signal colocalization Paraffin-embedded histological sections stained with antibodies against GFP and RFP (which cross-reacts with the tdTomato protein) and DAPI were used for quantitative analysis of signal overlap. The images were taken with a Carl Zeiss AxioImager.Z2 wide-field microscope with a Plan-Apochromat 40x/1.2 oil immersion objective. For each image, three channels were acquired using DAPI, GFP, and TexasRed filter cubes. Image analysis was performed using Imaris software (version 10.1, Bitplane, Oxford Instruments). Individual cells were segmented based on the DAPI channel using the Surface module. The segmented cells were classified as positive or negative according to the mean intensity of GFP and RFP. The total numbers of GFP-positive (green), RFP-positive (red), and double-positive cells (yellow) were counted. Various statistical analyzes, in particular normality and lognormality tests, unpaired t-tests, Welch’s t-test or Mann-Whitney test, were used to determine the co-expression of GFP and tdTomato using GraphPad Prism software. 3D imaging of Lgr5-positive cells To reveal the spatial distribution of Lgr5-positive cells and their progeny within the developing molar, we analyzed mandibles from Lgr5-EGFP-IRES-CreERT2 x Rosa26-tdTomato embryos. Embryos were induced at E12.5 and tissue harvested at E15.5. Complete mandibles without tongue were extracted by scissors and fixed for 24 hours in 4% PFA buffered to pH 7.35. After the permeabilization by 0,01% Triton X-100 (Merck, Germany) at 4°C for 16 hours, the tissue was counterstained by 1μg/ml DAPI at 4°C for 16 hours and cleared according to CUBIC protocol 20 . Endogenous fluorescent signal and nuclear DAPI signal from the whole molar area were captured in tiled Z-stack images using Z1 light sheet microscope (Zeiss) with objective 20x. Recorded datasets were deconvolved using Huygens Professional software (SVI), stitched and assembled to maximal projections or movies in Imaris software (Oxford instruments). Gene expression analyses by RNAScope Mice embryos were collected at E18.5. RNAScope Multiplex Fluorescent v2 assay for formalin-fixed paraffin-embedded tissue (323 110, Advanced Cell Diagnostics, Newark, California, USA) was used for detection of several different RNA transcripts. Several probes were used according to the manufacturer’s protocol (Supplementary Table 3) . Before hybridization at 40 °C for 2 hours, individual slides were boiled in retrieval buffer (322 001, Advanced Cell Diagnostics) at 97 °C for 10 minutes and pretreated with hydrogen peroxide at RT for 10 minutes and Protease Plus (322 331, Advanced Cell Diagnostics) at 40°C for 15 minutes. To visualize hybridized probes, a TSA-Plus Cyanine 3/Fluorescein system was used (NEL741001KT, Perkin-Elmer, Waltham, Massachusetts). Samples were counterstained with DAPI (323 108, Advanced Cell Diagnostics) and mounted in Fluoroshied TM (F6182-20ML, Sigma-Aldrich). Collection of tissues for single-cell analyses Mice embryos at E18.5 were used for cell isolation from the mandibular molar area for scRNAseq. Mice were euthanized by decapitation. Eleven mice heads were collected and lower jaws were dissected. Molar areas of lower jaws (epithelium and surrounding mesenchyme) were carefully isolated and cut into small pieces. Next, samples were transferred to 1.5 ml tube with collagenase type I (LS0004196, Worthington, USA) dissolved in DMEM/F12 (D8437, Sigma-Aldrich) and incubated for 3 hours at 37°C. During enzymatic digestion, samples were pipetted up and down seven times using a 1 ml pipette. At the end of incubation, 10% filtered FBS was added to each sample. Single-cell suspension was centrifuged in 4°C precooled centrifuge for 5 min at 200 x g. Then, the supernatant was removed and the pellet was resuspended in 1 ml DMEM/F12. Suspension was filtered using a 70 µm strainer into 50 ml falcon tubes (431751, Corning) and transferred into 15 ml tubes. Next, samples were centrifuged in 4°C precooled centrifuge for 5 min at 200 x g. Pellet was homogenized in 200µl 0.04% BSA in filtered phosphate-buffered saline (PBS). Sorting of cells for scRNA-seq LGR5-positive mice embryos were collected at E16.5 and E18.5 (15 embryos at E16.5 and 22 embryos at E18.5). These embryos were decapitated, and the heads were divided into upper and lower jaws. The M1 and closely surrounding area was isolated from the lower jaws. The isolated areas of each jaw were collected and cut into small pieces, and transferred to plates with 2.5 ml Collagenase P (3 U/mL; Colla-RO, col. No. 10103578001, Roche, USA) dissolved in Hanks’ balanced salts (HBBS, cat. No. 88284, Thermo Fisher Scientific, USA) and incubated for at least 1 hour at 37°C. During the enzymatic digestion, tissue pieces were gently pipetted up and down four times using a 1 ml pipette. After incubation, the cell suspension was centrifuged for 5 min at 600 x g. The supernatant was removed, and the pellet was resuspended in 500–1000 ul HBBS. Cell suspensions were filtered through a 70 µm sieve and stained for cell viability with Hoechst 33258 (cat. No. H3569, Thermo Fisher Scientific). Cells were analysed by flow cytometry using a FACSAria IIu cell sorter (BD Biosciences, USA) and an Influx high-speed cell sorter (BD Biosciences, USA). Viable single-cell EGFP-positive or EGFP-negative populations were sorted. Single-cell RNA-seq The collected samples were processed in two separate scRNA-seq runs: The first run analyzed whole molars at E18.5, while the second run focused on LGR5-positive sorted cells at E16.5 and E18.5. The first scRNA-seq run was performed on two independent biological samples, each comprising a pair of M1 from opposite sides of the mandible of two embryos. A total of 4,502 cells were sequenced in one sample (2,961 after filtering out duplicates and low-quality cells) and 3,109 cells in the other sample (2,016 after filtering out). The experimental setup and a summary of the first data set are shown in Fig. 1 . In the second scRNA-seq run using LGR5-positive sorted cells, 4,023 cells were obtained from 15 embryos at E16.5, and 4,684 cells were collected from 22 embryos at E18.5. Prior to loading onto the Chromium Controller (10× Genomics), cell suspensions were adjusted to 700 cells/μl and mixed with nuclease-free water and master mix (10× Genomics). Gel Bead-In Emulsions (GEMs) were generated, followed by reverse transcription, homogenization, washing, and cDNA amplification (BioRad C1000 Touch Thermal Cycler). Library preparation followed the Chromium Next GEM Single Cell 3′ Reagent Kit v3 (1st run) and v3.1 (2nd run) protocols (10× Genomics). All libraries passed quality control and were sequenced on the NextSeq 500 (Illumina) using the High Output Kit v2.5 (150 cycles), with 28 cycles for read 1 (cell barcode) and 130 (1st run) or 119 (2nd run) cycles for read 2 (cDNA). Single-cell RNA-seq data processing Both scRNA-seq datasets were pre-processed using the standardized pipeline from the Cell Ranger Single-Cell Software Suite (v3.1.0; 10× Genomics) 21 . Sequencing reads were aligned to the mouse reference genome (GRCm39, Ensembl annotation release 104), and gene expression was quantified at the single-cell level using UMI counts. Raw and filtered Cell Ranger outputs were imported into RStudio (R version 4.3.3) to assess and correct for ambient RNA contamination using the SoupX package (v1.6.2) 22 , applying a contamination rate of 0.1. The corrected count matrices were used to create Seurat objects (Seurat v4.4.0) 23 , followed by several quality control steps. Cells with >10% mitochondrial gene content or expressing fewer than 500 or more than 8000 genes were excluded. Data normalization and scaling were performed using the SCTransform algorithm 24 . For the E18.5 molar dataset, cell cycle regression was included during SCTransform. Dimensionality reduction was carried out using principal component analysis (PCA) with default parameters 25 , followed by Uniform Manifold Approximation and Projection (UMAP) for visualization 26 . Cell clustering was performed using the Louvain algorithm with a resolution of 0.8, based on the cell proximity matrix from the FindNeighbors function. Doublets were identified and removed using the DoubletFinder package (v2.0.4) 27 . UMAP visualizations were further customized using the ggplot2 package 28 , with additional graphical edits (e.g., color, font) applied in Adobe Photoshop Studio 2022. Cluster-specific marker genes were identified using Seurat’s FindMarkers function, considering only positive markers expressed in at least 25% of cells, with a log fold-change threshold of 0.5 and statistical testing via the Wilcoxon rank-sum test. Top five markers for each major cell type or subcluster were visualized using Seurat's DotPlot (integrated with ggplot2), while additional expression patterns were shown via FeaturePlot. Density visualizations were created using the Nebulosa package (v1.12.1) 29 , employing the 'wide' method. Signaling pathway activity was inferred using multivariate linear modeling with the decoupleR (v2.8.0) 30 and OmnipathR (v3.10.1) 31 packages. Heatmaps were generated using ggplot2 and pheatmap (v1.0.12). Transcription factor activities were estimated using a weighted mean approach via the Dorothea (v1.14.1) 32 , decoupleR, OmnipathR, and pheatmap packages. Spliced and unspliced RNA ratios were computed using the Velocyto package (v0.17.17) 33 in Python (v3.11.9), and RNA velocity analyses were further processed using scVelo (v0.3.2) 34 to generate latent time plots based on a dynamic model. Differentially expressed genes (up- and down-regulated) related to WNT signaling in the GFP+ dental epithelium and labial mesenchyme were extracted from the pathway activity analysis described above and visualized using the textshape (v1.7.5) and ggplot2 (v3.4.4) packages in R. Isolation, cultivation, and preparation of cells for organoid analysis Lower jaws from E18.5 mouse embryos were collected, and molar regions were isolated and transferred into Petri dishes containing 2 ml of collagenase P (3 U/mL; Colla-RO, 10103578001, Roche) dissolved in Hanks' Balanced Salt Solution (HBSS; cat. no. 88284, Thermo Fisher Scientific). Samples were incubated at 37 °C for 15 minutes. Following incubation, the molar epithelium was carefully separated from the surrounding mesenchyme and bone and dissected into M1 and M2. Genotyping was processed separately. The epithelial tissues were enzymatically dissociated in TrypLE Express (cat. no. 12604039, Thermo Fisher Scientific) for 5 minutes at 37 °C. After washing in PBS, cell clusters were pelleted by centrifugation (250 × g, 5 min, 4 °C) and resuspended in Matrigel (cat. no. 356239, Corning). Matrigel droplets containing molar epithelium were allowed to polymerize at 37 °C for 10 minutes and subsequently overlaid with organoid culture medium: Advanced DMEM/F-12 (cat. no. 12634010, Thermo Fisher Scientific) supplemented with 10 mM HEPES (15630080), 1× Penicillin-Streptomycin (15070063), 1× GlutaMAX (35050061), 1× B27 (12587-010), 1× N2 (17502-048), RSPO1 (200 ng/ml; 120-38), Noggin (100 ng/ml; 120-10C), FGF2 (20 ng/ml; 234-FSE), FGF10 (100 ng/ml; 100-26), EGF (20 ng/ml; AF-100-15), and Wnt surrogate-Fc Fusion protein (200 ng/ml; PHG0401), all from Thermo Fisher Scientific; and 0.5 μM A83-01 (SML0788), 1.25 mM N-acetyl-L-cysteine (A7250), 10 mM Nicotinamide (N0636), and 10 μM SB202190 (1264), all from Merck. Organoids were cultured at 37 °C in a humidified atmosphere with 5% CO₂. Medium was changed three times per week. Organoids were passaged every two weeks by dissociation in TrypLE, following Matrigel removal via vigorous pipetting in PBS. For imaging, organoids were either visualized live in Matrigel using a Dragonfly spinning disk microscope (Andor) with Hoechst 34580 staining (Merck) or processed for histology. In the latter case, Matrigel was removed, and organoids were embedded in 4% low-melting agarose (Merck) and paraffin-sectioned for standard histological analysis. For fluorescence-activated cell sorting (FACS) analysis, organoids were collected, Matrigel was removed by thorough pipetting in PBS, and the cells were dissociated into single-cell suspension using TrypLE for 15 minutes at 37 °C. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of molar epithelium and organoids Molar epithelium was dissected from the mandibular region at embryonic stage E18.5 and separated into M1 and M2 regions, as described in the previous section. Tissue samples were lysed in RLT buffer (Qiagen). Molar organoids were collected from two wells of a 24-well plate by vigorous pipetting in PBS, pelleted by centrifugation, and lysed in RLT buffer. From this point forward, all samples were processed identically using the RNeasy Mini Kit (Qiagen) for total RNA isolation. Reverse transcription was performed using Maxima Reverse Transcriptase (cat. no. EP0742, Thermo Fisher Scientific) according to the manufacturer’s protocol, using random hexamer primers (cat. no. 48190011, Thermo Fisher Scientific). Quantitative PCR was carried out on a LightCycler® 480 system using SYBR Green I Master Mix (Roche) in three technical replicates, following the manufacturer’s instructions. Two biological replicates of each M1 and M2 tissue sample, along with three biological replicates of each M1 and M2 organoid sample ( Lgr5 ⁺/⁺ , Lgr5 ⁺/⁻ , or Lgr5 ⁻/⁻ genotypes), were analyzed for the expression of selected genes (listed in Supplementary Table 4 ). For each gene, the average Ct value from the three technical replicates was first normalized to β-actin and then to the average value of the corresponding molar tissue sample (ΔΔCt method) 35 . A gene enrichment coefficient was calculated by averaging ΔΔCt values across all organoid replicates for each gene. A population enrichment coefficient was then derived by averaging the expression changes across all genes used to define a given cell subpopulation. For clarity in visualization, the average expression change is presented as –ΔΔCt, so that upregulation is shown as positive values and downregulation as negative. Immunoprecipitation and Western Blotting HEK 293T cells were cultured in two separate 15-cm dishes. The first dish was co-transfected with a plasmid encoding human hemagglutinin (HA)-tagged PTK7 and a plasmid encoding LGR5 tagged with STREP-FLAG. The second dish was transfected with the HA-tagged PTK7 plasmid and an empty control vector lacking LGR5. Forty-eight hours post-transfection, cells were washed with PBS, lysed, and scraped in 2 ml of lysis buffer containing: 150 mM NaCl, 50 mM Tris, 0.4% Triton X-100, 2 mM CaCl₂, 2 mM MgCl₂, and 1 mM EDTA. The buffer was supplemented with protease and phosphatase inhibitors (NaF, PMSF, TLCK, TPCK, Na₃VO₄, DTT). Lysates were clarified by centrifugation at 30,000 × g for 30 minutes at 4 °C. For immunoprecipitation, 900 μl of each lysate was incubated with 1 μg of anti-HA antibody (#3724, Cell Signaling, USA) on ice. After 1 hour, 15 μl of Protein G-Sepharose beads (#17-0886-01, GE Healthcare, USA) was added and the mixture was rotated on a carousel at 4 °C for an additional 2 hours. In parallel, another 900 μl of lysate was incubated with 15 μl of Strep-Tactin Sepharose beads (#2-1201-025, IBA, Germany) under identical conditions. Following incubation, beads were collected by centrifugation (200 × g, 2 min, 4 °C) and washed six times with lysis buffer. Both immunoprecipitates and input lysates were mixed with denaturing, reducing Laemmli buffer and boiled before electrophoresis. Proteins were separated on 8–15% SDS-PAGE gels and transferred to Immobilon-P membranes (Millipore). Detection was performed using anti-HA antibody (#3724, Cell Signaling) or M2 anti-FLAG antibody (#F1804, Sigma-Aldrich). Western blot signals were visualized using the Uvitec Alliance Q9 Atom imaging system and analyzed with Q9 Alliance software. RESULTS AND DISCUSSION Single-cell transcriptome profiling of molar development in mice identifies markers of the RSDL Teeth originate from the DL, a band of epithelial tissue that initiates tooth development. Within this structure, two functionally and evolutionarily distinct types of DL can be distinguished. The RSDL is a specialized epithelial extension arising from the dental epithelium. It serves as a source of replacement teeth and maintains the capacity for further tooth formation during development. In species such as the mouse, which have only one generation of teeth, a RSDL forms temporarily (Fig. 1A) . This structure normally regresses without further teeth developing and is considered a vestigial remnant of evolutionary history. By contrast, the sequential dental lamina (SeDL) is an active structure that drives the formation of posterior molars (Fig. 1A) . In this process, each new tooth germ emerges as an epithelial outgrowth from the preceding molar, enabling the progressive extension of the dental arch and initiation of successive molar germs. To investigate the characteristics of cells involved in this process, we isolated the lower M1 at E18.5 for scRNA-seq (Fig. 1B ), collecting dental and adjacent soft tissues while carefully excluding the alveolar bone. By comparing the dental stalk (DS) and lamina with other epithelial populations, this approach provided potential insights into signaling mechanisms associated with the underlying mesenchyme. Single-cell RNA-seq was performed on lower M1 from two independent biological samples ( Fig. 1; Supplementary Fig. 1 ). As overall quality and sequencing depth were comparable, both datasets were merged for further analysis to maximize data coverage (Supplementary Fig. 1A) . However, it is necessary to mention that some mesenchymal cell clusters, particularly in the outlying dental follicle and undifferentiated osteoblasts, were enriched due to technical variations of tissues removal between bone and follicle ( Supplementary Fig. 1A ). Unbiased clustering with four parameters identified eight main cell subpopulations, including epithelial and mesenchymal dental clusters, as well as immune, blood, pericyte, muscle, and glial compartments ( Fig. 1C, D; Supplementary material 1 ). Differential expression analysis revealed cluster-specific genes and pathways ( Fig. 1C; Supplementary Fig. 1B ). In order to capture the underlying biological dynamics, the cell cycle effect was initially retained in the visualization of the individual clusters (Fig. 1E) . Cycling cells identified by Ki67 and Cdk1 expression (Fig. 1F, F’; Supplementary Fig. 1C) were primarily enriched in a cluster corresponding to the dental mesenchyme, but also formed distinct subclusters within broader populations such as the dental papilla (DP) and dental epithelium. We also detected cells corresponding to different phases of the cell cycle, as shown by the expression of Ung (G1 and S phase), Top2a (S–G2 transition), Cdc20 (G2–M transition) and Ccnb2 (M–G1 transition), which were distributed across several clusters ( Supplementary Fig. 1D, E and data not shown). To validate the spatial distribution of the identified cell populations in mouse molars, we performed RNAScope at E18.5 WT embryos (Fig. 1G–I’; Supplementary Fig. 2A–C) . Non-dental clusters included muscle cells expressing Myod1 , Myf5 , or Ttn , which showed upregulation of signaling pathways such as PI3K and VEGF (Fig. 1H, H’; Supplementary Fig. 1B, 2A; Supplementary Table 5) . Immune cells, characterized by Ptprc , C1qa , and Tyrobp , exhibited increased activity of JAK-STAT and NFκB signaling, while Wnt signaling was downregulated in this cluster (Fig. 1G, G’; Supplementary Fig. 1B, 2B; Supplementary Table 5) . Pericytes expressing Epas1 , Rgs5 , and Higd1b showed upregulation of EGFR and TNFα signaling pathways ( Supplementary Fig. 1B, 2C; Supplementary Table 5) . Endothelial cells, marked by Cldn5 , Plvap , and Epas1 , displayed enriched JAK-STAT and VEGF signaling (Fig. 1I, I’; Supplementary Fig. 1B, 2C; Supplementary Table 5) . Neuroglial cells, identified by the expression of Sox10 , Gfra3 , and Fabp7 , were characterized by increased VEGF signaling and reduced Wnt pathway activity (Supplementary Fig. 1B; Supplementary Table 5) . Single-cell analysis of dental mesenchyme identifies a distinct Lgr5⁺ progenitor-like population Next, we analyzed dental mesenchyme cells in more detail. Consistent with their mesenchymal identity, populations located outside the tooth germ expressed general mesenchymal markers, including vimentin ( Vim ), Col1a1 or Twist1 ( Fig. 2A, A’; Supplementary Table 6; Supplementary material 2 ). Further subclustering revealed distinct mesenchymal populations surrounding the tooth ( Fig. 2B, D ). Unspecified oral fibroblasts near the oral epithelium expressed Bhlhe22 , Cachd1 , and Tbx15 ( Fig. 2D, E, J, J’; Supplementary Fig. 2D-G; Supplementary Table 6 ). A cluster near the alveolar bone contained undifferentiated osteoblasts marked by Mfap5 , Meis2 , and Pamr1 ( Fig. 2D, F, K, K’; Supplementary Fig. 2H; Supplementary Table 6 ). Another clusters were identified as the outlying dental follicle (oDF) and the surrounding dental follicle (sDF). Markers of the oDF cluster, located in follicle layers distant from the tooth epithelium, included Aldh1a2, Alpl , and Runx2 ( Fig. 1D, H, M, M’; Supplementary Fig. 2I, J; Supplementary Table 6 ). The sDF cluster was marked by the cell adhesion protein Spon1 , along with Tfap2c and Ptch1 (Fig. 2D, I, N, N’; Supplementary Fig. 2K, L; Supplementary Fig. 4E; Supplementary Table 6) . The dental mesenchyme cluster identified at E18.5 in our study shares gene expression pattern with both earlier (E16.5, e.g., Aspn, Supplementary material 1 ) and later (P3.5, e.g., Igf1, Supplementary material 1 ) developmental stages also described previously 15 . However, this cluster also contains a substantial population of undifferentiated cells with limited transcriptional specificity, making them difficult to distinguish into defined subtypes. At E18.5, the dental follicle formed a well-defined cluster that could be further subdivided into two distinct populations located just adjacent to tooth or in larger distance: the surrounding follicle and the outlying follicle ( Fig. 2H, I ). This contrasts with earlier findings at E16.5, where follicular populations were classified just as lateral and apical 15 . Notably, some marker genes, such as Aldh1a2 , were expressed at both these stages; however, their spatial distribution differed. At E16.5, Aldh1a2 expression was restricted to the apical aspect of the tooth bell 15 , whereas by E18.5, its expression expanded to encircle the entire tooth bell without direct interaction with the tooth epithelium (Supplementary Fig. 2J) . Additionally, a mesenchymal cluster including Lgr5 + cells was identified outside the tooth germ, mainly on the labial side of the DS, with key markers including Tcea3 or Emb (Fig. 2D, G, L, L’; Supplementary Fig. 3A, B; Supplementary Table 6), which are usuallyassociated with cell fate determination during development or maintaining cell identity. Latent time analysis 36 revealed that the least differentiated cells were mainly located in the mesenchymal cluster enriched with Lgr5-positive cells (Fig. 1C) , suggesting that this population could serve as a stem cell reservoir. This finding aligns with the well-established role of LGR5 as a marker of stem and progenitor cells in various tissues 4,10–12 . Notably, despite their undifferentiated state, these cells exhibited minimal proliferative activity, as indicated by the low expression of proliferation-associated genes such as Ki67 and Cdk1 ( Supplementary Fig. 3C, D ). This observation suggests that these Lgr5 -positive mesenchymal cells may exist in a relatively quiescent state, a characteristic commonly associated with tissue-resident stem cells 37 . The low proliferative activity of these cells may be crucial for maintaining a reservoir of progenitors that can be activated in response to specific developmental or regenerative cues. Previously, LGR5-positive cells have been detected in the developing mouse dentition as early as at E14.5 38 , where they play a crucial role in the continuous growth of mouse incisors 4 . In these ever-growing teeth, stem and progenitor cells are essential for maintaining self-renewal and ensuring sustained development. The niche of these stem/progenitor cells was found to be located within the labial cervical loop (LCL), a specialized epithelial structure critical for incisor renewal 39 . However, our findings reveal a distinct population of LGR5-positive cells located in the mesenchyme, suggesting a broader role for these cells in dental development beyond their previously characterized niche. While significant attention is typically given to progenitor cells in the incisor epithelium, little is known about the potential presence of stem cells in mesenchymal populations. Therefore, we also examined LGR5-positive cells in the tooth surrounding tissues. This unexpected localization raises intriguing questions about the functional diversity of LGR5-positive cells in tooth morphogenesis and their potential involvement in epithelial-mesenchymal interactions. To sum up, the molar tooth and its surrounding tissues are composed of a variety of cell populations, including endothelial, glial, immune, and pre-osteoblastic cells. Among these, Lgr5 -positive cells form a distinct mesenchymal cluster localized on the labial side of the DS, with bioinformatic analyses supporting their identity as potential progenitor cells. Cellular subpopulations and molecular signatures of the DP during molar development Next, we focused on the DP cluster, which was clearly distinguishable from other mesenchymal populations while retaining the expression of core mesenchymal markers such as Msx1 ( Fig. 1D, 3A; Supplementary material 3 ). Notably, a subset of genes—including Rspo2 , Dlx4 , Dlx5 , Dkk1 , and Scube1 —was specifically enriched in the DP, with minimal or no expression in the surrounding mesenchyme, indicating a unique gene expression signature for this compartment ( Fig. 2A’; Supplementary Fig. 2M-P ). Further re-clustering of DP cells, performed at a resolution of 0.8—a parameter that controls the “granularity” of clustering 40 —revealed four distinct mesenchymal subpopulations (Fig. 3A, B) . The DP can be functionally divided into coronal, lateral, and apical zones ( Fig. 3B ), with regions of the coronal papilla displaying distinct expression profiles that reflect the differentiation status of their cells. The apical papilla (AP) is situated in the lower region of the papilla, adjacent to the follicular cell layer (Fig. 3B, C, G, G’; Supplementary Fig. 4A, E; Supplementary Table 7) . It expressed extracellular matrix (ECM) proteins such as Spon1 (a dental follicle marker), as well as Postn and Lhx6 . Additionally, we observed upregulation of JAK-STAT, TRAIL, and TNFα signaling pathways in the AP, whereas Wnt signaling was downregulated (Supplementary Fig. 4A) . The coronal papilla medium (CPm) was characterized by the expression of the transcriptional regulator Lmo1 , as well as Piezo2 and Crym (Fig. 3B, C, D, D’; Supplementary Fig. 4A, B; Supplementary Table 7) . This region also showed upregulation of signaling pathways such as VEGF, EGFR, and MAPK (Supplementary Fig. 4A) . In contrast, the upper coronal papilla (CPu) exhibited increased activity of the hypoxia pathway and expressed genes such as the cytoskeletal component Tubb3 , along with Gfra1 and Sct (Fig. 3B, C, E, E’; Supplementary Fig. 4A, C; Supplementary Table 7) . The lateral papilla (LP) was defined by the expression of Tac1 (encoding the precursor of the peptide hormone substance P), Snai2 , and Rgs3 , and by upregulation of Wnt and TGFβ signaling pathways (Fig. 3B, C, F, F’; Supplementary Fig. 4A, D; Supplementary Table 7) . Latent time analysis identified the LP and AP clusters as the most differentiated, whereas the coronal papilla cluster contained the least differentiated cell population (Supplementary Fig. 4B) . Our bioinformatic analysis of the DP subclusters revealed gene expression patterns that align with previously reported findings in mouse incisors. Notably, genes such as Dlx5 and Scube1 , previously identified in the dental pulp of mouse incisors 13 , were also prominently expressed in the molar DP, highlighting conserved molecular signatures across different tooth types. Similarly, markers of pre-odontoblasts, including Dkk1 and Fgf3 , were detected in both incisors and molars, suggesting shared early differentiation programs. In contrast, we did not observe expression of mature odontoblast markers in the molar, which is consistent with the E18.5 developmental stage, when odontoblasts have not yet fully differentiated. Interestingly, Tac1 , previously described as a specific marker of dental pulp adjacent to the lingual cervical loop in mouse incisors 13 , was also detected in a comparable region of the molar, near both cervical loops—suggesting a conserved spatial expression pattern across tooth types. Furthermore, consistent with earlier scRNA-seq data from E16.5 15 , which identified apical and coronal DP populations based on spatial location and transcriptional profiles, our analysis at E18.5 revealed an even greater degree of cellular heterogeneity, reflecting advanced differentiation and tissue specialization. In summary, our findings reveal a dynamic cellular hierarchy within the developing DP. The AP contains less differentiated cells, which appear to be translocated inward as development proceeds, while the coronal region harbors the most differentiated populations, showing molecular features consistent with a continuum from pre-odontoblasts to mature odontoblasts. Notably, we did not identify a distinct Lgr5 -positive population within the DP, suggesting that Lgr5 is not a marker of progenitor cells in this compartment. Re-clustering of molar epithelial cells identifies subpopulations with progenitor state The epithelial compartment of the molar formed a distinct cluster, clearly separated from other cell types (Fig. 1D; Supplementary material 4) . These cells contribute to key epithelial structures, including the enamel organ, the DS that connects the tooth to the oral epithelium, and the RSDL. Due to strong intercellular junctions, epithelial cells are typically underrepresented in scRNA-seq datasets compared to mesenchymal cells. Nonetheless, the epithelial clusters (Fig. 3H; Supplementary material 4) showed robust expression of epithelium-specific genes, including various keratins, Epcam , desmocollins, and cadherins (Fig. 3H’) . Re-clustering of epithelial cells at E18.5 revealed six distinct subpopulations (Fig. 3H, I; Supplementary material 4). Notably, canonical ameloblast markers such as Enam , Klk4 , and Gm17660, which are characteristic of mature ameloblasts in adult incisors 41 , were not detected, indicating that ameloblast differentiation in the molar epithelium remains incomplete at this developmental stage. Instead, we identified two major populations of Shh -positive cells corresponding to the stratum intermedium (SI) and pre-ameloblasts. The SI subcluster, localized to the coronal region, was defined by the expression of Shh , Maf , Hopx , Rhov , and Notch1 (Fig. 3I, J, K, K’, N, N’; Supplementary Fig. 4G, I, J; Supplementary Table 8). In contrast, the pre-ameloblast subcluster, positioned more apically, expressed Tubb3 , Wfdc2 , and Shh , suggesting a distinct yet closely related stage within the ameloblast lineage (Fig. 3I, J, K, K’; Supplementary Fig. 4G; Supplementary Table 8). Both epithelial subclusters (SI and pre-ameloblasts) exhibited upregulation of EGFR and hypoxia signaling pathways. Additionally, SI cells showed increased activity of Wnt and PI3K signaling pathways, suggesting their distinct functional roles during ameloblast lineage progression ( Supplementary Fig. 4F ). The stellate reticulum (SR) subcluster was marked by the expression of Eif1b , Smyd3 , and Pfn2 , though it exhibited greater transcriptional heterogeneity compared to other epithelial populations. This subcluster also showed upregulation of TRAIL and JAK-STAT signaling pathways, suggesting active involvement in immune-related and stress-responsive signaling during tooth development ( Fig. 3I, J, L, L’; Supplementary Fig. 4F, H; Supplementary Table 8 ). The Tyms-positive subcluster (Tyms+) was defined by the expression of Tyms , Birc5 , Pclaf , and various histone genes ( Fig. 3H, J; Supplementary Table 8 ). As expected, the proliferating cell subcluster was enriched for genes such as Mki67, Lars2 , and Arid1a , which are involved in cell cycle regulation, DNA replication, and transcriptional control ( Fig. 3H, J, O, O’; Supplementary Fig. 3D; Supplementary Table 8 ). The subcluster encompassing the outer enamel epithelium (OEE), DS, and RSDL displayed a shared transcriptional signature characterized by the expression of Notch2 , Frem2 , Vcan , Notch1 , and Maf , the latter also found in the SI. This subcluster showed specific activation of TGFβ, PI3K and MAPK signaling pathways, while Wnt signaling activity was markedly downregulated, suggesting a different regulatory environment controlling epithelial organization and signaling in these regions (Fig. 3I, J, M - N’; Supplementary Fig. 4F, I, J; Supplementary Table 8) . Latent time analysis revealed that most epithelial cells remain in an undifferentiated state, making it challenging to clearly distinguish individual dental epithelial subclusters (Supplementary Fig. 4H) . Next, we identified a small cluster of Lgr5 -positive epithelial cells (Fig. 3P) , characterized by elevated expression of ECM components, including Col8a1 , Lama3 , and Col4a5 (Supplementary Fig. 5A–D) . In addition to these ECM-related genes, this population also expressed several components of the Wnt signaling pathway, encompassing both canonical and non-canonical signaling branches such as Ptk7 , Tc f7l2 , Sfrp2 , Rock2 , Gpc3 , and Cdc42ep3 ( Supplementary Fig. 5E–L ) 58, 62 . Spatially, these Lgr5 -positive epithelial cells were positioned at the interface of three major epithelial subclusters: the SI, SR, and the cluster the OEE, DS, and RSDL. This molecular signature is consistent with their transitional location within the tooth germ, suggesting a specialized role in mediating signaling and structural integration across epithelial compartments. In summary, the developing tooth epithelium comprises diverse cell types defined by position and function. Our dataset captured key epithelial populations, including SR and DS cells, but lacked fully differentiated ameloblasts, as markers like Enam and Amelx were not expressed—likely reflecting the earlier developmental stage of molars compared to incisors. At E18.5, we primarily observed pre-ameloblasts and undifferentiated epithelial cells expressing markers such as Krt17 and Epcam ( Supplementary material 4 ), similar to profiles reported at E16.5, in contrast to the mature ameloblast expression signatures found at postnatal stages like P3.5 15 . Analysis of stem and progenitor cell markers expression in Lgr5-positive epithelial cells of mouse molars Given the presence of Lgr5 -positive cells across distinct epithelial and mesenchymal subclusters, and their unique localization within the tooth germ, we explored whether these cells might represent a stem or progenitor cell population—analogous to those identified in the LCL of continuously growing mouse incisors 39,42 . To date, a comparable stem cell population in the molar region has not been described, prompting us to investigate its potential existence. Notably, stem cells in the LCL are known to co-express Lgr5 , Lrig1 , and Sox2 13 . SOX2 has been identified as a marker of epithelial progenitor cells within the DL, where it plays a key role in the sequential development of mouse molars (M1, M2, and M3), as well as in the formation of the SDL and DS in human primary molars and in the DL of reptiles 38 . In the LCL of mouse incisors, LGR5- and SOX2-positive cells have been identified in a region known to support the continuous growth of these teeth 4,38 . Supporting its essential role in tooth development, mutations in SOX2 have been associated with dental anomalies in humans, including supernumerary teeth and the prolonged retention of deciduous teeth 43 . Single-cell RNA-seq analysis uncovered a Lgr5 -positive epithelial cluster enriched for stem cell signature genes such as Sox2 , Gli1 , Lrig1 , and Igfbp5 (Supplementary Fig. 5M–P) . To determine whether Lgr5 expression overlaps with the stem cell marker SOX2 at the protein level, we performed co-localization analysis in dental tissues. Since there are no suitable antibodies for the immunohistochemical detection of LGR5 in mouse tissue sections, we used EGFP expression as a surrogate marker for the identification of cells with an active Lgr5 locus. For this purpose, we used the LGR5-EGFP-IRES-CreERT2 mouse strain. We observed partial co-expression in the basal layers of the lingual part of the DS. SOX2 was detected as early as E14.5, corresponding with the onset of RSDL formation, and its expression expanded into the thickening rudimentary RSDL at E16.5–E18.5 (Fig. 3P-S) . In contrast, EGFP protein extended beyond the shared basal domain into deeper DS cells and the SR (Fig. 3Q-S, 4H’) . Taken together, these findings indicate that co-expression of LGR5 and SOX2 was restricted to small regions within the epithelium. This observation is consistent with previous studies suggesting that LGR5-positive cells represent only a subset of the broader SOX2-positive cell population 44 . Notably, LGR5 (EGFP)-positive epithelial cells in developing molars were found to be non-proliferative, reflecting the behavior of the adjacent LGR5-positive mesenchymal population. This was evidenced by the absence of Ki67 expression in both epithelial and mesenchymal LGR5-expressing domains (Supplementary Fig. 3C, D) . To further assess proliferative activity, we performed short-term BrdU labeling (2-hour pulse), which revealed only sparse BrdU incorporation within the LGR5-positive region of M1, suggesting that these cells are largely quiescent at this stage. In contrast, more pronounced BrdU labeling was observed in the DS and the forming RSDL of the developing M2, indicating higher proliferative activity in these earlier or less differentiated structures (Supplementary Fig. 3E–F’) . Together, these findings suggest that although LGR5 marks a subset of epithelial cells, they are not actively cycling and may instead represent a more specialized or quiescent population, rather than classical progenitor or transit-amplifying cells. Spatio-temporal mapping of Lgr5 expression during molar odontogenesis Next, we mapped the occurrence and spatial distribution of Lgr5-positive cells from early tooth initiation (E11.5) to postnatal stage (P4) covering key stages of molar odontogenesis using the LGR5-EGFP-IRES-CreERT2 mouse strain and used EGFP as a surrogate marker for Lgr5 expression (Fig. 4A–H’’). At E11.5, the EGFP signal was detected near epithelial thickening but not in the developing tooth placode (Fig. 4A–A’’) . At the bud stage (E12.5–E13.5), EGFP-positive cells appeared on the lingual side of the dental epithelium and in the buccal mesenchyme (Fig. 4B–C’’) . At the cap stage (E14.5–E15.5), expression was restricted to the lingual epithelium of the DS, the RSDL and the labial mesenchyme below the oral epithelium (Fig. 4D–E’’) . This pattern was maintained until the bell stage (E16.5–E18.5) (Fig. 4F–G’’) , although at E18.5 EGFP expression could no longer be detected in the rudimentary dental lamina. At P4, expression decreased further and was restricted to the epithelium of the degenerating DS and the adjacent labial mesenchyme (Fig. 4H-H’’) . Sagittal sections of the molar region confirmed a uniform expression pattern in both M1 and M2, with EGFP-positive cells mainly located in the posterior epithelial tail — an area implicated in sequential molar formation (Supplementary Fig. 6G) . To confirm that EGFP expression accurately reflected Lgr5 -expressing cells, we performed RNAScope analysis of Lgr5 mRNA. Consistent with the reporter signal, Lgr5 transcripts exhibited an asymmetric distribution in the DS and superficial mesenchyme at E16.5 and E18.5 (Supplementary Fig. 3A, B) , which was very similar to the EGFP pattern in LGR5-EGFP-IRES-CreERT2 mice. In summary, analysis of Lgr5 expression during molar odontogenesis - in a broader craniofacial context - reveals a dynamic and tightly regulated spatio-temporal pattern. Several previous studies have shown that LGR5-positive cells are not found in the classical epithelial stem cell niches of the craniofacial region, but predominantly in mesenchymal compartments adjacent to folding epithelial structures 4,37,45 . This suggests that LGR5 also marks a population of mesenchymal cells that are involved in the control of epithelial patterning and do not serve as direct epithelial progenitors. The distribution of LGR5-positive cells in the molar region possibly indicates their role in morphogenesis, particularly within the posterior epithelial tail. Lineage tracing reveals region-specific contribution of LGR5⁺ cells to molar development To assess whether LGR5-positive cells contribute to molar lineages as they exhibit their low proliferative activity typical for stem cells, we performed lineage tracing using Lgr5-EGFP-IRES-CreERT2 mice crossed with the Rosa26-tdTomato reporter line (Fig. 5A) . Expression of the red fluorescent protein tdTomato was induced by tamoxifen administration at E12.5, a stage when EGFP expression driven from the Lgr5 locus was detectable in the epithelial thickening of the tooth and in the labial mesenchyme (Fig. 2S, S’; LGR5⁺ cells, green signal) and embryos were harvested at two distinct developmental stages of M2 development (E15.5 and E18.5 - induction and progression of M2). By E18.5, a few LGR5-D) cells were observed (red signal) in the RSDL and SI of M1, whereas in M2, these cells were more prominent within the SR and RSDL (Fig. 5B’–B’’’, C’–C’’’) . Additionally, scattered LGR5-D cells were detected in the mesenchyme surrounding both M1 and M2, particularly in the region adjacent to the DS (Fig. 5B’, B’’, C’, C’’) . A shorter tracing interval (analysis at E15.5), revealed a higher number of LGR5-D cells in the DS, RSDL, and labial mesenchyme, with a spatial distribution similar to that observed at E18.5 (Supplementary Fig. 6A-F’’) . Interestingly, over time, an increasing number of LGR5-D cells were detected in the SR, inner enamel epithelium (IEE), and RSDL of M2, although these cells had lost EGFP expression by E18.5, indicating downregulation of Lgr5 . At E15.5, nearly all LGR5-D cells still expressed EGFP, confirming their recent origin from Lgr5 -expressing progenitors (Supplementary Fig. 6B-F’’) . Three-dimensional analysis further demonstrated a continuous LGR5 signal along the jaw, with the highest density of LGR5-D cells in the posterior region near M2 (Supplementary Fig. 6G) . Quantification of LGR5⁺ (EGFP⁺, green), LGR5-D (tdTomato⁺, red), and double-positive cells at two tracing intervals (E12.5–E15.5 and E12.5–E18.5) (Fig. 5D–G) revealed stable LGR5 expression within the epithelial compartments of both M1 and M2. In contrast, a significant reduction in LGR5⁺ cells was observed in the mesenchyme of M1 by E18.5 (Fig. 5F) . Over time, the proportion of LGR5-descendant cells increased, accompanied by a rise in double-positive cells in both molars. These results suggest that most LGR5⁺ cells remain within their original tissue niche (Fig. 5E–G) . To uncover temporal contributions of Lg5+ cells to molar development, we also label cells at E13, when individual tooth buds are advanced, and analyzed the outcome at E18.5. The results were consistent with previous findings (Supplementary Fig. 7A) , showing LGR5-D cells localized at the tip of the RSDL extending toward M2, as well as in the developing M3 region (Supplementary Fig. 7B, C) . Sagittal sections further revealed LGR5-D cells at the leading edge of the RSDL directed toward the second molar (Supplementary Fig. 7D, D’’) and in the distal tip of the lamina associated with M2, corresponding to the future third molar (Supplementary Fig. 7E, E’’) . Notably, the distribution of LGR5-positive cells (green, red, and double-positive) within the DS differed between the M1 and M2 regions, with more superficial localization observed near M2 (Supplementary Fig. 7D’, E’) . In summary , LGR5-positive cells primarily contribute to the DS and RSDL, with only a few descendants observed in the IEE of M1. Lineage tracing revealed a more substantial contribution of these cells to the formation of M2. While a subset of LGR5-positive descendants migrates posteriorly along the jaw, the majority remain confined to their original niche over time. This restricted contribution contrasts with fate-mapping studies in other organs, where LGR5-positive stem cells give rise to all epithelial cell types 17 . These findings are particularly relevant in the context of sequential tooth development. The posterior extension of LGR5-positive cells corresponds to the sites of future molar initiation (e.g., M2 and M3), supporting the hypothesis that LGR5-positive cells in the epithelial tail region may contribute to the formation of new tooth buds. Interestingly, while some LGR5-positive cells appear to remain stationary 37 , others exhibit limited migratory behavior—suggesting a dual function as both localized signaling centers and potential progenitors for emerging dental structures. Sorting of Lgr5-positive cells and subsequent scRNA-seq revealed several distinct subpopulations of Lgr5 + mesenchymal cells To further investigate the molecular characteristics of LGR5-positive cells, we isolated EGFP-positive cells from Lgr5-EGFP-IRES-CreERT2 mice at two developmental stages using flow cytometry: E16.5, when the RSDL is just beginning to form, and E18.5, when the RSDL has matured into a well-defined epithelial protrusion. Single-cell RNA-seq was performed separately for each sample ( Fig. 6A; Supplementary material 5) . Analysis of the individual datasets revealed that unspecified oral fibroblasts and early pre-osteoblastic clusters were more prevalent at E16.5. In contrast, E18.5 samples exhibited a notable expansion of dental follicle populations, and most GFP-positive cells at this stage showed signs of active proliferation (Fig. 6B, C) . To gain a comprehensive overview, we integrated both datasets and performed unsupervised clustering, identifying 11 distinct cell subpopulations. Among them, a prominent dental epithelial cluster was defined by the expression of canonical epithelial markers such as Epcam , various keratins, and other intermediate filament genes essential for epithelial integrity ( Supplementary material ). Gene Set Enrichment Analysis (GSEA) revealed upregulation of VEGF and EGFR signaling pathways in this epithelial population (Fig. 6D, E; Supplementary Table 9) . Moreover, comparison of signaling pathway activity between the E16.5 and E18.5 datasets revealed increased JAK-STAT signaling and a concomitant decrease in WNT pathway activity (Fig. 5G, H) . In agreement with our first "unbiased" E18.5 scRNA-seq analysis, two distinct dental follicle clusters were identified: the sDF, which comprised cells located close to the dental epithelium, and the oDF, which consists of cells located further away. These clusters differed not only in their spatial positioning within the molar region, but also in their gene expression profiles. The sDF cluster was characterized by the expression of Ptch1 , Car2 and Tnmd as well as by the upregulation of the TGFβ pathway. In contrast, the oDF cluster expressed Npnt , Gas6 and Mgp and showed enrichment of the Wnt, TRAIL and VEGF signaling pathways. Both clusters expressed canonical markers for dental follicles such as Runx2 and Alpl 46,47 . The TNFα signaling pathway was also upregulated in both follicle populations (Fig. 6D, E; Supplementary Table 9) . Another cluster consisted of actively proliferating cells, as shown by the expression of cell cycle–related genes such as Ki67 , Top2a and Pclaf (Fig. 6C, D; Supplementary Table 9) . A distinct labial mesenchymal cluster was enriched with LGR5-positive cells and expressed markers such as Sostdc1 , Tcea3 and Tfap2b . This cluster also showed activation of the Wnt and TGFβ signaling pathways, consistent with the increased expression of Lgr5. The remaining clusters consisted of different populations of differentiating osteoblasts and unspecified oral fibroblasts (Fig. 6D, E; Supplementary Table 9) . Latent time analysis also identified a population of less differentiated tooth-associated cells within the labial mesenchyme (Fig. 6F) , where most LGR5-expressing cells were localized. This supports the hypothesis that LGR5-positive cells exhibit an undifferentiated, progenitor-like phenotype and emphasizes the potential importance of mesenchymal cells in the early stages of tooth development. Comparison of pathway activity between the E16.5 and E18.5 data sets in the dental epithelium showed increased JAK-STAT signaling (data not shown) and a concomitant decrease in WNT pathway activity ( Fig. 6G, H ). Moreover, comparison of signaling pathway activity between the E16.5 and E18.5 datasets in the dental mesenchyme revealed a reduction in WNT signaling activity at E18.5 (Fig. 6I, J) . The transcriptome of Lgr5⁺ epithelial cells reflects their epithelial identity and their partially immature state Next, we focused specifically on the cluster representing Lgr5-positive cells within the dental epithelium. Since our sorting strategy selectively targeted LGR5-positive cells, we obtained enough of these cells, allowing for a more detailed characterization. This enrichment also enabled us to compare and distinguish the molecular features of epithelial Lgr5 -positive cells from their mesenchymal counterparts. Lgr5 -positive epithelial cells displayed a transcriptional signature consistent with epithelial identity, marked by high expression of multiple keratins ( Krt5 , Krt7 , Krt14 ), along with key epithelial markers such as Dsp , Dsc , Dapl1 , and Asprv1 . These cells were also defined by strong desmosome-mediated adhesion signatures, marked by elevated expression of Pkp3 , Dsp , Rab25 , and Cdh1 . Among the actin-associated genes, Fermt1 was particularly highly expressed, suggesting a role in cytoskeletal organization and integrin-mediated signal transduction. In addition, genes associated with ion channel activity, such as Tacstd2 , S100A14 and Fxyd3 , were increasingly expressed in this population, suggesting active membrane-associated signaling dynamics. Within this epithelial cluster, we also detected several stem cell-associated transcription factors, such as Sox2 , Sox6 , and Pitx2 , along with key cell cycle regulators including Sfn and Serpinb5 , suggesting a progenitor-like state with both proliferative potential and epithelial-specific functionality ( Supplementary Table 10 ). Likely reflecting their close proximity to the basement membrane, LGR5-positive epithelial cells showed strong expression of core basement membrane components, including Lama5 , Lamb3 , Lamc2 , and Col17a1 . Additionally, several signaling pathway regulators were specifically upregulated in epithelial LGR5-positive cells compared to their mesenchymal counterparts. Notably, these included Esrp1 (a regulator of epithelial-specific splicing), Spint2 (a serine protease inhibitor involved in maintaining epithelial integrity), and Jag2 (a Notch pathway ligand) (Supplementary Table 10) . In summary, scRNA-seq analysis revealed different transcriptional profiles in LGR5-positive cells, with clear differences between epithelial and mesenchymal populations. Epithelial LGR5-positive cells exhibited a relatively homogeneous molecular signature characterized by the enrichment of genes involved in basal membrane composition, cell adhesion, ion channel activity, integrin signaling, and extracellular matrix organization. In contrast, LGR5-positive mesenchymal cells formed several transcriptionally distinct subpopulations, indicating functional heterogeneity within this compartment. Overall, these results suggest that epithelial LGR5-positive cells play a key role in the maintenance of epithelial identity, structural integrity and specialized functions during tooth development. Lgr5 deficiency disrupts sequential molar development To investigate the functional role of LGR5 during molar development, we used a mouse model in which the Lgr5 gene was constitutively deleted. The Lgr5 knockout mice exhibit perinatal lethality primarily due to severe ankyloglossia of the tongue — a developmental defect characterized by impaired tongue motility — and associated dilatations of the gastrointestinal tract, as previously reported 45 . These phenotypic abnormalities result in early postnatal death, which limits the window of analysis to the embryonic and late prenatal stages. Therefore, our study focused on molar development before birth so that we could assess the morphologic and cellular consequences of Lgr5 deficiency at key stages of odontogenesis. For our analysis, we used the non-functional ( null ) Lgr5-EGFP-IRES-CreERT2 allele, which does not produce functional LGR5 protein, crossed into a homozygous background. (Fig. 7A) . We performed microscopic analysis of Lgr5 ⁻/⁻ embryos at E16.5 and E18.5, focusing on tooth morphology. Despite the early and localized expression of Lgr5 in M1 during odontogenesis, we did not observe any overt morphological abnormalities in this tooth germ (Fig. 7B–C’) . In contrast, M2 exhibited several morphological defects, including a shortened DS (Fig. 6L–O’) . The enamel organ appeared nearly fused with the oral epithelium, suggesting a disruption in the process of sequential molar formation. Additionally, in the labial region of the DS, we observed multiple epithelial outgrowths and finger-like projections extending into the mesenchyme, accompanied by a loss of structural compactness in the DS (Fig. 7L–O’). Furthermore, the expression of the stem cell marker SOX2 was markedly reduced in the DS region of Lgr5 -deficient mice in both M1 and M2, suggesting a potential loss of stem cell characteristics (Fig. 7D–E’, P–Q’) . We also detected ectopically localized SOX2-positive cells on the labial side of the DS, indicating a disruption of the normal polarized expression pattern of this stem cell marker (Fig. 7Q’) . Interestingly, previous studies have shown that LGR5 expression suppresses the formation of cellular extensions, while Lgr5 -deficient cells show the formation of cytopodia. In the colon, loss of Lgr5 impairs cell–cell adhesion and disrupts epithelial architecture 48 , while overexpression of LGR5 increases intercellular adhesion and decreases motility of colon cancer cells 49 . These results are consistent with our observations in Lgr5 -deficient embryos, in which we detected abnormal epithelial protrusions, finger-like extensions into the mesenchyme, and loss of structural integrity of the dental stalk, suggesting a conserved role of LGR5 in maintaining epithelial cohesion and organization. Lgr4 expression partially overlaps with Lgr5 in molars To investigate whether the different phenotypic results between M1 and M2 were due to functional redundancy between Lgr5 and its related paralogs Lgr4 and Lgr6, we analyzed their expression patterns in the developing molars (Fig. 7F, G, J, K, R, S, V, W; Supplementary Fig. 8) . In E16.5, Lgr4 was broadly expressed in the DS, surrounding mesenchyme, RSDL, IEE and SI of M1. In M2, Lgr4 was additionally detected in the SR, although this expression decreased until E18.5 (Supplementary Fig. 8A–C; G–I″) . Overall, the expression of Lgr4 was broader and more diffuse than that of Lgr5 , with comparable or even higher signal intensity. In contrast, the expression of Lgr6 was significantly weaker. At E16.5, Lgr6 -positive cells were sparsely distributed in the DS epithelium, surrounding mesenchyme and IEE of M1, while expression in the M2 epithelium was minimal (Supplementary Fig. 8D–F″) . At E18.5, Lgr6 expression had disappeared from the M1 epithelium and was only present as scattered signals in the mesenchyme, with weak expression in the IEE of M2 (Supplementary Fig. 8J–L″) . Minimal overlap between Lgr5 and Lgr6 was observed and was restricted to the labial mesenchyme and DS epithelium at E16.5 (Supplementary Fig. 8D″, E″, F″) . Because the expression of Lgr4 and Lgr5 partially overlap, we examined also the expression of Lgr4 in Lgr5-deficient mice. While the spatial pattern of Lgr4 remained largely unchanged, the expression of Lgr4 was reduced - especially in the second molar (Fig. 7R, S, V, W) . Overall, these results suggest that Lgr4 is the only Lgr5 paralog with overlapping expression in the DS, particularly in M1, where its presence may partially compensate for the loss of Lgr5. This is consistent with our phenotypic observations: In Lgr5-deficient mice, M1 appeared morphologically normal, whereas the defects were restricted to the M2. A similar compensatory role was hypothesized for Lgr4 in M3: in keratinocyte-specific Lgr4 knockout mice, developmental defects in the M3 occurred as early as at E14. In addition, Lgr4 knockout mice frequently exhibited underdeveloped or missing M3 50 . In addition, Lgr4 was expressed in SOX2-positive cells at the posterior end of M2 and in early M3 tooth germs, suggesting that it may support tooth initiation and compensate for Lgr5 loss. This functional redundancy is consistent with findings from other tissues such as the intestine, where deletion of Lgr5 does not affect epithelial self-renewal, presumably due to compensation by other members of the LGR family 51,52 . These parallels highlight the extensive functional overlap between the LGR receptors and their context-dependent role in development. Downstream Wnt signaling is impaired in the Lgr5-deficient dental epithelium LGR5 was originally identified as a Wnt target gene that is upregulated in human cancer cells with aberrant Wnt/β-catenin activity 53 . In adult tissues, Wnt signaling plays a critical role in the maintenance of somatic stem cells and committed progenitor compartments, as described in detail by Cadigan and Peifer (2009) 54 . Importantly, Lgr5 serves both as a transcriptional target of the canonical Wnt/β-catenin pathway and as a positive regulator that enhances Wnt signaling activity 52 . In the context of tooth development, Wnt signaling has been shown to have stage- and tissue-specific effects. For example, forced activation of Wnt signaling in the embryonic mesenchyme inhibits the formation of posterior molars (M2 and M3) 55 , while epithelial-specific activation of the Wnt pathway can stimulate successional dental lamina and lead to ectopic formation of second-generation molars 56 . These findings highlight the balance of Wnt signaling required for proper molar formation and suggest that disruption of components of the Wnt signaling pathway — such as Lgr5— can alter the normal course of molar development. To investigate the molecular mechanisms underlying disrupted tooth development in Lgr5-deficient mice, we examined whether the LGR5-Wnt signaling axis is activated by analyzing the expression of LGR4/5/6 ligands—R-spondin family members ( Rspo1 , Rspo2 , and Rspo3 )—at E18.5, a developmental stage at which the dental phenotype is already evident. Expression analysis of Rspo1 revealed a partial overlap with Lgr5 in the labial mesenchyme of the DS (Supplementary Fig. 9A–D) . The expression of Rspo2 was restricted to the DP and did not overlap with Lgr5 (Supplementary Fig. 2M) . In contrast, Rspo3 was expressed in the mesenchyme on the lingual side of the DS, adjacent to but not overlapping with Lgr5-positive cells in the DS and the RSDL (Supplementary Fig. 9E, G) . In Lgr5-deficient embryos, the expression of Rspo3 was markedly altered and exhibited a more diffuse distribution in both the dental mesenchyme and epithelium. Importantly, the distinct border of Rspo3 expression around the DS and RSDL observed in wild-type (WT) embryos was lost in the mutants. These changes were observed in both M1 and M2 (Supplementary Fig. 9E–H) . To investigate further changes in downstream Wnt signaling, we analyzed the expression of Axin2 , a known target of the Wnt/β-catenin pathway 57,58 . In WT embryos, Axin2 was mainly expressed in the mesenchyme surrounding the tooth at the bell stage and in the DP and SR of M1 (Fig. 7H, J) . In contrast, Axin2 expression was almost absent in the SR of M2 (Fig. 7T, V) . We also observed a partial overlap between Axin2 and Lgr4 and LGR5 expression, especially in the DS adjacent to the dental mesenchyme (Fig. 7D-U; Fig. 3W–Y’) . In both molars, Lgr5-deficient embryos showed significantly reduced Axin2 expression. In the mutants, Axin2 -positive cells formed only a thin layer surrounding the dental epithelium, whereas they were more widely distributed in WT embryos (Fig. 7I, K, U, W) . These results suggest that the absence of Lgr5 leads to a disruption of downstream Wnt signaling, which likely contributes to the observed defects in dental development. The altered expression patterns of Rspo3 and Axin2 indicate a dysregulated signaling environment and underscore the role of LGR5 in enhancing Wnt activity during molar morphogenesis. Lgr5 deficiency impairs the integrity of the dental epithelium by disrupting cell adhesion and ECM organization Previous studies have shown that increased Lgr5 expression inhibits the formation of cellular projections, while its absence promotes the formation of cytopodia 48 . Furthermore, overexpression of Lgr5 in colon cancer cells enhances intercellular adhesion and suppresses cell motility 49 . Given these findings and the epithelial protrusions observed in the DS of Lgr5-deficient embryos, we investigated whether loss of Lgr5 affects epithelial compactness. First, we examined the expression of ECM components that are important for epithelial cohesion and basement membrane stability (Supplementary Fig. 9) . In Lgr5-deficient embryos, laminin expression was significantly reduced along the developing tooth germ, suggesting that perturbations in ECM composition contribute to impaired structural cohesion within the DS (Supplementary Fig. 9I–L) . Next, we examined cell-cell adhesion molecules such as E-cadherin and β-catenin. In WT embryos, E-cadherin was strongly expressed in the DS and RSDL, with signal extending into the SR in both M1 and M2 (Supplementary Fig. 9M, O) . In contrast, in Lgr5-deficient embryos, E-cadherin expression was absent in the SR, indicating a loss of epithelial cohesion (Supplementary Fig. 9N, P) . A similar trend was observed for β-catenin (Supplementary Fig. 9Q–T) . Although β-catenin was present in both WT and Lgr5-deficient epithelium, its signal differed in terms of distribution and expression. In WT embryos, β-catenin was clearly localized at the cell membrane, whereas the signal was diffuse and poorly defined in both molars in the mutants (Supplementary Fig. 9Q–T) . Of note, β-catenin is also a major transcriptional activator of canonical Wnt signaling 59 , however, we did not detect nuclear localization of β-catenin, suggesting that the canonical Wnt signaling pathway is not strongly activated. These data suggest that Lgr5 plays a critical role in maintaining epithelial compactness by regulating ECM composition and intercellular adhesion. Loss of Lgr5 disrupts basement membrane organization and weakens epithelial integrity, likely contributing to the aberrant dental morphology in Lgr5-deficient embryos. However, the exact contribution of impaired Wnt signaling in the absence of Lgr5 to these epithelial defects will be necessary to follow in future. Candidate LGR5 interactors involved in epithelial-mesenchymal crosstalk during tooth development To identify potential LGR5 binding partners that could contribute to epithelial-mesenchymal adhesion and structural integrity in the dental stalk, we performed a comparative analysis. To this end, we compared our single-cell data from LGR5-positive cells — identified in small epithelial subclusters and in the dental mesenchyme (Fig. 6A) — with previously published RNA-seq data from LGR5-expressing craniofacial cells 37 . Given the structural homology and potential functional overlap between LGR5 and LGR4, we compared this list with known and putative LGR4 interactors identified by mass spectrometry (Supplementary Fig. 10A; V. Kriz and V. Korinek, unpublished results) . To refine the list of candidates, we filtered the genes based on predicted subcellular localization, prioritizing those associated with the plasma membrane or ECM. This analysis yielded several promising candidates, including Ptk7 , Hsp90b1 , Lgals1 (galectin-1) and Anxa1 , which were selected for further investigation (Supplementary Fig. 10) . We also included NID2, a basement membrane protein recently identified as an endogenous ligand of LGR4 and a regulator of vascular calcification 60 . Its paralog NID1 was also analyzed to investigate possible redundancies or complementary functions in the dental context (Supplementary Fig. 10O–W’) . We then examined the spatial expression patterns of these candidate genes within the dental epithelium and mesenchyme in our scRNA-seq dataset. The expression of Ptk7 was mainly detected in mesenchymal clusters, where it partially overlapped with the expression of Lgr5. Of note, Ptk7 was also found in a small epithelial LGR5-positive subcluster, suggesting that it may act at the epithelial-mesenchymal interface (Supplementary Fig. 10B, 11B, D) . In contrast, Lgals1 and Anxa1 were restricted to the epithelial LGR5-positive subcluster, suggesting a role within the epithelial compartment (Supplementary Fig. 10C–J) . Similarly, Hsp90b1 was expressed in both the LGR5-positive mesenchymal cluster and the epithelial subcluster, as shown by analyses 61 , suggesting a dual role in regulating epithelial and mesenchymal functions (Supplementary Fig. 10K–N) . Nid2 -positive cells were distributed in both epithelial and mesenchymal LGR5-positive populations. While NID2 was enriched in the IEE, some LGR5-positive cells in the DS and RSDL also expressed NID2 (Supplementary Fig. 10W, W’) . In contrast, the expression of NID1 was largely restricted to the mesenchyme and partially overlapped with Lgr5. Remarkably, the expression of NID1 in the DL region was localized at the interface between epithelium and mesenchyme, suggesting that signaling between these compartments is regulated (Supplementary Fig. 10V, V’) . Among these candidates, PTK7 stands out due to its established role in Wnt signaling, cell adhesion and tissue patterning. Its expression at the interface of LGR5-positive epithelial and mesenchymal domains makes it a strong candidate for future functional studies aimed at uncovering LGR5-mediated mechanisms of niche organization in the developing tooth. PTK7 as a downstream effector and binding partner of LGR5 in the regulation of dental tissue PTK7, a member of the receptor tyrosine kinase family, plays a central role in non-canonical Wnt signaling, particularly in the regulation of planar cell polarity and cell adhesion 62 . Although it has no intrinsic kinase activity, PTK7 can mediate extracellular signal transduction across the plasma membrane. The partial co-localization of PTK7 and LGR5 in the epithelium and mesenchyme of the DS suggests a possible functional interaction at the epithelial–mesenchymal interface. This observation in conjunction with the known functions of PTK7 makes it a strong candidate for LGR5-mediated signaling during tooth development (Supplementary Fig. 11A–B) . To investigate a possible direct interaction between LGR5 and PTK7, we performed in silico modeling using AlphaFold3 63 . The analysis predicted an interaction between the C-terminal domains of the two proteins — residues 563–907 of LGR5 and 601–1062 of PTK7 — that is stabilized by a disulfide bond. This binding likely occurs between Cys749 of LGR5 (Cys187 in the model) and Cys718 of PTK7 (Cys118 in the model), although an alternative pairing with Cys722 of PTK7 was also considered. The higher conservation of Cys718 suggests that it is the more likely interactor (Supplementary Fig. 11E) . We confirmed this prediction experimentally by performing co-immunoprecipitation assays with STREP-FLAG-tagged LGR5 and HA-tagged PTK7 in cell cultures. These experiments confirmed a physical connection between LGR5 and PTK7 in reciprocal pull-down experiments (Supplementary Fig. 11F) , providing direct biochemical evidence for their interaction. Immunohistochemistry at E18.5 showed that PTK7 was enriched in the epithelial compartment of M1 and M2, which overlapped with the LGR5 expression domain and extended from the DS into the RSDL (Supplementary Fig. 11G–L’) . The PTK7 protein was predominantly membrane-associated, particularly on the lateral and basal surfaces of the basal epithelial cells and was also detected in the adjacent mesenchyme. In Lgr5-deficient embryos, PTK7 expression was significantly reduced in the dental stalk epithelium—particularly in M2—in regions that normally express Lgr5. This suggests that LGR5 is required for proper membrane localization and stabilization of PTK7 during molar development (Supplementary Fig. 11H, H’, J–L’) . PTK7 is also known to interact with MMP14 (MT1-MMP), a membrane-associated matrix metalloproteinase. Proteolytic cleavage by MMP14 generates a soluble N-terminal fragment (~70 kDa) and a membrane-bound C-terminal fragment (~50 kDa) that modulates the function of PTK7 in Wnt signaling 64 . In agreement, we found strong co-localization of Lgr5- and MMP14-expressing cells in both mesenchymal and epithelial compartments, particularly in SOX2-positive Lgr5⁺ epithelial cells (Supplementary Fig. 11C–D) . Moreover, MMP14 expression was upregulated in Lgr5-deficient embryos, particularly in the DS and RSDL of M2, with de novo expression observed in the labial region (Supplementary Fig. 5M–P’) . These results suggest that LGR5 spatially restricts MMP14 activity and prevents excessive PTK7 cleavage. Taken together, our data support a model in which PTK7 functions as a critical LGR5-dependent effector at the epithelial–mesenchymal interface of the developing tooth. Its proper localization appears to be dependent on LGR5, and its activity seems to be tightly associated with MMP14-mediated cleavage. Disruption of this regulatory axis in Lgr5-deficient embryos compromises the integrity of the tissue within the dental stalk and contributes to the observed morphological abnormalities. These results reveal a novel mechanism by which LGR5 regulates cell adhesion, signal transduction and niche organization during early tooth development. The growth of molar organoids is independent of Lgr5⁺ epithelial cells Given the different cellular and molecular behavior of LGR5-positive cells in M1 and M2, we investigated whether these epithelial populations exhibit intrinsic differences relevant to sequential tooth development. To determine this, we genetically labeled Lgr5-positive cells and their progeny in utero by inducing pregnant Lgr5-EGFP-CreERT2 x Rosa26-tdTomato females at E12.5 with tamoxifen. We then isolated M1 and M2 tissues from E18.5 embryos and generated single-cell suspensions. We used one half of each sample to establish 3D dental organoid cultures of Lgr5 WT and heterozygous siblings (Fig. 8A) . The other half of each sample was used to determine the percentage of EGFP- and tdTomato-labeled epithelial cells in the isolated tissues by FACS analysis. In the heterozygous embryos, the percentage of labeled epithelial cells, i.e. EpCam-positive cells, varied, but we were able to detect EGFP-positive and EGFP/tdTomato double-positive cells in all samples. In general, the percentage of labeled cells was higher in the M2 epithelium, at E18.5 there were more EGFP single-positive than EGFP/tdTomato double-positive cells, and we could detect only a few tdTomato single-positive cells (Supplementary Fig. 12A) . Interestingly, after reconstitution of organoids from single cells in vitro, we could no longer detect EGFP-positive cells microscopically (Fig. 8B-I) and by FACS analysis (data not shown). The tdTomato-positive organoids, originally derived from EGFP/tdTomato double-positive cells, were maintained in constant numbers in culture over several passages. This observation suggests that epithelial cells forming molar organoids have lost their Lgr5 expression but are still able to proliferate and maintain the in vitro structures. To confirm that Lgr5 is dispensable for organoid formation and growth, we performed a lineage tracing in vitro with tamoxifen induction, followed by microscopic and flow cytometric analyzes of Lgr5 WT, heterozygous and knock-out cells. We isolated M1 and M2 tissues from E18.5 embryos of different genotypes, prepared 3D molar organoids and treated them with 4-hydroxytamoxifen (4-OHT) to induce CreERT2-mediated recombination. Organoids from Lgr5-deficient molars exhibited comparable growth dynamics, morphology and structural organization to their wild-type and heterozygous counterparts. Epithelial sphere formation — a hallmark of organoid integrity and viability — was maintained in all genotypes, with no significant differences in size, budding or compaction (Fig. 8J, L, N) . After 48 h of 4-OHT treatment, no EGFP- or tdTomato-positive cells were detected in the M1 organoids regardless of Lgr5 genotype (data not shown). In contrast, M2 organoids contained only a small number of tdTomato⁺/EGFP cells (<1% of live cells), which were observed in both heterozygous and Lgr5-deficient samples (Fig. 8K, M, O) . This confirms that while LGR5-positive progeny persists, the original LGR5⁺ population does not expand or maintain its identity in vitro. Immunostaining for Ki67 and SOX2 also confirmed that proliferation and stemness are similar in Lgr5-deficient and control organoids (Fig. 8P–S’) . These results suggest that Lgr5-expressing cells are not required for organoid formation or self-renewal in vitro and are likely lost during culture propagation. To confirm this, we compared the gene expression profiles between freshly isolated molar epithelium and organoid cultures by RT-qPCR. Relative quantification of signature genes associated with specific epithelial subclusters, as defined by our scRNA-seq data, revealed that the subpopulation comprising Lgr5-positive cells in the DS, RSDL and OEE is significantly underrepresented in organoids compared to native tissue (Supplementary Fig. 12B) . The maintenance of SOX2-positive cells, normal proliferative activity, and intact cellular organization in Lgr5-deficient organoids suggest that primary tooth germ development is largely independent of Lgr5-expressing cells. Rather than playing a central role in the formation of the first tooth germ, Lgr5 appears to regulate the development of subsequent tooth lamina. The organoid-based analyzes also suggest that Lgr5-positive epithelial cells differentiate into other functional epithelial subtypes at later stages of development. Lgr5 expression in the SDL of minipig embryos reveals its role in tooth replacement To investigate the spatial and temporal dynamics of Lgr5 expression during the development of the SDL in a species capable of full tooth replacement, we analyzed embryonic stages of the minipig. As a diphyodont mammal, the minipig offers a valuable model for studying the mechanisms underlying natural tooth succession. By examining the distribution of Lgr5 across different regions (replacing dental lamina and not replacing interdental area) (Fig. 9A - D) , we aimed to determine whether Lgr5 expression correlates with active SDL formation, epithelial organization, and potential sites of replacement tooth initiation. This cross-species comparison also allowed us to assess the conservation of Lgr5 -associated stem cell niches beyond the monophyodont mouse model (Fig. 9) . As in mice, Lgr5 expression in minipigs was observed in the mesenchyme adjacent to the DL in p3 and in the interdental area of the M1, particularly at the interface between the oral epithelium and the laminar structure (Fig. 9E, F) . Interestingly, Lgr5 exhibited region-specific variation along the jaw, closely mirroring the expression pattern of SOX2 and revealing two distinct expression profiles. In regions where the growing tip of the dental lamina lacked SOX2 expression, Lgr5 was robustly expressed ( Fig. 9G, J ). In contrast, areas where SOX2 expression extended to the laminar tip showed either weak or undetectable levels of Lgr5 ( Fig. 9E, H ), suggesting a reciprocal relationship between these two markers and potentially distinct functional states within the SDL. Furthermore, neither Lgr5 nor SOX2 expression was detected in the tip of lamina of the interdental region (Fig. 9F, I) . Given the distinct spatial relationship between Lgr5 and SOX2 within the SDL, we next assessed the localization of PTK7, a key regulator of planar cell polarity and Wnt signaling. PTK7-positive cells were prominently detected within the DL, particularly in regions exhibiting a compact epithelial morphology—hallmarks of sites poised to initiate the next generation of teeth ( Fig. 9M ). In contrast, small interdental laminae and those showing signs of regression lacked PTK7 expression within the epithelial compartment ( Fig. 9K–M ), further supporting its role in maintaining an active, tooth-inductive niche. Comparison with our mouse data revealed that Lgr5 was similarly absent in the rudimentary or regressing DL in both species. In minipigs, features such as disorganized interdental cells, absence of Lgr5 expression, and laminar regression closely mirrored the disrupted epithelial architecture seen in Lgr5 -deficient mice. These parallels support the idea that LGR5 is essential for maintaining the integrity of the epithelial stem cell niche within the SDL, underscoring its conserved role in tooth renewal across mammalian species. CONCLUSION Our study provides a comprehensive single-cell atlas of the developing mouse molar tooth and reveals distinct epithelial and mesenchymal populations, including a previously unrecognized, spatially restricted Lgr5⁺ cell domain on the lingual side of the DS. This domain partially overlaps with SOX2 expression and is conserved in diphyodontic minipigs, indicating the presence of a specialized epithelial tissue niche critical for sequential tooth formation. By combining scRNA-seq of FACS-sorted Lgr5⁺ cells at two key developmental stages — when the RSDL first emerges and when it becomes fully establishes — with lineage tracing approaches, we demonstrated that Lgr5 regulates tooth development through multiple mechanisms. In addition to its function as a Wnt target and modulator, Lgr5 contributes to epithelial cohesion and structural integrity via interactions with adhesion molecules such as PTK7. Functionally, Lgr5⁺ cells are important for the maintenance of RSDL stability, a key epithelial structure required for the formation of replacement teeth. Loss of Lgr5 in mice impairs Wnt signaling, resulting in decreased Axin2 expression, altered Rspo family distribution, structural defects in the RSDL, and ultimately failure of successive molar formation. In minipigs, which closely resemble human diphyodont dentition, Lgr5 expression is restricted to active, non-regressing SDL regions, correlating with SOX2 and PTK7 expression and supporting the role of Lgr5 in maintaining a tissue compartment suitable for tooth replacement. In humans, the canonical Wnt/β-catenin signaling pathway is fundamental for tooth initiation, morphogenesis and differentiation, with early perturbations causing congenital anomalies such as hypodontia and oligodontia 65 . Mutations in Wnt-associated genes, including AXIN2 , WNT10A , ROR2 , LEF1 and LRP6 , are closely associated with permanent tooth malformations, with WNT10A variants being the most commonly affected 66 . Although the role of LGR5 and LGR4 in human tooth anomalies is not yet fully understood, the detection of LGR5 in permanent odontoblasts — but not in the deciduous dental pulp — suggests a selective role in the second dentition 67 , which is consistent with our experimental results. Overall, our results suggest that Lgr5⁺ cells significantly regulate RSDL stability and sequential tooth formation. The parallels between the disruption of RSDL in Lgr5-deficient mice and laminar regression in minipigs suggest that LGR5-dependent signaling is an important component of tooth replacement biology in mammals. These results lay the foundation for future studies on the involvement of LGR5 in tooth replacement disorders in humans and make LGR5-positive stem cells promising targets for regenerative therapies aimed at restoring lost or defective teeth. Declarations A CKNOWLEDGEMENT This work was supported by the Czech Science Foundation (19-01205S, 22-02794S) and by the MEYS CR (CZ.02.1.01/0.0/0.0/15_003/0000460). MB research on odontogenesis is financed by Czech Ministry of Health (NW24-10-00204). PK and VB were supported by Czech Science Foundation (25-17360S). We also acknowledge the core facility CELLIM supported by the Czech-BioImaging large RI project (LM2023050 funded by MEYS CR) for their support with obtaining scientific data presented in this paper. We also acknowledge the Light Microscopy Core Facility, IMG, Prague, Czech Republic, supported by MEYS – LM2023050, MEYS – CZ.02.1.01/0.0/0.0/18_046/0016045 and MEYS – CZ.02.01.01/00/23_015/0008205, for their support with the light sheet and spinning disc imaging presented herein. ELIXIR CZ large RI is gratefully acknowledged, supported by MEYS - LM2023055, for bioinformatics support. Computational resources were provided by the e-INFRA CZ project (ID:90254), supported by the Ministry of Education, Youth and Sports of the Czech Republic Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Data availability statement The original contributions presented in the study are included in the article/Supplementary Material. All single-cell RNA sequencing data generated in this study have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession code GSEXXXXXX . The processed data and metadata supporting the findings of this study are available within the article and its Supplementary Information files. Additional information is available from the corresponding authors upon reasonable request. Ethics statement The mouse study was reviewed and approved by the Animal Care Committee of the Institute of Molecular Genetics, CAS, CR (Ref. 58/2017). All procedures on minipigs were conducted following a protocol approved by the Laboratory Animal Science Committee of IAPG (Approval No. 97/2011). References Juuri, E. et al. Sox2 marks epithelial competence to generate teeth in mammals and reptiles. Development (Cambridge) 140 , 1424–1432 (2013). Li, J. et al. BMP-SHH Signaling Network Controls Epithelial Stem Cell Fate via Regulation of Its Niche in the Developing Tooth. Dev Cell 33 , 125–135 (2015). Wang, X. P. et al. An integrated gene regulatory network controls stem cell proliferation in teeth. PLoS Biol 5 , 1324–1333 (2007). kawasaki, M. et al. R-spondins/Lgrs expression in tooth development. Developmental Dynamics 243 , 844–851 (2014). Putnová, I. et al. Angled growth of the dental lamina is accompanied by asymmetrical expression of the WNT pathway receptor frizzled 6. Front Physiol 8 , (2017). Grumolato, L. et al. Canonical and noncanonical Wnts use a common mechanism to activate completely unrelated coreceptors. Genes Dev 24 , 2517–2530 (2010). Grigoryan, T., Wend, P., Klaus, A. & Birchmeier, W. Deciphering the function of canonical Wnt signals in development and disease: Conditional loss- and gain-of-function mutations of β-catenin in mice. Genes Dev 22 , 2308–2341 (2008). Ahn, Y., Sanderson, B. W., Klein, O. D. & Krumlauf, R. Inhibition of Wnt signaling by wise (Sostdc1) and negative feedback from Shh controls tooth number and patterning. Development 137 , 3221–3231 (2010). Järvinen, E. et al. Continuous tooth generation in mouse is induced by activated epithelial Wnt/β-catenin signaling. Proc Natl Acad Sci U S A 103 , 18627–18632 (2006). Barker, N. & Clevers, H. Leucine-Rich Repeat-Containing G-Protein-Coupled Receptors as Markers of Adult Stem Cells. Gastroenterology 138 , 1681–1696 (2010). Huch, M. et al. In vitro expansion of single Lgr5 + liver stem cells induced by Wnt-driven regeneration. Nature 494 , 247–250 (2013). Koo, B. K. & Clevers, H. Stem cells marked by the r-spondin receptor LGR5. Gastroenterology 147 , 289–302 (2014). Krivanek, J. et al. Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth. Nat Commun 11 , (2020). Sharir, A. et al. A large pool of actively cycling progenitors orchestrates self-renewal and injury repair of an ectodermal appendage. Nat Cell Biol 21 , 1102–1112 (2019). Jing, J. et al. Spatiotemporal single-cell regulatory atlas reveals neural crest lineage diversification and cellular function during tooth morphogenesis. Nat Commun 13 , (2022). Hallikas, O. et al. System‐level analyses of keystone genes required for mammalian tooth development. J Exp Zool B Mol Dev Evol 336 , 7 (2020). Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449 , 1003–1007 (2007). Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13 , 133–140 (2010). Stancikova, J. et al. NKD1 marks intestinal and liver tumors linked to aberrant wnt signaling. Cell Signal 27 , 245–256 (2015). Susaki, E. A. et al. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc 10 , 1709–1727 (2015). Zheng, G. X. Y. et al. Massively parallel digital transcriptional profiling of single cells. Nat Commun 8 , (2017). Young, M. D. & Behjati, S. SoupX removes ambient RNA contamination from droplet-based single-cell RNA sequencing data. Gigascience 9 , (2020). Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184 , 3573-3587.e29 (2021). Hafemeister, C. & Satija, R. Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol 20 , (2019). Jollife, I. T. & Cadima, J. Principal component analysis: A review and recent developments. Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 374 , (2016). McInnes, L. et al. UMAP: Uniform Manifold Approximation and Projection for Dimension Reduction. ArXiv arXiv:1802.03426 (2018) doi:10.48550/ARXIV.1802.03426. McGinnis, C. S., Murrow, L. M. & Gartner, Z. J. DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors. Cell Syst 8 , 329-337.e4 (2019). Wickham, H. (2009) Ggplot2 Elegant Graphics for Data Analysis. 2nd Edition, Springer, New York. - References - Scientific Research Publishing. https://www.scirp.org/reference/referencespapers?referenceid=2097981. Alquicira-Hernandez, J. & Powell, J. E. Nebulosa recovers single-cell gene expression signals by kernel density estimation. Bioinformatics 37 , 2485–2487 (2021). Badia-I-Mompel, P. et al. decoupleR: ensemble of computational methods to infer biological activities from omics data. Bioinformatics Advances 2 , (2022). Türei, D. et al. Integrated intra‐ and intercellular signaling knowledge for multicellular omics analysis. Mol Syst Biol 17 , (2021). Garcia-Alonso, L., Holland, C. H., Ibrahim, M. M., Turei, D. & Saez-Rodriguez, J. Benchmark and integration of resources for the estimation of human transcription factor activities. Genome Res 29 , 1363–1375 (2019). La Manno, G. et al. RNA velocity of single cells. Nature 560 , 494–498 (2018). Bergen, V., Lange, M., Peidli, S., Wolf, F. A. & Theis, F. J. Generalizing RNA velocity to transient cell states through dynamical modeling. Nat Biotechnol 38 , 1408–1414 (2020). Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25 , 402–408 (2001). Abarda, A., Dakkon, M., Azhari, M., Zaaloul, A. & Khabouze, M. Latent Transition Analysis (LTA) : A Method for Identifying Differences in Longitudinal Change Among Unobserved Groups. Procedia Comput Sci 170 , 1116–1121 (2020). Olbertová, K. et al. Role of LGR5-positive mesenchymal cells in craniofacial development. Front Cell Dev Biol 10 , (2022). Juuri, E. et al. Sox2+ Stem Cells Contribute to All Epithelial Lineages of the Tooth via Sfrp5+ Progenitors. Dev Cell 23 , 317–328 (2012). Harada, H. et al. Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling. Journal of Cell Biology 147 , 105–120 (1999). Ding, S., Du, M. & Zhu, H. Survey on granularity clustering. Cogn Neurodyn 9 , 561 (2015). Hermans, F. et al. Establishment of inclusive single-cell transcriptome atlases from mouse and human tooth as powerful resource for dental research. Front Cell Dev Biol 10 , (2022). Thesleff, I., Wang, X. P. & Suomalainen, M. Regulation of epithelial stem cells in tooth regeneration. C R Biol 330 , 561–564 (2007). Numakura, C. et al. Supernumerary impacted teeth in a patient with SOX2 anophthalmia syndrome. Am J Med Genet A 152 , 2355–2359 (2010). Sanz-Navarro, J., Feal, C. & Dauden, E. tratamiento de la sarna en humanos con ivermectina oral. Erupciones eccematosas como nuevos efectos adversos no reportados. Actas Dermosifiliogr 108 , 643–649 (2017). Morita, H. et al. Neonatal Lethality of LGR5 Null Mice Is Associated with Ankyloglossia and Gastrointestinal Distension. Mol Cell Biol 24 , 9736–9743 (2004). Wen, Q. et al. Runx2 Regulates Mouse Tooth Root Development Via Activation of WNT Inhibitor NOTUM. Journal of Bone and Mineral Research 35 , 2252–2264 (2020). Kramer, K. et al. Dental defects in the primary dentition associated with hypophosphatasia from biallelic ALPL mutations. Bone 143 , (2021). Carmon, K. S. et al. LGR5 receptor promotes cell-cell adhesion in stem cells and colon cancer cells via the IQGAP1-Rac1 pathway. Journal of Biological Chemistry 292 , 14989–15001 (2017). Walker, F., Zhang, H. H., Odorizzi, A. & Burgess, A. W. LGR5 is a negative regulator of tumourigenicity, antagonizes wnt signalling and regulates cell adhesion in colorectal cancer cell lines. PLoS One 6 , (2011). Yamakami, Y. et al. LGR4 is required for sequential molar development. Biochem Biophys Rep 8 , 174 (2016). Fernandez Vallone, V. et al. LGR 5 controls extracellular matrix production by stem cells in the developing intestine . EMBO Rep 21 , (2020). De Lau, W. et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476 , 293–297 (2011). Van de Wetering, M. et al. The β-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 111 , 241–250 (2002). Cadigan, K. M. & Peifer, M. Wnt signaling from development to disease: insights from model systems. Cold Spring Harb Perspect Biol 1 , (2009). Järvinen, E., Shimomura-Kuroki, J., Balic, A., Jussila, M. & Thesleff, I. Mesenchymal Wnt/β-catenin signaling limits tooth number. Development (Cambridge) 145 , (2018). Popa, E. M., Buchtova, M. & Tucker, A. S. Revitalising the rudimentary replacement dentition in the mouse. Development (Cambridge) 146 , (2019). Leung, J. Y. et al. Activation of AXIN2 Expression by β-Catenin-T Cell Factor: A FEEDBACK REPRESSOR PATHWAY REGULATING Wnt SIGNALING. Journal of Biological Chemistry 277 , 21657–21665 (2002). Jho, E. et al. Wnt/β-Catenin/Tcf Signaling Induces the Transcription of Axin2, a Negative Regulator of the Signaling Pathway. Mol Cell Biol 22 , 1172–1183 (2002). Valenta, T., Hausmann, G. & Basler, K. The many faces and functions of Î 2-catenin. EMBO Journal 31 , 2714–2736 (2012). Chen, Y. et al. Nidogen-2 is a Novel Endogenous Ligand of LGR4 to Inhibit Vascular Calcification. Circ Res 131 , 1037–1054 (2022). Fan, J. et al. Characterizing transcriptional heterogeneity through pathway and gene set overdispersion analysis. Nat Methods 13 , 241 (2016). Kenny, T. C. & Germain, D. PTK7 Faces the Wnt in Development and Disease. Front Cell Dev Biol 5 , 31 (2017). Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630 , 493–500 (2024). Golubkov, V. S. et al. Internal Cleavages of the Autoinhibitory Prodomain Are Required for Membrane Type 1 Matrix Metalloproteinase Activation, although Furin Cleavage Alone Generates Inactive Proteinase. J Biol Chem 285 , 27726 (2010). Wang, B. et al. Expression patterns of WNT/β-CATENIN signaling molecules during human tooth development. J Mol Histol 45 , 487–496 (2014). van den Boogaard, M. J. et al. Mutations in wnt10a are present in more than half of isolated hypodontia cases. J Med Genet 49 , 327–331 (2012). Kim, J. H. et al. Distinctive genetic activity pattern of the human dental pulp between deciduous and permanent teeth. PLoS One 9 , (2014). Additional Declarations There is NO Competing Interest. Supplementary Files OlbertovaetalsupplementarymaterialSept13.2025.pdf Supplementary material Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7607583","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":518528098,"identity":"05ae4887-e25d-4f44-96a0-4a4431896c2e","order_by":0,"name":"Marcela Buchtova","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYBACAwYGNjCDn5mBgZl4LQeADMlmkrUYHCBWi7lE8rPHHyoOyxsfZ374uYDBzp6gFssZaeYGB84cNtx2mM1YegZDMmGbDG7ksEkcbEtj3HaYwYyZh+EAG9Fa7Dc3s38DaeEhVotN4gZmHrAtEoT90vPMTOLMGZvkGYd5iqV5DJINCGoxZ09+JlFRIWHb339842eeCiJCDN2dpGoYBaNgFIyCUYAVAACxdDNq7grSjwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-0262-6774","institution":"Institute of Animal Phisiology and Genetics, Czech Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Marcela","middleName":"","lastName":"Buchtova","suffix":""},{"id":518528099,"identity":"fc4d9381-660b-4854-8841-3114bb9165ed","order_by":1,"name":"Kristyna Olbertova","email":"","orcid":"","institution":"Institute of Animal Phisiology and Genetics, Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kristyna","middleName":"","lastName":"Olbertova","suffix":""},{"id":518528100,"identity":"eb9956cc-5604-40b8-8063-01e3fae74c76","order_by":2,"name":"Dusan Hrckulak","email":"","orcid":"https://orcid.org/0000-0002-1752-3612","institution":"Institute of Molecular Genetics, Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Dusan","middleName":"","lastName":"Hrckulak","suffix":""},{"id":518528101,"identity":"eb7b4921-6f36-4fad-ad1c-b2979cec7b65","order_by":3,"name":"Petra Kompanikova","email":"","orcid":"https://orcid.org/0000-0002-3742-4523","institution":"Masaryk University","correspondingAuthor":false,"prefix":"","firstName":"Petra","middleName":"","lastName":"Kompanikova","suffix":""},{"id":518528102,"identity":"fc997233-e677-4edf-a8cd-09d86fccde4c","order_by":4,"name":"Petr Taus","email":"","orcid":"","institution":"Masaryk University","correspondingAuthor":false,"prefix":"","firstName":"Petr","middleName":"","lastName":"Taus","suffix":""},{"id":518528103,"identity":"f536ec7f-7170-4a7d-92ed-dd483188a83f","order_by":5,"name":"Vitezslav Kriz","email":"","orcid":"https://orcid.org/0009-0002-9151-9889","institution":"Institute of Molecular Genetics, Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Vitezslav","middleName":"","lastName":"Kriz","suffix":""},{"id":518528104,"identity":"0376537a-30b8-411c-84da-ea8b568b17bc","order_by":6,"name":"Wojciech Jesionek","email":"","orcid":"","institution":"Masaryk University","correspondingAuthor":false,"prefix":"","firstName":"Wojciech","middleName":"","lastName":"Jesionek","suffix":""},{"id":518528105,"identity":"6cd50c57-ff2d-405c-b38d-737762549ff2","order_by":7,"name":"Marian Novotny","email":"","orcid":"","institution":"Charles University in Prague, Faculty of Science","correspondingAuthor":false,"prefix":"","firstName":"Marian","middleName":"","lastName":"Novotny","suffix":""},{"id":518528106,"identity":"9ae141b4-6fa5-4ed9-80cf-f9468e6d6fb9","order_by":8,"name":"Lucie Vrlikova","email":"","orcid":"","institution":"Institute of Animal Physiology and Genetics","correspondingAuthor":false,"prefix":"","firstName":"Lucie","middleName":"","lastName":"Vrlikova","suffix":""},{"id":518528107,"identity":"dc507369-5ecb-44a3-b8e4-46f4637da06c","order_by":9,"name":"Eva Hruba","email":"","orcid":"","institution":"Masaryk University, Department of Histology and Embryology, Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Eva","middleName":"","lastName":"Hruba","suffix":""},{"id":518528108,"identity":"226c3a89-7364-49e8-9092-4aadc8d566a4","order_by":10,"name":"Denisa Luskova","email":"","orcid":"","institution":"Institute of Animal Physiology and Genetics","correspondingAuthor":false,"prefix":"","firstName":"Denisa","middleName":"","lastName":"Luskova","suffix":""},{"id":518528109,"identity":"80c2648e-2615-408f-beb2-fbef85649056","order_by":11,"name":"Monika Stastna","email":"","orcid":"","institution":"Institute of Molecular Genetics","correspondingAuthor":false,"prefix":"","firstName":"Monika","middleName":"","lastName":"Stastna","suffix":""},{"id":518528110,"identity":"dc8cbf8a-e246-4856-b1ec-0707c1cf5506","order_by":12,"name":"Veronika Jakesova","email":"","orcid":"","institution":"Institute of Animal Physiology and Genetics","correspondingAuthor":false,"prefix":"","firstName":"Veronika","middleName":"","lastName":"Jakesova","suffix":""},{"id":518528111,"identity":"333a72c7-9e71-474a-b009-b55073c91eeb","order_by":13,"name":"Linda Berkova","email":"","orcid":"","institution":"Institute of Molecular Genetics","correspondingAuthor":false,"prefix":"","firstName":"Linda","middleName":"","lastName":"Berkova","suffix":""},{"id":518528112,"identity":"c159a366-9d9d-4898-bb0b-7f2cd8a6fa1b","order_by":14,"name":"Karla Plevova","email":"","orcid":"","institution":"Masaryk University","correspondingAuthor":false,"prefix":"","firstName":"Karla","middleName":"","lastName":"Plevova","suffix":""},{"id":518528113,"identity":"acd26f9a-6115-4074-ba3b-c6cf56f8c1d0","order_by":15,"name":"Jan Krivanek","email":"","orcid":"https://orcid.org/0000-0002-7590-187X","institution":"Faculty of Medicine, Masaryk University","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Krivanek","suffix":""},{"id":518528114,"identity":"6b9eb674-dbb0-44b5-9e45-86bf40a05fea","order_by":16,"name":"Jan Kubovčiak","email":"","orcid":"https://orcid.org/0000-0001-6446-9797","institution":"Laboratory of genomics and Bioinformatics","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Kubovčiak","suffix":""},{"id":518528115,"identity":"dbc856df-9b1f-4880-a8e7-b789b71659be","order_by":17,"name":"Michal Kolar","email":"","orcid":"https://orcid.org/0000-0002-4593-1525","institution":"Institute of Molecular Genetics of the Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Michal","middleName":"","lastName":"Kolar","suffix":""},{"id":518528116,"identity":"d8758e38-33dd-4990-89ef-69330d87c187","order_by":18,"name":"Vítězslav Bryja","email":"","orcid":"https://orcid.org/0000-0002-9136-5085","institution":"Masaryk University","correspondingAuthor":false,"prefix":"","firstName":"Vítězslav","middleName":"","lastName":"Bryja","suffix":""},{"id":518528117,"identity":"20e3a4f7-f3fe-4394-96cd-1d2720176a54","order_by":19,"name":"Milan Esner","email":"","orcid":"https://orcid.org/0009-0002-7659-7600","institution":"Central European Institute of Technology (CEITEC), Celluar Imaging Facility","correspondingAuthor":false,"prefix":"","firstName":"Milan","middleName":"","lastName":"Esner","suffix":""},{"id":518528118,"identity":"47c9f2e5-4113-4da7-a438-7d0f72e44a4b","order_by":20,"name":"Vladimir Korinek","email":"","orcid":"","institution":"Institute of Molecular Genetics, Academy of Sciences of the Czech Republic","correspondingAuthor":false,"prefix":"","firstName":"Vladimir","middleName":"","lastName":"Korinek","suffix":""}],"badges":[],"createdAt":"2025-09-13 13:15:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7607583/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7607583/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":92113794,"identity":"ebba2b33-c9a1-48e1-864d-452aed44f7c1","added_by":"auto","created_at":"2025-09-24 18:59:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2801763,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-cell RNA sequencing (scRNA-seq) analysis of the mouse molar region at embryonic day (E) 18.5.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematic illustration depicting the formation of the rudimental successional dental lamina and sequential dental lamina \u003cstrong\u003e(A)\u003c/strong\u003e. Diagram outlining the experimental workflow, from sample collection to single-cell processing and transcriptomic analysis \u003cstrong\u003e(B)\u003c/strong\u003e. Dot plot showing the expression patterns of selected marker genes across identified cell clusters \u003cstrong\u003e(C)\u003c/strong\u003e. Uniform Manifold Approximation and Projection (UMAP) plot displaying the distribution of transcriptomes from eight primary cell clusters, with each dot representing a single cell \u003cstrong\u003e(D)\u003c/strong\u003e. UMAP visualization of cell cycle phases across all clusters \u003cstrong\u003e(E)\u003c/strong\u003e. Distribution of \u003cem\u003eMki67\u003c/em\u003eexpression within the dataset, indicating proliferative cells \u003cstrong\u003e(F)\u003c/strong\u003e. Immunohistochemical validation of MKI67 expression in the first lower molar \u003cstrong\u003e(F’)\u003c/strong\u003e. \u003cstrong\u003e(G–I’)\u003c/strong\u003e Validation and spatial characterization of peripheral non-dental populations using expression of selected markers: immune cells (\u003cem\u003ePtprc\u003c/em\u003e, \u003cstrong\u003eG, G’\u003c/strong\u003e; white arrows), muscle cells (\u003cem\u003eMyod1\u003c/em\u003e, \u003cstrong\u003eH, H’\u003c/strong\u003e), and endothelial cells (\u003cem\u003eEpas1\u003c/em\u003e, \u003cstrong\u003eI, I’\u003c/strong\u003e; white arrows).\u003c/p\u003e\n\u003cp\u003eDf, dental follicle; dm, dental mesenchyme; dp, dental papilla; ds, dental stalk; La, labial; Li, lingual; rsdl, rudimental successional dental lamina; SeDL, sequential dental lamina; sr, stellate reticulum; scale bar: 100μm.\u003c/p\u003e","description":"","filename":"FIGURE1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7607583/v1/245d138acf229919117c5216.jpg"},{"id":92113795,"identity":"986c38d7-7319-458f-bd62-4700e6269b97","added_by":"auto","created_at":"2025-09-24 18:59:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9066475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClustering and characterization of the dental mesenchyme region.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUMAP visualization of single-cell transcriptomes highlighting dental mesenchyme clusters, with vimentin mRNA expression (\u003cem\u003eVim\u003c/em\u003e,\u003cem\u003e \u003c/em\u003e\u003cstrong\u003eA\u003c/strong\u003e) and vimentin protein staining (VIM, \u003cstrong\u003eA’\u003c/strong\u003e) marking mesenchymal cells. UMAP plot showing distinct dental mesenchymal subpopulations, excluding the DP \u003cstrong\u003e(B)\u003c/strong\u003e. Latent time analysis of dental mesenchyme indicating less differentiated cells (purple) and progressively more differentiated cells (yellow) \u003cstrong\u003e(C)\u003c/strong\u003e. Dot plot displaying the expression profiles of selected marker genes across mesenchymal subpopulations \u003cstrong\u003e(D)\u003c/strong\u003e. \u003cstrong\u003e(E–I)\u003c/strong\u003e Schematic illustration showing the spatial distribution of identified mesenchymal clusters in the molar region: unspecified oral fibroblasts \u003cstrong\u003e(E)\u003c/strong\u003e, undifferentiated osteoblasts \u003cstrong\u003e(F)\u003c/strong\u003e, Lgr5⁺ cells \u003cstrong\u003e(G)\u003c/strong\u003e, outlying dental follicle \u003cstrong\u003e(H)\u003c/strong\u003e, and surrounding follicular tissue \u003cstrong\u003e(I)\u003c/strong\u003e. \u003cstrong\u003e(J–N’)\u003c/strong\u003e Validation of individual subclusters based on marker gene expression: \u003cem\u003eTbx15\u003c/em\u003e (unspecified oral fibroblasts, \u003cstrong\u003eJ, J’\u003c/strong\u003e), \u003cem\u003ePamr1\u003c/em\u003e(undifferentiated osteoblasts, \u003cstrong\u003eK, K’\u003c/strong\u003e), \u003cem\u003eLgr5\u003c/em\u003e (Lgr5-positive cluster, \u003cstrong\u003eL, L’\u003c/strong\u003e), \u003cem\u003eAldh1a2\u003c/em\u003e (outlying dental follicle, \u003cstrong\u003eM, M’\u003c/strong\u003e), and \u003cem\u003eSpon1\u003c/em\u003e (surrounding dental follicle, \u003cstrong\u003eN, N’\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eDm, dental mesenchyme; dp, dental papilla; ds, dental stalk; lm, labial mesenchyme; OBs, osteoblasts; odf, outlying dental follicle; rsdl, rudimental successional lamina; sdf, surrounding dental follicle; sr, stellate reticulum; scale bar: 100μm.\u003c/p\u003e","description":"","filename":"FIGURE2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7607583/v1/86423df4737e2c90ac1789b7.jpg"},{"id":92112831,"identity":"60ecf00b-0435-4e79-97aa-6807a0f02dd3","added_by":"auto","created_at":"2025-09-24 18:51:50","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6299835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClustering and characterization of the DP and dental epithelium regions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUMAP visualization of DP subpopulations, including coronal papilla upper, coronal papilla medium, apical papilla, and lateral papilla, identified based on their spatial localization within the papilla \u003cstrong\u003e(A)\u003c/strong\u003e. Expression of \u003cem\u003eScube1\u003c/em\u003e as a marker of DP cells \u003cstrong\u003e(A’)\u003c/strong\u003e. Schematic representation of the spatial arrangement of DP subclusters in the molar region \u003cstrong\u003e(B)\u003c/strong\u003e. Dot plot showing expression profiles of selected marker genes across DP subpopulations \u003cstrong\u003e(C)\u003c/strong\u003e. \u003cstrong\u003e(D–G’)\u003c/strong\u003e Validation of distinct papilla subclusters using marker gene expression: \u003cem\u003eLmo1\u003c/em\u003e \u003cstrong\u003e(D, D’)\u003c/strong\u003e, \u003cem\u003eTubb3\u003c/em\u003e \u003cstrong\u003e(E)\u003c/strong\u003e, TUBB3 \u003cstrong\u003e(E’)\u003c/strong\u003e, \u003cem\u003eTac1\u003c/em\u003e \u003cstrong\u003e(F, F’)\u003c/strong\u003e, and \u003cem\u003eSpon1\u003c/em\u003e \u003cstrong\u003e(G, G’)\u003c/strong\u003e. UMAP visualization of dental epithelial subpopulations \u003cstrong\u003e(H’)\u003c/strong\u003e. Schematic representation of dental epithelial subclusters in the molar region \u003cstrong\u003e(I)\u003c/strong\u003e. Dot plot displaying expression of selected genes across epithelial populations \u003cstrong\u003e(J)\u003c/strong\u003e.\u003cbr\u003e\n \u003cstrong\u003e(K–O’)\u003c/strong\u003e Validation of distinct epithelial subclusters based on marker gene expression: \u003cem\u003eShh\u003c/em\u003e \u003cstrong\u003e(K, K’),\u003c/strong\u003e \u003cem\u003ePfn2\u003c/em\u003e \u003cstrong\u003e(L, L’)\u003c/strong\u003e, \u003cem\u003eNotch2\u003c/em\u003e \u003cstrong\u003e(M)\u003c/strong\u003e, NOTCH \u003cstrong\u003e(M’)\u003c/strong\u003e, \u003cem\u003eMaf\u003c/em\u003e \u003cstrong\u003e(N)\u003c/strong\u003e, MAF \u003cstrong\u003e(N’)\u003c/strong\u003e, \u003cem\u003eMki67 \u003c/em\u003e\u003cstrong\u003e(O)\u003c/strong\u003e, MKI67 \u003cstrong\u003e(O’)\u003c/strong\u003e. Distribution of LGR5- and SOX2-positive cells in the dental epithelium \u003cstrong\u003e(P)\u003c/strong\u003e. \u003cstrong\u003e(Q–S)\u003c/strong\u003e Immunohistochemical analysis of LGR5 and SOX2 co-expression at E14.5 \u003cstrong\u003e(Q)\u003c/strong\u003e, E16.5 \u003cstrong\u003e(R)\u003c/strong\u003e, and E18.5 \u003cstrong\u003e(S)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eAp, apical papilla; cpm, coronal papilla medium; cpu, coronal papilla upper; de, dental epithelium; dp, dental papilla; ds, dental stalk; lm, labial mesenchyme; lp, lateral papilla; oee, oral enamel epithelium; pre-Am, pre-Ameloblasts; rsdl, rudimental successional dental lamina; si, stratum intermedium; sr, stellate reticulum; scale bars: 100 μm.\u003c/p\u003e","description":"","filename":"FIGURE3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7607583/v1/1849f62c5292cad2bb193142.jpg"},{"id":92112824,"identity":"fd6591d6-fd55-49e4-9f18-19e7130fcf2a","added_by":"auto","created_at":"2025-09-24 18:51:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4789443,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatio-temporal distribution of LGR5-positive cells during molar odontogenesis in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluorescence detection of LGR5 expression (green) in the developing lower molars of mice at key embryonic and early postnatal stages (E11.5–P4). The nuclei are counterstained with DAPI; (blue). Upper panels \u003cstrong\u003e(A–H)\u003c/strong\u003e: Overview images of transversal jaw sections highlighting regions with LGR5-positive cells. Middle panels \u003cstrong\u003e(A’-H’)\u003c/strong\u003e: Magnified views showing the specific localization of LGR5-expressing cells within epithelial and mesenchymal structures The dashed lines indicate the border of the dental epithelium. Bottom \u003cstrong\u003e(A’’-H’’)\u003c/strong\u003e: Schematic diagrams showing the position of LGR5-positive cells (green dots) at the corresponding developmental stages of molar formation.\u003c/p\u003e\n\u003cp\u003eDe, dental epithelium; dm, dental mesenchyme; dp, dental papilla; ds, dental stalk; dt, epithelium thickening; ek, enamel knot; etb, early tooth bell; lm, labial mesenchyme; ltb, late tooth bell; oc, oral cavity; ps, palatal shelve; rsdl, rudimental successional dental lamina; sr, stellate reticulum; tbu, tooth bud; tc, tooth cap; scale bars: 100 μm.\u003c/p\u003e","description":"","filename":"FIGURE4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7607583/v1/34d93ec8858c9bfd2c62a584.jpg"},{"id":92112829,"identity":"06d3fe0f-d63b-407b-8cd3-a54cbde68495","added_by":"auto","created_at":"2025-09-24 18:51:50","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3533307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLineage tracing of LGR5⁺ cell populations during molar development.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental design for lineage tracing using \u003cem\u003eLgr5-EGFP-IRES-CreERT2 x Rosa26-tdTomato\u003c/em\u003e mice. LGR5-expressing cells (EGFP⁺, green) activate CreER2 upon tamoxifen administration (E12.5), excising the STOP cassette in the \u003cem\u003eRosa26-tdTomato\u003c/em\u003e locus and permanently labeling the cell and its descendants with tdTomato (red). Cells that continue to express \u003cem\u003eLgr5\u003c/em\u003e are double-labeled (EGFP⁺/tdTomato⁺, yellow). Cells that stop to express \u003cem\u003eLgr5\u003c/em\u003e are single-labeled (EGFP\u003csup\u003e-\u003c/sup\u003e/tdTomato⁺, red). Samples were collected at E18.5 for analysis \u003cstrong\u003e(A)\u003c/strong\u003e. \u003cstrong\u003e(B–C’’’)\u003c/strong\u003e Sagittal sections of the developing M1 and M2 molar at E18.5 showing the spatial distribution of LGR5⁺ cells (EGFP, green), LGR5-D cells (tdTomato, red), and double-positive cells (yellow). In M1, LGR5⁺ and LGR5-D cells were detected in the epithelium of the lingual side of the DS and RSDL \u0026nbsp;\u003cstrong\u003e(B, B’’, B’’’, E)\u003c/strong\u003e and in the labial mesenchyme adjacent to the stalk \u003cstrong\u003e(B, B’’, C, C’’, F)\u003c/strong\u003e. In M2, most labeled cells were found in the DS epithelium and RSDL, with occasional descendants detected in the SR and IEE \u003cstrong\u003e(C’, C’’, C’’’, G)\u003c/strong\u003e. Example of cell segmentation used for quantification of LGR5⁺, LGR5-D, and double-positive cells \u003cstrong\u003e(D)\u003c/strong\u003e. \u003cstrong\u003e(E–G)\u003c/strong\u003e Transversal sections and quantification of labeled cells across two tracing intervals (E12.5–E15.5 and E12.5–E18.5) in the epithelium of M1 \u003cstrong\u003e(E)\u003c/strong\u003e, the mesenchyme of M1 \u003cstrong\u003e(F)\u003c/strong\u003e, and M2 \u003cstrong\u003e(G)\u003c/strong\u003e. Data are shown as a percentage of total cells per region.\u003c/p\u003e\n\u003cp\u003eDp, dental papilla; ds, dental stalk; lm, labial mesenchyme; M1, first molar; M2, second molar; rsdl, rudimental successional dental lamina; sr, stellate reticulum; scale bars: 100 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis of GFP, RFP, and double-positive cell populations in M1 and M2 epithelium and mesenchyme. \u003c/strong\u003eNormality and lognormality were tested using the Shapiro–Wilk test. \u003cstrong\u003eM1 epithelium:\u003c/strong\u003e Comparison of GFP signal between E12.5–E18.5 and E12.5–E15.5 (unpaired \u003cem\u003et\u003c/em\u003e-test) showed no significant difference (\u003cem\u003eP\u003c/em\u003e = 0.1118). RFP signal (unpaired \u003cem\u003et\u003c/em\u003e-test with Welch’s correction) was significantly different (*\u003cem\u003eP\u003c/em\u003e = 0.0019). Overlap analysis (Mann–Whitney test) was also significantly different (*\u003cem\u003eP\u003c/em\u003e = 0.0072). \u003cstrong\u003eM1 mesenchyme:\u003c/strong\u003e GFP signal (Mann–Whitney test) was significantly different (**\u003cem\u003eP\u003c/em\u003e = 0.0004). RFP signal (unpaired \u003cem\u003et\u003c/em\u003e-test) was significantly different (*\u003cem\u003eP\u003c/em\u003e = 0.0049). Overlap analysis (Mann–Whitney test) showed no significant difference (\u003cem\u003eP\u003c/em\u003e = 0.0709). \u003cstrong\u003eM2 epithelium:\u003c/strong\u003e GFP signal (Mann–Whitney test) showed no significant difference (\u003cem\u003eP\u003c/em\u003e = 0.0874). RFP signal (unpaired \u003cem\u003et\u003c/em\u003e-test with Welch’s correction) was significantly different (**\u003cem\u003eP\u003c/em\u003e = 0.0005). Overlap analysis (unpaired \u003cem\u003et\u003c/em\u003e-test) was significantly different (*\u003cem\u003eP\u003c/em\u003e = 0.0007).\u003c/p\u003e","description":"","filename":"FIGURE5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7607583/v1/e7c7ba2bb136ed83a5bbe96d.jpg"},{"id":92112825,"identity":"4522f18b-f1e0-415c-96fc-971c0ff7241e","added_by":"auto","created_at":"2025-09-24 18:51:50","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2369586,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-cell RNA-seq of GFP⁺ cells from the molar region at E16.5 and E18.5\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental workflow illustrating the collection of molar regions, dissociation into single-cell suspensions, FACS isolation of GFP⁺ cells, and subsequent scRNA-seq analysis \u003cstrong\u003e(A)\u003c/strong\u003e. UMAP visualization showing the merged datasets of GFP⁺ cells from embryonic day (E) 16.5 (blue) and E18.5 (red) \u003cstrong\u003e(B)\u003c/strong\u003e. UMAP representation of cell cycle phases (G1, S, G2/M) among GFP⁺ cells \u003cstrong\u003e(C)\u003c/strong\u003e. Clustering of transcriptomes reveals 11 major cellular populations, including dental epithelium, labial mesenchyme, proliferating cells, fibroblasts, and pre-osteoblast subclusters \u003cstrong\u003e(D)\u003c/strong\u003e. Heatmap displaying the activity of key signaling pathways across identified clusters (rows: pathways; columns: cell types) \u003cstrong\u003e(E)\u003c/strong\u003e. Latent time analysis indicating cellular differentiation states, with early progenitor-like cells localized in labial mesenchyme (purple; enriched for Lgr5 expression) and more differentiated populations in pre-osteoblast clusters (yellow) \u003cstrong\u003e(F)\u003c/strong\u003e. \u003cstrong\u003e(G, I)\u003c/strong\u003e Heatmaps comparing pathway activity scores in dental epithelial cells \u003cstrong\u003e(G)\u003c/strong\u003e and labial mesenchyme \u003cstrong\u003e(I)\u003c/strong\u003e between E16.5 and E18.5. \u003cstrong\u003e(H, J)\u003c/strong\u003e Genes driving changes in WNT signaling activity (log₂ fold change), such as \u003cem\u003eTiam1\u003c/em\u003e, which is downregulated at E16.5 compared to E18.5, and \u003cem\u003eCdk5rap3\u003c/em\u003e and \u003cem\u003eSh3bp4\u003c/em\u003e, which are downregulated at E18.5 compared to E16.5, are involved in the dental epithelium \u003cstrong\u003e(H)\u003c/strong\u003e. In the labial mesenchyme \u003cstrong\u003e(J)\u003c/strong\u003e, \u003cem\u003eFhl1\u003c/em\u003e is downregulated at E16.5 relative to E18.5; additionally, \u003cem\u003eβ-catenin\u003c/em\u003eand \u003cem\u003eNcam\u003c/em\u003e are upregulated at E18.5, while \u003cem\u003eMsx1\u003c/em\u003e is downregulate at E18.5 compared to E16.5.\u003c/p\u003e","description":"","filename":"FIGURE6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7607583/v1/0838469516cb0a285265ba3a.jpg"},{"id":92112827,"identity":"cfbe46b5-6e85-4bd2-bcf8-fc773f8999aa","added_by":"auto","created_at":"2025-09-24 18:51:50","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2708886,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular and molecular alterations in molars of Lgr5-deficient mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematic representation of the breeding strategy used to generate Lgr5-deficient (KO) mice by crossing heterozygous \u003cem\u003eLgr5-EGFP-IRES-CreERT2\u003c/em\u003e animals \u003cstrong\u003e(A)\u003c/strong\u003e. \u003cstrong\u003e(B–C’)\u003c/strong\u003e Hematoxylin–Eosin staining of transverse sections of M1 at E18.5 in wild-type (WT) and Lgr5-deficient embryos showing no overt morphological defects. \u003cstrong\u003e(D–E’)\u003c/strong\u003e Immunohistochemistry for SOX2 demonstrates reduced and spatially altered expression in the DS epithelium of Lgr5-deficient M1 compared to WT controls. \u003cstrong\u003e(F–G)\u003c/strong\u003e RNA scope analysis of \u003cem\u003eLgr4\u003c/em\u003e shows comparable expression between WT and mutant M1. \u003cstrong\u003e(H–I)\u003c/strong\u003e RNA scope analysis of \u003cem\u003eAxin2\u003c/em\u003ereveals strong and widespread expression in WT M1, including the DS, RSDL and SR, whereas expression is markedly reduced and limited in mutants. \u003cstrong\u003e(J–K)\u003c/strong\u003e Schematic summary of \u003cem\u003eLgr4\u003c/em\u003e and \u003cem\u003eAxin2\u003c/em\u003e expression patterns in WT and Lgr5-deficient M1. \u003cstrong\u003e(L–M)\u003c/strong\u003e Diagram illustrating morphological abnormalities in M2 of Lgr5-deficient embryos, including a shorter, thicker DS and abnormal epithelial protrusions on the labial side. \u003cstrong\u003e(N–O’)\u003c/strong\u003e Hematoxylin–Eosin staining of M2 transverse sections highlighting morphological differences between WT and mutant embryos. \u003cstrong\u003e(P–Q’)\u003c/strong\u003e SOX2 expression in Lgr5-deficient M2 is reduced and ectopically localized on the labial side of the DS compared to WT controls. \u003cstrong\u003e(R–S)\u003c/strong\u003e \u003cem\u003eLgr4\u003c/em\u003eexpression shows no marked differences between WT and KO M2. \u003cstrong\u003e(T–U)\u003c/strong\u003e \u003cem\u003eAxin2\u003c/em\u003eexpression is reduced in Lgr5-deficient M2 compared to WT, where it is normally detected in the surface epithelium of the DS, RSDL, and oral epithelium. \u003cstrong\u003e(V–W)\u003c/strong\u003e Schematic representation summarizing \u003cem\u003eLgr4\u003c/em\u003e and \u003cem\u003eAxin2\u003c/em\u003e expression patterns in M2 of WT and Lgr5-deficient embryos.\u003c/p\u003e\n\u003cp\u003eDf, dental follicle; dp, dental papilla; ds, dental stalk; lm, labial mesenchyme; oe, oral epithelium; rsdl, rudimental successional dental lamina; sr, stellate reticulum; scale bars: 100 μm.\u003c/p\u003e","description":"","filename":"FIGURE7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7607583/v1/117efc0036c3616473220b64.jpg"},{"id":92113796,"identity":"ae70f4f8-de7e-419d-b8c2-db9e801a5949","added_by":"auto","created_at":"2025-09-24 18:59:50","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":10400352,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression and functional role of Lgr5 in molar organoid cultures.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematic representation of lineage tracing of Lgr5-positive cells from in vivo recombined molar epithelium used to establish organoid cultures (\u003cstrong\u003eA\u003c/strong\u003e). \u003cstrong\u003e(B–I\u003c/strong\u003e) Organoids were derived from single-cell suspensions of M1 and M2 molar epithelium dissected from E18.5 \u003cem\u003eLgr5-EGFP-CreERT2 x Rosa26-tdTomato\u003c/em\u003eembryos 6 days after tamoxifen induction. tdTomato fluorescence was detected in wild-type (WT) and heterozygous (HET) samples after the first passage, whereas EGFP signal was absent. Detailed imaging of individual organoids was performed using spinning disc microscopy. \u003cstrong\u003e(J–O)\u003c/strong\u003e In vitro lineage tracing was performed by 4-OHT induction in \u003cem\u003eLgr5\u003c/em\u003e WT, HET, and knockout (KO) organoids. Bright-field microscopy showed no morphological differences between genotypes. FACS analysis 48 hours post-induction revealed no EGFP-positive cells and only a few tdTomato-positive cells in HET and KO samples. \u003cstrong\u003e(P–S’)\u003c/strong\u003eImmunostaining for MKI67 (proliferation marker) and SOX2 (stem cell marker) showed comparable expression intensity and spatial distribution between HET and KO organoids.\u003c/p\u003e","description":"","filename":"FIGURE8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7607583/v1/81257dbdc26374469c07c871.jpg"},{"id":92114195,"identity":"cd1c119b-38c0-4f6e-86bc-e4ea5c6af5a9","added_by":"auto","created_at":"2025-09-24 19:07:50","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":3837555,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLgr5\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, SOX2 and PTK7 in the different areas of the dental lamina in the diphyodontic dentition of the minipig.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe diagram shows the successive tooth development in the diphyodontic dentition of the minipig at E50 \u003cstrong\u003e(A)\u003c/strong\u003e. Hematoxylin-Eosin staining of sagittal section of the p3 \u003cstrong\u003e(B)\u003c/strong\u003e, the interdental area of the M1 \u003cstrong\u003e(C)\u003c/strong\u003eand the dental area of the M2 \u003cstrong\u003e(D)\u003c/strong\u003e. \u003cem\u003eLgr5\u003c/em\u003e expression in the epithelium of the dental lamina and in the surrounding mesenchyme of the third premolar \u003cstrong\u003e(E)\u003c/strong\u003e. Low expression of \u003cem\u003eLgr5\u003c/em\u003e is found in the mesenchyme of the interdental region between M1 and M2 \u003cstrong\u003e(F)\u003c/strong\u003e, while high \u003cem\u003eLgr5\u003c/em\u003e expression is observed in the mesenchyme and at the tip of the epithelium of the dental lamina of the second molar region \u003cstrong\u003e(G)\u003c/strong\u003e. SOX2 expression in oral epithelium and at the tip of the dental lamina of the third premolar \u003cstrong\u003e(H).\u003c/strong\u003e Several SOX2-positive cells are observed in the dental lamina of the interdental are of the M1 \u003cstrong\u003e(I)\u003c/strong\u003e, with SOX2 expression present in the epithelium of the dental area of M2, but not at its tip \u003cstrong\u003e(J)\u003c/strong\u003e. PTK7 expression in the mesenchyme of the dental lamina is observed in the third premolar \u003cstrong\u003e(K)\u003c/strong\u003e and interdental area of the M1 \u003cstrong\u003e(L)\u003c/strong\u003e. In addition, PTK7 expression in the second molar region is found both in the mesenchyme surrounding the dental lamina and in the oral basal layer of the dental lamina \u003cstrong\u003e(M)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eDl, dental lamina; M1/2/3, permanent molar 1/2/3; oe, oral epithelium; p3, deciduous premolar 3; p4, deciduous premolar 4; scale bars: 100 μm.\u003c/p\u003e","description":"","filename":"FIGURE9.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7607583/v1/4152111d7b86cd4be11cd610.jpg"},{"id":92114961,"identity":"1ad1008e-8705-4b7a-ac62-9384b7400d59","added_by":"auto","created_at":"2025-09-24 19:16:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":50277212,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7607583/v1/1ccd03ec-bd9b-48cc-9eae-e26c7ef68060.pdf"},{"id":92112832,"identity":"5db299a6-9a46-4dd5-9e58-8541fea8f1e3","added_by":"auto","created_at":"2025-09-24 18:51:50","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6930557,"visible":true,"origin":"","legend":"Supplementary material","description":"","filename":"OlbertovaetalsupplementarymaterialSept13.2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7607583/v1/3922590a52e4df7d78d8b165.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"LGR5 regulates sequential tooth development: evidence from single-cell transcriptomics and a gene inactivation model","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eFunctional tooth germs in vertebrates originate from the dental lamina (DL), whose longevity influences the number of tooth generations. Monophyodont species (e.g., mice) possess a rudimentary successional dental lamina (RSDL), while polyphyodont species (e.g., snakes) maintain a permanent DL. Diphyodont species (e.g., humans, pigs) initiate a second generation of teeth before the lamina fragments and regresses. However, the mechanisms establishing species-specific tooth generation remain unclear.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoreover, three molars are initiated by a sequential developmental process in mice. Tooth germs of the second (M2) and third (M3) molars are formed as the protrusion of the tooth epithelia of the first molar (M1)\u003csup\u003e\u0026nbsp;1\u003c/sup\u003e. Dental epithelial stem cells (DESCs) can transiently reside in the cervical loop of M1 and contribute to the subsequent tooth germ. Using mouse molars, we can therefore not only assess the presence of stem cells in the DL, but also their contribution to later tooth formation\u003csup\u003e\u0026nbsp;2.\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eRegulation of tooth renewal also occurs through DESCs that reside in specialized niches. While mammalian tooth regeneration is limited, continuously growing mouse incisors provide a model for stem cell proliferation and differentiation. In these, Wnt, BMP, and FGF signaling regulate stem cell activity\u003csup\u003e3\u003c/sup\u003e. Here, we investigate the role of Wnt signaling in the formation of successional dental laminae (SDLs) in the molar region \u0026mdash; an area that has received relatively little attention to date.\u003c/p\u003e\n\u003cp\u003eWnt signaling is critical for multiple stages of odontogenesis, with Wnt ligands exhibiting distinct expression patterns\u003csup\u003e4,5\u003c/sup\u003e. Canonical Wnt/\u0026beta;-catenin signaling maintains stem/progenitor cell renewal, while non-canonical, i.e. b-catenin-independent, Wnt signaling promotes differentiation\u003csup\u003e6\u003c/sup\u003e. The asymmetric expression of Wnt ligands could regulate dental progenitor cells, like in other tissues such as skin or gut\u003csup\u003e7\u003c/sup\u003e. Wnt inhibition arrests tooth development, while Wnt pathway upregulation induces ectopic teeth\u003csup\u003e8,9\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAs the most of\u0026nbsp;previous studies have focused on incisor development, molar formation has received comparatively little attention yet\u003csup\u003e13,14,15,16\u003c/sup\u003e. To address this gap, we investigated the heterogeneity of mesenchymal and epithelial cell populations in the molar region at embryonic day (E)\u0026nbsp;18.5\u0026mdash;a critical stage corresponding to the maximal projection of the RSDL. By integrating single-cell transcriptomics with histochemistry and in situ RNA hybridization, we provide a detailed characterization of the epithelial\u0026ndash;mesenchymal interactions that support the maintenance and function of this transient developmental structure.\u003c/p\u003e\n\u003cp\u003eTo investigate the existence of a stem cell niche in the rudimentary successional dental lamina (RSDL) of mouse molars, we combined single-cell transcriptomics and lineage tracing to analyze the localization, identity, and behavior of stem/progenitor cells. We focused on \u003cem\u003eLgr5\u003c/em\u003e, a Wnt target gene and established marker of epithelial stem cells in various tissues\u003csup\u003e4,10\u0026ndash;12\u003c/sup\u003e, whose role in molar development remains unresolved. Using \u003cem\u003eLgr5-EGFP-IRES-CreERT2\u003c/em\u003e and \u003cem\u003eRosa26-tdTomato\u003c/em\u003e reporter mice, we visualized Lgr5-expressing cells and traced their progeny during the formation of M1 and M2. Single-cell RNA sequencing (scRNA-seq) revealed \u003cem\u003eLgr5\u003c/em\u003e expression in both epithelial and mesenchymal compartments, highlighting distinct Lgr5\u003csup\u003e+\u003c/sup\u003e populations. In Lgr5-deficient mice, we investigated the functional effects of gene loss on molar development and the activity of the Wnt signaling pathway. These approaches enable to follow spatial restriction of Lgr5 expression within the DL and evaluate its role in regulating sequential tooth formation.\u003c/p\u003e\n\u003cp\u003eTaken together, our data reveal distinct epithelial and mesenchymal subpopulations with specialized molecular signatures, suggesting a more complex regulation of RSDL development than previously appreciated. The consistency in marker gene expression across studies underscores the robustness and biological relevance of our dataset.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eExperimental animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyze gene expression of LGR5\u0026nbsp;during odontogenesis we used \u003cem\u003eLgr5-EGFP-IRES-CreERT2\u003c/em\u003e mice [B6.129P2-Lgr5tm1(cre/ERT2)Cle/J]\u003csup\u003e17\u003c/sup\u003e.\u0026nbsp;These mice express fluorescent protein EGFP (for full list of abbreviations see \u003cstrong\u003eSupplementary Table 1)\u0026nbsp;\u003c/strong\u003eand tamoxifen-regulated variant of Cre recombinase from the endogenous \u003cem\u003eLgr5\u003c/em\u003e locus. Simultaneously, the “knock-in” allele disrupts \u003cem\u003eLgr5\u0026nbsp;\u003c/em\u003egene function. To follow the fate of Lgr5-positive cells, we combined \u003cem\u003eLgr5-EGFP-IRES-CreERT2\u003c/em\u003e mice with \u003cem\u003eRosa26-tdTomato\u003c/em\u003e mice [Jackson Laboratory, strain: 129S6-\u003cem\u003eGt(ROSA)26Sor\u003c/em\u003e \u003cem\u003e\u003csup\u003etm14(CAG-tdTomato)Hze\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e/J mice]\u003csup\u003e18\u003c/sup\u003e. Under the control of ubiquitously active \u003cem\u003eRosa26\u003c/em\u003e promoter, the \u003cem\u003eRosa26-tdTomato\u0026nbsp;\u003c/em\u003eallele encodes tandem dimer of red fluorescent Tomato (tdTomato) protein following transcriptional stop signal that is flanked by \u003cem\u003eloxP\u003c/em\u003e sites and sensitive to Cre-mediated excision. Therefore, Lgr5-positive cells were permanently labeled by tdTomato fluorescence after tamoxifen treatment in compound \u003cem\u003eLgr5-EGFP-CreERT2 x\u003c/em\u003e \u003cem\u003eRosa26-tdTomato\u0026nbsp;\u003c/em\u003emice. The Cre activity and labeling were induced in embryos by a single dose of tamoxifen in corn oil (2 mg in 100ul) that was gavaged orally into pregnant females at different time points of embryonic development (E12.5; E13.5). Samples were collected at different developmental stages, including E11.5 to 18.5 (E11.5–E18.5) and postnatal day 4 (P4). At\u0026nbsp;selected time points, embryos were euthanized by decapitation and fixed in 4-10% PFA at least overnight depending on developmental stage. Animal care and experimental procedures were approved by the Animal Care Committee of the Institute of Molecular Genetics, Prague, Czech Republic (Approval No. 26/2019).\u003c/p\u003e\n\u003cp\u003eTo evaluate diphyodont dentition, we selected the E50 developmental stage of the minipig, when the successional dental lamina is already well developed. Minipig embryos were obtained from the Liběchov animal facility (IAPG, Liběchov, Czech Republic). The day after insemination was established as day 1 of gestation. Staged embryos were obtained by hysterectomy, fixed in 10% neutral formaldehyde. Sections were stained with Haematoxylin-Eosin and alternative slides were used for immunohistochemical labeling or gene expression analyses by RNAScope. All procedures were conducted following a protocol approved by the Laboratory Animal Science Committee of IAPG (Approval No. 97/2011).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProcessing of embryos for histological analyses\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecimens for histological and immunohistochemical analysis were decalcified in 12.5% EDTA in 4% PFA in the fridge and then embedded into paraffin. Paraffin embedded tissues were cut in the transverse plane to get serial histological sections. These sections were stained with Haematoxylin-Eosin and alternative slides were used for immunohistochemical analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll photos were taken using the fluorescence microscope Leica DM LB2 (Leica Microsystems, Germany) unless otherwise mentioned. Pictures were processed by Adobe Photoshop (Adobe Systems Incorporated, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical\u0026nbsp;labeling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlternative slides were\u0026nbsp;deparaffinized and rehydrated through a series of ethanol. Water bath (97°C) in Dako solution (pH=6) or citrate buffer for 10 min was used for antigen retrieval. To prevent nonspecific binding of antibodies, blocking serum was applied on the samples for 30 min. Next, slides were incubated with primary antibody \u003cstrong\u003e(Supplementary Table 2)\u003c/strong\u003e for 1 hour or overnight, alternatively. The secondary antibody \u003cstrong\u003e(Supplementary Table 2)\u003c/strong\u003e was applied for 30 min and Fluoroshied with 4′,6-diamidino-2-phenylindole (DAPI; P36935, Invitrogen, USA) was used for the counterstaining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalyses of label-retaining cells by\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e5-Bromo-2'-Deoxyuridine\u0026nbsp;(BrdU\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePregnant mice (embryos E18.5) were injected peritoneally with 10 nM BrdU (50 mg/kg, Sigma-Aldrich, USA) two hours before collection and then embryos were euthanized by decapitation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImmunohistochemical analyses were performed on alternate slides using 2NHCl and 0.1 % trypsin as a pre-treatment. The sections were incubated at 37 °C for 10 minutes in both solutions. Next, the protocol was processed the same way as was described previously.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of cell fate of LGR5-positive cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell fate mapping was performed in \u003cem\u003eLgr5-EGFP-IRES-CreERT2\u003c/em\u003e mice crossed with \u003cem\u003eRosa26-tdTomato\u003c/em\u003e reporter mice. The combination of the two alleles allowed us to study the relationship between LGR5-expressing cells (which produce EGFP, green fluorescence) and LGR5-descendant (LGR5-D) cells (which produce tdTomato, red fluorescence). Tamoxifen was applied intraperitoneally\u0026nbsp;as described previously\u003csup\u003e19\u003c/sup\u003e at E12.5 or E13.5. Animals were collected at three time points (E15.5, E16.5, E18.5) to cover transition through different developmental stages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalyses of signal colocalization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParaffin-embedded histological sections stained with antibodies against GFP and RFP (which cross-reacts with the tdTomato protein) and DAPI were used for quantitative analysis of signal overlap.\u0026nbsp;The images were taken with a Carl Zeiss AxioImager.Z2 wide-field microscope with a Plan-Apochromat 40x/1.2 oil immersion objective. For each image, three channels were acquired using DAPI, GFP, and TexasRed filter cubes.\u003c/p\u003e\n\u003cp\u003eImage analysis was performed using Imaris software (version 10.1, Bitplane, Oxford Instruments). Individual cells were segmented based on the DAPI channel using the Surface module. The segmented cells were classified as positive or negative according to the mean intensity of GFP and RFP. The total numbers of GFP-positive (green), RFP-positive (red), and double-positive cells (yellow) were counted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVarious statistical analyzes, in particular normality and lognormality tests, unpaired t-tests, Welch’s t-test or Mann-Whitney test, were used to determine the co-expression of GFP and tdTomato using GraphPad Prism software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3D imaging of Lgr5-positive cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo reveal the spatial distribution of Lgr5-positive cells and their progeny within the developing molar, we analyzed mandibles from \u003cem\u003eLgr5-EGFP-IRES-CreERT2 x Rosa26-tdTomato\u003c/em\u003e embryos. Embryos were induced at E12.5 and tissue harvested at E15.5. Complete mandibles without tongue were extracted by scissors and fixed for 24 hours in 4% PFA buffered to pH 7.35. After the permeabilization by 0,01% Triton X-100 (Merck, Germany) at 4°C for 16 hours, the tissue was counterstained by 1μg/ml DAPI at 4°C for 16 hours and cleared according to CUBIC protocol\u003csup\u003e20\u003c/sup\u003e. Endogenous fluorescent signal and nuclear DAPI signal from the whole molar area were captured in tiled Z-stack images using Z1 light sheet microscope (Zeiss) with objective 20x. Recorded datasets were deconvolved using Huygens Professional software (SVI), stitched and assembled to maximal projections or movies in Imaris software (Oxford instruments).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression analyses by RNAScope\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice embryos were collected at E18.5.\u0026nbsp;RNAScope Multiplex Fluorescent v2 assay for formalin-fixed paraffin-embedded tissue (323 110, Advanced Cell Diagnostics, Newark, California, USA) was used for detection of several different RNA transcripts. Several probes were used according to the manufacturer’s protocol \u003cstrong\u003e(Supplementary Table 3)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eBefore hybridization at 40 °C for 2 hours, individual slides were boiled in retrieval buffer (322 001, Advanced Cell Diagnostics) at 97 °C for 10 minutes and pretreated with hydrogen peroxide at RT for 10 minutes and Protease Plus (322 331, Advanced Cell Diagnostics) at 40°C for 15 minutes. To visualize hybridized probes, a TSA-Plus Cyanine 3/Fluorescein system was used (NEL741001KT, Perkin-Elmer, Waltham, Massachusetts). Samples were counterstained with DAPI (323 108, Advanced Cell Diagnostics) and mounted in Fluoroshied\u003csup\u003eTM\u003c/sup\u003e (F6182-20ML, Sigma-Aldrich).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollection of tissues for single-cell analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice embryos at E18.5 were used for cell isolation from the mandibular molar area for scRNAseq. Mice were euthanized by decapitation. Eleven mice heads were collected and lower jaws were dissected. Molar areas of lower jaws (epithelium and surrounding mesenchyme) were carefully isolated and cut into small pieces. Next, samples were transferred to 1.5 ml tube with collagenase type I (LS0004196, Worthington, USA) dissolved in DMEM/F12 (D8437, Sigma-Aldrich) and incubated for 3 hours at 37°C. During enzymatic digestion, samples were pipetted up and down seven times using a 1 ml pipette. At the end of incubation, 10% filtered FBS was added to each sample. Single-cell suspension was centrifuged in 4°C precooled centrifuge for 5 min at 200 x g. Then, the supernatant was removed and the pellet was resuspended in 1 ml DMEM/F12. Suspension was filtered using a 70 µm strainer into 50 ml falcon tubes (431751, Corning) and transferred into 15 ml tubes. Next, samples were centrifuged in 4°C precooled centrifuge for 5 min at 200 x g. Pellet was homogenized in 200µl 0.04% BSA in filtered phosphate-buffered saline (PBS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSorting of cells for scRNA-seq\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLGR5-positive mice embryos were collected at E16.5 and E18.5 (15 embryos at E16.5 and 22 embryos at E18.5). These embryos were decapitated, and the heads were divided into upper and lower jaws. The M1 and closely surrounding area was isolated from the lower jaws. The isolated areas of each jaw were collected and cut into small pieces, and transferred to plates with 2.5 ml Collagenase P (3 U/mL; Colla-RO, col. No. 10103578001, Roche, USA) dissolved in Hanks’ balanced salts (HBBS, cat. No. 88284, Thermo Fisher Scientific, USA) and incubated for at least 1 hour at 37°C. During the enzymatic digestion, tissue pieces were gently pipetted up and down four times using a 1 ml pipette. After incubation, the cell suspension was centrifuged for 5 min at 600 x g. The supernatant was removed, and the pellet was resuspended in 500–1000 ul HBBS. Cell suspensions were filtered through a 70 µm sieve and stained for cell viability with Hoechst 33258 (cat. No. H3569, Thermo Fisher Scientific). Cells were analysed by flow cytometry using a FACSAria IIu cell sorter (BD Biosciences, USA) and an Influx high-speed cell sorter (BD Biosciences, USA). Viable single-cell EGFP-positive or EGFP-negative populations were sorted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-cell RNA-seq\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collected samples were processed in two separate scRNA-seq runs: The first run analyzed whole molars at E18.5, while the second run focused on LGR5-positive sorted cells at E16.5 and E18.5. The first scRNA-seq run was performed on two independent biological samples, each comprising a pair of M1 from opposite sides of the mandible of two embryos. A total of 4,502 cells were sequenced in one sample (2,961 after filtering out duplicates and low-quality cells) and 3,109 cells in the other sample (2,016 after filtering out). The experimental setup and a summary of the first data set are shown in \u003cstrong\u003eFig. 1\u003c/strong\u003e.\u0026nbsp;In the second scRNA-seq run using LGR5-positive sorted cells, 4,023 cells were obtained from 15 embryos at E16.5, and 4,684 cells were collected from 22 embryos at E18.5.\u003c/p\u003e\n\u003cp\u003ePrior to loading onto the Chromium Controller (10× Genomics), cell suspensions were adjusted to 700 cells/μl and mixed with nuclease-free water and master mix (10× Genomics). Gel Bead-In Emulsions (GEMs) were generated, followed by reverse transcription, homogenization, washing, and cDNA amplification (BioRad C1000 Touch Thermal Cycler). Library preparation followed the Chromium Next GEM Single Cell 3′ Reagent Kit v3 (1st run) and v3.1 (2nd run) protocols (10× Genomics). All libraries passed quality control and were sequenced on the NextSeq 500 (Illumina) using the High Output Kit v2.5 (150 cycles), with 28 cycles for read 1 (cell barcode) and 130 (1st run) or 119 (2nd run) cycles for read 2 (cDNA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-cell RNA-seq data processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth scRNA-seq datasets were pre-processed using the standardized pipeline from the Cell Ranger Single-Cell Software Suite (v3.1.0; 10× Genomics)\u003csup\u003e21\u003c/sup\u003e. Sequencing reads were aligned to the mouse reference genome (GRCm39, Ensembl annotation release 104), and gene expression was quantified at the single-cell level using UMI counts.\u003c/p\u003e\n\u003cp\u003eRaw and filtered Cell Ranger outputs were imported into RStudio (R version 4.3.3) to assess and correct for ambient RNA contamination using the SoupX package (v1.6.2)\u003csup\u003e22\u003c/sup\u003e, applying a contamination rate of 0.1. The corrected count matrices were used to create Seurat objects (Seurat v4.4.0)\u003csup\u003e23\u003c/sup\u003e, followed by several quality control steps. Cells with \u0026gt;10% mitochondrial gene content or expressing fewer than 500 or more than 8000 genes were excluded.\u003c/p\u003e\n\u003cp\u003eData normalization and scaling were performed using the SCTransform algorithm\u003csup\u003e24\u003c/sup\u003e. For the E18.5 molar dataset, cell cycle regression was included during SCTransform. Dimensionality reduction was carried out using principal component analysis (PCA) with default parameters\u003csup\u003e25\u003c/sup\u003e, followed by Uniform Manifold Approximation and Projection (UMAP) for visualization\u003csup\u003e26\u003c/sup\u003e. Cell clustering was performed using the Louvain algorithm with a resolution of 0.8, based on the cell proximity matrix from the FindNeighbors function. Doublets were identified and removed using the DoubletFinder package (v2.0.4)\u003csup\u003e27\u003c/sup\u003e. UMAP visualizations were further customized using the ggplot2 package\u003csup\u003e28\u003c/sup\u003e, with additional graphical edits (e.g., color, font) applied in Adobe Photoshop Studio 2022.\u003c/p\u003e\n\u003cp\u003eCluster-specific marker genes were identified using Seurat’s FindMarkers function, considering only positive markers expressed in at least 25% of cells, with a log fold-change threshold of 0.5 and statistical testing via the Wilcoxon rank-sum test. Top five markers for each major cell type or subcluster were visualized using Seurat's DotPlot (integrated with ggplot2), while additional expression patterns were shown via FeaturePlot. Density visualizations were created using the Nebulosa package (v1.12.1)\u003csup\u003e29\u003c/sup\u003e, employing the 'wide' method.\u003c/p\u003e\n\u003cp\u003eSignaling pathway activity was inferred using multivariate linear modeling with the decoupleR (v2.8.0)\u003csup\u003e30\u003c/sup\u003e and OmnipathR (v3.10.1)\u003csup\u003e31\u003c/sup\u003e packages. Heatmaps were generated using ggplot2 and pheatmap (v1.0.12). Transcription factor activities were estimated using a weighted mean approach via the Dorothea (v1.14.1)\u003csup\u003e32\u003c/sup\u003e, decoupleR, OmnipathR, and pheatmap packages.\u003c/p\u003e\n\u003cp\u003eSpliced and unspliced RNA ratios were computed using the Velocyto package (v0.17.17)\u003csup\u003e33\u003c/sup\u003e in Python (v3.11.9), and RNA velocity analyses were further processed using scVelo (v0.3.2)\u003csup\u003e34\u003c/sup\u003e to generate latent time plots based on a dynamic model.\u003c/p\u003e\n\u003cp\u003eDifferentially expressed genes (up- and down-regulated) related to WNT signaling in the GFP+ dental epithelium and labial mesenchyme were extracted from the pathway activity analysis described above and visualized using the textshape (v1.7.5) and ggplot2 (v3.4.4) packages in R.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation, cultivation, and preparation of cells for organoid analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLower jaws from E18.5 mouse embryos were collected, and molar regions were isolated and transferred into Petri dishes containing 2 ml of collagenase P (3 U/mL; Colla-RO, 10103578001, Roche) dissolved in Hanks' Balanced Salt Solution (HBSS; cat. no. 88284, Thermo Fisher Scientific). Samples were incubated at 37 °C for 15 minutes. Following incubation, the molar epithelium was carefully separated from the surrounding mesenchyme and bone and dissected into M1 and M2. Genotyping was processed separately.\u003c/p\u003e\n\u003cp\u003eThe epithelial tissues were enzymatically dissociated in TrypLE Express (cat. no. 12604039, Thermo Fisher Scientific) for 5 minutes at 37 °C. After washing in PBS, cell clusters were pelleted by centrifugation (250 × g, 5 min, 4 °C) and resuspended in Matrigel (cat. no. 356239, Corning). Matrigel droplets containing molar epithelium were allowed to polymerize at 37 °C for 10 minutes and subsequently overlaid with organoid culture medium: Advanced DMEM/F-12 (cat. no. 12634010, Thermo Fisher Scientific) supplemented with 10 mM HEPES (15630080), 1× Penicillin-Streptomycin (15070063), 1× GlutaMAX (35050061), 1× B27 (12587-010), 1× N2 (17502-048), RSPO1 (200 ng/ml; 120-38), Noggin (100 ng/ml; 120-10C), FGF2 (20 ng/ml; 234-FSE), FGF10 (100 ng/ml; 100-26), EGF (20 ng/ml; AF-100-15), and Wnt surrogate-Fc Fusion protein (200 ng/ml; PHG0401), all from Thermo Fisher Scientific; and 0.5 μM A83-01 (SML0788), 1.25 mM N-acetyl-L-cysteine (A7250), 10 mM Nicotinamide (N0636), and 10 μM SB202190 (1264), all from Merck.\u003c/p\u003e\n\u003cp\u003eOrganoids were cultured at 37 °C in a humidified atmosphere with 5% CO₂. Medium was changed three times per week. Organoids were passaged every two weeks by dissociation in TrypLE, following Matrigel removal via vigorous pipetting in PBS.\u003c/p\u003e\n\u003cp\u003eFor imaging, organoids were either visualized live in Matrigel using a Dragonfly spinning disk microscope (Andor) with Hoechst 34580 staining (Merck) or processed for histology. In the latter case, Matrigel was removed, and organoids were embedded in 4% low-melting agarose (Merck) and paraffin-sectioned for standard histological analysis.\u003c/p\u003e\n\u003cp\u003eFor fluorescence-activated cell sorting (FACS)\u0026nbsp;analysis, organoids were collected, Matrigel was removed by thorough pipetting in PBS, and the cells were dissociated into single-cell suspension using TrypLE for 15 minutes at 37 °C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of molar epithelium and organoids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolar epithelium was dissected from the mandibular region at embryonic stage E18.5 and separated into M1 and M2 regions, as described in the previous section. Tissue samples were lysed in RLT buffer (Qiagen). Molar organoids were collected from two wells of a 24-well plate by vigorous pipetting in PBS, pelleted by centrifugation, and lysed in RLT buffer. From this point forward, all samples were processed identically using the RNeasy Mini Kit (Qiagen) for total RNA isolation.\u003c/p\u003e\n\u003cp\u003eReverse transcription was performed using Maxima Reverse Transcriptase (cat. no. EP0742, Thermo Fisher Scientific) according to the manufacturer’s protocol, using random hexamer primers (cat. no. 48190011, Thermo Fisher Scientific). Quantitative PCR was carried out on a LightCycler® 480 system using SYBR Green I Master Mix (Roche) in three technical replicates, following the manufacturer’s instructions.\u003c/p\u003e\n\u003cp\u003eTwo biological replicates of each M1 and M2 tissue sample, along with three biological replicates of each M1 and M2 organoid sample (\u003cem\u003eLgr5\u003csup\u003e⁺/⁺\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003eLgr5\u003csup\u003e⁺/⁻\u003c/sup\u003e\u003c/em\u003e, or \u003cem\u003eLgr5\u003csup\u003e⁻/⁻\u003c/sup\u003e\u003c/em\u003e genotypes), were analyzed for the expression of selected genes (listed in\u0026nbsp;\u003cstrong\u003eSupplementary Table 4\u003c/strong\u003e). For each gene, the average Ct value from the three technical replicates was first normalized to β-actin and then to the average value of the corresponding molar tissue sample (ΔΔCt method)\u003csup\u003e35\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eA gene enrichment coefficient was calculated by averaging ΔΔCt values across all organoid replicates for each gene. A population enrichment coefficient was then derived by averaging the expression changes across all genes used to define a given cell subpopulation. For clarity in visualization, the average expression change is presented as –ΔΔCt, so that upregulation is shown as positive values and downregulation as negative.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoprecipitation and Western Blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK 293T cells were cultured in two separate 15-cm dishes. The first dish was co-transfected with a plasmid encoding human hemagglutinin (HA)-tagged PTK7 and a plasmid encoding LGR5 tagged with STREP-FLAG. The second dish was transfected with the HA-tagged PTK7 plasmid and an empty control vector lacking LGR5. Forty-eight hours post-transfection, cells were washed with PBS, lysed, and scraped in 2 ml of lysis buffer containing: 150 mM NaCl, 50 mM Tris, 0.4% Triton X-100, 2 mM CaCl₂, 2 mM MgCl₂, and 1 mM EDTA. The buffer was supplemented with protease and phosphatase inhibitors (NaF, PMSF, TLCK, TPCK, Na₃VO₄, DTT). Lysates were clarified by centrifugation at 30,000 × g for 30 minutes at 4 °C.\u003c/p\u003e\n\u003cp\u003eFor immunoprecipitation, 900 μl of each lysate was incubated with 1 μg of anti-HA antibody (#3724, Cell Signaling, USA) on ice. After 1 hour, 15 μl of Protein G-Sepharose beads (#17-0886-01, GE Healthcare, USA) was added and the mixture was rotated on a carousel at 4 °C for an additional 2 hours. In parallel, another 900 μl of lysate was incubated with 15 μl of Strep-Tactin Sepharose beads (#2-1201-025, IBA, Germany) under identical conditions. Following incubation, beads were collected by centrifugation (200 × g, 2 min, 4 °C) and washed six times with lysis buffer. Both immunoprecipitates and input lysates were mixed with denaturing, reducing Laemmli buffer and boiled before electrophoresis. Proteins were separated on 8–15% SDS-PAGE gels and transferred to Immobilon-P membranes (Millipore). Detection was performed using anti-HA antibody (#3724, Cell Signaling) or M2 anti-FLAG antibody (#F1804, Sigma-Aldrich). Western blot signals were visualized using the Uvitec Alliance Q9 Atom imaging system and analyzed with Q9 Alliance software.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003eSingle-cell transcriptome profiling of molar development in mice identifies markers of the RSDL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTeeth originate from the DL, a band of epithelial tissue that initiates tooth development. Within this structure, two functionally and evolutionarily distinct types of DL can be distinguished. The RSDL is a specialized epithelial extension arising from the dental epithelium. It serves as a source of replacement teeth and maintains the capacity for further tooth formation during development. In species such as the mouse, which have only one generation of teeth, a RSDL forms temporarily \u003cstrong\u003e(Fig. 1A)\u003c/strong\u003e. This structure normally regresses without further teeth developing and is considered a vestigial remnant of evolutionary history. By contrast, the sequential dental lamina (SeDL) is an active structure that drives the formation of posterior molars \u003cstrong\u003e(Fig. 1A)\u003c/strong\u003e. In this process, each new tooth germ emerges as an epithelial outgrowth from the preceding molar, enabling the progressive extension of the dental arch and initiation of successive molar germs. \u003c/p\u003e\n\u003cp\u003eTo investigate the characteristics of cells involved in this process, we isolated the lower M1 at E18.5 for scRNA-seq \u003cstrong\u003e(Fig. 1B\u003c/strong\u003e), collecting dental and adjacent soft tissues while carefully excluding the alveolar bone. By comparing the dental stalk (DS) and lamina with other epithelial populations, this approach provided potential insights into signaling mechanisms associated with the underlying mesenchyme. \u003c/p\u003e\n\u003cp\u003eSingle-cell RNA-seq was performed on lower M1 from two independent biological samples (\u003cstrong\u003eFig. 1; Supplementary Fig. 1\u003c/strong\u003e). As overall quality and sequencing depth were comparable, both datasets were merged for further analysis to maximize data coverage\u003cstrong\u003e (Supplementary Fig. 1A)\u003c/strong\u003e. However, it is necessary to mention that some mesenchymal cell clusters, particularly in the outlying dental follicle and undifferentiated osteoblasts, were enriched due to technical variations of tissues removal between bone and follicle (\u003cstrong\u003eSupplementary Fig. 1A\u003c/strong\u003e). \u003c/p\u003e\n\u003cp\u003eUnbiased clustering with four parameters identified eight main cell subpopulations, including epithelial and mesenchymal dental clusters, as well as immune, blood, pericyte, muscle, and glial compartments (\u003cstrong\u003eFig. 1C, D; Supplementary material 1\u003c/strong\u003e). Differential expression analysis revealed cluster-specific genes and pathways (\u003cstrong\u003eFig. 1C; Supplementary Fig. 1B\u003c/strong\u003e). In order to capture the underlying biological dynamics, the cell cycle effect was initially retained in the visualization of the individual clusters \u003cstrong\u003e(Fig. 1E)\u003c/strong\u003e. Cycling cells identified by \u003cem\u003eKi67\u003c/em\u003e and \u003cem\u003eCdk1\u003c/em\u003e expression \u003cstrong\u003e(Fig. 1F, F’; Supplementary Fig. 1C)\u003c/strong\u003e were primarily enriched in a cluster corresponding to the dental mesenchyme, but also formed distinct subclusters within broader populations such as the dental papilla (DP) and dental epithelium. We also detected cells corresponding to different phases of the cell cycle, as shown by the expression of \u003cem\u003eUng\u003c/em\u003e (G1 and S phase), \u003cem\u003eTop2a\u003c/em\u003e (S–G2 transition), \u003cem\u003eCdc20\u003c/em\u003e (G2–M transition) and \u003cem\u003eCcnb2\u003c/em\u003e (M–G1 transition), which were distributed across several clusters (\u003cstrong\u003eSupplementary Fig. 1D, E\u003c/strong\u003e and data not shown).\u003c/p\u003e\n\u003cp\u003eTo validate the spatial distribution of the identified cell populations in mouse molars, we performed RNAScope at E18.5 WT embryos \u003cstrong\u003e(Fig. 1G–I’; Supplementary Fig. 2A–C)\u003c/strong\u003e. Non-dental clusters included muscle cells expressing \u003cem\u003eMyod1\u003c/em\u003e, \u003cem\u003eMyf5\u003c/em\u003e, or \u003cem\u003eTtn\u003c/em\u003e, which showed upregulation of signaling pathways such as PI3K and VEGF \u003cstrong\u003e(Fig. 1H, H’; Supplementary Fig. 1B, 2A; Supplementary Table 5)\u003c/strong\u003e. Immune cells, characterized by \u003cem\u003ePtprc\u003c/em\u003e, \u003cem\u003eC1qa\u003c/em\u003e, and \u003cem\u003eTyrobp\u003c/em\u003e, exhibited increased activity of JAK-STAT and NFκB signaling, while Wnt signaling was downregulated in this cluster \u003cstrong\u003e(Fig. 1G, G’; Supplementary Fig. 1B, 2B; Supplementary Table 5)\u003c/strong\u003e. Pericytes expressing \u003cem\u003eEpas1\u003c/em\u003e, \u003cem\u003eRgs5\u003c/em\u003e, and \u003cem\u003eHigd1b\u003c/em\u003e showed upregulation of EGFR and TNFα signaling pathways (\u003cstrong\u003eSupplementary Fig. 1B, 2C; Supplementary Table 5)\u003c/strong\u003e. Endothelial cells, marked by \u003cem\u003eCldn5\u003c/em\u003e, \u003cem\u003ePlvap\u003c/em\u003e, and \u003cem\u003eEpas1\u003c/em\u003e, displayed enriched JAK-STAT and VEGF signaling \u003cstrong\u003e(Fig. 1I, I’; Supplementary Fig. 1B, 2C; Supplementary Table 5)\u003c/strong\u003e. Neuroglial cells, identified by the expression of \u003cem\u003eSox10\u003c/em\u003e, \u003cem\u003eGfra3\u003c/em\u003e, and \u003cem\u003eFabp7\u003c/em\u003e, were characterized by increased VEGF signaling and reduced Wnt pathway activity \u003cstrong\u003e(Supplementary Fig. 1B; Supplementary Table 5)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-cell analysis of dental mesenchyme identifies a distinct Lgr5⁺ progenitor-like population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we analyzed dental mesenchyme cells in more detail. Consistent with their mesenchymal identity, populations located outside the tooth germ expressed general mesenchymal markers, including vimentin (\u003cem\u003eVim\u003c/em\u003e), \u003cem\u003eCol1a1\u003c/em\u003e or \u003cem\u003eTwist1\u003c/em\u003e (\u003cstrong\u003eFig. 2A, A’; Supplementary Table 6; Supplementary material 2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFurther subclustering revealed distinct mesenchymal populations surrounding the tooth (\u003cstrong\u003eFig. 2B, D\u003c/strong\u003e). Unspecified oral fibroblasts near the oral epithelium expressed \u003cem\u003eBhlhe22\u003c/em\u003e, \u003cem\u003eCachd1\u003c/em\u003e, and \u003cem\u003eTbx15\u003c/em\u003e (\u003cstrong\u003eFig. 2D, E, J, J’; Supplementary Fig. 2D-G; Supplementary Table 6\u003c/strong\u003e). A cluster near the alveolar bone contained undifferentiated osteoblasts marked by \u003cem\u003eMfap5\u003c/em\u003e, \u003cem\u003eMeis2\u003c/em\u003e, and \u003cem\u003ePamr1\u003c/em\u003e (\u003cstrong\u003eFig. 2D, F, K, K’; Supplementary Fig. 2H; Supplementary Table 6\u003c/strong\u003e). Another clusters were identified as the outlying dental follicle (oDF) and the surrounding dental follicle (sDF). Markers of the oDF cluster, located in follicle layers distant from the tooth epithelium, included \u003cem\u003eAldh1a2, Alpl\u003c/em\u003e, and \u003cem\u003eRunx2\u003c/em\u003e (\u003cstrong\u003eFig. 1D, H, M, M’; Supplementary Fig. 2I, J; Supplementary Table 6\u003c/strong\u003e). The sDF cluster was marked by the cell adhesion protein \u003cem\u003eSpon1\u003c/em\u003e, along with \u003cem\u003eTfap2c\u003c/em\u003e and \u003cem\u003ePtch1\u003c/em\u003e \u003cstrong\u003e(Fig. 2D, I, N, N’; Supplementary Fig. 2K, L; Supplementary Fig. 4E; Supplementary Table 6)\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003eThe dental mesenchyme cluster identified at E18.5 in our study shares gene expression pattern with both earlier (E16.5, e.g., \u003cem\u003eAspn, \u003c/em\u003e\u003cstrong\u003eSupplementary material 1\u003c/strong\u003e) and later (P3.5, e.g., \u003cem\u003eIgf1, \u003c/em\u003e\u003cstrong\u003eSupplementary material 1\u003c/strong\u003e) developmental stages also described previously\u003csup\u003e15\u003c/sup\u003e. However, this cluster also contains a substantial population of undifferentiated cells with limited transcriptional specificity, making them difficult to distinguish into defined subtypes.\u003c/p\u003e\n\u003cp\u003eAt E18.5, the dental follicle formed a well-defined cluster that could be further subdivided into two distinct populations located just adjacent to tooth or in larger distance: the surrounding follicle and the outlying follicle (\u003cstrong\u003eFig. 2H, I\u003c/strong\u003e). This contrasts with earlier findings at E16.5, where follicular populations were classified just as lateral and apical\u003csup\u003e15\u003c/sup\u003e. Notably, some marker genes, such as \u003cem\u003eAldh1a2\u003c/em\u003e, were expressed at both these stages; however, their spatial distribution differed. At E16.5, \u003cem\u003eAldh1a2\u003c/em\u003e expression was restricted to the apical aspect of the tooth bell\u003csup\u003e15\u003c/sup\u003e, whereas by E18.5, its expression expanded to encircle the entire tooth bell without direct interaction with the tooth epithelium\u003cstrong\u003e (Supplementary Fig. 2J)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eAdditionally, a mesenchymal cluster including \u003cem\u003eLgr5\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e cells was identified outside the tooth germ, mainly on the labial side of the DS, with key markers including \u003cem\u003eTcea3\u003c/em\u003e or \u003cem\u003eEmb \u003c/em\u003e\u003cstrong\u003e(Fig. 2D, G, L, L’; Supplementary Fig. 3A, B; Supplementary Table 6), \u003c/strong\u003ewhich are usuallyassociated with cell fate determination during development or maintaining cell identity. Latent time analysis\u003csup\u003e36\u003c/sup\u003e revealed that the least differentiated cells were mainly located in the mesenchymal cluster enriched with Lgr5-positive cells \u003cstrong\u003e(Fig. 1C)\u003c/strong\u003e, suggesting that this population could serve as a stem cell reservoir. This finding aligns with the well-established role of LGR5 as a marker of stem and progenitor cells in various tissues\u003csup\u003e 4,10–12\u003c/sup\u003e. Notably, despite their undifferentiated state, these cells exhibited minimal proliferative activity, as indicated by the low expression of proliferation-associated genes such as \u003cem\u003eKi67\u003c/em\u003e and \u003cem\u003eCdk1\u003c/em\u003e (\u003cstrong\u003eSupplementary Fig. 3C, D\u003c/strong\u003e). This observation suggests that these \u003cem\u003eLgr5\u003c/em\u003e-positive mesenchymal cells may exist in a relatively quiescent state, a characteristic commonly associated with tissue-resident stem cells\u003csup\u003e37\u003c/sup\u003e. The low proliferative activity of these cells may be crucial for maintaining a reservoir of progenitors that can be activated in response to specific developmental or regenerative cues.\u003c/p\u003e\n\u003cp\u003ePreviously, LGR5-positive cells have been detected in the developing mouse dentition as early as at E14.5\u003csup\u003e38\u003c/sup\u003e, where they play a crucial role in the continuous growth of mouse incisors\u003csup\u003e4\u003c/sup\u003e. In these ever-growing teeth, stem and progenitor cells are essential for maintaining self-renewal and ensuring sustained development. The niche of these stem/progenitor cells was found to be located within the labial cervical loop (LCL), a specialized epithelial structure critical for incisor renewal\u003csup\u003e39\u003c/sup\u003e. However, our findings reveal a distinct population of LGR5-positive cells located in the mesenchyme, suggesting a broader role for these cells in dental development beyond their previously characterized niche. While significant attention is typically given to progenitor cells in the incisor epithelium, little is known about the potential presence of stem cells in mesenchymal populations. Therefore, we also examined LGR5-positive cells in the tooth surrounding tissues. This unexpected localization raises intriguing questions about the functional diversity of LGR5-positive cells in tooth morphogenesis and their potential involvement in epithelial-mesenchymal interactions. \u003c/p\u003e\n\u003cp\u003eTo sum up, the molar tooth and its surrounding tissues are composed of a variety of cell populations, including endothelial, glial, immune, and pre-osteoblastic cells. Among these, \u003cem\u003eLgr5\u003c/em\u003e-positive cells form a distinct mesenchymal cluster localized on the labial side of the DS, with bioinformatic analyses supporting their identity as potential progenitor cells. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCellular subpopulations and molecular signatures of the DP during molar development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we focused on the DP cluster, which was clearly distinguishable from other mesenchymal populations while retaining the expression of core mesenchymal markers such as \u003cem\u003eMsx1\u003c/em\u003e (\u003cstrong\u003eFig. 1D, 3A; Supplementary material 3\u003c/strong\u003e). Notably, a subset of genes—including \u003cem\u003eRspo2\u003c/em\u003e, \u003cem\u003eDlx4\u003c/em\u003e, \u003cem\u003eDlx5\u003c/em\u003e, \u003cem\u003eDkk1\u003c/em\u003e, and \u003cem\u003eScube1\u003c/em\u003e—was specifically enriched in the DP, with minimal or no expression in the surrounding mesenchyme, indicating a unique gene expression signature for this compartment (\u003cstrong\u003eFig. 2A’; Supplementary Fig. 2M-P\u003c/strong\u003e). Further re-clustering of DP cells, performed at a resolution of 0.8—a parameter that controls the “granularity” of clustering\u003csup\u003e40\u003c/sup\u003e—revealed four distinct mesenchymal subpopulations \u003cstrong\u003e(Fig. 3A, B)\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003eThe DP can be functionally divided into coronal, lateral, and apical zones (\u003cstrong\u003eFig. 3B\u003c/strong\u003e), with regions of the coronal papilla displaying distinct expression profiles that reflect the differentiation status of their cells. The apical papilla (AP) is situated in the lower region of the papilla, adjacent to the follicular cell layer \u003cstrong\u003e(Fig. 3B, C, G, G’; Supplementary Fig. 4A, E; Supplementary Table 7)\u003c/strong\u003e. It expressed extracellular matrix (ECM) proteins such as \u003cem\u003eSpon1\u003c/em\u003e (a dental follicle marker), as well as \u003cem\u003ePostn\u003c/em\u003e and \u003cem\u003eLhx6\u003c/em\u003e. Additionally, we observed upregulation of JAK-STAT, TRAIL, and TNFα signaling pathways in the AP, whereas Wnt signaling was downregulated \u003cstrong\u003e(Supplementary Fig. 4A)\u003c/strong\u003e. The coronal papilla medium (CPm) was characterized by the expression of the transcriptional regulator \u003cem\u003eLmo1\u003c/em\u003e, as well as \u003cem\u003ePiezo2\u003c/em\u003e and \u003cem\u003eCrym\u003c/em\u003e \u003cstrong\u003e(Fig. 3B, C, D, D’; Supplementary Fig. 4A, B; Supplementary Table 7)\u003c/strong\u003e. This region also showed upregulation of signaling pathways such as VEGF, EGFR, and MAPK \u003cstrong\u003e(Supplementary Fig. 4A)\u003c/strong\u003e. In contrast, the upper coronal papilla (CPu) exhibited increased activity of the hypoxia pathway and expressed genes such as the cytoskeletal component \u003cem\u003eTubb3\u003c/em\u003e, along with \u003cem\u003eGfra1\u003c/em\u003e and \u003cem\u003eSct\u003c/em\u003e \u003cstrong\u003e(Fig. 3B, C, E, E’; Supplementary Fig. 4A, C; Supplementary Table 7)\u003c/strong\u003e. The lateral papilla (LP) was defined by the expression of \u003cem\u003eTac1\u003c/em\u003e (encoding the precursor of the peptide hormone substance P), \u003cem\u003eSnai2\u003c/em\u003e, and \u003cem\u003eRgs3\u003c/em\u003e, and by upregulation of Wnt and TGFβ signaling pathways \u003cstrong\u003e(Fig. 3B, C, F, F’; Supplementary Fig. 4A, D; Supplementary Table 7)\u003c/strong\u003e. Latent time analysis identified the LP and AP clusters as the most differentiated, whereas the coronal papilla cluster contained the least differentiated cell population \u003cstrong\u003e(Supplementary Fig. 4B)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eOur bioinformatic analysis of the DP subclusters revealed gene expression patterns that align with previously reported findings in mouse incisors. Notably, genes such as \u003cem\u003eDlx5\u003c/em\u003e and \u003cem\u003eScube1\u003c/em\u003e, previously identified in the dental pulp of mouse incisors\u003csup\u003e13\u003c/sup\u003e, were also prominently expressed in the molar DP, highlighting conserved molecular signatures across different tooth types. Similarly, markers of pre-odontoblasts, including \u003cem\u003eDkk1\u003c/em\u003e and \u003cem\u003eFgf3\u003c/em\u003e, were detected in both incisors and molars, suggesting shared early differentiation programs. In contrast, we did not observe expression of mature odontoblast markers in the molar, which is consistent with the E18.5 developmental stage, when odontoblasts have not yet fully differentiated.\u003c/p\u003e\n\u003cp\u003eInterestingly, \u003cem\u003eTac1\u003c/em\u003e, previously described as a specific marker of dental pulp adjacent to the lingual cervical loop in mouse incisors\u003csup\u003e13\u003c/sup\u003e, was also detected in a comparable region of the molar, near both cervical loops—suggesting a conserved spatial expression pattern across tooth types. Furthermore, consistent with earlier scRNA-seq data from E16.5\u003csup\u003e15\u003c/sup\u003e, which identified apical and coronal DP populations based on spatial location and transcriptional profiles, our analysis at E18.5 revealed an even greater degree of cellular heterogeneity, reflecting advanced differentiation and tissue specialization.\u003c/p\u003e\n\u003cp\u003eIn summary, our findings reveal a dynamic cellular hierarchy within the developing DP. The AP contains less differentiated cells, which appear to be translocated inward as development proceeds, while the coronal region harbors the most differentiated populations, showing molecular features consistent with a continuum from pre-odontoblasts to mature odontoblasts. Notably, we did not identify a distinct \u003cem\u003eLgr5\u003c/em\u003e-positive population within the DP, suggesting that \u003cem\u003eLgr5\u003c/em\u003e is not a marker of progenitor cells in this compartment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRe-clustering of molar epithelial cells identifies subpopulations with\u003c/strong\u003e\u003cstrong\u003e progenitor state \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe epithelial compartment of the molar formed a distinct cluster, clearly separated from other cell types \u003cstrong\u003e(Fig. 1D; Supplementary material 4)\u003c/strong\u003e. These cells contribute to key epithelial structures, including the enamel organ, the DS that connects the tooth to the oral epithelium, and the RSDL. Due to strong intercellular junctions, epithelial cells are typically underrepresented in scRNA-seq datasets compared to mesenchymal cells. Nonetheless, the epithelial clusters \u003cstrong\u003e(Fig. 3H; \u003c/strong\u003e\u003cstrong\u003eSupplementary material 4)\u003c/strong\u003e showed robust expression of epithelium-specific genes, including various keratins, \u003cem\u003eEpcam\u003c/em\u003e, desmocollins, and cadherins \u003cstrong\u003e(Fig. 3H’)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRe-clustering of epithelial cells at E18.5 revealed six distinct subpopulations (Fig. 3H, I; Supplementary material 4). Notably, canonical ameloblast markers such as \u003c/strong\u003e\u003cem\u003eEnam\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003eKlk4\u003c/em\u003e\u003cstrong\u003e, and \u003c/strong\u003e\u003cem\u003eGm17660, \u003c/em\u003e\u003cstrong\u003ewhich are characteristic of mature ameloblasts in adult incisors\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e\u003cstrong\u003e41\u003c/strong\u003e\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e, were not detected, indicating that ameloblast differentiation in the molar epithelium remains incomplete at this developmental stage. Instead, we identified two major populations of \u003c/strong\u003e\u003cem\u003eShh\u003c/em\u003e\u003cstrong\u003e-positive cells corresponding to the stratum intermedium (SI) and pre-ameloblasts. The SI subcluster, localized to the coronal region, was defined by the expression of \u003c/strong\u003e\u003cem\u003eShh\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003eMaf\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003eHopx\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003eRhov\u003c/em\u003e\u003cstrong\u003e, and \u003c/strong\u003e\u003cem\u003eNotch1\u003c/em\u003e\u003cstrong\u003e (Fig. 3I, J, K, K’, N, N’; Supplementary Fig. 4G, I, J; Supplementary Table 8). In contrast, the pre-ameloblast subcluster, positioned more apically, expressed \u003c/strong\u003e\u003cem\u003eTubb3\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003eWfdc2\u003c/em\u003e\u003cstrong\u003e, and \u003c/strong\u003e\u003cem\u003eShh\u003c/em\u003e\u003cstrong\u003e, suggesting a distinct yet closely related stage within the ameloblast lineage (Fig. 3I, J, K, K’; Supplementary Fig. 4G; Supplementary Table 8).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth epithelial subclusters (SI and pre-ameloblasts) exhibited upregulation of EGFR and hypoxia signaling pathways. Additionally, SI cells showed increased activity of Wnt and PI3K signaling pathways, suggesting their distinct functional roles during ameloblast lineage progression (\u003cstrong\u003eSupplementary Fig. 4F\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe stellate reticulum (SR) subcluster was marked by the expression of \u003cem\u003eEif1b\u003c/em\u003e, \u003cem\u003eSmyd3\u003c/em\u003e, and \u003cem\u003ePfn2\u003c/em\u003e, though it exhibited greater transcriptional heterogeneity compared to other epithelial populations. This subcluster also showed upregulation of TRAIL and JAK-STAT signaling pathways, suggesting active involvement in immune-related and stress-responsive signaling during tooth development (\u003cstrong\u003eFig. 3I, J, L, L’; Supplementary Fig. 4F, H; Supplementary Table 8\u003c/strong\u003e). The Tyms-positive subcluster (Tyms+) was defined by the expression of \u003cem\u003eTyms\u003c/em\u003e, \u003cem\u003eBirc5\u003c/em\u003e, \u003cem\u003ePclaf\u003c/em\u003e, and various histone genes (\u003cstrong\u003eFig. 3H, J; Supplementary Table 8\u003c/strong\u003e). As expected, the proliferating cell subcluster was enriched for genes such as \u003cem\u003eMki67, \u003c/em\u003e\u003cem\u003eLars2\u003c/em\u003e, and \u003cem\u003eArid1a\u003c/em\u003e, which are involved in cell cycle regulation, DNA replication, and transcriptional control (\u003cstrong\u003eFig. 3H, J, O, O’; Supplementary Fig. 3D; Supplementary Table 8\u003c/strong\u003e). The subcluster encompassing the outer enamel epithelium (OEE), DS, and RSDL displayed a shared transcriptional signature characterized by the expression of \u003cem\u003eNotch2\u003c/em\u003e, \u003cem\u003eFrem2\u003c/em\u003e, \u003cem\u003eVcan\u003c/em\u003e, \u003cem\u003eNotch1\u003c/em\u003e, and \u003cem\u003eMaf\u003c/em\u003e, the latter also found in the SI. This subcluster showed specific activation of TGFβ, PI3K and MAPK signaling pathways, while Wnt signaling activity was markedly downregulated, suggesting a different regulatory environment controlling epithelial organization and signaling in these regions \u003cstrong\u003e(Fig. 3I, J, M - N’; Supplementary Fig. 4F, I, J; Supplementary Table 8)\u003c/strong\u003e. Latent time analysis revealed that most epithelial cells remain in an undifferentiated state, making it challenging to clearly distinguish individual dental epithelial subclusters \u003cstrong\u003e(Supplementary Fig. 4H)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eNext, we identified a small cluster of \u003cem\u003eLgr5\u003c/em\u003e-positive epithelial cells \u003cstrong\u003e(Fig. 3P)\u003c/strong\u003e, characterized by elevated expression of ECM components, including \u003cem\u003eCol8a1\u003c/em\u003e, \u003cem\u003eLama3\u003c/em\u003e, and \u003cem\u003eCol4a5\u003c/em\u003e \u003cstrong\u003e(Supplementary Fig. 5A–D)\u003c/strong\u003e. In addition to these ECM-related genes, this population also expressed several components of the Wnt signaling pathway, encompassing both canonical and non-canonical signaling branches such as \u003cem\u003ePtk7\u003c/em\u003e, \u003cem\u003eTc\u003c/em\u003e\u003cem\u003ef7l2\u003c/em\u003e, \u003cem\u003eSfrp2\u003c/em\u003e, \u003cem\u003eRock2\u003c/em\u003e, \u003cem\u003eGpc3\u003c/em\u003e, and \u003cem\u003eCdc42ep3\u003c/em\u003e (\u003cstrong\u003eSupplementary Fig. 5E–L\u003c/strong\u003e)\u003csup\u003e58,\u003cem\u003e62\u003c/em\u003e\u003c/sup\u003e. Spatially, these \u003cem\u003eLgr5\u003c/em\u003e-positive epithelial cells were positioned at the interface of three major epithelial subclusters: the SI, SR, and the cluster the OEE, DS, and RSDL. This molecular signature is consistent with their transitional location within the tooth germ, suggesting a specialized role in mediating signaling and structural integration across epithelial compartments.\u003c/p\u003e\n\u003cp\u003eIn summary, the developing tooth epithelium comprises diverse cell types defined by position and function. Our dataset captured key epithelial populations, including SR and DS cells, but lacked fully differentiated ameloblasts, as markers like \u003cem\u003eEnam\u003c/em\u003e and \u003cem\u003eAmelx\u003c/em\u003e were not expressed—likely reflecting the earlier developmental stage of molars compared to incisors. At E18.5, we primarily observed pre-ameloblasts and undifferentiated epithelial cells expressing markers such as \u003cem\u003eKrt17\u003c/em\u003e and \u003cem\u003eEpcam \u003c/em\u003e(\u003cstrong\u003eSupplementary material 4\u003c/strong\u003e), similar to profiles reported at E16.5, in contrast to the mature ameloblast expression signatures found at postnatal stages like P3.5\u003csup\u003e15\u003c/sup\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of stem and progenitor cell markers expression in Lgr5-positive epithelial cells of mouse molars\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the presence of \u003cem\u003eLgr5\u003c/em\u003e-positive cells across distinct epithelial and mesenchymal subclusters, and their unique localization within the tooth germ, we explored whether these cells might represent a stem or progenitor cell population—analogous to those identified in the LCL of continuously growing mouse incisors\u003csup\u003e39,42\u003c/sup\u003e. To date, a comparable stem cell population in the molar region has not been described, prompting us to investigate its potential existence. \u003c/p\u003e\n\u003cp\u003eNotably, stem cells in the LCL are known to co-express \u003cem\u003eLgr5\u003c/em\u003e, \u003cem\u003eLrig1\u003c/em\u003e, and \u003cem\u003eSox2\u003c/em\u003e\u003cem\u003e\u003csup\u003e13\u003c/sup\u003e\u003c/em\u003e. SOX2 has been identified as a marker of epithelial progenitor cells within the DL, where it plays a key role in the sequential development of mouse molars (M1, M2, and M3), as well as in the formation of the SDL and DS in human primary molars and in the DL of reptiles\u003csup\u003e38\u003c/sup\u003e. In the LCL of mouse incisors, LGR5- and SOX2-positive cells have been identified in a region known to support the continuous growth of these teeth\u003csup\u003e4,38\u003c/sup\u003e. Supporting its essential role in tooth development, mutations in \u003cem\u003eSOX2\u003c/em\u003e have been associated with dental anomalies in humans, including supernumerary teeth and the prolonged retention of deciduous teeth\u003csup\u003e43\u003c/sup\u003e. \u003c/p\u003e\n\u003cp\u003eSingle-cell RNA-seq analysis uncovered a \u003cem\u003eLgr5\u003c/em\u003e-positive epithelial cluster enriched for stem cell signature genes such as \u003cem\u003eSox2\u003c/em\u003e, \u003cem\u003eGli1\u003c/em\u003e, \u003cem\u003eLrig1\u003c/em\u003e, and \u003cem\u003eIgfbp5\u003c/em\u003e \u003cstrong\u003e(Supplementary Fig. 5M–P)\u003c/strong\u003e. To determine whether \u003cem\u003eLgr5\u003c/em\u003e expression overlaps with the stem cell marker \u003cstrong\u003eSOX2\u003c/strong\u003e at the protein level, we performed co-localization analysis in dental tissues. Since there are no suitable antibodies for the immunohistochemical detection of LGR5 in mouse tissue sections, we used EGFP expression as a surrogate marker for the identification of cells with an active \u003cem\u003eLgr5\u003c/em\u003e locus. For this purpose, we used the\u003cem\u003e LGR5-EGFP-IRES-CreERT2\u003c/em\u003e mouse strain.\u003c/p\u003e\n\u003cp\u003eWe observed partial co-expression in the basal layers of the lingual part of the DS. \u003cem\u003eSOX2\u003c/em\u003ewas detected as early as E14.5, corresponding with the onset of RSDL formation, and its expression expanded into the thickening rudimentary RSDL at E16.5–E18.5 \u003cstrong\u003e(Fig. 3P-S)\u003c/strong\u003e. In contrast, EGFP protein extended beyond the shared basal domain into deeper DS cells and the SR \u003cstrong\u003e(Fig. 3Q-S, 4H’)\u003c/strong\u003e. Taken together, these findings indicate that co-expression of LGR5 and SOX2 was restricted to small regions within the epithelium. This observation is consistent with previous studies suggesting that LGR5-positive cells represent only a subset of the broader SOX2-positive cell population\u003csup\u003e44\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eNotably, LGR5 (EGFP)-positive epithelial cells in developing molars were found to be non-proliferative, reflecting the behavior of the adjacent LGR5-positive mesenchymal population. This was evidenced by the absence of Ki67 expression in both epithelial and mesenchymal LGR5-expressing domains \u003cstrong\u003e(Supplementary Fig. 3C, D)\u003c/strong\u003e. To further assess proliferative activity, we performed short-term BrdU labeling (2-hour pulse), which revealed only sparse BrdU incorporation within the LGR5-positive region of M1, suggesting that these cells are largely quiescent at this stage. In contrast, more pronounced BrdU labeling was observed in the DS and the forming RSDL of the developing M2, indicating higher proliferative activity in these earlier or less differentiated structures \u003cstrong\u003e(Supplementary Fig. 3E–F’)\u003c/strong\u003e. Together, these findings suggest that although LGR5 marks a subset of epithelial cells, they are not actively cycling and may instead represent a more specialized or quiescent population, rather than classical progenitor or transit-amplifying cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpatio-temporal mapping of Lgr5 expression during molar odontogenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we mapped the occurrence and spatial distribution of Lgr5-positive cells from early tooth initiation (E11.5) to postnatal stage (P4) covering key stages of molar odontogenesis using the \u003cem\u003eLGR5-EGFP-IRES-CreERT2\u003c/em\u003e mouse strain and used EGFP as a surrogate marker for Lgr5 expression \u003cstrong\u003e(Fig. 4A–H’’).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt E11.5, the EGFP signal was detected near epithelial thickening but not in the developing tooth placode \u003cstrong\u003e(Fig. 4A–A’’)\u003c/strong\u003e. At the bud stage (E12.5–E13.5), EGFP-positive cells appeared on the lingual side of the dental epithelium and in the buccal mesenchyme \u003cstrong\u003e(Fig. 4B–C’’)\u003c/strong\u003e. At the cap stage (E14.5–E15.5), expression was restricted to the lingual epithelium of the DS, the RSDL and the labial mesenchyme below the oral epithelium \u003cstrong\u003e(Fig. 4D–E’’)\u003c/strong\u003e. This pattern \u003c/p\u003e\n\u003cp\u003ewas maintained until the bell stage (E16.5–E18.5) \u003cstrong\u003e(Fig. 4F–G’’)\u003c/strong\u003e, although at E18.5 EGFP expression could no longer be detected in the rudimentary dental lamina. At P4, expression decreased further and was restricted to the epithelium of the degenerating DS and the adjacent labial mesenchyme \u003cstrong\u003e(Fig. 4H-H’’)\u003c/strong\u003e. Sagittal sections of the molar region confirmed a uniform expression pattern in both M1 and M2, with EGFP-positive cells mainly located in the posterior epithelial tail — an area implicated in sequential molar formation \u003cstrong\u003e(Supplementary Fig. 6G)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo confirm that EGFP expression accurately reflected \u003cem\u003eLgr5\u003c/em\u003e-expressing cells, we performed RNAScope analysis of \u003cem\u003eLgr5\u003c/em\u003e mRNA. Consistent with the reporter signal, \u003cem\u003eLgr5\u003c/em\u003e transcripts exhibited an asymmetric distribution in the DS and superficial mesenchyme at E16.5 and E18.5 \u003cstrong\u003e(Supplementary Fig. 3A, B)\u003c/strong\u003e, which was very similar to the EGFP pattern in \u003cem\u003eLGR5-EGFP-IRES-CreERT2\u003c/em\u003e mice.\u003c/p\u003e\n\u003cp\u003eIn summary, analysis of Lgr5 expression during molar odontogenesis - in a broader craniofacial context - reveals a dynamic and tightly regulated spatio-temporal pattern. Several previous studies have shown that LGR5-positive cells are not found in the classical epithelial stem cell niches of the craniofacial region, but predominantly in mesenchymal compartments adjacent to folding epithelial structures\u003csup\u003e4,37,45\u003c/sup\u003e. This suggests that LGR5 also marks a population of mesenchymal cells that are involved in the control of epithelial patterning and do not serve as direct epithelial progenitors. The distribution of LGR5-positive cells in the molar region possibly indicates their role in morphogenesis, particularly within the posterior epithelial tail. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLineage tracing reveals region-specific contribution of LGR5⁺ cells to molar development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess whether LGR5-positive cells contribute to molar lineages as they exhibit their low proliferative activity typical for stem cells, we performed lineage tracing using \u003cem\u003eLgr5-EGFP-IRES-CreERT2\u003c/em\u003e mice crossed with the \u003cem\u003eRosa26-tdTomato\u003c/em\u003e reporter line \u003cstrong\u003e(Fig. 5A)\u003c/strong\u003e. Expression of the red fluorescent protein tdTomato was induced by tamoxifen administration at E12.5, a stage when EGFP expression driven from the \u003cem\u003eLgr5\u003c/em\u003e locus was detectable in the epithelial thickening of the tooth and in the labial mesenchyme \u003cstrong\u003e(Fig. 2S, S’; \u003c/strong\u003eLGR5⁺ cells, green signal) and embryos were harvested at two distinct developmental stages of M2 development (E15.5 and E18.5 - induction and progression of M2).\u003c/p\u003e\n\u003cp\u003eBy E18.5, a few LGR5-D) cells were observed (red signal) in the RSDL and SI of M1, whereas in M2, these cells were more prominent within the SR and RSDL \u003cstrong\u003e(Fig. 5B’–B’’’, C’–C’’’)\u003c/strong\u003e. Additionally, scattered LGR5-D cells were detected in the mesenchyme surrounding both M1 and M2, particularly in the region adjacent to the DS \u003cstrong\u003e(Fig. 5B’, B’’, C’, C’’)\u003c/strong\u003e. A shorter tracing interval (analysis at E15.5), revealed a higher number of LGR5-D cells in the DS, RSDL, and labial mesenchyme, with a spatial distribution similar to that observed at E18.5 \u003cstrong\u003e(Supplementary Fig. 6A-F’’)\u003c/strong\u003e. Interestingly, over time, an increasing number of LGR5-D cells were detected in the SR, inner enamel epithelium (IEE), and RSDL of M2, although these cells had lost EGFP expression by E18.5, indicating downregulation of \u003cem\u003eLgr5\u003c/em\u003e. At E15.5, nearly all LGR5-D cells still expressed EGFP, confirming their recent origin from \u003cem\u003eLgr5\u003c/em\u003e-expressing progenitors \u003cstrong\u003e(Supplementary Fig. 6B-F’’)\u003c/strong\u003e. Three-dimensional analysis further demonstrated a continuous LGR5 signal along the jaw, with the highest density of LGR5-D cells in the posterior region near M2 \u003cstrong\u003e(Supplementary Fig. 6G)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eQuantification of LGR5⁺ (EGFP⁺, green), LGR5-D (tdTomato⁺, red), and double-positive cells at two tracing intervals (E12.5–E15.5 and E12.5–E18.5) \u003cstrong\u003e(Fig. 5D–G) \u003c/strong\u003erevealed stable LGR5 expression within the epithelial compartments of both M1 and M2. In contrast, a significant reduction in LGR5⁺ cells was observed in the mesenchyme of M1 by E18.5 \u003cstrong\u003e(Fig. 5F)\u003c/strong\u003e. Over time, the proportion of LGR5-descendant cells increased, accompanied by a rise in double-positive cells in both molars. These results suggest that most LGR5⁺ cells remain within their original tissue niche \u003cstrong\u003e(Fig. 5E–G)\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003eTo uncover temporal contributions of Lg5+ cells to molar development, we also label cells at E13, when individual tooth buds are advanced, and analyzed the outcome at E18.5. The results were consistent with previous findings \u003cstrong\u003e(Supplementary Fig. 7A)\u003c/strong\u003e, showing LGR5-D cells localized at the tip of the RSDL extending toward M2, as well as in the developing M3 region \u003cstrong\u003e(Supplementary Fig. 7B, C)\u003c/strong\u003e. Sagittal sections further revealed LGR5-D cells at the leading edge of the RSDL directed toward the second molar \u003cstrong\u003e(Supplementary Fig. 7D, D’’)\u003c/strong\u003e and in the distal tip of the lamina associated with M2, corresponding to the future third molar \u003cstrong\u003e(Supplementary Fig. 7E, E’’)\u003c/strong\u003e. Notably, the distribution of LGR5-positive cells (green, red, and double-positive) within the DS differed between the M1 and M2 regions, with more superficial localization observed near M2 \u003cstrong\u003e(Supplementary Fig. 7D’, E’)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn summary\u003c/strong\u003e, LGR5-positive cells primarily contribute to the DS and RSDL, with only a few descendants observed in the IEE of M1. Lineage tracing revealed a more substantial contribution of these cells to the formation of M2. While a subset of LGR5-positive descendants migrates posteriorly along the jaw, the majority remain confined to their original niche over time. This restricted contribution contrasts with fate-mapping studies in other organs, where LGR5-positive stem cells give rise to all epithelial cell types\u003csup\u003e17\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThese findings are particularly relevant in the context of sequential tooth development. The posterior extension of LGR5-positive cells corresponds to the sites of future molar initiation (e.g., M2 and M3), supporting the hypothesis that LGR5-positive cells in the epithelial tail region may contribute to the formation of new tooth buds. Interestingly, while some LGR5-positive cells appear to remain stationary\u003csup\u003e37\u003c/sup\u003e, others exhibit limited migratory behavior—suggesting a dual function as both localized signaling centers and potential progenitors for emerging dental structures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSorting of Lgr5-positive cells and subsequent scRNA-seq revealed several distinct subpopulations of Lgr5\u003csup\u003e+\u003c/sup\u003e mesenchymal cells \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the molecular characteristics of LGR5-positive cells, we isolated EGFP-positive cells from \u003cem\u003eLgr5-EGFP-IRES-CreERT2\u003c/em\u003e mice at two developmental stages using flow cytometry: E16.5, when the RSDL is just beginning to form, and E18.5, when the RSDL has matured into a well-defined epithelial protrusion. Single-cell RNA-seq was performed separately for each sample (\u003cstrong\u003eFig. 6A; Supplementary material 5)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eAnalysis of the individual datasets revealed that unspecified oral fibroblasts and early pre-osteoblastic clusters were more prevalent at E16.5. In contrast, E18.5 samples exhibited a notable expansion of dental follicle populations, and most GFP-positive cells at this stage showed signs of active proliferation \u003cstrong\u003e(Fig. 6B, C)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo gain a comprehensive overview, we integrated both datasets and performed unsupervised clustering, identifying 11 distinct cell subpopulations. Among them, a prominent dental epithelial cluster was defined by the expression of canonical epithelial markers such as \u003cem\u003eEpcam\u003c/em\u003e, various keratins, and other intermediate filament genes essential for epithelial integrity (\u003cstrong\u003eSupplementary material\u003c/strong\u003e). Gene Set Enrichment Analysis (GSEA) revealed upregulation of VEGF and EGFR signaling pathways in this epithelial population \u003cstrong\u003e(Fig. 6D, E; Supplementary Table 9)\u003c/strong\u003e. Moreover, comparison of signaling pathway activity between the E16.5 and E18.5 datasets revealed increased JAK-STAT signaling and a concomitant decrease in WNT pathway activity \u003cstrong\u003e(Fig. 5G, H)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eIn agreement with our first \"unbiased\" E18.5 scRNA-seq analysis, two distinct dental follicle clusters were identified: the sDF, which comprised cells located close to the dental epithelium, and the oDF, which consists of cells located further away. These clusters differed not only in their spatial positioning within the molar region, but also in their gene expression profiles. The sDF cluster was characterized by the expression of \u003cem\u003ePtch1\u003c/em\u003e, \u003cem\u003eCar2\u003c/em\u003e and \u003cem\u003eTnmd\u003c/em\u003e as well as by the upregulation of the TGFβ pathway. In contrast, the oDF cluster expressed \u003cem\u003eNpnt\u003c/em\u003e, \u003cem\u003eGas6\u003c/em\u003e and \u003cem\u003eMgp\u003c/em\u003e and showed enrichment of the Wnt, TRAIL and VEGF signaling pathways. Both clusters expressed canonical markers for dental follicles such as \u003cem\u003eRunx2\u003c/em\u003e and \u003cem\u003eAlpl\u003csup\u003e46,47\u003c/sup\u003e\u003c/em\u003e. The TNFα signaling pathway was also upregulated in both follicle populations \u003cstrong\u003e(Fig.\u003c/strong\u003e\u003cstrong\u003e6D,\u003c/strong\u003e\u003cstrong\u003eE;\u003c/strong\u003e\u003cstrong\u003eSupplementary\u003c/strong\u003e\u003cstrong\u003eTable\u003c/strong\u003e\u003cstrong\u003e9)\u003c/strong\u003e. Another cluster consisted of actively proliferating cells, as shown by the expression of cell cycle–related genes such as \u003cem\u003eKi67\u003c/em\u003e, \u003cem\u003eTop2a\u003c/em\u003e and \u003cem\u003ePclaf\u003c/em\u003e \u003cstrong\u003e(Fig.\u003c/strong\u003e\u003cstrong\u003e6C,\u003c/strong\u003e\u003cstrong\u003eD;\u003c/strong\u003e\u003cstrong\u003eSupplementary\u003c/strong\u003e\u003cstrong\u003eTable\u003c/strong\u003e\u003cstrong\u003e9)\u003c/strong\u003e. A distinct labial mesenchymal cluster was enriched with LGR5-positive cells and expressed markers such as \u003cem\u003eSostdc1\u003c/em\u003e, \u003cem\u003eTcea3\u003c/em\u003e and \u003cem\u003eTfap2b\u003c/em\u003e. This cluster also showed activation of the Wnt and TGFβ signaling pathways, consistent with the increased expression of Lgr5. The remaining clusters consisted of different populations of differentiating osteoblasts and unspecified oral fibroblasts \u003cstrong\u003e(Fig.\u003c/strong\u003e\u003cstrong\u003e6D,\u003c/strong\u003e\u003cstrong\u003eE;\u003c/strong\u003e\u003cstrong\u003eSupplementary\u003c/strong\u003e\u003cstrong\u003eTable\u003c/strong\u003e\u003cstrong\u003e9)\u003c/strong\u003e. Latent time analysis also identified a population of less differentiated tooth-associated cells within the labial mesenchyme \u003cstrong\u003e(Fig.\u003c/strong\u003e\u003cstrong\u003e6F)\u003c/strong\u003e, where most LGR5-expressing cells were localized. This supports the hypothesis that LGR5-positive cells exhibit an undifferentiated, progenitor-like phenotype and emphasizes the potential importance of mesenchymal cells in the early stages of tooth development. Comparison of pathway activity between the E16.5 and E18.5 data sets in the dental epithelium showed increased JAK-STAT signaling (data not shown) and a concomitant decrease in WNT pathway activity (\u003cstrong\u003eFig. 6G, H\u003c/strong\u003e). Moreover, comparison of signaling pathway activity between the E16.5 and E18.5 datasets in the dental mesenchyme revealed a reduction in WNT signaling activity at E18.5 \u003cstrong\u003e(Fig. 6I, J)\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe transcriptome of Lgr5⁺ epithelial cells reflects their epithelial identity and their partially immature state\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we focused specifically on the cluster representing Lgr5-positive cells within the dental epithelium. Since our sorting strategy selectively targeted LGR5-positive cells, we obtained enough of these cells, allowing for a more detailed characterization. This enrichment also enabled us to compare and distinguish the molecular features of epithelial \u003cem\u003eLgr5\u003c/em\u003e-positive cells from their mesenchymal counterparts.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLgr5\u003c/em\u003e-positive epithelial cells displayed a transcriptional signature consistent with epithelial identity, marked by high expression of multiple keratins (\u003cem\u003eKrt5\u003c/em\u003e, \u003cem\u003eKrt7\u003c/em\u003e, \u003cem\u003eKrt14\u003c/em\u003e), along with key epithelial markers such as \u003cem\u003eDsp\u003c/em\u003e, \u003cem\u003eDsc\u003c/em\u003e, \u003cem\u003eDapl1\u003c/em\u003e, and \u003cem\u003eAsprv1\u003c/em\u003e. These cells were also defined by strong desmosome-mediated adhesion signatures, marked by elevated expression of \u003cem\u003ePkp3\u003c/em\u003e, \u003cem\u003eDsp\u003c/em\u003e, \u003cem\u003eRab25\u003c/em\u003e, and \u003cem\u003eCdh1\u003c/em\u003e. Among the actin-associated genes, \u003cem\u003eFermt1\u003c/em\u003e was particularly highly expressed, suggesting a role in cytoskeletal organization and integrin-mediated signal transduction. In addition, genes associated with ion channel activity, such as \u003cem\u003eTacstd2\u003c/em\u003e, \u003cem\u003eS100A14\u003c/em\u003e and \u003cem\u003eFxyd3\u003c/em\u003e, were increasingly expressed in this population, suggesting active membrane-associated signaling dynamics. Within this epithelial cluster, we also detected several stem cell-associated transcription factors, such as \u003cem\u003eSox2\u003c/em\u003e, \u003cem\u003eSox6\u003c/em\u003e, and \u003cem\u003ePitx2\u003c/em\u003e, along with key cell cycle regulators including \u003cem\u003eSfn\u003c/em\u003e and \u003cem\u003eSerpinb5\u003c/em\u003e, suggesting a progenitor-like state with both proliferative potential and epithelial-specific functionality (\u003cstrong\u003eSupplementary Table 10\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eLikely reflecting their close proximity to the basement membrane, LGR5-positive epithelial cells showed strong expression of core basement membrane components, including \u003cem\u003eLama5\u003c/em\u003e, \u003cem\u003eLamb3\u003c/em\u003e, \u003cem\u003eLamc2\u003c/em\u003e, and \u003cem\u003eCol17a1\u003c/em\u003e. Additionally, several signaling pathway regulators were specifically upregulated in epithelial LGR5-positive cells compared to their mesenchymal counterparts. Notably, these included \u003cem\u003eEsrp1\u003c/em\u003e (a regulator of epithelial-specific splicing), \u003cem\u003eSpint2\u003c/em\u003e (a serine protease inhibitor involved in maintaining epithelial integrity), and \u003cem\u003eJag2\u003c/em\u003e (a Notch pathway ligand) \u003cstrong\u003e(Supplementary Table 10)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eIn summary, scRNA-seq analysis revealed different transcriptional profiles in LGR5-positive cells, with clear differences between epithelial and mesenchymal populations. Epithelial LGR5-positive cells exhibited a relatively homogeneous molecular signature characterized by the enrichment of genes involved in basal membrane composition, cell adhesion, ion channel activity, integrin signaling, and extracellular matrix organization. In contrast, LGR5-positive mesenchymal cells formed several transcriptionally distinct subpopulations, indicating functional heterogeneity within this compartment. Overall, these results suggest that epithelial LGR5-positive cells play a key role in the maintenance of epithelial identity, structural integrity and specialized functions during tooth development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLgr5 deficiency disrupts sequential molar development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the functional role of LGR5 during molar development, we used a mouse model in which the \u003cem\u003eLgr5\u003c/em\u003e gene was constitutively deleted. The \u003cem\u003eLgr5\u003c/em\u003e knockout mice exhibit perinatal lethality primarily due to severe ankyloglossia of the tongue — a developmental defect characterized by impaired tongue motility — and associated dilatations of the gastrointestinal tract, as previously reported\u003csup\u003e45\u003c/sup\u003e. These phenotypic abnormalities result in early postnatal death, which limits the window of analysis to the embryonic and late prenatal stages. Therefore, our study focused on molar development before birth so that we could assess the morphologic and cellular consequences of Lgr5 deficiency at key stages of odontogenesis. For our analysis, we used the non-functional (\u003cem\u003enull\u003c/em\u003e) \u003cem\u003eLgr5-EGFP-IRES-CreERT2\u003c/em\u003e allele, which does not produce functional LGR5 protein, crossed into a homozygous background. \u003cstrong\u003e(Fig. 7A)\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003eWe performed microscopic analysis of \u003cstrong\u003e\u003cem\u003eLgr5\u003csup\u003e⁻/⁻\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e embryos at E16.5 and E18.5, focusing on tooth morphology. Despite the early and localized expression of \u003cstrong\u003eLgr5\u003c/strong\u003e in M1 during odontogenesis, we did not observe any overt morphological abnormalities in this tooth germ \u003cstrong\u003e(Fig. 7B–C’)\u003c/strong\u003e. In contrast, M2 exhibited several morphological defects, including a shortened DS \u003cstrong\u003e(Fig. 6L–O’)\u003c/strong\u003e. The enamel organ appeared nearly fused with the oral epithelium, suggesting a disruption in the process of sequential molar formation. Additionally, in the labial region of the DS, we observed multiple epithelial outgrowths and finger-like projections extending into the mesenchyme, accompanied by a loss of structural compactness in the DS \u003cstrong\u003e(Fig. 7L–O’).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurthermore, the expression of the stem cell marker \u003cstrong\u003eSOX2\u003c/strong\u003e was markedly reduced in the DS region of \u003cstrong\u003e\u003cem\u003eLgr5\u003c/em\u003e\u003c/strong\u003e-deficient mice in both M1 and M2, suggesting a potential loss of stem cell characteristics \u003cstrong\u003e(Fig. 7D–E’, P–Q’)\u003c/strong\u003e. We also detected ectopically localized SOX2-positive cells on the labial side of the DS, indicating a disruption of the normal polarized expression pattern of this stem cell marker \u003cstrong\u003e(Fig. 7Q’)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eInterestingly, previous studies have shown that LGR5 expression suppresses the formation of cellular extensions, while \u003cem\u003eLgr5\u003c/em\u003e-deficient cells show the formation of cytopodia. In the colon, loss of Lgr5 impairs cell–cell adhesion and disrupts epithelial architecture\u003csup\u003e48\u003c/sup\u003e, while overexpression of LGR5 increases intercellular adhesion and decreases motility of colon cancer cells\u003csup\u003e49\u003c/sup\u003e. These results are consistent with our observations in \u003cem\u003eLgr5\u003c/em\u003e-deficient embryos, in which we detected abnormal epithelial protrusions, finger-like extensions into the mesenchyme, and loss of structural integrity of the dental stalk, suggesting a conserved role of LGR5 in maintaining epithelial cohesion and organization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLgr4\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e expression partially overlaps with \u003cem\u003eLgr5\u003c/em\u003e in molars\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether the different phenotypic results between M1 and M2 were due to functional redundancy between Lgr5 and its related paralogs Lgr4 and Lgr6, we analyzed their expression patterns in the developing molars \u003cstrong\u003e(Fig. 7F, G, J, K, R, S, V, W; Supplementary Fig. 8)\u003c/strong\u003e. In E16.5, \u003cem\u003eLgr4\u003c/em\u003e was broadly expressed in the DS, surrounding mesenchyme, RSDL, IEE and SI of M1. In M2, \u003cem\u003eLgr4\u003c/em\u003e was additionally detected in the SR, although this expression decreased until E18.5 \u003cstrong\u003e(Supplementary Fig. 8A–C; G–I″)\u003c/strong\u003e. Overall, the expression of \u003cem\u003eLgr4 \u003c/em\u003ewas broader and more diffuse than that of \u003cem\u003eLgr5\u003c/em\u003e, with comparable or even higher signal intensity.\u003c/p\u003e\n\u003cp\u003eIn contrast, the expression of Lgr6 was significantly weaker. At E16.5, \u003cem\u003eLgr6\u003c/em\u003e-positive cells were sparsely distributed in the DS epithelium, surrounding mesenchyme and IEE of M1, while expression in the M2 epithelium was minimal \u003cstrong\u003e(Supplementary Fig. 8D–F″)\u003c/strong\u003e. At E18.5, \u003cem\u003eLgr6\u003c/em\u003e expression had disappeared from the M1 epithelium and was only present as scattered signals in the mesenchyme, with weak expression in the IEE of M2 \u003cstrong\u003e(Supplementary Fig. 8J–L″)\u003c/strong\u003e. Minimal overlap between \u003cem\u003eLgr5\u003c/em\u003e and \u003cem\u003eLgr6\u003c/em\u003e was observed and was restricted to the labial mesenchyme and DS epithelium at E16.5 \u003cstrong\u003e(Supplementary Fig. 8D″, E″, F″)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eBecause the expression of \u003cem\u003eLgr4\u003c/em\u003e and Lgr5 partially overlap, we examined also the expression of \u003cem\u003eLgr4\u003c/em\u003e in Lgr5-deficient mice. While the spatial pattern of \u003cem\u003eLgr4\u003c/em\u003e remained largely unchanged, the expression of \u003cem\u003eLgr4\u003c/em\u003e was reduced - especially in the second molar \u003cstrong\u003e(Fig. 7R, S, V, W)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eOverall, these results suggest that Lgr4 is the only Lgr5 paralog with overlapping expression in the DS, particularly in M1, where its presence may partially compensate for the loss of Lgr5. This is consistent with our phenotypic observations: In Lgr5-deficient mice, M1 appeared morphologically normal, whereas the defects were restricted to the M2. A similar compensatory role was hypothesized for \u003cem\u003eLgr4\u003c/em\u003e in M3: in keratinocyte-specific Lgr4 knockout mice, developmental defects in the M3 occurred as early as at E14. In addition, Lgr4 knockout mice frequently exhibited underdeveloped or missing M3\u003csup\u003e50\u003c/sup\u003e. In addition, \u003cem\u003eLgr4 \u003c/em\u003ewas expressed in SOX2-positive cells at the posterior end of M2 and in early M3 tooth germs, suggesting that it may support tooth initiation and compensate for Lgr5 loss.\u003c/p\u003e\n\u003cp\u003eThis functional redundancy is consistent with findings from other tissues such as the intestine, where deletion of Lgr5 does not affect epithelial self-renewal, presumably due to compensation by other members of the LGR family\u003csup\u003e51,52\u003c/sup\u003e. These parallels highlight the extensive functional overlap between the LGR receptors and their context-dependent role in development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDownstream Wnt signaling is impaired in the Lgr5-deficient dental epithelium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLGR5 was originally identified as a Wnt target gene that is upregulated in human cancer cells with aberrant Wnt/β-catenin activity\u003csup\u003e53\u003c/sup\u003e. In adult tissues, Wnt signaling plays a critical role in the maintenance of somatic stem cells and committed progenitor compartments, as described in detail by Cadigan and Peifer (2009)\u003csup\u003e54\u003c/sup\u003e. Importantly, Lgr5 serves both as a transcriptional target of the canonical Wnt/β-catenin pathway and as a positive regulator that enhances Wnt signaling activity\u003csup\u003e52\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn the context of tooth development, Wnt signaling has been shown to have stage- and tissue-specific effects. For example, forced activation of Wnt signaling in the embryonic mesenchyme inhibits the formation of posterior molars (M2 and M3)\u003csup\u003e55\u003c/sup\u003e, while epithelial-specific activation of the Wnt pathway can stimulate successional dental lamina and lead to ectopic formation of second-generation molars\u003csup\u003e56\u003c/sup\u003e. These findings highlight the balance of Wnt signaling required for proper molar formation and suggest that disruption of components of the Wnt signaling pathway — such as Lgr5— can alter the normal course of molar development.\u003c/p\u003e\n\u003cp\u003eTo investigate the molecular mechanisms underlying disrupted tooth development in Lgr5-deficient mice, we examined whether the LGR5-Wnt signaling axis is activated by analyzing the expression of LGR4/5/6 ligands—R-spondin family members (\u003cem\u003eRspo1\u003c/em\u003e, \u003cem\u003eRspo2\u003c/em\u003e, and \u003cem\u003eRspo3\u003c/em\u003e)—at E18.5, a developmental stage at which the dental phenotype is already evident.\u003c/p\u003e\n\u003cp\u003eExpression analysis of \u003cem\u003eRspo1\u003c/em\u003e revealed a partial overlap with \u003cem\u003eLgr5\u003c/em\u003e in the labial mesenchyme of the DS \u003cstrong\u003e(Supplementary Fig. 9A–D)\u003c/strong\u003e. The expression of \u003cem\u003eRspo2\u003c/em\u003e was restricted to the DP and did not overlap with \u003cem\u003eLgr5\u003c/em\u003e \u003cstrong\u003e(Supplementary Fig. 2M)\u003c/strong\u003e. In contrast, \u003cem\u003eRspo3\u003c/em\u003e was expressed in the mesenchyme on the lingual side of the DS, adjacent to but not overlapping with Lgr5-positive cells in the DS and the RSDL \u003cstrong\u003e(Supplementary Fig. 9E, G)\u003c/strong\u003e. In Lgr5-deficient embryos, the expression of \u003cem\u003eRspo3\u003c/em\u003e was markedly altered and exhibited a more diffuse distribution in both the dental mesenchyme and epithelium. Importantly, the distinct border of \u003cem\u003eRspo3\u003c/em\u003e expression around the DS and RSDL observed in wild-type (WT) embryos was lost in the mutants. These changes were observed in both M1 and M2 \u003cstrong\u003e(Supplementary Fig. 9E–H)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo investigate further changes in downstream Wnt signaling, we analyzed the expression of \u003cem\u003eAxin2\u003c/em\u003e, a known target of the Wnt/β-catenin pathway\u003csup\u003e57,58\u003c/sup\u003e. In WT embryos, \u003cem\u003eAxin2\u003c/em\u003e was mainly expressed in the mesenchyme surrounding the tooth at the bell stage and in the DP and SR of M1 \u003cstrong\u003e(Fig.\u003c/strong\u003e\u003cstrong\u003e7H,\u003c/strong\u003e\u003cstrong\u003eJ)\u003c/strong\u003e. In contrast, \u003cem\u003eAxin2\u003c/em\u003e expression was almost absent in the SR of M2 \u003cstrong\u003e(Fig.\u003c/strong\u003e\u003cstrong\u003e7T,\u003c/strong\u003e\u003cstrong\u003eV)\u003c/strong\u003e. We also observed a partial overlap between \u003cem\u003eAxin2\u003c/em\u003e and \u003cem\u003eLgr4 \u003c/em\u003eand LGR5 expression, especially in the DS adjacent to the dental mesenchyme \u003cstrong\u003e(Fig. 7D-U; Fig.\u003c/strong\u003e\u003cstrong\u003e3W–Y’)\u003c/strong\u003e. In both molars, Lgr5-deficient embryos showed significantly reduced \u003cem\u003eAxin2\u003c/em\u003e expression. In the mutants, \u003cem\u003eAxin2\u003c/em\u003e-positive cells formed only a thin layer surrounding the dental epithelium, whereas they were more widely distributed in WT embryos \u003cstrong\u003e(Fig.\u003c/strong\u003e\u003cstrong\u003e7I,\u003c/strong\u003e\u003cstrong\u003eK,\u003c/strong\u003e\u003cstrong\u003eU,\u003c/strong\u003e\u003cstrong\u003eW)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThese results suggest that the absence of Lgr5 leads to a disruption of downstream Wnt signaling, which likely contributes to the observed defects in dental development. The altered expression patterns of \u003cem\u003eRspo3\u003c/em\u003e and \u003cem\u003eAxin2\u003c/em\u003e indicate a dysregulated signaling environment and underscore the role of LGR5 in enhancing Wnt activity during molar morphogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLgr5 deficiency impairs the integrity of the dental epithelium by disrupting cell adhesion and ECM organization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that increased Lgr5 expression inhibits the formation of cellular projections, while its absence promotes the formation of cytopodia\u003csup\u003e48\u003c/sup\u003e. Furthermore, overexpression of Lgr5 in colon cancer cells enhances intercellular adhesion and suppresses cell motility\u003csup\u003e49\u003c/sup\u003e. Given these findings and the epithelial protrusions observed in the DS of Lgr5-deficient embryos, we investigated whether loss of Lgr5 affects epithelial compactness.\u003c/p\u003e\n\u003cp\u003eFirst, we examined the expression of ECM components that are important for epithelial cohesion and basement membrane stability \u003cstrong\u003e(Supplementary\u003c/strong\u003e\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e9)\u003c/strong\u003e. In Lgr5-deficient embryos, laminin expression was significantly reduced along the developing tooth germ, suggesting that perturbations in ECM composition contribute to impaired structural cohesion within the DS \u003cstrong\u003e(Supplementary\u003c/strong\u003e\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e9I–L)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eNext, we examined cell-cell adhesion molecules such as E-cadherin and β-catenin. In WT embryos, E-cadherin was strongly expressed in the DS and RSDL, with signal extending into the SR in both M1 and M2 \u003cstrong\u003e(Supplementary\u003c/strong\u003e\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e9M,\u003c/strong\u003e\u003cstrong\u003eO)\u003c/strong\u003e. In contrast, in Lgr5-deficient embryos, E-cadherin expression was absent in the SR, indicating a loss of epithelial cohesion \u003cstrong\u003e(Supplementary\u003c/strong\u003e\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e9N,\u003c/strong\u003e\u003cstrong\u003eP)\u003c/strong\u003e. A similar trend was observed for β-catenin \u003cstrong\u003e(Supplementary\u003c/strong\u003e\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e9Q–T)\u003c/strong\u003e. Although β-catenin was present in both WT and Lgr5-deficient epithelium, its signal differed in terms of distribution and expression. In WT embryos, β-catenin was clearly localized at the cell membrane, whereas the signal was diffuse and poorly defined in both molars in the mutants \u003cstrong\u003e(Supplementary\u003c/strong\u003e\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e9Q–T)\u003c/strong\u003e. Of note, β-catenin is also a major transcriptional activator of canonical Wnt signaling\u003csup\u003e59\u003c/sup\u003e, however, we did not detect nuclear localization of β-catenin, suggesting that the canonical Wnt signaling pathway is not strongly activated.\u003c/p\u003e\n\u003cp\u003eThese data suggest that Lgr5 plays a critical role in maintaining epithelial compactness by regulating ECM composition and intercellular adhesion. Loss of Lgr5 disrupts basement membrane organization and weakens epithelial integrity, likely contributing to the aberrant dental morphology in Lgr5-deficient embryos. However, the exact contribution of impaired Wnt signaling in the absence of Lgr5 to these epithelial defects will be necessary to follow in future.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCandidate LGR5 interactors involved in epithelial-mesenchymal crosstalk during tooth development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify potential LGR5 binding partners that could contribute to epithelial-mesenchymal adhesion and structural integrity in the dental stalk, we performed a comparative analysis. To this end, we compared our single-cell data from LGR5-positive cells — identified in small epithelial subclusters and in the dental mesenchyme \u003cstrong\u003e(Fig. 6A)\u003c/strong\u003e — with previously published RNA-seq data from LGR5-expressing craniofacial cells\u003csup\u003e37\u003c/sup\u003e. Given the structural homology and potential functional overlap between LGR5 and LGR4, we compared this list with known and putative LGR4 interactors identified by mass spectrometry \u003cstrong\u003e(Supplementary Fig. 10A; V. Kriz and V. Korinek, unpublished results)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo refine the list of candidates, we filtered the genes based on predicted subcellular localization, prioritizing those associated with the plasma membrane or ECM. This analysis yielded several promising candidates, including \u003cem\u003ePtk7\u003c/em\u003e, \u003cem\u003eHsp90b1\u003c/em\u003e, \u003cem\u003eLgals1\u003c/em\u003e (galectin-1) and \u003cem\u003eAnxa1\u003c/em\u003e, which were selected for further investigation \u003cstrong\u003e(Supplementary Fig. 10)\u003c/strong\u003e. We also included NID2, a basement membrane protein recently identified as an endogenous ligand of LGR4 and a regulator of vascular calcification\u003csup\u003e60\u003c/sup\u003e. Its paralog NID1 was also analyzed to investigate possible redundancies or complementary functions in the dental context \u003cstrong\u003e(Supplementary Fig. 10O–W’)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eWe then examined the spatial expression patterns of these candidate genes within the dental epithelium and mesenchyme in our scRNA-seq dataset. The expression of \u003cem\u003ePtk7\u003c/em\u003e was mainly detected in mesenchymal clusters, where it partially overlapped with the expression of Lgr5. Of note, \u003cem\u003ePtk7\u003c/em\u003e was also found in a small epithelial LGR5-positive subcluster, suggesting that it may act at the epithelial-mesenchymal interface \u003cstrong\u003e(Supplementary Fig. 10B, 11B, D)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eIn contrast, \u003cem\u003eLgals1\u003c/em\u003e and \u003cem\u003eAnxa1\u003c/em\u003e were restricted to the epithelial LGR5-positive subcluster, suggesting a role within the epithelial compartment \u003cstrong\u003e(Supplementary Fig. 10C–J)\u003c/strong\u003e. Similarly, \u003cem\u003eHsp90b1\u003c/em\u003e was expressed in both the LGR5-positive mesenchymal cluster and the epithelial subcluster, as shown by analyses\u003csup\u003e61\u003c/sup\u003e, suggesting a dual role in regulating epithelial and mesenchymal functions \u003cstrong\u003e(Supplementary Fig. 10K–N)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNid2\u003c/em\u003e-positive cells were distributed in both epithelial and mesenchymal LGR5-positive populations. While NID2 was enriched in the IEE, some LGR5-positive cells in the DS and RSDL also expressed NID2 \u003cstrong\u003e(Supplementary Fig. 10W, W’)\u003c/strong\u003e. In contrast, the expression of NID1 was largely restricted to the mesenchyme and partially overlapped with Lgr5. Remarkably, the expression of NID1 in the DL region was localized at the interface between epithelium and mesenchyme, suggesting that signaling between these compartments is regulated \u003cstrong\u003e(Supplementary Fig. 10V, V’)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eAmong these candidates, PTK7 stands out due to its established role in Wnt signaling, cell adhesion and tissue patterning. Its expression at the interface of LGR5-positive epithelial and mesenchymal domains makes it a strong candidate for future functional studies aimed at uncovering LGR5-mediated mechanisms of niche organization in the developing tooth.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePTK7 as a downstream effector and binding partner of LGR5 in the regulation of dental tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePTK7, a member of the receptor tyrosine kinase family, plays a central role in non-canonical Wnt signaling, particularly in the regulation of planar cell polarity and cell adhesion\u003csup\u003e62\u003c/sup\u003e. Although it has no intrinsic kinase activity, PTK7 can mediate extracellular signal transduction across the plasma membrane. The partial co-localization of PTK7 and LGR5 in the epithelium and mesenchyme of the DS suggests a possible functional interaction at the epithelial–mesenchymal interface. This observation in conjunction with the known functions of PTK7 makes it a strong candidate for LGR5-mediated signaling during tooth development \u003cstrong\u003e(Supplementary Fig. 11A–B)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo investigate a possible direct interaction between LGR5 and PTK7, we performed in silico modeling using AlphaFold3\u003csup\u003e63\u003c/sup\u003e. The analysis predicted an interaction between the C-terminal domains of the two proteins — residues 563–907 of LGR5 and 601–1062 of PTK7 — that is stabilized by a disulfide bond. This binding likely occurs between Cys749 of LGR5 (Cys187 in the model) and Cys718 of PTK7 (Cys118 in the model), although an alternative pairing with Cys722 of PTK7 was also considered. The higher conservation of Cys718 suggests that it is the more likely interactor \u003cstrong\u003e(Supplementary Fig. 11E)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eWe confirmed this prediction experimentally by performing co-immunoprecipitation assays with STREP-FLAG-tagged LGR5 and HA-tagged PTK7 in cell cultures. These experiments confirmed a physical connection between LGR5 and PTK7 in reciprocal pull-down experiments \u003cstrong\u003e(Supplementary Fig. 11F)\u003c/strong\u003e, providing direct biochemical evidence for their interaction.\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry at E18.5 showed that PTK7 was enriched in the epithelial compartment of M1 and M2, which overlapped with the LGR5 expression domain and extended from the DS into the RSDL \u003cstrong\u003e(Supplementary Fig. 11G–L’)\u003c/strong\u003e. The PTK7 protein was predominantly membrane-associated, particularly on the lateral and basal surfaces of the basal epithelial cells and was also detected in the adjacent mesenchyme.\u003c/p\u003e\n\u003cp\u003eIn Lgr5-deficient embryos, PTK7 expression was significantly reduced in the dental stalk epithelium—particularly in M2—in regions that normally express Lgr5. This suggests that LGR5 is required for proper membrane localization and stabilization of PTK7 during molar development \u003cstrong\u003e(Supplementary Fig. 11H, H’, J–L’)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003ePTK7 is also known to interact with MMP14 (MT1-MMP), a membrane-associated matrix metalloproteinase. Proteolytic cleavage by MMP14 generates a soluble N-terminal fragment (~70 kDa) and a membrane-bound C-terminal fragment (~50 kDa) that modulates the function of PTK7 in Wnt signaling\u003csup\u003e64\u003c/sup\u003e. In agreement, we found strong co-localization of Lgr5- and MMP14-expressing cells in both mesenchymal and epithelial compartments, particularly in SOX2-positive Lgr5⁺ epithelial cells \u003cstrong\u003e(Supplementary Fig. 11C–D)\u003c/strong\u003e. Moreover, MMP14 expression was upregulated in Lgr5-deficient embryos, particularly in the DS and RSDL of M2, with de novo expression observed in the labial region \u003cstrong\u003e(Supplementary Fig. 5M–P’)\u003c/strong\u003e. These results suggest that LGR5 spatially restricts MMP14 activity and prevents excessive PTK7 cleavage.\u003c/p\u003e\n\u003cp\u003eTaken together, our data support a model in which PTK7 functions as a critical LGR5-dependent effector at the epithelial–mesenchymal interface of the developing tooth. Its proper localization appears to be dependent on LGR5, and its activity seems to be tightly associated with MMP14-mediated cleavage. Disruption of this regulatory axis in Lgr5-deficient embryos compromises the integrity of the tissue within the dental stalk and contributes to the observed morphological abnormalities. These results reveal a novel mechanism by which LGR5 regulates cell adhesion, signal transduction and niche organization during early tooth development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe growth of molar organoids is independent of Lgr5⁺ epithelial cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the different cellular and molecular behavior of LGR5-positive cells in M1 and M2, we investigated whether these epithelial populations exhibit intrinsic differences relevant to sequential tooth development. To determine this, we genetically labeled Lgr5-positive cells and their progeny in utero by inducing pregnant \u003cem\u003eLgr5-EGFP-CreERT2 x Rosa26-tdTomato\u003c/em\u003e females at E12.5 with tamoxifen. We then isolated M1 and M2 tissues from E18.5 embryos and generated single-cell suspensions. We used one half of each sample to establish 3D dental organoid cultures of Lgr5 WT and heterozygous siblings \u003cstrong\u003e(Fig. 8A)\u003c/strong\u003e. The other half of each sample was used to determine the percentage of EGFP- and tdTomato-labeled epithelial cells in the isolated tissues by FACS analysis. In the heterozygous embryos, the percentage of labeled epithelial cells, i.e. EpCam-positive cells, varied, but we were able to detect EGFP-positive and EGFP/tdTomato double-positive cells in all samples. In general, the percentage of labeled cells was higher in the M2 epithelium, at E18.5 there were more EGFP single-positive than EGFP/tdTomato double-positive cells, and we could detect only a few tdTomato single-positive cells \u003cstrong\u003e(Supplementary Fig. 12A)\u003c/strong\u003e. Interestingly, after reconstitution of organoids from single cells in vitro, we could no longer detect EGFP-positive cells microscopically \u003cstrong\u003e(Fig. 8B-I)\u003c/strong\u003e and by FACS analysis (data not shown). The tdTomato-positive organoids, originally derived from EGFP/tdTomato double-positive cells, were maintained in constant numbers in culture over several passages. This observation suggests that epithelial cells forming molar organoids have lost their Lgr5 expression but are still able to proliferate and maintain the in vitro structures.\u003c/p\u003e\n\u003cp\u003eTo confirm that Lgr5 is dispensable for organoid formation and growth, we performed a lineage tracing in vitro with tamoxifen induction, followed by microscopic and flow cytometric analyzes of Lgr5 WT, heterozygous and knock-out cells. We isolated M1 and M2 tissues from E18.5 embryos of different genotypes, prepared 3D molar organoids and treated them with 4-hydroxytamoxifen (4-OHT) to induce CreERT2-mediated recombination. Organoids from Lgr5-deficient molars exhibited comparable growth dynamics, morphology and structural organization to their wild-type and heterozygous counterparts. Epithelial sphere formation — a hallmark of organoid integrity and viability — was maintained in all genotypes, with no significant differences in size, budding or compaction \u003cstrong\u003e(Fig. 8J, L, N)\u003c/strong\u003e. After 48 h of 4-OHT treatment, no EGFP- or tdTomato-positive cells were detected in the M1 organoids regardless of \u003cem\u003eLgr5\u003c/em\u003e genotype (data not shown). In contrast, M2 organoids contained only a small number of tdTomato⁺/EGFP cells (\u0026lt;1% of live cells), which were observed in both heterozygous and Lgr5-deficient samples \u003cstrong\u003e(Fig. 8K, M, O)\u003c/strong\u003e. This confirms that while LGR5-positive progeny persists, the original LGR5⁺ population does not expand or maintain its identity in vitro. Immunostaining for Ki67 and SOX2 also confirmed that proliferation and stemness are similar in Lgr5-deficient and control organoids \u003cstrong\u003e(Fig. 8P–S’)\u003c/strong\u003e. These results suggest that Lgr5-expressing cells are not required for organoid formation or self-renewal in vitro and are likely lost during culture propagation.\u003c/p\u003e\n\u003cp\u003eTo confirm this, we compared the gene expression profiles between freshly isolated molar epithelium and organoid cultures by RT-qPCR. Relative quantification of signature genes associated with specific epithelial subclusters, as defined by our scRNA-seq data, revealed that the subpopulation comprising Lgr5-positive cells in the DS, RSDL and OEE is significantly underrepresented in organoids compared to native tissue \u003cstrong\u003e(Supplementary Fig. 12B)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe maintenance of SOX2-positive cells, normal proliferative activity, and intact cellular organization in Lgr5-deficient organoids suggest that primary tooth germ development is largely independent of Lgr5-expressing cells. Rather than playing a central role in the formation of the first tooth germ, Lgr5 appears to regulate the development of subsequent tooth lamina. The organoid-based analyzes also suggest that Lgr5-positive epithelial cells differentiate into other functional epithelial subtypes at later stages of development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLgr5\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e expression in the SDL of minipig embryos reveals its role in tooth replacement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the spatial and temporal dynamics of \u003cem\u003eLgr5\u003c/em\u003e expression during the development of the SDL in a species capable of full tooth replacement, we analyzed embryonic stages of the minipig. As a diphyodont mammal, the minipig offers a valuable model for studying the mechanisms underlying natural tooth succession. By examining the distribution of \u003cem\u003eLgr5\u003c/em\u003e across different regions (replacing dental lamina and not replacing interdental area)\u003cstrong\u003e (Fig. 9A - D)\u003c/strong\u003e, we aimed to determine whether \u003cem\u003eLgr5\u003c/em\u003e expression correlates with active SDL formation, epithelial organization, and potential sites of replacement tooth initiation. This cross-species comparison also allowed us to assess the conservation of \u003cem\u003eLgr5\u003c/em\u003e-associated stem cell niches beyond the monophyodont mouse model \u003cstrong\u003e(Fig. 9)\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003eAs in mice, \u003cem\u003eLgr5\u003c/em\u003e expression in minipigs was observed in the mesenchyme adjacent to the DL in p3 and in the interdental area of the M1, particularly at the interface between the oral epithelium and the laminar structure \u003cstrong\u003e(Fig. 9E, F)\u003c/strong\u003e. Interestingly, \u003cem\u003eLgr5\u003c/em\u003e exhibited region-specific variation along the jaw, closely mirroring the expression pattern of SOX2 and revealing two distinct expression profiles. In regions where the growing tip of the dental lamina lacked SOX2 expression, \u003cem\u003eLgr5\u003c/em\u003e was robustly expressed (\u003cstrong\u003eFig. 9G, J\u003c/strong\u003e). In contrast, areas where SOX2 expression extended to the laminar tip showed either weak or undetectable levels of \u003cem\u003eLgr5\u003c/em\u003e (\u003cstrong\u003eFig. 9E, H\u003c/strong\u003e), suggesting a reciprocal relationship between these two markers and potentially distinct functional states within the SDL. Furthermore, neither \u003cem\u003eLgr5\u003c/em\u003e nor SOX2 expression was detected in the tip of lamina of the interdental region \u003cstrong\u003e(Fig. 9F, I)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eGiven the distinct spatial relationship between \u003cem\u003eLgr5\u003c/em\u003e and SOX2 within the SDL, we next assessed the localization of PTK7, a key regulator of planar cell polarity and Wnt signaling. PTK7-positive cells were prominently detected within the DL, particularly in regions exhibiting a compact epithelial morphology—hallmarks of sites poised to initiate the next generation of teeth (\u003cstrong\u003eFig. 9M\u003c/strong\u003e). In contrast, small interdental laminae and those showing signs of regression lacked PTK7 expression within the epithelial compartment (\u003cstrong\u003eFig. 9K–M\u003c/strong\u003e), further supporting its role in maintaining an active, tooth-inductive niche.\u003c/p\u003e\n\u003cp\u003eComparison with our mouse data revealed that \u003cem\u003eLgr5\u003c/em\u003e was similarly absent in the rudimentary or regressing DL in both species. In minipigs, features such as disorganized interdental cells, absence of \u003cem\u003eLgr5\u003c/em\u003e expression, and laminar regression closely mirrored the disrupted epithelial architecture seen in \u003cem\u003eLgr5\u003c/em\u003e-deficient mice. These parallels support the idea that LGR5 is essential for maintaining the integrity of the epithelial stem cell niche within the SDL, underscoring its conserved role in tooth renewal across mammalian species.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eOur study provides a comprehensive single-cell atlas of the developing mouse molar tooth and reveals distinct epithelial and mesenchymal populations, including a previously unrecognized, spatially restricted Lgr5⁺ cell domain on the lingual side of the DS. This domain partially overlaps with SOX2 expression and is conserved in diphyodontic minipigs, indicating the presence of a specialized epithelial tissue niche critical for sequential tooth formation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBy combining scRNA-seq of FACS-sorted Lgr5⁺ cells at two key developmental stages \u0026mdash; when the RSDL first emerges and when it becomes fully establishes \u0026mdash; with lineage tracing approaches, we demonstrated that Lgr5 regulates tooth development through multiple mechanisms. In addition to its function as a Wnt target and modulator, Lgr5 contributes to epithelial cohesion and structural integrity via interactions with adhesion molecules such as PTK7.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunctionally, Lgr5⁺ cells are important for the maintenance of RSDL stability, a key epithelial structure required for the formation of replacement teeth. Loss of \u003cem\u003eLgr5\u003c/em\u003e in mice impairs Wnt signaling, resulting in decreased \u003cem\u003eAxin2\u003c/em\u003e expression, altered Rspo family distribution, structural defects in the RSDL, and ultimately failure of successive molar formation. In minipigs, which closely resemble human diphyodont dentition, \u003cem\u003eLgr5\u003c/em\u003e expression is restricted to active, non-regressing SDL regions, correlating with SOX2 and PTK7 expression and supporting the role of Lgr5 in maintaining a tissue compartment suitable for tooth replacement.\u003c/p\u003e\n\u003cp\u003eIn humans, the canonical Wnt/\u0026beta;-catenin signaling pathway is fundamental for tooth initiation, morphogenesis and differentiation, with early perturbations causing congenital anomalies such as hypodontia and oligodontia\u003csup\u003e65\u003c/sup\u003e. Mutations in Wnt-associated genes, including \u003cem\u003eAXIN2\u003c/em\u003e, \u003cem\u003eWNT10A\u003c/em\u003e, \u003cem\u003eROR2\u003c/em\u003e, \u003cem\u003eLEF1\u003c/em\u003e and \u003cem\u003eLRP6\u003c/em\u003e, are closely associated with permanent tooth malformations, with WNT10A variants being the most commonly affected\u003csup\u003e66\u003c/sup\u003e. Although the role of LGR5 and LGR4 in human tooth anomalies is not yet fully understood, the detection of LGR5 in permanent odontoblasts \u0026mdash; but not in the deciduous dental pulp \u0026mdash; suggests a selective role in the second dentition\u003csup\u003e67\u003c/sup\u003e, which is consistent with our experimental results.\u003c/p\u003e\n\u003cp\u003eOverall, our results suggest that Lgr5⁺ cells significantly regulate RSDL stability and sequential tooth formation. The parallels between the disruption of RSDL in Lgr5-deficient mice and laminar regression in minipigs suggest that LGR5-dependent signaling is an important component of tooth replacement biology in mammals. These results lay the foundation for future studies on the involvement of LGR5 in tooth replacement disorders in humans and make LGR5-positive stem cells promising targets for regenerative therapies aimed at restoring lost or defective teeth.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003eCKNOWLEDGEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Czech Science Foundation (19-01205S, 22-02794S) and by the MEYS CR (CZ.02.1.01/0.0/0.0/15_003/0000460). MB research on odontogenesis is financed by Czech Ministry of Health (NW24-10-00204). PK and VB were supported by Czech Science Foundation (25-17360S).\u003c/p\u003e\n\u003cp\u003eWe also acknowledge the core facility CELLIM supported by the Czech-BioImaging large RI project (LM2023050 funded by MEYS CR) for their support with obtaining scientific data presented in this paper. We also acknowledge the Light Microscopy Core Facility, IMG, Prague, Czech Republic, supported by MEYS – LM2023050, MEYS – CZ.02.1.01/0.0/0.0/18_046/0016045 and MEYS – CZ.02.01.01/00/23_015/0008205, for their support with the light sheet and spinning disc imaging presented herein.\u0026nbsp;ELIXIR CZ large RI is gratefully acknowledged, supported by MEYS - LM2023055, for bioinformatics support. Computational resources were provided by the e-INFRA CZ project (ID:90254), supported by the Ministry of Education, Youth and Sports of the Czech Republic\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article/Supplementary Material. All single-cell RNA sequencing data generated in this study have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession code \u003cstrong\u003eGSEXXXXXX\u003c/strong\u003e. The processed data and metadata supporting the findings of this study are available within the article and its Supplementary Information files. Additional information is available from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mouse study was reviewed and approved by the Animal Care Committee of the Institute of Molecular Genetics, CAS, CR (Ref. 58/2017). All procedures on minipigs were conducted following a protocol approved by the Laboratory Animal Science Committee of IAPG (Approval No. 97/2011).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJuuri, E. \u003cem\u003eet al.\u003c/em\u003e Sox2 marks epithelial competence to generate teeth in mammals and reptiles. \u003cem\u003eDevelopment (Cambridge)\u003c/em\u003e \u003cstrong\u003e140\u003c/strong\u003e, 1424\u0026ndash;1432 (2013).\u003c/li\u003e\n\u003cli\u003eLi, J. \u003cem\u003eet al.\u003c/em\u003e BMP-SHH Signaling Network Controls Epithelial Stem Cell Fate via Regulation of Its Niche in the Developing Tooth. \u003cem\u003eDev Cell\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 125\u0026ndash;135 (2015).\u003c/li\u003e\n\u003cli\u003eWang, X. P. \u003cem\u003eet al.\u003c/em\u003e An integrated gene regulatory network controls stem cell proliferation in teeth. \u003cem\u003ePLoS Biol\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 1324\u0026ndash;1333 (2007).\u003c/li\u003e\n\u003cli\u003ekawasaki, M. \u003cem\u003eet al.\u003c/em\u003e R-spondins/Lgrs expression in tooth development. \u003cem\u003eDevelopmental Dynamics\u003c/em\u003e \u003cstrong\u003e243\u003c/strong\u003e, 844\u0026ndash;851 (2014).\u003c/li\u003e\n\u003cli\u003ePutnov\u0026aacute;, I. \u003cem\u003eet al.\u003c/em\u003e Angled growth of the dental lamina is accompanied by asymmetrical expression of the WNT pathway receptor frizzled 6. \u003cem\u003eFront Physiol\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, (2017).\u003c/li\u003e\n\u003cli\u003eGrumolato, L. \u003cem\u003eet al.\u003c/em\u003e Canonical and noncanonical Wnts use a common mechanism to activate completely unrelated coreceptors. \u003cem\u003eGenes Dev\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 2517\u0026ndash;2530 (2010).\u003c/li\u003e\n\u003cli\u003eGrigoryan, T., Wend, P., Klaus, A. \u0026amp; Birchmeier, W. Deciphering the function of canonical Wnt signals in development and disease: Conditional loss- and gain-of-function mutations of \u0026beta;-catenin in mice. \u003cem\u003eGenes Dev\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 2308\u0026ndash;2341 (2008).\u003c/li\u003e\n\u003cli\u003eAhn, Y., Sanderson, B. W., Klein, O. D. \u0026amp; Krumlauf, R. Inhibition of Wnt signaling by wise (Sostdc1) and negative feedback from Shh controls tooth number and patterning. \u003cem\u003eDevelopment\u003c/em\u003e \u003cstrong\u003e137\u003c/strong\u003e, 3221\u0026ndash;3231 (2010).\u003c/li\u003e\n\u003cli\u003eJ\u0026auml;rvinen, E. \u003cem\u003eet al.\u003c/em\u003e Continuous tooth generation in mouse is induced by activated epithelial Wnt/\u0026beta;-catenin signaling. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e103\u003c/strong\u003e, 18627\u0026ndash;18632 (2006).\u003c/li\u003e\n\u003cli\u003eBarker, N. \u0026amp; Clevers, H. Leucine-Rich Repeat-Containing G-Protein-Coupled Receptors as Markers of Adult Stem Cells. \u003cem\u003eGastroenterology\u003c/em\u003e \u003cstrong\u003e138\u003c/strong\u003e, 1681\u0026ndash;1696 (2010).\u003c/li\u003e\n\u003cli\u003eHuch, M. \u003cem\u003eet al.\u003c/em\u003e In vitro expansion of single Lgr5 + liver stem cells induced by Wnt-driven regeneration. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e494\u003c/strong\u003e, 247\u0026ndash;250 (2013).\u003c/li\u003e\n\u003cli\u003eKoo, B. K. \u0026amp; Clevers, H. Stem cells marked by the r-spondin receptor LGR5. \u003cem\u003eGastroenterology\u003c/em\u003e \u003cstrong\u003e147\u003c/strong\u003e, 289\u0026ndash;302 (2014).\u003c/li\u003e\n\u003cli\u003eKrivanek, J. \u003cem\u003eet al.\u003c/em\u003e Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, (2020).\u003c/li\u003e\n\u003cli\u003eSharir, A. \u003cem\u003eet al.\u003c/em\u003e A large pool of actively cycling progenitors orchestrates self-renewal and injury repair of an ectodermal appendage. \u003cem\u003eNat Cell Biol\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 1102\u0026ndash;1112 (2019).\u003c/li\u003e\n\u003cli\u003eJing, J. \u003cem\u003eet al.\u003c/em\u003e Spatiotemporal single-cell regulatory atlas reveals neural crest lineage diversification and cellular function during tooth morphogenesis. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eHallikas, O. \u003cem\u003eet al.\u003c/em\u003e System‐level analyses of keystone genes required for mammalian tooth development. \u003cem\u003eJ Exp Zool B Mol Dev Evol\u003c/em\u003e \u003cstrong\u003e336\u003c/strong\u003e, 7 (2020).\u003c/li\u003e\n\u003cli\u003eBarker, N. \u003cem\u003eet al.\u003c/em\u003e Identification of stem cells in small intestine and colon by marker gene Lgr5. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e449\u003c/strong\u003e, 1003\u0026ndash;1007 (2007).\u003c/li\u003e\n\u003cli\u003eMadisen, L. \u003cem\u003eet al.\u003c/em\u003e A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. \u003cem\u003eNat Neurosci\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 133\u0026ndash;140 (2010).\u003c/li\u003e\n\u003cli\u003eStancikova, J. \u003cem\u003eet al.\u003c/em\u003e NKD1 marks intestinal and liver tumors linked to aberrant wnt signaling. \u003cem\u003eCell Signal\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 245\u0026ndash;256 (2015).\u003c/li\u003e\n\u003cli\u003eSusaki, E. A. \u003cem\u003eet al.\u003c/em\u003e Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. \u003cem\u003eNat Protoc\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1709\u0026ndash;1727 (2015).\u003c/li\u003e\n\u003cli\u003eZheng, G. X. Y. \u003cem\u003eet al.\u003c/em\u003e Massively parallel digital transcriptional profiling of single cells. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, (2017).\u003c/li\u003e\n\u003cli\u003eYoung, M. D. \u0026amp; Behjati, S. SoupX removes ambient RNA contamination from droplet-based single-cell RNA sequencing data. \u003cem\u003eGigascience\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, (2020).\u003c/li\u003e\n\u003cli\u003eHao, Y. \u003cem\u003eet al.\u003c/em\u003e Integrated analysis of multimodal single-cell data. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e184\u003c/strong\u003e, 3573-3587.e29 (2021).\u003c/li\u003e\n\u003cli\u003eHafemeister, C. \u0026amp; Satija, R. Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. \u003cem\u003eGenome Biol\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, (2019).\u003c/li\u003e\n\u003cli\u003eJollife, I. T. \u0026amp; Cadima, J. Principal component analysis: A review and recent developments. \u003cem\u003ePhilosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences\u003c/em\u003e \u003cstrong\u003e374\u003c/strong\u003e, (2016).\u003c/li\u003e\n\u003cli\u003eMcInnes, L. \u003cem\u003eet al.\u003c/em\u003e UMAP: Uniform Manifold Approximation and Projection for Dimension Reduction. \u003cem\u003eArXiv\u003c/em\u003e arXiv:1802.03426 (2018) doi:10.48550/ARXIV.1802.03426.\u003c/li\u003e\n\u003cli\u003eMcGinnis, C. S., Murrow, L. M. \u0026amp; Gartner, Z. J. DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors. \u003cem\u003eCell Syst\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 329-337.e4 (2019).\u003c/li\u003e\n\u003cli\u003eWickham, H. (2009) Ggplot2 Elegant Graphics for Data Analysis. 2nd Edition, Springer, New York. - References - Scientific Research Publishing. https://www.scirp.org/reference/referencespapers?referenceid=2097981.\u003c/li\u003e\n\u003cli\u003eAlquicira-Hernandez, J. \u0026amp; Powell, J. E. Nebulosa recovers single-cell gene expression signals by kernel density estimation. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 2485\u0026ndash;2487 (2021).\u003c/li\u003e\n\u003cli\u003eBadia-I-Mompel, P. \u003cem\u003eet al.\u003c/em\u003e decoupleR: ensemble of computational methods to infer biological activities from omics data. \u003cem\u003eBioinformatics Advances\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eT\u0026uuml;rei, D. \u003cem\u003eet al.\u003c/em\u003e Integrated intra‐ and intercellular signaling knowledge for multicellular omics analysis. \u003cem\u003eMol Syst Biol\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eGarcia-Alonso, L., Holland, C. H., Ibrahim, M. M., Turei, D. \u0026amp; Saez-Rodriguez, J. Benchmark and integration of resources for the estimation of human transcription factor activities. \u003cem\u003eGenome Res\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 1363\u0026ndash;1375 (2019).\u003c/li\u003e\n\u003cli\u003eLa Manno, G. \u003cem\u003eet al.\u003c/em\u003e RNA velocity of single cells. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e560\u003c/strong\u003e, 494\u0026ndash;498 (2018).\u003c/li\u003e\n\u003cli\u003eBergen, V., Lange, M., Peidli, S., Wolf, F. A. \u0026amp; Theis, F. J. Generalizing RNA velocity to transient cell states through dynamical modeling. \u003cem\u003eNat Biotechnol\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 1408\u0026ndash;1414 (2020).\u003c/li\u003e\n\u003cli\u003eLivak, K. J. \u0026amp; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-\u0026Delta;\u0026Delta;CT method. \u003cem\u003eMethods\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 402\u0026ndash;408 (2001).\u003c/li\u003e\n\u003cli\u003eAbarda, A., Dakkon, M., Azhari, M., Zaaloul, A. \u0026amp; Khabouze, M. Latent Transition Analysis (LTA) : A Method for Identifying Differences in Longitudinal Change Among Unobserved Groups. \u003cem\u003eProcedia Comput Sci\u003c/em\u003e \u003cstrong\u003e170\u003c/strong\u003e, 1116\u0026ndash;1121 (2020).\u003c/li\u003e\n\u003cli\u003eOlbertov\u0026aacute;, K. \u003cem\u003eet al.\u003c/em\u003e Role of LGR5-positive mesenchymal cells in craniofacial development. \u003cem\u003eFront Cell Dev Biol\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eJuuri, E. \u003cem\u003eet al.\u003c/em\u003e Sox2+ Stem Cells Contribute to All Epithelial Lineages of the Tooth via Sfrp5+ Progenitors. \u003cem\u003eDev Cell\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 317\u0026ndash;328 (2012).\u003c/li\u003e\n\u003cli\u003eHarada, H. \u003cem\u003eet al.\u003c/em\u003e Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling. \u003cem\u003eJournal of Cell Biology\u003c/em\u003e \u003cstrong\u003e147\u003c/strong\u003e, 105\u0026ndash;120 (1999).\u003c/li\u003e\n\u003cli\u003eDing, S., Du, M. \u0026amp; Zhu, H. Survey on granularity clustering. \u003cem\u003eCogn Neurodyn\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 561 (2015).\u003c/li\u003e\n\u003cli\u003eHermans, F. \u003cem\u003eet al.\u003c/em\u003e Establishment of inclusive single-cell transcriptome atlases from mouse and human tooth as powerful resource for dental research. \u003cem\u003eFront Cell Dev Biol\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eThesleff, I., Wang, X. P. \u0026amp; Suomalainen, M. Regulation of epithelial stem cells in tooth regeneration. \u003cem\u003eC R Biol\u003c/em\u003e \u003cstrong\u003e330\u003c/strong\u003e, 561\u0026ndash;564 (2007).\u003c/li\u003e\n\u003cli\u003eNumakura, C. \u003cem\u003eet al.\u003c/em\u003e Supernumerary impacted teeth in a patient with SOX2 anophthalmia syndrome. \u003cem\u003eAm J Med Genet A\u003c/em\u003e \u003cstrong\u003e152\u003c/strong\u003e, 2355\u0026ndash;2359 (2010).\u003c/li\u003e\n\u003cli\u003eSanz-Navarro, J., Feal, C. \u0026amp; Dauden, E. tratamiento de la sarna en humanos con ivermectina oral. Erupciones eccematosas como nuevos efectos adversos no reportados. \u003cem\u003eActas Dermosifiliogr\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 643\u0026ndash;649 (2017).\u003c/li\u003e\n\u003cli\u003eMorita, H. \u003cem\u003eet al.\u003c/em\u003e Neonatal Lethality of LGR5 Null Mice Is Associated with Ankyloglossia and Gastrointestinal Distension. \u003cem\u003eMol Cell Biol\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 9736\u0026ndash;9743 (2004).\u003c/li\u003e\n\u003cli\u003eWen, Q. \u003cem\u003eet al.\u003c/em\u003e Runx2 Regulates Mouse Tooth Root Development Via Activation of WNT Inhibitor NOTUM. \u003cem\u003eJournal of Bone and Mineral Research\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 2252\u0026ndash;2264 (2020).\u003c/li\u003e\n\u003cli\u003eKramer, K. \u003cem\u003eet al.\u003c/em\u003e Dental defects in the primary dentition associated with hypophosphatasia from biallelic ALPL mutations. \u003cem\u003eBone\u003c/em\u003e \u003cstrong\u003e143\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eCarmon, K. S. \u003cem\u003eet al.\u003c/em\u003e LGR5 receptor promotes cell-cell adhesion in stem cells and colon cancer cells via the IQGAP1-Rac1 pathway. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e292\u003c/strong\u003e, 14989\u0026ndash;15001 (2017).\u003c/li\u003e\n\u003cli\u003eWalker, F., Zhang, H. H., Odorizzi, A. \u0026amp; Burgess, A. W. LGR5 is a negative regulator of tumourigenicity, antagonizes wnt signalling and regulates cell adhesion in colorectal cancer cell lines. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, (2011).\u003c/li\u003e\n\u003cli\u003eYamakami, Y. \u003cem\u003eet al.\u003c/em\u003e LGR4 is required for sequential molar development. \u003cem\u003eBiochem Biophys Rep\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 174 (2016).\u003c/li\u003e\n\u003cli\u003eFernandez Vallone, V. \u003cem\u003eet al.\u003c/em\u003e LGR 5 controls extracellular matrix production by stem cells in the developing intestine . \u003cem\u003eEMBO Rep\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, (2020).\u003c/li\u003e\n\u003cli\u003eDe Lau, W. \u003cem\u003eet al.\u003c/em\u003e Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e476\u003c/strong\u003e, 293\u0026ndash;297 (2011).\u003c/li\u003e\n\u003cli\u003eVan de Wetering, M. \u003cem\u003eet al.\u003c/em\u003e The \u0026beta;-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e111\u003c/strong\u003e, 241\u0026ndash;250 (2002).\u003c/li\u003e\n\u003cli\u003eCadigan, K. M. \u0026amp; Peifer, M. Wnt signaling from development to disease: insights from model systems. \u003cem\u003eCold Spring Harb Perspect Biol\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, (2009).\u003c/li\u003e\n\u003cli\u003eJ\u0026auml;rvinen, E., Shimomura-Kuroki, J., Balic, A., Jussila, M. \u0026amp; Thesleff, I. Mesenchymal Wnt/\u0026beta;-catenin signaling limits tooth number. \u003cem\u003eDevelopment (Cambridge)\u003c/em\u003e \u003cstrong\u003e145\u003c/strong\u003e, (2018).\u003c/li\u003e\n\u003cli\u003ePopa, E. M., Buchtova, M. \u0026amp; Tucker, A. S. Revitalising the rudimentary replacement dentition in the mouse. \u003cem\u003eDevelopment (Cambridge)\u003c/em\u003e \u003cstrong\u003e146\u003c/strong\u003e, (2019).\u003c/li\u003e\n\u003cli\u003eLeung, J. Y. \u003cem\u003eet al.\u003c/em\u003e Activation of AXIN2 Expression by \u0026beta;-Catenin-T Cell Factor: A FEEDBACK REPRESSOR PATHWAY REGULATING Wnt SIGNALING. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e277\u003c/strong\u003e, 21657\u0026ndash;21665 (2002).\u003c/li\u003e\n\u003cli\u003eJho, E. \u003cem\u003eet al.\u003c/em\u003e Wnt/\u0026beta;-Catenin/Tcf Signaling Induces the Transcription of Axin2, a Negative Regulator of the Signaling Pathway. \u003cem\u003eMol Cell Biol\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 1172\u0026ndash;1183 (2002).\u003c/li\u003e\n\u003cli\u003eValenta, T., Hausmann, G. \u0026amp; Basler, K. The many faces and functions of \u0026Icirc; 2-catenin. \u003cem\u003eEMBO Journal\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 2714\u0026ndash;2736 (2012).\u003c/li\u003e\n\u003cli\u003eChen, Y. \u003cem\u003eet al.\u003c/em\u003e Nidogen-2 is a Novel Endogenous Ligand of LGR4 to Inhibit Vascular Calcification. \u003cem\u003eCirc Res\u003c/em\u003e \u003cstrong\u003e131\u003c/strong\u003e, 1037\u0026ndash;1054 (2022).\u003c/li\u003e\n\u003cli\u003eFan, J. \u003cem\u003eet al.\u003c/em\u003e Characterizing transcriptional heterogeneity through pathway and gene set overdispersion analysis. \u003cem\u003eNat Methods\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 241 (2016).\u003c/li\u003e\n\u003cli\u003eKenny, T. C. \u0026amp; Germain, D. PTK7 Faces the Wnt in Development and Disease. \u003cem\u003eFront Cell Dev Biol\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 31 (2017).\u003c/li\u003e\n\u003cli\u003eAbramson, J. \u003cem\u003eet al.\u003c/em\u003e Accurate structure prediction of biomolecular interactions with AlphaFold 3. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e630\u003c/strong\u003e, 493\u0026ndash;500 (2024).\u003c/li\u003e\n\u003cli\u003eGolubkov, V. S. \u003cem\u003eet al.\u003c/em\u003e Internal Cleavages of the Autoinhibitory Prodomain Are Required for Membrane Type 1 Matrix Metalloproteinase Activation, although Furin Cleavage Alone Generates Inactive Proteinase. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e285\u003c/strong\u003e, 27726 (2010).\u003c/li\u003e\n\u003cli\u003eWang, B. \u003cem\u003eet al.\u003c/em\u003e Expression patterns of WNT/\u0026beta;-CATENIN signaling molecules during human tooth development. \u003cem\u003eJ Mol Histol\u003c/em\u003e \u003cstrong\u003e45\u003c/strong\u003e, 487\u0026ndash;496 (2014).\u003c/li\u003e\n\u003cli\u003evan den Boogaard, M. J. \u003cem\u003eet al.\u003c/em\u003e Mutations in wnt10a are present in more than half of isolated hypodontia cases. \u003cem\u003eJ Med Genet\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 327\u0026ndash;331 (2012).\u003c/li\u003e\n\u003cli\u003eKim, J. H. \u003cem\u003eet al.\u003c/em\u003e Distinctive genetic activity pattern of the human dental pulp between deciduous and permanent teeth. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, (2014).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"LGR5, SOX2, PTK7, Wnt signaling, stem cells, odontogenesis, organoids, single cell RNA sequencing","lastPublishedDoi":"10.21203/rs.3.rs-7607583/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7607583/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Tooth replacement in vertebrates depends on the persistence of the dental lamina, yet the molecular mechanisms that determine species-specific regenerative capacity remain poorly understood. Here we combine single-cell transcriptomics, lineage tracing, and genetic inactivation to define the role of LGR5, a canonical epithelial stem cell marker, in sequential molar development. We identify an unrecognized epithelial Lgr5⁺ population in the dental stalk and rudimentary successional dental lamina, together with a distinct mesenchymal Lgr5⁺ progenitor pool outside the tooth germ. Lineage tracing demonstrates that epithelial Lgr5⁺ cells contribute regionally to second molar formation, implicating them in sequential tooth initiation. Loss of Lgr5 disrupts lamina architecture, leading to shortened stalks, reduced SOX2 expression, impaired basement membrane integrity, and altered Wnt signaling, including downregulation of the LGR5-interacting protein PTK7. Organoid assays further show that Lgr5⁺ epithelial cells function as niche stabilizers rather than classical proliferative progenitors, providing structural and signaling support for replacement tooth development. Comparative analysis in diphyodont minipigs reveals conserved Lgr5 expression in non-regressing lamina domains, linking Lgr5 activity with species capable of multiple tooth generations.\r\nTogether, these results identify Lgr5 as a key regulator of dental lamina stability and sequential tooth development, uncovering molecular mechanisms that couple Wnt signaling with epithelial integrity and establishing a framework for Lgr5-based regenerative strategies in dentistry.","manuscriptTitle":"LGR5 regulates sequential tooth development: evidence from single-cell transcriptomics and a gene inactivation model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-24 18:51:45","doi":"10.21203/rs.3.rs-7607583/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a08c0381-5b08-463c-a6fb-71493d0036a4","owner":[],"postedDate":"September 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":55094233,"name":"Biological sciences/Developmental biology/Stem-cell niche"},{"id":55094234,"name":"Biological sciences/Anatomy"}],"tags":[],"updatedAt":"2025-10-31T07:05:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-24 18:51:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7607583","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7607583","identity":"rs-7607583","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}