Distinct BMP-Smad Signaling Outputs Confer Diverse Functions in Dental Mesenchyme

Distinct BMP-Smad Signaling Outputs Confer Diverse Functions in Dental Mesenchyme | 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 Distinct BMP-Smad Signaling Outputs Confer Diverse Functions in Dental Mesenchyme Qinghuang Tang, Liwen Li, Yihong Li, Amy Wang, Hua Li, Linyan Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5188541/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The canonical bone morphogenetic protein (BMP) signaling pathway plays a crucial regulatory role in tooth development by activating Smad proteins to regulate gene expression. Our previous research identified an atypical canonical BMP signaling in dental mesenchyme that is Smad4-independent but Smad1/5-dependent. This study demonstrates that phosphorylated Smad1/5 (pSmad1/5) and Smad4 transcriptionally regulate distinct gene sets in dental mesenchyme. Real-time monitoring of BMP-Smad transcriptional activity revealed that Smad4-dependent canonical BMP signaling is restricted to neurovascular cells surrounding the condensed dental mesenchymal cells where pSmad1/5 is present. Notably, we found that pSmad1/5 in dental mesenchymal cells form complexes with pSmad3 to prevent canonical BMP signaling. CUT&RUN assays revealed genome-wide co-occupancy of pSmad1/5 and pSmad3, indicating that pSmad1/5-pSmad3 complexes function as transcriptional regulation units. Integrative analyses of their transcriptional targets with RNA-seq demonstrated that the atypical canonical BMP signaling regulates tooth sensory innervation and is temporally required for maintaining odontogenic inductive potential in the dental mesenchyme. This enabled the identification of potentially critical genes for maintaining tooth inductive capability. Our findings elucidate the operating mechanism of atypical canonical BMP signaling in dental mesenchymal cells and clarify how BMP-Smad signaling exerts diverse functions across different cell types, shedding light on future tooth bioengineering strategies. Biological sciences/Developmental biology/Stem-cell niche Biological sciences/Cell biology/Cell signalling/Growth factor signalling Biological sciences/Molecular biology/Transcription/Transcriptional regulatory elements Atypical BMP-Smad signaling Dental mesenchyme Tooth innervation Odontogenic inductive potential Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Mammalian odontogenesis, the complex process of tooth formation and integration with supporting tissues, including the neurovascular systems 1 , serves as a model for studying essential developmental mechanisms of tissue interactions and genetic regulation 2 . In mice, tooth development begins at embryonic day 11.5 (E11.5) when the presumptive dental epithelium thickens at future tooth-forming site, creating dental placodes. These placodes act as early signaling centers with ‘odontogenic inductive potential’, capable of inducing tooth formation even when recombined with non-dental embryonic mesenchyme 3 . At E12.5, this potential shifts from the dental placode to the underlying dental mesenchyme 4 , initiating sequential morphogenesis of individual tooth germs through the bud, cap, and bell stages. Concurrently, a blood vessel plexus forms around the developing tooth germ 5 , while trigeminal nerve fibers extend towards the developing maxillary or mandibular tooth germs 6 , diverging into buccal and lingual branches adjacent to the condensed dental mesenchyme 7 . Although the structure, distribution and function of the dental neurovascular system are well documented in murine and human teeth 5,8 , the molecular mechanisms underlying the initial establishment of tooth blood supply and innervation, as well as their integration with advancing tooth morphogenesis, remain elusive. Bone morphogenetic proteins (BMPs), a subclass of the TGFβ signaling superfamily, are widely involved in a broad spectrum of developmental processes, including neurogenesis 9 , hematopoiesis 10 , and odontogenesis 11 . BMP4, in particular, is a critical regulator at various stages of early tooth development 12 . Mice with neural crest-specific inactivation of either Bmp4 or Bmpr1a exhibit arrested tooth development at the bud stage 12,13 . During tooth development initiation, epithelial BMP4 induces mesenchymal expression of Msx1 , which in turn promotes mesenchymal Bmp4 expression, forming a positive regulatory loop that governs the transition from bud to cap stage 14 . Notably, ectopic expression of Bmp4 driven by an Msx1 promoter in the dental mesenchyme can rescue tooth defects in Msx1 mutants, allowing development to progress to the cap stage 15 . Moreover, BMP4 also plays roles in determining the tooth-forming site 16 and tooth type 17 . The canonical BMP signaling begins with ligand binding, which assembles a heteromeric complex of type II and type I receptors 18 . This binding triggers phosphorylation of the type I receptor, activating intracellular mediators Smad1, Smad5, and Smad8 19 . These receptor-regulated Smads (R-Smads) form complexes with Smad4 and translocate to the nucleus. There, Smad proteins serve as signaling hubs, integrating regulatory inputs and cooperating with specific transcription factors to regulate target gene expression in a context-dependent manner 19 . BMPs can also activate non-canonical signaling pathways through kinases such as mitogen-activated protein kinase, p38, ERK and JNK, independent of Smad proteins. Other TGFβ superfamily ligands homologous to BMPs, such as TGFβs 1–3 and Activin, also play crucial roles in early tooth development. This is evidenced by delayed tooth initiation and development following specific inactivation of the type I receptor ALK5 in cranial neural crest derived cells 20 . These ligands bind distinct receptor complexes of type I and type II receptors 21 , activating the canonical Smad2/3 pathway and non-canonical pathways that overlap with those activated by BMPs. In addition to shared non-canonical pathways, crosstalk between BMP and TGFβ signaling pathways occurs at multiple levels 22 , resulting in diverse cellular responses from a limited number of signaling inputs. While R-Smads and Smad4 complexes have been long considered the principal functional units in canonical BMP signaling transcriptional regulation, recent observations challenge this view. In several developing organs, Smad1/5 inactivation causes severe defects, whereas Smad4 inactivation results in mild or no defects 23–25 . This suggests that R-Smads and Smad4 may exert diverse regulatory functions independently. Indeed, we have previously identified an atypical canonical BMP signaling pathway in human 26 and mouse 27 dental mesenchyme that regulates Msx1 expression in a Smad1/5-dependent but Smad4-independent manner. In this study, we further elucidated the cellular and molecular mechanisms underlying the atypical canonical BMP signaling in dental mesenchymal cells. Our findings reveal that BMP-Smad signaling confers diverse functions to ensure proper early tooth innervation and to maintain the odontogenic inductive potential in dental mesenchyme. These results provide compelling evidence for the versatility of BMP-Smad signaling, challenging the canonical BMP signaling dogma. Additionally, our findings have potential implications for future tooth bioengineering applications. Results Genome-wide profiling of pSmad1/5 and Smad4 binding sites in dental mesenchyme Our previous study identified an atypical canonical BMP-Smad signaling pathway in early developing dental mesenchyme of humans 26 and mice 27 , operating independently of Smad4. We confirmed this finding by using LacZ staining on embryonic stage 13.5 (E13.5) mandibles (Fig. 1 b) from BMP response element ( BRE ) -LacZ transgenic reporter mice 28 . E13.5 limb (Fig. 1 a), where canonical BMP signaling is known to operate, served as a biological control. As expected, LacZ signal was observed in the limb but not in the mandibular molar tooth germ (Fig. 1 a, b, red arrows). To investigate underlying molecular mechanisms, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) assay of phosphorylated Smad1/5 (pSmad1/5) and Smad4 on E13.5 limbs and molar tooth germs (Fig. 1 c, d). Results showed markedly different binding patterns of pSmad1/5 and Smad4 in the molar (Fig. 1 d), contrasting with similar patterns in the limb (Fig. 1 c). Intersection analysis revealed only 222 peaks are co-bound by pSmad1/5 and Smad4 in the molar (Fig. 1 f), compared to 3788 in the limb (Fig. 1 e), providing genome-wide evidence for the operation of atypical canonical BMP signaling pathway during early tooth development. Further characterization of pSmad1/5 and Smad4 binding peaks in the molar showed that pSmad1/5 preferentially bind to promoter regions (Fig. 1 g) as compared to Smad4 (Fig. 1 h), while both predominantly bind to cis-regulatory elements, such as distal intergenic and other intron regions (Fig. 1 g, h). Gene ontology (GO) analysis of pSmad1/5- and Smad4-specific peak target genes revealed their involvements in distinct biological processes, with shared roles in signaling transduction and neurogenesis (Fig. 1 i, j, black boxes). pSmad1/5 target genes are predominantly involved in ‘mesenchyme development’ and axonogenesis-related activities (Fig. 1 i), while Smad4 target genes are associated with ‘muscle cell differentiation’ and ‘sensory system development’ (Fig. 1 j). Intriguingly, target genes linked to pSmad1/5 and Smad4 co-bound peaks showed a predominant association with ‘sensory system development’ and ‘dendrite development’ (Fig. 1 k), which are also enriched in Smad4-specific target genes (Fig. 1 j, k, red boxes). Taken together, these results support the existence of a functional atypical canonical BMP-Smad signaling pathway during early tooth development and highlight the versatility of BMP-Smad signaling in transcriptional regulation. For brevity and distinction from canonical BMP signaling, we will refer to this pathway as “atypical BMP signaling” hereafter. Canonical BMP signaling activity is restricted to neurovascular cells The diverse BMP-Smad signaling outputs (Fig. 1 i-k) prompted us to examine its transcriptional activity during early tooth development more closely. Using a BRE-GFP transgenic reporter mouse line 29 , which allows real-time monitoring of canonical BMP-Smad transcriptional activity during development, we comprehensively examined the transcriptional activities of BMP-Smad signaling in early developing tooth germs. At E11.5, BRE-GFP activity was observed in only a few cells surrounding the dental mesenchyme (Fig. 2 a, j). By E12.5, BRE-GFP activity appeared in the dental epithelium (Fig. 2 b, k) and became pronounced in the enamel knot by E14.5 (Fig. 2 c, l). Interestingly, the emergence of BRE-GFP activity in the dental epithelium coincides with the shift out of the odontogenic inductive potential from the dental epithelium to the dental mesenchyme 3 . In the dental mesenchyme, BRE-GFP positive cells began to appear at the edges of the condensed dental mesenchyme at E12.5 (Fig. 2 b, k). By E14.5, these cells were primarily localized in the dental follicle, with some extending into the dental papilla, exhibiting neurovascular cell-like morphology (Fig. 2 c, l). Based on these observations and insights from GO analysis of pSmad1/5 and Smad4 co-bound genes, we hypothesized that BRE-GFP positive cells are neurovascular cells. To test this, we performed immunostaining for neurofilament (2H3, Fig. 2 d-f), a sensory nerve fiber specific marker 30 , and Pecam-1(CD31, Fig. 2 m-o), an endothelial cell marker 31 , on the developing molar tooth germs. BRE-GFP positive cells began to co-localize with CD31 at E11.5 (Fig. 2 m-r) and with 2H3 at E12.5 (Fig. 2 d-i), consistent with previous findings that blood vessel formation around the tooth germ precedes neurites ingrowth 5 . These results indicate that canonical BMP signaling is confined to neurovascular cells during early tooth development and is not active in dental mesenchymal cells. Formation of pSmad3-pSmad1/5 complexes in dental mesenchymal cells The presence of canonical BMP signaling pathway in neurovascular cells and its absence in dental mesenchymal cells raises questions about how the BMP-Smad signaling pathway achieves diverse transcriptional outputs. Previous research has shown that TGF-β signaling can inhibit canonical BMP signaling-induced transcription through the formation of pSmad3-pSmad1/5 complexes in multiple cell lines 32 . We investigated whether this mechanism is applied to early tooth development for inhibiting canonical BMP signaling in dental mesenchymal cells. We examined the expression patterns of phosphorylated Smads (pSmad1/5, pSmad2, pSmad3) and Smad4 in developing molar tooth germs from the BRE-GFP mouse line. Smad4 was widely expressed in both dental epithelial and dental mesenchymal cells at E12.5, E13.5 and E14.5 (Fig. 3 a, g, m). pSmad2 was weakly present in most of the dental mesenchyme, with moderate expression in a few scattered cells in the buccal dental mesenchyme (Fig. 3 d, j, p). Notably, pSmad1/5 (Fig. 3 b, h, n) and pSmad3 (Fig. 3 e, k, p) exhibited similar expression patterns during early tooth development. At E12.5, they were present in the buccal dental mesenchyme and some of the condensed dental mesenchyme (Fig. 3 b, e), becoming intense in the dental mesenchyme at E13.5 (Fig. 3 h, k) and E14.5 (Fig. 3 n, q). In neurovascular cells where canonical BMP signaling operates, pSmad1/5 co-localized with BRE-GFP signals (Fig. 3 c, i, o), while pSmad3 was absent (Fig. 3 f, l, r). This finding, along with the similar expression patterns of pSmad1/5 and pSmad3 suggests that the atypical BMP signaling in dental mesenchymal cells probably operates through the formation of pSmad1/5-pSmad3 complexes. To verify this, we performed co-immunoprecipitation (Co-IP) analyses on E13.5 dental mesenchymal cells using pSmad3 antibody. Western blotting of the pSmad3 immunoprecipitation confirmed that pSmad3 forms a novel complex with pSmad1/5 (Fig. 3 s). TGFβ-BMP antagonism is responsible for the operation of atypical BMP signaling in dental mesenchymal cells To investigate whether the formation of pSmad1/5-pSmad3 complexes in dental mesenchymal cells inhibits canonical BMP signaling, we suppressed Smad3 phosphorylation by treating tooth germs with SIS3 33 at the bud stage. Inhibition of pSmad3 (Fig. 4 a, e) induced ectopic BRE-GFP activities in dental mesenchymal cells (Fig. 4 f, g) compared to DMSO-treated controls (Fig. 4 b, c). This result confirmed that the formation of pSmad1/5-pSmad3 complexes in dental mesenchymal cells inhibits canonical BMP signaling. Given that pSmad3 is a well-known intracellular effector of TGFβ signaling, we examined the expressions of several TGFβ superfamily members in molars at the bud stage (Fig. S1 a-j). Bmp4 was intensively expressed in the condensed dental mesenchyme (Fig. S1 a, b). Tgfβ1 , Tgfβ3, and Tgfβr1 were widely expressed throughout dental mesenchymal cells at E12.5 and E13.5 (Fig. S1 c-h). Specifically, Tgfβ1 showed scattered expression in the dental mesenchyme (Fig. S1 c, d), while Tgfβ3 exhibited intensive expression in the presumptive dental follicle (Fig. S1 g, h). Notably, Inhba (InhibinβA, a component of Activin A) was strongly expressed in the dental mesenchyme at E12.5 and E13.5 (Fig. S1 i, j). To determine whether TGFβ family members inhibit canonical BMP signaling in dental mesenchymal cells, we applied SB431542, a selective inhibitor of TGFβ superfamily signaling 34 , to E13.5 tooth germs. This treatment activated ectopic BRE-GFP in dental mesenchymal cells (Fig. 4 j, k) compared to DMSO treatment (Fig. 4 b, c). SB431542 treatment decreased pSmad3 expression (Fig. 4 i) but did not affect pSmad1/5 expression in dental mesenchymal cells (Fig. 4 p-r). Importantly, the expression of Msx1, a readout of the atypical BMP signaling 27 , was reduced after both SIS3 and SB431542 treatment (Fig. 4 h, l). This indicates that the pSmad3-mediated TGFβ inhibition is responsible for the operation of atypical BMP signaling in dental mesenchymal cells. When dorsomorphin 35 , an inhibitor of type I BMP receptor, was applied together with SB431452 to E13.5 tooth germs, both endogenous BRE-GFP activities in neurovascular cells and ectopic BRE-GFP activities induced by the TGFβ signaling inhibition in dental mesenchymal cells disappeared (Fig. 4 s-u). This result indicates that BMP signaling is essential for Smad1/5 phosphorylation in both neurovascular and dental mesenchymal cells. Taken together, these results demonstrate that BMP signaling exerts both canonical and atypical signaling outputs in the dental mesenchyme. Canonical BMP signaling operates in neurovascular cells, while atypical BMP signaling operates in dental mesenchymal cells, where the canonical BMP signaling was inhibited by TGFβ signaling through the formation of pSmad1/5-pSmad3 complexes. Genome-wide mapping of atypical BMP signaling targets in dental mesenchyme Given the predominant operation of the atypical BMP signaling in the dental mesenchyme, to investigate its transcriptional regulatory function, we performed CUT&RUN (Cleavage Under Targets and Release Using Nuclease) 36 of pSmad1/5 and pSmad3 on the E13.5 tooth germs, a time the dental mesenchyme has gained sustained odontogenic inductive capability. Two independent biological replicates were used to maximize site discovery and remove sequencing artifacts (Fig. 5 a). Using the bedtools intersect function 37 , we identified 10741 common pSmad1/5 binding peaks and 7905 common pSmad3 binding peaks across replicates (Fig. S2 a). Genome-wide distributions of pSmad15 and pSmad3 peaks relative to gene transcription start site (TSS) were highly similar (Fig. 5 a and Fig. S2 b). pSmad1/5 and pSmad3 peaks showed similar binding preference, both exhibiting the highest abundance in distal intergenic (33.46% vs. 28.97%) and other intron regions (16.26% vs. 13.27%) (Fig. 5 b). This indicates that both pSmad1/5 and pSmad3 primarily function on distal enhancers, consistent with the consensus that activated Smad complexes regulate transcription via chromatin remodeling 18 . Functional classification of pSmad1/5 and pSmad3 binding genes into Biological Process (BP, Fig. 5 c), Cellular Component (CC, Fig. S2 c) and Molecular Function (MF, Fig. S2 d) categories revealed enrichment in similar GO terms across all three categories. These results further substantiate the formation of pSmad1/5-pSmad3 complexes in dental mesenchyme at a genome-wide level, suggesting that they act as transcriptional regulatory units. Smad complexes, due to their weak DNA binding affinity, frequently cooperate with context-specific transcription factors (TFs) to enhance and stabilize their DNA binding 38 . To understand how pSmad1/5-pSmad3 complexes act as transcriptional regulatory units, and identify their cooperating TFs, we performed comprehensive motif analyses on the pSmad1/5 and pSmad3 bound peaks. Unbiased motif discovery using STREME 39 yielded 10 recurring ungapped motifs in each sequence dataset (Fig. 6 a). Motif comparison with a Mouse Embryonic Stem Cell TF motif database 40 showed that the majority of motifs from both pSmad1/5 (8/10) and pSmad3 (8/10) peaks matched with the SMAD1 motif (Fig. S3 a, b), confirming the specificity and accuracy of the CUT&RUN. Interestingly, pairwise comparison of pSmad1/5 and pSmad3 motifs revealed striking similarities (Fig. 6 a, heatmap). Optimal alignment on paired pSmad1/5 and pSmad3 motifs with the highest similarity scores (Fig. 6 a, middle) enabled the identification of overlapping motif sequences potentially bound by their cooperating TFs. Querying these sequences with the HOCOMOCO Mouse v11 full TF motif database 41 allowed us to infer 9 TFs that can recognize them (Fig. 6 a), including TEAD4, a known Smad-cooperating TF that can form a complex with SMAD2/3 on mesenchymal enhancers upon TGFβ stimulation 42 . This observation suggests a possible mechanism by which the pSmad1/5-pSmad3 complex acts as transcriptional unit in dental mesenchymal cells, where TFs recognizing these overlapping motif sequences assemble the two R-Smads into a novel mixed complex for transcriptional regulation. Other inferred TFs include FOS, FOXJ3, FOXA3, NR5A2, SP1, BATF (B-ATF, basic leucine zipper ATF-like TF) and ASCL1 (Fig. 6 a). Many of these TFs are associated with neuron differentiation or axonogenesis 43 , implicating a potential role for atypical BMP signaling in tooth innervation. Interestingly, the overlapping motif sequence shared by pSmad1/5 motif-5 and pSmad3 motif-10 can be recognized by both SMAD1 and SMAD3 (Fig. 6 a), suggesting physical interaction of pSmad1/5 and pSmad3 on genomic loci. Given the widespread expression of Smad4 in dental mesenchymal cells (Fig. 3 ), we then explored why pSmad1/5 preferentially forms a transcriptional regulatory unit with pSmad3, but not Smad4. Spaced motif analysis 44 on the pSmad1/5 bound DNA sequences revealed SMAD3 as the most favored cooperating TF (Fig. 6 b), followed by TFs belonging to the AP2B cluster (Fig. S3 c), which is essential for functional axonogenesis in hippocampal neurons 45 . Above all, our genome-wide mapping of pSmad1/5 and pSmad3 suggests that the similarity of motifs utilized by these two R-Smads enables the formation of a novel mixed R-Smads complex via synergistic effects of their shared TFs in dental mesenchymal cells. This mixed complex confers atypical BMP signaling output by converging the TGFβ and BMP signaling pathways at the intracellular effector level. We then set out to assess the transcriptional regulatory functions of atypical BMP signaling. Intersection of 5160 pSmad1/5 and 4952 pSmad3 target genes found 2777 co-target genes (Fig. 6 c). Notably, both pSmad1/5 and pSmad3 also have their own specific target genes (Fig. 6 c), suggesting additional regulatory mechanisms. Pathway analysis of their co-target genes showed involvement in TGFβ and BMP signaling pathway transduction, as well as processes such as ‘Neuronal System’ and ‘Axon guidance’ (Fig. 6 d), consistent with previous studies on the roles of TGFβ and BMP signaling in neurogenesis 9,46–48 and axon development 49,50 . Atypical BMP signaling regulates tooth innervation during early tooth development Tooth development is regulated by epithelial-mesenchymal interactions and coordinated with innervation to form a functional sensory organ 6,51 . Previous studies have shown that target-derived BMP and TGFβ signaling can regulate peripheral innervation by limiting sensory neuron number 52 or guiding neurite outgrowth 47 . Importantly, BMP4 has been identified as a major target-derived factor that retrogradely controls differential gene expression in trigeminal ganglia through both Smad4-independent and Smad4-dependent pathways 9 . Our genome-wide binding profiles of pSmad1/5 and pSmad3 suggest the involvement of mixed R-Smads in axon development regulation (Fig. 5 , 6 ). Based on these findings, we hypothesize that atypical BMP signaling regulates tooth innervation during early tooth development. To test it, we investigated axonogenesis in mouse embryonic heads and mandibles following the disruption of atypical BMP signaling using SIS3 (Fig. S4 a, b). As expected, disruption of the atypical BMP signaling in the head at E11.5 (Fig. S4 c-f), in the mandible at E12.5 (Fig. S4 g, h) and E13.5 (Fig. S4 i-l) all resulted in reduced peripheral axon growth, including axons adjacent to the developing tooth germ (Fig. S4 j, l), in a stage-dependent manner. The effect on tooth innervation was more pronounced with earlier disruption of atypical BMP signaling. To understand how the atypical BMP signaling regulates tooth innervation, we conducted RNA-seq analysis on in vitro cultured tooth germs after disrupting atypical BMP signaling with SIS3 at E12.5 for one day (Fig. 7 a). Intersecting the differentially expressed genes (DEGs) with pSmad1/5 and pSmad3 target genes (Fig. 6 ) revealed 560 genes bound by both pSmad1/5 and pSmad3, while 444 (428 + 15 + 1) genes were co-bound (Fig. 7 b). This result indicates that BMP and TGFβ signaling have both specific and converged regulatory functions on axon development, consistent with previous studies 47,52 , and suggests discrete regulatory roles for pSmad1/5 and pSmad3. Functional classification of the 444 genes co-bound by pSmad1/5 and pSmad3 into axonogenesis-associated GO terms (Fig. 7 c) revealed 27 axonogenesis-associated genes directly regulated by pSmad1/5 and pSmad3 co-binding (Fig. 7 d). Among them, Nefl, a protein forming neurofilaments in axonal cytoplasm 53 , showed decreased expression after SIS3 treatment (Fig. 7 d), consistent with 2H3 immunofluorescence staining results (Fig. S4 ). Conversely, Wnt3, an early axonal guidance cue 54 , showed increased expression after SIS3 treatment (Fig. 7 d). CUT&RUN-qPCR verified the direct regulation of these genes (Fig. 7 e). Together, these results indicated that the pSmad1/5-pSmad3 complex-mediated atypical BMP signaling regulates tooth innervation (Fig. 7 f) by directly modulating the expression of axonogenesis-associated genes. Atypical BMP signaling is required for maintaining odontogenic inductive potential in dental mesenchyme To investigate the biological significance of atypical BMP signaling in dental mesenchyme, we transplanted tooth germs with ectopically activated canonical BMP signaling into mouse kidney capsule to allow for further tooth development. All transplanted tooth germs developed normally after either SB431542 treatment (n = 10/10) (Fig. S5 c, d) or DMSO treatment (n = 10/10) (Fig. S5 a-b), consistent with the previous report that Smad3 null mouse exhibit normal early tooth development 55 . The shift in BMP4 expression pattern coincides with the shift of odontogenic inductive potential between dental epithelium and dental mesenchyme 3 . Our observations that both the dental epithelium at the initiation stage and the dental mesenchyme at the bud stage, where the odontogenic inductive potential resides, lack canonical BMP signaling, prompted us to investigate whether the atypical BMP signaling sustained by TGFβ in the dental mesenchyme is necessary for odontogenic inductive potential. To address this question, we inhibited TGFβ signaling using SB431542 at E12.5 or E13.5 for one day to disrupt the atypical BMP signaling in the dental mesenchyme (Fig. 8 a). The SB431542-treated dental mesenchyme was subsequently recombined with E10.5 secondary pharyngeal arch epithelia. Continued SB431542 treatment on the recombinants of E12.5 dental mesenchyme and the secondary pharyngeal arch epithelia for one day prior to kidney capsule transplantation resulted in failed tooth formation (0/20, Fig. 8 e). In contrast, control recombinants with E12.5 dental mesenchyme treated with DMSO for one day prior to transplantation formed teeth (10/20, Fig. 8 d). Interestingly, E13.5 dental mesenchyme retained odontogenic inductive potential, manifested by a high rate of tooth formation (8/10) in SB431542-treated recombinants compared to DMSO-treated controls (Fig. 8 b, c). These results indicated that restricted operation of atypical BMP signaling in dental mesenchymal cells is temporally required for maintaining odontogenic inductive potential in the dental mesenchyme within a short time window between E12.5 and E13.5. We then investigated which genes directly regulated by atypical BMP signaling are crucial for maintaining odontogenic inductive potential in dental mesenchyme. Using RNA-seq analysis on in vitro cultured E12.5 tooth germs after disrupting atypical BMP signaling with SB431542 for one day (Fig. S6 a), we identified DEGs between control tooth germs at E12.5 and E13.5 and intersected them with DEGs resulting from atypical BMP signaling disruption (Fig. S6 b). Given that pSmad1/5 can function independently of both Smad4 and pSmad3 (Fig. 6 c), we further intersected the overlapped DEGs with pSmad1/5 target genes, identifying 611 genes directly regulated by atypical BMP signaling after SB431542 treatment (Fig. S6 b). Hierarchical clustering of enriched terms for these 611 genes demonstrated high association with mesenchymal development and the WNT signaling pathway (Fig. S6 c), which plays an essential role in tooth initiation 56 . We classified these genes into mesenchyme development and odontogenesis associated GO terms, identifying 41 potentially important genes for maintaining odontogenic inductive potential in dental mesenchyme (Fig. S6 d). Interestingly, atypical BMP signaling regulates these genes in three different ways: pSmad1/5 binding alone (Fig. S6 e); pSmad1/5 binding with pSmad3, but not at the same position (Fig. S6 f); and pSmad1/5 co-binding with pSmad3 at the same genomic loci (Fig. S6 g). This observation enhances our understanding of the diversity of TGFβ/BMP signaling through Smads. Together, our results suggest that TGFβ-sustained atypical BMP-Smad signaling confers diverse functions to ensure proper tooth innervation during early tooth development, thereby maintaining odontogenic inductive potential in dental mesenchyme. Discussion Building on our previous studies 26,27 , this investigation provide genome-wide evidence supporting the functional operation of an atypical canonical BMP signaling pathway in the dental mesenchyme. This pathway confers compartmentalized distributions of distinct BMP-Smad signaling outputs during early tooth development. While canonical BMP signaling output is restricted to neurovascular cells, the atypical canonical BMP signaling output in dental mesenchymal cells represents a distinct output. We demonstrate that this atypical pathway in dental mesenchymal cells operates through the formation of a novel mixed pSmad1/5-pSmad3 complex. This complex functions as a transcriptional regulatory unit, ensuring proper tooth innervation and maintaining odontogenic inductive potential in the dental mesenchyme. Research over recent decades has revealed the repeated use of a relatively small number of signaling pathways driving various developmental decisions during early tooth development 57 . The TGFβ/BMP signaling pathway is such conserved pathway employed throughout this process. To achieve biological efficiency, different cells must respond differently to the same signal received multiple times. Understanding the genetic regulation network and functional distinctions of diverse signaling outputs in early tooth development is therefore of great interest. The classic view of TGFβ/BMP signaling pathways holds that upon activation, phosphorylated R-Smads form an obligate heterotrimer with Smad4 to regulate target gene transcription 18 . However, this dogma has been challenged by studies observing divergent phenotypes in Smad1/5 CKO mice when compared to Smad4 CKO mice 23–25 , suggesting R-Smads can transduce BMP signaling independently of Smad4. Our previous study demonstrated that BMP4-induced R-Smads (pSmad1/5/8) can regulate Msx1 expression in the dental mesenchyme of both humans 26 and mice 27 independent of Smad4. In this study, ChIP-seq analysis of pSmad1/5 and Smad4 in molar tooth germs revealed that pSmad1/5 and Smad4 exert distinct functions during early tooth development except shared roles in signal transduction and neurogenesis (Fig. 1 i-k). Consistently, real-time monitoring of canonical BMP-Smad transcriptional activity (Fig. 2 ) using transgenic mouse line BRE-GFP 29 revealed compartmentalized distributions of distinct BMP-Smad signaling outputs during early tooth development. GFP presence, indicating canonical BMP signaling, was restricted to neurovascular cells, while its absence in dental mesenchymal cells represented atypical canonical BMP signaling. This compartmentalized and diverse BMP-Smad signaling output resembles phenomena observed in endothelial networks 58 . This atypical BMP signaling in dental mesenchymal cells operates through the formation of a novel mixed pSmad1/5-pSmad3 complex, as evidenced by pharmacological inhibition and Co-IP experiments (Fig. 3 ), consistent with previous in vitro 32 and in vivo studies 59 . Genome-wide mapping of pSmad1/5 and pSmad3 targets using CUT&RUN assay revealed similar genomic distribution patterns and biological processes for their target genes (Fig. 5 ). This suggests the complex acts as a genome-wide transcriptional regulatory unit during early tooth development. Motif analyses of pSmad1/5 and pSmad3 binding peaks showed high similarity between their motifs, further supporting this notion (Fig. 6 ). Using pairwise motif alignment, we identified 9 shared TFs as potential regulatory partners of the pSmad1/5-pSmad3 complex (Fig. 6 a), many of which are known regulators of neurogenesis 43,60–63 . Functional classification of pSmad1/5 and pSmad3 co-target genes (Fig. 6 c, d) and examination of neurite outgrowth after disrupting atypical BMP signaling (Fig. S4 ) verified their role in regulating tooth innervation. These results align with previous studies demonstrating that target-derived BMP4 52 and TGFβ signaling 47 can regulate the extent of peripheral innervation in vivo . BMP4 has been identified as a major target-derived factor that retrogradely regulates gene expression in developing trigeminal sensory neurons through both Smad4-independent and Smad4-dependent pathways 9 . Notably, peripheral sensory innervation remains normal after conditional deletion of Smad4 with Advillin Cre/+ , a sensory neuron-specific Cre mouse line 64 . Collectively, these previous findings and our studies suggest that TGFβ and BMP signaling converge on a novel pSmad1/5-pSmad3 complex to regulate tooth innervation independently of Smad4. The formation of mixed R-Smads has also been observed in TGFβ signaling in various epithelial cells and fibroblasts 32,59,65 . The prevalence of mixed pSmad1/5-pSmad3 complex in dental mesenchymal cells, where Smad4 is widely expressed (Fig. 3 ), raises an important question: why does pSmad1/5 prefer to form complexes with pSmad3 instead of Smad4? Two possible mechanisms for this preferential formation can be envisioned from our results. First, TGFβ signaling leads to Smad4 saturation through the formation of pSmad2/3-Smad4 complexes; Second, pSmad3 or other unidentified TFs compete with Smad4 to form novel complexes comprising pSmad1/5 but not Smad4. Given Smad4’s ubiquitous expression in dental mesenchyme (Fig. 3 ) and a previous study showing that Smad4 overexpression did not affect TGFβ’s ability to inhibit BMP7-induced transcription 32 , our results suggest that pSmad3 or candidate partner TFs competes with Smad4 to form novel complexes. This competition mechanism has been previously described as a means of mutual antagonism between Nodal and BMP signaling 66 . Several observations support this competition mechanism: First, pSmad3 is absent in BRE-GFP positive neurovascular cells (Fig. 3 ); Second, inhibition of pSmad3 expression results in ectopic BRE-GFP expression in dental mesenchymal cells (Fig. 4 ); Third, spaced motif analysis shows that SMAD3 is the most favored TF to form complexes with pSmad1/5 STREME-1 motif (Fig. 6 ). Recent research has shown that weak TF-TF contacts guided by DNA mediate the selectivity of cooperating partners 67 . We propose that the preferential formation of pSmad1/5-pSmad3 complexes in dental mesenchymal cells occurs through a similar mechanism 67 , where pSmad1/5-bound DNA guides the selection of pSmad3 over Smad4. We further demonstrated the biological significance of TGFβ-sustained atypical BMP signaling through tissue recombination experiments (Fig. 8 ). These experiments revealed that atypical BMP signaling is crucial for maintaining odontogenic inductive potential in dental mesenchyme within a short time window between E12.5 and E13.5. To dissect how atypical BMP signaling regulates target genes for maintaining this potential, we intersected DEGs from RNA-seq after pSmad3 inhibition using SIS3 (Fig. 7 ) with the pSmad1/5 and pSmad3 co-bound genes (Fig. 6 ). This analysis demonstrated that TGFβ-sustained atypical BMP signaling regulates tooth innervation (Fig. S4 ) through direct control of axonogenesis-associated gene expression (Fig. 7 c-f). Given that identifying ‘odontogenic inducer’ is a major challenge for in vitro production of implantable bioengineering tooth germs 68 , we sought to identify genes potentially important for maintaining odontogenic inductive potential in dental mesenchyme (Fig. 9). Integrative analyses of DEGs between E13.5 and E12.5 stages, DEGs after disrupting atypical BMP signaling with SB431542, and pSmad1/5 bound genes identified 611 genes essential for mesenchymal development (Fig. S6 b). Further functional classification revealed 41 genes important for mesenchyme development and odontogenesis (Fig. S6 c, d). A deeper analysis uncovered three distinct mechanisms by which TGFβ-sustained atypical BMP signaling regulates gene expression: pSmad1/5 co-binding with pSmad3 on the same genomic loci (Fig. S6 g); pSmad1/5 and pSmad3 both binding but on different genomic positions (Fig. S6 f), and pSmad1/5 binding alone (Fig. S6 e), possibly with unidentified Smad-cooperating TFs warranting future investigation. Notably, Msx1 , a readout gene of atypical BMP signaling 27 , is regulated by pSmad1/5 alone (Fig. S6 e). Given that Msx1 is also expressed in endothelial cells at artery branching sites 69 , and canonical BMP signaling operates in vascular cells in the dental mesenchyme (Fig. 2 ), this suggests multiple regulatory mechanisms for Msx1 expression. Collectively, our results suggest that the TGFβ-sustained atypical BMP-Smad pathway generates diverse signaling outputs to ensure proper tooth innervation and maintain odontogenic inductive potential in dental mesenchyme. This diversity may explain how mutations in components of the same pathway can cause different phenotypic disorders. While studies on tooth development typically focus on epithelial-mesenchymal interactions, our investigation of intra-mesenchymal interactions between axonal nerves and dental mesenchymal cells expands our understanding of early tooth development. This study provides valuable insights into the functions of peripheral nerves in odontogenesis, potentially informing strategies for producing implantable bioengineered tooth germs in vitro . Materials and methods Animals The generation and genotyping protocols of BRE-LacZ and BRE-GFP mice have been described previously 28,29 . Both mouse lines were maintained on CD1 background. All animal experiments in this study were approved by the Institutional Animal Care and Use Committee, Tulane University. Organ culture, inhibitor treatment and tissue recombination Heads and mandibles from BRE-LacZ mice at embryonic stage 13.5 (E13.5) were dissected and set for roller cultured as previously described 70 prior to SIS3 treatment. The molar tooth germs dissected from BRE-GFP mouse embryos at indicated stages were first cultured in vitro in the DMEM supplemented with 10% FBS (Cytiva, SH30070.02) and GlutaMAXTM-1(Gibco, 35050-061), treated with the indicated inhibitors, including SB431542 (Sigma-Aldrich 616461, 50µM) and SIS3 (Tocris #5291, 300 µM), and then were subjected to the subsequent experiments. For tissue recombination experiments, the dental mesenchyme from indicated stages after treatment with SB431542 for 1 or 2 days were separated from their original dental epithelia and recombined with the E10.5 secondary pharyngeal arch epithelia, which were then further cultured with DMSO or SB431542 for 1 day prior to kidney capsule transplantation in mice. All surgical procedures were performed under anesthesia that administered by intra-peritoneal injection. After one month, the host mice were sacrificed for harvest of grafted recombinants, followed with morphogenetic and histological analyses. X-gal staining, immunofluorescence, RNAscope in situ hybridization, and co-immunoprecipitation (Co-IP) Standard X-gal staining was conducted on the E13.5 embryonic limb and mandible from BRE-LacZ mice as previously described 28 . Molar tooth germs harvested from embryonic BRE-GFP mice at indicated stages, or in vitro cultured tooth germs were fixed in 4% paraformaldehyde (PFA) at 4°C overnight, paraffin-embedded, and sectioned at 5µm for immunofluorescence staining. Sections were incubated with the primary antibody and the secondary antibodies conjugated to fluorophores (Life Technologies), and then counterstained with DAPI to reveal nuclei. Images were collected through Nikon Eclipse Ti2 Confocal Microscope. The primary antibodies used in this study included pSmad1/5 (Cell signaling technology, #9516), pSmad2 (Abcam, ab188334), pSmad3 (Abcam, ab52903), Smad4 (cell signaling technology, #46535), GFP (Novus Biologicals, NB100-1614) and CD133 (Abcam, Ab19898). RNAscope in situ hybridization was performed with the RNAscope 2.5 HD Reagent Kit Brown (Advanced Cell Diagnostics, 322300) following the manufacturer’s instructions. The signal was detected by Alexa Fluor™ 596 Tyramide SuperBoost™ Kits (ThermoFisher, USA). Probe details are listed in Supplementary Table 1. Traditional in situ hybridization of Bmp4 was conducted as previously described 13 . Co-IP assays were performed on the freshly dissected tooth germs at E13.5 using Pierce™ Co-Immunoprecipitation Kit (Thermo Scientific, USA). Chromatin immunoprecipitation sequencing (ChIP-seq) ChIP-seq analyses of pSmad1/5 and Smad4 were performed as previously described 71 on the tooth germs from E13.5 mouse embryos using Active Motif HS ChIP Kit, with the same stage embryonic limb tissues as biological controls. ChIPed DNAs were subjected to ChIP-seq. Library preparation and sequencing were performed at Active Motif (Carlsbad, CA) and BGI (Hong Kong, China). The 50 bp single-end sequencing reads were then aligned to the mouse reference genome mm10 using bowtie2 72 . The resulting SAM files were converted into BAM files with samtools 73 , which were then subjected to the BCP 74 for peak calling. Cleavage Under Targets & Release Using Nuclease (CUT&RUN) and quantitative PCR (qPCR) CUT&RUN experiments on E13.5 tooth germs were carried out as described by manufacturer’s manual (Cell Signaling Technology, #86652). Briefly, E13.5 tooth germs were harvested and bound to concanavalin A-coated magnetic beads. Digitonin was then used to permeabilize the cell membranes, allowing the phosphorylated Smad1/5 (Cell Signaling Technology, #9516) and Smad3 (Cell Signaling Technology, #9520) antibody to enter cells and find their targets. After that, the beads were briefly washed, and then incubated with pA-MNase enzyme. When the bead-bound cells were chilled to 0℃, CaCl 2 was added to start the digestion for 30 min. After stopping the reactions by chelation, the DNA fragments released into solution by cleavage were then extracted for qPCR or library construction. The qPCR primers are listed in Supplementary Table 1. To construct the CUT&RUN DNA library, we adopted the protocol of NEBNext Ultra II DNA Library Prep Kit, NEB (USA). The constructed DNA libraries were sequenced on the Illumina NextSeq 500 platform using NextSeq 500/550 High Output Kit v2 in a 100 bp paired-end mode. The CUT&RUN data were then processed by following the pipeline as previously described 36 with custom modified scripts. Briefly, paired-end sequencing reads were aligned to the mouse reference genome mm10 using bowtie2 72 . The resulting BAM alignment files were then subjected to samtools 73 for filtering low-quality reads, reads in the ENCODE blacklist regions, unmapped mates, and PCR duplicates. The filtered BAM files were converted into bedGraph files using the bamtobed tool from bedtools 37 . These bedGraph files were then used as input for SEACR 75 to call peaks with the -non - stringent setting. pSmad1/5 and pSmad3 peaks from two replicates were intersected using bedtools 37 to get their common peaks. Motif discovery analysis was performed on ± 100bp sequences from the pSmad1/5 and pSmad3 common peak summits with MEME Suite ( http://meme-suite.org ) 76 . Motif comparison, motif alignment and pairwise motif similarity scoring were performed using TomTom 77 from MEME Suite 76 . The pairwise motif similarity scores were visualized using custom R script. RNA preparation and RNA sequencing (RNA-seq) E12.5 tooth germs after treatment with SIS3 or SB431542 for 1 day, and E13.5 control tooth germs were collected for RNA-seq assays. DMSO treated tooth germs were used as controls. Total RNAs were extracted using RNeasy Micro Plus Kit (Qiagen, USA), and quantified using a Qubit 2.0 Fluorometric Quantitation system (Life Technologies, USA). Library preparation was performed as previously described 78 . The prepared libraries were then pooled and sequenced on the Illumina HiSeq4000 platform using 75bp pair-end reads mode. To avoid batch effect, all RNA-seq experiments were performed in duplicate. The RNA-seq sequencing reads were mapped to the mm10 transcriptome using Salmon quant for quantification of gene expression at transcript level 79 . Salmon quantification files were then imported into R with the tximport package 80 to summarize gene-level expression. Genes with fewer than 10 counts across all samples were excluded from the downstream analysis. Differentially expressed gene (DEGs) were identified using DESeq2 81 , with genes having an adjusted p-value (padj) < 0.01 considered as significant DEGs. These significant DEGs were subsequently imported into R for intersection with CUT&RUN peak target genes and functional classification. Peak annotation, Functional classification and Data visualization ChIP-seq peaks and the common peaks from CUT&RUN replicates were annotated using ChIPseeker R package 82 . The annotated peak target genes were then imported into the clusterProfiler R package 83 for gene ontology (GO) and pathway enrichment analysis. Enriched GO terms and pathways were then visualized by the implemented functions of clusterProfiler, excluding general and redundant terms. To characterize the read distributions of ChIP-seq and CUT&RUN sequencing, filtered BAM files were used as input for the deepTools 84 . Heatmaps of signals centered on transcription start sites (TSS) were generated using the plotHeatmap subcommand. For UCSC genome browser visualization of RNA-seq reads, sequencing reads were aligned to the mouse reference genome (mm10) using Hisat2 85 with default parameters. BAM files from two replicates in each group were merged with samtools 73 and then converted into bigwig format using the bamCoverage subcommand from deepTools 84 . For UCSC genome browser visualization of CUT&RUN sequencing reads, redundant reads in the samtools-merged BAM files from two replicates were first filtered using the intersect function of bedtools 37 and then converted into bigwig format. Declarations Data availability All of the RNA-Seq, ChIP-Seq and CUT&RUN datasets were deposited into the GEO with SuperSeries accession number xxxxx. Acknowledgements This research was supported by the National Institutes of Health grant R01DE024152 to Y.C.. H.L. was supported in part by a fellowship from Fujian Normal University and by a grant (2021J01681) from the Natural Science Foundation of Fujian Province, P.R. China. L.W. received a fellowship from the China Scholarship Council during the initial phase of the studies. We thank Kejing Song and Genevieve Pierre at the Tulane Center for Translational Research in Infection and Inflammation’s NextGen Sequencing Core for assistance with initial bioinformatic analyses. High performance computing (HPC) resources provided by Tulane University Technology Services also contributed to this research. Conflict of interests The authors declare that there are no conflicts of interest regarding the research, authorship, and publication of this manuscript. Contributions Q.T., L.L., and Y.C. conceived the project. Q.T. and L.L. performed most experiments, collected and analyzed data, and prepared figures. Q.T. and L.L. also drafted the initial manuscript. Y.L., A.W., L.W. and C.G. assisted with genotyping, histology, immunohistochemistry, Co-IP, CUT&RUN-qPCR, and tissue recombination experiments. Q.T., with support from Z.W., conducted all bioinformatic analyses. 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Nat Biotechnol 37 , 907-915 (2019). https://doi.org/10.1038/s41587-019-0201-4 Additional Declarations (Not answered) Supplementary Files SupplementalTable1.docx Supplemental Table 1 FigS1toFig4.jpg Supplemental Figure S1 FigS2toFig5.jpg Supplemental Figure S2 FigS3toFig6.jpg Supplemental Figure S3 FigS4toFig6.jpg Supplemental Figure S4 FigS5toFig8.jpg Supplemental Figure S5 FigS6toFig8.jpg Supplemental Figure S6 SuppLegends.docx Cite Share Download PDF Status: Posted 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. 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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-5188541","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":373322020,"identity":"fb73a0b5-6fde-44d2-bcc7-260cf7f3fddd","order_by":0,"name":"Qinghuang Tang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYDACZjBiYOCXYGD4UMFgAWIbEKdFcgYD44wzDBJEaGGAajG4QawWvuO8h18XttnlGd/uMWw4UCMhx8DevE0CnxbJw3xp1jPbkovN7pwBajkmYczAc6wMrxaDwzxmxrxtzInbbuSYP/7AJpHYIJFjRoyW+sTNM3KAtvwDapF/Q1CL8WPetsOJGySAWg62gWzhwa9FEmgLM8+544kz7hwrbDjYJ2HMxpNWbIFPC9/5M8afecqqE/tnN29sOPDNRo6f/fDGG/i0MBxgYEN1Bhte5RAtzB8IKhoFo2AUjIKRDQDR5kqruvahBQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-9436-8628","institution":"University at Buffalo","correspondingAuthor":true,"prefix":"","firstName":"Qinghuang","middleName":"","lastName":"Tang","suffix":""},{"id":373322021,"identity":"c1f0194a-4a92-43de-83f1-83d195952d5a","order_by":1,"name":"Liwen Li","email":"","orcid":"","institution":"University at Buffalo","correspondingAuthor":false,"prefix":"","firstName":"Liwen","middleName":"","lastName":"Li","suffix":""},{"id":373322022,"identity":"d25241a2-9a7c-4859-80ee-69bb57356562","order_by":2,"name":"Yihong Li","email":"","orcid":"","institution":"Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yihong","middleName":"","lastName":"Li","suffix":""},{"id":373322023,"identity":"cb264922-0ef2-4513-95b8-b33006f91d47","order_by":3,"name":"Amy Wang","email":"","orcid":"","institution":"Tulane University","correspondingAuthor":false,"prefix":"","firstName":"Amy","middleName":"","lastName":"Wang","suffix":""},{"id":373322024,"identity":"a2bc6417-123a-423b-b093-05615dcab85c","order_by":4,"name":"Hua Li","email":"","orcid":"","institution":"Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Li","suffix":""},{"id":373322025,"identity":"93c62c97-9529-4545-8598-5fd0892a6524","order_by":5,"name":"Linyan Wang","email":"","orcid":"","institution":"Chengdu Second People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Linyan","middleName":"","lastName":"Wang","suffix":""},{"id":373322026,"identity":"b443ba79-67f5-4cd4-b8f1-69da44a0e0f8","order_by":6,"name":"Cong Gu","email":"","orcid":"","institution":"Tulane University","correspondingAuthor":false,"prefix":"","firstName":"Cong","middleName":"","lastName":"Gu","suffix":""},{"id":373322027,"identity":"4b2967f7-8f71-4978-9f68-e9c67ff9d61c","order_by":7,"name":"Jung-Mi Lee","email":"","orcid":"","institution":"University at Buffalo","correspondingAuthor":false,"prefix":"","firstName":"Jung-Mi","middleName":"","lastName":"Lee","suffix":""},{"id":373322028,"identity":"a935489b-0612-498d-b33d-d559c1153bb8","order_by":8,"name":"Zhaoming Wu","email":"","orcid":"https://orcid.org/0000-0002-0131-2423","institution":"Faculty of Dentistry, The University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Zhaoming","middleName":"","lastName":"Wu","suffix":""},{"id":373322029,"identity":"7d82f39f-f467-4a70-a372-3107d524aab5","order_by":9,"name":"Hyuk-Jae Kwon","email":"","orcid":"","institution":"University at Buffalo","correspondingAuthor":false,"prefix":"","firstName":"Hyuk-Jae","middleName":"","lastName":"Kwon","suffix":""},{"id":373322030,"identity":"027540e8-f4be-47bb-9be7-cbf2d55e1ada","order_by":10,"name":"YiPing Chen","email":"","orcid":"","institution":"Tulane University","correspondingAuthor":false,"prefix":"","firstName":"YiPing","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-10-01 16:05:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5188541/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5188541/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68238847,"identity":"64358676-7226-4158-9192-b4f3831f1f94","added_by":"auto","created_at":"2024-11-05 07:43:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":306851,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenome-wide profiling of pSmad1/5 and Smad4 binding sites in molar and limb. \u003c/strong\u003e(\u003cstrong\u003ea-b\u003c/strong\u003e) LacZ staining of mandible and limb from E13.5 BRE-LacZ mouse embryos shows that canonical BMP signaling is present in the limb (\u003cstrong\u003ea\u003c/strong\u003e, arrows) but absent in the molar tooth germ (\u003cstrong\u003eb\u003c/strong\u003e, arrows). Dotted lines outline molar tooth germs. (\u003cstrong\u003ec, d\u003c/strong\u003e) Heatmap plots of pSmad1/5 and Smad4 ChIP-seq signals in the limb and the molar tooth germ. pSmad1/5 and Smad4 ChIP-seq signals exhibit similar distribution patterns in the limb (\u003cstrong\u003ec\u003c/strong\u003e) but show markedly distinct patterns in the molar tooth germ (\u003cstrong\u003ed\u003c/strong\u003e).\u003cstrong\u003e \u003c/strong\u003eThe heatmaps display regions of 3kb up- and down-stream of all TSSs annotated in RefSeq genes. (\u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e) Venn diagrams showing the overlapping peak numbers of pSmad1/5 and Smad4 in the limb (\u003cstrong\u003ee\u003c/strong\u003e) and the molar tooth germ (\u003cstrong\u003ef\u003c/strong\u003e). Numbers inside the circles represent the count of specific peaks bound by the respective Smad. (\u003cstrong\u003eg, h\u003c/strong\u003e) Pie charts from peak genomic annotation reveal that pSmad1/5 (\u003cstrong\u003eg\u003c/strong\u003e) and Smad4 (\u003cstrong\u003eh\u003c/strong\u003e) peaks exhibit different binding preferences in the molar tooth germ. Both pSmad1/5 and Smad4 peaks predominantly bind to cis-regulatory elements (such as distal intergenic and other intron regions), with pSmad1/5 peaks showing a preferential binding on the promoter regions compared to Smad4. (\u003cstrong\u003ei-k\u003c/strong\u003e) GO analysis of genes specifically bound by pSmad1/5 (\u003cstrong\u003ei\u003c/strong\u003e) or Smad4 (\u003cstrong\u003ej\u003c/strong\u003e), or co-bound by pSmad1/5 and Smad4 (\u003cstrong\u003ek\u003c/strong\u003e) in the molar tooth germ. Scale bar, 1mm.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/9580e69b33041fe6623a2f77.jpg"},{"id":68240134,"identity":"524aba82-066b-4718-bbd2-6b8c64e0d8c2","added_by":"auto","created_at":"2024-11-05 07:59:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":278130,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBRE-GFP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e positive cells in the dental mesenchyme differentiate into neurovascular cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ec, j-l\u003c/strong\u003e) Distribution of \u003cem\u003eBRE-GFP\u003c/em\u003epositive cells during molar tooth development at E11.5 (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e), E12.5 (\u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ek\u003c/strong\u003e) and E14.5 (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003el\u003c/strong\u003e). At E11.5, \u003cem\u003eBRE-GFP\u003c/em\u003epositive cells were detected at a considerable distance from the dental epithelium (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e). At E12.5, \u003cem\u003eBRE-GFP\u003c/em\u003e positive cells were present in the outer border of the condensed dental mesenchyme (\u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ek\u003c/strong\u003e). At E14.5, \u003cem\u003eBRE-GFP\u003c/em\u003e positive cells were primarily localized in the dental follicle, with a few \u003cem\u003eBRE-GFP\u003c/em\u003e positive branching cells beginning to enter the dental papilla (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003el\u003c/strong\u003e). \u003cem\u003eBRE-GFP\u003c/em\u003e positive cells also emerged in the enamel knot of the dental epithelium at this stage (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003el\u003c/strong\u003e). (\u003cstrong\u003ed\u003c/strong\u003e-\u003cstrong\u003ei, m-r\u003c/strong\u003e) Co-localization of \u003cem\u003eBRE-GFP\u003c/em\u003e positive cells with neurovascular cells in the dental mesenchyme (\u003cstrong\u003eg\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003ep\u003c/strong\u003e-\u003cstrong\u003er\u003c/strong\u003e), as evidenced by the immunofluorescence staining of 2H3(\u003cstrong\u003ed\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e) and CD31(\u003cstrong\u003em\u003c/strong\u003e-\u003cstrong\u003eo\u003c/strong\u003e), respectively. Merged images (g-i, p-r) reveal that \u003cem\u003eBRE-GFP\u003c/em\u003epositive cells begin to co-localize with 2H3 at E12.5 (\u003cstrong\u003eg\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e) and with CD31 at E11.5 (\u003cstrong\u003ep\u003c/strong\u003e-\u003cstrong\u003er\u003c/strong\u003e). Scale bar, 100μm.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/642c19dd9f43ab0853d1ce16.jpg"},{"id":68239080,"identity":"1915a3eb-6715-4314-84ec-97388044cfdd","added_by":"auto","created_at":"2024-11-05 07:51:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":306845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFormation of pSmad1/5-pSmad3 complexes in the dental mesenchyme.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea-r\u003c/strong\u003e) Immunofluorescence staining of Smad4 (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003em\u003c/strong\u003e), pSmad1/5 (\u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003en\u003c/strong\u003e), pSmad2 (\u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e, \u003cstrong\u003ep\u003c/strong\u003e), pSmad3 (\u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003ek\u003c/strong\u003e, \u003cstrong\u003eq\u003c/strong\u003e), and GFP in the frontal sections of molar tooth germs from BRE-GFP mouse embryos at E12.5 (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e), E13.5 (\u003cstrong\u003eg\u003c/strong\u003e-\u003cstrong\u003el\u003c/strong\u003e) and E14.5 (\u003cstrong\u003em\u003c/strong\u003e-\u003cstrong\u003er\u003c/strong\u003e). GFP fluorescence (by \u003cem\u003eBRE-GFP\u003c/em\u003e), indicative of canonical BMP signaling activity, co-localized with pSmad1/5 (red, \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003eo\u003c/strong\u003e) but not pSmad3 (red, \u003cstrong\u003ef\u003c/strong\u003e, \u003cstrong\u003el\u003c/strong\u003e, \u003cstrong\u003er\u003c/strong\u003e) in neurovascular cells. (\u003cstrong\u003es\u003c/strong\u003e) Co-immunoprecipitation (Co-IP) analysis on E13.5 molar dental mesenchyme using pSmad3 antibody. Immunoblotting of the pSmad3 immunoprecipitants with pSmad1/5 (left) and pSmad3 (right) antibodies revealed that pSmad3 forms complexes with pSmad1/5 (\u003cstrong\u003es\u003c/strong\u003e). Input and IgG control experiments were performed in parallel to ensure antibody specificity and validate the results (\u003cstrong\u003es\u003c/strong\u003e). Scale bar, 200μm.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/dd3804c4be9a4556870f8e51.jpg"},{"id":68238855,"identity":"88523d0f-a151-4d02-92b5-a33549e431d0","added_by":"auto","created_at":"2024-11-05 07:43:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":278405,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of TGFβ-pSmad3 signaling leads to ectopic activation of canonical BMP signaling in the dental mesenchyme.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea-l\u003c/strong\u003e) Immunofluorescence staining of GFP (\u003cem\u003eBRE-GFP\u003c/em\u003e), pSmad3, pSmad1/5 and Msx1 in \u003cem\u003ein vitro\u003c/em\u003e cultured tooth germs after treatment with SIS3 or SB431542 for 2 days. SIS3 (\u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003eh\u003c/strong\u003e) or SB431542 (\u003cstrong\u003ei\u003c/strong\u003e-\u003cstrong\u003el\u003c/strong\u003e) treatment led to decreased expression of pSmad3 (\u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003ek\u003c/strong\u003e), induced ectopic \u003cem\u003eBRE-GFP\u003c/em\u003e activity in dental mesenchymal cells (\u003cstrong\u003ef\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e, \u003cstrong\u003eq\u003c/strong\u003e, \u003cstrong\u003er\u003c/strong\u003e), and resulted in reduced expression of Msx1 (\u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003el\u003c/strong\u003e) compared to the DMSO controls (a-d). (\u003cstrong\u003em\u003c/strong\u003e-\u003cstrong\u003er\u003c/strong\u003e) SB431542 treatment did not affect the expression of pSmad1/5 (\u003cstrong\u003ep\u003c/strong\u003e-\u003cstrong\u003er\u003c/strong\u003e) when compared to DMSO treatment (\u003cstrong\u003em\u003c/strong\u003e-\u003cstrong\u003eo\u003c/strong\u003e). (\u003cstrong\u003es-u\u003c/strong\u003e) Immunofluorescence staining of pSmad1/5 and GFP on tooth germs after co-treatment with SB431542 and Dorsomorphin shows abrogated activities of both pSmad1/5 (\u003cstrong\u003es\u003c/strong\u003e, \u003cstrong\u003eu\u003c/strong\u003e) and \u003cem\u003eBRE-GFP\u003c/em\u003e (\u003cstrong\u003et\u003c/strong\u003e, \u003cstrong\u003eu\u003c/strong\u003e). Scale bar, 100μm.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/24106847a26814faa9f56c52.jpg"},{"id":68238102,"identity":"451d13d4-d0c3-41bc-a1f0-21de75bdf360","added_by":"auto","created_at":"2024-11-05 07:35:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":298663,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenome-wide mapping of pSmad1/5 and pSmad3 targets in the dental mesenchyme. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Heatmap plots showing that pSmad1/5 and pSmad3 CUT\u0026amp;RUN signals in the molar tooth germs exhibit similar distribution patterns. The heatmaps display regions of 3kb up- and down-stream of all TSS annotated in RefSeq genes. (\u003cstrong\u003eb\u003c/strong\u003e) Pie charts demonstrate a similar genomic distribution between pSmad1/5- and pSmad3-bound peaks. (\u003cstrong\u003ec\u003c/strong\u003e) GO analysis of the pSmad1/5- and pSmad3-bound genes shows a similar enrichment of biological process (BP) GO terms between the two gene sets.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/1e71793669ded3241c612dcd.jpg"},{"id":68238105,"identity":"8d3af025-e7bd-4348-a121-03240eca2c12","added_by":"auto","created_at":"2024-11-05 07:35:52","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":298305,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epSmad1/5 and pSmad3 act as a transcriptional unit in dental mesenchymal cells. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Pairwise motif comparison on the \u003cem\u003ede novo\u003c/em\u003emotifs of pSmad1/5 and pSmad3. The DNA sequences of pSmad1/5 and pSmad3 motifs are highly similar. Pairwise motif alignment on these motifs revealed potential shared TFs may recognize their overlapping DNA sequences. (\u003cstrong\u003eb\u003c/strong\u003e) Spaced motif analysis unveils that Smad3 is the most preferential motif physically interacting with pSmad1/5 top 1 motif. (\u003cstrong\u003ec\u003c/strong\u003e) Venn diagram showing the overlapping targets of pSmad1/5 and pSmad3 in the molar tooth germ. (\u003cstrong\u003ed\u003c/strong\u003e) Pathway enrichment analysis on the overlapping targets of pSmad1/5 and pSmad3 showing that axonogenesis is one of the highly enriched biological processes.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/8a267acf5fb62a078be177bb.jpg"},{"id":68239081,"identity":"351f937d-3707-4ae5-8897-065f8a092622","added_by":"auto","created_at":"2024-11-05 07:51:52","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":306225,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAtypical BMP signaling regulates tooth sensory innervation during early molar tooth development.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Heatmap showing the representative significant differentially expressed genes (DEGs) in E12.5 tooth germs after treatment with SIS3 for 1 day. (\u003cstrong\u003eb\u003c/strong\u003e) Venn diagrams showing the numbers of overlapping genes among pSmad1/5 targets, pSmad3 targets, pSmads co-bound genes and significant DEGs after SIS3 treatment. (\u003cstrong\u003ec\u003c/strong\u003e) Heatplot showing the pSmads complex directly targeted genes that are classified into axonogenesis-associated biological processes. (\u003cstrong\u003ed\u003c/strong\u003e) UCSC genome browser visualization of CUT\u0026amp;RUN and RNA-seq signals for the representative genes that are directly regulated by pSmads complex. (\u003cstrong\u003ee\u003c/strong\u003e) CUT\u0026amp;RUN qPCR assay showing that the relative binding affinity of pSmad1/5 (in comparison to IgG) on the pSmads co-bound genes are significantly decreased after SIS3 treatment. (\u003cstrong\u003ef\u003c/strong\u003e) A schematic diagram demonstrating that atypical BMP signaling is essential for sensory innervation in early tooth development. I, Incisor; M, Molar; TG, Trigeminal Ganglion.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/667de94517e2fdffd53154a4.jpg"},{"id":68238856,"identity":"5948f5ca-745b-4ad6-b92a-459586a9dd1b","added_by":"auto","created_at":"2024-11-05 07:43:52","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":299021,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of odontogenic inductive potential in the dental mesenchyme after disruption of the atypical BMP signaling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ee\u003c/strong\u003e) Tissue recombination and transplantation of dental mesenchyme treated with SB431542. Dental mesenchyme from tooth germs treated with SB431542 at either E12.5 or E13.5 for 1 day (\u003cstrong\u003ea\u003c/strong\u003e) was separated from the epithelium and recombined with the E10.5 2\u003csup\u003end\u003c/sup\u003e pharyngeal arch (PA) epithelium (epi). The tissue recombinants were cultured\u003cem\u003e in vitro\u003c/em\u003e for 1 day and then transplanted into mouse kidney capsule to allow their further growth for 3 weeks (\u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003ee\u003c/strong\u003e). Teeth formed in the recombinants of E13.5 dental mesenchyme and the 2\u003csup\u003end\u003c/sup\u003e PA epithelium, regardless of whether they were treated with SB431542 treatment (\u003cstrong\u003ec\u003c/strong\u003e) or not (\u003cstrong\u003eb\u003c/strong\u003e) in organ culture prior to kidney capsule grafting. However, the tissue recombinants of E12.5 dental mesenchyme and the 2\u003csup\u003end\u003c/sup\u003e PA epithelium failed to develop into teeth when treated with SB431542, instead showing a keratinizing epithelial cyst with bone tissue (\u003cstrong\u003ee\u003c/strong\u003e), compared to the DMSO treatment group (\u003cstrong\u003ed\u003c/strong\u003e). n, number of biological replicates. Scale bar, 200μm.\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/11c2cc1fdef3fb61d48fc481.jpg"},{"id":68240472,"identity":"b26cfa6a-38d3-4a82-ad3e-8c927aacdb28","added_by":"auto","created_at":"2024-11-05 08:07:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3643568,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/d4d003b8-1236-4ce9-b36f-a5cbf0565da3.pdf"},{"id":68238091,"identity":"3cea5195-c43d-49af-8070-15862164e714","added_by":"auto","created_at":"2024-11-05 07:35:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18505,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Table 1\u003c/p\u003e","description":"","filename":"SupplementalTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/b2d639f222cbacedea67f8c0.docx"},{"id":68238093,"identity":"ad1d0186-7215-408a-8c83-58b0f126d756","added_by":"auto","created_at":"2024-11-05 07:35:52","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":269070,"visible":true,"origin":"","legend":"Supplemental Figure S1","description":"","filename":"FigS1toFig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/4c8f740b7015984ad08cc223.jpg"},{"id":68238848,"identity":"34cb786d-0c22-444e-aae2-a3ad373b1e1d","added_by":"auto","created_at":"2024-11-05 07:43:52","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":205461,"visible":true,"origin":"","legend":"Supplemental Figure S2","description":"","filename":"FigS2toFig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/0702b17bbbcfe50f9f12b0da.jpg"},{"id":68238853,"identity":"5e7f2828-4fdd-4eca-b7d8-d6d20a78de26","added_by":"auto","created_at":"2024-11-05 07:43:52","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":261215,"visible":true,"origin":"","legend":"Supplemental Figure S3","description":"","filename":"FigS3toFig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/2566d41a8d73b29debeb7e4f.jpg"},{"id":68238107,"identity":"5d9efd6e-7c4c-4231-809d-7fc8e013acdf","added_by":"auto","created_at":"2024-11-05 07:35:52","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":300651,"visible":true,"origin":"","legend":"Supplemental Figure S4","description":"","filename":"FigS4toFig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/cc37dfa0b0e3c9ceec5fec34.jpg"},{"id":68238850,"identity":"aa5b709d-49d0-4334-b451-d63be191dde9","added_by":"auto","created_at":"2024-11-05 07:43:52","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":111960,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Figure S5\u003c/p\u003e","description":"","filename":"FigS5toFig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/6b3592a0a86cddc46aa7d89f.jpg"},{"id":68238104,"identity":"d65c0ea8-5200-4450-aaea-32516a75fe98","added_by":"auto","created_at":"2024-11-05 07:35:52","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":304816,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Figure S6\u003c/p\u003e","description":"","filename":"FigS6toFig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/167bdeae0dda859f7f5777a3.jpg"},{"id":68238097,"identity":"0a0e9115-de4d-477f-8492-45d74479ed7d","added_by":"auto","created_at":"2024-11-05 07:35:52","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":17718,"visible":true,"origin":"","legend":"","description":"","filename":"SuppLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-5188541/v1/3cf3ec0d388aeaa2762621db.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Distinct BMP-Smad Signaling Outputs Confer Diverse Functions in Dental Mesenchyme","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMammalian odontogenesis, the complex process of tooth formation and integration with supporting tissues, including the neurovascular systems\u003csup\u003e1\u003c/sup\u003e, serves as a model for studying essential developmental mechanisms of tissue interactions and genetic regulation\u003csup\u003e2\u003c/sup\u003e. In mice, tooth development begins at embryonic day 11.5 (E11.5) when the presumptive dental epithelium thickens at future tooth-forming site, creating dental placodes. These placodes act as early signaling centers with \u0026lsquo;odontogenic inductive potential\u0026rsquo;, capable of inducing tooth formation even when recombined with non-dental embryonic mesenchyme\u003csup\u003e3\u003c/sup\u003e. At E12.5, this potential shifts from the dental placode to the underlying dental mesenchyme\u003csup\u003e4\u003c/sup\u003e, initiating sequential morphogenesis of individual tooth germs through the bud, cap, and bell stages. Concurrently, a blood vessel plexus forms around the developing tooth germ\u003csup\u003e5\u003c/sup\u003e, while trigeminal nerve fibers extend towards the developing maxillary or mandibular tooth germs\u003csup\u003e6\u003c/sup\u003e, diverging into buccal and lingual branches adjacent to the condensed dental mesenchyme\u003csup\u003e7\u003c/sup\u003e. Although the structure, distribution and function of the dental neurovascular system are well documented in murine and human teeth\u003csup\u003e5,8\u003c/sup\u003e, the molecular mechanisms underlying the initial establishment of tooth blood supply and innervation, as well as their integration with advancing tooth morphogenesis, remain elusive.\u003c/p\u003e \u003cp\u003eBone morphogenetic proteins (BMPs), a subclass of the TGFβ signaling superfamily, are widely involved in a broad spectrum of developmental processes, including neurogenesis\u003csup\u003e9\u003c/sup\u003e, hematopoiesis\u003csup\u003e10\u003c/sup\u003e, and odontogenesis\u003csup\u003e11\u003c/sup\u003e. BMP4, in particular, is a critical regulator at various stages of early tooth development\u003csup\u003e12\u003c/sup\u003e. Mice with neural crest-specific inactivation of either \u003cem\u003eBmp4\u003c/em\u003e or \u003cem\u003eBmpr1a\u003c/em\u003e exhibit arrested tooth development at the bud stage\u003csup\u003e12,13\u003c/sup\u003e. During tooth development initiation, epithelial BMP4 induces mesenchymal expression of \u003cem\u003eMsx1\u003c/em\u003e, which in turn promotes mesenchymal \u003cem\u003eBmp4\u003c/em\u003e expression, forming a positive regulatory loop that governs the transition from bud to cap stage\u003csup\u003e14\u003c/sup\u003e. Notably, ectopic expression of \u003cem\u003eBmp4\u003c/em\u003e driven by an \u003cem\u003eMsx1\u003c/em\u003e promoter in the dental mesenchyme can rescue tooth defects in \u003cem\u003eMsx1\u003c/em\u003e mutants, allowing development to progress to the cap stage\u003csup\u003e15\u003c/sup\u003e. Moreover, BMP4 also plays roles in determining the tooth-forming site\u003csup\u003e16\u003c/sup\u003e and tooth type\u003csup\u003e17\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe canonical BMP signaling begins with ligand binding, which assembles a heteromeric complex of type II and type I receptors\u003csup\u003e18\u003c/sup\u003e. This binding triggers phosphorylation of the type I receptor, activating intracellular mediators Smad1, Smad5, and Smad8\u003csup\u003e19\u003c/sup\u003e. These receptor-regulated Smads (R-Smads) form complexes with Smad4 and translocate to the nucleus. There, Smad proteins serve as signaling hubs, integrating regulatory inputs and cooperating with specific transcription factors to regulate target gene expression in a context-dependent manner\u003csup\u003e19\u003c/sup\u003e. BMPs can also activate non-canonical signaling pathways through kinases such as mitogen-activated protein kinase, p38, ERK and JNK, independent of Smad proteins. Other TGFβ superfamily ligands homologous to BMPs, such as TGFβs 1\u0026ndash;3 and Activin, also play crucial roles in early tooth development. This is evidenced by delayed tooth initiation and development following specific inactivation of the type I receptor ALK5 in cranial neural crest derived cells\u003csup\u003e20\u003c/sup\u003e. These ligands bind distinct receptor complexes of type I and type II receptors\u003csup\u003e21\u003c/sup\u003e, activating the canonical Smad2/3 pathway and non-canonical pathways that overlap with those activated by BMPs. In addition to shared non-canonical pathways, crosstalk between BMP and TGFβ signaling pathways occurs at multiple levels\u003csup\u003e22\u003c/sup\u003e, resulting in diverse cellular responses from a limited number of signaling inputs.\u003c/p\u003e \u003cp\u003eWhile R-Smads and Smad4 complexes have been long considered the principal functional units in canonical BMP signaling transcriptional regulation, recent observations challenge this view. In several developing organs, Smad1/5 inactivation causes severe defects, whereas Smad4 inactivation results in mild or no defects\u003csup\u003e23\u0026ndash;25\u003c/sup\u003e. This suggests that R-Smads and Smad4 may exert diverse regulatory functions independently. Indeed, we have previously identified an atypical canonical BMP signaling pathway in human\u003csup\u003e26\u003c/sup\u003e and mouse\u003csup\u003e27\u003c/sup\u003e dental mesenchyme that regulates \u003cem\u003eMsx1\u003c/em\u003e expression in a Smad1/5-dependent but Smad4-independent manner. In this study, we further elucidated the cellular and molecular mechanisms underlying the atypical canonical BMP signaling in dental mesenchymal cells. Our findings reveal that BMP-Smad signaling confers diverse functions to ensure proper early tooth innervation and to maintain the odontogenic inductive potential in dental mesenchyme. These results provide compelling evidence for the versatility of BMP-Smad signaling, challenging the canonical BMP signaling dogma. Additionally, our findings have potential implications for future tooth bioengineering applications.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGenome-wide profiling of pSmad1/5 and Smad4 binding sites in dental mesenchyme\u003c/h2\u003e \u003cp\u003eOur previous study identified an atypical canonical BMP-Smad signaling pathway in early developing dental mesenchyme of humans\u003csup\u003e26\u003c/sup\u003e and mice\u003csup\u003e27\u003c/sup\u003e, operating independently of Smad4. We confirmed this finding by using LacZ staining on embryonic stage 13.5 (E13.5) mandibles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) from BMP response element (\u003cem\u003eBRE\u003c/em\u003e)\u003cem\u003e-LacZ\u003c/em\u003e transgenic reporter mice\u003csup\u003e28\u003c/sup\u003e. E13.5 limb (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), where canonical BMP signaling is known to operate, served as a biological control. As expected, LacZ signal was observed in the limb but not in the mandibular molar tooth germ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b, red arrows). To investigate underlying molecular mechanisms, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) assay of phosphorylated Smad1/5 (pSmad1/5) and Smad4 on E13.5 limbs and molar tooth germs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d). Results showed markedly different binding patterns of pSmad1/5 and Smad4 in the molar (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), contrasting with similar patterns in the limb (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Intersection analysis revealed only 222 peaks are co-bound by pSmad1/5 and Smad4 in the molar (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef), compared to 3788 in the limb (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), providing genome-wide evidence for the operation of atypical canonical BMP signaling pathway during early tooth development. Further characterization of pSmad1/5 and Smad4 binding peaks in the molar showed that pSmad1/5 preferentially bind to promoter regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg) as compared to Smad4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh), while both predominantly bind to cis-regulatory elements, such as distal intergenic and other intron regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, h). Gene ontology (GO) analysis of pSmad1/5- and Smad4-specific peak target genes revealed their involvements in distinct biological processes, with shared roles in signaling transduction and neurogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei, j, black boxes). pSmad1/5 target genes are predominantly involved in \u0026lsquo;mesenchyme development\u0026rsquo; and axonogenesis-related activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei), while Smad4 target genes are associated with \u0026lsquo;muscle cell differentiation\u0026rsquo; and \u0026lsquo;sensory system development\u0026rsquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej). Intriguingly, target genes linked to pSmad1/5 and Smad4 co-bound peaks showed a predominant association with \u0026lsquo;sensory system development\u0026rsquo; and \u0026lsquo;dendrite development\u0026rsquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek), which are also enriched in Smad4-specific target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej, k, red boxes). Taken together, these results support the existence of a functional atypical canonical BMP-Smad signaling pathway during early tooth development and highlight the versatility of BMP-Smad signaling in transcriptional regulation. For brevity and distinction from canonical BMP signaling, we will refer to this pathway as \u0026ldquo;atypical BMP signaling\u0026rdquo; hereafter.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCanonical BMP signaling activity is restricted to neurovascular cells\u003c/h3\u003e\n\u003cp\u003eThe diverse BMP-Smad signaling outputs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei-k) prompted us to examine its transcriptional activity during early tooth development more closely. Using a \u003cem\u003eBRE-GFP\u003c/em\u003e transgenic reporter mouse line\u003csup\u003e29\u003c/sup\u003e, which allows real-time monitoring of canonical BMP-Smad transcriptional activity during development, we comprehensively examined the transcriptional activities of BMP-Smad signaling in early developing tooth germs. At E11.5, \u003cem\u003eBRE-GFP\u003c/em\u003e activity was observed in only a few cells surrounding the dental mesenchyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, j). By E12.5, \u003cem\u003eBRE-GFP\u003c/em\u003e activity appeared in the dental epithelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, k) and became pronounced in the enamel knot by E14.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, l). Interestingly, the emergence of \u003cem\u003eBRE-GFP\u003c/em\u003e activity in the dental epithelium coincides with the shift out of the odontogenic inductive potential from the dental epithelium to the dental mesenchyme\u003csup\u003e3\u003c/sup\u003e. In the dental mesenchyme, \u003cem\u003eBRE-GFP\u003c/em\u003e positive cells began to appear at the edges of the condensed dental mesenchyme at E12.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, k). By E14.5, these cells were primarily localized in the dental follicle, with some extending into the dental papilla, exhibiting neurovascular cell-like morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, l). Based on these observations and insights from GO analysis of pSmad1/5 and Smad4 co-bound genes, we hypothesized that \u003cem\u003eBRE-GFP\u003c/em\u003e positive cells are neurovascular cells. To test this, we performed immunostaining for neurofilament (2H3, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-f), a sensory nerve fiber specific marker\u003csup\u003e30\u003c/sup\u003e, and Pecam-1(CD31, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003em-o), an endothelial cell marker\u003csup\u003e31\u003c/sup\u003e, on the developing molar tooth germs. \u003cem\u003eBRE-GFP\u003c/em\u003e positive cells began to co-localize with CD31 at E11.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003em-r) and with 2H3 at E12.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-i), consistent with previous findings that blood vessel formation around the tooth germ precedes neurites ingrowth\u003csup\u003e5\u003c/sup\u003e. These results indicate that canonical BMP signaling is confined to neurovascular cells during early tooth development and is not active in dental mesenchymal cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eFormation of pSmad3-pSmad1/5 complexes in dental mesenchymal cells\u003c/h3\u003e\n\u003cp\u003eThe presence of canonical BMP signaling pathway in neurovascular cells and its absence in dental mesenchymal cells raises questions about how the BMP-Smad signaling pathway achieves diverse transcriptional outputs. Previous research has shown that TGF-β signaling can inhibit canonical BMP signaling-induced transcription through the formation of pSmad3-pSmad1/5 complexes in multiple cell lines\u003csup\u003e32\u003c/sup\u003e. We investigated whether this mechanism is applied to early tooth development for inhibiting canonical BMP signaling in dental mesenchymal cells. We examined the expression patterns of phosphorylated Smads (pSmad1/5, pSmad2, pSmad3) and Smad4 in developing molar tooth germs from the \u003cem\u003eBRE-GFP\u003c/em\u003e mouse line. Smad4 was widely expressed in both dental epithelial and dental mesenchymal cells at E12.5, E13.5 and E14.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, g, m). pSmad2 was weakly present in most of the dental mesenchyme, with moderate expression in a few scattered cells in the buccal dental mesenchyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, j, p). Notably, pSmad1/5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, h, n) and pSmad3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, k, p) exhibited similar expression patterns during early tooth development. At E12.5, they were present in the buccal dental mesenchyme and some of the condensed dental mesenchyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, e), becoming intense in the dental mesenchyme at E13.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh, k) and E14.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003en, q). In neurovascular cells where canonical BMP signaling operates, pSmad1/5 co-localized with \u003cem\u003eBRE-GFP\u003c/em\u003e signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, i, o), while pSmad3 was absent (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, l, r). This finding, along with the similar expression patterns of pSmad1/5 and pSmad3 suggests that the atypical BMP signaling in dental mesenchymal cells probably operates through the formation of pSmad1/5-pSmad3 complexes. To verify this, we performed co-immunoprecipitation (Co-IP) analyses on E13.5 dental mesenchymal cells using pSmad3 antibody. Western blotting of the pSmad3 immunoprecipitation confirmed that pSmad3 forms a novel complex with pSmad1/5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003es).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eTGFβ-BMP antagonism is responsible for the operation of atypical BMP signaling in dental mesenchymal cells\u003c/h3\u003e\n\u003cp\u003eTo investigate whether the formation of pSmad1/5-pSmad3 complexes in dental mesenchymal cells inhibits canonical BMP signaling, we suppressed Smad3 phosphorylation by treating tooth germs with SIS3\u003csup\u003e33\u003c/sup\u003e at the bud stage. Inhibition of pSmad3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, e) induced ectopic \u003cem\u003eBRE-GFP\u003c/em\u003e activities in dental mesenchymal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, g) compared to DMSO-treated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c). This result confirmed that the formation of pSmad1/5-pSmad3 complexes in dental mesenchymal cells inhibits canonical BMP signaling. Given that pSmad3 is a well-known intracellular effector of TGFβ signaling, we examined the expressions of several TGFβ superfamily members in molars at the bud stage (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea-j). \u003cem\u003eBmp4\u003c/em\u003e was intensively expressed in the condensed dental mesenchyme (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea, b). \u003cem\u003eTgfβ1\u003c/em\u003e, \u003cem\u003eTgfβ3, and Tgfβr1\u003c/em\u003e were widely expressed throughout dental mesenchymal cells at E12.5 and E13.5 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec-h). Specifically, \u003cem\u003eTgfβ1\u003c/em\u003e showed scattered expression in the dental mesenchyme (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec, d), while \u003cem\u003eTgfβ3\u003c/em\u003e exhibited intensive expression in the presumptive dental follicle (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eg, h). Notably, Inhba (InhibinβA, a component of Activin A) was strongly expressed in the dental mesenchyme at E12.5 and E13.5 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ei, j). To determine whether TGFβ family members inhibit canonical BMP signaling in dental mesenchymal cells, we applied SB431542, a selective inhibitor of TGFβ superfamily signaling\u003csup\u003e34\u003c/sup\u003e, to E13.5 tooth germs. This treatment activated ectopic \u003cem\u003eBRE-GFP\u003c/em\u003e in dental mesenchymal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej, k) compared to DMSO treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c). SB431542 treatment decreased pSmad3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei) but did not affect pSmad1/5 expression in dental mesenchymal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ep-r). Importantly, the expression of Msx1, a readout of the atypical BMP signaling\u003csup\u003e27\u003c/sup\u003e, was reduced after both SIS3 and SB431542 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh, l). This indicates that the pSmad3-mediated TGFβ inhibition is responsible for the operation of atypical BMP signaling in dental mesenchymal cells. When dorsomorphin\u003csup\u003e35\u003c/sup\u003e, an inhibitor of type I BMP receptor, was applied together with SB431452 to E13.5 tooth germs, both endogenous \u003cem\u003eBRE-GFP\u003c/em\u003e activities in neurovascular cells and ectopic \u003cem\u003eBRE-GFP\u003c/em\u003e activities induced by the TGFβ signaling inhibition in dental mesenchymal cells disappeared (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003es-u). This result indicates that BMP signaling is essential for Smad1/5 phosphorylation in both neurovascular and dental mesenchymal cells. Taken together, these results demonstrate that BMP signaling exerts both canonical and atypical signaling outputs in the dental mesenchyme. Canonical BMP signaling operates in neurovascular cells, while atypical BMP signaling operates in dental mesenchymal cells, where the canonical BMP signaling was inhibited by TGFβ signaling through the formation of pSmad1/5-pSmad3 complexes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eGenome-wide mapping of atypical BMP signaling targets in dental mesenchyme\u003c/h3\u003e\n\u003cp\u003eGiven the predominant operation of the atypical BMP signaling in the dental mesenchyme, to investigate its transcriptional regulatory function, we performed CUT\u0026amp;RUN (Cleavage Under Targets and Release Using Nuclease)\u003csup\u003e36\u003c/sup\u003e of pSmad1/5 and pSmad3 on the E13.5 tooth germs, a time the dental mesenchyme has gained sustained odontogenic inductive capability. Two independent biological replicates were used to maximize site discovery and remove sequencing artifacts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Using the bedtools intersect function\u003csup\u003e37\u003c/sup\u003e, we identified 10741 common pSmad1/5 binding peaks and 7905 common pSmad3 binding peaks across replicates (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea). Genome-wide distributions of pSmad15 and pSmad3 peaks relative to gene transcription start site (TSS) were highly similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and Fig.\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003eS2\u003c/span\u003eb). pSmad1/5 and pSmad3 peaks showed similar binding preference, both exhibiting the highest abundance in distal intergenic (33.46% vs. 28.97%) and other intron regions (16.26% vs. 13.27%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). This indicates that both pSmad1/5 and pSmad3 primarily function on distal enhancers, consistent with the consensus that activated Smad complexes regulate transcription via chromatin remodeling\u003csup\u003e18\u003c/sup\u003e. Functional classification of pSmad1/5 and pSmad3 binding genes into Biological Process (BP, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), Cellular Component (CC, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ec) and Molecular Function (MF, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ed) categories revealed enrichment in similar GO terms across all three categories. These results further substantiate the formation of pSmad1/5-pSmad3 complexes in dental mesenchyme at a genome-wide level, suggesting that they act as transcriptional regulatory units. Smad complexes, due to their weak DNA binding affinity, frequently cooperate with context-specific transcription factors (TFs) to enhance and stabilize their DNA binding\u003csup\u003e38\u003c/sup\u003e. To understand how pSmad1/5-pSmad3 complexes act as transcriptional regulatory units, and identify their cooperating TFs, we performed comprehensive motif analyses on the pSmad1/5 and pSmad3 bound peaks. Unbiased motif discovery using STREME\u003csup\u003e39\u003c/sup\u003e yielded 10 recurring ungapped motifs in each sequence dataset (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Motif comparison with a Mouse Embryonic Stem Cell TF motif database\u003csup\u003e40\u003c/sup\u003e showed that the majority of motifs from both pSmad1/5 (8/10) and pSmad3 (8/10) peaks matched with the SMAD1 motif (Fig.\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003eS3\u003c/span\u003ea, b), confirming the specificity and accuracy of the CUT\u0026amp;RUN. Interestingly, pairwise comparison of pSmad1/5 and pSmad3 motifs revealed striking similarities (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, heatmap). Optimal alignment on paired pSmad1/5 and pSmad3 motifs with the highest similarity scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, middle) enabled the identification of overlapping motif sequences potentially bound by their cooperating TFs. Querying these sequences with the HOCOMOCO Mouse v11 full TF motif database\u003csup\u003e41\u003c/sup\u003e allowed us to infer 9 TFs that can recognize them (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), including TEAD4, a known Smad-cooperating TF that can form a complex with SMAD2/3 on mesenchymal enhancers upon TGFβ stimulation\u003csup\u003e42\u003c/sup\u003e. This observation suggests a possible mechanism by which the pSmad1/5-pSmad3 complex acts as transcriptional unit in dental mesenchymal cells, where TFs recognizing these overlapping motif sequences assemble the two R-Smads into a novel mixed complex for transcriptional regulation. Other inferred TFs include FOS, FOXJ3, FOXA3, NR5A2, SP1, BATF (B-ATF, basic leucine zipper ATF-like TF) and ASCL1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Many of these TFs are associated with neuron differentiation or axonogenesis\u003csup\u003e43\u003c/sup\u003e, implicating a potential role for atypical BMP signaling in tooth innervation. Interestingly, the overlapping motif sequence shared by pSmad1/5 motif-5 and pSmad3 motif-10 can be recognized by both SMAD1 and SMAD3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), suggesting physical interaction of pSmad1/5 and pSmad3 on genomic loci.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven the widespread expression of Smad4 in dental mesenchymal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), we then explored why pSmad1/5 preferentially forms a transcriptional regulatory unit with pSmad3, but not Smad4. Spaced motif analysis\u003csup\u003e44\u003c/sup\u003e on the pSmad1/5 bound DNA sequences revealed SMAD3 as the most favored cooperating TF (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), followed by TFs belonging to the AP2B cluster (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ec), which is essential for functional axonogenesis in hippocampal neurons\u003csup\u003e45\u003c/sup\u003e. Above all, our genome-wide mapping of pSmad1/5 and pSmad3 suggests that the similarity of motifs utilized by these two R-Smads enables the formation of a novel mixed R-Smads complex via synergistic effects of their shared TFs in dental mesenchymal cells. This mixed complex confers atypical BMP signaling output by converging the TGFβ and BMP signaling pathways at the intracellular effector level. We then set out to assess the transcriptional regulatory functions of atypical BMP signaling. Intersection of 5160 pSmad1/5 and 4952 pSmad3 target genes found 2777 co-target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Notably, both pSmad1/5 and pSmad3 also have their own specific target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), suggesting additional regulatory mechanisms. Pathway analysis of their co-target genes showed involvement in TGFβ and BMP signaling pathway transduction, as well as processes such as \u0026lsquo;Neuronal System\u0026rsquo; and \u0026lsquo;Axon guidance\u0026rsquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ed), consistent with previous studies on the roles of TGFβ and BMP signaling in neurogenesis\u003csup\u003e9,46\u0026ndash;48\u003c/sup\u003e and axon development\u003csup\u003e49,50\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAtypical BMP signaling regulates tooth innervation during early tooth development\u003c/h2\u003e \u003cp\u003eTooth development is regulated by epithelial-mesenchymal interactions and coordinated with innervation to form a functional sensory organ\u003csup\u003e6,51\u003c/sup\u003e. Previous studies have shown that target-derived BMP and TGFβ signaling can regulate peripheral innervation by limiting sensory neuron number\u003csup\u003e52\u003c/sup\u003e or guiding neurite outgrowth\u003csup\u003e47\u003c/sup\u003e. Importantly, BMP4 has been identified as a major target-derived factor that retrogradely controls differential gene expression in trigeminal ganglia through both Smad4-independent and Smad4-dependent pathways\u003csup\u003e9\u003c/sup\u003e. Our genome-wide binding profiles of pSmad1/5 and pSmad3 suggest the involvement of mixed R-Smads in axon development regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Based on these findings, we hypothesize that atypical BMP signaling regulates tooth innervation during early tooth development. To test it, we investigated axonogenesis in mouse embryonic heads and mandibles following the disruption of atypical BMP signaling using SIS3 (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003ea, b). As expected, disruption of the atypical BMP signaling in the head at E11.5 (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003ec-f), in the mandible at E12.5 (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eg, h) and E13.5 (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003ei-l) all resulted in reduced peripheral axon growth, including axons adjacent to the developing tooth germ (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003ej, l), in a stage-dependent manner. The effect on tooth innervation was more pronounced with earlier disruption of atypical BMP signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo understand how the atypical BMP signaling regulates tooth innervation, we conducted RNA-seq analysis on \u003cem\u003ein vitro\u003c/em\u003e cultured tooth germs after disrupting atypical BMP signaling with SIS3 at E12.5 for one day (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Intersecting the differentially expressed genes (DEGs) with pSmad1/5 and pSmad3 target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e) revealed 560 genes bound by both pSmad1/5 and pSmad3, while 444 (428\u0026thinsp;+\u0026thinsp;15\u0026thinsp;+\u0026thinsp;1) genes were co-bound (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). This result indicates that BMP and TGFβ signaling have both specific and converged regulatory functions on axon development, consistent with previous studies\u003csup\u003e47,52\u003c/sup\u003e, and suggests discrete regulatory roles for pSmad1/5 and pSmad3. Functional classification of the 444 genes co-bound by pSmad1/5 and pSmad3 into axonogenesis-associated GO terms (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003ec) revealed 27 axonogenesis-associated genes directly regulated by pSmad1/5 and pSmad3 co-binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). Among them, Nefl, a protein forming neurofilaments in axonal cytoplasm\u003csup\u003e53\u003c/sup\u003e, showed decreased expression after SIS3 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003ed), consistent with 2H3 immunofluorescence staining results (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Conversely, Wnt3, an early axonal guidance cue\u003csup\u003e54\u003c/sup\u003e, showed increased expression after SIS3 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). CUT\u0026amp;RUN-qPCR verified the direct regulation of these genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). Together, these results indicated that the pSmad1/5-pSmad3 complex-mediated atypical BMP signaling regulates tooth innervation (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003ef) by directly modulating the expression of axonogenesis-associated genes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAtypical BMP signaling is required for maintaining odontogenic inductive potential in dental mesenchyme\u003c/h3\u003e\n\u003cp\u003eTo investigate the biological significance of atypical BMP signaling in dental mesenchyme, we transplanted tooth germs with ectopically activated canonical BMP signaling into mouse kidney capsule to allow for further tooth development. All transplanted tooth germs developed normally after either SB431542 treatment (n\u0026thinsp;=\u0026thinsp;10/10) (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003ec, d) or DMSO treatment (n\u0026thinsp;=\u0026thinsp;10/10) (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003ea-b), consistent with the previous report that \u003cem\u003eSmad3\u003c/em\u003e null mouse exhibit normal early tooth development\u003csup\u003e55\u003c/sup\u003e. The shift in BMP4 expression pattern coincides with the shift of odontogenic inductive potential between dental epithelium and dental mesenchyme\u003csup\u003e3\u003c/sup\u003e. Our observations that both the dental epithelium at the initiation stage and the dental mesenchyme at the bud stage, where the odontogenic inductive potential resides, lack canonical BMP signaling, prompted us to investigate whether the atypical BMP signaling sustained by TGFβ in the dental mesenchyme is necessary for odontogenic inductive potential. To address this question, we inhibited TGFβ signaling using SB431542 at E12.5 or E13.5 for one day to disrupt the atypical BMP signaling in the dental mesenchyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). The SB431542-treated dental mesenchyme was subsequently recombined with E10.5 secondary pharyngeal arch epithelia. Continued SB431542 treatment on the recombinants of E12.5 dental mesenchyme and the secondary pharyngeal arch epithelia for one day prior to kidney capsule transplantation resulted in failed tooth formation (0/20, Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003ee). In contrast, control recombinants with E12.5 dental mesenchyme treated with DMSO for one day prior to transplantation formed teeth (10/20, Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). Interestingly, E13.5 dental mesenchyme retained odontogenic inductive potential, manifested by a high rate of tooth formation (8/10) in SB431542-treated recombinants compared to DMSO-treated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, c). These results indicated that restricted operation of atypical BMP signaling in dental mesenchymal cells is temporally required for maintaining odontogenic inductive potential in the dental mesenchyme within a short time window between E12.5 and E13.5. We then investigated which genes directly regulated by atypical BMP signaling are crucial for maintaining odontogenic inductive potential in dental mesenchyme. Using RNA-seq analysis on \u003cem\u003ein vitro\u003c/em\u003e cultured E12.5 tooth germs after disrupting atypical BMP signaling with SB431542 for one day (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003ea), we identified DEGs between control tooth germs at E12.5 and E13.5 and intersected them with DEGs resulting from atypical BMP signaling disruption (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eb). Given that pSmad1/5 can function independently of both Smad4 and pSmad3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), we further intersected the overlapped DEGs with pSmad1/5 target genes, identifying 611 genes directly regulated by atypical BMP signaling after SB431542 treatment (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eb). Hierarchical clustering of enriched terms for these 611 genes demonstrated high association with mesenchymal development and the WNT signaling pathway (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003ec), which plays an essential role in tooth initiation\u003csup\u003e56\u003c/sup\u003e. We classified these genes into mesenchyme development and odontogenesis associated GO terms, identifying 41 potentially important genes for maintaining odontogenic inductive potential in dental mesenchyme (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003ed). Interestingly, atypical BMP signaling regulates these genes in three different ways: pSmad1/5 binding alone (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003ee); pSmad1/5 binding with pSmad3, but not at the same position (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003ef); and pSmad1/5 co-binding with pSmad3 at the same genomic loci (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eg). This observation enhances our understanding of the diversity of TGFβ/BMP signaling through Smads. Together, our results suggest that TGFβ-sustained atypical BMP-Smad signaling confers diverse functions to ensure proper tooth innervation during early tooth development, thereby maintaining odontogenic inductive potential in dental mesenchyme.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBuilding on our previous studies\u003csup\u003e26,27\u003c/sup\u003e, this investigation provide genome-wide evidence supporting the functional operation of an atypical canonical BMP signaling pathway in the dental mesenchyme. This pathway confers compartmentalized distributions of distinct BMP-Smad signaling outputs during early tooth development. While canonical BMP signaling output is restricted to neurovascular cells, the atypical canonical BMP signaling output in dental mesenchymal cells represents a distinct output. We demonstrate that this atypical pathway in dental mesenchymal cells operates through the formation of a novel mixed pSmad1/5-pSmad3 complex. This complex functions as a transcriptional regulatory unit, ensuring proper tooth innervation and maintaining odontogenic inductive potential in the dental mesenchyme.\u003c/p\u003e \u003cp\u003eResearch over recent decades has revealed the repeated use of a relatively small number of signaling pathways driving various developmental decisions during early tooth development\u003csup\u003e57\u003c/sup\u003e. The TGFβ/BMP signaling pathway is such conserved pathway employed throughout this process. To achieve biological efficiency, different cells must respond differently to the same signal received multiple times. Understanding the genetic regulation network and functional distinctions of diverse signaling outputs in early tooth development is therefore of great interest.\u003c/p\u003e \u003cp\u003eThe classic view of TGFβ/BMP signaling pathways holds that upon activation, phosphorylated R-Smads form an obligate heterotrimer with Smad4 to regulate target gene transcription\u003csup\u003e18\u003c/sup\u003e. However, this dogma has been challenged by studies observing divergent phenotypes in \u003cem\u003eSmad1/5\u003c/em\u003e\u003csup\u003eCKO\u003c/sup\u003e mice when compared to \u003cem\u003eSmad4\u003c/em\u003e\u003csup\u003eCKO\u003c/sup\u003e mice\u003csup\u003e23\u0026ndash;25\u003c/sup\u003e, suggesting R-Smads can transduce BMP signaling independently of Smad4. Our previous study demonstrated that BMP4-induced R-Smads (pSmad1/5/8) can regulate \u003cem\u003eMsx1\u003c/em\u003e expression in the dental mesenchyme of both humans\u003csup\u003e26\u003c/sup\u003e and mice\u003csup\u003e27\u003c/sup\u003e independent of Smad4. In this study, ChIP-seq analysis of pSmad1/5 and Smad4 in molar tooth germs revealed that pSmad1/5 and Smad4 exert distinct functions during early tooth development except shared roles in signal transduction and neurogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei-k). Consistently, real-time monitoring of canonical BMP-Smad transcriptional activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) using transgenic mouse line \u003cem\u003eBRE-GFP\u003c/em\u003e\u003csup\u003e29\u003c/sup\u003e revealed compartmentalized distributions of distinct BMP-Smad signaling outputs during early tooth development. GFP presence, indicating canonical BMP signaling, was restricted to neurovascular cells, while its absence in dental mesenchymal cells represented atypical canonical BMP signaling. This compartmentalized and diverse BMP-Smad signaling output resembles phenomena observed in endothelial networks\u003csup\u003e58\u003c/sup\u003e. This atypical BMP signaling in dental mesenchymal cells operates through the formation of a novel mixed pSmad1/5-pSmad3 complex, as evidenced by pharmacological inhibition and Co-IP experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), consistent with previous \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e32\u003c/sup\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies\u003csup\u003e59\u003c/sup\u003e. Genome-wide mapping of pSmad1/5 and pSmad3 targets using CUT\u0026amp;RUN assay revealed similar genomic distribution patterns and biological processes for their target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This suggests the complex acts as a genome-wide transcriptional regulatory unit during early tooth development. Motif analyses of pSmad1/5 and pSmad3 binding peaks showed high similarity between their motifs, further supporting this notion (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Using pairwise motif alignment, we identified 9 shared TFs as potential regulatory partners of the pSmad1/5-pSmad3 complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), many of which are known regulators of neurogenesis\u003csup\u003e43,60\u0026ndash;63\u003c/sup\u003e. Functional classification of pSmad1/5 and pSmad3 co-target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d) and examination of neurite outgrowth after disrupting atypical BMP signaling (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e) verified their role in regulating tooth innervation. These results align with previous studies demonstrating that target-derived BMP4\u003csup\u003e52\u003c/sup\u003e and TGFβ signaling\u003csup\u003e47\u003c/sup\u003e can regulate the extent of peripheral innervation \u003cem\u003ein vivo\u003c/em\u003e. BMP4 has been identified as a major target-derived factor that retrogradely regulates gene expression in developing trigeminal sensory neurons through both Smad4-independent and Smad4-dependent pathways\u003csup\u003e9\u003c/sup\u003e. Notably, peripheral sensory innervation remains normal after conditional deletion of \u003cem\u003eSmad4\u003c/em\u003e with \u003cem\u003eAdvillin\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre/+\u003c/em\u003e\u003c/sup\u003e\u003csub\u003e,\u003c/sub\u003e a sensory neuron-specific Cre mouse line\u003csup\u003e64\u003c/sup\u003e. Collectively, these previous findings and our studies suggest that TGFβ and BMP signaling converge on a novel pSmad1/5-pSmad3 complex to regulate tooth innervation independently of Smad4. The formation of mixed R-Smads has also been observed in TGFβ signaling in various epithelial cells and fibroblasts\u003csup\u003e32,59,65\u003c/sup\u003e. The prevalence of mixed pSmad1/5-pSmad3 complex in dental mesenchymal cells, where Smad4 is widely expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), raises an important question: why does pSmad1/5 prefer to form complexes with pSmad3 instead of Smad4? Two possible mechanisms for this preferential formation can be envisioned from our results. First, TGFβ signaling leads to Smad4 saturation through the formation of pSmad2/3-Smad4 complexes; Second, pSmad3 or other unidentified TFs compete with Smad4 to form novel complexes comprising pSmad1/5 but not Smad4. Given Smad4\u0026rsquo;s ubiquitous expression in dental mesenchyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and a previous study showing that \u003cem\u003eSmad4\u003c/em\u003e overexpression did not affect TGFβ\u0026rsquo;s ability to inhibit BMP7-induced transcription\u003csup\u003e32\u003c/sup\u003e, our results suggest that pSmad3 or candidate partner TFs competes with Smad4 to form novel complexes. This competition mechanism has been previously described as a means of mutual antagonism between Nodal and BMP signaling\u003csup\u003e66\u003c/sup\u003e. Several observations support this competition mechanism: First, pSmad3 is absent in \u003cem\u003eBRE-GFP\u003c/em\u003e positive neurovascular cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e); Second, inhibition of pSmad3 expression results in ectopic \u003cem\u003eBRE-GFP\u003c/em\u003e expression in dental mesenchymal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e); Third, spaced motif analysis shows that SMAD3 is the most favored TF to form complexes with pSmad1/5 STREME-1 motif (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Recent research has shown that weak TF-TF contacts guided by DNA mediate the selectivity of cooperating partners\u003csup\u003e67\u003c/sup\u003e. We propose that the preferential formation of pSmad1/5-pSmad3 complexes in dental mesenchymal cells occurs through a similar mechanism\u003csup\u003e67\u003c/sup\u003e, where pSmad1/5-bound DNA guides the selection of pSmad3 over Smad4.\u003c/p\u003e \u003cp\u003eWe further demonstrated the biological significance of TGFβ-sustained atypical BMP signaling through tissue recombination experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These experiments revealed that atypical BMP signaling is crucial for maintaining odontogenic inductive potential in dental mesenchyme within a short time window between E12.5 and E13.5. To dissect how atypical BMP signaling regulates target genes for maintaining this potential, we intersected DEGs from RNA-seq after pSmad3 inhibition using SIS3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003e) with the pSmad1/5 and pSmad3 co-bound genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This analysis demonstrated that TGFβ-sustained atypical BMP signaling regulates tooth innervation (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e) through direct control of axonogenesis-associated gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003ec-f). Given that identifying \u0026lsquo;odontogenic inducer\u0026rsquo; is a major challenge for \u003cem\u003ein vitro\u003c/em\u003e production of implantable bioengineering tooth germs\u003csup\u003e68\u003c/sup\u003e, we sought to identify genes potentially important for maintaining odontogenic inductive potential in dental mesenchyme (Fig.\u0026nbsp;9). Integrative analyses of DEGs between E13.5 and E12.5 stages, DEGs after disrupting atypical BMP signaling with SB431542, and pSmad1/5 bound genes identified 611 genes essential for mesenchymal development (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eb). Further functional classification revealed 41 genes important for mesenchyme development and odontogenesis (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003ec, d). A deeper analysis uncovered three distinct mechanisms by which TGFβ-sustained atypical BMP signaling regulates gene expression: pSmad1/5 co-binding with pSmad3 on the same genomic loci (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eg); pSmad1/5 and pSmad3 both binding but on different genomic positions (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003ef), and pSmad1/5 binding alone (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003ee), possibly with unidentified Smad-cooperating TFs warranting future investigation. Notably, \u003cem\u003eMsx1\u003c/em\u003e, a readout gene of atypical BMP signaling\u003csup\u003e27\u003c/sup\u003e, is regulated by pSmad1/5 alone (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003ee). Given that \u003cem\u003eMsx1\u003c/em\u003e is also expressed in endothelial cells at artery branching sites\u003csup\u003e69\u003c/sup\u003e, and canonical BMP signaling operates in vascular cells in the dental mesenchyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), this suggests multiple regulatory mechanisms for \u003cem\u003eMsx1\u003c/em\u003e expression. Collectively, our results suggest that the TGFβ-sustained atypical BMP-Smad pathway generates diverse signaling outputs to ensure proper tooth innervation and maintain odontogenic inductive potential in dental mesenchyme. This diversity may explain how mutations in components of the same pathway can cause different phenotypic disorders. While studies on tooth development typically focus on epithelial-mesenchymal interactions, our investigation of intra-mesenchymal interactions between axonal nerves and dental mesenchymal cells expands our understanding of early tooth development. This study provides valuable insights into the functions of peripheral nerves in odontogenesis, potentially informing strategies for producing implantable bioengineered tooth germs \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eThe generation and genotyping protocols of \u003cem\u003eBRE-LacZ\u003c/em\u003e and \u003cem\u003eBRE-GFP\u003c/em\u003e mice have been described previously\u003csup\u003e28,29\u003c/sup\u003e. Both mouse lines were maintained on CD1 background. All animal experiments in this study were approved by the Institutional Animal Care and Use Committee, Tulane University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOrgan culture, inhibitor treatment and tissue recombination\u003c/h2\u003e \u003cp\u003eHeads and mandibles from \u003cem\u003eBRE-LacZ\u003c/em\u003e mice at embryonic stage 13.5 (E13.5) were dissected and set for roller cultured as previously described\u003csup\u003e70\u003c/sup\u003e prior to SIS3 treatment. The molar tooth germs dissected from \u003cem\u003eBRE-GFP\u003c/em\u003e mouse embryos at indicated stages were first cultured \u003cem\u003ein vitro\u003c/em\u003e in the DMEM supplemented with 10% FBS (Cytiva, SH30070.02) and GlutaMAXTM-1(Gibco, 35050-061), treated with the indicated inhibitors, including SB431542 (Sigma-Aldrich 616461, 50\u0026micro;M) and SIS3 (Tocris #5291, 300 \u0026micro;M), and then were subjected to the subsequent experiments. For tissue recombination experiments, the dental mesenchyme from indicated stages after treatment with SB431542 for 1 or 2 days were separated from their original dental epithelia and recombined with the E10.5 secondary pharyngeal arch epithelia, which were then further cultured with DMSO or SB431542 for 1 day prior to kidney capsule transplantation in mice. All surgical procedures were performed under anesthesia that administered by intra-peritoneal injection. After one month, the host mice were sacrificed for harvest of grafted recombinants, followed with morphogenetic and histological analyses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eX-gal staining, immunofluorescence, RNAscope\u003c/b\u003e \u003cb\u003ein situ\u003c/b\u003e \u003cb\u003ehybridization, and co-immunoprecipitation (Co-IP)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eStandard X-gal staining was conducted on the E13.5 embryonic limb and mandible from \u003cem\u003eBRE-LacZ\u003c/em\u003e mice as previously described\u003csup\u003e28\u003c/sup\u003e. Molar tooth germs harvested from embryonic \u003cem\u003eBRE-GFP\u003c/em\u003e mice at indicated stages, or \u003cem\u003ein vitro\u003c/em\u003e cultured tooth germs were fixed in 4% paraformaldehyde (PFA) at 4\u0026deg;C overnight, paraffin-embedded, and sectioned at 5\u0026micro;m for immunofluorescence staining. Sections were incubated with the primary antibody and the secondary antibodies conjugated to fluorophores (Life Technologies), and then counterstained with DAPI to reveal nuclei. Images were collected through Nikon Eclipse Ti2 Confocal Microscope. The primary antibodies used in this study included pSmad1/5 (Cell signaling technology, #9516), pSmad2 (Abcam, ab188334), pSmad3 (Abcam, ab52903), Smad4 (cell signaling technology, #46535), GFP (Novus Biologicals, NB100-1614) and CD133 (Abcam, Ab19898). RNAscope in situ hybridization was performed with the RNAscope 2.5 HD Reagent Kit Brown (Advanced Cell Diagnostics, 322300) following the manufacturer\u0026rsquo;s instructions. The signal was detected by Alexa Fluor\u0026trade; 596 Tyramide SuperBoost\u0026trade; Kits (ThermoFisher, USA). Probe details are listed in Supplementary Table\u0026nbsp;1. Traditional \u003cem\u003ein situ\u003c/em\u003e hybridization of \u003cem\u003eBmp4\u003c/em\u003e was conducted as previously described\u003csup\u003e13\u003c/sup\u003e. Co-IP assays were performed on the freshly dissected tooth germs at E13.5 using Pierce\u0026trade; Co-Immunoprecipitation Kit (Thermo Scientific, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation sequencing (ChIP-seq)\u003c/h2\u003e \u003cp\u003eChIP-seq analyses of pSmad1/5 and Smad4 were performed as previously described\u003csup\u003e71\u003c/sup\u003e on the tooth germs from E13.5 mouse embryos using Active Motif HS ChIP Kit, with the same stage embryonic limb tissues as biological controls. ChIPed DNAs were subjected to ChIP-seq.\u0026nbsp;Library preparation and sequencing were performed at Active Motif (Carlsbad, CA) and BGI (Hong Kong, China). The 50 bp single-end sequencing reads were then aligned to the mouse reference genome mm10 using bowtie2\u003csup\u003e72\u003c/sup\u003e. The resulting SAM files were converted into BAM files with samtools\u003csup\u003e73\u003c/sup\u003e, which were then subjected to the BCP\u003csup\u003e74\u003c/sup\u003e for peak calling.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCleavage Under Targets \u0026amp; Release Using Nuclease (CUT\u0026amp;RUN) and quantitative PCR (qPCR)\u003c/h2\u003e \u003cp\u003eCUT\u0026amp;RUN experiments on E13.5 tooth germs were carried out as described by manufacturer\u0026rsquo;s manual (Cell Signaling Technology, #86652). Briefly, E13.5 tooth germs were harvested and bound to concanavalin A-coated magnetic beads. Digitonin was then used to permeabilize the cell membranes, allowing the phosphorylated Smad1/5 (Cell Signaling Technology, #9516) and Smad3 (Cell Signaling Technology, #9520) antibody to enter cells and find their targets. After that, the beads were briefly washed, and then incubated with pA-MNase enzyme. When the bead-bound cells were chilled to 0℃, CaCl\u003csub\u003e2\u003c/sub\u003e was added to start the digestion for 30 min. After stopping the reactions by chelation, the DNA fragments released into solution by cleavage were then extracted for qPCR or library construction. The qPCR primers are listed in Supplementary Table\u0026nbsp;1. To construct the CUT\u0026amp;RUN DNA library, we adopted the protocol of NEBNext Ultra II DNA Library Prep Kit, NEB (USA). The constructed DNA libraries were sequenced on the Illumina NextSeq 500 platform using NextSeq 500/550 High Output Kit v2 in a 100 bp paired-end mode. The CUT\u0026amp;RUN data were then processed by following the pipeline as previously described\u003csup\u003e36\u003c/sup\u003e with custom modified scripts. Briefly, paired-end sequencing reads were aligned to the mouse reference genome mm10 using bowtie2\u003csup\u003e72\u003c/sup\u003e. The resulting BAM alignment files were then subjected to samtools\u003csup\u003e73\u003c/sup\u003e for filtering low-quality reads, reads in the ENCODE blacklist regions, unmapped mates, and PCR duplicates. The filtered BAM files were converted into bedGraph files using the bamtobed tool from bedtools\u003csup\u003e37\u003c/sup\u003e. These bedGraph files were then used as input for SEACR\u003csup\u003e75\u003c/sup\u003e to call peaks with the -non - stringent setting. pSmad1/5 and pSmad3 peaks from two replicates were intersected using bedtools\u003csup\u003e37\u003c/sup\u003e to get their common peaks. Motif discovery analysis was performed on \u0026plusmn;\u0026thinsp;100bp sequences from the pSmad1/5 and pSmad3 common peak summits with MEME Suite (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme-suite.org\u003c/span\u003e\u003cspan address=\"http://meme-suite.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e76\u003c/sup\u003e. Motif comparison, motif alignment and pairwise motif similarity scoring were performed using TomTom\u003csup\u003e77\u003c/sup\u003e from MEME Suite\u003csup\u003e76\u003c/sup\u003e. The pairwise motif similarity scores were visualized using custom R script.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRNA preparation and RNA sequencing (RNA-seq)\u003c/h2\u003e \u003cp\u003eE12.5 tooth germs after treatment with SIS3 or SB431542 for 1 day, and E13.5 control tooth germs were collected for RNA-seq assays. DMSO treated tooth germs were used as controls. Total RNAs were extracted using RNeasy Micro Plus Kit (Qiagen, USA), and quantified using a Qubit 2.0 Fluorometric Quantitation system (Life Technologies, USA). Library preparation was performed as previously described\u003csup\u003e78\u003c/sup\u003e. The prepared libraries were then pooled and sequenced on the Illumina HiSeq4000 platform using 75bp pair-end reads mode. To avoid batch effect, all RNA-seq experiments were performed in duplicate. The RNA-seq sequencing reads were mapped to the mm10 transcriptome using Salmon quant for quantification of gene expression at transcript level\u003csup\u003e79\u003c/sup\u003e. Salmon quantification files were then imported into R with the tximport package\u003csup\u003e80\u003c/sup\u003e to summarize gene-level expression. Genes with fewer than 10 counts across all samples were excluded from the downstream analysis. Differentially expressed gene (DEGs) were identified using DESeq2\u003csup\u003e81\u003c/sup\u003e, with genes having an adjusted p-value (padj)\u0026thinsp;\u0026lt;\u0026thinsp;0.01 considered as significant DEGs. These significant DEGs were subsequently imported into R for intersection with CUT\u0026amp;RUN peak target genes and functional classification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePeak annotation, Functional classification and Data visualization\u003c/h2\u003e \u003cp\u003eChIP-seq peaks and the common peaks from CUT\u0026amp;RUN replicates were annotated using ChIPseeker R package\u003csup\u003e82\u003c/sup\u003e. The annotated peak target genes were then imported into the clusterProfiler R package\u003csup\u003e83\u003c/sup\u003e for gene ontology (GO) and pathway enrichment analysis. Enriched GO terms and pathways were then visualized by the implemented functions of clusterProfiler, excluding general and redundant terms. To characterize the read distributions of ChIP-seq and CUT\u0026amp;RUN sequencing, filtered BAM files were used as input for the deepTools\u003csup\u003e84\u003c/sup\u003e. Heatmaps of signals centered on transcription start sites (TSS) were generated using the plotHeatmap subcommand. For UCSC genome browser visualization of RNA-seq reads, sequencing reads were aligned to the mouse reference genome (mm10) using Hisat2\u003csup\u003e85\u003c/sup\u003e with default parameters. BAM files from two replicates in each group were merged with samtools\u003csup\u003e73\u003c/sup\u003e and then converted into bigwig format using the bamCoverage subcommand from deepTools\u003csup\u003e84\u003c/sup\u003e. For UCSC genome browser visualization of CUT\u0026amp;RUN sequencing reads, redundant reads in the samtools-merged BAM files from two replicates were first filtered using the intersect function of bedtools\u003csup\u003e37\u003c/sup\u003e and then converted into bigwig format.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll of the RNA-Seq, ChIP-Seq and CUT\u0026amp;RUN datasets were deposited into the GEO with SuperSeries accession number xxxxx.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Institutes of Health grant R01DE024152 to Y.C.. H.L. was supported in part by a fellowship from Fujian Normal University and by a grant (2021J01681) from the Natural Science Foundation of Fujian Province, P.R. China. \u0026nbsp;L.W. received a fellowship from the China Scholarship Council during the initial phase of the studies.\u003c/p\u003e\n\u003cp\u003eWe thank Kejing Song and Genevieve Pierre at the Tulane Center for Translational Research in Infection and Inflammation’s NextGen Sequencing Core for assistance with initial bioinformatic analyses. High performance computing (HPC) resources provided by Tulane University Technology Services also contributed to this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest regarding the research, authorship, and publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQ.T., L.L., and Y.C. conceived the project. Q.T. and L.L. performed most experiments, collected and analyzed data, and prepared figures. Q.T. and L.L. also drafted the initial manuscript. \u0026nbsp;Y.L., A.W., L.W. and C.G. assisted with genotyping, histology, immunohistochemistry, Co-IP, CUT\u0026amp;RUN-qPCR, and tissue recombination experiments. Q.T., with support from Z.W., conducted all bioinformatic analyses. H.L., J.L., Z.W. and H.K. provided valuable insights and contributed to manuscript preparation and editing. Y.C. performed the final revision and editing of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLuukko, K. \u0026amp; Kettunen, P. Coordination of tooth morphogenesis and neuronal development through tissue interactions: lessons from mouse models. \u003cem\u003eExp Cell Res\u003c/em\u003e \u003cstrong\u003e325\u003c/strong\u003e, 72-77 (2014). https://doi.org/10.1016/j.yexcr.2014.02.029\u003c/li\u003e\n\u003cli\u003eThesleff, I. 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Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. \u003cem\u003eNat Biotechnol\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 907-915 (2019). https://doi.org/10.1038/s41587-019-0201-4\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Atypical BMP-Smad signaling, Dental mesenchyme, Tooth innervation, Odontogenic inductive potential","lastPublishedDoi":"10.21203/rs.3.rs-5188541/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5188541/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe canonical bone morphogenetic protein (BMP) signaling pathway plays a crucial regulatory role in tooth development by activating Smad proteins to regulate gene expression. Our previous research identified an atypical canonical BMP signaling in dental mesenchyme that is Smad4-independent but Smad1/5-dependent. This study demonstrates that phosphorylated Smad1/5 (pSmad1/5) and Smad4 transcriptionally regulate distinct gene sets in dental mesenchyme. Real-time monitoring of BMP-Smad transcriptional activity revealed that Smad4-dependent canonical BMP signaling is restricted to neurovascular cells surrounding the condensed dental mesenchymal cells where pSmad1/5 is present. Notably, we found that pSmad1/5 in dental mesenchymal cells form complexes with pSmad3 to prevent canonical BMP signaling. CUT\u0026amp;RUN assays revealed genome-wide co-occupancy of pSmad1/5 and pSmad3, indicating that pSmad1/5-pSmad3 complexes function as transcriptional regulation units. Integrative analyses of their transcriptional targets with RNA-seq demonstrated that the atypical canonical BMP signaling regulates tooth sensory innervation and is temporally required for maintaining odontogenic inductive potential in the dental mesenchyme. This enabled the identification of potentially critical genes for maintaining tooth inductive capability. Our findings elucidate the operating mechanism of atypical canonical BMP signaling in dental mesenchymal cells and clarify how BMP-Smad signaling exerts diverse functions across different cell types, shedding light on future tooth bioengineering strategies.\u003c/p\u003e","manuscriptTitle":"Distinct BMP-Smad Signaling Outputs Confer Diverse Functions in Dental Mesenchyme","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-05 07:35:45","doi":"10.21203/rs.3.rs-5188541/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5309df40-0cbb-4eef-818a-a4c05dfc99d1","owner":[],"postedDate":"November 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":39737377,"name":"Biological sciences/Developmental biology/Stem-cell niche"},{"id":39737378,"name":"Biological sciences/Cell biology/Cell signalling/Growth factor signalling"},{"id":39737379,"name":"Biological sciences/Molecular biology/Transcription/Transcriptional regulatory elements"}],"tags":[],"updatedAt":"2024-11-05T07:35:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-05 07:35:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5188541","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5188541","identity":"rs-5188541","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}