Villification of the intestinal epithelium is driven by Foxl1

Villification of the intestinal epithelium is driven by Foxl1 | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Article Villification of the intestinal epithelium is driven by Foxl1 Klaus Kaestner, Guoli Zhu, Deeksha Lahori, Jonathan Schug This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4882679/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Feb, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The primitive gut tube of mammals initially forms as a simple cylinder consisting of the endoderm-derived, pseudostratified epithelium and the mesoderm-derived surrounding mesenchyme. During mid-gestation a dramatic transformation occurs in which the epithelium is both restructured into its final cuboidal form and simultaneously folded and refolded to create intestinal villi and intervillus regions, the incipient crypts. Here we show that the mesenchymal winged helix transcription factor Foxl1, itself induced by epithelial hedgehog signaling, controls villification by activating BMP and PDGFRa as well as planar cell polarity genes in epithelial-adjacent telocyte progenitors, both directly and in a feed- forward loop with Foxo3. In the absence of Foxl1-dependent mesenchymal signaling, villus formation is delayed, the separation of epithelial cells into mitotic intervillus and postmitotic villus cells impaired, and the differentiation of secretory progenitors blocked. Thus, Foxl1 orchestrates key events during the epithelial transition of the fetal mammalian gut. Biological sciences/Developmental biology/Stem-cell niche Biological sciences/Developmental biology/Organogenesis Villus formation epithelial-mesenchymal cross talk telocyte transcription factor networks Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The surface area of the human small intestine measures about 30 m 2 , or the size of half a badminton court 1 . This large area is required to enable effective digestion and absorption of nutrients. The major factor responsible for increasing the size of the intestinal epithelium is villification, or the formation of intestinal villi that project into the gut lumen, which increase the surface area compared to a flat epithelium by close to 100-fold 1 . Remarkably, while villi are a feature of the small intestine in mammals and birds, they arise by divergent morphogenetic mechanisms. Thus, villus formation in birds is dependent on the sequential formation of first inner circular, then longitudinal, and finally muscularis mucosa muscles which coincide with the appearance of epithelial ridges, zigzags, and villi, respectively 2 . In contrast, in mammals, villus formation can proceed in the absence of tensile forces generated by intestinal muscles; rather, it is dependent on PDGFRa/BMP positive mesenchymal cell clusters 3 , which themselves are induced by epithelial hedgehog signaling 4 . At the onset of villification, also termed ‘epithelial transition’, the endoderm-derived epithelium forms a simple tube of pseudostratified cells with a layer thickness of ~ 50 µm. This pseudostratified epithelium, characterized by cell cycle-dependent interkinetic nuclear migration 5 , is then changed to a columnar epithelium coincident with villus formation. BMP signals from villus cluster mesenchymal cells restrict proliferation in the overlying epithelium so that cycling cells become restricted to intervillus regions, the precursors of the small intestinal proliferative lcrypts present in the adult. The critical role of BMP signaling in villus cluster formation was established by explant cultures of presumptive small intestine from 13.5 dpc (days post conception) embryos treated with localized sources of BMP or the pan-BMP inhibitor dorsomorphin 6 . The induction of villus formation and the formation of villus clusters in the underlying mesenchyme are dependent on epithelial to mesenchymal hedgehog signaling. Thus, both Sonic Hedgehog (shh) and Indian Hedgehog (Ihh) are expressed in the epithelium, while the hedgehog receptors (Ptch1 and Ptch2) as well as the downstream transcription factor Gli1 are expressed in the mesenchyme 7 , 8 . Because the two epithelial hedgehog proteins are partially redundant, Madison and colleagues addressed the role of hedgehog signaling in the developing intestine using epithelial expression of the pan-hedgehog inhibitor Hhip 9 . Suppression of hedgehog signaling impaired villus formation, which was partially due to decreased BMP expression in the underlying mesenchyme. Previously, the winged helix transcription factor Foxl1 (formerly termed Fhh6 ) was shown to be expressed in the first one to two cell layers of the mesenchyme juxtaposed to the epithelium before villus formation by mRNA in situ hybridization 10 , which was recently confirmed by immunofluorescence staining 11 , making it an excellent candidate as regulator of key developmental processes. Mice null for Foxl1 exhibit delayed villus formation and a defect in the inhibition of epithelial proliferation in nascent villi, suggesting Foxl1 as an important transcription factor controlling mesenchymal to epithelial signaling 10 . A link to epithelial hedgehog signaling was subsequently established by the identification of functionally relevant binding sites for the hedgehog-dependent Gli transcription factors within an evolutionarily ultra-conserved enhancer at the Foxl1 locus 12 . Foxl1 expression was induced in fetal gut mesenchyme explants treated with Shh, while its mRNA levels were reduced in mice deficient for the hedgehog dependent transcription factors Gli2 and Gli3 . Taken together, these findings suggested that Foxl1 is a critical mediator of epithelial to mesenchymal cross-talk during villus formation. Here, we set out to determine the molecular targets and pathways controlled by Foxl1 during intestinal villification. Results Histological analysis of the small intestine shows that as villus formation is well on its way in wild type fetuses at 15.5 dpc, while this process has barely been initiated in Foxl1 deficient mice (Fig. 1 A,B). Even two days later, at 17.5 dpc, villification is abnormal in mutant mice, with apparent bridging of nascent villi across the gut lumen, likely reflecting persistent epithelial ridges (Fig. 1 C, D), a phenotype that persists at 18.5 dpc (Fig. 1 E,F). In order to investigate this phenotype in three dimensions, we performed scanning electron microscopy. As shown in Fig. 1 G-L, in control mice villification occurs through the formation of regularly spaced short invaginations into the gut lumen, which by 18.5 dpc have progressed to form elongated villi. This process is dramatically altered by loss of Foxl1, with absence of the regularly spaced nascent villi and presence instead of long epithelial ridges at 15.5 dpc (yellow arrow in Fig. 1 H), which persist until late gestation (Fig. 1 J, red arrow). The presence of epithelial ridges instead of villi is reminiscent of the phenotype seen in small intestinal explants treated with the pan-BMP inhibitor dorsomorphin 6 , a connection we explore further below. Fetal gut telocytes progenitors exist in two subpopulations The inclusion of tdTomato in the recently developed Foxl1 mutant allele employed here 13 enabled us to follow the fate and determine the molecular properties of Foxl1-positive cells embryos heterozygous and homozygous for this Foxl1 null allele. Of note, the Foxl1 phenotype is recessive, as Foxl1 heterozygous mice are indistinguishable from wild type controls 10 , 14 – 16 . We performed scRNAseq on the proximal half of the small intestine of 15.5 dpc FoxL1 CreER-tdTom/+ fetuses (Fig. 2 A,B). As shown in the UMAP analysis in Fig. 2 C and D, Foxl1 + cells segregate into two closely related cell clusters, which we termed ‘telocyte progenitors 1 and 2’. A list of marker genes for all cell populations shown in Fig. 2 C is given in Supplementary Table 1. Telocyte progenitors 1 express high levels of Pdgfra and multiple Bmp mRNAs (Fig. 1 E), and thus most likely correspond to ‘villus cluster cells’, i.e the PDGFRa positive cells important in villus formation originally identified by Karlsson and colleagues 3 . By exclusion, the telocyte progenitor 2 population likely represents Foxl1 + cells directly adjacent to the intervillus epithelium, i.e. the presumptive future crypt cells. Interestingly, we identified Glp2r , encoding the GLP2 receptor, as another gene predominantly expressed in telocyte progenitor 1 cells, and confirm the localization of the Glp2r mRNA to villus cluster cells by RNAscope analysis (Fig. 2 F,G). A further telocyte progenitor 1 marker is the Zinc-finger transcription factor Spalt like transcription factor 1 ( Sall1 ), which is part of the NuRD transcriptional repressor complex (Fig. 2 H,I). Sall1/Foxl1 double positive cells can be seen to also mark a cluster of cells that appears to be in the process of initiating a new villus (Fig. 2 I). Importantly, this single cell analysis clearly shows that Foxl1 + cells are distinct from myofibroblast, interstitial cells of Cajal, and pericytes, identified by the expression of Acta2 med /Myh11 med /Des med /Tagln med , Etv1 + /Kit + /Acta low , and Cspg4 + /Pdgfrb + /Abcc9 + or Abcc9 + /Ndufa4l2 + , respectively (Fig. 2 C). Major hallmarks of the telocyte progenitor 1 and 2 populations are summarized in the graphic shown in Fig. 2 J. Expression of PDGFRa in telocyte progenitors is dependent on Foxl1 Next, we analyzed the expression profile of the fetal small intestine of Foxl1 null ( FoxL1 CreER-tdTom/CreER-tdTom ) mice and compared them to those of heterozygous fetuses. Note that in both control and Foxl1 null fetuses, Foxl1-expressing cells are localized to the mesodermal cell layer that is directly juxtaposed to the developing epithelium, and loss of Foxl1 does not affect telocyte progenitor number (Fig. 3 A). The UMAP plot shown in Fig. 3 B clearly demonstrates that the telocyte progenitor 1 population is dramatically reduced in abundance in the Foxl1 null intestine. As mentioned above, among the markers of the telocyte progenitor 1 cells is PDGFRa. The scRNAseq data shown in Fig. 3 C as well as the immunofluorescence staining presented in Fig. 3 D demonstrate that PDGFRa expression is Foxl1 dependent. To determine if Pdgfra is a direct target of Foxl1, we performed Cut-and-Run assays on telocytes from 15.5 dpc fetal gut. However, we found no Foxl1 binding event within 10 kb of the Pdgfra promoter (data not shown). It was previously reported that the winged helix transcription factor Foxo3 is a transcriptional activator of Pdgfra , which binds to an evolutionarily conserved cis -regulatory element in its proximal promoter 17 . Therefore, we hypothesized that Foxl1 might indirectly control Pdgfra via activation of Foxo3 . Using Cut-and-Run of sorted fetal telocytes, we indeed found several Foxl1 binding sites in the Foxo3 promoter (Fig. 3 E). In addition, our scRNAseq data show a reduction of Foxo3 transcript levels in the telocyte progenitor 1 population of Foxl1 null fetuses (Fig. 3 F). These data suggest indirect regulation of the Pdgfra gene by Foxl1 through a transcription factor cascade via Foxo3. Foxl1 controls multiple Bmp genes in telocyte progenitor cells Next, we focused on BMP proteins, as BMP signaling from the mesoderm to the epithelium is critical for villus cluster formation 6 . Telocyte-produced BMPs enriched in the villus cluster cells signal to the overlaying endoderm to inhibit Wnt signaling and limit proliferation (Fig. 4 A). High expression of several BMP genes was present in particular in villus custer telocyte-progenitor 1 cells (Fig. 4 B). In case of BMP4, expression also extends to Foxl1 negative GPX3 + FLCs; however, its levels are clearly reduced specifically in Foxl1-deficient telocytes (Fig. 4 B). To determine if BMP signaling to the epithelium is impaired by this reduction in telocyte BMP expression, we performed immunostaining for phosphorylated SMAD1/5 (Fig. 4 C). Nuclear pSMAD1/5 is clearly detectable in epithelial cells in the villus tip but not intervillus regions in control embryos. In contrast, epithelial cells in Foxl1 null mice are devoid of signal, confirming loss of active BMP signaling to the epithelium. When analyzing the pSMAD1/5 staining, we also noticed signal in nuclei of mesenchymal cells within the invaginating villi, with most of them negative for the Foxl1-tdTomato signal. These findings suggest unexpected bi-directional signaling of villus tip telocytes to both epithelium and neighboring mesenchymal cells. At present, the significance of this observation is unknown; however, the pSMAD1/5 signal in mesenchymal cells is also Foxl1-dependent (Fig. 4 C). Loss of mesenchymal BMP signals is expected to result in de-inhibition of Wnt signaling in the epithelium overlying villus cluster telocytes. Indeed, we found expression of the Wnt target gene Sox9 expanded from the developing crypts to nascent villi in the Foxl1 null fetal intestine (Fig. 4 D). Likewise, epithelial proliferation was not confined to the nascent crypts but extended to the villus epithelium in mutant mice (Fig. 4 D). Thus, Foxl1 is a critical factor required for the demarcation of the postmitotic villus from the mitotic intervillus epithelium. The schema in Fig. 4 E summarizes these findings. Foxl1 is required for mesenchymal expression of planar cell polarity genes Patterning of the developing gut epithelium is clearly perturbed in the absence of Foxl1 , and the hyperproliferation of the epithelium due to lack of BMP signaling documented above regionally leads to an apparent multilayered epithelium, in which mesenchyme-distal cells undergo apoptosis as indicated by cleaved caspase 3 staining (Fig. 5 A). Recently, planar cell polarity genes were identified among the mesenchymal Gli targets in the fetal gut, and it was demonstrated further that the GLI2 target gene Fat4 is required for villus development during the epithelial transition 18 . The discovery that the PCP pathway acts within the mesenchymal compartment to structure stromal cells was surprising, as typically PCP pathway function has been reported within epithelial cell layers 19 . As documented above, before the epithelial transition, Foxl1 + cells form a uniform cell layer with a depth of only one to two cells surrounding the primitive gut tube, which is then patterned into the telocyte progenitor 1 (villus cluster) and telocyte progenitor 2 (crypt base) cells, possibly with the involvement of the PCP system. We found that several PCP genes ( Fat4 , Wnt5a , Vangl1 and 2 ) exhibit reduced expression in the absence of Foxl1 (Fig. 5 B). Using our Cut and Run data, we found Foxl1 binding in the promoter of Fat4 , suggesting a direct regulatory relationship (Fig. 5 C). Rao-Bhatia and colleagues had found villification defects in mice with mutations in the PCP gene Fat4 , which were worsened by simultaneous heterozygous loss of Vangl2 18 . This defect was preceded by a reduction in the number of epithelial T-folds, characteristic invaginations of the epithelium that form the boundaries of developing villi and that can be visualized by staining with the apical membrane marker Ezrin. In order to evaluate if the reduced expression of PCP genes in Foxl1 null mice impacts epithelial remodeling, we stained small intestinal sections from fetuses at developmental stages spanning villification (13.5 to 15.5 dpc) for Ezrin to identify T-folds and PDGFRa to label villus cluster cells. As shown in Fig. 5 D-F, the number of T-folds is clearly reduced in the Foxl1-deficient intestine, coinciding with the loss of PDGFRa expression in telocyte progenitors. Next, we employed staining for F-actin to assess the orientation of stromal cells in the developing intestine. As shown in Fig. 5 G, while mesenchymal cells in the control fetal gut reorient their major axis to be parallel to the invaginating villi, this process fails to occur in Foxl1 null mice, supporting the notion of failed planar cell polarity in stromal cells. Loss of Foxl1 impacts epithelial gene expression profiles As shown above, Foxl1 deficiency impacts the patterning of the overlying epithelium, with many villus tip epithelial cells remaining in the cell cycle (Fig. 4 D). We had also noted a shift in the UMAP pattern of epithelial cells between control and Foxl1 null cells in 15.5 dpc embryos (green box in Fig. 3 B). To address this issue further, we reclustered the epithelial cells via UMAP. Figure 6 A shows that fetal gut epithelial cells of control embryos partition into two major groups, which we identified as ‘secretory progenitors’ and ‘undifferentiated epithelial cells’ based on their expression profile. The heatmap in Fig. 6 B shows the 225 most differentially expressed genes between these two clusters (false discovery rate 2), while Fig. 6 C indicates selected markers genes for each cell type. Fetal secretory progenitors are characterized by high levels of the mRNAs for transcription factors Klf4 , Spdef , and Sox4 , known to be critical for secretory cell differentiation 20 – 22 . Undifferentiated epithelial cells in contrast exhibit strong expression of Sox9 (which marks them as proliferating intervilllus cells as seen in Fig. 4 D) as well as markers of the absorptive enterocyte lineage ( Alpi , the gene for intestinal alkaline phosphatase, Fabp1 , encoding fatty acid binding protein 1, Apoa4 , encoding Apolipoprotein A4 which is important in intestinal cholesterol absorption, and Slc16a1 , encoding the monocarboxylic acid transporter for lactate). Next, we added epithelial cells from Foxl1 null embryos to the UMAP plot and found that they are largely confined to the undifferentiated epithelial cell cluster (Fig. 6 D). When we quantified the proportion of cells in each cluster, we found a striking loss of secretory progenitor cells in Foxl1 null embryos (Fig. 6 E). Finally, we performed gene set enrichment analysis to search for pathways that are differentially regulated in the absence of Foxl1. As shown in Fig. 6 F, the response to BMP signaling, negative regulation of epithelial proliferation, and establishment of planar cell polarity were all strongly enriched among the genes more highly expressed in the control gut epithelium, confirming that loss of telocyte Foxl1 has a major impact on the development of the fetal intestinal epithelium. Finally, we confirmed these findings by immunofluorescent staining for markers of the secretory cell lineage. Staining for Agr2 (Anterior gradient protein 2 homolog), a protein disulfide isomerase required for the formation of mixed disulfides in intestinal mucins (Fig. 6 G), and its substrate Muc2 (Mucin2), the major mucin of intestinal goblet cells (Fig. 6 H), are both expressed in the embryonic day 15.5 control intestine in secretory cell progenitors, but completely absent from the Foxl1 null gut, confirming the findings from our single cell RNAseq analysis. Discussion Villus formation is a fascinating and obviously essential process in vertebrate gut development and depends on reciprocal epithelial-to-mesenchymal cross talk. Epithelial hedgehog proteins are among the earliest signals emanating from the endoderm during organogenesis of the gut. Consequently, inhibition of hedgehog signaling with neutralizing antibodies or ablation of either Shh (sonic hedgehog) or Ihh (Indian hedgehog) causes impairment of gut development and villus formation 7 , 8 . Hedgehog proteins signal via their receptor Ptch1 (patched 1), expressed exclusively in the gut mesoderm, to stabilize the DNA-binding transcription factors Gli2 and Gli3. In 2006, computational analysis of evolutionarily conserved enhancers led to the identification of an ultra-conserved putative enhancer located between the neighboring Foxl1 and Foxf1 genes 23 . We identified seven Gli binding sites in this genomic region, some conserved from Fugu to human, and showed through in vitro and in vivo studies that both Foxl1 and Foxf1 are Gli target genes 12 . Here, we demonstrate that Foxl1-expressing telocyte progenitors are partitioned into two major subpopulations with distinct gene expression profiles. Telocyte progenitors 1 and 2 correspond to telocytes in the villus clusters and those adjacent to developing crypts, respectively. Villification is strongly impaired in the absence of Foxl1 , and abnormal proliferation persist in epithelial cells in the developing villi. We attribute this to the loss of BMP signaling as multiple Bmp genes exhibit reduced expression in absence of Foxl1. BMP signaling had been established by Walton and colleagues as key factor of villus formation 6 . Recently, planar cell polarity genes were identified among the mesenchymal Gli2 targets in the fetal gut, and it was demonstrated that the Gli2 target Fat4 is required for villus development during the epithelial transition 18 . The discovery that the PCP pathway acts within the mesenchymal compartment to structure stromal cells is novel and exciting, as typically PCP pathway function had been reported within epithelial cell layers 19 . The model of villification proposed by Rao-Bhatia and colleagues states that activation of Gli2 in telocyte progenitors opposite to the hedgehog-secreting epithelium is sufficient to activate Fat4 and other planar cell polarity genes directly 18 . Our data indicate that PCP induction in the developing gut mesenchyme is more complex than proposed by Rao-Bhatia and colleagues and depends not only on GLI proteins but also on winged helix transcription factors active in telocyte progenitors. We document by scRNAseq analysis of the proximal small intestine of 15.5 dpc fetuses that expression of planar cell polarity genes including Fat4 is enriched in Foxl1 + telocyte progenitors and reduced dramatically in the absence of Foxl1 (Fig. 5 ). Therefore, we propose a feed- forward loop for the regulation of stromal planar cell polarity genes, in which Gli2 activates both Foxl1 and Fat4 (and related targets), while Foxl1 also activates planar cell polarity gene expression in epithelium-adjacent stromal cells in a coherent feed-forward loop (Fig. 7 ). Loss of the telocyte transcription factor Foxl1 has a major secondary impact on the overlying epithelium, were reduced BMP signaling leads to loss of secretory progenitor differentiation and retention of proliferating epithelial cells overlying villus cluster cells. In conclusion, we have shown that the winged helix transcription factor, expressed in endoderm-adjacent mesodermal telocytes, is critical for the epithelial transition, epithelial gene expression, and ordered villus formation via the regulation of BMP, PDGFRa, and PCP signaling molecules. Materials and Methods Mice All animal procedures were approved by Institutional Animal Care and Use Committee of the Office of Animal Welfare at the University of Pennsylvania and conducted under protocol 804436. The mice used in this study were housed in a Specific Pathogen Free (SPF) facility at the University of Pennsylvania's animal center. They were individually housed in ventilated cages and provided with controlled temperature, humidity, a 12-hour light-dark cycle, a standard rodent chow diet, and constant access to water.C57BL/6 wild-type mice were obtained from Jackson Laboratory (Stock number: 000664). The Foxl1CreERT2-TdTomato gene replacement allele was described previously 13 . For embryonic studies, embryonic day 0.5 (0.5 dpc) was defined as noon on the day when the copulatory plug was observed. Since homozygotes of the Foxl1CreERT2-TdTomato gene replacement allele are viable and fertile in adulthood, the Foxl1 null embryos were generated by either crossing two heterozygotes or crossing one homozygote with a heterozygote. Single cells for the Cut & Run experiment were isolated from dissected embryonic small intestines at 15.5 dpc, which were obtained by crossing Foxl1CreERT2- tdTomato heterozygous mice with C57BL/6 wild-type mice. Embryonic data were collected from developmental stages before (13.5 dpc), during (14.5 dpc), and after (15.5–18.5 dpc) the onset of villification in the developing small intestines. Due to the indistinguishable sex of the embryos and the absence of reported sex differences in villus morphogenesis, mice of both sexes were included in all experimental procedures. Therefore, random assignment of mice from either sex was conducted within each experimental group. Tissue isolation and sectioning Mouse embryonic small intestinal samples from different stages were carefully dissected and harvested from the body. All samples used for histological sectioning and subsequent staining were taken from duodenal sections. For paraffin section, samples were fixed overnight at 4° C temperature in 4% paraformaldehyde in PBS buffer (Invitrogen) and incubated in 70% ethanol solution after three times’ wash with PBS buffer for paraffin section, and then were submitted to the Molecular Pathology and Imaging Core of the Center for Molecular Studies in Digestive and Liver Diseases (P30 DK050306) for further process, embed and sectioning for sectioning at tum thickness and then sectioned slices were kept at room temperature. For frozen section, samples were incubated overnight in 30% sucrose in PBS buffer after overnighted fixation at 4°C in 4% paraformaldehyde in PBS buffer (Invitrogen) until the intestinal tissues completely sink to the tube bottom, and then were embed in OCT for quick- frozen and stored at -80°C. OCT-embedded samples were performed the cryo-sectioning though a using a cryostat (Cryostar NX50, ThermoFisher Scientific) at 10 µm, dried for 10 minutes at room temperature before the further immunofluorescent staining or stored at -80°C. Scanning Electron Microscopy The scanning electron microscope (SEM) experiments were conducted at the microscopy core facility of the UPenn Department of Cell and Developmental Biology. After dissection, the presumptive duodenal sections of the 15.5 and 18.5 dpc fetuses were washed three times with 50 mM Na-cacodylate buffer and fixed overnight using a solution consisting of 2.5% glutaraldehyde in 50 mM Na-cacodylate buffer at a pH of 7.3. Subsequently, samples were dehydrated using a graded series of ethanol, gradually reaching 100% ethanol over a span of 1.5 hours. After dehydration, samples were incubated for 20 minutes in a solution containing 50% HMDS (Sigma-Aldrich) in ethanol, followed by three changes of 100% HMDS. After air-drying overnight, samples were mounted on stubs and coated with a layer of gold palladium using the sputter coating technique. Finally, we observed and photographed the specimens utilizing a Quanta 250 FEG scanning electron microscope manufactured by FEI (Hillsboro, OR, USA) with a 10 kV accelerating voltage. Histology and Immunofluorescence Paraffin sections were deparaffinized and rehydrated using xylene and descending ethanol gradients. For H&E staining, tissues were stained with Harris’ Hematoxylin and alcoholic Eosin Y. For IF staining, paraffin sections were subjected to antigen retrieval. For the frozen slices, OCT was directly removed in sterile water. Then the slices were performed the incubation with primary antibodies overnight at 4°C after serum blocking for 1 hour at room temperature and then with appropriate secondary antibodies at room temperature in the dark for 1 hour. The following antibodies were used: goat anti-PDGFR alpha 1: 200 (R&D Systems, AF1062), mouse anti-EZRIN 1:1000 (Sigma-Aldrich, E8897), rat anti-E-Cadherin antibody [DECMA-1] 1: 200 (Abcam, ab11512), goat anti-E-cadherin 1: 200 (R&D systems, AF-648), rabbit anti-Sox9 1: 200 (Abcam, ab185966), rabbit anti-Ki67 1: 200 (Abcam, ab16667), RFP antibody 1: 500 (Rockland, 600-401-379), Anterior gradient protein homolog 2 1: 200 (Abcam, ab209224), Muc2 1: 200 (Abcam, ab272692), Phospho-Smad1/5 1: 50 (Cell Signaling Technology, 9516), Alexa Fluor 488 Phalloidin 1: 400 (Thermo Fisher Scientific, A12379), Sal-like protein 1 1: 200 (Abcam, ab41974), Cleaved Caspase-3 1: 200 (Cell Signaling Technology), and Alexa Fluor 488-, 594- and 647-conjugated secondary antibodies, obtained from Life Technologies. Subsequently, slides were stained with DAPI (Sigma-Aldrich, D9542) to label nuclei. For F-actin staining, tissues were frozen-sectioned, treated with PBS followed by PBST (1:1000 Triton X-100 in PBS), followed by 1 hour of staining with Alexa Fluor 488-conjugated Phalloidin diluted 1:1000 into the blocking buffer at room temperature. Slides were imaged using a Leica Stellaris 5 confocal microscope. Single-cell capturing and cDNA library construction Timed embryos were obtained by crossing homozygous male mice with heterozygous female mice. The small intestine was obtained through dissection, and half of the portion close to the stomach was collected after folding it in half. Additionally, the tail of each embryo was collected for genomic DNA extraction using the KAPA Mouse Genotyping Kit HotStart (Kapa Biosystems, KK7352). Subsequently, genotyping was performed to distinguish between heterozygotes and homozygotes. Isolated intestinal cells were digested with collagenase type II and DNase I to prepare the single-cell suspension and then sorted in phosphate-buffered saline with 0.05% BSA to enrich the living cells through FACS sorting (MoFlo Astrios Sorter). Then the cells obtained were measured for cell concentration and viability with Trypan blue using a Countess II Automated Cell Counter from Life Technologies. Then the single-cell suspension was diluted to appropriate concentration and loaded on a 10x Genomics Chromium Single Cell Controller (Pleasanton, CA) with a target of about 5,000 cells per sample. Single-cell library preparation was completed using the 10x Genomics Chromium Single Cell 3’ Library & Gel Bead Kit v2 strictly following manufacturer’s protocol. The quality and quantity testing of obtained short cDNA fragment libraries using an Agilent 2100 Bioanalyzer and Invitrogen Qubit Fluorometer. Finally, the single-cell cDNA Libraries were sequenced on an Illumina Novaseq 6000 instrument. Cut & Run and DNA product sequencing Timed embryos were obtained by crossing homozygous or heterozygous male mice with C57BL/6 female mice. The entire length of the small intestine was digested with collagenase type II and DNase I to reach the single-cell state. Subsequently, the cells were sorted in phosphate-buffered saline with 0.05% BSA to enrich the tdTomato-positive cells using FACS (MoFlo Astrios Sorter). Isolated tdTomato-positive telocytes from the embryonic small intestines at 15.5 dpc were subjected to CUT&RUN experiments. CUT&RUN experiments were performed using the CUT&RUN Assay Kit (EpiCypher, Catalog No. 14-1048) following the manufacturer’s Instructions using 40,000 telocyte progenitors. Anti-Foxl1, H3K4me3 positive control and rabbit IgG negative control antibodies (13-0042k) were used in these experiments. Purified CUT&RUN DNA products were subjected to the CUT&RUN Library Prep Kit (EpiCypher, Catalog No. 14-1002) for library construction, and the libraries sequenced on an Illumina Hiseq X Ten instrument. Single-molecule RNA fluorescent in situ hybridization Proximal small intestinal sections from the Foxl1 null mice and controls from the same litter were fixed and gradient dehydrated following the methods described above. To perform the single-molecule RNA fluorescence in situ hybridization (smFISH) we employed the RNAScope Multiplex Fluorescent Reagent Kit v2 (323270) from Advanced Cell Diagnostics following the manufacturer’s recommendations. smFISH imaging was performed using a confocal Leica Stellaris 5 microscope. The following RNAscope probes were obtained from Advanced Cell Diagnostics: Foxl1(C3), and Glp2r (C1). Declarations Competing Interest Statement The authors declare no competing financial interests. Acknowledgements We thank members of the Kaestner lab for helpful discussions and Mark Tigue for maintaining our mouse colony. This work was supported by NIH grants R37DK053839 and R01DK139049. We thank the UPenn Center for Molecular Studies in Digestive and Liver Diseases (P30 DK050306) for the use of the Molecular Pathology and Imaging Core (MPIC) for tissue processing, the UPenn Diabetes Research Center Functional Genomics Core (P30 DK019125) for help with data analysis, and the Cell & Developmental Biology Microscopy Core for the use of their confocal imaging services. Author Contributions: GZ and KHK – Conceptualization and writing. GZ, HDM, and DL – Methodology. GZ and JS - Data curation and visualization. KHK - Supervision. KHK – Funding acquisition. References Helander, H.F. & Fandriks, L. Surface area of the digestive tract - revisited. Scand J Gastroenterol 49 , 681-689 (2014). Walton, K.D., Mishkind, D., Riddle, M.R., Tabin, C.J. & Gumucio, D.L. 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Ramalho-Santos, M., Melton, D.A. & McMahon, A.P. Hedgehog signals regulate multiple aspects of gastrointestinal development. Development 127 , 2763-2772 (2000). Madison, B.B. et al. Epithelial hedgehog signals pattern the intestinal crypt-villus axis. Development 132 , 279-289 (2005). Kaestner, K.H., Silberg, D.G., Traber, P.G. & Schutz, G. The mesenchymal winged helix transcription factor Fkh6 is required for the control of gastrointestinal proliferation and differentiation. Genes Dev 11 , 1583-1595 (1997). Kondo, A. & Kaestner, K.H. FoxL1(+) mesenchymal cells are a critical source of Wnt5a for midgut elongation during mouse embryonic intestinal development. Cells Dev 165 , 203662 (2021). Madison, B.B., McKenna, L.B., Dolson, D., Epstein, D.J. & Kaestner, K.H. FoxF1 and FoxL1 link hedgehog signaling and the control of epithelial proliferation in the developing stomach and intestine. J Biol Chem 284 , 5936-5944 (2009). Kolev, H.M. et al. A FoxL1-CreERT-2A-tdTomato Mouse Labels Subepithelial Telocytes. Cell Mol Gastroenterol Hepatol 12 , 1155-1158 e1154 (2021). Katz, J.P. et al. Foxl1 null mice have abnormal intestinal epithelia, postnatal growth retardation, and defective intestinal glucose uptake. Am J Physiol Gastrointest Liver Physiol 287 , G856-864 (2004). Perreault, N., Katz, J.P., Sackett, S.D. & Kaestner, K.H. Foxl1 controls the Wnt/beta-catenin pathway by modulating the expression of proteoglycans in the gut. J Biol Chem 276 , 43328-43333 (2001). Perreault, N., Sackett, S.D., Katz, J.P., Furth, E.E. & Kaestner, K.H. Foxl1 is a mesenchymal Modifier of Min in carcinogenesis of stomach and colon. Genes Dev 19 , 311-315 (2005). Mei, Y. et al. Regulation of neuroblastoma differentiation by forkhead transcription factors FOXO1/3/4 through the receptor tyrosine kinase PDGFRA. Proc Natl Acad Sci U S A 109 , 4898-4903 (2012). Rao-Bhatia, A. et al. Hedgehog-Activated Fat4 and PCP Pathways Mediate Mesenchymal Cell Clustering and Villus Formation in Gut Development. Dev Cell 52 , 647-658 e646 (2020). Butler, M.T. & Wallingford, J.B. Planar cell polarity in development and disease. Nat Rev Mol Cell Biol 18 , 375-388 (2017). Gracz, A.D. et al. Sox4 Promotes Atoh1-Independent Intestinal Secretory Differentiation Toward Tuft and Enteroendocrine Fates. Gastroenterology 155 , 1508-1523 e1510 (2018). Gregorieff, A. et al. The ets-domain transcription factor Spdef promotes maturation of goblet and paneth cells in the intestinal epithelium. Gastroenterology 137 , 1333-1345 e1331-1333 (2009). Katz, J.P. et al. The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon. Development 129 , 2619-2628 (2002). Hallikas, O. et al. Genome-wide prediction of mammalian enhancers based on analysis of transcription-factor binding affinity. Cell 124 , 47-59 (2006). Supplementary Table Supplementary Table 1 is not available with this version Additional Declarations There is NO Competing Interest. Supplementary Files supplementoryfig1.png Figure S1: Single nuclei RNA-sequencing of adult mouse colon. A brief description of sample processing and data analysis. Colon tissue from two control mice was used to generate marker genes of the cell types indicated. Cite Share Download PDF Status: Published Journal Publication published 24 Feb, 2026 Read the published version in Nature Communications → 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 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-4882679","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":339911524,"identity":"1d8d1fd1-8418-41e9-accb-ec4ec4632203","order_by":0,"name":"Klaus Kaestner","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-1228-021X","institution":"University of Pennsylvania","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Klaus","middleName":"","lastName":"Kaestner","suffix":""},{"id":339911525,"identity":"29c6891e-731e-4559-8db5-2b4cf751bb6d","order_by":1,"name":"Guoli Zhu","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guoli","middleName":"","lastName":"Zhu","suffix":""},{"id":339911526,"identity":"b6ea18f5-06ba-4b74-82ce-752029590c90","order_by":2,"name":"Deeksha Lahori","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Deeksha","middleName":"","lastName":"Lahori","suffix":""},{"id":339911527,"identity":"0d57b3f3-9346-4b37-b9b0-21ef78e8959f","order_by":3,"name":"Jonathan Schug","email":"","orcid":"","institution":"University of Pennsylvania Perelman School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"","lastName":"Schug","suffix":""}],"badges":[],"createdAt":"2024-08-08 17:30:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4882679/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4882679/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-026-69791-5","type":"published","date":"2026-02-24T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62623516,"identity":"d9e7ba3c-b332-4674-99a0-8a36269b8ee1","added_by":"auto","created_at":"2024-08-16 14:40:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":689930,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntestinal villification is dependent on the winged helix transcription factor FOXL1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-F) Hematoxylin and eosin stained small intestine from control (A,C,E), or Foxl1 null (B,D,F) fetuses at the developmental stages indicated. The yellow arrow in B indicates lack of invagination, and the red arrow in D marks persistent epithelial ridges.\u003c/p\u003e\n\u003cp\u003e(G-L) Scanning electron micrographs of small intestine from control (G,I,K), or Foxl1 null (H,J,L) fetuses at the developmental stages indicated. In all panels, larger magnification images shown on the right correspond to the areas outlined on the left. Magnification is indicated by scale bars.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4882679/v1/8fc29daaf446027e1b7c55ad.png"},{"id":62622759,"identity":"7c46d8d5-eedd-4723-9e9d-05caf51e4d99","added_by":"auto","created_at":"2024-08-16 14:32:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":431333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFetal FOXL1-positive telocyte progenitors partition into villus cluster and intervillus populations.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) FOXL1-positive cells labeled by tdTomato expression restructure during the epithelial transition, when the pseudostratified epithelium (E14.5) is converted to a simple cuboidal epithelium (E16.5). Epithelial cells are labeled with Ezrin or E-cadherin (E-cad) in green, Foxl1-positive cells by tdTomato in red.\u003c/p\u003e\n\u003cp\u003e(B) Experimental outline for single cell RNAseq study. The proximal half of the E15.5 small intestine was used for the analysis.\u003c/p\u003e\n\u003cp\u003e(C) UMAP plot of scRNAseq data identifies more than a dozen cell types. Foxl1-positive cells are labeled as telocyte progenitors 1 and 2.\u003c/p\u003e\n\u003cp\u003e(D) UMAP plot showing mRNA levels of Foxl1.\u003c/p\u003e\n\u003cp\u003e(E) UMAP plot of mRNA expression of multiple genes with differential activity between the two telocyte progenitor populations. Note the high expression of multiple BMP mRNAs in telocyte progenitor 1 cells.\u003c/p\u003e\n\u003cp\u003e(F) UMAP plot of mRNA abundance for Glp2r, encoding the receptor for the intestinotrophic hormone GLP-2. Glp2r expression is highly enriched in telocyte progenitor 1 cells.\u003c/p\u003e\n\u003cp\u003e(G) RNAscope analysis of fetal mouse intestine from E15.5 fetuses localizes Glp2r transcripts (green) and Foxl1 mRNA (purple). The Glp2r mRNA is highly enriched in villus tip telocyte progenitors.\u003c/p\u003e\n\u003cp\u003e(H) UMAP plot of mRNA abundance for Sall1, encoding the Spalt Like Transcription Factor 1, a zinc finger transcriptional repressor. Sall1 expression is highly enriched in telocyte progenitor 1 cells.\u003c/p\u003e\n\u003cp\u003e(I) Immunofluorescence staining fetal mouse intestine from E15.5 fetuses with antibodies specific to Sall1 (green) and Foxl1 (red) show localized expression in villus tip telocytes, as well as in a forming villus cluster (white arrow). DAPI (blue) was used to visualize nuclei.\u003c/p\u003e\n\u003cp\u003e(J) Model of relative positioning and prominent marker genes of telocyte progenitors 1 and 2 during intestinal villification.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4882679/v1/7ec27f7080304e64004fe9b8.png"},{"id":62622763,"identity":"30fd2ec5-65a9-428a-8efa-f8c45c37c6a1","added_by":"auto","created_at":"2024-08-16 14:32:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":505557,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVillus cluster PDGFRa expression is Foxl1 dependent.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Foxl1-positive telocytes, labeled by tdTomato expression (red) are retained in number in \u003cem\u003eFoxl1\u003c/em\u003e null mice. Epithelial cells are labeled by E-cadherin (green).\u003c/p\u003e\n\u003cp\u003e(B) UMAP plot of scRNAseq data comparing control and \u003cem\u003eFoxl1\u003c/em\u003e null fetal gut shows dramatic shifts in both telocyte progenitor (blue box) as well as in epithelial cells (green box). Single cells are labeled by genotype\u003c/p\u003e\n\u003cp\u003e(C) UMAP plot showing PDGFRa expression in control and Foxl1 null telocyte progenitors.\u003c/p\u003e\n\u003cp\u003e(D) Immunofluorescence labeling confirms dramatic reduction in PDGFRa expression (red) in the absence of Foxl1. The apical membrane of epithelial cells is stained for Ezrin (green). The persistence of telocyte progenitors in \u003cem\u003eFoxl1\u003c/em\u003e null mice is confirmed by staining for the tdTomato transgene (yellow) which replaces the \u003cem\u003eFoxl1\u003c/em\u003ecoding region in this \u003cem\u003eFoxl1\u003c/em\u003e null allele.\u003c/p\u003e\n\u003cp\u003e(E) Cut and Run analysis of fetal telocytes indicates that the \u003cem\u003eFoxo3\u003c/em\u003e promoter is in an active state, as indicated by the strong H3K4me3 signal (red trace). Foxl1 is bound to the proximal promoter of \u003cem\u003eFoxo3\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(F) Foxo3 expression in fetal telocyte progenitors is Foxl1-dependent as shown by reduced expression levels in the \u003cem\u003eFoxl1\u003c/em\u003enull intestine by scRNAseq analysis.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4882679/v1/9a568d48a8ea80d6b2667deb.png"},{"id":62623515,"identity":"e8b85ebb-f2a6-4419-81a8-4bd9acce5d25","added_by":"auto","created_at":"2024-08-16 14:40:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":411002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of BMP expression and abnormal epithelial proliferation in the Foxl1-deficient fetal small intestine.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Model for partitioning of small intestinal epithelial cells into postmitotic villus cells and proliferating intervillus cells through BMP signals emanating from villus cluster cells.\u003c/p\u003e\n\u003cp\u003e(B) Expression of multiple \u003cem\u003eBmp\u003c/em\u003egenes is reduced in telocyte progenitor 1 cells in absence of Foxl1.\u003c/p\u003e\n\u003cp\u003e(C) Determination of BMP signaling pathway activation by immunofluorescence staining for phosphorylated Smad1/5 (p-Smad1/5), downstream mediators of BMP receptor activation, in the fetal gut (E15.5). In control mice, p-Smad1/5 is detected in both villus tip epithelial cells as well as mesenchymal cells. In the Foxl1 null gut, p-Smad1/5 is dramatically reduced in both populations, indicating reduced BMP signaling.\u003c/p\u003e\n\u003cp\u003e(D) Expression of the Wnt target gene \u003cem\u003eSox9\u003c/em\u003e(purple) is restricted to intervillus regions in the E15.5 control small intestine, but maintained in a subset of villus epithelial cells in \u003cem\u003eFoxl1\u003c/em\u003enull mice. Likewise, proliferating epithelial cells, marked by Ki67 (purple) become restricted to intervillus regions in control mice, but extend over the emerging villus tip in absence of Foxl1. Epithelial cells are marked by E-cadherin (green).\u003c/p\u003e\n\u003cp\u003e(E) Model for BMP signaling defect in the \u003cem\u003eFoxl1\u003c/em\u003e null small intestine. While in control fetuses, BMPs produced by Foxl1-positive telocyte progenitors activate SMAD1/5 in the villus tip epithelium as well as in mesenchymal cells, this pathway is interrupted in the absence of Foxl1. Loss of BMP signaling leads to over-proliferation of villus tip epithelial cells.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4882679/v1/614f87f5e676ce8e901be3fa.png"},{"id":62622765,"identity":"153038c5-7a6b-4b74-bcbf-75d70a7ca1e0","added_by":"auto","created_at":"2024-08-16 14:32:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":367668,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFoxl1 is required for full activation of planar cell polarity genes in small intestinal telocyte progenitors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Small intestinal epithelial structure of the fetal small intestine in E15.5 fetuses shows cuboidal organization of a single-cell layer epithelium. In \u003cem\u003eFoxl1\u003c/em\u003enull mice, epithelial organization is disrupted, and the epithelium is partially multilayered, with frequent apoptotic cells, as visualized by cleaved caspase 3 immunostaining (green). The continued presence of telocytes is evidenced by Foxl1-tdTomato staining (red). DAPI (blue) was employed to visualize nuclei, and E-cadherin to label epithelial cells.\u003c/p\u003e\n\u003cp\u003e(B) UMAP plots show reduced expression of planar cell polarity genes \u003cem\u003eFat4\u003c/em\u003e, \u003cem\u003eVangl1\u003c/em\u003e, \u003cem\u003eVangl2\u003c/em\u003e, and \u003cem\u003eWnt5a\u003c/em\u003ein Foxl1-deficient telocyte progenitors.\u003c/p\u003e\n\u003cp\u003e(C) Cut-and-Run analysis shows binding of Foxl1 at the \u003cem\u003eFat4\u003c/em\u003e promoter.\u003c/p\u003e\n\u003cp\u003e(D) Staining with the apical membrane marker Ezrin was used to identify T-folds, the sites of emerging villi, in the fetal (E14.5) small intestine.\u003c/p\u003e\n\u003cp\u003e(E) Quantification of T-fold number per linear distance of the small intestine shows a dramatic reduction in the density of T-folds in the \u003cem\u003eFoxl1\u003c/em\u003e null small intestine. ***, p\u0026lt;0.001, n=4\u003c/p\u003e\n\u003cp\u003e(F) Model for villification in control and \u003cem\u003eFoxl1\u003c/em\u003e null mice.\u003c/p\u003e\n\u003cp\u003e(G) \u0026nbsp;Staining of the fetal (E15.5) small intestine with F-actin was employed to determine the orientation of the dominant cellular axis in the mesenchyme of the developing intestine. While in control mice, villus cluster mesenchymal cells are re-oriented to be parallel with the direction of the emerging villus, this process is impaired in the absence of Foxl1.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4882679/v1/019d063c862056a90c3bf0d5.png"},{"id":62622758,"identity":"727e70e5-fe65-4747-9d35-96e7938c6b1a","added_by":"auto","created_at":"2024-08-16 14:32:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":394852,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFoxl1-dependent telocyte signals are required for epithelial gene expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) UMAP plot of control epithelial cells from E15.5 embryos. The cells partition into two major clusters of secretory progenitors (red) and undifferentiated epithelial cells (blue).\u003c/p\u003e\n\u003cp\u003e(B) Heatmap of the 255 genes differentially expressed between secretory progenitors and undifferentiated epithelial cells. Each row represents a gene, each column a cell.\u003c/p\u003e\n\u003cp\u003e(C) Expression of key marker genes in secretory progenitors and undifferentiated epithelial cells\u003c/p\u003e\n\u003cp\u003e(D) UMAP plot of control (green) and \u003cem\u003eFoxl1\u003c/em\u003enull (orange) epithelial cells. Note that only very few \u003cem\u003eFoxl1\u003c/em\u003e null cells cluster with secretory progenitor cells.\u003c/p\u003e\n\u003cp\u003e(E) Proportion of secretory progenitors and undifferentiated epithelial cells in control and Foxl1 null embryos.\u003c/p\u003e\n\u003cp\u003e(F) Gene set enrichment analysis (GSEA) identifies critical pathways as Foxl1 dependent. The normalized enrichment scores (NES) were 2.18 for the ‘response to BMP’, 1.97 for ‘negative regulation of epithelial proliferation’, and 1.90 for ‘establishment of planar polarity of embryonic epithelium.\u003c/p\u003e\n\u003cp\u003e(G) Staining of the fetal (E15.5) small intestine with an antibody specific to Agr2 (Anterior gradient protein 2 homolog), a marker of the secretory lineage, was used to localize secretory progenitor cells. In control mice, these cells are distributed throughout the gut epithelium, while their number is dramatically reduced in the absence of \u003cem\u003eFoxl1\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(H) Staining of the fetal (E15.5) small intestine with an antibody specific to Muc2 (Mucin 2), a marker of the goblet cell lineage, was used to localize goblet cell progenitors. In control mice, these cells are distributed throughout the gut epithelium, while their number is dramatically reduced in the absence of \u003cem\u003eFoxl1\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4882679/v1/19bd51356645d85bfd628ce9.png"},{"id":62623882,"identity":"7a097582-c6dc-46ff-aa5f-bc41c4a0691e","added_by":"auto","created_at":"2024-08-16 14:48:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":154669,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStromal Foxl1 is required for villification of the fetal intestine.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Foxl1-positive telocytes (green) are partitioned into villus cluster and intervillus cells in the control mouse small intestine at E15.5. A fraction of the overlying epithelial cells already express markers of the secretory lineage (pink).\u003c/p\u003e\n\u003cp\u003e(B) Hedgehog signals from the overlaying epithelium locally induce expression of Foxl1 via Gli transcription factors. Foxl1 activates PDGFRa, BMP and PCP gene expression, the latter in a coherent feed-forward loop together with Gli proteins.\u003c/p\u003e\n\u003cp\u003e(C,D) In the absence of Foxl1, telocyte progenitors do not respond properly to epithelial hedgehog signaling, leading in turn to defects in reciprocal telocyte to epithelium signaling. As consequence, the epithelium cannot be properly patterned into a cuboidal, postmitotic villus epithelium, and differentiation into secretory cell progenitors is impaired.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4882679/v1/eb15459a17e1638305679a0e.png"},{"id":105982858,"identity":"9951e27d-238f-440d-b611-3d1513659060","added_by":"auto","created_at":"2026-04-02 07:05:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3725004,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4882679/v1/000920f0-3951-4a16-bab1-e190f075ab6f.pdf"},{"id":62622764,"identity":"fb2a800a-fc9c-4b88-8071-660aae7d0bdd","added_by":"auto","created_at":"2024-08-16 14:32:37","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":160974,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1: Single nuclei RNA-sequencing of adult mouse colon.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA brief description of sample processing and data analysis. Colon tissue from two control mice was used to generate marker genes of the cell types indicated.\u003c/p\u003e","description":"","filename":"supplementoryfig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4882679/v1/0a41f789e2c288859da42551.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Villification of the intestinal epithelium is driven by Foxl1","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe surface area of the human small intestine measures about 30 m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, or the size of half a badminton court\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. This large area is required to enable effective digestion and absorption of nutrients. The major factor responsible for increasing the size of the intestinal epithelium is villification, or the formation of intestinal villi that project into the gut lumen, which increase the surface area compared to a flat epithelium by close to 100-fold\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Remarkably, while villi are a feature of the small intestine in mammals and birds, they arise by divergent morphogenetic mechanisms. Thus, villus formation in birds is dependent on the sequential formation of first inner circular, then longitudinal, and finally muscularis mucosa muscles which coincide with the appearance of epithelial ridges, zigzags, and villi, respectively\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In contrast, in mammals, villus formation can proceed in the absence of tensile forces generated by intestinal muscles; rather, it is dependent on PDGFRa/BMP positive mesenchymal cell clusters\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, which themselves are induced by epithelial hedgehog signaling\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAt the onset of villification, also termed \u0026lsquo;epithelial transition\u0026rsquo;, the endoderm-derived epithelium forms a simple tube of pseudostratified cells with a layer thickness of ~\u0026thinsp;50 \u0026micro;m. This pseudostratified epithelium, characterized by cell cycle-dependent interkinetic nuclear migration\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, is then changed to a columnar epithelium coincident with villus formation. BMP signals from villus cluster mesenchymal cells restrict proliferation in the overlying epithelium so that cycling cells become restricted to intervillus regions, the precursors of the small intestinal proliferative lcrypts present in the adult. The critical role of BMP signaling in villus cluster formation was established by explant cultures of presumptive small intestine from 13.5 dpc (days post conception) embryos treated with localized sources of BMP or the pan-BMP inhibitor dorsomorphin\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe induction of villus formation and the formation of villus clusters in the underlying mesenchyme are dependent on epithelial to mesenchymal hedgehog signaling. Thus, both Sonic Hedgehog (shh) and Indian Hedgehog (Ihh) are expressed in the epithelium, while the hedgehog receptors (Ptch1 and Ptch2) as well as the downstream transcription factor Gli1 are expressed in the mesenchyme\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Because the two epithelial hedgehog proteins are partially redundant, Madison and colleagues addressed the role of hedgehog signaling in the developing intestine using epithelial expression of the pan-hedgehog inhibitor Hhip\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Suppression of hedgehog signaling impaired villus formation, which was partially due to decreased BMP expression in the underlying mesenchyme.\u003c/p\u003e \u003cp\u003ePreviously, the winged helix transcription factor Foxl1 (formerly termed \u003cem\u003eFhh6\u003c/em\u003e) was shown to be expressed in the first one to two cell layers of the mesenchyme juxtaposed to the epithelium before villus formation by mRNA in situ hybridization\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, which was recently confirmed by immunofluorescence staining\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, making it an excellent candidate as regulator of key developmental processes. Mice null for \u003cem\u003eFoxl1\u003c/em\u003e exhibit delayed villus formation and a defect in the inhibition of epithelial proliferation in nascent villi, suggesting Foxl1 as an important transcription factor controlling mesenchymal to epithelial signaling\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. A link to epithelial hedgehog signaling was subsequently established by the identification of functionally relevant binding sites for the hedgehog-dependent Gli transcription factors within an evolutionarily ultra-conserved enhancer at the \u003cem\u003eFoxl1\u003c/em\u003e locus\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Foxl1 expression was induced in fetal gut mesenchyme explants treated with Shh, while its mRNA levels were reduced in mice deficient for the hedgehog dependent transcription factors \u003cem\u003eGli2\u003c/em\u003e and \u003cem\u003eGli3\u003c/em\u003e. Taken together, these findings suggested that Foxl1 is a critical mediator of epithelial to mesenchymal cross-talk during villus formation. Here, we set out to determine the molecular targets and pathways controlled by Foxl1 during intestinal villification.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eHistological analysis of the small intestine shows that as villus formation is well on its way in wild type fetuses at 15.5 dpc, while this process has barely been initiated in \u003cem\u003eFoxl1\u003c/em\u003e deficient mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,B). Even two days later, at 17.5 dpc, villification is abnormal in mutant mice, with apparent bridging of nascent villi across the gut lumen, likely reflecting persistent epithelial ridges (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D), a phenotype that persists at 18.5 dpc (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE,F). In order to investigate this phenotype in three dimensions, we performed scanning electron microscopy. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-L, in control mice villification occurs through the formation of regularly spaced short invaginations into the gut lumen, which by 18.5 dpc have progressed to form elongated villi. This process is dramatically altered by loss of Foxl1, with absence of the regularly spaced nascent villi and presence instead of long epithelial ridges at 15.5 dpc (yellow arrow in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), which persist until late gestation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ, red arrow). The presence of epithelial ridges instead of villi is reminiscent of the phenotype seen in small intestinal explants treated with the pan-BMP inhibitor dorsomorphin\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, a connection we explore further below.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eFetal gut telocytes progenitors exist in two subpopulations\u003c/h3\u003e\n\u003cp\u003eThe inclusion of tdTomato in the recently developed \u003cem\u003eFoxl1\u003c/em\u003e mutant allele employed here\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e enabled us to follow the fate and determine the molecular properties of Foxl1-positive cells embryos heterozygous and homozygous for this \u003cem\u003eFoxl1\u003c/em\u003e null allele. Of note, the \u003cem\u003eFoxl1\u003c/em\u003e phenotype is recessive, as \u003cem\u003eFoxl1\u003c/em\u003e heterozygous mice are indistinguishable from wild type controls\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. We performed scRNAseq on the proximal half of the small intestine of 15.5 dpc \u003cem\u003eFoxL1\u003c/em\u003e\u003csup\u003eCreER-tdTom/+\u003c/sup\u003e fetuses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B). As shown in the UMAP analysis in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D, Foxl1\u003csup\u003e+\u003c/sup\u003e cells segregate into two closely related cell clusters, which we termed \u0026lsquo;telocyte progenitors 1 and 2\u0026rsquo;. A list of marker genes for all cell populations shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC is given in Supplementary Table\u0026nbsp;1. Telocyte progenitors 1 express high levels of \u003cem\u003ePdgfra\u003c/em\u003e and multiple \u003cem\u003eBmp\u003c/em\u003e mRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), and thus most likely correspond to \u0026lsquo;villus cluster cells\u0026rsquo;, i.e the PDGFRa positive cells important in villus formation originally identified by Karlsson and colleagues\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. By exclusion, the telocyte progenitor 2 population likely represents Foxl1\u003csup\u003e+\u003c/sup\u003e cells directly adjacent to the intervillus epithelium, i.e. the presumptive future crypt cells. Interestingly, we identified \u003cem\u003eGlp2r\u003c/em\u003e, encoding the GLP2 receptor, as another gene predominantly expressed in telocyte progenitor 1 cells, and confirm the localization of the Glp2r mRNA to villus cluster cells by RNAscope analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF,G). A further telocyte progenitor 1 marker is the Zinc-finger transcription factor Spalt like transcription factor 1 (\u003cem\u003eSall1\u003c/em\u003e), which is part of the NuRD transcriptional repressor complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH,I). Sall1/Foxl1 double positive cells can be seen to also mark a cluster of cells that appears to be in the process of initiating a new villus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Importantly, this single cell analysis clearly shows that Foxl1\u003csup\u003e+\u003c/sup\u003e cells are distinct from myofibroblast, interstitial cells of Cajal, and pericytes, identified by the expression of Acta2\u003csup\u003emed\u003c/sup\u003e/Myh11\u003csup\u003emed\u003c/sup\u003e/Des\u003csup\u003emed\u003c/sup\u003e/Tagln\u003csup\u003emed\u003c/sup\u003e, Etv1\u003csup\u003e+\u003c/sup\u003e/Kit\u003csup\u003e+\u003c/sup\u003e/Acta\u003csup\u003elow\u003c/sup\u003e, and Cspg4\u003csup\u003e+\u003c/sup\u003e/Pdgfrb\u003csup\u003e+\u003c/sup\u003e/Abcc9\u003csup\u003e+\u003c/sup\u003e or Abcc9\u003csup\u003e+\u003c/sup\u003e/Ndufa4l2\u003csup\u003e+\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Major hallmarks of the telocyte progenitor 1 and 2 populations are summarized in the graphic shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExpression of PDGFRa in telocyte progenitors is dependent on Foxl1\u003c/h2\u003e \u003cp\u003eNext, we analyzed the expression profile of the fetal small intestine of \u003cem\u003eFoxl1\u003c/em\u003e null (\u003cem\u003eFoxL1\u003c/em\u003e\u003csup\u003eCreER-tdTom/CreER-tdTom\u003c/sup\u003e) mice and compared them to those of heterozygous fetuses. Note that in both control and \u003cem\u003eFoxl1\u003c/em\u003e null fetuses, Foxl1-expressing cells are localized to the mesodermal cell layer that is directly juxtaposed to the developing epithelium, and loss of Foxl1 does not affect telocyte progenitor number (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The UMAP plot shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB clearly demonstrates that the telocyte progenitor 1 population is dramatically reduced in abundance in the \u003cem\u003eFoxl1\u003c/em\u003e null intestine. As mentioned above, among the markers of the telocyte progenitor 1 cells is PDGFRa. The scRNAseq data shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC as well as the immunofluorescence staining presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD demonstrate that PDGFRa expression is Foxl1 dependent. To determine if \u003cem\u003ePdgfra\u003c/em\u003e is a direct target of Foxl1, we performed Cut-and-Run assays on telocytes from 15.5 dpc fetal gut. However, we found no Foxl1 binding event within 10 kb of the \u003cem\u003ePdgfra\u003c/em\u003e promoter (data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt was previously reported that the winged helix transcription factor Foxo3 is a transcriptional activator of \u003cem\u003ePdgfra\u003c/em\u003e, which binds to an evolutionarily conserved \u003cem\u003ecis\u003c/em\u003e-regulatory element in its proximal promoter\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Therefore, we hypothesized that Foxl1 might indirectly control \u003cem\u003ePdgfra\u003c/em\u003e via activation of \u003cem\u003eFoxo3\u003c/em\u003e. Using Cut-and-Run of sorted fetal telocytes, we indeed found several Foxl1 binding sites in the \u003cem\u003eFoxo3\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). In addition, our scRNAseq data show a reduction of Foxo3 transcript levels in the telocyte progenitor 1 population of \u003cem\u003eFoxl1\u003c/em\u003e null fetuses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). These data suggest indirect regulation of the \u003cem\u003ePdgfra\u003c/em\u003e gene by Foxl1 through a transcription factor cascade via Foxo3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFoxl1 controls multiple Bmp genes in telocyte progenitor cells\u003c/h2\u003e \u003cp\u003eNext, we focused on BMP proteins, as BMP signaling from the mesoderm to the epithelium is critical for villus cluster formation\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Telocyte-produced BMPs enriched in the villus cluster cells signal to the overlaying endoderm to inhibit Wnt signaling and limit proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). High expression of several BMP genes was present in particular in villus custer telocyte-progenitor 1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In case of BMP4, expression also extends to Foxl1 negative GPX3\u0026thinsp;+\u0026thinsp;FLCs; however, its levels are clearly reduced specifically in Foxl1-deficient telocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). To determine if BMP signaling to the epithelium is impaired by this reduction in telocyte BMP expression, we performed immunostaining for phosphorylated SMAD1/5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Nuclear pSMAD1/5 is clearly detectable in epithelial cells in the villus tip but not intervillus regions in control embryos. In contrast, epithelial cells in \u003cem\u003eFoxl1\u003c/em\u003e null mice are devoid of signal, confirming loss of active BMP signaling to the epithelium.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen analyzing the pSMAD1/5 staining, we also noticed signal in nuclei of mesenchymal cells within the invaginating villi, with most of them negative for the Foxl1-tdTomato signal. These findings suggest unexpected bi-directional signaling of villus tip telocytes to both epithelium and neighboring mesenchymal cells. At present, the significance of this observation is unknown; however, the pSMAD1/5 signal in mesenchymal cells is also Foxl1-dependent (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eLoss of mesenchymal BMP signals is expected to result in de-inhibition of Wnt signaling in the epithelium overlying villus cluster telocytes. Indeed, we found expression of the Wnt target gene \u003cem\u003eSox9\u003c/em\u003e expanded from the developing crypts to nascent villi in the \u003cem\u003eFoxl1\u003c/em\u003e null fetal intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Likewise, epithelial proliferation was not confined to the nascent crypts but extended to the villus epithelium in mutant mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Thus, Foxl1 is a critical factor required for the demarcation of the postmitotic villus from the mitotic intervillus epithelium. The schema in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE summarizes these findings.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFoxl1 is required for mesenchymal expression of planar cell polarity genes\u003c/h2\u003e \u003cp\u003ePatterning of the developing gut epithelium is clearly perturbed in the absence of \u003cem\u003eFoxl1\u003c/em\u003e, and the hyperproliferation of the epithelium due to lack of BMP signaling documented above regionally leads to an apparent multilayered epithelium, in which mesenchyme-distal cells undergo apoptosis as indicated by cleaved caspase 3 staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Recently, planar cell polarity genes were identified among the mesenchymal Gli targets in the fetal gut, and it was demonstrated further that the GLI2 target gene \u003cem\u003eFat4\u003c/em\u003e is required for villus development during the epithelial transition\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The discovery that the PCP pathway acts within the mesenchymal compartment to structure stromal cells was surprising, as typically PCP pathway function has been reported within epithelial cell layers\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. As documented above, before the epithelial transition, Foxl1\u0026thinsp;+\u0026thinsp;cells form a uniform cell layer with a depth of only one to two cells surrounding the primitive gut tube, which is then patterned into the telocyte progenitor 1 (villus cluster) and telocyte progenitor 2 (crypt base) cells, possibly with the involvement of the PCP system. We found that several PCP genes (\u003cem\u003eFat4\u003c/em\u003e, \u003cem\u003eWnt5a\u003c/em\u003e, \u003cem\u003eVangl1\u003c/em\u003e and \u003cem\u003e2\u003c/em\u003e) exhibit reduced expression in the absence of Foxl1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Using our Cut and Run data, we found Foxl1 binding in the promoter of \u003cem\u003eFat4\u003c/em\u003e, suggesting a direct regulatory relationship (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRao-Bhatia and colleagues had found villification defects in mice with mutations in the PCP gene \u003cem\u003eFat4\u003c/em\u003e, which were worsened by simultaneous heterozygous loss of \u003cem\u003eVangl2\u003c/em\u003e \u003csup\u003e18\u003c/sup\u003e. This defect was preceded by a reduction in the number of epithelial T-folds, characteristic invaginations of the epithelium that form the boundaries of developing villi and that can be visualized by staining with the apical membrane marker Ezrin. In order to evaluate if the reduced expression of PCP genes in \u003cem\u003eFoxl1\u003c/em\u003e null mice impacts epithelial remodeling, we stained small intestinal sections from fetuses at developmental stages spanning villification (13.5 to 15.5 dpc) for Ezrin to identify T-folds and PDGFRa to label villus cluster cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F, the number of T-folds is clearly reduced in the Foxl1-deficient intestine, coinciding with the loss of PDGFRa expression in telocyte progenitors. Next, we employed staining for F-actin to assess the orientation of stromal cells in the developing intestine. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, while mesenchymal cells in the control fetal gut reorient their major axis to be parallel to the invaginating villi, this process fails to occur in \u003cem\u003eFoxl1\u003c/em\u003e null mice, supporting the notion of failed planar cell polarity in stromal cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eLoss of Foxl1 impacts epithelial gene expression profiles\u003c/h2\u003e \u003cp\u003eAs shown above, Foxl1 deficiency impacts the patterning of the overlying epithelium, with many villus tip epithelial cells remaining in the cell cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). We had also noted a shift in the UMAP pattern of epithelial cells between control and \u003cem\u003eFoxl1\u003c/em\u003e null cells in 15.5 dpc embryos (green box in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To address this issue further, we reclustered the epithelial cells via UMAP. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA shows that fetal gut epithelial cells of control embryos partition into two major groups, which we identified as \u0026lsquo;secretory progenitors\u0026rsquo; and \u0026lsquo;undifferentiated epithelial cells\u0026rsquo; based on their expression profile. The heatmap in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB shows the 225 most differentially expressed genes between these two clusters (false discovery rate\u0026thinsp;\u0026lt;\u0026thinsp;10%; absolute fold-change\u0026thinsp;\u0026gt;\u0026thinsp;2), while Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC indicates selected markers genes for each cell type. Fetal secretory progenitors are characterized by high levels of the mRNAs for transcription factors \u003cem\u003eKlf4\u003c/em\u003e, \u003cem\u003eSpdef\u003c/em\u003e, and \u003cem\u003eSox4\u003c/em\u003e, known to be critical for secretory cell differentiation\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Undifferentiated epithelial cells in contrast exhibit strong expression of Sox9 (which marks them as proliferating intervilllus cells as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) as well as markers of the absorptive enterocyte lineage (\u003cem\u003eAlpi\u003c/em\u003e, the gene for intestinal alkaline phosphatase, \u003cem\u003eFabp1\u003c/em\u003e, encoding fatty acid binding protein 1, \u003cem\u003eApoa4\u003c/em\u003e, encoding Apolipoprotein A4 which is important in intestinal cholesterol absorption, and \u003cem\u003eSlc16a1\u003c/em\u003e, encoding the monocarboxylic acid transporter for lactate).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we added epithelial cells from \u003cem\u003eFoxl1\u003c/em\u003e null embryos to the UMAP plot and found that they are largely confined to the undifferentiated epithelial cell cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). When we quantified the proportion of cells in each cluster, we found a striking loss of secretory progenitor cells in \u003cem\u003eFoxl1\u003c/em\u003e null embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Finally, we performed gene set enrichment analysis to search for pathways that are differentially regulated in the absence of Foxl1. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, the response to BMP signaling, negative regulation of epithelial proliferation, and establishment of planar cell polarity were all strongly enriched among the genes more highly expressed in the control gut epithelium, confirming that loss of telocyte Foxl1 has a major impact on the development of the fetal intestinal epithelium. Finally, we confirmed these findings by immunofluorescent staining for markers of the secretory cell lineage. Staining for Agr2 (Anterior gradient protein 2 homolog), a protein disulfide isomerase required for the formation of mixed disulfides in intestinal mucins (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG), and its substrate Muc2 (Mucin2), the major mucin of intestinal goblet cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH), are both expressed in the embryonic day 15.5 control intestine in secretory cell progenitors, but completely absent from the Foxl1 null gut, confirming the findings from our single cell RNAseq analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eVillus formation is a fascinating and obviously essential process in vertebrate gut development and depends on reciprocal epithelial-to-mesenchymal cross talk. Epithelial hedgehog proteins are among the earliest signals emanating from the endoderm during organogenesis of the gut. Consequently, inhibition of hedgehog signaling with neutralizing antibodies or ablation of either \u003cem\u003eShh\u003c/em\u003e (sonic hedgehog) or \u003cem\u003eIhh\u003c/em\u003e (Indian hedgehog) causes impairment of gut development and villus formation\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Hedgehog proteins signal via their receptor Ptch1 (patched 1), expressed exclusively in the gut mesoderm, to stabilize the DNA-binding transcription factors Gli2 and Gli3. In 2006, computational analysis of evolutionarily conserved enhancers led to the identification of an ultra-conserved putative enhancer located between the neighboring \u003cem\u003eFoxl1\u003c/em\u003e and \u003cem\u003eFoxf1\u003c/em\u003e genes\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. We identified seven Gli binding sites in this genomic region, some conserved from \u003cem\u003eFugu\u003c/em\u003e to human, and showed through \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies that both \u003cem\u003eFoxl1\u003c/em\u003e and \u003cem\u003eFoxf1\u003c/em\u003e are Gli target genes\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Here, we demonstrate that Foxl1-expressing telocyte progenitors are partitioned into two major subpopulations with distinct gene expression profiles. Telocyte progenitors 1 and 2 correspond to telocytes in the villus clusters and those adjacent to developing crypts, respectively.\u003c/p\u003e \u003cp\u003eVillification is strongly impaired in the absence of \u003cem\u003eFoxl1\u003c/em\u003e, and abnormal proliferation persist in epithelial cells in the developing villi. We attribute this to the loss of BMP signaling as multiple \u003cem\u003eBmp\u003c/em\u003e genes exhibit reduced expression in absence of Foxl1. BMP signaling had been established by Walton and colleagues as key factor of villus formation\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecently, planar cell polarity genes were identified among the mesenchymal Gli2 targets in the fetal gut, and it was demonstrated that the Gli2 target \u003cem\u003eFat4\u003c/em\u003e is required for villus development during the epithelial transition\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The discovery that the PCP pathway acts within the mesenchymal compartment to structure stromal cells is novel and exciting, as typically PCP pathway function had been reported within epithelial cell layers\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The model of villification proposed by Rao-Bhatia and colleagues states that activation of Gli2 in telocyte progenitors opposite to the hedgehog-secreting epithelium is sufficient to activate \u003cem\u003eFat4\u003c/em\u003e and other planar cell polarity genes directly\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Our data indicate that PCP induction in the developing gut mesenchyme is more complex than proposed by Rao-Bhatia and colleagues and depends not only on GLI proteins but also on winged helix transcription factors active in telocyte progenitors. We document by scRNAseq analysis of the proximal small intestine of 15.5 dpc fetuses that expression of planar cell polarity genes including \u003cem\u003eFat4\u003c/em\u003e is enriched in Foxl1\u0026thinsp;+\u0026thinsp;telocyte progenitors and reduced dramatically in the absence of Foxl1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Therefore, we propose a feed- forward loop for the regulation of stromal planar cell polarity genes, in which Gli2 activates both \u003cem\u003eFoxl1\u003c/em\u003e and \u003cem\u003eFat4\u003c/em\u003e (and related targets), while Foxl1 also activates planar cell polarity gene expression in epithelium-adjacent stromal cells in a coherent feed-forward loop (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Loss of the telocyte transcription factor Foxl1 has a major secondary impact on the overlying epithelium, were reduced BMP signaling leads to loss of secretory progenitor differentiation and retention of proliferating epithelial cells overlying villus cluster cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, we have shown that the winged helix transcription factor, expressed in endoderm-adjacent mesodermal telocytes, is critical for the epithelial transition, epithelial gene expression, and ordered villus formation via the regulation of BMP, PDGFRa, and PCP signaling molecules.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eMice\u003c/h2\u003e\n \u003cp\u003eAll animal procedures were approved by Institutional Animal Care and Use Committee of the Office of Animal Welfare at the University of Pennsylvania and conducted under protocol 804436. The mice used in this study were housed in a Specific Pathogen Free (SPF) facility at the University of Pennsylvania\u0026apos;s animal center. They were individually housed in ventilated cages and provided with controlled temperature, humidity, a 12-hour light-dark cycle, a standard rodent chow diet, and constant access to water.C57BL/6 wild-type mice were obtained from Jackson Laboratory (Stock number: 000664). The Foxl1CreERT2-TdTomato gene replacement allele was described previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eFor embryonic studies, embryonic day 0.5 (0.5 dpc) was defined as noon on the day when the copulatory plug was observed. Since homozygotes of the Foxl1CreERT2-TdTomato gene replacement allele are viable and fertile in adulthood, the Foxl1 null embryos were generated by either crossing two heterozygotes or crossing one homozygote with a heterozygote. Single cells for the Cut \u0026amp; Run experiment were isolated from dissected embryonic small intestines at 15.5 dpc, which were obtained by crossing Foxl1CreERT2- tdTomato heterozygous mice with C57BL/6 wild-type mice.\u003c/p\u003e\n \u003cp\u003eEmbryonic data were collected from developmental stages before (13.5 dpc), during (14.5 dpc), and after (15.5\u0026ndash;18.5 dpc) the onset of villification in the developing small intestines. Due to the indistinguishable sex of the embryos and the absence of reported sex differences in villus morphogenesis, mice of both sexes were included in all experimental procedures. Therefore, random assignment of mice from either sex was conducted within each experimental group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eTissue isolation and sectioning\u003c/h2\u003e\n \u003cp\u003eMouse embryonic small intestinal samples from different stages were carefully dissected and harvested from the body. All samples used for histological sectioning and subsequent staining were taken from duodenal sections. For paraffin section, samples were fixed overnight at 4\u0026deg; C temperature in 4% paraformaldehyde in PBS buffer (Invitrogen) and incubated in 70% ethanol solution after three times\u0026rsquo; wash with PBS buffer for paraffin section, and then were submitted to the Molecular Pathology and Imaging Core of the Center for Molecular Studies in Digestive and Liver Diseases (P30 DK050306) for further process, embed and sectioning for sectioning at tum thickness and then sectioned slices were kept at room temperature. For frozen section, samples were incubated overnight in 30% sucrose in PBS buffer after overnighted fixation at 4\u0026deg;C in 4% paraformaldehyde in PBS buffer (Invitrogen) until the intestinal tissues completely sink to the tube bottom, and then were embed in OCT for quick- frozen and stored at -80\u0026deg;C. OCT-embedded samples were performed the cryo-sectioning though a using a cryostat (Cryostar NX50, ThermoFisher Scientific) at 10 \u0026micro;m, dried for 10 minutes at room temperature before the further immunofluorescent staining or stored at -80\u0026deg;C.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eScanning Electron Microscopy\u003c/h2\u003e\n \u003cp\u003eThe scanning electron microscope (SEM) experiments were conducted at the microscopy\u003c/p\u003e\n \u003cp\u003ecore facility of the UPenn Department of Cell and Developmental Biology. After dissection, the presumptive duodenal sections of the 15.5 and 18.5 dpc fetuses were washed three times with 50 mM Na-cacodylate buffer and fixed overnight using a solution consisting of 2.5% glutaraldehyde in 50 mM Na-cacodylate buffer at a pH of 7.3. Subsequently, samples were dehydrated using a graded series of ethanol, gradually reaching 100% ethanol over a span of 1.5 hours. After dehydration, samples were incubated for 20 minutes in a solution containing 50% HMDS (Sigma-Aldrich) in ethanol, followed by three changes of 100% HMDS. After air-drying overnight, samples were mounted on stubs and coated with a layer of gold palladium using the sputter coating technique. Finally, we observed and photographed the specimens utilizing a Quanta 250 FEG scanning electron microscope manufactured by FEI (Hillsboro, OR, USA) with a 10 kV accelerating voltage.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eHistology and Immunofluorescence\u003c/h2\u003e\n \u003cp\u003eParaffin sections were deparaffinized and rehydrated using xylene and descending ethanol gradients. For H\u0026amp;E staining, tissues were stained with Harris\u0026rsquo; Hematoxylin and alcoholic Eosin Y. For IF staining, paraffin sections were subjected to antigen retrieval. For the frozen slices, OCT was directly removed in sterile water. Then the slices were performed the incubation with primary antibodies overnight at 4\u0026deg;C after serum blocking for 1 hour at room temperature and then with appropriate secondary antibodies at room temperature in the dark for 1 hour.\u003c/p\u003e\n \u003cp\u003eThe following antibodies were used: goat anti-PDGFR alpha 1: 200 (R\u0026amp;D Systems, AF1062), mouse anti-EZRIN 1:1000 (Sigma-Aldrich, E8897), rat anti-E-Cadherin antibody [DECMA-1] 1: 200 (Abcam, ab11512), goat anti-E-cadherin 1: 200 (R\u0026amp;D systems, AF-648), rabbit anti-Sox9 1: 200 (Abcam, ab185966), rabbit anti-Ki67 1: 200 (Abcam, ab16667), RFP antibody 1: 500 (Rockland, 600-401-379), Anterior gradient protein homolog 2 1: 200 (Abcam, ab209224), Muc2 1: 200 (Abcam, ab272692), Phospho-Smad1/5 1: 50 (Cell Signaling Technology, 9516), Alexa Fluor 488 Phalloidin 1: 400 (Thermo Fisher Scientific, A12379), Sal-like protein 1 1: 200 (Abcam, ab41974), Cleaved Caspase-3 1: 200 (Cell Signaling Technology), and Alexa Fluor 488-, 594- and 647-conjugated secondary antibodies, obtained from Life Technologies. Subsequently, slides were stained with DAPI (Sigma-Aldrich, D9542) to label nuclei. For F-actin staining, tissues were frozen-sectioned, treated with PBS followed by PBST (1:1000 Triton X-100 in PBS), followed by 1 hour of staining with Alexa Fluor 488-conjugated Phalloidin diluted 1:1000 into the blocking buffer at room temperature. Slides were imaged using a Leica Stellaris 5 confocal microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eSingle-cell capturing and cDNA library construction\u003c/h2\u003e\n \u003cp\u003eTimed embryos were obtained by crossing homozygous male mice with heterozygous female mice. The small intestine was obtained through dissection, and half of the portion close to the stomach was collected after folding it in half. Additionally, the tail of each embryo was collected for genomic DNA extraction using the KAPA Mouse Genotyping Kit HotStart (Kapa Biosystems, KK7352). Subsequently, genotyping was performed to distinguish between heterozygotes and homozygotes.\u003c/p\u003e\n \u003cp\u003eIsolated intestinal cells were digested with collagenase type II and DNase I to prepare the single-cell suspension and then sorted in phosphate-buffered saline with 0.05% BSA to enrich the living cells through FACS sorting (MoFlo Astrios Sorter). Then the cells obtained were measured for cell concentration and viability with Trypan blue using a Countess II Automated Cell Counter from Life Technologies. Then the single-cell suspension was diluted to appropriate concentration and loaded on a 10x Genomics Chromium Single Cell Controller (Pleasanton, CA) with a target of about 5,000 cells per sample. Single-cell library preparation was completed using the 10x Genomics Chromium Single Cell 3\u0026rsquo; Library \u0026amp; Gel Bead Kit v2 strictly following manufacturer\u0026rsquo;s protocol. The quality and quantity testing of obtained short cDNA fragment libraries using an Agilent 2100 Bioanalyzer and Invitrogen Qubit Fluorometer. Finally, the single-cell cDNA Libraries were sequenced on an Illumina Novaseq 6000 instrument.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eCut \u0026amp; Run and DNA product sequencing\u003c/h2\u003e\n \u003cp\u003eTimed embryos were obtained by crossing homozygous or heterozygous male mice with C57BL/6 female mice. The entire length of the small intestine was digested with collagenase type II and DNase I to reach the single-cell state. Subsequently, the cells were sorted in phosphate-buffered saline with 0.05% BSA to enrich the tdTomato-positive cells using FACS (MoFlo Astrios Sorter).\u003c/p\u003e\n \u003cp\u003eIsolated tdTomato-positive telocytes from the embryonic small intestines at 15.5 dpc were subjected to CUT\u0026amp;RUN experiments. CUT\u0026amp;RUN experiments were performed using the CUT\u0026amp;RUN Assay Kit (EpiCypher, Catalog No. 14-1048) following the manufacturer\u0026rsquo;s\u003c/p\u003e\n \u003cp\u003eInstructions using 40,000 telocyte progenitors. Anti-Foxl1, H3K4me3 positive control and rabbit IgG negative control antibodies (13-0042k) were used in these experiments. Purified CUT\u0026amp;RUN DNA products were subjected to the CUT\u0026amp;RUN Library Prep Kit (EpiCypher, Catalog No. 14-1002) for library construction, and the libraries sequenced on an Illumina Hiseq X Ten instrument.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eSingle-molecule RNA fluorescent in situ hybridization\u003c/h2\u003e\n \u003cp\u003eProximal small intestinal sections from the \u003cem\u003eFoxl1\u003c/em\u003e null mice and controls from the same litter were fixed and gradient dehydrated following the methods described above. To perform the single-molecule RNA fluorescence in situ hybridization (smFISH) we employed the RNAScope Multiplex Fluorescent Reagent Kit v2 (323270) from Advanced Cell Diagnostics following the manufacturer\u0026rsquo;s recommendations. smFISH imaging was performed using a confocal Leica Stellaris 5 microscope. The following RNAscope probes were obtained from Advanced Cell Diagnostics: Foxl1(C3), and Glp2r (C1).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe\u0026nbsp;thank\u0026nbsp;members\u0026nbsp;of\u0026nbsp;the\u0026nbsp;Kaestner lab\u0026nbsp;for\u0026nbsp;helpful\u0026nbsp;discussions\u0026nbsp;and\u0026nbsp;Mark\u0026nbsp;Tigue\u0026nbsp;for\u0026nbsp;maintaining\u0026nbsp;our\u0026nbsp;mouse\u0026nbsp;colony.\u0026nbsp;This\u0026nbsp;work\u0026nbsp;was supported by NIH grants R37DK053839 and R01DK139049.\u0026nbsp;We thank the UPenn Center for Molecular Studies in Digestive and Liver Diseases (P30 DK050306) for the use of the Molecular Pathology and Imaging Core (MPIC) for tissue processing, the UPenn Diabetes Research Center Functional Genomics Core (P30 DK019125) for help with data analysis, and\u0026nbsp;the\u0026nbsp;Cell\u0026nbsp;\u0026amp;\u0026nbsp;Developmental\u0026nbsp;Biology\u0026nbsp;Microscopy\u0026nbsp;Core\u0026nbsp;for\u0026nbsp;the\u0026nbsp;use\u0026nbsp;of\u0026nbsp;their\u0026nbsp;confocal\u0026nbsp;imaging services.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eGZ and KHK \u0026ndash; Conceptualization and writing. GZ, HDM, and DL \u0026ndash; Methodology. GZ and JS - Data curation and visualization. KHK - Supervision. KHK \u0026ndash; Funding acquisition.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHelander, H.F. \u0026amp; Fandriks, L. Surface area of the digestive tract - revisited. \u003cem\u003eScand J Gastroenterol\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 681-689 (2014).\u003c/li\u003e\n\u003cli\u003eWalton, K.D., Mishkind, D., Riddle, M.R., Tabin, C.J. \u0026amp; Gumucio, D.L. Blueprint for an intestinal villus: Species-specific assembly required. \u003cem\u003eWiley Interdiscip Rev Dev Biol\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, e317 (2018).\u003c/li\u003e\n\u003cli\u003eKarlsson, L., Lindahl, P., Heath, J.K. \u0026amp; Betsholtz, C. 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During mid-gestation a dramatic transformation occurs in which the epithelium is both restructured into its final cuboidal form and simultaneously folded and refolded to create intestinal villi and intervillus regions, the incipient crypts. Here we show that the mesenchymal winged helix transcription factor Foxl1, itself induced by epithelial hedgehog signaling, controls villification by activating BMP and PDGFRa as well as planar cell polarity genes in epithelial-adjacent telocyte progenitors, both directly and in a feed- forward loop with Foxo3. In the absence of Foxl1-dependent mesenchymal signaling, villus formation is delayed, the separation of epithelial cells into mitotic intervillus and postmitotic villus cells impaired, and the differentiation of secretory progenitors blocked. Thus, Foxl1 orchestrates key events during the epithelial transition of the fetal mammalian gut.\u003c/p\u003e","manuscriptTitle":"Villification of the intestinal epithelium is driven by Foxl1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-16 14:32:32","doi":"10.21203/rs.3.rs-4882679/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3dea9f16-c9b0-4257-b1c0-0320a962e1eb","owner":[],"postedDate":"August 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":36011837,"name":"Biological sciences/Developmental biology/Stem-cell niche"},{"id":36011838,"name":"Biological sciences/Developmental biology/Organogenesis"}],"tags":[],"updatedAt":"2026-04-02T07:05:48+00:00","versionOfRecord":{"articleIdentity":"rs-4882679","link":"https://doi.org/10.1038/s41467-026-69791-5","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-02-24 05:00:00","publishedOnDateReadable":"February 24th, 2026"},"versionCreatedAt":"2024-08-16 14:32:32","video":"","vorDoi":"10.1038/s41467-026-69791-5","vorDoiUrl":"https://doi.org/10.1038/s41467-026-69791-5","workflowStages":[]},"version":"v1","identity":"rs-4882679","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4882679","identity":"rs-4882679","version":["v1"]},"buildId":"cTy_lsJlmDsVRNrSptgXS","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}