Villification of the intestinal epithelium is driven by Foxl1 through activation of PDGFRα and BMPs

Domain: chemistry
Published: 2026-02-24
Editorial outcome: major_revision

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. Here, we show that the mesenchymal winged helix transcription factor Foxl1, itself induced by epithelial hedgehog signaling, controls villification by activating BMP and PDGFRα and the planar cell polarity factor Fat4 in epithelial-adjacent telocyte progenitors either directly or indirectly. In the absence of Foxl1-dependent mesenchymal signaling, villus formation and the separation of epithelial cells into mitotic intervillus and postmitotic villus are delayed, and the differentiation of secretory progenitors temporarily blocked. Thus, Foxl1 orchestrates key events during the epithelial transition of the fetal mammalian gut.

Introduction
The surface area of the human small intestine measures about 30 m2, or the size of half a badminton court1. This large area is required to enable effective digestion and absorption of nutrients. Villification, or the formation of intestinal villi that project into the gut lumen, is the major factor responsible for this increase in surface area (compared to a flat epithelium by close to 100-fold)1. Remarkably, while villi are a feature of the small intestine in both mammals and birds, they arise by divergent morphogenetic mechanisms. 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, respectively2–4. In contrast, mammalian villus formation can proceed in the absence of tensile forces generated by intestinal muscles; rather, it is dependent on PDGFRα (platelet-derived growth factor receptor alpha)/BMP (bone morphogenetic protein) positive mesenchymal cell clusters5, which themselves are induced by epithelial hedgehog signaling6,7.

While initially epithelial progenitors are spread uniformly along the gut tube, as villi form, stem cells become restricted to intervillus regions driven by reciprocal signaling between the epithelium and the underlying mesenchyme8,9. Sonic hedgehog (Shh) signals become locally concentrated through epithelial folding, which has been proposed to involve intraepithelial compressive forces from rounding of mitotic cells8. Locally confined Shh signals induce the aforementioned BMP-expressing villus clusters in the mesenchyme2. 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 dorsomorphin10. More recently, it was demonstrated that the symmetry of the primitive gut tube is broken by mesenchymal tissue fluidization enabled by matrix metalloproteases, which initiate epithelial folding at the interface between PDGFRαHigh and PDGFRαLow mesenchyme11. In this model, increased surface tension in the PDGFRαHigh tissue provides the driving force to establish mesenchymal villus clusters described previously5,7,12.

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 migration9, then changes to a columnar epithelium coincident with villus formation. Of note, adult Lgr5-positive intestinal stem/progenitor cells surprisingly have their origin in both nascent intervillus and villus regions, as the fetal epithelium undergoes massive remodeling and fission, which relocates epithelial cells from the non-proliferative pre-villus zone into the proliferative intervillus regions13.

As introduced above, 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 mesenchyme14,15. Because the two epithelial hedgehog proteins are partially redundant, Madison and colleagues used the pan-hedgehog inhibitor Hhip to demonstrate that suppression of hedgehog signaling impaired villus formation, partially due to decreased BMP expression in the underlying mesenchyme14.

The winged helix transcription factor Foxl1 (formerly termed Fkh6) is expressed in the first one to two cell layers of the mesenchyme juxtaposed to the epithelium before villus formation, originally shown by mRNA in situ hybridization15 and recently confirmed by immunofluorescence staining16. In addition, mice null for Foxl1 exhibit delayed villus formation and a defect in the inhibition of epithelial proliferation in nascent villi, suggesting Foxl1 is an important transcription factor controlling mesenchymal to epithelial signaling15. A link to epithelial hedgehog signaling was subsequently established via identification of functionally relevant binding sites for the hedgehog-dependent Gli transcription factors within an evolutionarily ultra-conserved enhancer at the Foxl1 locus17. Furthermore, 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 Gli317. Taken together, these findings suggest that Foxl1 is a critical mediator of epithelial to mesenchymal crosstalk during villus formation. Here, we set out to determine the molecular targets and pathways controlled by Foxl1 during intestinal villification.

Results
Our study comprised histological, ultrastructural, immunostaining, and molecular analyses during various stages of fetal intestinal development at the proximal, middle, and distal areas of the developing small intestine, as outlined in Fig. 1A. Note that overall intestinal length is reduced in the 15.5 dpc Foxl1 null fetus (Fig. 1B), while the overall size of the fetus is not changed (data not shown). Immunostaining confirmed the absence of any Foxl1 protein in the Foxl1 null fetus, as expected (Fig. S1A). Histological analysis of the small intestine showed that while villus formation is progressing as expected in the anterior half of the small intestine in wild type fetuses at 15.5 dpc, this process is lagging in Foxl1 deficient mice (Fig. 1C, D). The delay in villus formation is evident throughout the small intestine, though at 15.5 dpc it is most pronounced in the proximal and middle portions of the tissue, reflecting the anterior to posterior wave of villus formation (Fig. S1B). Even two days later, at 17.5 dpc, villification is abnormal in mutant mice (Fig. 1E, F), a phenotype that persists at 18.5 dpc (Figs. 1G, H; S2A). In order to investigate this phenotype in three dimensions, we performed scanning electron microscopy. As shown in Fig. 1I-N, 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 altered by loss of Foxl1, with absence of the regularly spaced nascent villi and the presence of occasional long epithelial ridges at 15.5 dpc (yellow arrow in Fig. 1J), which persist until late gestation (Fig. 1L, N, red arrow; see also Fig. S3A). Abnormal epithelial ridges in the Foxl1 null gut were further confirmed by immunostaining of small intestinal sections, which showed flat stretches of epithelial cells with the presence of Olfm4-positive cell clusters, which likely represent late induction of new proliferative intervillus regions (Fig. S3B). The presence of epithelial ridges instead of villi are reminiscent of the phenotype seen in small intestinal explants treated with the pan-BMP inhibitor dorsomorphin10, a connection we explore further below. By postnatal day 5, villus formation has mostly normalized in the mutant mice, with the occasional abnormally formed villus remaining (Figs. S2B, S4).

Fetal gut telocytes progenitors exist in two subpopulations
The inclusion of tdTomato in the recently developed Foxl1 mutant allele employed here18 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 controls15,19–21. We performed scRNAseq on the proximal half of the small intestine of 15.5 dpc Foxl1CreER-tdTom/+ fetuses (Fig. 2A, B). As shown in the UMAP analysis in Fig. 2C 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. 2C is given in Supplementary Data 1. To investigate potential pseudotemporal relationships between the telocyte progenitor 2 and progenitor 1 populations, we performed RNA velocity analysis22. This approach estimates the future transcriptional state of individual cells by leveraging the ratio of spliced to unspliced transcripts, thereby inferring likely trajectories of transcriptional change (see “Methods” for details). This analysis suggests that telocyte 2 progenitors are precursors to both telocyte 1 progenitors as well as other, Foxl1-negative mesenchymal progenitor populations (Fig. 2E). Further support for this notion comes from analysis of the proliferation genes Top2a, Birc5, Mki67 and Pcna, which are all expressed at higher levels in telocyte 2 progenitors (Fig. S5).

Telocyte progenitors 1 express high levels of Pdgfra and multiple Bmp mRNAs (Fig. 2F), and thus most likely correspond to ‘villus cluster cells’, i.e. the PDGFRα-positive cells important in villus formation originally identified by Karlsson and colleagues5. By exclusion, the telocyte progenitor 2 population likely represents Foxl1+ cells directly adjacent to the intervillus epithelium. Interestingly, we identified Glp2r, encoding the GLP-2 receptor, as another gene predominately expressed in telocyte progenitor 1 cells, and confirmed the localization of the Glp2r mRNA to villus cluster cells by RNAscope analysis (Fig. 2G-I). An additional 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. 2J-L). 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. 2L). Importantly, this single cell analysis clearly shows that Foxl1+ cells are distinct from myofibroblasts, interstitial cells of Cajal, and pericytes, identified by the expression of Acta2med/Myh11med/Desmed/Taglnmed, Etv1+/Kit+/Actalow, and Cspg4+/Pdgfrb+/Abcc9+ or Abcc9+/Ndufa4l2+, respectively (Fig. 2C). Key marker genes used to identify the various cell clusters are highlighted in Figs. S6 and S7. Major hallmarks of the telocyte progenitor 1 and 2 populations are summarized in the graphic shown in Fig. 2M.

Expression of PDGFRα in telocyte progenitors is dependent on Foxl1
Next, we analyzed the expression profile of the fetal small intestine of Foxl1 null (Foxl1CreER-tdTom/CreER-tdTom) mice and compared them to those of heterozygous fetuses. The number of cells in each cluster by genotype is given in Supplementary Data 2, and the complete list of differentially expressed genes contained in Supplementary Data 3. Note that in the Foxl1 null fetuses, the cis-regulatory elements CreER-tdTomato expression are still active, enabling us to visualize Foxl1 telocyte progenitors, which are still localized to the mesodermal cell layer that is directly juxtaposed to the developing epithelium. Loss of Foxl1 does not significantly affect telocyte progenitor number (Fig. 3A). The UMAP plots shown in Figs. 3B and S8 clearly demonstrate that the telocyte progenitor 1 population is shifted in the Foxl1 null intestine reflecting its altered expression profile; however, all cell types are present in all fetuses analyzed (Figure S8). Two of the telocyte progenitor 1 marker genes mentioned above, Glp2r and Sall1, are Foxl1-dependent, either directly or indirectly (Fig. S9A-D). As mentioned above, among the markers of the telocyte progenitor 1 cells is PDGFRα. The scRNAseq data shown in Fig. 3C, D as well as the immunofluorescence staining presented in Fig. 3E demonstrate that PDGFRα expression is reduced in Foxl1-deficient telocyte 1 progenitors. Further support for a critical role for Foxl1 as either direct or indirect upstream regulator of Pdgfra comes from whole-mount immunofluorescence staining (Fig. 3F), which shows the localization of PDGFRα high cells to villus cluster cells and their absence in Foxl1 null fetuses. The full three-dimensional view of PDGFRα expression is visualized by the supplementary videos 1 and 2. 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), suggesting that regulation might be indirect. Because villus formation is delayed but not completely blocked in Foxl1 null mice, we hypothesized that PDGFRα high villus clusters might reappear at later stages of gestation. This is indeed the case, as shown by immunofluorescence staining of E18.5 embryos for PDGFRα (Fig. S10), suggesting the possibility that other transcription factors can activate the Pdgfra gene at later stages of development.

Foxl1 is required for activation of 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 formation10. Telocyte-produced BMPs enriched in villus cluster cells signal to the adjacent endoderm to inhibit WNT signaling and limit proliferation (Fig. 4A). High expression of several BMP genes was present particularly in villus cluster telocyte-progenitor 1 cells (Fig. 4B). In the case of BMP4, expression also extends to Foxl1-negative mesenchymal progenitor cells; however, its levels are clearly reduced specifically in Foxl1-deficient telocytes (Fig. 4B). Transcript levels of Bmp2, Bmp3, Bmp4, Bmp5 and Bmp6 were all significantly reduced in the Foxl1 null telocyte progenitor 1 population (Fig. 4B). 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. 4C). Nuclear pSMAD1/5 is clearly detectable in epithelial cells in the villus tip, but not intervillus regions in control embryos. In contrast, most epithelial cells in Foxl1 null mice are devoid of signal, confirming a reduction of active BMP signaling to the epithelium. When analyzing the pSMAD1/5 staining, we also noted signal in nuclei of mesenchymal cells within the invaginating villi, with most of them negative for Foxl1-tdTomato. These findings are expected given the previous report demonstrating that BMP-driven villus cluster formation occurs even in the absence of epithelial BMP receptors and thus has to rely on BMP signaling within the mesenchymal compartment10.

Loss of mesenchymal BMP signals is expected to result in de-inhibition of WNT signaling in the epithelium overlying villus cluster telocytes23. Indeed, we found expression of the WNT target gene Sox9 expanded from the developing intervillus regions to nascent villi in the Foxl1 null fetal intestine (Fig. 4D). Likewise, epithelial proliferation was not confined to the nascent intervillus regions but extended to the villus epithelium in mutant mice (Fig. 4D). This likely reflects the developmental delay in these mice, as prior to villus demarcation, all epithelial cells are proliferative. Of note, at later stages of development, both the Sox9-positive epithelial domain as well as the proliferative zone normalize in Foxl1 null mice, consistent with the overall normalization of the histological phenotype documented above (Fig. S11).

Foxl1 contributes to regulation of planar cell polarity genes
Patterning of the developing gut epithelium is temporarily perturbed in the absence of Foxl1, and the hyperproliferation of the epithelium due to lack of BMP signaling documented above regionally causes epithelial cells to be more than 100 μm distal to the underlying stroma (Figs. 5A, S12A). A subset of the luminal-most epithelial cells undergoes apoptosis as indicated by cleaved caspase 3 staining (Fig. 5A) as well as the TUNEL assay (Fig. S12B). It was shown previously that after mitosis of epithelial cells, one daughter cell inherits the connection to the basement membrane from the mother cell, while the other daughter cell has to develop a new basal filopodium, a process that is dependent on Wnt5a24. When this does not occur, the ‘connection-less’ epithelial cells undergo apoptosis.

Recently, planar cell polarity (PCP) genes were identified among mesenchymal Gli targets in the fetal gut, and it was further demonstrated that the GLI2 target gene Fat4 is required for villus development during the epithelial transition25. Rao-Bhatia and colleagues had found that the villification defects in mice with mutations in the PCP gene Fat4 were 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 Ezrin25. In order to evaluate if loss of Foxl1 delays epithelial remodeling, we stained small intestinal sections from fetuses at developmental stages spanning villification (13.5 to 15.5 dpc; Fig. S12C) for Ezrin to identify T-folds and PDGFRα to label villus cluster cells. As shown in Fig. 5C, the number of T-folds is reduced in the Foxl1-deficient intestine of 14.5 dpc embryos, coinciding with the loss of PDGFRα 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. 5D, 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 impaired planar cell polarity in stromal cells. When analyzing expression of PCP genes using our scRNAseq data, we found that expression of Fat4 was significantly reduced in Foxl1 null telocyte progenitor 1 cells, while that of the PCP genes Vangl1 and 2 was not affected (Fig. 5E). We also found Fat4 mRNA to be more abundant in smooth muscle cells; the significance of this observation is unknown (Fig. 5E). Using our Cut and Run data, we found Foxl1 binding in the promoter of Fat4, suggesting a direct regulatory relationship (Fig. 5F).

Loss of Foxl1 impacts epithelial gene expression profiles
As shown above, Foxl1 deficiency transiently impacts the patterning of the overlying epithelium, with many presumptive villus tip epithelial cells remaining in the cell cycle (Fig. 4D). 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. 3B). To address this issue further, we re-clustered the epithelial cells from control mice only via UMAP. Fig. 6A 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 profiles. The heatmap in Fig. 6B shows the 225 most differentially expressed genes between these two clusters (false discovery rate <10%; absolute fold-change >2), while Fig. 6C indicates selected markers genes for each cell type. Fetal secretory progenitors are characterized by high mRNA levels for transcription factors Klf4, Spdef, and Sox4, known to be critical for secretory cell differentiation26–28. Undifferentiated epithelial cells, in contrast, exhibit strong expression of Sox9 (which marks them as proliferating intervillus cells as seen in Fig. 4D) 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. 6D). When we quantified the proportion of cells in each cluster, we found a striking loss of secretory progenitor cells in Foxl1 null embryos (Fig. 6E). Finally, we performed gene set enrichment analysis to search for pathways that are differentially regulated in epithelial cells in the absence of telocyte Foxl1. As shown in Fig. 6F, 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, if transient, impact on the development of the fetal intestinal epithelium at this time-point. Finally, we confirmed these findings by immunofluorescent staining for markers of the secretory cell lineage. Antibody staining to detect AGR2 (Anterior gradient protein 2 homolog), a protein disulfide isomerase required for the formation of mixed disulfides in intestinal mucins (Fig. 6G, I), and its substrate MUC2 (Mucin2), the major mucin of intestinal goblet cells (Fig. 6H, J), demonstrated that both are expressed in the embryonic day 15.5 control intestine in secretory cell progenitors. However, the proportion of AGR2+ and MUC2+ cells is significantly reduced in the Foxl1 null gut, confirming the findings from our single cell RNA seq analysis. The fact that the percentage of secretory cells labeled by MUC2 and AGR2 immunofluorescence staining is smaller than the percentage of secretory progenitors identified by RNA seq (compare Fig. 6H, J–E) is likely because the protein markers only detect the most mature secretory lineage cells, while RNA seq, using hundreds of transcript measurements, can assign lineage even to very immature secretory progenitors. Of note, this defect in secretory lineage specification in the Foxl1 null gut is only transitory, as by embryonic day 18.5 the frequency of AGR2 and MUC2 positive cells has normalized (Fig. S13).

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 individually housed in ventilated cages and provided with controlled temperature, humidity, a 12 h 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 previously18.

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 small intestinal samples from different stages were carefully dissected. All samples used for histological sectioning and subsequent staining were taken from the anterior small intestine, from the stomach to the duodenal flexure, unless noted otherwise. For paraffin sections, samples were fixed overnight at 4 °C in 4% paraformaldehyde in PBS buffer (Invitrogen), washed three times with PBS before dehydration in a graded ethanol series and paraffin embedding. Tissues were sectioned at 5 μm thickness by the Molecular Pathology and Imaging Core of the UPenn Center for Molecular Studies in Digestive and Liver Diseases. For frozen sections, samples were incubated overnight in 30% sucrose in PBS buffer after overnight fixation at 4 °C in 4% paraformaldehyde in PBS buffer (Invitrogen) until the intestinal tissues had settled at the bottom of the tube, and were embedded in OCT for quick freezing and storage at −80 °C. OCT-embedded samples were sectioned at 10 µm using a cryostat (Cryostar NX50, ThermoFisher Scientific), dried for 10 minutes at room temperature, and then stored at −80 °C.

Scanning electron microscopy
The scanning electron microscope (SEM) experiments were conducted by the microscopy core facility of the UPenn Department of Cell and Developmental Biology. After dissection, the presumptive duodenal sections of 15.5 and 18.5 dpc fetuses and postnatal day 5 newborns 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 h. After dehydration, samples were incubated for 20 min 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 technique35. 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, immunofluorescence and TUNEL staining
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 pressure cooker antigen retrieval with Tris/EDTA buffer (Abcam, ab93684). For the frozen slices, OCT was directly removed in sterile water. The slices were then incubated with primary antibodies overnight at 4°C after blocking with blocking buffer (Abcam, ab126587) for 1 h at room temperature and then with appropriate secondary antibodies at room temperature in the dark for 1 h. The following antibodies were used: goat anti-PDGFRalpha 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 h 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.

For the detection of Foxl1 protein, paraffin-embedded sections were deparaffinized in xylene and rehydrated through graded ethanol to PBS. Antigen retrieval was performed in Tris-EDTA buffer (pH 9.0) using a pressure cooker, followed by cooling and washing in PBS. Sections were permeabilized in PBST (PBS containing 0.1–0.2% Triton X-100) for 10–15 min and washed three times with PBS. Endogenous peroxidase was quenched with 3% H₂O₂ for 10–15 min, followed by PBS washes. Sections were blocked with blocking buffer (Abcam, ab126587) for 1 hour at room temperature.

Primary antibody against Foxl1 (guinea pig36;) was applied at 1:1500 dilution and incubated overnight at 4°C. The antibody was detected using a biotinylated goat anti-guinea pig secondary antibody, followed by signal amplification using the biotin–avidin system (Vector Labs, SP-2001) and tyramide-based amplification (PerkinElmer, NEL700A001KT) according to the manufacturer’s instructions. Sections were then incubated with fluorescent labels, including SA-Cy5 (1:300), for 2–3 h at room temperature in the dark. Nuclei were counterstained with DAPI, and slides were mounted with antifade medium for imaging.

Apoptotic cells in frozen tissue sections were detected using the In Situ Cell Death Detection Kit, TMR red (Roche, 12156792910) according to the manufacturer’s instructions. Sections were pre-treated with Proteinase K for 15 min at room temperature, incubated with the TUNEL reaction mixture for 1 h at 37°C in the dark, counterstained with DAPI, and mounted with anti-fade medium before imaging by fluorescence microscopy.

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 the anterior half collected for processing. 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.

Intestinal tissue was digested with collagenase type II and DNase I dissolved in ‘Dispase in DMEM/F-12’ (Stemcell, 07923) to prepare a single-cell suspension and then FACS sorted in phosphate-buffered saline with 0.5% BSA to enrich for live cells (MoFlo Astrios Sorter). Cell number and viability were determined using a Countess II Automated Cell Counter from Life Technologies after staining with Trypan blue.

Single-cell suspensions were diluted to the appropriate concentration and loaded on a 10x Genomics Chromium Single Cell Controller with a target of 5000 cells per sample. Single-cell library preparation was completed using the 10x Genomics Chromium Single Cell 3’ Library & Gel Bead Kit v2 following the manufacturer’s protocol. Quality and quantity of the cDNA libraries were assessed using an Agilent 2100 Bioanalyzer and Invitrogen Qubit Fluorometer. Finally, the libraries were sequenced on an Illumina Novaseq 6000 instrument. The single-cell RNA-seq data generated in this study have been deposited in GEO under accession number GSE302290.

Single-cell analysis workflow
Single-cell RNA sequencing (scRNAseq) libraries were prepared using the 10x Genomics Chromium platform and sequenced on a NovaSeq 6000 system with 150bp paired-end reads. Each library was sequenced to a minimum depth of 361 million raw paired reads to ensure sufficient coverage for downstream analyses. Adapter trimming and read preprocessing were performed using Cutadapt37, following the standard 10x Genomics configuration of 28 bp (cell barcode and unique molecular identifier [UMI]) and 90 bp (transcript read). For consistency, only the first 361 million raw paired reads for each sample were used for subsequent analyses. Trimmed reads were aligned to the mouse reference genome (mm10 release) using the CellRanger pipeline (v7.0.0)38 with the -include-introns false parameter to exclude intronic reads. Gene-level expression matrices were generated for each sample.

To reduce technical noise and potential confounders, several stringent filtering steps were applied. Genes located on sex chromosomes (chrX and chrY) were excluded to minimize sex-specific expression effects. Ambient RNA contamination was estimated and removed using SoupX39. Low-quality cells and potential doublets were filtered out by retaining only cells with total RNA counts (nCount_RNA) between 4,096 and 65,536. Cells with high mitochondrial gene expression (>10% of total RNA counts) were also excluded as indicators of cell stress or death.

Filtered expression matrices were integrated using the Seurat R package (v4.3.0)40. Integration features were identified using SelectIntegrationFeatures and PrepSCTIntegration, and integration anchors were determined with FindIntegrationAnchors. The datasets were then merged using IntegrateData to correct for batch effects. Dimensionality reduction was conducted via principal component analysis (PCA). Clustering was performed using Uniform Manifold Approximation and Projection (UMAP) based on the top 20 principal components (as determined by elbow plots), followed by cluster identification with FindClusters (resolution=0.5, method=‘igraph’). Cell type identities were assigned using scSorter41 with a manually curated list of canonical marker genes. Assignments were refined by visual inspection of marker gene expression across clusters.

Cluster-specific differential gene expression analysis was performed using the FindMarkers function in Seurat with the Wilcoxon rank-sum test (test.use=“wilcox”). Expression levels of selected marker genes across telocyte progenitor cell clusters and smooth muscle cell clusters were visualized using violin plots. Statistical comparisons were performed using the stat_compare_means function from the rstatix R package42, applying Wilcoxon rank-sum tests for significance testing.

For RNA velocity analysis, spliced and unspliced reads were counted using Velocyto (version 0.17.17)43 based on the CellRanger output files. RNA velocity was then calculated with scVelo (version 0.3.3)22 using the stochastic model for gene-specific velocity estimation. Neighborhood graphs were computed using principal components derived from the integrated Seurat pipeline described above, and velocities were projected onto the same UMAP embedding to visualize predicted transitions between cell states. To better highlight the transcriptional dynamics within the telocyte progenitor compartment, velocity calculations were recomputed on selected clusters.

After QC filtering, the full dataset contained 8120 control and 7548 Foxl1-null cells. We also performed gene-expression analyses on selected combinations of mesenchymal clusters. The numbers of cells included in these clusters were as follows: Telocyte progenitor 1 (918 control, 502 mutant), Telocyte progenitor 2 (468 control, 478 mutant), Mesenchymal progenitor 1 (642 control, 827 mutant), Mesenchymal progenitor 2 (440 control, 302 mutant), and Smooth muscle (1293 control, 1375 mutant; combining Smooth muscle-1 and Smooth muscle-2). The specific cluster combinations analyzed are indicated in the corresponding figure panels. The analysis workflow used here is available via Zenodo (https://zenodo.org/records/17713171).

Cut&run and DNA product sequencing
Timed embryos were obtained by crossing Foxl1 heterozygous mice with C57BL/6 female mice. The entire length of the small intestine was digested with collagenase type II and DNase I dissolved in ‘Dispase in DMEM/F-12’ (Stemcell, 07923) to reach the single-cell state. Subsequently, the cells were sorted in phosphate-buffered saline with 0.5% BSA to enrich for 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, 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).

Whole-mount immunostaining
Fetal small intestine segments were fixed in 4% PFA at 4 °C overnight, then rehydrated in PBS. Tissues were permeabilized and blocked in 1× blocking buffer (ab126587) for 24 h at 4 °C. Tissues were incubated with the primary antibody (anti-Pdgfra, R&D Systems, AF1062) in the same buffer for 48 h, followed by overnight washes in PBS containing 0.5% Triton X-100. Secondary antibodies were applied for 48 h, followed by overnight washes with 0.5% Triton X-100. Nuclei were counterstained with DAPI where indicated. Tissues were then dehydrated in methanol and cleared in a 1:2 mixture of benzyl alcohol and benzyl benzoate (BABB) through a series of brief exchanges. Cleared samples were mounted in Fast Wells (FW20 or 25 × 25 mm, Grace Biolabs) and imaged by z-stack confocal microscopy (Leica Stellaris 5).

Statistical analysis
For comparisons between two groups (WT vs. Foxl1 null), we used a two-tailed unpaired t-test with Welch’s correction using GraphPad Prism 10. Data are presented as mean±SEM unless otherwise indicated. Expression levels of selected marker genes across specific cell clusters were visualized using violin plots. Statistical comparisons were performed using the stat_compare_means function from the rstatix R package42, applying Wilcoxon rank-sum tests for significance testing.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Zhu et al. examine the role of FOXL1 in mouse intestinal villification. To do so, they revisit a previously described Foxl1 null mutant, which has defects in villus morphogenesis. While this study provides greater detail to the cellular and molecular underpinnings of the Foxl1 mutant phenotype and puts Foxl1 function into the context of recent literature, many results are difficult to interpret based on the data provided, leading to the overstatement of several conclusions. For example, many conclusions rely on single micrographs without quantification or descriptions regarding the number of replicates. Nonetheless, if the manuscript is sufficiently improved, it would be an important contribution to the field by adding another genetic link to the network of those contributing to villus formation – of which there are currently few. Beyond re-working the manuscript to incorporate adequate data quantification and the reporting of replicates, I have several major concerns that should be addressed prior to publication:\n1. Throughout the manuscript’s figures, it is not always apparent what region of the small intestine is being examined (e.g., proximal, middle, or distal). Given that villification happens in a wave along the length (Walton et al., 2012), it is essential that equivalent regions are being compared. Accordingly, the authors should adequately examine whether the defects in villification are consistent across the length, or whether specific regions are more affected by Foxl1 loss. Along these lines, there seems to be a degree of variation in phenotypes that is not discussed. For example, villi look more normal 2E, while completely dysmorphic in 5C. Thus, the cartoon schematic in Figure 7 is somewhat misleading, and the source of this variation should be discussed. Finally, the plane of section of micrographs can often lead to misinterpretation of phenotypes, and the SEM data shown is also difficult to interpret. The authors should consider using whole mount imaging to more adequately quantify villification (e.g., Maimets et al., Nature Comm 2022).\n2. There is insufficient information provided on how the scRNA-seq data was collected and processed. For example: downstream of the 10x collection, what softwares/pipelines were used to clean-up, process the data, and perform cell clustering? Was cell clustering performed on the combined mutant and control intestines, or just the controls? Is each sample intestine equally represented in the UMAPs (i.e., a supplemental figure should be included to show distribution of cells from each replicate)? Were the intestines derived from embryos of the same litter, and were they all collected on the same day? Are the UMAPs portraying both controls and mutants together? These points should be clearly stated in the text, figure, and legends. Finally, will these data be available to the public?\n3. Several potential misinterpretations are made from interpreting UMAPs. For example, telocyte prog 2 is labeled as not having Bmp expression, but this seems incorrect upon referencing the UMAP. Moreover, many claims are made about absolute gene expression from the UMAPs, but this is challenging to visualize when plotted this way. Other methods should be use to graphically represent these data (e.g. violin plots). Additionally, where relevant comparisons are being made, expression levels should be validated by other methods such as in situ hybridization, RNA scope, or qPCR.\n4. Figure 3A The statement, “and loss of Foxl1 does not affect telocyte progenitor number” is not adequately supported without quantification. It is challenging to decipher from a single micrograph, and moreover is confusing when the following figure contradicts this statement by showing a reduction in telocyte progenitors by scRNA-seq.\n5. Figure 3 – the indirect evidence of Foxl1 regulating Pdgfra is not convincing, and the statement “Expression of PDGFRa in telocyte progenitors is dependent on Foxl1” is not fully supported by the evidence evidence. Indeed, Pdgfra is seemingly still expressed in many of the cells, including telocyte prog 2 cells, based on the UMAP shown. If assumed that the scale is equivalent for both the null and the control, the levels of Pdgfra expression look similar, but that instead there are fewer cells that reach the state of high levels of Pdgfra expression. Thus, it seems that Foxl1 null mutants lack fully differentiated Pdgfra-high cells (Walton et al. 2012 and Huycke et al., 2024), and a graphical representation such as that shown in Figure 6E – but quantifying relative amounts of telocyte prog 1 vs. prog 2 in controls and mutants, might be more appropriate here. Similarly, it appears that Foxo3 levels are relatively similar between conditions. As noted above The authors should evaluate RNA expression by other means, such as in situ hybridization, RNA scope, or qPCR of Foxl1+ cells to draw a stronger correlation here.\n6. Figure 4C,D and E – The pSmad and Sox9 staining is very dim and difficult to see for interpretation. The authors should include the pSmad and Sox9-only channels with increased brightness. Cartoon schematics in E are difficult to accept from single images without quantification. The mutants should be examined at later stages to determine whether it is a delay in Bmp activity in the epithelium or remains absent.\n7. The authors propose that telocyte prog 1 and prog 2 are distinct cell types (Figure 7). As an alternative, the authors should consider that, instead, telocyte prog2 are precursors to prog1, linked to the multiple rounds of villificaiton that occur (Walton 2012), the first of which has occurred ed at E15.5. RNA velocity analysis using the latest Monocle package could be used to gain insight into the pseudotemporal relationship between these populations.\n8. If the epithelial proliferation and differentiation defects in Foxl1 mutants is due to the repression of the BMP pathway, as the authors claim, then they should test whether the application of exogenous BMP ligands can rescue these phenotypes in explant cultures of Foxl1 mutants.\n9. Model figure 7C make is look like the epithelium stratifies – is this true? In many of the images shown the epithelium appears columnar. This relates back to the earlier comment about phenotypic variation and differences across stages.\n10. The paper does not cite many highly relevant original articles investigating villification. To name a few: Paper should cite relevant primary literature and include in discussion of how the results fit together. These should certainly be included in the introduction and/or discussion and to give proper credit to many of the works that this study draws from. For example: 1) Walton et al., 2012 PNAS; 2) Shyer et al., 2013 Science; 3) Shyer et al., 2015 Cell; 4) Freddo et al. 2016 Integr. Biol 5)Guiu et al. 2019 Nature 6) Maimets et al., 2022 Nature Comm; 7) Huycke et al., 2024 Cell; ) 8) Hinant et al., 2024 PLOS\nAdditional comments:\n1. The graphic in Figure 4A is strikingly similar to a graphic from Shyer et al., 2015, yet this work is not referenced. Credit should be given to the original work that it is adapted from.\n2. Gene vs. Protein nomenclature should follow established standards for italicization and capitalization.\n3. The following statement is confusing, because the null fetuses should not express functional Foxl1: 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”. It would be more appropriate to say that the Foxl1 enhancers used in the reporter line are still active. “\n4. Figure 2I – seems to have epithelial marker but it’s not labeled in the figure.\n5. Figure 3D – it looks like Foxl1 expression is missing from the controls. Is this a real finding or a poor representative sample?\n6. Figure 4E – the colors in the figure should be labeled.\n7. Mentions multiple times that the role of PCP within the mesenchyme is novel and exciting, but this has been reported many times in other contexts. For example, work from Yamada et al., 2010 Dev Dynamics (intestine); Parmore et al., 2024 Dev Cell (lung); Gros et al., 2010 Current Biology (Limb); Zhang et al., 2020 Elife (lung).\n8. Figure S1 is listed as a supplemental figure and legend about the colon, but I can’t find anything about this referenced in the main text. What is supplemental figure 1, or is this an error?\n9. Figure 4 - What are “GPX3+ FLCs” in the following statement? Also, it is not clear that Bmp4 is reduced “In case of BMP4, expression also extends to Foxl1 negative GPX3+ FLCs; however, its levels are clearly reduced specifically in Foxl1-deficient telocytes” . GPX3 and FLC are never spelled out, and are also included in the UMAP plot in Figure 2.\n10. “These findings suggest unexpected bi-directional signaling of villus tip telocytes to both epithelium and neighboring mesenchymal cells.” I disagree that this is an unexpected finding. Walton et al., 2016 previously reported Bmp signaling in the mesenchyme to be important for villus formation.", "reviewer_2": "Foxl1 has been appreciated as a marker of telocytes which play key roles in mesenchymal-mesenchymal signaling as well as epithelial mesenchymal signaling that are critical in the process of villus patterning and formation. This manuscript examines the role of Foxl1 in this process by comparing Foxl1 null mutants with heterozygous littermates and begins to tease apart the mechanism of Foxl1 and how Foxl1 interacts with other key signaling pathways in villus development and maintenance. In addition, Foxl1 is noted to mark two telocyte progenitor populations which localize to the villus tip mesenchyme and to the mesenchyme adjacent to the intervillus epithelium. Multiple roles were identified for Foxl1 including indirectly stimulating Pdgfra expression, Bmp ligand expression and planar cell polarity transcripts like Wnt5a and Fat4. Additionally, Foxl1 binding in the promoter of Fat4 in regions marked by H3K4 methylation indicate a direct role for Foxl1 in PCP in the mesenchyme. The interactions above in the epithelium and mesenchyme combine to result in the lack of patterned distinct villi in the small intestine in Foxl1 null fetal intestines.\nThis manuscript adds to the understanding of villus patterning and emergence and findings help unite our understanding of mesenchymal cluster formation and mesenchymal organization with changes in the epithelium resulting in villus emergence, outgrowth and maturation.\nThe majority of claims are supported, minor suggested changes/additions would lend further support (noted in the list below).\nThe only major request is to add later stages of analysis to some portions to be sure that a developmental delay does not explain some of the phenotypes.\nThe methodology is sound and meets the expected standards in the field.\nDetail on where single cell data is stored, computational processing and algorithms used for single cell analysis and GSEA should be added.\nMinor points:\nOverall check capitalization and italics for proteins vs RNA or genes\nLine 63 shh --> Shh\nLine 57 “lcrypts”- likely typo --> crypts\nLine 89 & paragraph: the discussion of ridges is confusing. Ridges do not form in mouse intestines as they do in chicks, therefore “persistant ridges” past late gestation is not possible. Abnormal ridge formation seems a better description however these ridges are still not equivalent to those that form in chicks—based on the SEM panels, the majority of the ridge-like structures do not run around the entire circumference like they do in chicks.\nFigure 1, panel D and magnification- is this two villi that connect at the tips or is this plane of section where you’ve captured the center of a structure more like a ridge?\nRecognizing that it may be difficult to collect post-natal stages as most Foxl1 mutants die before weaning, did you collect mutant intestines after E18.5? If so, do villi manage to extend or do they always stay short? Are the abnormal ridges maintained after birth? Is crypt emergence also affected?\nFigure 1, Panel H & J- please note in figure legend what the yellow and red arrows indicate. These SEM images nicely show the lack of distinct, organized villi and the presence of seemingly unorganized ridge-like structures.\nLine 108- Supplementary Table 1 was missing, but has been provided\nLine 109- I think this should say Fig 2E\nFigure 2, panel E—Lama5, Lama2, Rspo2 and Rspo3 are not mentioned in the manuscript text. Perhaps the authors could elaborate on these panels or remove to focus on Pdgfra and the Bmp ligands?\nFigure 2, panel G: Foxl1 RNAscope is very difficult to see—perhaps a larger magnification/inset (like in I) would make it easier to see or using gray or a more contrasting color than red on the black background?\nFigure 2, panel I: First panel may be mislabeled as Glpr? Seems it should be DAPI/Sal1/Foxl1tdTomato\nFigure 2, panel J: Please make panel J larger—this is a very helpful graphic, but it is hard to read the small text and distinguish the small green text on the tan epithelium\nGlp1r is specific to telocyte progenitor -1 and Sal1 is expressed in both, did you find any markers specific to telocyte progenitor-2?\nIt might be helpful to show RNAscope or other localization of Gpx3, Col1a1 or another Gpx3 cluster marker to show if that is expanded in the Foxl1 null mice (ie is there a shift to Gpx3+ cells with decrease in telocyte progenitors?).\nLines 128-130- Please clarify conflicting statements: “loss of Foxl1 does not affect telocyte progenitor number (Fig. 3A). The UMAP plot shown in Fig. 3B clearly demonstrates that the telocyte progenitor 1 population is dramatically reduced in abundance in the Foxl1 null intestine.”\nFigure 3. It is hard to tell if there are similar number of telocyte progenitors between control and mutant intestines—because the mutants are delayed, it is difficult to compare the two. It also looks like there may be fewer Foxl1 negative mesenchymal cells next to the epithelium in mutants (still plenty of tdTomato expression from FoxL1 promoter, but maybe fewer cells overall). Is it possible that mesenchyme overall is not as expanded with loss of Foxl1 (like in the PDGFRa mutants, the early proliferation fails so there are fewer cells)? Perhaps quantifying would help clarify these questions- ex. Tomato+/DAPI+ vs vimentin+/DAPI+ and quantification of single cell (number of cells in each cluster as a proportion of total cells in the sample)?\nFigure 3, panel B- the blue and green boxes are not mentioned here in the text and epithelial changes (referred to in figure legend, green box) are also not mentioned here in the text. It might be helpful to have insets of control and null umaps as separate plots. It is difficult to see dots of each if they are overlapping.\nFigure 3- Is the intensity scale set the same in the feature plots for PDGFRa and Foxo3 in the control and the mutant in panels C and F and are the same number of cells compared for control and mutant?\nFigure 3, panel C- Assuming same numbers of cells were compared, I agree that there appear to be fewer cells expressing high levels of PDGFRa transcript in the mutant (quantification would make this more convincing), but it also seems that many of the cells expressing high PDGFRa have shifted to the GPX3+ cluster?\nFigure 3, panel D- These panels show very nice histology and overall pattern, but Foxl1-tdTomato is difficult to see. Perhaps an inset with magnified panel would help plus arrows pointing to villus cluster progenitor 1 and intervillus progenitor 2.\nFigure 3, panel F: Are the Foxo3+ cells shifted to the telocyte progenitor-2 and Gpx3 clusters?\nFigure 3. Are Glp2r and Sall1 expressed in Foxl1 mutants (in the RNA-seq or by RNAscope)?\nFigure 3, E. If you have a good Foxo3 antibody for cut and run, were you able to confirm Foxo3 binding in the Pdgfra promoter in mouse fetal intestines?\nFigure 3, F. You could also use RNAscope to confirm a reduction of Foxo3 localization in telocytes in the Foxl1 null intestines.\nLine 146- Bmp signaling within the mesenchyme is critical for cluster formation, not Bmp signaling to the epithelium. Clusters will still form even with Bmpr1a receptor deleted in the epithelium (reference 6) and when epithelium is removed entirely (as long as Hh is supplemented in the media) see Huycke et al., 2024 https://doi.org/10.1016/j.cell.2024.04.039\nFigure 4, panel A- one “Wnt” is reflected.\nFigure 4, panel B- Bmp1 is not part of the TGFb superfamily. Maybe leave it out here unless you will discuss its role further? Bmp1 is also expressed in the Gpx3 cluster. Bmp4 appears to be reduced and possibly shifted to the Gpx3 cluster. Quantification of the Bmp+ cells within each cluster as a proportion of total cells in the sample might help make these changes more obvious to appreciate.\nFigure 4, panel C- please add a magnified inset for the mutant in the top panel.\nLines 157-161 Bmp signaling within the mesenchyme is the basis of the manuscript in reference 6 and therefore is not an unexpected finding.\nFigure 4, panel D- Sox9 staining is very nice. Ki67 is overexposed- please replace the Ki67 panels with images with less exposure.\nS10 is nice!\n195- I was confused by the term “distal” here and was looking for a proximal-distal difference. Perhaps revising to note the epithelial thickness expands from the usual ~50 micron to ~100 micron thickness and then saying the luminal most cells (or apical side cells) undergo apoptosis?\nFigure 5C- Since the epithelium is flat (no villi) in both the control and mutant at this stage, the number of cells per 100 um should be comparable. You could count folds/DAPI, but I don’t think it is necessary.\nFigure 6G- It seems these counts are proportion relative to DAPI, please note proportion of what? Scale bars are missing.\nLine 202- it is not clear whether expression of Ki67 throughout the epithelium is because the null intestines are delayed in development, are abnormally continuing to divide and not withdrawing from the cell cycle as they would with villus emergence/lengthening, or because they are stuck in the cell cycle. You could test the rate of the cycle with spaced injections of BrdU and IdU, EdU or CldU to see how many positive nuclei there are in controls vs mutants. You could also look at various markers of cell division stages.\nLine 203-206 and 216-219 (Figure 6D&E)- The shift that is noted could be the result of delayed differentiation rather than a loss of these cell types. At later stages do the null intestines develop more differentiated epithelial cell types and are these cell types generated at proportions similar to controls?\nFigure 6, panel G- Please add panels for later stages to show whether the epithelial development is delayed in the null and they eventually form secretory and absorptive cell types or they continue to be absent even at later stages.\nLine 244-245- Is there a marker specific for the telocyte 2 progenitor that you can use to validate RNAscope localization of this cluster to the crypt adjacent mesenchymal domain?"}, "author_response": "Response to Reviewers’ Comments:\nWe thank the reviewers for the time and effort that have taken to carefully review our\nmanuscript. This is a case where scientific review clearly improved the manuscript. We\nhave performed multiple additional experiments, added 12 supplementary figures, and\nmodified all main figures and extensively revise the text. We also apologize for not\nhaving taken into account all of the relevant recent literature, and thank the reviewers\nfor enabling us to correct our error. We hope that you will find the manuscript\nsignificantly improved.\nFor the authors,\nKlaus H. Kaestner\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this manuscript, Zhu et al. examine the role of FOXL1 in mouse intestinal villification.\nTo do so, they revisit a previously described Foxl1 null mutant, which has defects in\nvillus morphogenesis. While this study provides greater detail to the cellular and\nmolecular underpinnings of the Foxl1 mutant phenotype and puts Foxl1 function into the\ncontext of recent literature, many results are difficult to interpret based on the data\nprovided, leading to the overstatement of several conclusions. For example, many\nconclusions rely on single micrographs without quantification or descriptions regarding\nthe number of replicates. Nonetheless, if the manuscript is sufficiently improved, it would\nbe an important contribution to the field by adding another genetic link to the network of\nthose contributing to villus formation – of which there are currently few.\nWe have added additional data and quantification to multiple figures, and added twelve\nsupplementary figures to support our conclusions.\nBeyond re-working the manuscript to incorporate adequate data quantification and the\nreporting of replicates, I have several major concerns that should be addressed prior to\npublication:\n1. Throughout the manuscript’s figures, it is not always apparent what region of the\nsmall intestine is being examined (e.g., proximal, middle, or distal). Given that\nvillification happens in a wave along the length (Walton et al., 2012), it is essential that\nequivalent regions are being compared. Accordingly, the authors should adequately\nexamine whether the defects in villification are consistent across the length, or whether\nspecific regions are more affected by Foxl1 loss. Along these lines, there seems to be a\ndegree of variation in phenotypes that is not discussed. For example, villi look more\nnormal 2E, while completely dysmorphic in 5C. Thus, the cartoon schematic in Figure 7\nis somewhat misleading, and the source of this variation should be discussed. Finally,\nthe plane of section of micrographs can often lead to misinterpretation of phenotypes,\n1\nand the SEM data shown is also difficult to interpret. The authors should consider using\nwhole mount imaging to more adequately quantify villification (e.g., Maimets et al.,\nNature Comm 2022).\nThe reviewer is of course correct that villification occurs in an anterior-posterior wave.\nWe have carefully matched the anatomical locations of our control and Foxl1 deficient\nsections. To address this issue specifically, we have added a new Supplemental Figure\n1, which compares proximal, middle, and distal small intestine from day 15.5 dpc\nfetuses between the two genotypes. This figure reproduces the AP wave of\ndifferentiation in control mice the reviewer pointed out, and shows further that Foxl1\nmutant mice show a delay in villification is most prominent in the proximal and middle\nportions of the small intestine, while no difference is seen in the distal small intestine,\nwhere villification has not yet commenced in the 15.5 dpc fetus. In addition,\nSupplementary Figure 2 documents villification from proximal to distal small intestine at\n18.5 dpc and postnatal day 5. We state in the manuscript that the deletion of Foxl1\ndelays but ultimately does not block villus formation. We have also added whole mount\nstaining of 15.5 dpc small intestine for Pdgfra, and included these data as new Fig. 3F.\n2. There is insufficient information provided on how the scRNA-seq data was collected\nand processed. For example: downstream of the 10x collection, what\nsoftwares/pipelines were used to clean-up, process the data, and perform cell\nclustering? Was cell clustering performed on the combined mutant and control\nintestines, or just the controls? Is each sample intestine equally represented in the\nUMAPs (i.e., a supplemental figure should be included to show distribution of cells from\neach replicate)? Were the intestines derived from embryos of the same litter, and were\nthey all collected on the same day? Are the UMAPs portraying both controls and\nmutants together? These points should be clearly stated in the text, figure, and legends.\nFinally, will these data be available to the public?\nWe apologize for our oversight. We have added full detail to the method section as\nrequested. Cell clustering was performed on the combined mutant and control\nintestines (Figure 2C, New Supplementary Figure 7). Fig. S7 also shows new UMAP\nplots of control only and mutant only cells. All six embryos came from the same litter\nand were processed on the same day to limit technical variability. The data will be\nmade available by December 2025, or sooner should the paper be accepted for\npublication.\n3. Several potential misinterpretations are made from interpreting UMAPs. For example,\ntelocyte prog 2 is labeled as not having Bmp expression, but this seems incorrect upon\nreferencing the UMAP. Moreover, many claims are made about absolute gene\nexpression from the UMAPs, but this is challenging to visualize when plotted this way.\nOther methods should be use to graphically represent these data (e.g. violin plots).\nAdditionally, where relevant comparisons are being made, expression levels should be\nvalidated by other methods such as in situ hybridization, RNA scope, or qPCR.\nplots to multiple figures (Fig. 2H, 2K; Fig. 3D; Fig. 4B; Fig. 5E; Fig. S6 and Fig. S8). We\nhave also validated differences in expression for the most relevant genes by either\nRNAscope or immunostaining (Sall1, Glp2r, Agr2, Muc2, Pdgfra).\n4. Figure 3A The statement, “and loss of Foxl1 does not affect telocyte progenitor\nnumber” is not adequately supported without quantification. It is challenging to decipher\nfrom a single micrograph, and moreover is confusing when the following figure\ncontradicts this statement by showing a reduction in telocyte progenitors by scRNA-seq.\nWe apologize for our oversight. We have quantified the number of Foxl1-TdTomato\npositive cells over multiple control and mutant animals, and provided a quantification in\nthe new panel Figure 3A.\nOf note, the UMAP leaves the impression that telocyte 1 progenitors are reduced\nin number, but they are actually only shifted in phenotype. Figure 7E, which quantifies\nthe various cell clusters for each scRNAseq data set also shows that, while the\nproportion of the two telocyte progenitor cells populations varies from fetus to fetus,\nthere is no systematic effect of genotype.\n5. Figure 3 – the indirect evidence of Foxl1 regulating Pdgfra is not convincing, and the\nstatement “Expression of PDGFRa in telocyte progenitors is dependent on Foxl1” is not\nfully supported by the evidence evidence. Indeed, Pdgfra is seemingly still expressed in\nmany of the cells, including telocyte prog 2 cells, based on the UMAP shown. If\nassumed that the scale is equivalent for both the null and the control, the levels of\nPdgfra expression look similar, but that instead there are fewer cells that reach the state\nof high levels of Pdgfra expression. Thus, it seems that Foxl1 null mutants lack fully\ndifferentiated Pdgfra-high cells (Walton et al. 2012 and Huycke et al., 2024), and a\ngraphical representation such as that shown in Figure 6E – but quantifying relative\namounts of telocyte prog 1 vs. prog 2 in controls and mutants, might be more\nappropriate here. Similarly, it appears that Foxo3 levels are relatively similar between\nconditions. As noted above The authors should evaluate RNA expression by other\nmeans, such as in situ hybridization, RNA scope, or qPCR of Foxl1+ cells to draw a\nstronger correlation here.\nFollowing the reviewer’s suggestion, we have changed the figure and modified our\nconclusions. Thus, we have replaced the UMAP in panel 3C with a new one that shows\nthe spread of the data values better, which makes the loss of Pdgfra high telocyte 1\nprogenitors more apparent. Secondly, we have added a new panel 3D which shows the\ndata as violin plots, which confirm the suspicion of the reviewer that Pdgfra expression\nis reduced only in the telocyte1 progenitor population, i.e. the Pdgfra high cells, but is\nnot significantly changed in the telocyte progenitor 2 population. Our claim that Pdgfra\nexpression is reduced in absence of Foxl1 is further supported by immunofluorescence\nstaining analysis, formerly panel D, now panel 3E, as well as whole mount staining of\nthe anterior small intestine with an antibody to Pdgfra, the new Figure 3F. Additional\n3\ndata on Pdgfra protein staining in 18.5 dpc fetuses confirm the reduced number of\nPdgfra-high cell clusters in the Foxl1 null mice.\nRegarding Foxo3, we were in error. We have now performed qRT-PCR analysis of\nFoxo3 and found no change in its expression level by genotype. Therefore, the\nproposed regulatory cascade – Foxl1 to Foxo3 to Pdgfra can no longer be supported.\nWe have deleted all related figures and text. We thank the reviewer from preventing us\nfrom publishing misleading information.\n6. Figure 4C,D and E – The pSmad and Sox9 staining is very dim and difficult to see for\ninterpretation. The authors should include the pSmad and Sox9-only channels with\nincreased brightness. Cartoon schematics in E are difficult to accept from single images\nwithout quantification. The mutants should be examined at later stages to determine\nwhether it is a delay in Bmp activity in the epithelium or remains absent.\nFollowing the reviewer’s suggestion, we have improved on our immunofluorescence\nimages. We find that the pSmad1/5 signal present in mesenchymal cells is higher than\nthat in villus epithelial cells. To aid visualization, we have added a Z-stack projection\nwithout DAPI co-staining of nuclei to make the pSmad1/5 more apparent. We stand by\nour original statement that pSmad1/5 signaling to the epithelium is reduced transiently\nin the absence of Foxl1, consistent with the well published role of BMP signaling during\ngut development, and the fact that several BMP mRNAsare significantly reduced in\ntelocyte progenitor 1 cells, as now documented by the new violin plots in panels 4B.\nWe have also improved on the Sox9 IF staining shown in panel 4D.\nOf note, as with other phenotypes, Sox9 staining becomes normalized as\ndevelopment progresses, as now shown in the new Fig. S10. By 18.5 dpc, Sox9\npositive cells extend only partly up the developing villi, and by postnatal day 5, Sox9\nexpression is confined to the nascent crypts, just as it is in control mice.\n7. The authors propose that telocyte prog 1 and prog 2 are distinct cell types (Figure 7).\nAs an alternative, the authors should consider that, instead, telocyte prog2 are\nprecursors to prog1, linked to the multiple rounds of villificaiton that occur (Walton\n2012), the first of which has occurred ed at E15.5. RNA velocity analysis using the latest\nMonocle package could be used to gain insight into the pseudotemporal relationship\nbetween these populations.\nWe thank the reviewer for this suggestion, and have performed pseudotime analysis\nbetween the two telocyte progenitor populations as suggested. These new data are\nnow included as Figure 2E. As predicted by the reviewer, telocyte 2 are indeed\nprogenitors to telocyte 1 cells. We thank the reviewer for this valuable suggestion.\n8. If the epithelial proliferation and differentiation defects in Foxl1 mutants is due to the\nrepression of the BMP pathway, as the authors claim, then they should test whether the\napplication of exogenous BMP ligands can rescue these phenotypes in explant cultures\nof Foxl1 mutants.\nFoxl1 deficient gut ‘self-normalizes’ with time, thus addition of BMP had no further\neffect.\n9. Model figure 7C make is look like the epithelium stratifies – is this true? In many of\nthe images shown the epithelium appears columnar. This relates back to the earlier\ncomment about phenotypic variation and differences across stages.\nAfter analyzing dozens of embryos, we conclude that some portions of the epithelium\nindeed remain pseudo-stratified in the absence of Foxl1 for longer than they do in wild\ntype mice. Eventually, the epithelium resolves into a columnar structure, as shown in\nSupplemental Figures 1 and 2. We have removed model Figure 7 in order not to\noversimplify our findings, and thus mislead the reader.\n10. The paper does not cite many highly relevant original articles investigating\nvillification. To name a few: Paper should cite relevant primary literature and include in\ndiscussion of how the results fit together. These should certainly be included in the\nintroduction and/or discussion and to give proper credit to many of the works that this\nstudy draws from. For example: 1) Walton et al., 2012 PNAS; 2) Shyer et al., 2013\nScience; 3) Shyer et al., 2015 Cell; 4) Freddo et al. 2016 Integr. Biol 5)Guiu et al. 2019\nNature 6) Maimets et al., 2022 Nature Comm; 7) Huycke et al., 2024 Cell; ) 8) Hinant et\nal., 2024 PLOS\nWe apologize for our oversight and have added these papers to the introduction, and\nexpanded the text to better approximate the state of the field..\nAdditional comments:\n1. The graphic in Figure 4A is strikingly similar to a graphic from Shyer et al., 2015, yet\nthis work is not referenced. Credit should be given to the original work that it is adapted\nfrom.\nWe apologize for our oversight and have now referenced this paper in the legend to\nFigure 4A.\n2. Gene vs. Protein nomenclature should follow established standards for italicization\nand capitalization.\nWe have improved on gene/protein nomenclature.\n3. The following statement is confusing, because the null fetuses should not express\nfunctional Foxl1: Note that in both control and Foxl1 null fetuses, Foxl1-expressing cells\nare localized to the mesodermal cell layer that is directly juxtaposed to the developing\n5\nepithelium”. It would be more appropriate to say that the Foxl1 enhancers used in the\nreporter line are still active. “\nThis is of course correct. We apologize for our error and have corrected the text.\n4. Figure 2I – seems to have epithelial marker but it’s not labeled in the figure.\nWe have removed the epithelial marker to enhance visualization of the Glp2r and Foxl1\nsignals.\n5. Figure 3D – it looks like Foxl1 expression is missing from the controls. Is this a real\nfinding or a poor representative sample?\nWe apologize for the poor quality of the figure, where overlay of multiple colors\nobscured the signal. As we have demonstrated Foxl1 expression elsewhere multiple\ntimes, we decided to remove this signal from the figure for clarity, which is now panel\n3E.\n6. Figure 4E – the colors in the figure should be labeled.\nWe have added the color scheme as requested.\n7. Mentions multiple times that the role of PCP within the mesenchyme is novel and\nexciting, but this has been reported many times in other contexts. For example, work\nfrom Yamada et al., 2010 Dev Dynamics (intestine); Parmore et al., 2024 Dev Cell\n(lung); Gros et al., 2010 Current Biology (Limb); Zhang et al., 2020 Elife (lung).\nWe apologize for our oversight. Rao-Bhatia and colleagues had represented their\nfindings as completely novel. We have removed any mention of the role of PCP within\nthe mesenchyme as novel and exciting.\n8. Figure S1 is listed as a supplemental figure and legend about the colon, but I can’t\nfind anything about this referenced in the main text. What is supplemental figure 1, or is\nthis an error?\nWe apologize for our error. This figure did not exist. It has no relationship to the 12 new\nsupplementary figures now added to the paper.\n9. Figure 4 - What are “GPX3+ FLCs” in the following statement? Also, it is not clear that\nBmp4 is reduced “In case of BMP4, expression also extends to Foxl1-negative mesenchymal\nprogenitor cells; however, its levels are clearly reduced specifically in Foxl1-deficient\ntelocytes” . GPX3 and FLC are never spelled out, and are also included in the UMAP plot in Figure\n2.\nclusters in the UMAP in panel 2C as “Mesenchymal progenitor 1 and 2”. While we are\ncertain of their mesenchymal nature and the fact that they do not express Foxl1, their\nfurther fate is unknown at this time. In all later panels and supplemental figures, this\ndesignation has replaced “GPX3+ FLCs”.\nThe point we attempted to make, albeit clumsily, was that while Foxl1+ telocytes\nexpress BMP proteins, there are additional Foxl1- mesenchymal cells that also\nelaborate some BMP mRNAs. By definition, only BMP production in Foxl1+ cells can\nbe Foxl1 dependent.\nWe have revised Figure 4 to include violin plots and statistical analysis to more\nclearly document where expression of certain BMPs is Foxl1 dependent, and where it is\nnot. We have included a non-relevant cell type (smooth muscle cells) and telocyte 2\nprogenitor cells to indicate that this is not a global effect, but specific to mostly telocyte 1\nprogenitors, or PdgfraHigh cells.\n10. “These findings suggest unexpected bi-directional signaling of villus tip telocytes to\nboth epithelium and neighboring mesenchymal cells.” I disagree that this is an\nunexpected finding. Walton et al., 2016 previously reported Bmp signaling in the\nmesenchyme to be important for villus formation.\nWe apologize for our oversight. Of course, Walton and colleagues had shown that villus\nclusters still form in the absence of epithelial BMP receptor. We have changed the text\naccordingly.\nReviewer #2 (Remarks to the Author):\nFoxl1 has been appreciated as a marker of telocytes which play key roles in\nmesenchymal-mesenchymal signaling as well as epithelial mesenchymal signaling that\nare critical in the process of villus patterning and formation. This manuscript examines\nthe role of Foxl1 in this process by comparing Foxl1 null mutants with heterozygous\nlittermates and begins to tease apart the mechanism of Foxl1 and how Foxl1 interacts\nwith other key signaling pathways in villus development and maintenance. In addition,\nFoxl1 is noted to mark two telocyte progenitor populations which localize to the villus tip\nmesenchyme and to the mesenchyme adjacent to the intervillus epithelium. Multiple\nroles were identified for Foxl1 including indirectly stimulating Pdgfra expression, Bmp\nligand expression and planar cell polarity transcripts like Wnt5a and Fat4. Additionally,\nFoxl1 binding in the promoter of Fat4 in regions marked by H3K4 methylation indicate a\ndirect role for Foxl1 in PCP in the mesenchyme. The interactions above in the\nepithelium and mesenchyme combine to result in the lack of patterned distinct villi in the\nsmall intestine in Foxl1 null fetal intestines.\nThis manuscript adds to the understanding of villus patterning and emergence and\nfindings help unite our understanding of mesenchymal cluster formation and\nmesenchymal organization with changes in the epithelium resulting in villus emergence,\noutgrowth and maturation.\nfurther support (noted in the list below).\nWe thank the reviewer for the overall positive assessment of our study.\nThe only major request is to add later stages of analysis to some portions to be sure\nthat a developmental delay does not explain some of the phenotypes.\nWe have added additional data points (New supplementary figures 2-4). In keeping\nwith the first publication on the Foxl1 null phenotype, postnatally the intestinal\ndevelopment ‘catches up’, thus, the developmental defect is not absolute.\nThe methodology is sound and meets the expected standards in the field.\nWe thank the reviewer for the overall positive assessment of our study.\nDetail on where single cell data is stored, computational processing and algorithms\nused for single cell analysis and GSEA should be added.\nWe have added the requested information.\nMinor points:\nOverall check capitalization and italics for proteins vs RNA or genes\nWe have addressed this issue.\nLine 63 shh --> Shh\nFixed.\nLine 57 “lcrypts”- likely typo --> crypts\nFixed.\nLine 89 & paragraph: the discussion of ridges is confusing. Ridges do not form in mouse\nintestines as they do in chicks, therefore “persistant ridges” past late gestation is not\npossible. Abnormal ridge formation seems a better description however these ridges are\nstill not equivalent to those that form in chicks—based on the SEM panels, the majority\nof the ridge-like structures do not run around the entire circumference like they do in\nchicks.\nplane of section where you’ve captured the center of a structure more like a ridge?\nRecognizing that it may be difficult to collect post-natal stages as most Foxl1 mutants\ndie before weaning, did you collect mutant intestines after E18.5? If so, do villi manage\nto extend or do they always stay short? Are the abnormal ridges maintained after birth?\nIs crypt emergence also affected?\nThis is an important issue. We have altered the text to refer instead to “aberrant ridge\nformation”. This phenomenon is transient, which we have also clarified in the text.\nFigure 1, Panel H & J- please note in figure legend what the yellow and red arrows\nindicate. These SEM images nicely show the lack of distinct, organized villi and the\npresence of seemingly unorganized ridge-like structures.\nWe have added an explanation of the arrows to the legend. Thank you for noticing our\noversight.\nLine 108- Supplementary Table 1 was missing, but has been provided\nFixed.\nLine 109- I think this should say Fig 2E\nWe have addressed this issue.\nFigure 2, panel E—Lama5, Lama2, Rspo2 and Rspo3 are not mentioned in the\nmanuscript text. Perhaps the authors could elaborate on these panels or remove to\nfocus on Pdgfra and the Bmp ligands?\nWe have removed the four genes from Figure 2, and focused instead on Pdgra and\nBmp ligands as suggested.\nFigure 2, panel G: Foxl1 RNAscope is very difficult to see—perhaps a larger\nmagnification/inset (like in I) would make it easier to see or using gray or a more\ncontrasting color than red on the black background?\nThank you for the suggestion. We have updated the panel to make the signal more\nobvious.\nFigure 2, panel I: First panel may be mislabeled as Glpr? Seems it should be\nDAPI/Sal1/Foxl1tdTomato\nWe apologize for our error and have fixed the issue. Thank you.\nhard to read the small text and distinguish the small green text on the tan epithelium\nGlp1r is specific to telocyte progenitor -1 and Sal1 is expressed in both, did you find any\nmarkers specific to telocyte progenitor-2?\nWe have increased the font size and the panel, which is now panel M. Unfortunately, we\nhave not found any marker specific for the telocyte progenitor 2 population.\nIt might be helpful to show RNAscope or other localization of Gpx3, Col1a1 or another\nGpx3 cluster marker to show if that is expanded in the Foxl1 null mice (ie is there a shift\nto Gpx3+ cells with decrease in telocyte progenitors?).\nTwo comments here. First, as suggested by reviewer 1, we have removed Gpx3 and\nCol1a1 as cluster markers, and replaced these with ‘mesenchymal progenitors 1 and 2’.\nBy quantification of the cell percentages in the scRNAseq data for each individual\nembryo (New Supplementary Figure 7E), we did not observe any change in the\nfrequency of these cell populations.\nLines 128-130- Please clarify conflicting statements: “loss of Foxl1 does not affect\ntelocyte progenitor number (Fig. 3A). The UMAP plot shown in Fig. 3B clearly\ndemonstrates that the telocyte progenitor 1 population is dramatically reduced in\nabundance in the Foxl1 null intestine.”\nReviewer 1 makes a related point. We have quantified the number of Foxl1-TdTomato\npositive cells over multiple control and mutant animals, and provided a quantification in\nthe new panel added to Figure 3A.\nOf note, the UMAP leaves the impression that telocyte 1 progenitors are reduced in\nnumber, but they are actually only shifted in phenotype. Cell frequency quantification\n(Supplementary Figure 7) shows variability of the telocyte progenitor cells by embryo,\nbut no systematic depletion in the Foxl1 null mice.\nFigure 3. It is hard to tell if there are similar number of telocyte progenitors between\ncontrol and mutant intestines—because the mutants are delayed, it is difficult to\ncompare the two. It also looks like there may be fewer Foxl1 negative mesenchymal\ncells next to the epithelium in mutants (still plenty of tdTomato expression from FoxL1\npromoter, but maybe fewer cells overall). Is it possible that mesenchyme overall is not\nas expanded with loss of Foxl1 (like in the PDGFRa mutants, the early proliferation fails\nso there are fewer cells)? Perhaps quantifying would help clarify these questions- ex.\nTomato+/DAPI+ vs vimentin+/DAPI+ and quantification of single cell (number of cells in\neach cluster as a proportion of total cells in the sample)?\nand mutant animals and provided a quantification in the new panel Figure 3A.\nFigure 3, panel B- the blue and green boxes are not mentioned here in the text and\nepithelial changes (referred to in figure legend, green box) are also not mentioned here\nin the text. It might be helpful to have insets of control and null umaps as separate plots.\nIt is difficult to see dots of each if they are overlapping.\nWe have added description of the two boxes to the text, and have added UMAPs for\ncontrol and mutant mice separately as supplemental figure 7B, C.\nFigure 3- Is the intensity scale set the same in the feature plots for PDGFRa and Foxo3\nin the control and the mutant in panels C and F and are the same number of cells\ncompared for control and mutant?\nThe reviewer likely refers to panels D and G. We have removed any data related to\nFoxo3, as qRT-PCR analysis did not confirm a difference in gene expression. For\nPdgfra, we have also added violin plots and statistical analysis (new Figure 3D) to show\nthat its expression in telocyte progenitor 1, but not 2, cells is decreased in the Foxl1\nmutants.\nFigure 3, panel C- Assuming same numbers of cells were compared, I agree that there\nappear to be fewer cells expressing high levels of PDGFRa transcript in the mutant\n(quantification would make this more convincing), but it also seems that many of the\ncells expressing high PDGFRa have shifted to the GPX3+ cluster?\nWe have adjusted these panels, and also added violin plots to make the quantitative\ndifference in gene expression more clear. We have also added whole mount staining\nfor Pdgfra in Figure 3F. We do not see the appearance of any other Pdgfra high cells in\nthe Foxl1 null intestine.\nFigure 3, panel D- These panels show very nice histology and overall pattern, but Foxl1-\ntdTomato is difficult to see. Perhaps an inset with magnified panel would help plus\narrows pointing to villus cluster progenitor 1 and intervillus progenitor 2.\nThis point was also made by Reviewer 1. We apologize for the poor quality of the figure,\nwhere overlay of multiple colors obscured the signal. As we have demonstrated Foxl1\nexpression elsewhere multiple times, we decided to remove this signal from the figure\nfor clarity, which is now panel 3E.\nFigure 3, panel F: Are the Foxo3+ cells shifted to the telocyte progenitor-2 and Gpx3\nclusters?\nconclusion that Foxl1 regulates Pdgfra via Foxo3.\nFigure 3. Are Glp2r and Sall1 expressed in Foxl1 mutants (in the RNA-seq or by\nRNAscope)?\nWe thank the reviewer for this suggestion. We find that both Glp2r and Sall1 expression\nare reduced specifically in telocyte progenitor 1 cells. These new data are included as\nSupplementary Figure 8.\nFigure 3, E. If you have a good Foxo3 antibody for cut and run, were you able to confirm\nFoxo3 binding in the Pdgfra promoter in mouse fetal intestines?\nWe tested multiple commercially available antibodies to Foxo3, unfortunately, none of\nthem worked for cut and run.\nFigure 3, F. You could also use RNAscope to confirm a reduction of Foxo3 localization\nin telocytes in the Foxl1 null intestines.\nWe have attempted RNAscope for Foxo3 but did not obtain a good signal, likely due to\nits low expression level. We quantified Foxo3 expression by qRT-PCR, and found that it\nis not changed in the absence of Foxl1. As stated above, we have removed all\nreference to Foxo3 from the paper.\nLine 146- Bmp signaling within the mesenchyme is critical for cluster formation, not Bmp\nsignaling to the epithelium. Clusters will still form even with Bmpr1a receptor deleted in\nthe epithelium (reference 6) and when epithelium is removed entirely (as long as Hh is\nsupplemented in the media) see Huycke et al.,\n2024 https://doi.org/10.1016/j.cell.2024.04.039\nWe apologize for our error and have revised the text.\nFigure 4, panel A- one “Wnt” is reflected.\nWe have modified the figure.\nFigure 4, panel B- Bmp1 is not part of the TGFb superfamily. Maybe leave it out here\nunless you will discuss its role further? Bmp1 is also expressed in the Gpx3 cluster.\nBmp4 appears to be reduced and possibly shifted to the Gpx3 cluster. Quantification of\nthe Bmp+ cells within each cluster as a proportion of total cells in the sample might help\nmake these changes more obvious to appreciate.\nWe have modified the figure, removed Bmp1, and added violin plots by cell cluster to\nmake the differences more clear. Statistical analysis also showed that Foxl1 is most\nimportant for Bmp expression in telocyte progenitor 1, or villus cluster cells.\nWe have modified the figure, now 4C, to include a violin plot and statistical analysis to\nmake the change in BMP4 mRNA in the telocyte 1 cluster more clear.\nLines 157-161 Bmp signaling within the mesenchyme is the basis of the manuscript in\nreference 6 and therefore is not an unexpected finding.\nWe have modified the text. Indeed, the critical role of Bmp signaling during gut\ndevelopment is well established. We apologize for our error.\nFigure 4, panel D- Sox9 staining in the mutant appears in this image to be mostly\nrestricted to the intervillus domain with just a few scattered cells outside the intervillus\ndomain (normal for delayed development?). Ki67 appears to not yet be restricted, but\nthis may be because development is delayed- it’s still not entirely restricted in the\ncontrols at E15.5. If you look at E18.5 (and later if you are able to collect some samples\nafter birth), do you still see Ki67 throughout the epithelium and scattered Sox9+ cells\noutside the intervillus domain in the mutants?\nWe have analyzed E18.5 embryos and postnatal day 5 newborns for Ki67 and Sox9. As\npredicted by the reviewer, by this time the epithelium has begun to normalize. These\nnew data are included as supplementary figure S10.\nLine 172- hyperproliferation may not be the only explanation for Ki67 expression\nthroughout the epithelium in the mutant. Ki67 in all epithelial cells may be reflective of a\ndevelopmental delay as the entire epithelium is Ki67+ before villus demarcation. This\nepithelium is also likely NOT truly multilayered. Grosse et al., 2011 and Yamada 2013\nshowed that the intestinal epithelium is pseudostratified and Wang et al., 2018 showed\nthat Wnt5a guides pathfinding of the basal process back to the basal side if it was not\nsplit evenly among daughter cells. The phenotype shown in Figure 5, panel A is similar\nto the Wnt5a mutants and the Cleaved Caspase 3 positive cells are likely cells being\nsloughed off after not reconnecting to the basement membrane- see Wang, 2018 Figure\n7, G-I.\nWe agree that the Ki67 expression in the presumptive villus epithelium at 15.5 in Foxl1\nmutants reflects the developmental delay in these mice. We have changed the wording\nto make this point clear.\nWe have added TUNEL staining (new Figure S11) to confirm the presence of apoptotic\ncells. Whether parts of the epithelium in Foxl1 null mice are transiently truly\n‘multilayered’ seems to be an issue of semantics. If a cell is not connected to the\nbasement membrane, at that moment the epithelium is multilayered. This is short-lived,\n13\nas the ‘connection-less’ epithelial cell undergoes apoptosis, similar to what was seen by\nWang et al. We have added the reference to Wang et al and described their key\nfindings in the text.\nLines 195-197- the lack of “re-orientation” in the mutants may reflect delayed\ndevelopment or just lack of organization overall in the mutants. Perhaps including an\nearlier stage to show orientation in control vs null would show whether there is\norientation/organization in the controls prior to cluster formation that the null intestines\nlack and including a later stage might show whether the mutant intestines are able to\norganize more as development proceeds.\nStaining for B-catenin may also help to clarify organization (outline the mesenchymal\ncells) and any differences in nuclear B-catenin might also illustrate altered Wnt signaling\nwith altered Bmp.\nWe agree that the phenotype of the Foxl1 null mice reflects a developmental delay.\nPostnatally, the epithelium is normalized, as shown in the new Supplemental Figures 2\nand 4.\nLine 202- it is not clear whether expression of Ki67 throughout the epithelium is\nbecause the null intestines are delayed in development, are abnormally continuing to\ndivide and not withdrawing from the cell cycle as they would with villus\nemergence/lengthening, or because they are stuck in the cell cycle. You could test the\nrate of the cycle with spaced injections of BrdU and IdU, EdU or CldU to see how many\npositive nuclei there are in controls vs mutants. You could also look at various markers\nof cell division stages.\nWe agree that the phenotype of the Foxl1 null mice reflects a developmental delay. We\nhave added the following statement to the Results section:\n“This likely reflects the developmental delay in these mice, as prior to villus\ndemarcation, all epithelial cells are proliferative. Of note, at later stages of development,\nboth the Sox9-positive epithelial domain as well as the proliferative zone normalize in\nFoxl1 null mice, consistent with the overall normalization of the histological phenotype\ndocumented above (Fig. S10).”\nWe have also performed staining of control and mutant fetal intestine with pHH3\nto mark cells in mitosis, and found no difference. In addition, we pulsed mice with\nspaced injections of IdU and CldU, as suggested by the reviewer, and found no\ndifference in the number of double positive or single positive epithelial cells. Given the\nlarge number of supplementary figures and the lack of differences, we opted not to\ninclude these data in the manuscript.\nLine 203-206 and 216-219 (Figure 6D&E)- The shift that is noted could be the result of\ndelayed differentiation rather than a loss of these cell types. At later stages do the null\nintestines develop more differentiated epithelial cell types and are these cell types\ngenerated at proportions similar to controls?\nmarkers in the 18.5 dpc small intestine, and found that by this time, the phenotype had\nnormalized. These new data are included as Supplementary Figure 12. We thank the\nreviewer for this suggestion.\nFigure 6, panel G- Please add panels for later stages to show whether the epithelial\ndevelopment is delayed in the null and they eventually form secretory and absorptive\ncell types or they continue to be absent even at later stages.\nWe have added two new panels to show AGR2 and MUC2 expression at E18.5 in\nSupplementary Figure 12.\nWe have also added new data on OLFM4 and CD44, markers of stem/progenitor cells\non both E15.5 and E18.5, which support the notion of transiently delayed separation of\nintervillus and villus epithelial cells in the Foxl1 mutant.\nLine 244-245- Is there a marker specific for the telocyte 2 progenitor that you can use to\nvalidate RNAscope localization of this cluster to the crypt adjacent mesenchymal\ndomain?\nUnfortunately, we could not identify a marker specific for telocyte progenitor 2 cells.\n1 had not further comments, below our responses to the comments by Reviewer 2.\nPrevious requests (from 1st submission):\n1) How many samples/embryos/litters are examined has not been included that I can\nfind in the text, figure legends or methods. Only 4 of the figure legends note numbers:\nSupplemental 9 notes N=3 and it’s not clear what the N refers to; Supplemental Figure\n12 notes N=4, but what does that mean? Same for Figure 3A and Figure 6J.\nWe apologize for our oversight and have added the sample number to all figures.\n2) The statistics used to determine significance are not described except for the single-\ncell violin plots.\nThank you for pointing this out. All WT versus Foxl1 null quantitative\ncomparisons were analyzed using a two-tailed unpaired t-test with Welch’s\ncorrection. We have added a description of the statistical methods to the Methods\nsection and indicated the specific test used in each figure legend.\n3) How many cells are included in each UMAP and analysis for control and mutant\nsamples?\nWe have added a new supplemental table 2 which includes this information. We\nincluded 8120 cells for control and 7548 cells for Foxl1 mutants.\n4) The code used to analyze the single-cell data should be shared somewhere (Zenodo\nfor example).\nThank you for the suggestion. The code has been shared via Zenodo;\nhttps://zenodo.org/records/17713171\n5) Scalebars are missing from many panels: In the main figures 4C, 4D, 5D, 6G-H\nWe apologize for our oversight and have added the missing scale bars.\n6) Wnt is still reflected in Figure 4A\nWe apologize for our oversight and have fixed this issue.\nRevision Requests:\nMajor issues:\n1) The data shown indicate that Foxl1 does NOT regulate PDGFRa. I am convinced that\nFoxl1 regulates the telocyte population which is marked by PDGFRa, but I think all of\nyour data is more indicative of PDGFRa expression itself being independent of Foxl1.\n• No Foxl1 binding sites found in cut and run\n• PDGFRa is expressed in more cells than FoxL1 (Supplemental Figure 5)\n• PDGFRa staining is seen in the Foxl1 mutants—if PDGFRa is Foxl1-dependent, it\nshould not be detected in the mutants (supplemental movies show strong PDGFRa\nstaining; figure 3 E and F show what appear to be lower levels, but stain is still present;\nSupplemental figure 9; Supplemental figure 11 (weaker, but still present).\nWith all due respect, we disagree with the reviewer on this point. Gene regulation in\nmammalian cells is complex, and frequently multiple transcription factors add in\ncombinatorial fashion to affect gene expression of their targets. Thus, it is common that\nif one upstream TF is deleted, the expression of downstream targets is not completely\nsilenced. The fact that we did not find Foxl1 binding sites by cut-and-run does not mean\nthat genetically, Foxl1 cannot be upstream of PDGFRa, as it is possible that Foxl1 acts\nthrough another TF. The fact that PDGFRa is expressed in other cell types also does\nnot preclude regulation by Foxl1 in telocyte progenitors, it simply means that in other\ncell types additional TFs can activate the gene. We do not agree with the last point of\nthe reviewer that “if PDGFRa is Foxl1-dependent, it should not be detected in the\nmutants”. As mentioned above, and also in the Discussion, other TFs, including the\nrelated Foxf1 protein, likely contribute to the complex regulation of PDGFRa. The data\nin Figure 3 clearly document reduced expression of PDGFRa on the mRNA and protein\nlevel in absence of Foxl1.\n2) Figure 3A. FoxF1-tdtomato+ cells should be normalized to DAPI. There are many\nmore cells per 100 um in the controls that have villi and therefore using 100 um long\nimages does not count equivalent numbers of cells between controls and mutants. The\nsingle-cell data seems to show decreased telocyte progenitors in the mutants\n(Supplemental Fig 7). It would also be good to count other telocyte markers as a\ncomparison-- are the Foxl1 mutant cells still telocyte progenitors or telocytes? Do you\nsee tdTomato (Foxl1 mutant cells) in both telocyte progenitor 1 and 2 populations or any\nother cell types?\nFollowing the reviewer’s suggestion, we have also normalized the FoxL1-tdTomato+\ncells per submucosal nuclei. We have added this new analysis of the data to Figure 3A\nas an additional panel.\n3) Line 152- 153: Sal1 appears to be Foxl1 independent as it is still expressed in the\nmutants (Supplemental 8).\nSimilar to Point 1, we disagree with the reviewer’s interpretation of the data. Yes, there\nis some residual expression of Sal1, likely driven by other TFs that bind the genes cis-\nregulatory elements, but the levels of Sal1 mRNA are clearly and significantly reduced\nin absence of Foxl1 (Figure S8D).\n4) Line 154- again, PDGFRa is shown to be independent of Foxl1 (noted in point 1\nabove).\nAnswered above, we disagree with the reviewer on this point. If PDGFRa were\nindependent of Foxl1, its mRNA and protein levels would be unchanged, but they are\nnot.\n5) The absence of clusters marked by PDGFRa indicates FoxL1 is affecting cluster\nformation. The presence of low PDGFRa expression in the mutants suggests that\nPDGFRa itself is independent of FoxL1 since it is still expressed in the absence of\nFoxl1. Again- PDGFRa high expression is seen when clusters form in the mutants at\nlater stages (S9).\nThis is one interpretation of the data, which we have added to the Discussion. However,\nanother likely possibility, as also stated in the text several times, that absence of Foxl1\nonly delays villus formation. This could be because other TFs, such as FoxF1, are\nactivated later during fetal development and can then compensate for the absence of\nFoxl1 and activate PDGFRa fully. There are many examples of partial redundancy and\noverlapping functions in mammalian transcription factor biology, for instance for the\nrelated FoxA clade of forkhead proteins.\nMinor issues/Questions/Comments:\nLine 108- proliferative areas are intervillus domains at the stages shown (E18.5)- crypts\nare not present yet.\nThank you for pointing this out, we have changed the text accordingly.\nA side, but interesting thought: the villi normalize in mutants around the time crypts are\nforming-- perhaps for future studies, this might indicate a switch in programming from\nvillus formation (program to emerge villi from a flat epithelium) to villus maintenance\n(migrating cells from the crypts)?\nIndeed an interesting suggestion for future studies. We are planning to perform\nconditional ablation of both Foxl1 and Foxf1, which might contribute to addressing this\nquestion.\nLine 122: How is Supplemental table 1 sorted- it seems to be alphabetical by cluster?\nShowing the mostly highly expressed genes per cluster at the top of each cluster list\nwould be helpful. Also showing the relative expression levels between the transcripts\nper cluster would be good. A table of differential gene expression between the controls\nand mutants per cluster (at least for the telocyte 1 and 2 populations) might be\ninformative.\nSupplementary table 1 is sorted by the degree of differential gene expression\n(enrichment of a given marker gene in a given cluster compared to all other cells.\nPlease also match the names of the clusters in the table to the UMAPs and other\nfigures in the manuscript. (Ex: GPX3+ FLC in table).\nYes, good suggestion. All of these were changed to ‘mesenchymal progenitor 1’ and\n‘mesenchymal progenitor 2’.\nIs proliferative state a possible delineator between clusters 1 and 2? Aggregated\ntelocytes (clusters) are non-proliferative while PDGFRa-high not yet clustered sub-\nepithelial cells are still proliferative—perhaps matches with proliferative marker\nexpression in single-cell data (Top2a, Birc5, Ki67, Pcna) would show this?\nNumber of cells analyzed for controls and mutants should be included throughout the\nsingle-cell analyses.\nThis is a good suggestion, and we have provided the expression levels of these four\nproliferation markers as new Supplementary Figure 5. Indeed, telocyte progenitor 2\ncells have much higher expression of the prolfieratin genes. This is also now mentioned\nin the results section. A new table with the number of cells analyzed has been added\nas Supplemental Table 2.\nFigure 2M- This schematic is confusing/ incomplete—perhaps showing a villus tip\ncluster and a nascent cluster will help clarify. Are you suggesting that emerged vs\nnascent clusters are the 2 telocyte progenitor populations or is it that aggregated vs\nunaggregated telocytes are the 2 populations? All markers noted are expressed in both\ndomains. Bmp localization is not shown in this manuscript. In other publications, Bmps\nare localized in the domains shown in the schematic as telocyte progenitor 2. Bmp\ntranscripts are also shown in the telocyte progenitor 2 population in panel F. Please\nremove Bmp+ and Bmp- from the schematic or show Bmp localization.\nWe have modified panel 2M following the reviewer’s suggestion.\nLine 150-151- please explain what you mean by the progenitor 1 population being\nshifted. Do you mean within the cluster that the mutant cells sit closer to the other\nclusters while the control cells are more towards the edge of the cluster? Or, do you\nmean the mutant progenitor 1 population is becoming progenitor 2 or moving to another\ncluster?\nThe position of the progenitor 1 population is slightly shifted within the UMAP plot only,\nsuggesting changes in transcriptome profile. We have altered the text to make this point\nmore clear.\nLine 152- the overall distribution in S7 looks different among samples and I am not\nconvinced from this figure that the overall distributions are not different between control\nand mutant. I think this is partially addressed in rebuttal comments noting that changes\nare not genotype specific, but rather there appears to be variability between all samples.\nPerhaps you could just say that all cell types noted in controls are also present in the\nmutants?\nAgreed, all cell types are still present in the mutants, it is just that they expression profile\nis slightly altered, hence their position in the UMAP plot is shifted. We have changed the\ntext as suggested by the reviewer to state that “all cell types are present in all fetuses\nanalyzed”.\nFigure 3 legend- “(A) Foxl1-positive telocytes, labeled by tdTomato expression (red),\nare retained in Foxl1 null mice” is confusing because Foxl1 is replaced by tdTomato and\nFoxl1 RNA/protein is not expressed.\nAgreed, we have changed the text of the legend to match what is written in the results\nsection.\nLine 169- you should add the references for where you built the model for Bmp\ninhibiting Wnt in the adjacent epithelium. Fig 4A- The lowest Wnt on the left is reversed.\n“In the case of BMP4, expression also extends to Foxl1-negative mesenchymal\nprogenitor cells; however, its levels are clearly reduced specifically in Foxl1-deficient\ntelocytes (Fig. 4B)” would be more convincing if you showed unchanged Bmp\nexpression in a cluster that express higher levels of Bmp4 in the violin plots (Bmp4 is\nvery low or not detected in smooth muscle from what I can see in the violin plot) or\nmaybe you could show Bmp4 expression in the whole UMAP that shows all the\nclusters?\nReference 10 is cited in the text. For Figure 4A, we have fixed the “Wnt”. For panel 4B\nwe have replaced the “smooth muscle cell’ cluster with the ‘mesenchymal progenitor 1’\ncluster, which has much higher BMP4 expression. BMP4 expression in ‘mesenchymal\nprogenitor 1’ is not affected by genotype as expected, as these cells are Foxl1 negative\neven in wild type embryos.\nFigure 4C should be shown with panels of individual channels. It is difficult to see the\ngreen p-SMAD stain with DAPI overlaid. It is still difficult to see in the lower panels\nwithout DAPI because of the ECAD. The bottom right panel for the Foxl1 null\nreconstruction is tilted so it appears blurry—please replace this one. I find the schematic\nmisleading as I see quite a few more p-SMAD1/5+ cells in the mutant mesenchyme\nthan the schematic would indicate. If you want to say there is less Bmp signaling there,\nyou need to count mesenchymal pSMAD1/5+ cells normalized to DAPI. There also\nseems to be green signal behind the epithelium in the mutants that is not shown in the\nschematic.\nIt would be nice to include the Foxl1-pSMAD1/5 co-expressing cells in the control\nmesenchyme in your model.\nAre the yellow and blue arrows indicating Bmp ligand being secreted from the FoxL1+\ncells? Please note in figure legend.\nWe have replaced Figure 4C with a better pSMAD stain and better panels and have\naltered the model as requested.\nLine 177- the mutant epithelium does not appear to be devoid of pSMAD1/5 signal, but\nwith edits above, you may be able to show that there are fewer pSMAD1/5+ cells in the\nepithelium.\nWe have changed the text to state that there are fewer pSMAD1/5 cells in the\nepithelium.\nLine 184- add reference for why you expected loss of Bmp  loss of Wnt inhibition\nWe have added the reference.\nFigure 4D- Sox9 staining is very nice. Ki67 is overexposed- please replace the Ki67\npanels with images with less exposure. S10 is nice!\nWe have replaced figure 4D as requested with new images.\n195- I was confused by the term “distal” here and was looking for a proximal-distal\ndifference. Perhaps revising to note the epithelial thickness expands from the usual ~50\nmicron to ~100 micron thickness and then saying the luminal most cells (or apical side\ncells) undergo apoptosis?\nCorrect, we meant luminal most and were not referring to anterior posterior aspect of\nthe gut. We have changed the text accordingly.\nFigure 5C- Since the epithelium is flat (no villi) in both the control and mutant at this\nstage, the number of cells per 100 um should be comparable. You could count\nfolds/DAPI, but I don’t think it is necessary.\nWe believe the data are quite clear that at E14.5 there are fewer T-folds.\nFigure 6G- It seems these counts are proportion relative to DAPI, please note\nproportion of what? Scale bars are missing.\nScale bars have been added, and yes, the proportion of proliferating cells is the fraction\nof DAPI/E-cadherin positive cells, which we have now noted in the legend."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have appropriately addressed my previous critiques.", "reviewer_2": "** I strongly recommend publication, but I believe the issues noted below should be addressed. All points should be minor\ntext revisions and/or re-analysis of existing data.**\nSorry this took me longer to review. There seem to be many additions and updates which greatly improve the manuscript,\nbut it took me a while to go through it all! Most of my concerns on the original submission have been addressed with this\nrevision. A few issues were noted to have been corrected in the rebuttal, but I’m not finding the corrections. These should be\ncorrected prior to publication:\nPrevious requests (from 1st submission):\n1) How many samples/embryos/litters are examined has not been included that I can find in the text, figure legends or\nmethods. Only 4 of the figure legends note numbers: Supplemental 9 notes N=3 and it’s not clear what the N refers to;\nSupplemental Figure 12 notes N=4, but what does that mean? Same for Figure 3A and Figure 6J.\n2) The statistics used to determine significance are not described except for the single-cell violin plots.\n3) How many cells are included in each UMAP and analysis for control and mutant samples?\n4) The code used to analyze the single-cell data should be shared somewhere (Zenodo for example).\n5) Scalebars are missing from many panels: In the main figures 4C, 4D, 5D, 6G-H\n6) Wnt is still reflected in Figure 4A\nRevision Requests:\nMajor issues:\n1) The data shown indicate that Foxl1 does NOT regulate PDGFRa. I am convinced that Foxl1 regulates the telocyte\npopulation which is marked by PDGFRa, but I think all of your data is more indicative of PDGFRa expression itself being\nindependent of Foxl1.\n• No Foxl1 binding sites found in cut and run\n• PDGFRa is expressed in more cells than FoxL1 (Supplemental Figure 5)\n• PDGFRa staining is seen in the Foxl1 mutants—if PDGFRa is Foxl1-dependent, it should not be detected in the mutants\n(supplemental movies show strong PDGFRa staining; figure 3 E and F show what appear to be lower levels, but stain is still\npresent; Supplemental figure 9; Supplemental figure 11 (weaker, but still present).\n2) Figure 3A. FoxF1-tdtomato+ cells should be normalized to DAPI. There are many more cells per 100 um in the controls\nthat have villi and therefore using 100 um long images does not count equivalent numbers of cells between controls and\nmutants. The single-cell data seems to show decreased telocyte progenitors in the mutants (Supplemental Fig 7). It would\nalso be good to count other telocyte markers as a comparison-- are the Foxl1 mutant cells still telocyte progenitors or\ntelocytes? Do you see tdTomato (Foxl1 mutant cells) in both telocyte progenitor 1 and 2 populations or any other cell types?\n3) Line 152- 153: Sal1 appears to be Foxl1 independent as it is still expressed in the mutants (Supplemental 8).\n4) Line 154- again, PDGFRa is shown to be independent of Foxl1 (noted in point 1 above).\n5) The absence of clusters marked by PDGFRa indicates FoxL1 is affecting cluster formation. The presence of low PDGFRa\nexpression in the mutants suggests that PDGFRa itself is independent of FoxL1 since it is still expressed in the absence of\nFoxl1. Again- PDGFRa high expression is seen when clusters form in the mutants at later stages (S9).\nMinor issues/Questions/Comments:\nLine 108- proliferative areas are intervillus domains at the stages shown (E18.5)- crypts are not present yet.\nA side, but interesting thought: the villi normalize in mutants around the time crypts are forming-- perhaps for future studies,\nthis might indicate a switch in programming from villus formation (program to emerge villi from a flat epithelium) to villus\nmaintenance (migrating cells from the crypts)?\nLine 122: How is Supplemental table 1 sorted- it seems to be alphabetical by cluster? Showing the mostly highly expressed\ngenes per cluster at the top of each cluster list would be helpful. Also showing the relative expression levels between the\ntranscripts per cluster would be good. A table of differential gene expression between the controls and mutants per cluster\n(at least for the telocyte 1 and 2 populations) might be informative.\nPlease also match the names of the clusters in the table to the UMAPs and other figures in the manuscript. (Ex: GPX3+ FLC\nin table).\nIs proliferative state a possible delineator between clusters 1 and 2? Aggregated telocytes (clusters) are non-proliferative\nwhile PDGFRa-high not yet clustered sub-epithelial cells are still proliferative—perhaps matches with proliferative marker\nexpression in single-cell data (Top2a, Birc5, Ki67, Pcna) would show this?\nNumber of cells analyzed for controls and mutants should be included throughout the\nsingle-cell analyses.\nFigure 2M- This schematic is confusing/ incomplete—perhaps showing a villus tip cluster and a nascent cluster will help\nclarify. Are you suggesting that emerged vs nascent clusters are the 2 telocyte progenitor populations or is it that aggregated\nvs unaggregated telocytes are the 2 populations? All markers noted are expressed in both domains. Bmp localization is not\nshown in this manuscript. In other publications, Bmps are localized in the domains shown in the schematic as telocyte\nprogenitor 2. Bmp transcripts are also shown in the telocyte progenitor 2 population in panel F. Please remove Bmp+ and\nBmp- from the schematic or show Bmp localization.\nLine 150-151- please explain what you mean by the progenitor 1 population being shifted. Do you mean within the cluster\nthat the mutant cells sit closer to the other clusters while the control cells are more towards the edge of the cluster? Or,\ndo you mean the mutant progenitor 1 population is becoming progenitor 2 or moving to another cluster?\nLine 152- the overall distribution in S7 looks different among samples and I am not convinced from this figure that the overall\ndistributions are not different between control and mutant. I think this is partially addressed in rebuttal comments noting that\nchanges are not genotype specific, but rather there appears to be variability between all samples. Perhaps you could just\nsay that all cell types noted in controls are also present in the mutants?\nFigure 3 legend- “(A) Foxl1-positive telocytes, labeled by tdTomato expression (red), are retained in Foxl1 null mice” is\nconfusing because Foxl1 is replaced by tdTomato and Foxl1 RNA/protein is not expressed.\nLine 169- you should add the references for where you built the model for Bmp inhibiting Wnt in the adjacent epithelium.\nFig 4A- The lowest Wnt on the left is reversed.\n“In the case of BMP4, expression also extends to Foxl1-negative mesenchymal progenitor cells; however, its levels are\nclearly reduced specifically in Foxl1-deficient telocytes (Fig. 4B)” would be more convincing if you showed unchanged Bmp\nexpression in a cluster that express higher levels of Bmp4 in the violin plots (Bmp4 is very low or not detected in smooth\nmuscle from what I can see in the violin plot) or maybe you could show Bmp4 expression in the whole UMAP that shows all\nthe clusters?\nFigure 4C should be shown with panels of individual channels. It is difficult to see the green p-SMAD stain with DAPI\noverlaid. It is still difficult to see in the lower panels without DAPI because of the ECAD. The bottom right panel for the Foxl1\nnull reconstruction is tilted so it appears blurry—please replace this one. I find the schematic misleading as I see quite a few\nmore p-SMAD1/5+ cells in the mutant mesenchyme than the schematic would indicate. If you want to say there is less Bmp\nsignaling there, you need to count mesenchymal pSMAD1/5+ cells normalized to DAPI. There also seems to be green\nsignal behind the epithelium in the mutants that is not shown in the schematic.\nIt would be nice to include the Foxl1-pSMAD1/5 co-expressing cells in the control mesenchyme in your model.\nAre the yellow and blue arrows indicating Bmp ligand being secreted from the FoxL1+ cells? Please note in figure legend.\nLine 177- the mutant epithelium does not appear to be devoid of pSMAD1/5 signal, but with edits above, you may be able to\nshow that there are fewer pSMAD1/5+ cells in the epithelium.\nLine 184- add reference for why you expected loss of Bmp   loss of Wnt inhibition\nFigure 4D- Sox9 staining is very nice. Ki67 is overexposed- please replace the Ki67 panels with images with less exposure.\nS10 is nice!\n195- I was confused by the term “distal” here and was looking for a proximal-distal difference. Perhaps revising to note the\nepithelial thickness expands from the usual ~50 micron to ~100 micron thickness and then saying the luminal most cells (or\napical side cells) undergo apoptosis?\nFigure 5C- Since the epithelium is flat (no villi) in both the control and mutant at this stage, the number of cells per 100 um\nshould be comparable. You could count folds/DAPI, but I don’t think it is necessary.\nFigure 6G- It seems these counts are proportion relative to DAPI, please note proportion of what? Scale bars are missing.", "reviewer_author_response": "We thank the reviewers for taken the time to evaluate our revised manuscript. Reviewer\n1 had not further comments, below our responses to the comments by Reviewer 2.\nPrevious requests (from 1st submission):\n1) How many samples/embryos/litters are examined has not been included that I can\nfind in the text, figure legends or methods. Only 4 of the figure legends note numbers:\nSupplemental 9 notes N=3 and it’s not clear what the N refers to; Supplemental Figure\n12 notes N=4, but what does that mean? Same for Figure 3A and Figure 6J.\nWe apologize for our oversight and have added the sample number to all figures.\n2) The statistics used to determine significance are not described except for the single-\ncell violin plots.\nThank you for pointing this out. All WT versus Foxl1 null quantitative\ncomparisons were analyzed using a two-tailed unpaired t-test with Welch’s\ncorrection. We have added a description of the statistical methods to the Methods\nsection and indicated the specific test used in each figure legend.\n3) How many cells are included in each UMAP and analysis for control and mutant\nsamples?\nWe have added a new supplemental table 2 which includes this information. We\nincluded 8120 cells for control and 7548 cells for Foxl1 mutants.\n4) The code used to analyze the single-cell data should be shared somewhere (Zenodo\nfor example).\nThank you for the suggestion. The code has been shared via Zenodo;\nhttps://zenodo.org/records/17713171\n5) Scalebars are missing from many panels: In the main figures 4C, 4D, 5D, 6G-H\nWe apologize for our oversight and have added the missing scale bars.\n6) Wnt is still reflected in Figure 4A\nWe apologize for our oversight and have fixed this issue.\nRevision Requests:\nMajor issues:\n1) The data shown indicate that Foxl1 does NOT regulate PDGFRa. I am convinced that\nFoxl1 regulates the telocyte population which is marked by PDGFRa, but I think all of\nyour data is more indicative of PDGFRa expression itself being independent of Foxl1.\n• No Foxl1 binding sites found in cut and run\n• PDGFRa is expressed in more cells than FoxL1 (Supplemental Figure 5)\n• PDGFRa staining is seen in the Foxl1 mutants—if PDGFRa is Foxl1-dependent, it\nshould not be detected in the mutants (supplemental movies show strong PDGFRa\nstaining; figure 3 E and F show what appear to be lower levels, but stain is still present;\nSupplemental figure 9; Supplemental figure 11 (weaker, but still present).\nWith all due respect, we disagree with the reviewer on this point. Gene regulation in\nmammalian cells is complex, and frequently multiple transcription factors add in\ncombinatorial fashion to affect gene expression of their targets. Thus, it is common that\nif one upstream TF is deleted, the expression of downstream targets is not completely\nsilenced. The fact that we did not find Foxl1 binding sites by cut-and-run does not mean\nthat genetically, Foxl1 cannot be upstream of PDGFRa, as it is possible that Foxl1 acts\nthrough another TF. The fact that PDGFRa is expressed in other cell types also does\nnot preclude regulation by Foxl1 in telocyte progenitors, it simply means that in other\ncell types additional TFs can activate the gene. We do not agree with the last point of\nthe reviewer that “if PDGFRa is Foxl1-dependent, it should not be detected in the\nmutants”. As mentioned above, and also in the Discussion, other TFs, including the\nrelated Foxf1 protein, likely contribute to the complex regulation of PDGFRa. The data\nin Figure 3 clearly document reduced expression of PDGFRa on the mRNA and protein\nlevel in absence of Foxl1.\n2) Figure 3A. FoxF1-tdtomato+ cells should be normalized to DAPI. There are many\nmore cells per 100 um in the controls that have villi and therefore using 100 um long\nimages does not count equivalent numbers of cells between controls and mutants. The\nsingle-cell data seems to show decreased telocyte progenitors in the mutants\n(Supplemental Fig 7). It would also be good to count other telocyte markers as a\ncomparison-- are the Foxl1 mutant cells still telocyte progenitors or telocytes? Do you\nsee tdTomato (Foxl1 mutant cells) in both telocyte progenitor 1 and 2 populations or any\nother cell types?\nFollowing the reviewer’s suggestion, we have also normalized the FoxL1-tdTomato+\ncells per submucosal nuclei. We have added this new analysis of the data to Figure 3A\nas an additional panel.\n3) Line 152- 153: Sal1 appears to be Foxl1 independent as it is still expressed in the\nmutants (Supplemental 8).\nSimilar to Point 1, we disagree with the reviewer’s interpretation of the data. Yes, there\nis some residual expression of Sal1, likely driven by other TFs that bind the genes cis-\nregulatory elements, but the levels of Sal1 mRNA are clearly and significantly reduced\nin absence of Foxl1 (Figure S8D).\n4) Line 154- again, PDGFRa is shown to be independent of Foxl1 (noted in point 1\nabove).\nAnswered above, we disagree with the reviewer on this point. If PDGFRa were\nindependent of Foxl1, its mRNA and protein levels would be unchanged, but they are\nnot.\n5) The absence of clusters marked by PDGFRa indicates FoxL1 is affecting cluster\nformation. The presence of low PDGFRa expression in the mutants suggests that\nPDGFRa itself is independent of Foxl1 since it is still expressed in the absence of\nFoxl1. Again- PDGFRa high expression is seen when clusters form in the mutants at\nlater stages (S9).\nThis is one interpretation of the data, which we have added to the Discussion. However,\nanother likely possibility, as also stated in the text several times, that absence of Foxl1\nonly delays villus formation. This could be because other TFs, such as FoxF1, are\nactivated later during fetal development and can then compensate for the absence of\nFoxl1 and activate PDGFRa fully. There are many examples of partial redundancy and\noverlapping functions in mammalian transcription factor biology, for instance for the\nrelated FoxA clade of forkhead proteins.\nMinor issues/Questions/Comments:\nLine 108- proliferative areas are intervillus domains at the stages shown (E18.5)- crypts\nare not present yet.\nThank you for pointing this out, we have changed the text accordingly.\nA side, but interesting thought: the villi normalize in mutants around the time crypts are\nforming-- perhaps for future studies, this might indicate a switch in programming from\nvillus formation (program to emerge villi from a flat epithelium) to villus maintenance\n(migrating cells from the crypts)?\nIndeed an interesting suggestion for future studies. We are planning to perform\nconditional ablation of both Foxl1 and Foxf1, which might contribute to addressing this\nquestion.\nLine 122: How is Supplemental table 1 sorted- it seems to be alphabetical by cluster?\nShowing the mostly highly expressed genes per cluster at the top of each cluster list\nwould be helpful. Also showing the relative expression levels between the transcripts\nper cluster would be good. A table of differential gene expression between the controls\nand mutants per cluster (at least for the telocyte 1 and 2 populations) might be\ninformative.\nSupplementary table 1 is sorted by the degree of differential gene expression\n(enrichment of a given marker gene in a given cluster compared to all other cells.\nPlease also match the names of the clusters in the table to the UMAPs and other\nfigures in the manuscript. (Ex: GPX3+ FLC in table).\nYes, good suggestion. All of these were changed to ‘mesenchymal progenitor 1’ and\n‘mesenchymal progenitor 2’.\nIs proliferative state a possible delineator between clusters 1 and 2? Aggregated\ntelocytes (clusters) are non-proliferative while PDGFRa-high not yet clustered sub-\nepithelial cells are still proliferative—perhaps matches with proliferative marker\nexpression in single-cell data (Top2a, Birc5, Ki67, Pcna) would show this?\nNumber of cells analyzed for controls and mutants should be included throughout the\nsingle-cell analyses.\nThis is a good suggestion, and we have provided the expression levels of these four\nproliferation markers as new Supplementary Figure 5. Indeed, telocyte progenitor 2\ncells have much higher expression of the prolfieratin genes. This is also now mentioned\nin the results section. A new table with the number of cells analyzed has been added\nas Supplemental Table 2.\nFigure 2M- This schematic is confusing/ incomplete—perhaps showing a villus tip\ncluster and a nascent cluster will help clarify. Are you suggesting that emerged vs\nnascent clusters are the 2 telocyte progenitor populations or is it that aggregated vs\nunaggregated telocytes are the 2 populations? All markers noted are expressed in both\ndomains. Bmp localization is not shown in this manuscript. In other publications, Bmps\nare localized in the domains shown in the schematic as telocyte progenitor 2. Bmp\ntranscripts are also shown in the telocyte progenitor 2 population in panel F. Please\nremove Bmp+ and Bmp- from the schematic or show Bmp localization.\nWe have modified panel 2M following the reviewer’s suggestion.\nLine 150-151- please explain what you mean by the progenitor 1 population being\nshifted. Do you mean within the cluster that the mutant cells sit closer to the other\nclusters while the control cells are more towards the edge of the cluster? Or, do you\nmean the mutant progenitor 1 population is becoming progenitor 2 or moving to another\ncluster?\nThe position of the progenitor 1 population is slightly shifted within the UMAP plot only,\nsuggesting changes in transcriptome profile. We have altered the text to make this point\nmore clear.\nLine 152- the overall distribution in S7 looks different among samples and I am not\nconvinced from this figure that the overall distributions are not different between control\nand mutant. I think this is partially addressed in rebuttal comments noting that changes\nare not genotype specific, but rather there appears to be variability between all samples.\nPerhaps you could just say that all cell types noted in controls are also present in the\nmutants?\nAgreed, all cell types are still present in the mutants, it is just that they expression profile\nis slightly altered, hence their position in the UMAP plot is shifted. We have changed the\ntext as suggested by the reviewer to state that “all cell types are present in all fetuses\nanalyzed”.\nFigure 3 legend- “(A) Foxl1-positive telocytes, labeled by tdTomato expression (red),\nare retained in Foxl1 null mice” is confusing because Foxl1 is replaced by tdTomato and\nFoxl1 RNA/protein is not expressed.\nAgreed, we have changed the text of the legend to match what is written in the results\nsection.\nLine 169- you should add the references for where you built the model for Bmp\ninhibiting Wnt in the adjacent epithelium. Fig 4A- The lowest Wnt on the left is reversed.\n“In the case of BMP4, expression also extends to Foxl1-negative mesenchymal\nprogenitor cells; however, its levels are clearly reduced specifically in Foxl1-deficient\ntelocytes (Fig. 4B)” would be more convincing if you showed unchanged Bmp\nexpression in a cluster that express higher levels of Bmp4 in the violin plots (Bmp4 is\nvery low or not detected in smooth muscle from what I can see in the violin plot) or\nmaybe you could show Bmp4 expression in the whole UMAP that shows all the\nclusters?\nReference 10 is cited in the text. For Figure 4A, we have fixed the “Wnt”. For panel 4B\nwe have replaced the “smooth muscle cell’ cluster with the ‘mesenchymal progenitor 1’\ncluster, which has much higher BMP4 expression. BMP4 expression in ‘mesenchymal\nprogenitor 1’ is not affected by genotype as expected, as these cells are Foxl1 negative\neven in wild type embryos.\nFigure 4C should be shown with panels of individual channels. It is difficult to see the\ngreen p-SMAD stain with DAPI overlaid. It is still difficult to see in the lower panels\nwithout DAPI because of the ECAD. The bottom right panel for the Foxl1 null\nreconstruction is tilted so it appears blurry—please replace this one. I find the schematic\nmisleading as I see quite a few more p-SMAD1/5+ cells in the mutant mesenchyme\nthan the schematic would indicate. If you want to say there is less Bmp signaling there,\nyou need to count mesenchymal pSMAD1/5+ cells normalized to DAPI. There also\nseems to be green signal behind the epithelium in the mutants that is not shown in the\nschematic.\nIt would be nice to include the Foxl1-pSMAD1/5 co-expressing cells in the control\nmesenchyme in your model.\nAre the yellow and blue arrows indicating Bmp ligand being secreted from the FoxL1+\ncells? Please note in figure legend.\nWe have replaced Figure 4C with a better pSMAD stain and better panels and have\naltered the model as requested.\nLine 177- the mutant epithelium does not appear to be devoid of pSMAD1/5 signal, but\nwith edits above, you may be able to show that there are fewer pSMAD1/5+ cells in the\nepithelium.\nWe have changed the text to state that there are fewer pSMAD1/5 cells in the\nepithelium.\nLine 184- add reference for why you expected loss of Bmp  loss of Wnt inhibition\nWe have added the reference.\nFigure 4D- Sox9 staining is very nice. Ki67 is overexposed- please replace the Ki67\npanels with images with less exposure. S10 is nice!\nWe have replaced figure 4D as requested with new images.\n195- I was confused by the term “distal” here and was looking for a proximal-distal\ndifference. Perhaps revising to note the epithelial thickness expands from the usual ~50\nmicron to ~100 micron thickness and then saying the luminal most cells (or apical side\ncells) undergo apoptosis?\nCorrect, we meant luminal most and were not referring to anterior posterior aspect of\nthe gut. We have changed the text accordingly.\nFigure 5C- Since the epithelium is flat (no villi) in both the control and mutant at this\nstage, the number of cells per 100 um should be comparable. You could count\nfolds/DAPI, but I don’t think it is necessary.\nWe believe the data are quite clear that at E14.5 there are fewer T-folds.\nFigure 6G- It seems these counts are proportion relative to DAPI, please note\nproportion of what? Scale bars are missing.\nScale bars have been added, and yes, the proportion of proliferating cells is the fraction\nof DAPI/E-cadherin positive cells, which we have now noted in the legend."}, "author_response": ""}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have addressed the minor points in their 2nd re-submission."}, "author_response": ""}]

Decision letter