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• W2801858122 abstract "•Identification of genes associated with crypt formation and villar differentiation•Myosin II-dependent apical constriction is required for initial crypt invagination•Hinges compartmentalize crypts and villi and pattern the intestine•Rac1 controls hinge formation through negative regulation of α6/β4 integrins The adult mammalian intestine is composed of two connected structures, the absorptive villi and the crypts, which house progenitor cells. Mouse crypts develop postnatally and are the architectural unit of the stem cell niche, yet the pathways that drive their formation are not known. Here, we combine transcriptomic, quantitative morphometric, and genetic analyses to identify mechanisms of crypt development. We uncover the upregulation of a contractility gene network at the earliest stage of crypt formation, which drives myosin II-dependent apical constriction and invagination of the crypt progenitor cells. Subsequently, hinges form, compartmentalizing crypts from villi. Hinges contain basally constricted cells, and this cell shape change was inhibited by increased hemidesmosomal adhesion in Rac1 null mice. Loss of hinges resulted in reduced villar spacing, revealing an unexpected role for crypts in tissue architecture and physiology. These studies provide a framework for studying crypt morphogenesis and identify essential regulators of niche formation. The adult mammalian intestine is composed of two connected structures, the absorptive villi and the crypts, which house progenitor cells. Mouse crypts develop postnatally and are the architectural unit of the stem cell niche, yet the pathways that drive their formation are not known. Here, we combine transcriptomic, quantitative morphometric, and genetic analyses to identify mechanisms of crypt development. We uncover the upregulation of a contractility gene network at the earliest stage of crypt formation, which drives myosin II-dependent apical constriction and invagination of the crypt progenitor cells. Subsequently, hinges form, compartmentalizing crypts from villi. Hinges contain basally constricted cells, and this cell shape change was inhibited by increased hemidesmosomal adhesion in Rac1 null mice. Loss of hinges resulted in reduced villar spacing, revealing an unexpected role for crypts in tissue architecture and physiology. These studies provide a framework for studying crypt morphogenesis and identify essential regulators of niche formation. The mammalian intestinal epithelium is arranged in a series of finger-like projections into the lumen called villi, and invaginations into the mesenchyme called crypts. The villi are composed of terminally differentiated cells, including absorptive enterocytes, goblet cells, and enteroendocrine cells, whereas the crypt contains stem and transit amplifying cells. The actively cycling adult intestinal stem cells, also known as crypt base columnar cells, sit at the base of the crypt between the terminally differentiated Paneth cells (Clevers, 2013Clevers H. The intestinal crypt, a prototype stem cell compartment.Cell. 2013; 154: 274-284Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar, Tan and Barker, 2014Tan D.W. Barker N. Intestinal stem cells and their defining niche.Curr. Top. Dev. Biol. 2014; 107: 77-107Crossref PubMed Scopus (81) Google Scholar). Crypt base columnar stem cells generate transit-amplifying cells that undergo four to five divisions as they move up the crypt axis (Marshman et al., 2002Marshman E. Booth C. Potten C.S. The intestinal epithelial stem cell.Bioessays. 2002; 24: 91-98Crossref PubMed Scopus (481) Google Scholar). Cells then exit the crypt compartment and simultaneously undergo differentiation as they enter the villus. Whether crypt exit and differentiation onset are necessarily linked is unknown. Villus formation occurs during embryogenesis. In mice, this is driven by the formation of mesenchymal cell clusters, which induce overlying epithelial cells to form villi; in chick, contraction of the underlying smooth muscle drives villus morphogenesis (Shyer et al., 2013Shyer A.E. Tallinen T. Nerurkar N.L. Wei Z. Gil E.S. Kaplan D.L. Tabin C.J. Mahadevan L. Villification: how the gut gets its villi.Science. 2013; 342: 212-218Crossref PubMed Scopus (338) Google Scholar, Shyer et al., 2015Shyer A.E. Huycke T.R. Lee C. Mahadevan L. Tabin C.J. Bending gradients: how the intestinal stem cell gets its home.Cell. 2015; 161: 569-580Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, Walton et al., 2012Walton K.D. Kolterud A. Czerwinski M.J. Bell M.J. Prakash A. Kushwaha J. Grosse A.S. Schnell S. Gumucio D.L. Hedgehog-responsive mesenchymal clusters direct patterning and emergence of intestinal villi.Proc. Natl. Acad. Sci. USA. 2012; 109: 15817-15822Crossref PubMed Scopus (99) Google Scholar). In either case, formation of the villi also results in the establishment of intervillar domains that contain progenitors. This is due to compartmentalization of signals, such as Shh and Bmp4, which repress progenitor fates in the villi (Walton et al., 2012Walton K.D. Kolterud A. Czerwinski M.J. Bell M.J. Prakash A. Kushwaha J. Grosse A.S. Schnell S. Gumucio D.L. Hedgehog-responsive mesenchymal clusters direct patterning and emergence of intestinal villi.Proc. Natl. Acad. Sci. USA. 2012; 109: 15817-15822Crossref PubMed Scopus (99) Google Scholar, Walton et al., 2016Walton K.D. Whidden M. Kolterud A. Shoffner S.K. Czerwinski M.J. Kushwaha J. Parmar N. Chandhrasekhar D. Freddo A.M. Schnell S. et al.Villification in the mouse: Bmp signals control intestinal villus patterning.Development. 2016; 143: 427-436Crossref PubMed Scopus (84) Google Scholar). Later events then transform the intervillar region from flat sheets of epithelial cells into cup-like crypts. However, the cell biological mechanisms driving crypt formation have not been reported. Although the crypt is the architectural unit of the intestinal stem cell niche, the function of this structure in stem cell establishment/maintenance and in organ physiology remains unknown. Possible functions include increasing the area available for the number of progenitor cells needed to fuel the rapid turnover of the intestinal epithelium, compartmentalizing signals between villi and crypts, and protecting stem cells from soluble signals in the lumen. Data supporting this last role have been reported for the colon, where a metabolite generated by the lumenal microbiota suppresses stem cell proliferation (Kaiko et al., 2016Kaiko G.E. Ryu S.H. Koues O.I. Collins P.L. Solnica-Krezel L. Pearce E.J. Pearce E.L. Oltz E.M. Stappenbeck T.S. The colonic crypt protects stem cells from microbiota-derived metabolites.Cell. 2016; 165: 1708-1720Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). However, because the mechanisms underlying crypt morphogenesis have not been defined, these hypotheses have not yet been directly tested, and we therefore lack insight into the functions of this niche architecture. To understand how crypt morphogenesis occurs, we combined quantitative morphometric and RNA sequencing (RNA-seq) analyses to identify architectural changes and molecular regulators of crypt morphogenesis. These analyses led us to identify two distinct pathways involved in crypt formation. First, myosin II-mediated apical constriction is required for the earliest stage of crypt invagination. Subsequently, we demonstrate that a hinge region forms between crypts and villi to morphologically compartmentalize them. The formation of this region requires the small GTPase Rac1, which acts to suppress hemidesmosomal integrins in nascent crypts. In the absence of Rac1, remodeling of the basal surface of cells, which is required for hinge cell formation, does not occur. Finally, our data demonstrate that crypt-villus compartmentalization is required for proper villar spacing and mesoscale patterning of the small intestine. To analyze postnatal crypt morphogenesis, we first needed to label this heterogeneous intervillar cell population. Prior studies focused on profiling adult Lgr5+ stem cells, which constitute a minority of cells within the crypt compartment. Thus, we searched for markers that labeled the entire developing crypt throughout its morphogenesis. We found that the hyaluronic acid receptor CD44v6 was robustly expressed in crypt progenitor units throughout the formation of the crypt, consistent with previous reports that it is expressed both embryonically (Shyer et al., 2015Shyer A.E. Huycke T.R. Lee C. Mahadevan L. Tabin C.J. Bending gradients: how the intestinal stem cell gets its home.Cell. 2015; 161: 569-580Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) and in adults (Alho and Underhill, 1989Alho A.M. Underhill C.B. The hyaluronate receptor is preferentially expressed on proliferating epithelial cells.J. Cell Biol. 1989; 108: 1557-1565Crossref PubMed Scopus (194) Google Scholar) (Figures 1A and 1B ). In both tissue sections and epithelial whole mounts, CD44v6 marked discrete cell populations between the villi. Even at postnatal day 0 (P0), there were clear intervillar units before any overt crypt morphogenesis had occurred (Figure 1B, inset). Therefore, we define crypt progenitor units by their CD44v6 expression. Notably, proliferation is confined to the CD44v6 population already before birth (Crosnier et al., 2006Crosnier C. Stamataki D. Lewis J. Organizing cell renewal in the intestine: stem cells, signals and combinatorial control.Nat. Rev. Genet. 2006; 7: 349-359Crossref PubMed Scopus (568) Google Scholar, Shyer et al., 2015Shyer A.E. Huycke T.R. Lee C. Mahadevan L. Tabin C.J. Bending gradients: how the intestinal stem cell gets its home.Cell. 2015; 161: 569-580Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Epithelial whole mounts from ZO1-GFP mice, which marks the apical tight junctions of the epithelial cells (Foote et al., 2013Foote H.P. Sumigray K.D. Lechler T. FRAP analysis reveals stabilization of adhesion structures in the epidermis compared to cultured keratinocytes.PLoS One. 2013; 8: e71491Crossref PubMed Scopus (19) Google Scholar, Huebner et al., 2014Huebner R.J. Lechler T. Ewald A.J. Developmental stratification of the mammary epithelium occurs through symmetry-breaking vertical divisions of apically positioned luminal cells.Development. 2014; 141: 1085-1094Crossref PubMed Scopus (39) Google Scholar), allowed us to quantitate both cell number and morphometry. Because intestinal morphogenesis occurs in a wave from anterior to posterior, we confined all analyses to the medial third of the small intestine. From P0 to P10, the CD44v6-positive (CD44v6+) compartment reorganized from a flat sheet of cells to a cup-like invagination (Figures 1A and 1B). Notably, the architecture of the developing crypt was preserved in epithelial whole mounts in which supporting mesenchymal structures were removed. While this does not rule out roles for surrounding tissue in crypt morphogenesis, it demonstrates that the maintenance of tissue structure is autonomous to the epithelium. Despite their change in appearance, the mean number of cells within these morphogenetic units was constant over the first 10 postnatal days (Figure 1C), thus demonstrating that proliferation does not drive this reorganization. While these cells are still actively proliferating, they give rise to differentiated cells rather than expanding the transit amplifying cell population. During initial morphogenesis (i.e., up to P10), the nascent crypt maintains just above 30 cells (±9 cells, SD). However, from about P15 onward it undergoes dramatic elongation and expansion of the transit amplifying cell population (Figure S1). Although the number of cells within each crypt did not change, there was a decrease in the 2D crypt area, as measured by rotating 3D reconstructions of crypts to view from the bottom, then manually outlining the perimeter of the CD44v6+ region (Figure 1D). The CD44v6+ area was just above 1 mm2 from P0 to P3, and then decreased to approximately half that size by P6. Concomitantly, crypt depth, measured as the distance from the crypt mouth (ZO1-labeled tight junctions of most lumenal CD44v6+ cell) to the crypt base (basal side of most distal CD44v6+ cell) increased as it invaginated into the underlying mesenchyme (Figure 1E). As crypts formed via invagination, we noted the formation of a pronounced border between crypts and villi. We term this region the “hinge,” as the crypt-villus axis contains a clear boundary in adults. This is a 3D structure that forms a rim or ridge at the top of the crypts. This results in a clear plateau between crypts and villi (Figure 2A, rightmost image). When cells were still arranged in a flat sheet (P0–P2), there was no obvious hinge region when viewing either basal (Figure 1F) or apical membranes (Figure 1H). However, a bend with a broad curvature appeared by P3 in approximately 60% of crypts (Figure 1J). We measured the radius of the inflection zone and found that the radius continued to decrease over development, as a sharper angle formed between the developing crypt and villus compartments (Figure 1K). By P7, 100% of crypts examined had defined hinge regions when examining either basal or apical markers (Figures 1G and 1I), and thus had morphologically compartmentalized their crypts and villi (Figure 1J). Crypt hinges have not been morphometrically characterized before, and the cell biological basis/machinery for their formation is currently unknown. To identify molecular regulators of the crypt morphogenetic program we characterized above, we developed a fluorescence-activated cell sorting isolation protocol to purify developing crypt and corresponding villar cells. Epithelial cell adhesion molecule (EpCAM) marked all epithelial cells, while CD44 marked the intervillar cells. We sorted EpCAM+/CD44– cells and EpCAM+/CD44+ cells from the medial small intestine of wild-type (WT) mice at four stages: P0, P3, P6, and P10 (Figures 2A, S2A, and S2B). qPCR analysis verified that the stem cell marker Ascl2 was enriched in CD44+/EpCAM+ cells, whereas the enterocyte marker Lactase was enriched in CD44–/EpCAM+ cells (Figure S2C). We performed RNA-seq analysis on the two cell populations at each stage. Principal component analysis revealed that biological replicates clustered together and that the variance between populations was greatest by developmental stage rather than cell compartment (Figure S2D). Interestingly, however, P0 and P10 populations were more similar to each other than either was to P3 or P6. Principal components 2 and 3 segregated crypts from villi more clearly (Figures S2D and S2E). We obtained core gene signatures of the crypt and villi (genes expressed and enriched at all time points). There was a core set of 299 genes that were enriched in crypts at all stages examined (Figure 2B). Pathway analysis revealed that developing crypts were enriched for genes regulating the cell cycle, DNA replication, and other processes associated with actively proliferating cells (Figures 2B and S2F). Additionally, the adult intestinal stem cell markers Lgr5, Smoc2, Olfm4, Msi1, Axin2, and Ascl2 were enriched in crypts at all stages. Interestingly, the core set of 299 crypt-enriched genes shared only 64 genes with a previously published adult intestinal stem cell signature (Munoz et al., 2012Munoz J. Stange D.E. Schepers A.G. van de Wetering M. Koo B.K. Itzkovitz S. Volckmann R. Kung K.S. Koster J. Radulescu S. et al.The Lgr5 intestinal stem cell signature: robust expression of proposed quiescent '+4' cell markers.EMBO J. 2012; 31: 3079-3091Crossref PubMed Scopus (538) Google Scholar) (data not shown). This likely reflects the heterogeneity of the crypt pool versus the purified Lgr5+ cells. We used antibody staining to validate our RNA-seq data and to identify novel crypt-enriched markers, including the protocadherin Pcdh8 (PAPC). In adults, Pcdh8 has been reported to be an intestinal stem cell marker (Merlos-Suarez et al., 2011Merlos-Suarez A. Barriga F.M. Jung P. Iglesias M. Cespedes M.V. Rossell D. Sevillano M. Hernando-Momblona X. da Silva-Diz V. Munoz P. et al.The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse.Cell Stem Cell. 2011; 8: 511-524Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar). However, Pcdh8 protein was broadly enriched in the crypt compartment throughout crypt morphogenesis, and was not restricted to the crypt base until initial crypt morphogenesis had completed (Figure 2C). These data highlight the difference in cellular composition of postnatal crypts compared with adult crypts, and suggest a maturation process of adult stem cells from a more homogeneous population of neonatal progenitors. We also identified genes that were specifically enriched at later stages of crypt development, such as Krt7 and L1cam. Consistent with transcriptome data, keratin 7 protein was absent from early neonatal crypts, though it was expressed in goblet cells in the villi. However, its expression was highly upregulated in crypts at later time points, beginning to be expressed by P8, with all crypts labeled by P15 (Figure 2D). In contrast, while L1CAM levels began to increase by RNAseq at P10, it was still difficult to detect by immunofluorescence. However, at later stages, L1CAM marked crypts (Figure 2E). These results demonstrate the dynamics of crypt marker expression during development and reveal that the entire structure, and not only stem cells, undergoes maturational changes in gene expression. In addition to crypt-enriched genes, we identified genes whose expression was upregulated in villi at all stages. As shown in the Venn diagram in Figure 2F, 293 genes were enriched in villi (≥2-fold compared with crypts, p < 0.05) at all stages examined. Villi were enriched in genes related to metabolism, transport, and digestion (Figures 2F and S2G). Krt20, a known structural component of villi (Zhou et al., 2003Zhou Q. Toivola D.M. Feng N. Greenberg H.B. Franke W.W. Omary M.B. Keratin 20 helps maintain intermediate filament organization in intestinal epithelia.Mol. Biol. Cell. 2003; 14: 2959-2971Crossref PubMed Scopus (74) Google Scholar), was also upregulated in our dataset (data not shown). Importantly, we found that the transcription factors Maf and Mafb were enriched in villi at all stages examined. We confirmed this by antibody staining and found that MafB was nuclear in the villus, but was not detectable in crypt cells (Figures 2G and 2H). In addition, MafB was specific for enterocytes, as enteroendocrine cells marked by Sox9-GFP and Goblet cells identified by morphology were negative for nuclear MafB staining (Figures 2I–2K). There are currently no known transcriptional markers that are specific for the enterocyte lineage and present at all developmental stages. Maf and MafB have been shown to be necessary for the differentiation of several tissues and cell types (Lopez-Pajares et al., 2015Lopez-Pajares V. Qu K. Zhang J. Webster D.E. Barajas B.C. Siprashvili Z. Zarnegar B.J. Boxer L.D. Rios E.J. Tao S. et al.A LncRNA-MAF: MAFB transcription factor network regulates epidermal differentiation.Dev. Cell. 2015; 32: 693-706Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, Miyai et al., 2016Miyai M. Hamada M. Moriguchi T. Hiruma J. Kamitani-Kawamoto A. Watanabe H. Hara-Chikuma M. Takahashi K. Takahashi S. Kataoka K. Transcription factor MafB coordinates epidermal keratinocyte differentiation.J. Invest. Dermatol. 2016; 136: 1848-1857Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, Artner et al., 2007Artner I. Blanchi B. Raum J.C. Guo M. Kaneko T. Cordes S. Sieweke M. Stein R. MafB is required for islet beta cell maturation.Proc. Natl. Acad. Sci. USA. 2007; 104: 3853-3858Crossref PubMed Scopus (185) Google Scholar). Its expression pattern in the differentiated cells of the small intestine suggests that it may play a similar role in enterocyte differentiation. In either case, it serves as a useful new marker for this cell population. In addition to compartment-specific genes, we were very interested in dynamic patterns of transcription that might reveal stage-specific regulators of crypt morphogenesis or differentiation/maturation pathways. We thus performed fuzzy c-means clustering and examined clusters containing genes whose levels increased as crypts developed (Figure S3A). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed enrichment for a number of signaling pathways during crypt development, including Wnt, Hippo, and phosphatidylinositol 3-kinase/AKT (Figure S3B). Consistent with this, we saw an increase in Wnt signaling over crypt morphogenesis using a Tcf/Lef-H2B-GFP reporter mouse (Figures S3C and S3D) and an Axin2-CreER reporter mouse line (Figure S3E). In addition, mRNA expression levels of the stem cell markers Lgr5 and Sox9 (both putative Wnt targets) became more highly expressed as crypts developed (Lgr5, 3.6-fold; Sox9, 2.0-fold). However, while Lgr5 mRNA levels increased, the number of Lgr5-GFP-labeled cells decreased over crypt development, becoming restricted to the crypt base by P13 (Figure S3G). Quantification showed that the percentage of Lgr5+ cells in each crypt remained constant from P0 to P6, then decreased by P10 (Figure S3F). These data are consistent with postnatal intestinal stem cells undergoing a maturation process to become adult intestinal stem cells, though specific profiling of these cells will be needed to confirm this. Importantly, these data demonstrate that dynamic transcriptional changes occur concomitant with morphological development. We therefore sought to better understand the cell biological basis of crypt formation. Crypt formation involves epithelial invagination, a common morphogenetic event that has diverse underlying mechanisms in different tissues (Pearl et al., 2017Pearl E.J. Li J. Green J.B. Cellular systems for epithelial invagination.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2017; 372Crossref PubMed Scopus (67) Google Scholar). To identify candidates that may be involved in the initiation of crypt formation and invagination, we examined genes that were enriched in P0 crypts compared with villi and compared with later stage crypts. Interestingly, we found that a large subset of myosin II-associated contractility genes (Zaidel-Bar et al., 2015Zaidel-Bar R. Zhenhuan G. Luxenburg C. The contractome–a systems view of actomyosin contractility in non-muscle cells.J. Cell Sci. 2015; 128: 2209-2217Crossref PubMed Scopus (59) Google Scholar) was enriched in crypts compared with villi (Figure 3A; Table S1), including the regulatory myosin light chain Myl9, the myosin heavy chain gene Myh9, the Rho GEF Ect2, and the actin nucleators Diaph2 and Diaph3. Several of these, including the myosin heavy chain genes, were also enriched in P0 crypts compared with P3 crypts (Myh9 3.7X enriched, Myh14 8X enriched at P0; Table S2). To examine whether there was evidence for myosin II-based contractility in the initiation of crypt formation, we examined the apical surfaces of intervillar cells from neonatal ZO1-GFP intestines. We found that these cells were apically constricted compared with villar cells (Figure 3B). We quantified the apical surface area of crypt and villar cells in 3D using MorphoGraphX software (Barbier de Reuille et al., 2015Barbier de Reuille P. Routier-Kierzkowska A.L. Kierzkowski D. Bassel G.W. Schupbach T. Tauriello G. Bajpai N. Strauss S. Weber A. Kiss A. et al.MorphoGraphX: A platform for quantifying morphogenesis in 4D.Elife. 2015; 4: 05864Crossref PubMed Scopus (289) Google Scholar, de Reuille et al., 2014de Reuille P.B. Robinson S. Smith R.S. Quantifying cell shape and gene expression in the shoot apical meristem using MorphoGraphX.Methods Mol. Biol. 2014; 1080: 121-134Crossref PubMed Scopus (23) Google Scholar) and found that crypt cell apical area (18.4 ± 0.5 μm2, SD) was smaller than villar cell apical area at P0 (49.5 ± 1.1 μm2). Furthermore, the apical area of crypt cells decreased by approximately 3-fold between P0 and P1 (P1: 6.2 ± 0.1 μm2; t test, p < 0.001) (Figure 3C). When we examined depth-coded images of an invaginating crypt, it was clear that the apical cell areas at the base of the crypt were smallest, and as the cells moved up and out of the crypt, their apical areas became larger and more regular in shape (Figure 3D). In addition to the constricted apical surfaces, we observed several parameters that suggested that crypt cells may be undergoing dynamic rearrangements prior to invagination. Villar cells were generally hexagonal in shape, with an aspect ratio (length of long axis/short axis) slightly over 1. However, there was a much larger range of aspect ratios in crypt cells, with 40% of cells having an aspect ratio over 2.0 (compared with 10%–15% in villar cells) (Figure 3E). Because villar cells were stereotypically hexagonal, they had five to eight neighbors, with approximately 5% of cells falling outside that range. In contrast, crypt cells had irregular shapes and number of neighbors, with over 20% of cells having fewer than five or more than eight neighbors at P0 (Figure 3F), consistent with the cells in the intervillar spaces undergoing dynamic rearrangements. Apical constrictions that occur in Drosophila gastrulation and Xenopus neural tube closure are driven by actomyosin contractility (Martin et al., 2009Martin A.C. Kaschube M. Wieschaus E.F. Pulsed contractions of an actin-myosin network drive apical constriction.Nature. 2009; 457: 495-499Crossref PubMed Scopus (828) Google Scholar, Rolo et al., 2009Rolo A. Skoglund P. Keller R. Morphogenetic movements driving neural tube closure in Xenopus require myosin IIB.Dev. Biol. 2009; 327: 327-338Crossref PubMed Scopus (77) Google Scholar). The upregulation of contractility genes, which converge on type II non-muscle myosins, suggested that this early morphogenetic change may be myosin II dependent and required for subsequent crypt invagination. This prompted us to examine whether myosin activation was differentially regulated in distinct intestinal compartments. We examined pMLC(18/19) localization in postnatal intestines and found that it was restricted to the zonula adherens in villi (Figure 3G, arrowheads). In contrast, pMLC was more uniform across the apical surface and down the lateral membranes of postnatal intervillar cells (Figure 3H). When observed en face, intervillar cells had pMLC+ puncta in the apicomedial region of the cells (Figure 3J), while there was little detectable pMLC in the apicomedial region of villar cells (Figure 3I). These data are consistent with myosin activity being differentially regulated in crypts and villi during crypt morphogenesis. Furthermore, the apicomedial localization of pMLC suggested that there may be an active contractile network at the apical side of intervillar cells. To determine whether crypt cell apical constriction is myosin II dependent, we examined the localization of the three type II myosins in the mouse intestine. Myosin heavy chain IIC (MyoIIC) was highly enriched at the apical surface of intestinal epithelial cells (Figure 4A). When observed en face, MyoIIC was localized to the apical cortex and in a medial meshwork (Figure 4B), similar to the medial myosin network observed in gastrulating Drosophila cells (Martin et al., 2009Martin A.C. Kaschube M. Wieschaus E.F. Pulsed contractions of an actin-myosin network drive apical constriction.Nature. 2009; 457: 495-499Crossref PubMed Scopus (828) Google Scholar). In contrast to MyoIIC, myosin heavy chain IIA (MyoIIA) primarily localized to lateral junctions (Figure 4C), whereas myosin IIB was not detectable in the intestinal epithelium by antibody staining, consistent with our RNA-seq data (Figure 4D, and data not shown). Due to its apical enrichment, we first examined the intestinal crypt architecture of MyoIIC−/− mice (MyoIIC knockout [KO]). However, consistent with the lack of overt phenotype of these null mice (Ma et al., 2010Ma X. Jana S.S. Conti M.A. Kawamoto S. Claycomb W.C. Adelstein R.S. Ablation of nonmuscle myosin II-B and II-C reveals a role for nonmuscle myosin II in cardiac myocyte karyokinesis.Mol. Biol. Cell. 2010; 21: 3952-3962Crossref PubMed Scopus (83) Google Scholar), the intestines appeared grossly normal (Figures 4E and 4F). Interestingly, myosin IIA relocalized to the apical surface of MyoIIC KO intestinal epithelial cells, suggesting that in the intestine, myosin IIA can compensate for the loss of myosin IIC (Figures 4G and 4H). Therefore, to determine whether type II myosins function in crypt morphogenesis, and particularly in intervillar cell apical constriction, we generated double myosin IIA/myosin IIC knockouts (MyoIIA/C dKO) using the Villin-CreER transgene and injecting with tamoxifen at P0. The apical area of MyoIIA/C dKO crypt cells was significantly larger than control crypt cells at P5 (6.0 ± 0.3 μm2 in control versus 17.8 ± 1.0 μm2 in MyoIIA/C dKO; p < 0.001), and was not statistically different from that of the apical areas of P0 control crypt cells (18.4 ± 0.5 μm2 in P0 control) (Figures 3C and 4K–4M). These data demonstrate that type II myosins are required for the apical constriction of intestinal crypt cells. In addition to loss of apical constriction, both the invagination (Figures 4I, 4J, and 4N) and the change in 2D area of the crypt (Figure 4O) were inhibited in the MyoIIA/C dKO intestine. These changes were not secondary to changes in number of crypt progenitor cells, as cell number was comparable with control crypts (Figure 4P). Furthermore, proliferation rates were normal, and we did not observe an increase in crypt cell apoptosis (data not shown). Together, these data demonstrate that type II myosins are required for the initiation of crypt invagination" @default.
• W2801858122 created "2018-05-17" @default.
• W2801858122 creator A5019702284 @default.
• W2801858122 creator A5031655339 @default.
• W2801858122 creator A5069853168 @default.
• W2801858122 date "2018-04-01" @default.
• W2801858122 modified "2023-10-15" @default.
• W2801858122 title "Morphogenesis and Compartmentalization of the Intestinal Crypt" @default.
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• W2801858122 doi "https://doi.org/10.1016/j.devcel.2018.03.024" @default.
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