Supplementary Materials1. of niche formation. Graphical abstract AMG-073 HCl (Cinacalcet HCl) Sumigray et al. uncover cell biological regulators of stem cell niche morphogenesis in the intestine. Using transcriptomic and genetic approaches, they find two key actions in crypt genesis: an initial apical constriction that is required for AMG-073 HCl (Cinacalcet HCl) invagination and a subsequent compartmentalization of crypts, which promote villar spacing and absorptive activity. Introduction The mammalian intestinal epithelium is usually 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, 2013; Tan and Barker, 2014). Crypt base columnar stem cells generate transit-amplifying cells that undergo 4-5 divisions as they move up the crypt axis (Marshman et al., 2002). 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 usually unknown. Villus formation occurs during embryogenesis. In mice, this is driven by the formation of mesenchymal cell clusters that induce CASP8 overlying epithelial cells to form villi; in chick, contraction of the underlying smooth muscle drives villus morphogenesis (Shyer et al., 2015; Shyer et al., 2013; Walton et al., 2012). 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., 2012; Walton et al., 2016). Later events then transform the intervillar region from flat linens of epithelial cells into cup-like crypts. However, the cell biological mechanisms driving crypt formation have not been reported. Although the AMG-073 HCl (Cinacalcet HCl) 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 has been reported for the colon, where a metabolite generated by the lumenal microbiota suppresses stem cell proliferation (Kaiko et al., 2016). 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 AMG-073 HCl (Cinacalcet HCl) architecture. To understand how crypt morphogenesis occurs, we combined quantitative morphometric and 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.