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The protective and absorptive functions of the intestinal epithelium rely on differentiated enterocytes in the villi. The differentiation of enterocytes is orchestrated by sub-epithelial mesenchymal cells producing distinct ligands along the villus axis, in particular Bmps and Tgfβ. Here we show that individual Bmp ligands and Tgfβ drive distinct enterocytic programs specific to villus zonation. Bmp4 is expressed mainly from the center to the upper part of the villus, and it activates preferentially genes connected to lipid uptake and metabolism. In contrast, Bmp2 is produced by villus-tip mesenchymal cells, and it influences the adhesive properties of villus-tip epithelial cells and the expression of immunomodulators. Hence, Bmp2 promotes the terminal enterocytic differentiation at the villus-tip. Additionally, Tgfβ induces epithelial gene expression programs similar to that triggered by Bmp2. The inhibition of Bmp receptor type Iin vivoand using intestinal organoids lacking Smad4 revealed that Bmp2-driven villus-tip program is activated by a canonical Smad-dependent mechanism. Finally, we established an organoid cultivation system that enriches for villus-tip enterocytes and thereby better mimics the cellular composition of the intestinal epithelium. Altogether our data suggest that not only Bmp gradient, but also the activity of individual Bmp drives specific enterocytic programs.

Goblet cells secrete mucin to create a protective mucus layer against invasive bacterial infection and are therefore essential for maintaining intestinal health. However, the molecular pathways that regulate goblet cell function remain largely unknown. Although GPR35 is highly expressed in colonic epithelial cells, its importance in promoting the epithelial barrier is unclear. In this study, we show that epithelial Gpr35 plays a critical role in goblet cell function. In mice, cell type-specific deletion of Gpr35 in epithelial cells but not in macrophages results in goblet cell depletion and dysbiosis, rendering these animals more susceptible to Citrobacter rodentium infection. Mechanistically, scRNA-seq analysis indicates that signaling of epithelial Gpr35 is essential to maintain normal pyroptosis levels in goblet cells. Our work shows that the epithelial presence of Gpr35 is a critical element for the function of goblet cell-mediated symbiosis between host and microbiota. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/437264v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1219637org.highwire.dtl.DTLVardef@cf67corg.highwire.dtl.DTLVardef@10a5026org.highwire.dtl.DTLVardef@13f787_HPS_FORMAT_FIGEXP M_FIG C_FIG

Intestinal organoids derived from single cells undergo complex crypt-villus patterning and morphogenesis. However, the nature and coordination of the underlying forces remains poorly characterized. Through light-sheet microscopy and mechanical perturbations, we demonstrate that organoid crypt formation coincides with stark lumen volume reduction, which works synergistically with actomyosin-generated crypt apical and villus basal tension to drive morphogenesis. We analyse these mechanical features in a quantitative 3D biophysical model and detect a critical point in actomyosin tensions, above which crypt becomes robust to volume changes. Finally, via single-cell RNA sequencing and pharmacological perturbations, we show that enterocyte-specific expressed sodium/glucose cotransporter modulates lumen volume reduction via promoting cell swelling. Altogether, our study reveals how cell fate-specific changes in osmotic and actomyosin forces coordinate robust organoid morphogenesis.One Sentence SummaryEmergence of region-specific cell fates drive actomyosin patterns and luminal osmotic changes in organoid development

Accurate lineage reconstruction of mammalian pre-implantation development is essential for inferring the earliest cell fate decisions of mammalian development. Lineage tracing using global labeling techniques is complicated by increasing cell density and rapid embryo rotation, impeding automatic alignment and rendering accurate cell tracking of obtained four-dimensional imaging data sets highly challenging. Here, we exploit the advantageous properties of primed convertible fluorescent proteins (pr-pcFPs) to simultaneously visualize the global green and the photoconverted red population to minimize tracking uncertainties over prolonged time windows. Confined primed conversion of H2B-pr-mEosFP labeled nuclei combined with light-sheet imaging greatly facilitates segmentation, classification, and tracking of individual nuclei from the 4-cell stage up to the blastocyst. Using green and red labels as fiducial markers, we computationally correct for rotational and translational drift and accomplish high fidelity lineage tracing combined with a reduced data size – addressing majors concerns in the field of volumetric embryo imaging.