Terminal differentiation of villus tip enterocytes is governed by distinct Tgfβ superfamily members

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 from the centre to the upper part of the villus and 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. Additionally, Tgfβ induces epithelial gene expression programs similar to those triggered by Bmp2. Bmp2-driven villus tip program is activated by a canonical Bmp receptor type I/Smad-dependent mechanism. Finally, we establish an organoid cultivation system that enriches villus tip enterocytes and thereby better mimics the cellular composition of the intestinal epithelium. Our data suggest that not only a Bmp gradient but also the activity of individual Bmp drives specific enterocytic programs

The Invading Anchor Cell Induces Lateral Membrane Constriction during Vulval Lumen Morphogenesis in C. elegans

During epithelial tube morphogenesis, linear arrays of cells are converted into tubular structures through actomyosin-generated intracellular forces that induce tissue invagination and lumen formation. We have investigated lumen morphogenesis in the C. elegans vulva. The first discernible event initiating lumen formation is the apical constriction of the two innermost primary cells (VulF). The VulF cells thereafter constrict their lateral membranes along the apicobasal axis to extend the lumen dorsally. Lateral, but not apical, VulF constriction requires the prior invasion of the anchor cell (AC). The invading AC extends actin-rich protrusions toward VulF, resulting in the formation of a direct AC-VulF interface. The recruitment of the F-BAR-domain protein TOCA-1 to the AC-VulF interface induces the accumulation of force-generating actomyosin, causing a switch from apical to lateral membrane constriction and the dorsal extension of the lumen. Invasive cells may induce shape changes in adjacent cells to penetrate their target tissues

Cell fate coordinates mechano-osmotic forces in intestinal crypt formation

10.1038/s41556-021-00700-2NATURE CELL BIOLOGY237733-74

Cell fate coordinates mechano-osmotic forces in intestinal crypt formation

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. Here, using light-sheet microscopy and large-scale imaging quantification, we demonstrate that crypt formation coincides with a stark reduction in lumen volume. We develop a 3D biophysical model to computationally screen different mechanical scenarios of crypt morphogenesis. Combining this with live-imaging data and multiple mechanical perturbations, we show that actomyosin-driven crypt apical contraction and villus basal tension work synergistically with lumen volume reduction to drive crypt morphogenesis, and demonstrate the existence of a critical point in differential tensions above which crypt morphology becomes robust to volume changes. Finally, we identified a sodium/glucose cotransporter that is specific to differentiated enterocytes that modulates lumen volume reduction through cell swelling in the villus region. Together, our study uncovers the cellular basis of how cell fate modulates osmotic and actomyosin forces to coordinate robust morphogenesis

An in vivo EGF receptor localization screen in C. elegans Identifies the Ezrin homolog ERM-1 as a temporal regulator of signaling

The subcellular localization of the epidermal growth factor receptor (EGFR) in polarized epithelial cells profoundly affects the activity of the intracellular signaling pathways activated after EGF ligand binding. Therefore, changes in EGFR localization and signaling are implicated in various human diseases, including different types of cancer. We have performed the first in vivo EGFR localization screen in an animal model by observing the expression of the EGFR ortholog LET-23 in the vulval epithelium of live C. elegans larvae. After systematically testing all genes known to produce an aberrant vulval phenotype, we have identified 81 genes regulating various aspects of EGFR localization and expression. In particular, we have found that ERM-1, the sole C. elegans Ezrin/Radixin/Moesin homolog, regulates EGFR localization and signaling in the vulval cells. ERM-1 interacts with the EGFR at the basolateral plasma membrane in a complex distinct from the previously identified LIN-2/LIN-7/LIN-10 receptor localization complex. We propose that ERM-1 binds to and sequesters basolateral LET-23 EGFR in an actin-rich inactive membrane compartment to restrict receptor mobility and signaling. In this manner, ERM-1 prevents the immediate activation of the entire pool of LET-23 EGFR and permits the generation of a long-lasting inductive signal. The regulation of receptor localization thus serves to fine-tune the temporal activation of intracellular signaling pathways

An In Vivo EGF Receptor Localization Screen in <i>C. elegans</i> Identifies the Ezrin Homolog ERM-1 as a Temporal Regulator of Signaling

<div><p>The subcellular localization of the epidermal growth factor receptor (EGFR) in polarized epithelial cells profoundly affects the activity of the intracellular signaling pathways activated after EGF ligand binding. Therefore, changes in EGFR localization and signaling are implicated in various human diseases, including different types of cancer. We have performed the first <i>in vivo</i> EGFR localization screen in an animal model by observing the expression of the EGFR ortholog LET-23 in the vulval epithelium of live <i>C. elegans</i> larvae. After systematically testing all genes known to produce an aberrant vulval phenotype, we have identified 81 genes regulating various aspects of EGFR localization and expression. In particular, we have found that ERM-1, the sole <i>C. elegans</i> Ezrin/Radixin/Moesin homolog, regulates EGFR localization and signaling in the vulval cells. ERM-1 interacts with the EGFR at the basolateral plasma membrane in a complex distinct from the previously identified LIN-2/LIN-7/LIN-10 receptor localization complex. We propose that ERM-1 binds to and sequesters basolateral LET-23 EGFR in an actin-rich inactive membrane compartment to restrict receptor mobility and signaling. In this manner, ERM-1 prevents the immediate activation of the entire pool of LET-23 EGFR and permits the generation of a long-lasting inductive signal. The regulation of receptor localization thus serves to fine-tune the temporal activation of intracellular signaling pathways.</p></div

Genes that control the localization or expression of LET-23::GFP.

<p>Genes that control the localization or expression of LET-23::GFP.</p

Identification of genes regulating LET-23 EGFR localization and signaling.

<p>(A) Schematic drawing of an L2 larva with the location of the VPCs and AC. P5.p, P6.p and P7.p get induced to form the mature vulva. P3.p, P4.p and P8.p divide once and fuse to the hypodermis. (B) Overview of the LET-23 EGFR and NOTCH signaling network controlling 1° and 2° vulval fate specification. (C) LET-23::GFP expression (green) in P6.p of a late L2 larva during vulval induction. The AC is labeled with an <i>mCherry</i>::<i>plcδ^PH</i> reporter (magenta) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004341#pgen.1004341-Ziel1" target="_blank">[35]</a>. Note the low LET-23::GFP levels in the 2° P5.p and P7.p. (D) Expression of the LET-23::GFP reporter in the 1° lineage at the Pn.px and (E) Pn.pxx stage. (F) Pie charts indicating the frequencies of the different classes of mislocalization phenotypes observed after RNAi and (G) the Clusters of Orthologous Groups (KOGs) of the 81 genes identified in the screen (H–M). Examples of different genes identified in the LET-23 localization screen. Left panels show the corresponding Nomarski images and right panels LET-23::GFP expression in the 1° cells and their neighbors (asterisks). (H) The negative empty vector control and (I) <i>lin-7</i> RNAi as positive control. (J) <i>erm-1</i> RNAi as an example for reduced basolateral (arrow) and increased apical localization (arrow head), (K) <i>sft-4</i> RNAi with normal localization in P6.p but persistent expression in P7.p (asterisk), and (L) <i>C11H1.3</i> RNAi (Pn.px stage) with punctate apical accumulation (arrow head). (M) <i>ego-2</i> RNAi with cytoplasmic accumulation of LET-23::GFP in P6.p (arrow head) and P5.p (asterisk). (N) perinuclear localization of SFT-4::GFP in the vulval cells and the AC and (O) intracellular punctate expression of C11H1.3::GFP in P6.p. (P) Cytoplasmic and nuclear expression of EGO-2::GFP in P6.p. (Q) Vulval induction in <i>let-60(n1046gf)</i> larvae treated with different RNAi clones. Vulval induction (VI) indicates the average number of induced VPCs per animal. “vulva i” indicates Pn.p cell-specific RNAi in the <i>rde-1(lf);let-60(n1046gf); [P_lin-31::rde-1]</i> background. %Muv (Multivulva) indicates the fraction of animals with VI>3. The numbers of animals scored are indicated in brackets. * Indicates p<0.05 as determined in a two tailed student's t-test - two-sample unequal variance. <i>t-test</i> values in <i>RNAi: C11H1.3</i> (0.003), <i>sft-4</i> (0.013), <i>mig-6</i> (0.002). Error bars represent the standard error of the mean. The scale bars are 10 µm.</p

ERM-1 negatively regulates vulval induction and binds to LET-23.

<p>(A) Genetic epistasis analysis between <i>erm-1</i> and components of the <i>egfr/ras/mapk</i> pathway. Vulval induction (VI) indicates the average numbers of induced VPCs in different double mutant combinations scored in <i>erm-1(tm677)</i> heterozygous (white bars) versus homozygous (gray bars) animals. %Vul indicates the fraction of animals with VI<3 and % Muv the fraction of animals with VI>3. The numbers of animals scored for each genotype are indicated in brackets. N.S: no significant change. *Indicates p<0.05 as determined in a two tailed student's t-test - two-sample unequal variance. (B) Structures of the GST::ERM-1 fusion proteins tested for LET-23 binding. (C) Interaction of LET-23 from wild-type extracts with different GST::ERM-1 fusion proteins detected on an anti-LET-23 Western blot. (D) Binding of LET-23 extracted from <i>lin-7(e1413)</i> and (E) from <i>let-23(sy1)</i> mutants to GST::ERM-1 proteins. The dashed lines indicate the approximate positions of the 180 kDa and 116 kDA protein standards.</p