Dush MK and Nascone-Yoder NM (2019), Vangl2 coordinates cell rearrangements during g...

XB-ART-55944

Dev Dyn 2019 Jul 01;2487:569-582. doi: 10.1002/dvdy.61.

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Vangl2 coordinates cell rearrangements during gut elongation.

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BACKGROUND: The embryonic gut tube undergoes extensive lengthening to generate the surface area required for nutrient absorption across the digestive epithelium. In Xenopus, narrowing and elongation of the tube is driven by radial rearrangements of its core of endoderm cells, a process that concomitantly opens the gut lumen and facilitates epithelial morphogenesis. How endoderm rearrangements are properly oriented and coordinated to achieve this complex morphogenetic outcome is unknown. RESULTS: We find that, prior to gut elongation, the core Wnt/PCP component Vangl2 becomes enriched at both the anterior and apical aspects of individual endoderm cells. In Vangl2-depleted guts, the cells remain unpolarized, down-regulate cell-cell adhesion proteins, and, consequently, fail to rearrange, leading to a short gut with an occluded lumen and undifferentiated epithelium. In contrast, endoderm cells with ectopic Vangl2 protein acquire abnormal polarity and adhesive contacts. As a result, endoderm cells also fail to rearrange properly and undergo ectopic differentiation, resulting in guts with multiple torturous lumens, irregular epithelial architecture, and variable intestinal topologies. CONCLUSIONS: Asymmetrical enrichment of Vangl2 in individual gut endoderm cells orients polarity and adhesion during radial rearrangements, coordinating digestive epithelial morphogenesis and lumen formation with gut tube elongation.

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Species referenced: Xenopus laevis
Genes referenced: cdh1 myh6 pmch prkci vangl2
GO keywords: foregut morphogenesis [+]
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	Figure 1 Model of endoderm dynamics during the morphogenesis of the Xenopus primitive gut tube. Between NF32 and NF42 (A,B), the endoderm cells of the prospective midgut gradually elongate and become oriented along the radii of the primitive gut tube. Subsequent radial intercalation (blue arrows in B) facilitates lumen expansion and the morphogenesis of a single‐layered digestive epithelium by NF46. This convergence of the rearranging endoderm also drives longitudinal tissue extension (black arrows in B), as the cells preferentially intercalate between anterior and posterior neighbors within the walls of the gut (red arrows), ultimately facilitating tube narrowing and elongation (C). (Adapted from Reed et al.22)
	Figure 2 Vangl2 localization becomes apically and anteriorly polarized during Xenopus gut morphogenesis. Low levels of Vangl2 mRNA were targeted to the gut endoderm to generate mosaic overexpression (see Experimental Procedures). Injected embryos (NF35 and NF39) were then sectioned frontally (as indicated by the dashed line in the cartoons) and immunostained to detect integrin (Int, green cell outlines, A‐F), Vangl2 (red, A‐F), beta‐catenin (ß‐cat; red, G,H) and/or alpha‐tubulin (αTub; green, G,H). The boxed region in A is magnified in C and E; the boxed region in B is magnified in D and F. At NF35, Vangl2 (red) is localized in puncta throughout endoderm cell membranes (A,C,E); the disorganized (nonparallel) microtubule architecture at this stage (G) indicates endoderm cells are still largely unpolarized. However, by NF39 (B,D,F), when cells are elongating along the radial axis of the gut tube and the parallel alignment of microtubule bundles reveals the cells' radial polarization (arrows, H), Vangl2 has become localized predominately at the apical (left) end and anterior (top) face of each cell (arrows, F). Scale bars A,B = 75 μM. Scale bars C‐H = 25 μM
	Figure 3 Endoderm‐specific knockdown of Vangl2 activity causes gut morphogenesis defects. While the gut has substantially elongated in control embryos injected with vangl2 gRNA alone (A; NF44), embryos injected with vangl2 gRNA plus Cas9 mRNA (B; NF44) are severely shortened and/or malrotated, correlating with indels at the vangl2 locus on both L (Δ4, Δ3, Δ11, Δ5) and S (Δ8) chromosomes. Likewise, while embryos injected with control morpholino (CoMO; D; NF46) develop long, coiled gut tubes, embryos injected with Vangl2 morpholino (Vangl2 MO) have abnormally short and malrotated guts (E). F: Western blotting for Xenopus Vangl2 protein confirms that Vangl2 is depleted in Vangl2 MO‐injected embryos, compared to CoMO‐injected controls (NF41); GAPDH was detected as a loading control. G‐H: Co‐injection of Vangl2 MO with a morpholino‐resistant WT vangl2 mRNA partially rescues the short gut phenotype (compare to E). H: Annotations (a,b,c) indicate injected groups significantly different from each other P < 0.05. (Error bars indicate SD)
	Figure 4 Vangl2 is required early in gut morphogenesis for endoderm cell shape, adhesion, and microtubule organization. Control morpholino (CoMO; A,D‐G,N,O) or Vangl2 morpholino (Vangl2 MO; B,I‐L,P,Q) was co‐injected with mRNA‐encoding GFP (green, D,I) targeting the gut endoderm. Compared to control guts (NF41‐42; A, Vangl2 MO‐injected guts are shorter, straighter, and less cohesive, occasionally with loose cells escaping from the gut tube (arrowhead in B). Transverse sections through injected guts (as indicated by the dashed line in the cartoon, C) were immunostained to reveal cell outlines (red, beta‐catenin, ßcat; D‐F,I‐K) and microtubules (MTs) (green, αTub; E,G,J,L). The boxed region in E is magnified in F,G; the boxed region in J is magnified in K,L. Vangl2 MO‐injected cells exhibit rounder cell shapes, as indicated by decreased length to width (L:W) ratios of individual cells (displayed as box and whiskers plot, H). In addition, ßcat levels in Vangl2 MO‐injected cells (K) are decreased compared to control cells (F). Moreover, whereas MTs in CoMO‐injected cells are oriented parallel to the radial axis of the gut tube (arrows, G), they appear randomly oriented in Vangl2 MO‐injected cells (arrows, L). M: Most MTs in CoMO‐injected cells are distributed within 0° to 20° angle of the apicobasal axis of the epithelium; in contrast, the MTs in Vangl2 MO‐injected cells are distributed more broadly, deviating from parallel. Endoderm cells were dissociated from CoMO‐ (N,O) or Vangl2 MO‐ (P,Q) injected gut tubes, then challenged to reaggregate for 30′ (see Experimental Procedures). While CoMO‐injected cells are able to condense into large clumps of cells that reform adherens junctions (O), Vangl2 MO‐injected cells remain almost completely dissociated (Q). R: The extent of endoderm adhesion is quantified as the percent reduction of the area of the culture dish still covered by loose cells after reaggregation challenge; error bars represent SE. , P < 0.05; *, P < 0.01. Scale bars D,E,I,J = 75 μM. Scale bars F,G,K,L = 25 μM
	Figure 5 Vangl2 is required for gut lumen formation and apicobasal polarity. Embryos were injected with CoMO (A,C,E) or Vangl2 MO (B,D,F) plus mRNA‐encoding mCherry (mCh, red in A,B; as a lineage tracer) targeting the gut endoderm. Frontal sections (as indicated by the dashed line in the cartoon diagram, NF46) were immunostained for apical (Par3, green in A,B; aPKC, red in C‐F) and basolateral (integrin, Int, green in C‐F) proteins to reveal cell polarity. Boxed regions in C and D are magnified in E and F, respectively. Unlike controls (A,C), Vangl2 MO‐injected guts do not form a central lumen; the unintercalated cells (asterisks in B; boxed region in D) exhibit abnormally rounded shapes and decreased apical markers (eg, aPKC; F) compared to controls (E). Nuclei, TO‐PRO‐3 (blue). Scale bars A‐D = 75 μM. Scale bars E,F = 25 μM
	Figure 6 Vangl2 is required during late gut morphogenesis for epithelial polarity and microtubule architecture. Embryos were injected with control morpholino (CoMO; A,C,E,G,I,K,M) or Vangl2 MO (B,D,F,H,J,L,N) plus mRNA‐encoding mCherry (mCh, red in A,B; as a lineage tracer) targeting the gut endoderm. Frontal sections (NF46) were immunostained for the apical marker MHCB (green in A,B), alpha‐tubulin (αTub, green in C‐F, to reveal microtubule architecture), and E‐cadherin (Ecad, green in G‐N, to outline cell surfaces and reveal adherens junctions). Boxed regions in C,D,G,H,K, and L are magnified in E,F,I,J,M, and N, respectively. Unlike controls (E), Vangl2 MO‐injected cells are unable to form properly oriented microtubule arrays (F). Likewise, intercellular adhesion is reduced dramatically in Vangl2 morphants (compare Ecad staining in I,J). Immunostaining for the mitotic marker phosphohistone H3 (pHH3, red in G‐J) suggests that the abnormal epithelial morphogenesis observed in Vangl2‐deficient guts is independent of early cell inviability. However, immunostaining for activated caspase (Casp, red in K‐N) shows that unintercalated cells in the Vangl2‐deficient gut lumen eventually die by apoptosis (L,N), an event rarely observed in controls (K,M). Nuclei, TO‐PRO‐3 (blue). Scale bars in A‐D,G,H,K,L = 75 μM; E,F,I,J,M,N = 25 μM
	Figure 7 Ectopic Vangl2 expression disrupts tissue‐level endoderm orientation during early gut morphogenesis. Embryos were injected with GFP mRNA alone (A,H,I,L‐O) or GFP plus vangl2 mRNA (B,D‐G,J‐K,P‐S) and allowed to develop to NF35 (D‐G) or NF41 (H‐S). By NF41, the gut tubes of embryos injected with vangl2 mRNA are short and wide, with a bulging topology (B) compared to controls (A). Frontal sections (as exemplified by cartoon diagram, C) were immunostained for the indicated proteins at either NF35 (D‐G) or NF41 (H‐S). The boxed regions in F,H,J,M, and Q are magnified in G,I,K,N‐O, and R‐S, respectively. At NF35, vangl2 mRNA‐injected endoderm cells, indicated by GFP expression (green in D), exhibit ectopic Vangl2 localization at the membrane (E‐G) compared to neighboring uninjected cells. By NF41‐NF42, GFP mRNA‐injected cells (red in H and L) are uniformly radially oriented, with apically enriched E‐cadherin (I) and parallel arrays of microtubules (arrows, O). In contrast, vangl2 mRNA‐injected cells (red in J and P) are not aligned with respect to the gut axes, exhibit variably localized E‐cadherin (K) and beta‐catenin (R), and form unusual tissue‐level configurations, including rosettes (marked by arrowheads in Q, asterisks in R; arrows in S indicate abnormal microtubule orientations). Scale bars D‐F,H,J,L,M,P,Q = 75 μM. Scale bars G,I,K,N,O,R,S = 25 μM
	Figure 8 Ectopic Vangl2 expression disrupts tissue‐level gut epithelial organization. Embryos were injected with mCherry (mCh) mRNA alone (A‐C) or mCherry plus vangl2 mRNA (two examples in E‐G and I‐K, respectively) to target the prospective gut tube and allowed to develop to NF46. Compared to the long, rotated guts of control embryos injected with mCh mRNA alone (A), the gut tubes of embryos injected with vangl2 mRNA (E,I) are shorter and wider than controls and often have unusual bulges and/or indentations (arrowheads in E,I). To analyze tissue architecture in injected embryos, transverse sections (eg, section plane approximated by horizontal line in A) were immunostained for various proteins, as indicated: beta‐catenin (ßcat; green) to reveal cell shape/adhesion; aPKC (red) or MHC (green) to reveal the apical/lumenal cell surface; and integrin (Int, green), which is enriched at basement membranes. Serial sections from the same embryo are shown sequentially in F‐F″ and J‐J”, with section planes approximated by the horizontal lines in E and I, respectively. B: In controls, the segments of the gut tube are comprised of a central lumen surrounded by a single layer of apicobasally polarized epithelium, as summarized in the cartoon (D; blue arrows represent the orientation of cell polarity with respect to basement membrane [green] and apical surface [red]). C: Higher‐magnification views (from serial sections) of the boxed region in B reveal uniform basolateral distribution of beta‐catenin, apically localized MHC/aPKC, and parallel arrays of apically nucleated microtubules (as indicated by αTub). F‐F″: In contrast to controls, the segments of a vangl2 mRNA‐injected gut appear thickened, with walls composed of multiple layers of disoriented epithelial tissue forming a torturous, branching, and/or noncontiguous lumen, as exemplified in the cartoon (H). G: Higher‐magnification views (from serial sections) of the boxed region in F′ reveal that vangl2 mRNA‐overexpressing cells retain beta‐catenin expression, although microtubule bundles are often short and/or obliquely oriented, consistent with the complex, multilayered epithelial organization created by the noncontiguous apical/lumenal surface delineated by MHC/aPKC. J‐J”: In another vangl2 mRNA‐injected gut, basement membrane, as indicated by Int expression, is detected internally, as represented in the cartoon (L). K: Higher‐magnification views (from serial sections) of the boxed region in J show both the discontinuous apical/lumenal surface (indicated by MHC/aPKC expression) and presumed basement membrane (Int) in the center of the gut tube, revealing the perturbed polarity of epithelial organization in the context of Vangl2 overexpression. Nuclei, TO‐PRO‐3 (blue). Scale bars B,F‐F″,J‐J” = 75 μM. Scale bars C,G,K = 25 μM

References [+] :

Adler, The genetic control of tissue polarity in Drosophila. 1992, Pubmed