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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 singleâlayered 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)
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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, AâF), Vangl2 (red, AâF), betaâcatenin (Ãâcat; red, G,H) and/or alphaâtubulin (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 = 75âμM. Scale bars CâH = 25âμM
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Figure 3
Endodermâspecific 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 (Î4, Î3, Î11, Î5) and S (Î8) 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 MOâinjected embryos, compared to CoMOâinjected controls (NF41); GAPDH was detected as a loading control. GâH: Coâinjection of Vangl2 MO with a morpholinoâresistant 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 Pâ<â0.05. (Error bars indicate SD)
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Figure 4
Vangl2 is required early in gut morphogenesis for endoderm cell shape, adhesion, and microtubule organization. Control morpholino (CoMO; A,DâG,N,O) or Vangl2 morpholino (Vangl2 MO; B,IâL,P,Q) was coâinjected with mRNAâencoding GFP (green, D,I) targeting the gut endoderm. Compared to control guts (NF41â42; A, Vangl2 MOâinjected 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, betaâcatenin, Ãcat; DâF,IâK) 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 MOâinjected 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, Ãcat levels in Vangl2 MOâinjected cells (K) are decreased compared to control cells (F). Moreover, whereas MTs in CoMOâinjected cells are oriented parallel to the radial axis of the gut tube (arrows, G), they appear randomly oriented in Vangl2 MOâinjected cells (arrows, L). M: Most MTs in CoMOâinjected cells are distributed within 0° to 20° angle of the apicobasal axis of the epithelium; in contrast, the MTs in Vangl2 MOâinjected cells are distributed more broadly, deviating from parallel. Endoderm cells were dissociated from CoMOâ (N,O) or Vangl2 MOâ (P,Q) injected gut tubes, then challenged to reaggregate for 30â² (see Experimental Procedures). While CoMOâinjected cells are able to condense into large clumps of cells that reform adherens junctions (O), Vangl2 MOâinjected 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. *, Pâ<â0.05; **, Pâ<â0.01. Scale bars D,E,I,J = 75âμM. Scale bars F,G,K,L = 25âμM
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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 mRNAâencoding 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 CâF) and basolateral (integrin, Int, green in CâF) proteins to reveal cell polarity. Boxed regions in C and D are magnified in E and F, respectively. Unlike controls (A,C), Vangl2 MOâinjected 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, TOâPROâ3 (blue). Scale bars AâD = 75âμM. Scale bars E,F = 25âμM
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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 mRNAâencoding 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), alphaâtubulin (αTub, green in CâF, to reveal microtubule architecture), and Eâcadherin (Ecad, green in GâN, 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 MOâinjected 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 GâJ) suggests that the abnormal epithelial morphogenesis observed in Vangl2âdeficient guts is independent of early cell inviability. However, immunostaining for activated caspase (Casp, red in KâN) shows that unintercalated cells in the Vangl2âdeficient gut lumen eventually die by apoptosis (L,N), an event rarely observed in controls (K,M). Nuclei, TOâPROâ3 (blue). Scale bars in AâD,G,H,K,L = 75âμM; E,F,I,J,M,N = 25âμM
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Figure 7
Ectopic Vangl2 expression disrupts tissueâlevel endoderm orientation during early gut morphogenesis. Embryos were injected with GFP mRNA alone (A,H,I,LâO) or GFP plus vangl2 mRNA (B,DâG,JâK,PâS) and allowed to develop to NF35 (DâG) or NF41 (HâS). 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 (DâG) or NF41 (HâS). The boxed regions in F,H,J,M, and Q are magnified in G,I,K,NâO, and RâS, respectively. At NF35, vangl2 mRNAâinjected endoderm cells, indicated by GFP expression (green in D), exhibit ectopic Vangl2 localization at the membrane (EâG) compared to neighboring uninjected cells. By NF41âNF42, GFP mRNAâinjected cells (red in H and L) are uniformly radially oriented, with apically enriched Eâcadherin (I) and parallel arrays of microtubules (arrows, O). In contrast, vangl2 mRNAâinjected cells (red in J and P) are not aligned with respect to the gut axes, exhibit variably localized Eâcadherin (K) and betaâcatenin (R), and form unusual tissueâlevel configurations, including rosettes (marked by arrowheads in Q, asterisks in R; arrows in S indicate abnormal microtubule orientations). Scale bars DâF,H,J,L,M,P,Q = 75 μM. Scale bars G,I,K,N,O,R,S = 25 μM
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Figure 8
Ectopic Vangl2 expression disrupts tissueâlevel gut epithelial organization. Embryos were injected with mCherry (mCh) mRNA alone (AâC) or mCherry plus vangl2 mRNA (two examples in EâG and IâK, 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: betaâcatenin (Ãcat; 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 FâFâ³ and JâJâ, 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: Higherâmagnification views (from serial sections) of the boxed region in B reveal uniform basolateral distribution of betaâcatenin, apically localized MHC/aPKC, and parallel arrays of apically nucleated microtubules (as indicated by αTub). FâFâ³: In contrast to controls, the segments of a vangl2 mRNAâinjected 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: Higherâmagnification views (from serial sections) of the boxed region in Fâ² reveal that vangl2 mRNAâoverexpressing cells retain betaâcatenin 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. JâJâ: In another vangl2 mRNAâinjected gut, basement membrane, as indicated by Int expression, is detected internally, as represented in the cartoon (L). K: Higherâmagnification 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, TOâPROâ3 (blue). Scale bars B,FâFâ³,JâJâ = 75âμM. Scale bars C,G,K = 25âμM
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