Wnt5a and Wnt11 regulate mammalian anterior-posterior axis elongation

Abstract

Mesoderm formation and subsequent anterior-posterior (A-P) axis elongation are fundamental aspects of gastrulation, which is initiated by formation of the primitive streak (PS). Convergent extension (CE) movements and epithelial-mesenchymal transition (EMT) are important for A-P axis elongation in vertebrate embryos. The evolutionarily conserved planar cell polarity (PCP) pathway regulates CE, and Wnts regulate many aspects of gastrulation including CE and EMT. However, the Wnt ligands that regulate A-P axis elongation in mammalian development remain unknown. Wnt11 and Wnt5a regulate axis elongation in lower vertebrates, but only Wnt5a, not Wnt11, regulates mammalian PCP signaling and A-P axis elongation in development. Here, by generating Wnt5a; Wnt11 compound mutants, we show that Wnt11 and Wnt5a play redundant roles during mouse A-P axis elongation. Both genes regulate trunk notochord extension through PCP-controlled CE of notochord cells, establishing a role for Wnt11 in mammalian PCP. We show that Wnt5a and Wnt11 are required for proper patterning of the neural tube and somites by regulating notochord formation, and provide evidence that both genes are required for the generation and migration of axial and paraxial mesodermal precursor cells by regulating EMT. Axial and paraxial mesodermal precursors ectopically accumulate in the PS at late gastrula stages in Wnt5a(-/-); Wnt11(-/-) embryos and these cells ectopically express epithelial cell adhesion molecules. Our data suggest that Wnt5a and Wnt11 regulate EMT by inducing p38 (Mapk14) phosphorylation. Our findings provide new insights into the role of Wnt5a and Wnt11 in mouse early development and also in cancer metastasis, during which EMT plays a crucial role.

Keywords: Convergent extension; EMT; Gastrulation; Notochord; Planar cell polarity; Wnt.

Fig. 1.

A-P axis and notochord defects in Wnt5a^−/−; Wnt11^−/− embryos. (A-L) Phenotypic analysis of mouse embryos of the indicated genotypes at the stages shown reveals severe shortening of the A-P axis in Wnt5a^−/−; Wnt11^−/− (D,H,L) as compared with Wnt5a^−/−; Wnt11^+/− (C,G,K) embryos. (M-P) Whole-mount in situ hybridization for the notochord and floor plate marker Shh. (M′-P′) Transverse sections of the embryos shown in M-P at the forelimb bud level. fp, floor plate; nc, notochord.

Fig. 2.

Defective CE and PCP signaling in the notochord of Wnt5a^−/−; Wnt11^−/− embryos. (A,B) Whole-mount in situ hybridization for brachyury (T) in ventral views of the notochord at E8.5. T expression outside of the notochord is indicated by an arrow in B. (C-D′) DiI labeling of PNC cells. Ventral (C,D) and distal (C′,D′) views of the embryos 12 h after labeling. (E,F) Vangl1 expression as shown by immunofluorescence in notochord cells of E8.5 embryos. Asymmetric localization of Vangl1 protein along the A-P axis is indicated by arrows in E. Cells with irregular morphology are indicated by arrowheads in F. (E′,F′) Co-staining of Vangl1 (red) and T (green) in notochord cells at E8.5. a, anterior; p, posterior. Scale bars: 25 µm in E-F′.

Fig. 3.

Wnt5a^−/−; Wnt11^−/− embryos display malformed heart and tail bud. (A-C) Heart formation is shown by whole-mount in situ hybridization for Nkx2.5 in E9.5 embryos. Wnt5a^−/−; Wnt11^−/− embryos fail to undergo heart looping. (D-L) Expression of Wnt3a (D-F), Fgf8 (G-I) and T (J-L) in the tail buds of E9.5 embryos as shown by whole-mount in situ hybridization. The line in D-F indicates extension of unsegmented mesoderm. (M-R) Expression of Tbx6 in the posterior of embryos at E8.5 in dorsal view (M-O) and in tail buds of E9.5 embryos (P-R).

Fig. 4.

Reduced and irregular somite formation in Wnt5a^−/−; Wnt11^−/− embryos. Whole-mount in situ hybridization with the indicated probes in E9.5 embryos. Dorsal views of the tail bud are shown. (A-C) Expression of Uncx4.1 is reduced in Wnt5a^−/−; Wnt11^−/− embryos. Note the loss of symmetric somite formation in Wnt5a^−/−; Wnt11^−/− embryos (n=3/6). Arrows mark caudalmost somites. (D-L) Reduced expression of Mesp2, Hes7 and Lfng in Wnt5a^−/−; Wnt11^−/− embryos (F,I,L). Asymmetric gene expression is indicated by arrows (F,L). (M-R) Notch1 and Notch2 expression is reduced in Wnt5a^−/−; Wnt11^−/− embryos (O,R, arrows), whereas expression of Dll1 and Dll3 is unchanged (S-X). L, left; R, right.

Fig. 5.

Ectopic cell accumulation in Wnt5a^−/−; Wnt11^−/− embryos. Whole-mount in situ hybridization showing expression of the indicated genes in E8.5 embryos. Sagittal sections show the expression of T (B,B′) but not of Noto (D,D′) in the ectopic cell accumulation in Wnt5a^−/−; Wnt11^−/− embryos (arrows in B′,D′). (E,F) Transverse sections of the embryos shown in Fig. 3M,R. Tbx6 expression was only found in the dorsal part of the ectopic cell accumulation (arrow in F). The ventral border of the ectopic cell accumulation is indicated by a dotted line (F). Boxes in A-D indicate the areas shown at higher magnification in A′-D′, respectively.

Fig. 6.

Defective EMT in Wnt5a^−/−; Wnt11^−/− embryos. Immunofluorescent staining of sagittal sections of posterior parts of E8.5 embryos. (A,B) T (green) expression is reduced in the dorsal PS region of the Wnt5a^−/−; Wnt11^−/− embryo, whereas Sox2 (red) expression is extended ventrally and posteriorly. T expression in the ectopic cell accumulation in Wnt5a^−/−; Wnt11^−/− embryos is marked by an arrow in B. Note that T staining was observed throughout PS cells but is overlaid by strong DAPI signals, resulting in the appearance of staining restricted to the cytosol. (C-D″) Ectopic expression of E-cadherin (red) in T (green) in Wnt5a^−/−; Wnt11^−/− embryos (arrows in D,D″). Boxes in C′,D′ indicate areas shown at higher magnification in C″,D″, respectively. (E,F) Reduced expression of vimentin in Wnt5a^−/−; Wnt11^−/− embryos (arrows in E,F). (G,H) Increased expression of ZO-2 in the ectopically accumulated cells in Wnt5a^−/−; Wnt11^−/− embryos (arrow in H). (I,J) Loss of FN in the ectopic cell accumulation of Wnt5a^−/−; Wnt11^−/− embryos (arrow in J). Scale bars: 50 µm.

Fig. 7.

Reduced p38 phosphorylation in the posterior of Wnt5a^−/−; Wnt11^−/− embryos and induction of p38 phosphorylation by Wnt5a and Wnt11. (A-B′) Immunofluorescent staining of phosphorylated p38 (P-p38) in sagittal sections of posterior embryos at E8.5. P-p38 is reduced in the PS region of Wnt5a^−/−; Wnt11^−/− embryos (B). Boxes in A,B indicate areas shown at higher magnification in A′,B′, respectively. (C-E) Western blot of P-p38 in MEF cells (C) showing that p38 phosphorylation and c-Jun phosphorylation are induced by recombinant Wnt5a and Wnt11 protein. Quantifications of three experiments reveal significant changes in phosphorylation levels induced by the addition of Wnt5a and Wnt11 (D,E). (F,G) Activation of RhoA is induced by Wnt11 (F). aRhoA, activated RhoA; tRhoA, total RhoA. Quantification of three experiments is shown in G. (H,I) Inhibition of RhoA activation using 1 µM Rhosin prevents Wnt5a/Wnt11-induced phosphorylation of p38 (H). Quantification of three experiments is shown in I. (D,E,G,I) Student's t-test; n.s., not significant. Error bars indicate s.d. Scale bars: 100 µm.