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In a microarray-based screen for genes that are involved in tissue separation downstream of Paraxial Protocadherin (PAPC) and Frizzled-7 (Fz7)-mediated signaling we identified xGit2 and xRhoGAP 11A, two GTPase-activating proteins (GAP) for small GTPases. xGit2 and xRhoGAP 11A are expressed in the dorsal ectoderm, and their transcription is downregulated in the involuting dorsal mesoderm by PAPC and Fz7. Overexpression of xGit2 and xRhoGAP 11A inhibits Rho activity and impairs convergent extension movements as well as tissue separation behaviour. We propose that Rho activity in the involuting mesoderm is enhanced through inhibition of xGit2 and xRhoGAP 11A transcription by PAPC and Fz7. By this mechanism xRhoGAP 11A and xGit2 are restricted to the dorsal ectoderm, while Rho signaling is inhibited.
Fig. 1. Gain of PAPC and Fz7 function alters gene transcription. (A) Ontology of upregulated and downregulated genes in PAPC and Fz7-expressing Xenopus animal caps (st.10.5) identified by microarray analysis. The intersections of those genes that were regulated in all three biological replicates (p = 0.01) are shown. Up and downregulated genes were categorized according to the predicted or established functions of the proteins acquired by similarity search against DNA and protein databases. (B) Downregulation of RhoGAP 11A and xGit2 was confirmed by qRT-PCR in PAPC and in PAPC and Fz7 injected animal caps. (C) Protein structure of xRhoGAP 11A and xGit2, two downregulated genes. (D) Expression of RhoGAP11A and xGit2 during early development by RT-PCR analysis (Stage (st.) 7: morula; st.8,: early blastula; st.9: late blastula; st.10: early gastula; st.11: mid gastrula; st.12: late gastrula; st.17: neurula; st.21: tailbud stage; st.33: tadpole stage). (E) Expression of PAPC, xRhoGAP 11A and xGit2 by whole mount in situ hybridization in hemisectioned Xenopus gastrula embryos (st.10.5).
Fig. 2. xGit2 and xRhoGAP 11A inhibit convergent extension movements. (A) Synthetic mRNA for xGit2 (800Â pg/embryo) and xRhoGAP 11A (60Â pg/embryo) was injected into Xenopus embryos at the 4-cell stage. Overexpression of both xGit2 and xRhoGAP 11A leads to gastrulation defects, such as shortened body axes and spina bifida. (B) Microinjection of xRhoGAP 11A (60Â pg/embryo), xGit2 (800Â pg/embryo) and xGit2 R39K (800Â pg/embryo), a protein with a mutated Arf-GAP domain into the dorsal blastomeres of 4-cell embryos inhibits convergent extension movements in dorsal marginal zone explants. (C) Overexpression of xRhoGAP 11A (60Â pgRNA/embryo) and xGit2 (800Â pg RNA/embryo) inhibits CE in Bvg1 (200Â pgRNA/embryo) induced animal cap explants. Elongation of the explants was scored when control siblings had reached st. 22.
Fig. 3. xGit2 and xRhoGAP 11A inhibit tissue separation. (A) Synthetic RNA for xRhoGAP 11A (60Â pg/embryo) and xGit2 (800Â pg/embryo) were injected into the dorsal blastomeres of 4-cell stage Xenopus embryos. Overexpression of both RhoGAP 11A and xGit2 abolished the posterior part of Brachet's cleft. Blue arrowheads indicate the posterior end of Brachet's cleft. (B) Experimental procedure of the blastocoel roof (BCR) assay. (C) xGit2 (800Â pg/embryo) and xRhoGAP 11A (60Â pg/embryo) reduced separation in the BCR assay in Bvg1-induced animal cap tissue (200Â pg/embryo). (D) Statistical evaluation of the BCR assays.
Fig. 5. xGit2 and xRhoGAP 11A reduce RhoA activity. (A) Synthetic mRNAs for xGit2 (800Â pg/embryo), xRhoGAP 11A (60Â pg/embryo) and xGit2 R39K (800Â pg/embryo), which lacks ArfGAP activity, were injected in combination with RhoA-myc (200Â pg/embryo) into the animal region of 4-cell-stage Xenopus embryos. GTP-bound Rho was recovered from embryo extracts at stage 11 by RBD-GST fusion protein and detected on a Western blot using an anti-myc antibody. RhoA activity was inhibited by xGit2, xGit2 R39k and xRhoGAP11A. (B) Quantification of the Westernblot shown in (A). Ratios of pulldown and input were taken. The relative Rho-activity of embryos injected only with RhoA-MT was set 100%. (C) RNA for xRhoGAP11a (30Â pg/embryo) and xGit2 (400Â pg/embryo) was injected in combination with Histone 2b-RFP RNA (200Â pg/embryo) into the dorsal rightblastomere of 4-cell stage Xenopus embryos. Dorsal marginal zones were explanted at stage 10.5 and incubated with RBD-GFP protein to visualize endogenous activated Rho. Injected cells were identified by nuclear Histone 2B-RFP. Endogenous RhoA activity was inhibited by xRhoGAP 11A and xGit2 compared to uninjected dorsal marginal zone tissue (WT).
Fig. 6. Knockdown of xRhoGAP 11A and xGit2 rescue knockdown of PAPC. (A) Mesoderm differentiation and elongation of animal caps was induced by injection of synthetic Bvg1 RNA (200Â pg/embryo) into the animal blastomeres of 4-cell-stage embryos. Elongation was blocked by coninjection of 0.4Â mM PAPC antisense morpholino oligonucleotide (MoPAPC). Knockdown of xRhoGAP and xGit2 after injection of antisense morpholino oligonucleotides (MoRhoGAP 0.5Â mM, MoGit2 0.5Â mM) rescued animal cap elongation in PAPC-depleted ACs. (B) Statistical evaluation of animal cap elongation. Standard error is indicated. (C) Knockdown of xRhoGAP 11A and xGit2 rescues tissue separation in PAPC-depleted embryos. Tissue separation behaviour was induced in animal cap cells by injection of 200Â pg Bvg1-RNA per embryo and injected either alone or in combination with MoPAPC (0.5Â mM), MoGit (0.5Â mM) and MoRhoGAP (0.5Â mM). (D) Morpholino oligonucleotid-mediated knockdown of xRhoGAP 11A rescues RhoA activity in PAPC-depleted embryos in RhoA-activity assay. RhoA-myc RNA (200Â pg/embryo) was either injected alone or in combination with MoPAPC (0.4Â mM), MoRhoGAP (0.5Â mM) or MoGit2 (0.5Â mM). GTP-bound RhoA-myc was recovered from embryos lysed at stage 11 and visualized using a Western blot and anti-myc antibody. (E) Quantification of Rho activity assay shown in (D).
