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Fig. 5. GPC4 regulates expression of dorsal forebrain markers. Molecular analysis of neural markers in CoMo- and Gpc4Mo-injected embryos. (A-D,G-N) Frontal views; (E,F) dorsal views; (O-R) side view. Anterior is to the left. (A,B) Otx2 expression (stage 21). Arrowhead indicates forebrain expression; asterisk indicates midbrain expression. (C,D) Bf1 expression (stage 21). Arrows indicate expression in the developing telencephalon; arrowhead indicates expression in the olfactory placodes. (E,F) Expression of the posterior neural markers Krox20 (bracket) and Hoxb9 (arrowhead) in stage 21 embryos. (G,H) Fgf8 expression (stage 17). Asterisk indicates anterior neural ridge; arrowhead indicates isthmus. (I,J) Nkx2.1 expression in the ventral forebrain (stage 21); note that Nkx2.1 expression persists in GPC4-depleted embryos (J). (K,L) Emx2 expression in the dorsal forebrain of developing embryos (stage 21); note that Emx2 expression is drastically reduced in GPC4-depleted embryos (L). (M) Emx2 expression in embryos co-injected with CoMo and mouse Gpc4 (mGpc4) mRNA; note that overexpression of mouse Gpc4 does not affect Emx2 expression (compare with K). (N) Rescue of Emx2 expression in embryos co-injected with Gpc4Mo and mouse Gpc4 mRNA (compare with L). (O) Emx2 expression in a tailbud embryo injected with CoMo. (P) Emx2 expression in a tailbud embryo co-injected with CoMo and mouse Gpc4 mRNA. (Q) Loss of Emx2 expression in a tailbud embryo injected with Gpc4Mo. (R) Rescue of Emx2 expression and forehead morphology in a tailbud embryo co-injected with Gpc4Mo and mouse Gpc4 mRNA. |
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Fig. 7. GPC4 modulates FGF signalling during neurulation. (A) Immunoblot analysis of GPC4/FGF2 complexes, as detected by anti-Myc antibodies. The `Input' lane contains NIH3T3 cells transfected with the Myc epitope-tagged mouse Gpc4 cDNA. The HS-GAG modified mouse GPC4 proteins have an apparent Mr of around 200 kDa (arrow), whereas the unmodified proteins run at 60 kDa (asterisk). In the `GST-FGF-2' lane only the modified 200 kDa GPC4 protein (arrow) binds to FGF2. Mouse GPC4 does not bind to GST (control; `GST' lane). (B) Immunoblot analysis of phosphorylated ERK (pERK) and SMAD1 (pSMAD1) proteins in Xenopus embryos (stage 15). Levels of phosphorylated proteins were determined in embryos that were: cultured in the presence of the FGF inhibitor SU5402 (0.1 mg/ml; lane `SU5402'); cultured with DMSO as a control (lane `DMSO'); injected with CoMo (lane `CoMo'); injected with Gpc4Mo (lane `Gpc4Mo'); or co-injected with Gpc4Mo and mouse Gpc4 mRNA (lane `Gpc4Mo + mGpc4'). TUB, α-Tubulin levels in the extracts were determined to normalize samples. (C-H) Molecular analysis of embryos cultured with DMSO (panels C,E,G) and with SU5402 (0.1 mg/ml; panels D,F,H). Arrows in panels C-F indicate Emx2 expression. (C) Emx2 expression in control embryos cultured with DMSO (stage 17). (D) Downregulation of Emx2 in embryos cultured with SU5402 (stage 17). (E) Emx2 and Krox20 (bracket) expression in control embryos cultured with DMSO (stage 22). (F) Downregulation of Emx2, but not Krox20 (bracket), in embryos cultured with SU5402 (stage 22). (G) Nkx2.1 expression in embryos cultured with DMSO (stage 22). (H) Nkx2.1 expression in embryos cultured with SU5402 (stage 22). (I) Lack of cell death in the forebrain region of an embryo cultured with DMSO (stage 20). (J) Apoptotic cells detected in the forebrain region of an embryo cultured with SU5402 (stage 20). |
