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Fig. 4. Apoc1 is required for neural border formation and neural crest emergence. (A) Whole mount in situ hybridization for the neural crest (NC) markers c-myc, sox9, snai2, twist1, id3. Control MO injected embryos are shown in upper panel and Apoc1 tbMO injected ones are shown in lower panel. (B) The reduction of sox9 and snai2 expression by the Apoc1 tbMO could be rescued by coinjection of 400 pg of apoc1 mRNA (upper panel) but not by coinjection of 400 pg of sp-apoc1 mRNA (lower panel). (C) Whole mount in situ hybridization for the neural plate border specifiers msx1, pax3, zic1, the neural marker sox2, and the non-neural ectoderm marker xk81a1. Control MO injected embryos are shown in upper panel and Apoc1 tbMO injected ones are shown in lower panel. One of the dorsal blastomeres was injected with 30 ng of either control MO (upper panels in A, C) or Apoc1 tbMO (lower panels in A, C) together with 100 pg lacZ mRNA. Embryos were fixed at stage 15. The injected side was traced by X-gal staining (blue). |
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Fig. 5. Apoc1 can substitute for wnt in the animal cap neural crest induction assay. (A) 200 pg of chrd, 200 pg wnt3a and/or 300 pg of apoc1 was injected into two animal blastomeres of 4- or 8-cell stage embryos. Animal caps were dissected at stage 9 and cultured until stage 19. Gene expression was assayed by RT-PCR. (B) 125 pg tBR (dominant negative form of bmpr1a) and 30 pg aN-HA-Ctnnb1 (constitutively active form of ctnnb1) mRNA was injected in two animal blastomeres at the 4- to 8-cell stage to induce neural crest cell marker expression. In addition, 40 ng of either control (ctrl) or translation blocking Apoc1 MO (tbMO) with or without 400 pg MO-resistant apoc1 mRNA was injected to determine if Apoc1 is required for neural crest induction. Animal caps were dissected at stage 9 and cultured until stage 16. Gene expression was assayed by RT-PCR. (C) Coinjection of apoc1 and zic1 into the ventral side of the embryo induced ectopic expression of sox9 and snai2. 50 pg of zic1 +/- 500 pg of apoc1 was injected into ventral marginal cells of 8 cell-stage embryos. The embryos were fixed at stage 14, and subjected to whole mount in situ hybridization. The blue arrows point to endogenous sox9 and snai2 expression. The yellow arrowheads show the area of ectopic sox9 and snai2 expression. |
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Fig. 6. Apoc1 and gbx2.2 are required for emergence of neural crest cells, but are independently regulated by Wnt signaling. (A-H) The expression of apoc1 and gbx2.2 is independently regulated, but both genes are required for neural crest induction. Apoc1 expression in Gbx2.2 (A) or control MO (B) injected embryos and gbx2.2 expression in Apoc1 (C) or control MO (D) injected embryos. The reduction of sox9 and snai2 expression was not rescued by coinjection of 100 pg gbx2.2 mRNA in Apoc1 (E,G) or 400 pg apoc1 mRNA in Gbx2.2 (F, H) depleted embryos. 30 ng of MO and 100 pg lacZ mRNA was injected into one side of a single dorsoanimal blastomere of 8-cell stage embryos. The embryos were fixed at stage 15. (I-R) Apoc1 and gbx2.2 are both required for Wnt-mediated neural crest induction in vivo. 200 pg of GR-ctnnb1 and 100 pg lacZ mRNA were injected into one side of a dorsoanimal blastomere of 8-cell stage embryos and the embryos were treated with DEX at stage 10.5. The embryos were fixed at stage 15 for whole mount in situ hybridization with snail2 (I), sox9 (J), apoc1 (K), gbx2.2 (L), msx1 (M), and pax3 (N). Ectopic expression of gbx2.2 in GR-ctnnb1 injected embryos was not reduced by coinjecting 30 ng Apoc1 tbMO (P), but the anterior expansion of snai2 was rescued (O). Ectopic expression of apoc1 in GR-ctnnb1 injected embryos was not reduced by coinjecting 30 ng Gbx2.2 MO (R), but the anterior expansion of snai2 was rescued (Q). Blue arrowhead indicates ectopic expression. |
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Fig. S2. Apoc1 is required for neural border formation and neural crest emergence. Whole mount in situ hybridization for the neural crest (NC) markers snai2, sox9 and twist1 and the neural plate border specifiers pax3 and zic1. Control MO (40ng) injected embryos are shown in upper panel and Apoc1 spMO (40ng) injected ones are shown in lower panel. Injection of Apoc1 spMO also results in a reduction of the expression of the NC markers as well as the neural plate border specifiers on the injected side. |
