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. 2018 Sep 12;115(39):E9135–E9144. doi: 10.1073/pnas.1812117115

Fig. 4.

Fig. 4.

Bighead is required for head development in a BMP-independent way. All embryo pictures were taken at 25× magnification. (A–D) Bighead knockdown inhibits head formation, which is rescued by coinjection of MO-resistant Bighead mRNA. Embryos were injected two times dorsal-marginal at the four-cell stage as indicated and collected at the tailbud stage. The dosages for MO and mRNA were as follows: 32 ng of Bighead (L+S) MOs (directed against the L and S Bighead genes) and 800 pg of Act-HA-Bighead.S mRNA. Numbers of embryos analyzed were as follows: Controls (Ctrl), n = 153, 100% normal; Bighead MOs, n = 122, 93% with a small head phenotype; MO-resistant HA-Bighead mRNA, n = 109, 95% with a dorsalized phenotype; rescue by coinjection of MO and Bighead mRNA, n = 94, 87% rescued. (E and F) In situ hybridization for Otx2 confirming that Bighead knockdown inhibits forebrain development. Embryos were injected two times dorsal-marginal at the four-cell stage with 32 ng of Bighead MO and collected at stage 15. Ctrl, n = 35, 100% normal; Bighead mRNA, n = 48, 83% with enlarged brain phenotype. (G) Expression of the organizer marker chordin at gastrula stage 10.5. (H) Chordin expression was decreased by Bighead MOs. (I) Chordin expression was increased by injection of 800 pg of Bighead mRNA into the animal pole. Note the reduction of Chordin by Bighead MO (n = 34, 100% with phenotype) and expansion of Chordin by Bighead mRNA (n = 47, 93% with increased chordin). (J–O) Experiments with β-catenin–depleted embryos demonstrating that Bighead is not a BMP antagonist but, instead, behaves as a Wnt antagonist. (K) Embryos were injected with 24 ng of β-catenin MO four times into the marginal-vegetal region at the two-cell stage. Then, at the four-cell stage, one dorsal-marginal cell was injected with 1 pg of xWnt8 mRNA (L), 100 pg of Chordin mRNA (M), 50 pg of Dkk1 mRNA (N), or 400 pg of Bighead mRNA (O). Embryos were collected for in situ hybridization with the neural marker Sox2 at stage 18. Note that β-catenin MO completely ventralized embryos and that only the BMP antagonist Chordin could rescue an axis. xWnt8 mRNA was entirely inactive in β-catenin–depleted embryos, as were Dkk1 and Bighead. Numbers of embryos analyzed were as follows: J, n = 47, 100%; K, n = 52, 100% with a ventralized phenotype; L, n = 29, 100% ventralized; M, n = 34, 92% with rescued CNS; N, n = 29, 97% ventralized; O, n = 32, 97% completely ventralized.