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Fig. 6 MSANTD2 CRISPR/Cas9 embryo phenotypes. Embryos injected with control (A–C, CTRL) or four MSANTD2-directed sgRNAs (D–F, MSANTD2). A/D and B/E present lateral views of whole embryos and of the head region, respectively. C/F show dorsal views of the embryo head region. Embryos injected with four sgRNAs showed developmental delays as well as tail and nervous system malformations compared to control embryos at 24hpf (A–F). Tail curvation and not well-defined somites are shown with black arrowheads (D). Default in neural tube folding and cell aggregates around the nervous system are indicated with white arrowheads and black arrows, respectively (E–F). Not well-formed MHB are shown with white stars (*) (E–F). (G) Proportions of F0 zebrafish phenotypes. Embryos were injected with sgRNA 1 + 2 + 3 + 4, sgRNA 1 + 4 and sgRNA 4 targeting MSANTD2, four scrambled sgRNAs or noninjected (WT), and were scored for phenotypes at 24hpf. Percentages with strong (red), intermediate (orange) or no phenotypes (gray–WT) are shown. Strong phenotypes: developmental delays, tail malformations, nervous system malformations, aggregates; intermediate phenotypes: developmental delays, nervous system malformations, no or few aggregates, no tail malformation. (H–O): Dextran Texas Red injection in brain ventricles of 24hpf (H–K) or 30hpf (L–O) embryos injected with control (H, L, CTRL) or four MSANTD2-directed sgRNAs (I–K, M–O, MSANTD2) (dorsal view). At each stage, three pictures of MSANTD2 CRISPR/Cas9 embryos were compared to a control picture in order to represent phenotype variability. The proportion of embryos presenting the same phenotypes are the following: I—31%, J—47%, K—22% at 24hpf and M—14%, N—43%, O—43% at 30hpf. All cases illustrate neural tubes misfolding (shown with white arrowheads) particularly in the MHB region. We also observed smaller red fluorescent areas in the forebrain and midbrain regions, indicating reduction of these ventricles. F, forebrain, M, midbrain; H, hindbrain. Scale bar 200 µm.