Fig. 7

Effects of MSANTD2 inactivation by CRISPR/Cas9 in zebrafish embryos. (A–D) Acridine orange staining of embryos injected with control (CTRL) or four MSANTD2-directed sgRNAs (MSANTD2) at 24hpf. Numerous dead cells were visible in MSANTD2 CRISPR/Cas9 embryos compared to control embryos. Regions of cell aggregates were indicated with white stars. (E–I) Embryos injected with control (CTRL) or four MSANTD2-directed sgRNAs (MSANTD2) in Tg(elavl3:GCaMP3) zebrafish embryos. At each stage, two pictures of MSANTD2 CRISPR/Cas9 embryos were compared to a control in order to represent the variability of impaired neuronal pattern. Differentiated neurons were marked by green fluorescence. The results showed general anatomical defects characterized by different patterns of fluorescence between MSANTD2-mutated and control embryos (shown with white arrowheads). Abnormal pattern of neuronal marking was not only explained by developmental delay, since MSANTD2 mutated embryos are still different from control embryos at later stages. (J–N): Expression of FGF8 (J, K), HER5 (L), PAX2A (M) and DLX2 (N) at 24hpf in embryos injected with control (CTRL, top) or four MSANTD2-directed sgRNAs (MSANTD2, bottom). J present lateral and K/L/M/N dorsal views of the head region of the embryos, respectively. Differences in gene expression patterns between MSANTD2-mutated and control embryos are indicated with black arrowheads. Dd, dorsal diencephalon; hn, hindbrain neurons; oc, otic capsule; os, optic stalks; ov, otic vesicle; pa, pharyngeal arches; pr, proximal part of retina; t, telencephalon; tp, thyroid primordium. For each picture at least n = 10 embryos were observed.