gas2l3 is a maternal and zygotic gene. Zebrafish embryos at 8-cell stage (A); 3, 6, and 9 hpf (B); and 13, 17, 21 and 24 hpf (C) were fixed for whole-mount in situ hybridization with gas2l3 antisense or sense probes. gas2l3 sense probe was used as a negative control. The three main regions of the brain, forebrain (FB), midbrain (MB) and hindbrain (HB), are depicted. Neural tube (NT), telencephalon (TL), and the region of the olfactory placodes (OP) are also indicated.

Gas2l3 is essential for brain morphogenesis and development. (A) The zebrafish gas2l3 gene consists of 9 exons interspersed with 8 introns. Black square indicates target sequence of gas2l3 splicing-block MO. Gray arrow indicates transcript direction. Black arrows indicate forward and reverse primers designed to test MO efficiency (see Fig. S2 for more information). (B) One-cell stage embryos were injected with 2 pmol gas2l3 MO or standard control MO. Representative images of MO-injected embryos (mild or severe morphants), alongside an uninjected embryo (normal), were taken at 24 hpf. Panels ii, iv, and vi are magnified images of panels i, iii, and v respectively. Matching dorsal views are depicted in panels vii, viii, and ix. FB, forebrain; MB, midbrain; HB, hindbrain. Black arrows point toward abnormal forebrain protrusions. (C) Embryos were injected with gas2l3 MO (1 or 2 pmol) or control MO (2 pmol) in combination with mock, gas2l3 RNA, EGFP-gas2l3 RNA, and EGFP RNA (100 pg). Injected embryos were categorized as normal or abnormal according to their brain morphology at 24 hpf. Values are shown as mean normal or abnormal percentages ± SEM of abnormal. We used the ANOVA Bonferroni test in order to determine the statistical significance of the results; Means with distinct letters on top of the chart are significantly different (p<0.001, F=25.846, df=5, 31). Following microinjection, dead, mechanically damaged embryos or embryos with unspecified morphology were excluded from the analysis.

Abnormal brain ventricles and proliferation in Gas2l3-knockdown embryos. (A and B) One-cell stage embryos were injected with 2 pmol gas2l3 or standard control MO. Images of the forebrain (A) and hindbrain (B) were taken 24 hpf using a stereomicroscope Leica M165 FC (representative images are depicted). Fore- and hindbrain maximal ventricle opening was measured using ImageJ software (black dotted lines). Results are shown as mean (N=10)±SEM. Different letters indicate significant differences (ANOVA, Bonferroni, p<0.001, df=2, 27). (C) One-cell stage embryos were injected with 2 pmol gas2l3 or standard control MO and 100 pg CAAX-GFP mRNA. Images of the forebrain (left) and mid- and hindbrains (right) were taken at 24 hpf (upper panels; representative images are depicted). Matching regions (A and B) of the mid- and hindbrain of the control and gas2l3 knockdown embryos are magnified for better visualization (lower panels). (D) One-cell-stage embryos were treated as described in (C). At 24 hpf, embryos were fixed and processed for whole-mount immunostaining with anti-phosphorylated histone H3 antibodies (P-H3). Z-sections covering either the fore/midbrain or hindbrains (x,y) and the complete dorsal ventral axis (z) were taken. Representative images are depicted (E). The number of mitotic cells in the two brain sections was derived from the mid z-section of control embryos and gas2l3 morphants (N=5). This number was normalized to the corresponding tissue area to calculate mitotic indexes. A similar approach was used to determine the mitotic index of the eye. Average mitotic index ratios between control embryos (wt) and morphants (MO) are shown for the 2 brain sections, as well as for the eye. A statistically significant reduction in the mitotic index was observed in the brain of gas2l3 morphants, but not in their eyes (Mann-Whitney U test). (F) TUNEL assay was performed on fixed 24 hpf embryos injected withgas2l3- or control-MO. The average apoptotic-index ratio between the two groups is shown (N=10). The Mann-Whitney U test was used. We used Zeiss LSM780 upright confocal microscope, and a 20× lens for imaging (C, D and F).