PKC overactivation during early development causes mild developmental delay and brain size deficit.

a Lateral views of representative larvae (3?dpf) exposed to DMSO, PMA or PMA+Go6983. Head size was calculated with the boundary shown in yellow dotted line. b Quantitative representation of body length and head size (3?dpf) normalized to DMSO (DMSO n?=?8, PMA n?=?10, PMA+Go6983 20?nM n?=?6, PMA+Go6983 100?nM n?=?10, **p?<?0.01, ***p?<?0.001, one-way ANOVA). c Lateral views of representative larvae (9?dpf) previously exposed to DMSO or PMA. Head size was calculated with the boundary shown in yellow dotted line. d Quantitative representation of body length and head size (9?dpf) normalized to DMSO (DMSO n?=?8, PMA n?=?6, *p?<?0.05, t-test analysis). e Dorsal views of zebrafish brains (3?dpf) visualized by Huc:GFP. Brain region was divided by yellow dotted line. Scale bar, 100??m. f Quantitative representation of different brain regions normalized to DMSO (n?=?16 per group, ***p?<?0.001, t-test analysis). g DAPI staining of zebrafish head sections (3?dpf). Scale bar, 100??m. h Nuclei number in forebrain and midbrain (n?=?6 per group, ***p?<?0.001, t-test)

Aberrant PKC activation impaired neurogenesis.

a, b Reduced mitotic cells in zebrafish head (3?dpf). a Fluorescence of pHH3-positive dots (the upper row) merged with brightfield images of zebrafish head (the lower row) (n?=?26 per group, ***p?<?0.001, t-test). Scale bar, 100??m. c Flow cytometry analysis of the different populations of cells. d Newborn neurons (Q2) are significantly reduced in PMA-exposed fish (n?=?9 per group, p?<?0.001, t-test)

Excessive PKC signaling promotes the degradation of ?-catenin.

a Western blot analysis of ?-catenin. b Quantification of the protein level of ?-catenin relative to wild-type (WT) group (n?=?3 per group, ***p?<?0.001, t-test). c The nuclear accumulation of ?-catenin is reduced by PMA treatment, d indicated by the quantification of ?-catenin⁺/DAPI⁺ dots in brain region (n?=?6 per group, ***p?<?0.001, t-test). e RT-qPCR analysis of the transcriptional target of ?-catenin, mycb, axin2 and jun (n?=?6 per group, *p?<?0.05, t-test)

Stabilizing of ?-catenin by gsk3? inhibition partially restores the head size deficit.

a Western blot analysis of ?-catenin. b Lateral views of representative larvae (3?dpf) exposed to DMSO, PMA or PMA+LiCl. Head size was calculated with the boundary shown in yellow dotted line. c Quantitative representation of body length and head size (3?dpf) normalized to DMSO (DMSO n?=?14, PMA n?=?18, PMA+LiCl 50?mM n?=?12, PMA+LiCl 100?mM n?=?18, *p?<?0.05, ***p?<?0.001, one-way ANOVA)

PKC over-activation induce a transient increase of cell apoptosis within the head. (a) TUNEL staining of zebrafish head. (b) Quantification of TUNEL signaling. (* p<0.05, t-test).

PKC hyper-activation has a mild effect on neurogenesis at 24hpf (a) pHH3 and TUNEL staining of zebrafish embryos. (b) Quantification of pHH3 and TUNEL staining within zebrafish head. (* p<0.05, t-test).