Transcriptomic profiling analysis of bcas3 KO zebrafish larvae. (A), Hierarchical clustering heat map of 920 upregulated and 998 downregulated genes from 3dpf bcas3−/− larvae comparison to WT controls (n = 30, per group). Color intensity indicating fold changes. Upregulation and downregulation are marked in pink and blue, respectively. WT: wild-type larvae; mut: bcas3−/− larvae. (B), ClueGO pathway analysis. (C), Real-time RT-PCR analysis showed that the rpl10 mRNAlevel in 24 hpf embryos and 36 hpf embryos from bcas3−/− zebrafish was reduced compared to WT controls. (n = 3, per group). The rpl13a gene was used as endogenous control. (D), Real-time RT-PCR analysis for cyfip2, erbb3b and eya4 mRNA in 36 hpf zebrafish. (n = 3, per group). The rpl13a gene was used as endogenous control. (E), GESA analysis revealed that bcas3 KO affected expression of genes involved in neuron migration. GESA, gene set enrichment analysis. (F), Real-time RT-PCR analysis for nr2f1b, prkg1b and ackr3b mRNA in 3 dpf zebrafish. (n = 3, per group). The rpl13a gene was used as endogenous control. (G-H), 36 hpf WT and bcas3−/− larvae were stained with acridine orange (AO) to label apoptosis cells in vivo. Representative images (G) of AO staining in the larvae brains as indicated. Scale bars: 200 μm. (H), Quantitative analysis of apoptotic cells in the brain areas of the WT (n = 16) and bcas3−/− larvae (n = 17). (I-J), Western-blot analysis for apoptosis- relative protein Bcl2 and Bax in 3 dpf zebrafish. (n = 5, per group). β-Tubulin was used as endogenous control. Data are shown as mean ± SEM. Unpaired Student’s t-test was used to analyze RT-qPCR, AO staining, and western blot. Significance levels are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant
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