Fig. 4

Tuba1a knockdown dramatically inhibits CNS development. Confocal images of zebrafish injected with tuba1a MO and rescue mRNAs. Top panels: Control (0.25 mM), tuba1a (0.125 mM) and tuba1b-targeting morpholinos (MO) (0.25 mM), along with bcl2 mRNA (25 pg) were injected into single cell zebrafish embryos. Embryos were analyzed at 24 h post fertilization for differentiating neurons by whole mount immunostaining with anti-acetylated tubulin antibody. Shown are representative images of immunostained embryos. Arrowheads point to the developing post-optic commissure (POC) and the anterior commissure (AC), while arrows point to the trigeminal ganglion (TG) and the motor neurons (MN). Note the lack of differentiating neurons in the Tuba1a knockdown embryos. Bottom panels: Control (0.25 mM) or tuba1a-targeting (0.125 mM) MOs with bcl2 mRNA (25 pg) or tuba1a mRNA (12.5 pg) were injected into single cell zebrafish embryos. Embryos were analyzed for differentiating neurons by whole mount immunostaining with anti-acetylated tubulin antibody. Shown are representative images of immunostained embryos focusing on the head/eye region. The majority of immunostaining detects sensory neurons on the head (black arrows), eye (white arrows) and running over the yolk (white arrowheads). Note that both the bcl2 and tuba1a mRNA rescued embryos show significant neural differentiation; however, tuba1a mRNA appears to rescue nervous system development much better than bcl2 mRNA.