Fig. 4

Increased Expression of Brain Marker Genes in crbn-overexpressing Embryos

(A–E) In situ hybridization analysis for various brain marker genes in uninjected embryos or those overexpressing gfp, crbn^ΔMid, or crbn^WT. (A) sox2 expression in the anterior brain field at 9 hpf. Dorsal view, anterior to the top. (B) c-mycexpression at 30 hpf in the tectal proliferating zone and in the ciliary marginal zone in retina. Lateral view, anterior to the left. (C) emx1 expression at 20 hpf in dorsal telencephalon. Lateral view, anterior to the left. (D) otx2a expression at 20 hpf in diencephalon. Lateral view, anterior to the left. (E) pax2.1 expression at 20 hpf in optic stalk and MHB. Lateral view, anterior to the left.

(F) The areas of expression domains for these genes, indicated with dashed lines in (A) to (E), were measured and normalized to the value of uninjected embryos and are shown as means ± SD. (n = 15 per group).

(G) One-cell-stage embryos of her5PAC:egfp transgenic zebrafish were left uninjected or injected with capped mRNA encoding mcherry, crbn^ΔMid, or crbn^WTand then analyzed at 24 hpf. Green fluorescence images (lower panels) and those overlaid with DAPI signals (upper panels) are shown.

(H) The number of her5PAC:EGFP-positive cells were counted and are shown as means ± SD (n = 4–6 per group).

(I and J) After mRNA microinjection, her5PAC:egfp transgenic embryos were grown in the presence of 0.1% DMSO or 400 μM thalidomide and analyzed at 24 hpf (I) or 30 hpf (J). Green fluorescence images overlaid with bright-field images are shown.

Scale bar, 100 μm. *p < 0.05, ***p < 0.001.