Thalidomide Retards Brain Development and Causes Microcephaly

(A) Head morphology of 72-hpf zebrafish that were grown in the presence of 0.1% DMSO (left), 200 μM thalidomide (middle), or 400 μM thalidomide (right). Thin vertical lines indicate the distance between the rostral-most tip of olfactory bulbs and the temporal-most edge of eyes. Otic vesicles (arrowheads) and pectoral fins (arrows) are also indicated.

(B) A schematic diagram depicting body length (black arrow), eye diameter (blue arrow) and head thickness (red arrow) that were used to determine ratios.

(C and D) The ratios of eye diameter (blue arrow) in (C) and head thickness (red arrow) in (D) to body length (black arrow) of zebrafish that were grown with or without 400 µM thalidomide were determined and are shown as means ± SEM (n = 20 per group).

(E) Primary neurons of 24-hpf embryos that were grown in the presence of 0.1% DMSO or 200 or 400 μM thalidomide. Where indicated, capped mRNA encoding crbn^WT or crbn^YW/AA was microinjected at the 1-cell stage before thalidomide treatment. Bright-field (BF) (upper panels) and fluorescence (lower panels) images of embryos immunostained with acetylated α-tubulin antibody are shown. Tel, telencephalon; SOC, supraoptic commissure; TPOC, tract of postoptic commissure; PC, posterior commissure.

(F) Fluorescence intensity of acetylated α-tubulin-positive neural clusters in telencephalon and TPOC. Fluorescence intensities of the regions indicated with rectangles in (E) were measured and normalized to the intensity of DMSO-treated embryos and are shown as means ± SD (n = 15 per group).

(G) Percent incidence of head phenotype (n = 100 per group in a single trial). The head sizes of 72-hpf embryos were classified based on the head-to-body ratio using the following criteria: ≥13%, normal; <13%, small.

Scale bar, 50 μm. *p < 0.05, ***p < 0.001.

Knockdown of crbn or cul4 Impairs Head Development

(A) Schematic structure of CRL4^CRBN binding to thalidomide.

(B) Immunoblot analysis (left) and RT-PCR analysis (right) of 24-hpf embryos injected with the indicated MOs against crbn. The indicated antibodies were used to analyze embryos injected with the translation-blocking MO. RT-PCR was performed using the indicated primer set to analyze embryos injected with the splice-blocking MO. The target site of the MO is indicated with red bar in the diagram.

(C and E) One-cell-stage embryos were left uninjected or injected with the indicated MOs against crbn (C) or cul4 (E) with or without corresponding mRNA and then subjected to immunostaining with anti-acetylated α-tubulin antibody at 24 hpf. Fluorescence images (lower panels) and those overlaid with bright-field images (upper panels) are shown.

(D and F) The ratios of head thickness to body length of embryos that were left uninjected or injected with MOs against crbn (D) or cul4 (F) are shown as means ± SD (n = 15 per group). Scale bar, 50 µm. ∗∗∗p < 0.001.

Overexpression of crbn Enlarges the Head

(A–D) Head enlargement in crbn^WT-overexpressing embryos at the 10-somite stage (14 hpf) (A), 24 hpf (B), and 48 hpf (C and D). One-cell-stage embryos were left uninjected or injected with approximately 300 pg of capped mRNA (300 ng/μL) encoding gfp, crbn^ΔMid, or crbn^WT.

(E) One-cell-stage embryos were left uninjected or injected with capped mRNA at the indicated concentration. The head sizes of 14-hpf embryos were classified into “large” and “normal” based on the head-to-body ratio using the following criteria: ≤8%, normal; >8%, large. The fractions of large head embryos are shown as means ± SD (n = 3 per group).

(F and G) The ratios of eye diameter (F) and head thickness (G) to body length were determined at 48 hpf as in Figures 1B–1D and are shown as means ± SD (n = 15 per group).

Scale bar, 50 μm in (A and B). *p < 0.05, **p < 0.01, ***p < 0.001.

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.