Fig. 1

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.