Fig. 5

Exposure to BN and BC194 results in vascular defects and mis-patterning.

(a) Representative confocal images (20x magnification) of zebrafish ISVs at 24 and 48 hpe. Zebrafish embryos were incubated in egg water at 25 °C until 8–12 somites of age. The embryos were then manually dechorionated and incubated in egg water containing the indicated compounds at 28.5 °C until the images were taken. Calibration bar represents 100 µm. (b,c) Quantification of ectopic branching (b) and aberrant patterning (c) of ISVs. Embryos were incubated with the indicated compounds (5 µM) or DMSO until images were taken by fluorescent microscopy at 24 and 48 hpe. Aberrant structures were then counted within a region encompassing 5 ISVs anterior and posterior from the end of the yolk extension; mean ± SEM, n ≥ 8, ^#*p < 0.0001 relative to DMSO at 24 and 48 hpe respectively (one-way ANOVA, Tukey Test). ^‡BN-treated fish rarely survived to 48 hpe. (d) RT-qPCR values for the expression amino acid starvation response genes asns, gpt2, and eif4ebp1. Dechorionated embryos (15 somites) were incubated in egg water containing the indicated compounds for 24 hours. Total RNA was extracted by trizol/chloroform and the expression levels relative to actb were determined using the ΔΔCT method; mean ± SEM, n = 3, ****p < 0.0001 relative to DMSO (two-way ANOVA, Tukey Test). See Supplementary Figure S7 for physiological and toxicity information.