Fig. 5

col4a1Δ20 mutant larvae have a milder neurovascular phenotype than crispants. A. Schematic representing the 20-base pair deletion at the exon 8/intron 8–9 boundary of the zebrafish col4a1 gene B. Representative maximum intensity projections of the developing cerebrovasculature in wildtype (col4a1+/+), heterozygous (col4a1+/Δ20) and homozygous (col4a1Δ20/Δ20) larvae on the Tg(kdrl: EGFP) background at 4 dpf. The zebrafish were live-imaged using light-sheet microscopy in a dorsal orientation with a 20x objective and 0.7x digital zoom. Images are a maximum intensity projection of a z-stack with an inverted greyscale image below. Different individual fish represent each genotype at each time point and the scale bar is 100 µm. Genotyping was performed after image acquisition. Abbreviations: PHBC- primordial hindbrain channel, PrA-porencephalic artery, PMBC- primordial midbrain channel. C. The distance between the left and right PMBC is reduced in col4a1Δ20/Δ20 larvae compared to col4a1+/+ siblings. D. Basilar artery (BA) width is increased in col4a1+/Δ20 and col4a1Δ20/Δ20 compared to wildtype col4a1+/+ siblings. E. The mean width of both PHBCs is increased in col4a1+/Δ20 and col4a1Δ20/Δ20 larvae compared to col4a1+/+ siblings. There is also a difference between col4a1+/Δ20 and col4a1Δ20/Δ20 siblings. F. The area of forebrain space is increased in col4a1Δ20/Δ20 larvae compared to col4a1+/+ siblings. C-F. Vascular measurements were made on maximum intensity projections from Z-stacks using FIJI. The larvae were live-imaged using light-sheet microscopy in a dorsal orientation with a 20x objective and 0.7x digital zoom. Genotyping was performed after image acquisition. n = 18–27 per group over 3 biological replicates with each larvae measurement plotted individually. Data were analysed with a one-way ANOVA. Asterisks signify means that are significantly different at the 5 % level using the Tukey HSD test ns P > 0.05, * P ≤ 0.05, **P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001. G. An o-dianisidine-stained col4a1Δ20/Δ20 larva at 3 days post fertilisation showing ICH (red arrows) and a 100 µm scale bar. H. Pie chart to show the expected Mendelian ratio of embryos from a heterozygous incross. I. Embryos from a col4a1+/Δ20 incross were treated with increasing concentrations of atorvastatin (ATV) at 1 dpf. At 2 dpf, embryos were stained with o-dianisidine to isolate ICH+ and ICH- embryos followed by genotyping. The percentage of ICH+ embryos from each genotype is written in white and the total number of ICH+ embryos are written below. Data are from two biological replicates.