FIGURE 1

VSOAC deficiency in swell1a^{twu0421/twu0421} and swell1b^{twu0422/twu0422} mutants. (A) Genomic lesions in swell1a^twu0421 (twu0421) and swell1b^twu0422 (twu0422) were generated using CRISPR/Cas9 with specific guideRNA (gRNA), while the swell1b^sa16642 (sa16642) mutant line was obtained from ZIRC. The gRNA-specific sequence is underlined, the protospacer adjacent motif (PAM) is shown in italics, deleted nucleotides in the mutant lines are highlighted in bold red, and a point mutation in sa16642 is shown in bold italic red. (B) Protein domain structure of swell1a and swell1b and corresponding mutant proteins. The transmembrane domain (TM) is shown as a blue box, and the leucine-rich repeat (LRR) domain is shown as a white box. The location of the missense amino acid mutation in the mutant line is indicated by a thick red line. (C) The inflation of the brain ventricle was not impaired in swell1a and swell1b mutant lines. The diencephalic/mesencephalic ventricle (DMv) area sizes are comparable between WT and swell1a/b single mutant, and the double knockout (dKO) line. Morpholinos specifically targeting swell1a (aMO) and swell1b (bMO) were used to validate gene knockdown specificity. The mutant lines resist the corresponding MO but remain susceptible to the other. Genotype: WT (wild-type); twu0421 (swell1a^twu0421); twu0422 (swell1b^twu0422); sa16642 (swell1b^sa16642); dKO (swell1a^twu0421; swell1b^twu0422). MO: a (swll1a morpholino); b (swell1b morpholino). The sample size (n) of each group is indicated at the top of the scatter plot. The data were collected from three biological replicates. ns, no significance; **p < 0.01; ***p < 0.001; ****p < 0.0001; #, no significant difference between each other. (D) Whole-cell patch clamp recordings under hypotonic conditions showed current changes in HAP1 cells but not in SWELL1-knockout cells (HAP1/SWELL1*). Overexpression of WT swell1a (HAP1/SWELL1* + swell1a) or swell1b (HAP1/SWELL1* + swell1b) restored VSOAC activity. However, overexpression of twu0421 (HAP1/SWELL1* + twu0421) or twu0422 (HAP1/SWELL1* + twu0422) failed to rescue VSOAC activity. The sample size (n) of each group is indicated at the top of the histogram. The data were collected from three biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001.