Figure 3

Stable CRISPR/Cas9 mutagenesis of six2b. (a) Schematic of mutagenesis target site. (b) PCR analysis of stable line six2b^T35-3 genotype differentiating between heterozygous and wild-type animals. (c) Amino acid alignment of wild-type and mutant Six2b. The mutant line is predicted to lose most of the Six domain and the entire homeodomain and C-terminus. Black bar covers Six domain, grey bar covers homeodomain. (d) Whole mount in situ hybridization of homozygous mutant embryos at 48 hpf for wt1a, cdh17, and slc20a1a. Proximal tubule defects were detected in homozygous embryos. (e) Compiled data of proximal tubule morphology defects observed in six2b homozygous mutants. For comparison, heterozygous embryos are displayed in Supplemental Fig. 3 (f) qPCR of six2a and six2b in heterozygous and homozygous six2b mutants at 15 somites. Expression is relative to wild-type embryos at the same developmental stage. Error bars are standard error of the mean. *p-value = 0.03.