Early expression of six2a and six2b paralogs. (a, c, e, g) Whole mount in situ hybridization for six2a. Expression is detected in the developing head and otic vesicles. Focal expression was observed adjacent to the pronephric tubule at 24 hpf (arrowhead in g). (b, d, f, h) Whole mount in situ hybridization for six2b. Expression was detected in the intermediate mesoderm and otic vesicles. At 18 somites, weak signal could be detected in the pronephric tubules. By 24 hpf six2b was predominantly found in cells adjacent to the tubule epithelium (arrowhead in h). Head is toward the left in all images. IM, intermediate mesoderm; OT, otic vesicle; P, pronephros.

Pronephric marker expression following injection of six2b MO1. (a) Expression of pronephric markers at 24 hpf following MO1 injection. No significant changes in gene expression or tissue morphology were observed. (b) Expression of pronephric markers at 48 hpf following MO1 injection. Knock-down of six2b resulted in glomerular defects as seen with wt1a, nphs1, and nphs2. Proximal tubule morphogenesis was defective using cdh17 and slc20a1a probes. The proximal straight tubule appeared unaffected using trpm7. (c) Compiled data of wt1a morphological defects following mouse Six2 mRNA injection alone or combined with MO1. Six2 mRNA co-injection partially rescued the MO1 induced wt1a phenotype. Arrowheads for 48 hpf pax2a expression denote neck region.

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.

Pronephric marker expression in F₀ embryos following six2b CRISPR/Cas9 reaction mix injection. (a) Pronephric marker expression at 24 hpf. No significant changes in gene expression or tissue morphology were observed. (b) Expression of pronephric markers at 48 hpf. Morphological defects in the glomerulus and proximal tubule are observed following genetic disruption of the six2b locus. The proximal straight tubule marked by trpm7 appeared unaffected. Arrowhead for pax2a denotes the neck region. (c) PCR detection of genomic DNA lesions in the six2b gene from two independently injected embryos displaying defective wt1a expression domain morphology. Bottom arrow points to wild-type allele amplicon. (d) Representative TIDE analysis of F₀ CRISPR/Cas9 injected embryo following in situ hybridization for wt1a.