Figure 3. osr1 knockdown results in segment-specific kidney defects.

(A) osr1 gene structure and morpholino oligo targeting exon 2 (ex2d). (B) RTPCR analysis of morpholino-induced osr1 mis-splicing. Blocking the exon 2 splice donor sequence resulted in the complete deletion of exon 2, which contains the ATG start codon as well as the entire coding sequence for the osr1 transcriptional regulatory domain and the first zinc finger. In embryos injected with 7.4 ng morpholino, no wild-type mRNA was detectable at 24 hpf, indicating that these morphant embryos were functionally null for osr1. A 5-base pair mismatch control morpholino did not cause any molecular or phenotypic defects. Co-injection of osr1 synthetic mRNA along with the exon 2 donor morpholino rescued the osr1 phenotype in 53% (9/17) of embryos as determined by pax2a expression (see results), demonstrating specificity of the morpholino knockdown. (C) Expression of ae2 in the proximal pronephros (white arrowhead) is absent in osr1 morphants (D) while the distal pronephros is unaffected. Immunofluorescence using anti-NaK ATPase alpha6F monoclonal antibody labels the entire pronephros in wild type embryos (E) while expression is specifically lost in the proximal nephron of osr1 morphants (white arrowhead, F) while the distal pronephros was unaffected (red arrowhead, F) .