osr1 knockdown results in segment-specific kidney defects. (A) osr1 gene structure and morpholino oligo targeting exon 2 (ex2d). (B) RT-PCR 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 and 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 five-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), whereas the distal pronephros is unaffected (black arrowhead). Immunofluorescence using anti-NaK ATPase alpha6F monoclonal antibody labels the entire pronephros in wild-type embryos (E), whereas expression is specifically lost (F) in the proximal nephron (white arrowhead) of osr1 morphants, leaving the distal pronephros unaffected (red arrowhead).
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