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Fig. 3. 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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