Maternally inherited Rad21 protein can sustain further cell division, but not gene expression; loss of one copy of rad21 affects transcription of runx1, ascl1a and ascl1b. (A,B) Rad21 protein is detectable in early rad21 morphant and mutant zebrafish embryos at stages when gene expression is compromised, but cell division can still occur. Protein was isolated from wild-type embryos and rad21ATGMO morphants (A) or nz171 homozygotes (B) at the stages indicated. Protein quantities were assessed by immunoblotting, with levels relative to α-tubulin. The graphs on the right show quantification of the immunoblots on the left. (C,D) Loss of one copy of rad21 reduces rad21 transcript and protein levels, and affects gene expression. (C) Protein was isolated from nz171 siblings (Sibs: includes +/+ and +/- embryos, which are phenotypically indistinguishable) and wild-type (+/+) controls at 48 h.p.f. Relative levels of Rad21 protein in nz171 siblings and wild-type embryos were quantified by immunoblotting and results are presented in the graph on the right (values represent the mean±s.e.m. of three independent experiments performed in triplicate. *P<0.01, Student′s t-test). A representative immunoblot appears on the left. (D) Quantitative RT-PCR of cDNA generated from pooled wild-type (+/+), nz171 sibling (Sibs) and nz171 mutant (-/-) embryos. Bar charts are representative results from three independent experiments. Values are relative to wild type, bars represent the mean of samples run in triplicate; all values are significant with P<0.05.
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