Fig. 5

Loss of function of def selectively up-regulates the expression of 113p53 in the def^hi429 mutant. (A) 5'-RACE reaction identified two p53 isoforms. The longer isoform corresponds to the wild-type p53 (p53) and was predominant in the wild-type embryos. The short isoform (113p53) was drastically increased in the def^hi429 mutant. (B) The short isoform contains 155 bp derived from intron 4 (letters in lowercase) immediately adjacent to exon 5 and encodes for an N-truncated p53 protein initiated at codon 113 of the wild-type p53. (C) RNA gel blot hybridization using probes for detecting both p53 and 113p53 isoforms (P1 probe, top panel), p53 only (P2 probe, second panel), and 113p53 only (P3 probe, third panel) showed that 113p53 but not p53 was selectively increased in the def-MO morphants (def-MO) and def^hi429 mutant (mu), respectively, when compared with the standard control MO morphants (SC-MO) and the wild-type control (wt). (Bottom panel) 28S rRNA was used as the loading control. (D) Whole-mount in situ hybridization using the P1 probe showed that 113p53 expression in the def^hi429 mutant (mu, right panels) was increased at 3 dpf, 4 dpf, and 5 dpf, and this increase was specifically limited to within digestive organs when compared with the expression of p53 in the wild type (left panels). (in) Intestine; (lv) liver; (p) pancreas; (sw) swimbladder. (E,F) At 4 dpf, sectioning in situ hybridization further confirmed that p53 expression was increased in the intestine (in), liver (data not shown), and exocrine pancreas (ex) but not in the endocrine pancreas (en) in the def^hi429 mutant.