Overexpression experiments with bach1b and nrf2a reveal their antagonism in the regulation of zymogen expression. (A) qRT-PCR analysis showed that bach1b overexpression resulted in the downregulation of six peptidase precursor genes – cpa5, ctr1l, ctrb1, ela2l, try and tryl – whereas nrf2a overexpression resulted in their upregulation. Overexpression experiments were performed by microinjecting bach1b or nrf2a capped mRNAs into one-cell stage embryos. The expression levels of the six peptidase precursor genes in the microinjected and control larvae at 84 hpf were determined by qRT-PCR analysis. (B) Representative images of in situ hybridization staining show that downregulation of cpa5 resulted from bach1b overexpression and that its upregulation resulted from nrf2a overexpression, both specifically in the exocrine pancreas. Dorsal view, anterior to the left. (C) Mean optic densities of in situ hybridization staining of a group of larvae (10–12 each) corresponding to Fig. 3B were quantified by using ImageJ. (D) cpa5 morphant phenotypes. For individual larvae (84 hpf) of the bach1b overexpression group, the optic density values lower than the mean optic density value of its own group were marked as ‘strong’, and the optic density values higher than the mean optic density of the control group were marked as ‘weak’. For individual larvae of the nrf2a overexpression group, the optic density values higher than the mean optic density value of its own group were marked as ‘strong’, the optic density values lower than the mean optic density of the control group were marked as ‘weak’, and the values of optic density between ‘weak’ and ‘strong’ marked as ‘medium’. The statistical significance of difference between means was determined by one-way ANOVA and Tukey’s multiple comparison test (n=9) by using SPSS10.0.1. *P<0.05, **P<0.01, ***P<0.001.
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