Fig. 4

Fig. 4 prep1.2 expression is positively regulated by retinoic acid. A, C and E: In situ hybridization experiments showing that prep1.2 mRNA in able to respond to RA treatment. In RA-treated embryos analyzed at 24 hpf the prep1.2 mRNA signal (blue, highlighted with a bar) is anteriorized and its intensity is significantly increased (C). In MO1-aldh1a2 injected embryos, prep1.2 expression is reduced, especially in the prospective branchial region (bar) (E). prep1.1 expression does not respond to RA (D) and is unaffected in aldh1a2 morphant embryos (F). G: qRT-PCR assay of prep1.2, hoxb1b and tbx1 expression in RA- and DEAB-treated embryos analyzed at 24 hpf. prep1.2 and hoxb1b are about 3-fold upregulated, while tbx1 is downregulated, in embryos treated with RA. DEAB treatment does not affect the expression of prep1.2 and tbx1 while hoxb1b is downregulated. The expression levels are compared with the housekeeping genes b-actin and eF1. H: Reporter analysis of the prep1.2 RARE. The basal promoter used in this study (p50-Luc) is unresponsive to RA. The 0.5 kbp intron-1 fragment bearing the putative prep1.2 RARE (p50-RARE-Luc) represses the basal promoter activity but confers a 7 fold RA-responsiveness. Mutagenesis of the RARE sequence of the 0.5 kbp intron-1 fragment restores the basal promoter activity.