Fig. 1

Identification of teleost ptf1a autoregulatory enhancers. (A) The diagram represents the predicted PTF1 binding sites upstream of the ptf1a coding sequence from the indicated teleost species; the red squares indicate E boxes (CANNTG) and the blue squares TC-boxes (TTCCC). (B) The transgenes generated for analysis of the autoregulatory enhancer are diagramed, including the exact sequences of the E and TC boxes of the two proximal sites, and the mutations introduced into them. Transgenes containing all three (C) or only the two proximal (D) PTF1 binding sites, in conjunction with the heterologous cFos minimal promoter, have similar activity to the entire ptf1a locus at 2 dpf (C), where they are active in the nervous system and pancreas (arrowhead), and at 5 dpf (D), where they show robust activity in the exocrine pancreas. The indicated mutations of the E-box and TC-box sequences abolish this activity (Supplementary Fig. 1). (E, F) A 90 bp synthetic enhancer containing the 3 predicted binding sites recapitulates this expression pattern at 2 dpf (E) and 5 dpf (F), but in addition is active in the retina, a site of endogenous ptf1a expression. medaka: O. latipes, stickleback: G. aculeatus, fugu: T. rubribes, tetraodon: T. nigroviridis, zebrafish: D. rerio. Scale bars: 200 μm.