<i>In vivo</i> assays of the functions of predicted binding sites of Tin, Doc, and Pnr in identified cardiac enhancers.

<p>(A1–F1) Positions, orientations, and scores of predicted binding sites within cardiac enhancers (identical Y-scales). <i>In vivo</i> results for functionality of binding sites are summarized to the right (large ✓: essential; small (✓): required for full activity or redundantly with motifs for other factors; × not required). (A2, A3) <i>EgfrE1s</i>-GFP activity in cardiac mesoderm (A2, stage 12) is lost upon mutation of Tin binding motifs shown in (A1). (A4–A6) Mutation of GATA motifs shown in (A1) leads to a mild reduction of <i>EgfrE1s</i>-GFP activity at stage 12 (A4) and a more significant reduction at stage 14 (A6). (B2, B3) <i>lin-28L64s</i>-GFP activity in cardioblasts (B2, stage 14) is lost upon mutation of Tin binding motifs. (B4) Mutation of GATA motifs leads to loss of <i>lin-28L64s</i>-GFP activity in both cardioblasts and amnioserosa cells. (B5) Mutation of Doc binding motifs affects neither cardioblast nor amnioserosa activity of <i>lin-28L64s</i>-GFP. (C1, C2) <i>midE19s</i>-GFP activity in cardioblasts (C2, stage 15) is lost upon mutation of Tin binding motifs (C3). (C4) Mutation of GATA motifs does not affect <i>midE19s</i>-GFP activity. (C5) Mutation of Doc binding motifs reduces cardioblast <i>midE19s</i>-GFP activity. (D2–D4) Mutation of the binding motifs for Tin (D3) or Pnr (D4) does not affect <i>RhoLE102s</i>-GFP activity in the cardiogenic and dorsal somatic mesoderm. (D5) Mutation of Doc binding motifs causes loss of <i>RhoLE102s</i>-GFP activity in cardiogenic and dorsal somatic mesoderm. (E2, E3) Mutation of Tin binding motifs causes loss of <i>tupE9s</i>-GFP activity in dorsal and cardiogenic mesoderm (ms). Ectopic <i>tupE9s</i>-GFP occurs in dorsal ectoderm (ec). (E4, E5) Mutation of GATA motifs (E4) or Doc binding motifs (E5) does not affect <i>tupE9s</i>-GFP activity. (F2–F5) Mutation of either the Tin binding motifs (F3), the GATA motifs (F4) or the Doc binding motifs (F5) does not affect <i>unc-5L25s</i>-GFP activity in cardioblasts and pericardial cells at stage 15. Three sequences, CCAAGGG, TCAATTG, TCGAGTG, poorly matching the Tin binding motif (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003195#pgen-1003195-g001" target="_blank">Figure 1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003195#pgen.1003195.s011" target="_blank">Table S5</a>), are still present but it is unknown whether they can bind Tin. (F6) Staining of stage 15 <i>unc-5L25s</i>-GFP control embryo for GFP and Tin identifies GFP-positive cells as cardioblasts (arrow heads) and Tin⁺ pericardial cells (arrows). (F7) Simultaneous mutation of Tin binding motifs and GATA motifs does not affect cardiac <i>unc-5L25s</i>-GFP activity. (F8) Simultaneous mutation of Tin and Doc binding motifs severely reduces <i>unc-5L25s</i>-GFP activity in cardioblasts but not in pericardial cells. (F9) Simultaneous mutation of binding motifs for Tin, Pnr, and Doc abrogates <i>unc-5L25s</i>-GFP activity (anti-GFP, green) in cardioblasts but not in pericardial cells (anti-Eve, red).