Dependency of early cardiac enhancer activities on <i>tin</i>.

<p>Shown are stage 11–12 embryos stained for enhancer activities (anti-βGal or anti-GFP) and Tin (green). (A–K) Enhancer activities in wild type backgrounds (left corner quadrants: anti-Tin omitted for better visualization of reporter patterns; arrow heads: early cardiac expression). (A′–K′) Enhancer activities in homozygous <i>tin</i>³⁴⁶ mutant backgrounds. (A, A′) <i>EgfrE1</i>-LacZ expression in cardiac mesoderm but not in somatic mesoderm (asterisks) requires <i>tin</i>. (B, B′) <i>fzL4-</i>GFP expression in cardioblast progenitors requires <i>tin</i>. (C, C′) High-level <i>HimL47</i>-GFP expression in cardiogenic mesoderm requires <i>tin</i> but somatic mesodermal expression does not. (D, D′) <i>lin-28L64</i>-GFP expression in cardiac mesoderm requires <i>tin</i>. Amnioserosa expression is unaffected in <i>tin</i> mutants. (E, E′) <i>midE19</i>-GFP expression in cardioblast progenitors requires <i>tin</i>. (F, F′) <i>RhoLE102</i>-GFP expression in cardiogenic mesoderm, but not in somatic mesoderm, requires <i>tin</i>. (G, G′) <i>tshL8</i>-LacZ expression in cardiogenic mesoderm, but not in somatic mesoderm, requires <i>tin</i>. (H, H′) <i>tupE9</i>-GFP expression in cardiogenic mesoderm requires <i>tin</i>. (I, I′) <i>unc-5L25</i>-GFP expression in cardiac mesoderm but not in somatic mesoderm requires <i>tin</i>. (J, J′) <i>CG3638L6</i>-GFP expression in cardioblast progenitors requires <i>tin</i>. (K, K′) <i>CG9973E15</i>-GFP expression in cardioblast progenitors requires <i>tin</i>.</p

<i>In vivo</i> assays of the function of the CEE motifs in selected cardiac enhancers.

<p>(A1, B1, C1) Schematic drawings of the predicted Tin, Pnr, and Doc binding sites within cardiac enhancers as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003195#pgen-1003195-g005" target="_blank">Figure 5</a> and the positions of the CEE motifs relative to these (green bars). (A2–A5) Mutation of the CEE motifs causes a strong reduction of <i>EgfrE1s</i>-GFP activity in cardioblast progenitors at stage 12 (A2, A3) and in cardioblasts at stage 14 (A4, A5). (B2–B5) Mutation of the CEE motifs leads to the absence of <i>lin-28L64s</i>-GFP activity in heart precursors (and amnioserosa cells) at stage 14 (B2, B3) and a reduced activity in cardioblasts and pericardial cells at stage 16 (B4, B5). (C2, C3) Mutation of the CEE motifs causes almost complete loss of <i>midE19s</i>-GFP activity in Tin⁺ cardioblasts (stage 16).</p

<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).</p

Tin binding and reporter activity of enhancers active in dorsal mesodermal cells, cardiac progenitors and heart progenitors.

<p>(A–P) show the Tin binding peaks (blue: 3–5.5 hrs: pink: 5–8 hrs AEL), the location of the tested enhancers (yellow bars) and the gene models (red: genes closest to tested fragment). Y axes are at identical scales whereas x axis scales are variable (see scale bars). A′ to P′ show enhancer activities at earliest stage of appearance (arrow or arrow heads: dorsal/cardiac mesoderm) and A″ to P″ at latest stage of detection in cardiac tissues (arrows or arrow heads). Shown are <i>GFP</i> or <i>lacZ in situ</i> hybridizations except in A″, I′, I″ and P″, which show anti-GFP antibody stainings (in A″, I″ and P″ perduring GFP from earlier expression). Genes are ordered alphabetically, with CGs last. (A′) Stage 12. Expression of <i>discoL10-GFP</i> in heart progenitors, segmental subsets of visceral muscle precursors and (A″), stage 14, perdurance of GFP in developing visceral muscles and heart. (B′, B″) Expression of <i>EgfrE1-lacZ</i> in cardioblast progenitors and subsets of somatic mesodermal cells (stage 12), and in cardioblasts (stage 14). (C′, C″) Expression of <i>fzL4-GFP</i> in cardioblast progenitors (stage 12) and in cardioblasts and amnioserosa (stage 14). (D′, D″) Expression of <i>HimL47-GFP</i> in cardiogenic mesoderm and somatic mesodermal cells (stage 12), and in cardioblasts and developing somatic muscles (stage 14). (E′, E″) Expression of <i>hthE54-GFP</i> in heart progenitors of Md, T1–T3 segments (arrow heads) and in somatic mesoderm (with A–P graded sizes of clusters) (stage 12) and in anterior developing heart cells (arrow head) and somatic muscles (stage 14). (F′, F″) Expression of <i>lin-28L64-GFP</i> in cardioblasts (stage 12, stage14). (G′, G″) Expression of <i>maL9-GFP</i> in cardiac and somatic mesoderm (stage 12) and in heart precursors (stage 14). (H′, H″) Expression of <i>midE19-GFP</i> in Tin⁺ cardioblasts (stage 12, stage 15). (I′, I″) Expression (I′, stage 12) and perdurance (I″ stage 14) of <i>nauL35</i>-GFP largely in dorsal somatic mesoderm (asterisks). (J′, J″) Expression of <i>nocL7-GFP</i> in pericardial cell progenitors (stage 12) and pericardial cells (stage 16). (K′, K″) Expression of <i>RhoLE102-GFP</i> in segmented dorsal mesoderm (stage 11) and cardioblast progenitors (stage 12). (L′, L″) Expression of <i>tshL8-lacZ</i> in cardiogenic mesoderm and thoracic ectoderm (stage 11) and in Tin⁺ pericardial cells (stage 15). (M′, M″) Expression of <i>tupE9-GFP</i> in cardiogenic mesoderm (stage 11) and cardioblasts (stage 14). (N′, N″) Expression of <i>unc-5L25-GFP</i> in cardioblast and pericardial cell progenitors (stage 12) and in cardioblasts and Tin⁺ pericardial cell (stage 16). (O′, O″) Expression of <i>CG3638L6-GFP</i> in cardioblast progenitors (stage 12) and cardioblasts (stage 15). (P′, P″) Expression of <i>CG9973E15-GFP</i> in cardiac mesoderm (stage 11) and perdurance of GFP in developing heart (stage 14).</p

Sequence motifs enriched in Tin binding regions.

<p>(A) Schematic drawings of the expression domains of Tinman (red), Doc (green, hatched), and Pnr (blue, hatched) during the embryonic stages used for chromatin preparations. Grey: mesoderm not expressing any of these factors. (B) Tin motif recovered using <i>de novo</i> motif discovery in the top 100 peaks and full Early and Late datasets. The Tin motifs discovered in the full Early and Late dataset are almost identical, while the ones from top 100 peaks show some differences. These Tin motifs closely resemble the motifs derived from published Tin binding sites with verified <i>in vivo</i> functions (bottom) and previously published Tinman/Nkx2-5 motifs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003195#pgen.1003195-Chen1" target="_blank">[80]</a>. The Tin motif from the full datasets is preferentially located in the center (0.5 on X-axis) of ChIP peaks (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003195#s4" target="_blank">Materials and Methods</a>). (C) The AGATAC motif is the most enriched sixmer in both Early and Late datasets. The core of this motif is the GATA sequence to which a number of TFs, including GATA factor Pnr, bind to. The AGATAC motif is preferentially located in the centre of ChIP peaks similarly to the Tin motif, but to a slightly lesser extent. (D) The binding motifs of the T-box factors Mid and Doc2 from SELEX experiments. The Doc2 motif is also located preferentially near the ChIP peak centre, but to a lesser extent than both Tin and GATA motifs.</p