Transcriptional profiling of SHF from <i>shh^−/−</i> embryos.

<p>(A) Microdissection for isolation of SHF tissues. E9.5 embryos were isolated (I). Thoracic tissues including the heart were removed from head and tail, kept for genotyping or non-cardiac controls (II). Neural tube was removed (III). SHF tissue was bisected and separated from the heart (IV). Microdissected tissue was kept as anterior SHF (Va), posterior SHF (Vb) or heart (Vc). (B) RT-PCR demonstrates decreased expression of <i>Shh</i>, <i>Gli1</i> and <i>Ptch1</i> in <i>shh</i> mutant SHF tissues isolated for transcriptional profiling (C) Gene Ontology biological processes (GOBPs) enriched in the transcriptional profile analysis of SHF tissue from wild-type and <i>Shh</i> mutant embryos identifies developmental terms. (D) 13 genes identified in the transcriptional profile were verified as Shh-dependent using RT-qPCR (relative quantitation, RQ). * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.</p

Model for Hedgehog/Tbx5 interaction.

<p>(A) Intersection of <i>Tbx5</i> expression, restricted to the posterior SHF and heart, and <i>Gli1</i> expression, broadly expressed in axial mesenchyme and brain but excluded from the heart, is the posterior SHF. Activation of TBX5/GLI1 responsive enhancer is observed principally in the overlap between the <i>Tbx5</i> and <i>Gli1</i> expression domains. (B) In the presence of GLI activator (GLIA) alone, the enhancer is weakly active. In the presence of both GLIA and TBX5 is transcription from the enhancer strongly activated. When the GLI binding site is mutated, GLIA alone is insufficient to activate strong expression, but GlLIA may interact with TBX5 to activate expression more strongly than TBX5 alone.</p

Analysis of ChIP-Seq data and its intersection with transcriptional profiling data.

<p>(A) Distribution of ChIP-seq peaks highlighted the modeled GLI3-binding centered in peak regions, using MACS2 software. (B) GLI3 ChIP-seq revealed 1316 peaks defining potential binding sites in the mouse genome. Intersection with <i>shh</i>-dependent transcriptional profiling identified 112 candidate direct Hedgehog-dependent target genes. (C) Summary of <i>de novo</i> and known motifs enriched in <i>shh</i>-dependent GLI3-bound regions (Top 2 sub-panels) compared with similar known GLI motifs from literature and TRANSFAC database (Bottom 3 sub-panels). (D) Among the 112 genes, 26 are transcription factors or regulators of transcription, a significant over-representation. (E) Among the 112 genes, 4 are FOX family transcription factors, a significant over-representation.</p

Integration of Hedgehog and Tbx5 activity on an enhancer at <i>Foxf1a</i>.

<p>(A) Integration of Hedgehog and Tbx5 activity on an enhancer at <i>Foxf1a</i>. ChIP-seq for GLI3 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004604#pgen-1004604-g002" target="_blank">Figure 2</a>) and TBX5 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004604#pgen.1004604-Kothary1" target="_blank">[35]</a> identified a candidate <i>Foxf1a</i> enhancer. (B) ChIP-PCR from microdissected pSHF for GLI3, GLI1 and TBX5 demonstrated <i>in vivo</i> binding of each factor to the candidate enhancer. (C) Luciferase assays demonstrated that GLI1 and TBX5 individually and together synergistically activated the enhancer. Activation of enhancer with mutated GLI binding sites was significantly reduced by GLI1; however, synergistic GLI1/TBX5 activity is largely maintained. Activation of enhancer with mutated TBX binding sites was reduced cells transfected with TBX5 alone, but activation in cells transfected with both GLI1 and TBX5 was still relatively high. (D) Representative images of the enhancer activated specific posterior SHF expression of <i>lacZ</i> in transient transgenic embryos at E9.5. Atria: At; Ventricle: V. P-values:, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.</p

Expression of <i>Foxf1a</i> and <i>Foxf2</i> in <i>shh^−/−</i> and <i>Tbx5^+/−</i> mutants embryos at E9.5.

<p>In-situ hybridization demonstrated SHF expression of both <i>Foxf1a</i> and <i>Foxf2</i>, with a loss of SHF expression of <i>Foxf1a</i> in <i>Shh</i> mutants, (A, B) and a near-complete loss of <i>Foxf2</i> in <i>Shh</i> mutants (E, F). <i>Tbx5</i> heterozygotes expressed <i>Foxf1a</i> at decreased levels specifically in the posterior SHF tissues (C, D), whereas <i>Foxf2</i> expression patterns were unchanged (G, H). Arrow: dorsal mesenchymal protrusion tissues in A′–H′, Brackets: SHF mesenchymal tissues. AT: Atrium, V: Ventricles.</p

Atrioventricular septal defects in <i>Foxf1a^+/−; Foxf2^+/−</i> compound heterozygote embryos at E14.5.

<p><i>Foxf1a^+/−; Foxf2^+/−</i> embryos displayed atrial septal defects including absence of the dorsal mesenchymal protrusion (D, D′, black arrows). Compound heterozygotes also displayed expanded mesenchymal cap of primary atrial septum (red arrow) (D, D′). Wild-type (A, A′), <i>Foxf1a^+/−</i> (B, B′), and <i>Foxf2^+/−</i> embryos (C, C′) showed no atrial septal defects. P-values (Fisher's exact test): <i>Foxf1a</i>^+/− (9 embryos) vs wild-type (4 embryos) = 1; <i>Foxf2</i>^+/− (2 embryos) vs wild-type = 0.33; <i>Foxf1a^+/−; Foxf2^+/−</i> (3 embryos) vs wild-type = 0.03.</p