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Foxf genes integrate tbx5 and hedgehog pathways in the second heart field for cardiac septation.

Hoffmann AD, Yang XH, Burnicka-Turek O, Bosman JD, Ren X, Steimle JD, Vokes SA, McMahon AP, Kalinichenko VV, Moskowitz IP - PLoS Genet. (2014)

Bottom Line: GLI1 and TBX5 synergistically activated transcription from this cis-regulatory element in vitro.This enhancer drove reproducible expression in vivo in the posterior SHF, the only region where Gli1 and Tbx5 expression overlaps.Our findings implicate Foxf genes in atrioventricular septation, describe the molecular underpinnings of the genetic interaction between Hedgehog signaling and Tbx5, and establish a molecular model for the selection of the SHF gene regulatory network for cardiac septation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics, Pathology, and Human Genetics, The University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
The Second Heart Field (SHF) has been implicated in several forms of congenital heart disease (CHD), including atrioventricular septal defects (AVSDs). Identifying the SHF gene regulatory networks required for atrioventricular septation is therefore an essential goal for understanding the molecular basis of AVSDs. We defined a SHF Hedgehog-dependent gene regulatory network using whole genome transcriptional profiling and GLI-chromatin interaction studies. The Forkhead box transcription factors Foxf1a and Foxf2 were identified as SHF Hedgehog targets. Compound haploinsufficiency for Foxf1a and Foxf2 caused atrioventricular septal defects, demonstrating the biological relevance of this regulatory network. We identified a Foxf1a cis-regulatory element that bound the Hedgehog transcriptional regulators GLI1 and GLI3 and the T-box transcription factor TBX5 in vivo. GLI1 and TBX5 synergistically activated transcription from this cis-regulatory element in vitro. This enhancer drove reproducible expression in vivo in the posterior SHF, the only region where Gli1 and Tbx5 expression overlaps. Our findings implicate Foxf genes in atrioventricular septation, describe the molecular underpinnings of the genetic interaction between Hedgehog signaling and Tbx5, and establish a molecular model for the selection of the SHF gene regulatory network for cardiac septation.

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Expression of Foxf1a and Foxf2 in shh−/− and Tbx5+/− mutants embryos at E9.5.In-situ hybridization demonstrated SHF expression of both Foxf1a and Foxf2, with a loss of SHF expression of Foxf1a in Shh mutants, (A, B) and a near-complete loss of Foxf2 in Shh mutants (E, F). Tbx5 heterozygotes expressed Foxf1a at decreased levels specifically in the posterior SHF tissues (C, D), whereas Foxf2 expression patterns were unchanged (G, H). Arrow: dorsal mesenchymal protrusion tissues in A′–H′, Brackets: SHF mesenchymal tissues. AT: Atrium, V: Ventricles.
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pgen-1004604-g003: Expression of Foxf1a and Foxf2 in shh−/− and Tbx5+/− mutants embryos at E9.5.In-situ hybridization demonstrated SHF expression of both Foxf1a and Foxf2, with a loss of SHF expression of Foxf1a in Shh mutants, (A, B) and a near-complete loss of Foxf2 in Shh mutants (E, F). Tbx5 heterozygotes expressed Foxf1a at decreased levels specifically in the posterior SHF tissues (C, D), whereas Foxf2 expression patterns were unchanged (G, H). Arrow: dorsal mesenchymal protrusion tissues in A′–H′, Brackets: SHF mesenchymal tissues. AT: Atrium, V: Ventricles.

Mentions: We investigated the hypothesis that Foxf1a and Foxf2 expression was downstream of Hedgehog signaling in cardiac development. Shh-dependent expression of both genes in the SHF was confirmed by qPCR: Foxf1a expression was reduced by 50% (p = 0.05) and Foxf2 was reduced by 80% in the SHF of Shh−/− versus wild-type controls (p = 0.01) (Figure 1D). In-situ hybridization to evaluate the patterning of expression showed that Foxf1a and Foxf2 were both expressed in the posterior SHF, but not in the heart, in wild-type embryos at E9.5, with Foxf1a expression extending more ventrally than Foxf2 to include the DMP (Figures 3A, A′, E, E′). Mesenchymal expression of both Foxf1a and Foxf2 demonstrated a severe decrement in shh−/− mutant embryos (Figures 3B, B′, F, F′).


Foxf genes integrate tbx5 and hedgehog pathways in the second heart field for cardiac septation.

Hoffmann AD, Yang XH, Burnicka-Turek O, Bosman JD, Ren X, Steimle JD, Vokes SA, McMahon AP, Kalinichenko VV, Moskowitz IP - PLoS Genet. (2014)

Expression of Foxf1a and Foxf2 in shh−/− and Tbx5+/− mutants embryos at E9.5.In-situ hybridization demonstrated SHF expression of both Foxf1a and Foxf2, with a loss of SHF expression of Foxf1a in Shh mutants, (A, B) and a near-complete loss of Foxf2 in Shh mutants (E, F). Tbx5 heterozygotes expressed Foxf1a at decreased levels specifically in the posterior SHF tissues (C, D), whereas Foxf2 expression patterns were unchanged (G, H). Arrow: dorsal mesenchymal protrusion tissues in A′–H′, Brackets: SHF mesenchymal tissues. AT: Atrium, V: Ventricles.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214600&req=5

pgen-1004604-g003: Expression of Foxf1a and Foxf2 in shh−/− and Tbx5+/− mutants embryos at E9.5.In-situ hybridization demonstrated SHF expression of both Foxf1a and Foxf2, with a loss of SHF expression of Foxf1a in Shh mutants, (A, B) and a near-complete loss of Foxf2 in Shh mutants (E, F). Tbx5 heterozygotes expressed Foxf1a at decreased levels specifically in the posterior SHF tissues (C, D), whereas Foxf2 expression patterns were unchanged (G, H). Arrow: dorsal mesenchymal protrusion tissues in A′–H′, Brackets: SHF mesenchymal tissues. AT: Atrium, V: Ventricles.
Mentions: We investigated the hypothesis that Foxf1a and Foxf2 expression was downstream of Hedgehog signaling in cardiac development. Shh-dependent expression of both genes in the SHF was confirmed by qPCR: Foxf1a expression was reduced by 50% (p = 0.05) and Foxf2 was reduced by 80% in the SHF of Shh−/− versus wild-type controls (p = 0.01) (Figure 1D). In-situ hybridization to evaluate the patterning of expression showed that Foxf1a and Foxf2 were both expressed in the posterior SHF, but not in the heart, in wild-type embryos at E9.5, with Foxf1a expression extending more ventrally than Foxf2 to include the DMP (Figures 3A, A′, E, E′). Mesenchymal expression of both Foxf1a and Foxf2 demonstrated a severe decrement in shh−/− mutant embryos (Figures 3B, B′, F, F′).

Bottom Line: GLI1 and TBX5 synergistically activated transcription from this cis-regulatory element in vitro.This enhancer drove reproducible expression in vivo in the posterior SHF, the only region where Gli1 and Tbx5 expression overlaps.Our findings implicate Foxf genes in atrioventricular septation, describe the molecular underpinnings of the genetic interaction between Hedgehog signaling and Tbx5, and establish a molecular model for the selection of the SHF gene regulatory network for cardiac septation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics, Pathology, and Human Genetics, The University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
The Second Heart Field (SHF) has been implicated in several forms of congenital heart disease (CHD), including atrioventricular septal defects (AVSDs). Identifying the SHF gene regulatory networks required for atrioventricular septation is therefore an essential goal for understanding the molecular basis of AVSDs. We defined a SHF Hedgehog-dependent gene regulatory network using whole genome transcriptional profiling and GLI-chromatin interaction studies. The Forkhead box transcription factors Foxf1a and Foxf2 were identified as SHF Hedgehog targets. Compound haploinsufficiency for Foxf1a and Foxf2 caused atrioventricular septal defects, demonstrating the biological relevance of this regulatory network. We identified a Foxf1a cis-regulatory element that bound the Hedgehog transcriptional regulators GLI1 and GLI3 and the T-box transcription factor TBX5 in vivo. GLI1 and TBX5 synergistically activated transcription from this cis-regulatory element in vitro. This enhancer drove reproducible expression in vivo in the posterior SHF, the only region where Gli1 and Tbx5 expression overlaps. Our findings implicate Foxf genes in atrioventricular septation, describe the molecular underpinnings of the genetic interaction between Hedgehog signaling and Tbx5, and establish a molecular model for the selection of the SHF gene regulatory network for cardiac septation.

Show MeSH
Related in: MedlinePlus