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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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Transcriptional profiling of SHF from shh−/− embryos.(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 Shh, Gli1 and Ptch1 in shh 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 Shh 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.
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pgen-1004604-g001: Transcriptional profiling of SHF from shh−/− embryos.(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 Shh, Gli1 and Ptch1 in shh 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 Shh 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.

Mentions: Progenitor cells for the atrial and atrioventricular septum require Shh signaling in the posterior SHF (pSHF) between embryonic day 8 and embryonic day 10 (E8–E10) to migrate into the heart to form the atrial septum between E9–E11 [4], [5]. To identify the Hedgehog-dependent gene regulatory networks required for this process, we compared transcriptional profiling of the posterior SHF from wild-type and Shh (MGI: 1932461) embryos at E9.5 to identify differentially expressed transcripts. We isolated the pSHF by microdissection including the dorsal mesenchymal protrusion and closely associated surrounding ventral lateral plate mesenchyme. Our dissection included the attached foregut, but excluded the heart, dorsal lateral plate mesenchyme and neural tube (Figure 1A). RNA was isolated and known Hedgehog-dependent transcripts were evaluated by RT-PCR to verify genotyping prior to whole genome transcriptional profiling. Shh, Ptch1 (19206) and Gli1 all demonstrated significantly reduced expression (p>0.05) in the Shh samples compared to wild-type micro-dissected samples (Figure 1B). Specifically, Shh was reduced more than 90%, while Ptch1 and Gli1 were each reduced approximately 50%, consistent with significantly reduced Hedgehog signaling in the mutant samples and confirming the genotypic fidelity of the isolated samples.


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)

Transcriptional profiling of SHF from shh−/− embryos.(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 Shh, Gli1 and Ptch1 in shh 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 Shh 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.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004604-g001: Transcriptional profiling of SHF from shh−/− embryos.(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 Shh, Gli1 and Ptch1 in shh 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 Shh 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.
Mentions: Progenitor cells for the atrial and atrioventricular septum require Shh signaling in the posterior SHF (pSHF) between embryonic day 8 and embryonic day 10 (E8–E10) to migrate into the heart to form the atrial septum between E9–E11 [4], [5]. To identify the Hedgehog-dependent gene regulatory networks required for this process, we compared transcriptional profiling of the posterior SHF from wild-type and Shh (MGI: 1932461) embryos at E9.5 to identify differentially expressed transcripts. We isolated the pSHF by microdissection including the dorsal mesenchymal protrusion and closely associated surrounding ventral lateral plate mesenchyme. Our dissection included the attached foregut, but excluded the heart, dorsal lateral plate mesenchyme and neural tube (Figure 1A). RNA was isolated and known Hedgehog-dependent transcripts were evaluated by RT-PCR to verify genotyping prior to whole genome transcriptional profiling. Shh, Ptch1 (19206) and Gli1 all demonstrated significantly reduced expression (p>0.05) in the Shh samples compared to wild-type micro-dissected samples (Figure 1B). Specifically, Shh was reduced more than 90%, while Ptch1 and Gli1 were each reduced approximately 50%, consistent with significantly reduced Hedgehog signaling in the mutant samples and confirming the genotypic fidelity of the isolated samples.

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