A Tbx1-Six1/Eya1-Fgf8 genetic pathway controls mammalian cardiovascular and craniofacial morphogenesis
Chaoshe Guo, Ye Sun, Bin Zhou, Rosalyn M. Adam, XiaoKun Li, William T. Pu, Bernice E. Morrow, Anne Moon, Xue Li
Chaoshe Guo, Ye Sun, Bin Zhou, Rosalyn M. Adam, XiaoKun Li, William T. Pu, Bernice E. Morrow, Anne Moon, Xue Li
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Research Article Cardiology

A Tbx1-Six1/Eya1-Fgf8 genetic pathway controls mammalian cardiovascular and craniofacial morphogenesis

Abstract

Shared molecular programs govern the formation of heart and head during mammalian embryogenesis. Development of both structures is disrupted in human chromosomal microdeletion of 22q11.2 (del22q11), which causes DiGeorge syndrome (DGS) and velo-cardio-facial syndrome (VCFS). Here, we have identified a genetic pathway involving the Six1/Eya1 transcription complex that regulates cardiovascular and craniofacial development. We demonstrate that murine mutation of both Six1 and Eya1 recapitulated most features of human del22q11 syndromes, including craniofacial, cardiac outflow tract, and aortic arch malformations. The mutant phenotypes were attributable in part to a reduction of fibroblast growth factor 8 (Fgf8), which was shown to be a direct downstream effector of Six1 and Eya1. Furthermore, we showed that Six1 and Eya1 genetically interacted with Fgf8 and the critical del22q11 gene T-box transcription factor 1 (Tbx1) in mice. Together, these findings reveal a Tbx1-Six1/Eya1-Fgf8 genetic pathway that is crucial for mammalian cardiocraniofacial morphogenesis and provide insights into the pathogenesis of human del22q11 syndromes.

Authors

Chaoshe Guo, Ye Sun, Bin Zhou, Rosalyn M. Adam, XiaoKun Li, William T. Pu, Bernice E. Morrow, Anne Moon, Xue Li

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Figure 4

Six1 and Eya1 directly regulate Fgf8 expression.

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Six1 and Eya1 directly regulate Fgf8 expression. (A–D) Fgf8 express...
(A–D) Fgf8 expression was dependent on Six1 and Eya1. Fgf8 was downregulated in the PA ectoderm (arrowhead) of E9.5 Six1–/–Eya1+/– (B) and Six1–/–Eya1–/– (D) mutants and other PA regions (bracket) of Six1+/–Eya1–/– (C) and Six1–/–Eya1–/– (D) mutants. (E–I) Six1hpAP/Cre efficiently turned on the Fgf8eGFP reporter allele in the pharynx (E, bracket) and OFT. eGFP+ (Fgf8/eGFP) cells were found in the pharyngeal ectoderm (F), endoderm (I), SHF/SpM (I), and OFT/RV (F and G). eGFP staining colocalized with Nkx2.5 in the OFT/RV (F and G) and Isl1 in the SHF/SpM (G and I). A, atrium; NT, neural tube. (J and K) Colabeling of Six1-expressing cells with Cre antibody (red) and Fgf8-expressing cells with eGFP antibody (green) of E9.5 Six1hpAP/CreFgf8eGFP embryos (sagittal section). Colabeling appeared white with blue DAPI counter staining. Boxed regions in, F, H, and J are enlarged in G, I, and K, respectively. Original magnification, ×100 (A–D); ×200 (E–K). (L) Potential murine Fgf8 enhancer. Dark boxes mark evolutionarily conserved regions (cons); putative Six1 binding sites and mutations (red) are listed. hu, human; ms, mouse. (M–O) Six1 and Eya1 synergistically regulated reporters containing WT Fgf8 enhancers (M and N), but not the putative Six1 binding site mutant enhancers (O), in transiently transfected HEK293 cells. Reporter constructs are as in L. (P) Quantitative PCR analyses of in vivo ChIP assays of E9.5 mouse PA/heart tissues. Six1 protein was selectively bound to the second conserved region of Fgf8 enhancers (cons2; ~4.4-fold relative to anti-Six1/IgG enrichment). Fgf8 coding region (exon5) served as a negative control.