Abstract
DiGeorge syndrome (DGS) is the most common microdeletion syndrome, and is characterized by congenital cardiac, craniofacial and immune system abnormalities. The cardiac defects in DGS patients include conotruncal and ventricular septal defects. Although the etiology of DGS is critically regulated by TBX1 gene, the molecular pathways underpinning TBX1's role in heart development are not fully understood. In this study, we characterized heart defects and downstream signaling in the zebrafish tbx1−/− mutant, which has craniofacial and immune defects similar to DGS patients. We show that tbx1−/− mutants have defective heart looping, morphology and function. Defective heart looping is accompanied by failure of cardiomyocytes to differentiate normally and failure to change shape from isotropic to anisotropic morphology in the outer curvatures of the heart. This is the first demonstration of tbx1's role in regulating heart looping, cardiomyocyte shape and differentiation, and may explain how Tbx1 regulates conotruncal development in humans. Next we elucidated tbx1's molecular signaling pathway guided by the cardiac phenotype of tbx1−/− mutants. We show for the first time that wnt11r (wnt11 related), a member of the non-canonical Wnt pathway, and its downstream effector gene alcama (activated leukocyte cell adhesion molecule a) regulate heart looping and differentiation similarly to tbx1. Expression of both wnt11r and alcama are downregulated in tbx1−/− mutants. In addition, both wnt11r −/− mutants and alcama morphants have heart looping and differentiation defects similar to tbx1−/− mutants. Strikingly, heart looping and differentiation in tbx1−/− mutants can be partially rescued by ectopic expression of wnt11r or alcama, supporting a model whereby heart looping and differentiation are regulated by tbx1 in a linear pathway through wnt11r and alcama. This is the first study linking tbx1 and non-canonical Wnt signaling and extends our understanding of DGS and heart development.
Highlights
DiGeorge syndrome (DGS) is the most common microdeletion syndrome occurring in 1/4000 live births [1]
We show that tbx1 is required for normal heart looping and differentiation of myocardium derived from the Primary Heart Fields (PHF) and Secondary Heart Field (SHF)
We investigated whether tbx1 is necessary for correct cardiac morphogenesis in zebrafish, similar to humans and mice
Summary
DiGeorge syndrome (DGS) is the most common microdeletion syndrome occurring in 1/4000 live births [1]. Mutations in Wnt cause cardiac OFT defects such as truncus arteriosus, similar to those observed in Tbx12/2 mutants [25]. Previous studies suggest that Tbx is expressed in SHF and that Tbx positively regulates SHF cell proliferation and contribution to the muscle layer of OFT [12,28,29] This vertebrate pattern of heart development is largely conserved in zebrafish. The simpler structure of the heart combined with transparency of embryos and availability of a tbx mutant, make zebrafish the ideal system to study cardiac defects associated with DGS. We use the zebrafish tbx12/2 mutant [35] to study cardiac defects associated with DGS and identify tbx target genes. In addition we provide evidence that tbx mediates its function at least in part via wnt11r, and one of its downstream mediators, alcama
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