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Tugging on the heartstrings: determining the function of Tbx5 in early cardiac development

Date

2012

Authors

Parrie, Lindsay E., author
Garrity, Deborah, advisor
Bouma, Gerrit, committee member
Mykles, Donald, committee member
Reddy, A. S. N., committee member

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Abstract

During cardiac morphogenesis, the vertebrate heart acquires a characteristic three dimensional shape well-suited for efficient function. The morphology of the developing cardiac organ reflects a series of changes in the cardiomyocytes themselves, which must become specified, migrate, proliferate, grow in size, alter their shape and adhesive properties, and develop ultrastructure, among other differentiated characteristics. Mutation of the T-box transcription factor tbx5 leads to embryonic lethal cardiac phenotypes and forelimb malformations in vertebrate models. Haploinsufficiency of Tbx5 results in Holt-Oram Syndrome (HOS), a human congenital disease characterized by cardiac and forelimb defects. Homozygous mutation of zebrafish tbx5a in heartstrings (hst) embryos also leads to lethal defects in cardiac looping morphogenesis and prevents initiation of pectoral fin formation. Here I describe a new hst mutant allele (tbx5as296) which encodes a premature stop codon within the tbx5a T-box region, a location likely to generate a full loss-of-function allele. Data from comparative genetics and immunoblot analyses indicate that both alleles are null. I find that mutants completely lacking Tbx5a generated normal cardiomyocyte numbers in early chamber morphogenesis stages. Moreover, in situ hybridization data and functional assays support the idea that venous differentiation is not seriously impaired in zebrafish mutants, in contrast to mouse. However, cardiac cell size was significantly smaller in both chambers of tbx5a mutants. Hearts stalled early in the process of cardiac looping, but cell shape changes associated with chamber ballooning surprisingly still occurred. These studies point to a critical role for Tbx5a in growth-related aspects of cardiac differentiation, and suggest that morphologic events of cardiac looping morphogenesis and chamber ballooning are genetically separable. A second zebrafish tbx5 paralog was recently described, termed tbx5b, which showed a lower amount of sequence conservation than is typical for a T-box gene. Based on overlapping expression patterns within the embryonic heart, I hypothesized that functional redundancy between tbx5a and tbx5b might reduce the severity of cardiac phenotypes for tbx5a mutant embryos. I here report that the cardiac phenotypes in tbx5b-depleted fish were similar, but not identical, to those of homozygous tbx5a mutants. In addition, tbx5b-depletion led to defects in the timing and morphogenesis of pectoral fin outgrowth. Somewhat surprisingly, simultaneous depletion of both Tbx5 gene paralogs did not lead to more severe cardiac phenotypes, and injection of wild-type mRNA was not sufficient to cross-rescue the phenotypes of the paralogous gene. In the heart, tbx5a and tbx5b appear to have related essential functions that are nevertheless independently required. In the fin, tbx5a alone was required for fin bud initiation, but both genes are independently required for patterning and morphogenesis. Therefore, this work demonstrates a functional divergence between the two zebrafish tbx5 paralogs.

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cardiac
zebrafish
tbx5
development

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