Wtip is required for proepicardial organ specification and cardiac left/right asymmetry in zebrafish.

Wilm's tumor 1 interacting protein (Wtip) was identified as an interacting partner of Wilm's tumor protein (WT1) in a yeast two-hybrid screen. WT1 is expressed in the proepicardial organ (PE) of the heart, and mouse and zebrafish wt1 knockout models appear to lack the PE. Wtip's role in the heart remains unexplored. In the present study, we demonstrate that wtip expression is identical in wt1a­, tcf21­, and tbx18­positive PE cells, and that Wtip protein localizes to the basal body of PE cells. We present the first genetic evidence that Wtip signaling in conjunction with WT1 is essential for PE specification in the zebrafish heart. By overexpressing wtip mRNA, we observed ectopic expression of PE markers in the cardiac and pharyngeal arch regions. Furthermore, wtip knockdown embryos showed perturbed cardiac looping and lacked the atrioventricular (AV) boundary. However, the chamber­specific markers amhc and vmhc were unaffected. Interestingly, knockdown of wtip disrupts early left­right (LR) asymmetry. Our studies uncover new roles for Wtip regulating PE cell specification and early LR asymmetry, and suggest that the PE may exert non­autonomous effects on heart looping and AV morphogenesis. The presence of cilia in the PE, and localization of Wtip in the basal body of ciliated cells, raises the possibility of cilia-mediated PE signaling in the embryonic heart.

Zebrafish tbx5 paralogs demonstrate independent essential requirements in cardiac and pectoral fin development.

BACKGROUND: T-box genes constitute a large family of transcriptional regulators involved in developmental patterning. Homozygous mutation of tbx5 leads to embryonic lethal cardiac phenotypes and forelimb malformations in vertebrate models. Haploinsufficiency of tbx5 results in Holt-Oram syndrome, a human congenital disease characterized by cardiac and forelimb defects. Homozygous mutation of zebrafish tbx5a leads to lethal defects in cardiac looping morphogenesis, blocks pectoral fin initiation, and impairs outgrowth. Recently, a second zebrafish tbx5 gene was described, termed tbx5b. RESULTS: Our phylogenetic analyses confirm tbx5b as a paralog that likely arose in the teleost-specific whole genome duplication ∼270 MYA. Using morpholino depletion studies, we find that tbx5b is required in the heart for embryonic survival, and influences the timing and morphogenesis of pectoral fin development. Because tbx5a hypomorphic mutations are embryonic lethal, tbx5a and tbx5b functions in the heart must not be completely redundant. Consistent with this hypothesis, simultaneous depletion of both tbx5 paralogs did not lead to more severe phenotypes, and injection of wild-type mRNA from one tbx5 paralog was not sufficient to cross-rescue phenotypes of the paralogous gene. CONCLUSIONS: Collectively, these data indicate that, despite similar spatio-temporal expression patterns, tbx5a and tbx5b have independent functions in heart and fin development.