Pdlim7 regulates nuclear/cytoplasmic localization and activity of Tbx5 during cardiac developmentPublic Deposited
The T-box transcription factor, Tbx5, is involved in heart development and congenital disease. For example mutations in human TBX5 lead to Holt-Oram syndrome, a disease characterized by a range of heart and arm malformations. Tbx5 gene activity has been perturbed in several animal models including the chicken, mouse, and zebrafish. In zebrafish, the tbx5 heartstrings (hst) mutation results in embryos that fail to develop pectoral fins (forelimbs) and form a non-looped, string-like heart. Despite the importance of Tbx5 in heart formation, little is known about its function and regulation. To gain insight into Tbx5 regulation, the Simon laboratory performed a yeast two-hybrid screen to identify interacting proteins. One candidate isolated from the screen was Pdlim7, a member of the PDZ-LIM protein family. I hypothesized that the binding of Tbx5 to Pdlim7 modifies its transcriptional activity and explored the consequences of Tbx5/Pdlim7 complex formation in vitro employing appropriate cultured cells and in vivo using the zebrafish embryo. Evidence from others in the laboratory using in vitro binding studies and in vivo protein localization in the chicken limb and heart suggested an interaction between Tbx5 and Pdlim7. However, what is the mechanistic action of the Tbx5/Pdlim7 complex on a cellular level? Using co-immunoprecipitations and confocal imaging I demonstrated that Tbx5 and Pdlim7 bind in the cell. In the presence of Pdlim7, employing FRAP, I was able to show that Tbx5 shuttles dynamically between the nucleus and cytoplasm and, in a complex with Pdlim7, localizes to the actin cytoskeleton. To test whether the relocalization from nuclear to cytoplasmic sites interfered with downstream gene expression, I used luciferase reporters and demonstrated that Pdlim7 acts as a repressor of Tbx5 activity. Several studies had shown the importance of Tbx5 in heart formation, however, nothing was known about Pdlim7 function in vivo. Therefore I asked, what is the role of Pdlim7 during development, specifically does it relate to heart formation and Tbx5 regulation? I performed functional studies to perturb Pdlim7 function in zebrafish embryos. First, I demonstrated overlapping expression domains for both tbx5 and pdlim7 in the developing heart. Next, inhibition of Pdlim7 function by injection of antisense morpholino (MO) oligonucleotides which resulted in a failure of cardiac looping, reminiscent of tbx5 hst mutants. Interestingly, tbx5 and pdlim7 also appeared to genetically interact, suggesting that Pdlim7 and Tbx5 operate in a common pathway during cardiac development. Loss of Pdlim7 function in zebrafish embryos did not affect early cardiac patterning or gene expression. However, changes in gene expression were observed at the atrioventricular boundary (AV). Loss of Pdlim7 resulted in a loss of gene expression at the AV boundary and in some cases blood regurgitation, suggesting valve defects. Using the AV boundary Tbx5 target gene tbx2b as a readout, I was able to show that Pdlim7 regulates Tbx5 in vivo. Misregulation of Pdlim7 in wild-type and hst/+ compromised embryos resulted in inappropriate tbx2b expression due to improper subcellular localization of Tbx5. Therefore, it appears that Tbx5 and Pdlim7 work together to regulate gene expression and cardiac shape at the AV boundary through a mechanism of modifying Tbx5 subcellular localization.