The proposed studies determine the biological function of Lmp4 in heart development, alone and in association with Tbx5. This may provide new explanations for the wide range of phenotypes observed in Holt-Oram syndrome patients. To address the functional role of Lmp4 in heart development, we genetically inactivated the gene in the mouse. Utilizing the Lmp4 knockout mouse model, the proposed research will specifically test the hypothesis that Lmp4 functions as a regulator of Tbx5 subcellular localization in heart and coronary vasculature development. Lmp4 and Tbx5 expression will be analyzed in Lmp4 mutant mice, in comparison to wild type mice, providing insight into whether Lmp4 has a direct influence on Tbx5 subcellular distribution in an Lmp4 dosage dependent manner. The morphological phenotypes associated with genetic Lmp4 inactivation in the developing mouse heart will be analyzed in conjunction with Lmp4 and Tbx5 expression domains. This will determine whether the phenotypes are a direct result of compromised Lmp4 expression, or an indirect effect via altered Tbx5 subcellular localization. Primary epicardial cells cultured from homozygous Lmp4 mouse hearts will be used to determine the mechanistic function of Lmp4 alone and in association with Tbx5. The migratory behavior of the Lmp4 mutant cells will be assessed following rescue by transfection with full-length Lmp4 and rescue by transfection with Lmp4 constructs that lack the LIM3 domain Tbx5 binds. Lmp4 is a member of the PDZ-LIM family of proteins that function in critical biological processes such as organ development. Tbx5 is a member of the T-box family of transcription factors that has critical roles in many aspects of organogenesis. The overall goal of this proposed research is to elucidate the functional role of the interaction of Lmp4 and Tbx5 in heart development, which may provide new insights into heart/limb phenotypes observed in Holt-Oram syndrome.
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