Heart development following genetic inactivation of Lmp4

描述（由申请人提供）：拟议的研究确定Lmp4在心脏发育中的生物学功能，单独或与Tbx5相关。这可能为在Holt-Oram综合征患者中观察到的广泛表型提供新的解释。为了解决Lmp4在心脏发育中的功能作用，我们在小鼠中基因灭活了该基因。利用Lmp4敲除小鼠模型，本研究将专门验证Lmp4在心脏和冠状动脉血管发育中作为Tbx5亚细胞定位调节剂的假设。我们将分析Lmp4突变小鼠与野生型小鼠中Lmp4和Tbx5的表达，以了解Lmp4是否以Lmp4剂量依赖性的方式直接影响Tbx5亚细胞分布。在发育中的小鼠心脏中，与Lmp4基因失活相关的形态学表型将结合Lmp4和Tbx5表达域进行分析。这将决定表型是Lmp4表达受损的直接结果，还是通过改变Tbx5亚细胞定位的间接影响。从纯合子Lmp4小鼠心脏培养的原代心外膜细胞将用于确定Lmp4单独和与Tbx5相关的机制功能。Lmp4突变体细胞的迁移行为将通过转染全长Lmp4和转染缺乏LIM3结构域Tbx5结合的Lmp4构建体来评估。Lmp4是PDZ-LIM蛋白家族的一员，在器官发育等关键生物过程中起作用。Tbx5是T-box转录因子家族的一员，在器官发生的许多方面起着关键作用。本研究的总体目标是阐明Lmp4和Tbx5相互作用在心脏发育中的功能作用，这可能为Holt-Oram综合征中观察到的心脏/肢体表型提供新的见解。

DESCRIPTION (provided by applicant): 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.