1 propose to elucidate mechanisms underlying cardiac chamber morphogenesis by determining contributions from the structure and composition of the nuclear envelope. Form determines function for our organs, yet little is known o the mechanisms that control morphogenesis. In 1975, Antonio Garcia-Bellido proposed a hierarchy for genes involved in morphogenesis: 'activators', 'selectors', and 'realizators'. However, the many roles 'realizators'have in multiple cell types hinder their study. One way to circumvent this problem is to examine what conducts information between the genome and 'realizators'. Connections exist where the cytoskeleton links the cell cortex and nuclear envelope. These connections are clearly important for heart function, because their disruption in a group of diseases called envelopathies cause congenital heart defects. For envelopathies, it is unclear whether the disruption of normal heart function begins during development. Here, I propose to test whether internal envelope structure or nuclear connections with the cytoskeleton are necessary for cardiac chamber morphogenesis. I will express dominant mutant versions of either inner or outer nuclear membrane proteins from human envelopathies in the nascent myocardium to determine the impact of nuclear envelope composition on chamber morphogenesis. Next, I will test the hypothesis that genome organization at the level of nuclear shape reorganizes in response to chamber shaping inputs. These studies will determine where and when gross changes in nuclear shape, and the underlying expression programs, are occurring during chamber morphogenesis. The third portion of this project involves the development of tools for analyzing subcellular events within the cells of the developing heart. By fluorescently highlighting subcellular behavior related to the cell's cortex, cytoskeleton, and nucleus, I will track and quantify how these organelles are coordinated and how their coordination is disrupted in the disease model developed in the first portion of the proposal. These tools will allow me to test the hypothesis that nuclear envelope reorganization coordinates subcellular changes during chamber morphogenesis. Not knowing how cardiac chambers obtain their characteristic shapes and how the process can be disrupted at the cellular and subcellular level hinders how we understand and treat congenital heart disease. As the mutant nuclear envelope proteins used in this study are derived from human cardiac myopathies, the findings will be relevant to human health. Any findings may also be relevant to understanding human aging because the processes disrupted in this disease model are disrupted in accelerated aging diseases.
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