Mural cells play an indispensable role in promoting long-term stabilization of the endothelial cell (EC) layer linin the vascular tube and are critical for proper maintenance of the circulatory system both during development and as an adult. Defects in mural cell differentiation or interactions with ECs are implicated in atherosclerosis/restenosis, stroke, retinopathies and other human vascular pathologies, and understanding the developmental origins of these cells and the genes regulating their behavior is of particular importance long- term to determine and understand dysfunction in disease. Using recently developed zebrafish transgenic lines marking restricted mural cell populations in vivo, time-lapse two-photon microscopy will be used for lineage tracing experiments to determine the ontogeny of mural cells populating various vascular beds. Additionally, previously described regulators of mural cell function, such as PDGFR?, will be manipulated through conditional gene suppression strategies to validate that these cells are responding to similar cues to those noted in mammals and to provide a broader understanding to the role these cues play in stages of mural cell development, such as delamination from their tissue site of origin, cell proliferation, cell recruitment to the vasculature and EC-mural cell heterotypic interactions (Aim 1).
Aim 2 will focus on the identification of novel gene programs regulated in mural cells by utilizing a combination of 'RiboTag' technology, to isolate and profile translating mRNAs from mural cells at various stages of development through RNA-Seq, with proteomics analysis of FACs sorted mural cell populations, to assess changes in total and phospho-protein levels during mural cell development (Aim 2). Finally, functional analysis will be carried out on identified regulated genes (with those that have implications in human disorders having highest priority for analysis) in addition to a genome wide ENU mutagenesis screen, to gain a broader understanding of gene programs directing mural cell behavior during both development and disease (Aim 3). Taking this multifaceted genetics based approach to understand signals governing mural cell behavior during development will broaden insights into 'normal' mural cell biology while providing a starting point for analysis of disease and provide me a unique niche in the vascular biology community to begin work as a tenure-track research faculty member.
Many disease states, including atherosclerosis, stroke and diabetic retinopathy, have been connected to disrupted mural cell function, leading to decreased stabilization of the blood vessel wall and the onset of disease pathology. By enhancing insights into the regulation and development of mural cells under normal conditions, we can establish a platform of gene regulation and cellular function from which to compare disease. Using a genetic approach in the zebrafish, this research plan aims to identify genes regulating mural cell function during development.
