As described in the goals and objectives section of this report, this project consists of three specific aims: Studying genes regulating vascular integrity We have used forward-genetic screens and CRISPR gene editing to generate zebrafish mutants that disrupt cranial vascular integrity in the zebrafish, using next-generation sequencing to perform rapid molecular cloning of the defective genes from mutants. We previously characterized the role of GDF6 (BMP13) in vascular integrity, demonstrating that this gene promotes maintenance of vascular integrity by suppressing excess VEGF signaling. We are currently characterizing the molecular nature of the defects in the important intracellular regulatory protein RhoA, for which we have generated an allelic series of mutations. These and other new vascular integrity mutants promise to bring to light new pathways important in the maintenance of vascular barrier function. Studying the acquisition and function of supporting vascular smooth muscle cells The vascular smooth cells (VSMC) that surround the endothelial tube play a critical role in regulating vascular tone and vascular integrity. We have recently been examining the acquisition and function of these cells using the zebrafish. We have developed a number of useful transgenic tools to visualize and experimentally manipulate this cell population in the zebrafish, and are using these and other tools to, among other things, (i) examine the origins of these cells from early sclerotome, (ii) demonstrate that VSMC interaction with the endothelium is required to maintain the vascular basement membrane and restrict vessel diameter, and (iii) elucidate the molecular pathways responsible for the selective acquisition of VSMC by arteries (as opposed to veins). Studying the vasculature and vascular-associated cells in the meninges The meninges are an external enveloping connective tissue that encases the brain, producing cerebrospinal fluid, acting as a cushion against trauma, nourishing the brain via nutrient circulation, and removing waste. Despite its importance, the cell types present in the meninges and the function and embryonic origins of this tissue are still not well understood. We recently discovered a novel perivascular cell population closely associated with blood vessels on the zebrafish brain. Based on similarities in their morphology, location, and highly unusual scavenger behavior, these cells appear to be the zebrafish equivalent of mammalian Fluorescent Granular Perithelial cells (FGPs), macrophage-like cells about which very little is known that likely play important roles in brain function and in a variety of CNS pathologies. Despite their macrophage-like morphology and their perivascular location these cells are molecularly most similar to lymphatic endothelial cells and they transdifferentiate from primitive endothelium deep inside the brain before migrating to the brain surface. Using forward-genetic screening of transgenic zebrafish for ENU-induced recessive mutations, we recently identified a mutant that appears to be specifically deficient in FGPs, providing a genetic model that we are using to further explore the important functional role of these cells. Our findings thus far provide the first report of a non-vessel forming, perivascular cell population in the brain that emerges by transdifferentiation from vascular endothelium. We are further exploring the structure, cellular composition, and functional roles of meningeal vascular-associated cell populations using the zebrafish model.
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