Browsing by Author "Whitesell, Thomas Richard"

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    Vascular Smooth Muscle Cell Development in Zebrafish
    (2018-09-20) Whitesell, Thomas Richard; Childs, Sarah Jane; Grewal, Savraj S.; Biernaskie, Jeff A.
    During the formation of blood vessels, the endothelium becomes progressively covered by vascular smooth muscle cells and pericytes, known collectively as vascular mural cells. The vascular mural cells of the head arise from migratory neural crest and lateral mesodermal populations, and differentiate into mural cells which surround the endothelium of blood vessels, providing support and contraction. Using zebrafish as a model, we created transgenic reporter lines for acta2/αsma (α-smooth muscle actin) that label mature vascular and visceral smooth muscle. Using these reporters, we study the cellular dynamics of mature vascular smooth cells along the ventral aorta and in the brain. These reporter lines only begin robust expression of vascular mural cells at 4 days post fertilization (dpf); however, there are peri-vascular cells present around the endothelium by 2 dpf. Therefore, there is a need to identify an earlier marker of the vascular mural cell lineage. One such marker is the forkhead box transcription factor foxc1b. Utilizing a foxc1b:EGFP reporter line, foxc1b labels groups of mesenchymal cells which undergo a morphological change to associate with the endothelium, and then later co-express the mature smooth muscle marker acta2. These vascular smooth muscle cells on the ventral aorta are the earliest identified smooth muscle cells in the zebrafish embryo. foxc1b:EGFP continues to label smooth muscle cells through the lifespan of zebrafish, but is not co-expressed with pdgfrβ (platelet derived growth factor β) and therefore likely does not label pericytes. Thus, foxc1b is an early smooth muscle marker, but not a pericyte marker. To assess if there are transcriptomic differences between mural cell populations, fluorescence-activated cell sorting and RNA Sequencing were used to identify differentially expressed genes. Further examination of genes from the RNA Sequencing datasets reveals novel vascular mural cell markers. One highly differentially expressed gene was Ras-like family protein member 12, rasl12. rasl12 is expressed in both smooth muscle cells and pericytes. rasl12 knockout mutants were created, but there were large phenotypic differences between two mutant alleles, requiring further study. Overall, this thesis addresses the development of vascular smooth muscle cells, by labelling and visualizing cellular behaviors in zebrafish.