Date of Award

4-1-1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Anatomy and Cell Biology

Abstract

Little is known concerning origins of vascular precursors and associated regulatory mechanisms involved in intramural coronary vessel vasculogenesis occurring in the subepicardial space of the embryonic vertebrate heart. This process appears to derive from reciprocal tissue interactions between the myocardium, dorsal mesocardium (DM) and continuous epicardium. Epicardial formation in the Japanese quail was investigated using scanning and transmission (TEM) electron microscopy, and light microscopy (LM). In situ indirect immunocytochemistry was utilized with monoclonal antibodies specific for cellular fibronectin, endothelial, and vascular smooth muscle precursors. The antibody QH1 (Peault et al., 1983) is specific for quail endothelial and hematopoietic cells, and their precursors. The vasculogenic potentials of the DM and epicardial epithelium (EE) were investigated in vitro with a three-dimensional collagen gel culture system.The EE originated from villous projections of the DM, which contacted the heart by stage 17 (52-64 hours). The EE completely invested the heart by stage 24 (4.5 days). Intramural coronary vessel vasculogenesis began during this period, thus stages 17-24 embryos were embedded in paraffin for LM immunofluorescence, and in Lowicryl K4M for TEM immunogold labeling. Fibronectin was associated with the EE, the outer myocardium, and subepicardial mesenchyme, suggesting that it plays a supportive role during intramural coronary vasculogenesis. QH1 and anti-$\alpha$-smooth muscle actin labeled endothelial and vascular smooth muscle precursors, respectively, which were derived from distinct, independent mesenchymal precursor cells within the connective tissue spaces of the DM and continuous subepicardium. The mesothelium of the DM and EE contained cells which labeled with these antibodies, suggesting that additional subpopulations of vascular precursors were derived from epithelial-mesenchymal transitions. QH1-positive endothelial precursors within the EE were also identified by immunogold electron microscopy. In vitro studies in which either DM or DM combined with EE was explanted onto hydrated collagen gels resulted in an epithelial monolayer covering the gel surface, and "seeding" the underlying gel with migratory mesenchymal cells, many of which differentially immunostained with QH1 and anti-$\alpha$-smooth muscle actin. These results indicated that these tissues were sources of vascular precursors supporting intramural coronary vessel vasculogenesis.

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