| Summary: | 碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 94 === The genetic mechanisms of vascular development during embryogenesis and post embryogenesis are largely unknown. We use transgenic zebrafish that express EGFP in blood vessel endothelial cells to study vascular development. Utilizing ENU mutagenesis, we isolated one mutation, called prp （for persistent plexus）that displayed defects in regulating vascular plexus formation in the fins.
In prp mutation, vessel plexuses appear constantly in the tips of each fin ray which is not observed in wild type fish. In order to understand the cause to this phenotype, we searched for the earliest morphological abnormality during caudal fin development. We first established the morphological stages of caudal fin development, which could be divided into three major morphological events. First, the blood vessels grow from the posterior end of the axial vessels and extend into the fin folds. Secondly, the vessels begin to proliferate rapidly and branch to lay out the fin ray pattern which is followed by the deposition of the dermal bone matrix of the fin rays. Finally, the vessels continue to grow and form the complete 16 to 18-fin-ray pattern. However, the vasculature of each fin ray at this moment only consists of two vessels. When comparing with wild type, we found that the prp vascular phenotypes appear soon after blood vessels develop into the fin folds during the second stage. The vessel phenotype is followed by fin ray abnormalities. By time-lapse, immunohistochemistry experiments, we concluded that the vessel phenotype is not due to developmental defects in the osteoblast patterning. Rather, our analysis with prp mutation reveals that blood vessel plays a role in guiding osteoblast and later dermal bone patterning in zebrafish caudal fins. We also found that the prp vessel phenotype is independent of sensory neuron or epidermis patterning. By simply comparing the vascular morphology and the number of endothelial cells of wild type and prp mutants, we also found that the plexus formation in prp mutation is not likely due to over-proliferation of vessel cells.
However, we found that prp mutation develop smaller fin fold prior to caudal fin development, suggesting defects in fin fold might result in vascular phenotypes in the caudal fin. The finfold is a clear, thin membrane around the entire trunk region containing epidermis and actinotrichia. By light microscopy and alcian blue staining, we observed abnormal patterning of actinotrichia in prp fin folds. By WMISH with col 1 α1（collagen Type Ι α1）gene, we found that prp mutation causes ectopic and high level expression of col 1 α1. Finally, by transmission electron microscopy（TEM）, we observed significant abnormalities in actinotrichia patterning in prp mutants, including loose organization , irregular orientation, matrix degeneration, and penetration into fin rays. These results suggest defects in regulation of collagen gene expression and collagen deposition and assembly and/or maintenance. In summary, our studies reveal that prp mutation causes abnormal actinotrichia patterning likely due to defects in collagen assembly resulting in persistent formation of vascular plexuses and sequential dermal bone abnormalities.
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