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Improvement on Ornamental Fish Species: Effects of Salinity, 5-bromouracil and 6-aminopurine on Zebrafish Development and Production of Interspecies Chimera between Zebrafish (Danio Rerio) and Rosy Barb (Puntius Conchonius)

【摘要】: Zebrafish (Danio rerio) is an important vertebrate model for studying embryology and developmental biology, developmental neuroscience and genetics. As zebrafish is hardy and easy to raise and mate at weekly intervals this fish was selected for our studies. The embryonic development and larval culture of zebrafish have been extensively studied, but the reports detailing the effects of salinity on its embryogenesis was unexpectedly lacking. In order to establish zebrafish as a model fish for the study of osmoregulatory mechanisms, we examined first the adaptive response by varying all combinations of developmental stages and pulse exposures of zebrafish embryos. The main findings in the present study are (1) Zebrafish embryos are only tolerant of salinity less than 4ppt; (2) The salinity-resistant ability of zebrafish embryos increased with development, i.e. the later the development, the more resistant to salinity; (3) The nuclear division of the embryonic cells were markedly impaired by higher salinity. This seems to be the first noted report on morphological and cytological observations on the effects of salinity on the development of zebrafish embryos. It is known that the base analogues, 5-bromouracil (BrUra), an analogue of thymine, and 6-aminopurine (APur), an analogue of adenine, cause transition mutations in various organisms. Most of our understanding about BrUra- and APur-induced mutagenesis has been largely derived from analysis of bacteria and cell culture systems to date. In order to study the nature and occurrence of mutations at a broader scale and to uncover additional loci of developmental importance, we have used zebrafish (Danio rerio) to test two base analogues 5-bromouracil and 6-aminopurine. The agents do not impair the early development processes of the embryos such as cleavage and epiboly, while the late development is impaired. We have identified several phenotypes from the morphogenetic characters that occur during zebrafish gastrulation, head formation and tail formation. We have identified a new mutant, no head (nh) displaying defects in mouth and eyes formation. Besides, we have isolated 16 mutants in our screen, which lack differentiated notochord and the caudal region (no tail; nt/). Four phenotypes affecting the mouth with lower jaw protruding upward have been isolated. These observations may represent cases of mutagenic effects of BrUra on zebrafish development. Interestingly, golden (go/) mutants showing deduced levels of pigmentation have also been found in our BrUra treated embryos. These indicate that BrUra and APur are effective to induce mutant morphogenetic characters in zebrafish organogenesis, especially in head and tail formation. The incorporation of BrUra and APur into the embryos may be a progressively accumulating process, and the incorporated BrUra and APur do not cause abnormal development until they reach the critical amount. The mutated features seen in BrUra- and APur-exposed embryos are often different, but the majority of them share RAPD banding patterns in common with the control. This suggests that most mutagenesis induced by BrUra and APur are most probably point mutations. The ability to induce point mutations by these base analogues will facilitate genetic screens to explain networks of interacting genes that control specific developmental pathways. However, it appears that BrUra can occasionally cause extensive changes in the genome as the fish with golden (go/) pigmentation have different band patterns compared with the control from the same parent. Establishment of a cell-mediated gene transfer system has potential as a new breeding technique for commercially valuable fishes. As an important step toward developing an inter-species chimera, we wished to determine whether cells from blastula-stage embryos of rosy barb (Puntius conchonius) could be transplanted into zebrafish (Danio rerio) blastula-stage embryos and participate in the development of the recipient. From the total of 473 transplants obtained only a fraction of 13 chimer

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