We generated a transgenic line Tg(k18:shh:RFP) with overexpression of Sonic hedgehog in the skin epidermis. By 5 day-post-fertilization (dpf), many epidermal lesions were clearly observed, including a swollen yolk sac, epidermis growth malformation around the eyes and at the basement of the pectoral fins. Skin histology revealed embryos derived from Tg(k18:shh:RFP) displayed an elevated Nuclear/Cytoplasmic ratio and pleomorphic nuclei compared to their wild type littermates, suggesting the abnormal growth pattern on the epidermis of Tg(k18:shh:RFP) embryos were dysplasia. Later (by 7 dpf), Tg(k18:shh:RFP) embryos displayed broader pectoral fins which are similar to the polydactyly phenotypes of Nevoid basal cell carcinoma syndrome (NBCCS)/Gorlin patients and polydactylous mice. In addition, treatment with cyclopamine is able to enhance and prolong the survival rates and survival durations of Tg(k18:shh:RFP) embryos. In conclusion, this unique Tg(k18:shh:RFP) fish line, should be an excellent experimental animal for screening for a lower toxicity level of the new Hh-inhibitor and can even be used as a new anti-cancer drug-screening platform.
This study generated a transgenic zebrafish line Tg(k18:Ptmaa-RFP) with overexpression of Prothymosin alpha type a (Ptmaa) in the skin epidermis. Red fluorescence first appears very weakly in the early stage, become stronger and mainly restricted in the nuclei of the epithelial cells from 3 dpf-larvae to adult fish. However, no evident morphological abnormalities were observed. Thus, overexpression of Ptmaa alone is not sufficient to cause disorganized growths or even cancer in zebrafish skin. Molecular and histological evidences showed that Tg(k18:Ptmaa-RFP) embryos have more proliferating cells in the pelvic fins [WT: 3.92 +/- 7.15; Tg(k18:Ptmaa-RFP): 38.00 +/- 10.87] and thicker skin [WT: 10.98 +/- 1.41 mum; Tg(k18:Ptmaa-RFP): 14.02 +/- 1.32 mum], indicating that overexpression of Ptmaa can promote proliferation. On the other hand, fewer apoptotic signals were found when Tg(k18:Ptmaa-RFP) embryos were exposed to UVB. Together with quantitative RT-PCR data, we suggest that UVB-induced epidermal cell apoptosis of zebrafish larvae can be attenuated by overexpression of Ptmaa through the enhancement of transcriptions of bcl2 mRNAs. Taken together, we conclude that overexpression of Ptmaa in zebrafish epidermal cells promotes proliferation and attenuates UVB-induced apoptosis but does not cause skin cancer.
We isolated two atp2a genes, atp2a1 and atp2a2a, from embryonic zebrafish. Amino acid sequences deduced from zebrafish atp2a genes are aligned with orthologue proteins from other species, the results showed that they share high percentage of identities (82%-94%) and acidic pIs (5.03-5.33). Whole mount in situ hybridization experiments showed that atp2a1 and atp2a2a are maternal inherited genes which can be detected at 1-cell stage embryos and express in the entire animal pole from 6 hours post-fertilization (hpf) to 12 hpf. At the later stages (48-96 hpf), expression of atp2a1 was restricted in head and trunk muscles as well as in some neurons. In contrast to the strongly expression of atp2a1 in head muscle, expression of atp2a2a was detected in head muscle in a fainter manner. In addition, transcripts of atp2a2a were observed in the developing heart during early cardiogenesis. The present studies not only help us to comparatively analyze atp2a genes across species, but also provide useful information about expressions during early embryogenesis that will help in further investigations of functional studies of Atp2a in the future.
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