Spatiotemporal expression of zebrafish keratin 18 during early embryogenesis and the establishment of a keratin 18:RFP transgenic line.
Zebrafish cytokeratin 18 (K18) is one of the type I keratin genes expressed the earliest after amputation of the zebrafish fin, but its spatiotemporal expression during early development is unclear. Whole-mount in situ hybridization revealed that k18 was a maternally inherited gene and that its expression is restricted to the single layer of enveloping cells on the surface of embryos during the gastrula stage. At later stages, K18 expression was detected in the epithelial cells, pronephric duct, digestive tract, dorsal aorta, and fins. By using microinjection, we generated the transgenic line Tg(k18(2.9):RFP), which carries an upstream 2.9-kb segment of k18 gene fused with a red fluorescent protein (RFP) reporter. The spatiotemporal distributions of red fluorescent signal of Tg(k18(2.9):RFP) line correlated well with endogenous k18 transcripts detected by whole-mount in situ hybridization, indicating that this line is capable of recapitulating endogenous k18 expression patterns. We noticed that the red fluorescence appeared strongly in the dorsal, pectoral, pelvic, anal, and caudal fins when transgenic fish became adults. Interestingly, we also found that when F1 female from the Tg(k18(2.9):RFP) line were mated with wild-type males, 100% (326/326) of F2 offspring expressed red fluorescence at the one-cell stage. In contrast, when F1 male from the Tg(k18(2.9):RFP) line were mated with wild-type females, only 49.8% (138/277) of F2 embryos exhibited red fluorescence. On the basis of these findings, we suggest that the transcript of zebrafish K18 is inherited as a maternal effect. We believe that Tg(k18(2.9):RFP) fish should be an excellent experimental animal for studying the zygotic regulatory mechanism of k18.
Wang
Yun-Hsin
YH
Institute of Molecular and Cellular Biology, National Taiwan University No. 1, Roosevelt Road, Section 4, Taipei 106, Taiwan, ROC.
Chen
Yau-Hung
YH
Lin
Yu-Ju
YJ
Tsai
Huai-Jen
HJ
D000818:Animals; D030801:Animals, Genetically Modified; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004625:Embryo, Nonmammalian; D047108:Embryonic Development; D005260:Female; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D017403:In Situ Hybridization; D053538:Keratin-18; D007633:Keratins / Q000235:genetics*; D008297:Male; D008845:Microinjections; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution; D015027:Zebrafish / Q000196:embryology / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000235:genetics*
D053538:Keratin-18; D029961:Zebrafish Proteins; C479873:krt18a.1 protein, zebrafish; D007633:Keratins; D004247:DNA