斑馬魚胚胎早期轉錄因子Mfy-5之生物特性:以繼代遺傳轉殖品系作為研究模式(1/2)

Myf-5, a transcription factor that controls muscle differentiation, is expressed in somites during early embryogenesis. However, gene regulation of myf-5 is poorly understood and detailed functional analysis of the regulatory cis-elements is needed. In zebrafish, the myf-5 upstream sequence from -82 to -62 (-82/-62) was fused with a basal promoter and transferred to fertilized zebrafish eggs. The -82/-62 cassette drove GFP reporter gene expression specifically in the somites. Moreover, GFP signals were detected exclusively in the somites of 28-hpf embryos derived from eggs injected with pCMV-5×(-82/-62), which contained 5 copies of the -82/-62 cassette inserted within CMV promoter/enhancer. Thus, the -82/-62 cassette, conserved in mouse myf-5, functions to drive somite-specific expression and to repress non-specific expression during the early development of zebrafish embryos. Mutated sequence analysis of -82/-62 cassette showed that the -70/-62 sequence was the key element for controlling myf-5 specificity. The putative CCAAT-like box, located at -66/-62, could not direct somite-specific expression. A DNA-protein complex was specifically formed between the –70/-62 probe and embryonic nuclear extracts. We conclude that the -70/-62 motif is essential for controlling somite-specific expression and the CCAAT-like box is essential for activating gene transcription. Moreover, comparing the GFP-expression patterns shown from 4 transgenic lines derived from injecting with the fragment of -9977/-1, -6212/-1, -2937/-1, and -290/-1, we conclude a) the -9977/-6213 segment contained several repressor-rich regions and a somite-specific enhancer; b) the -6212/-2938 segment contained a stage-specific repressor, tissue-specific elements (fin muscle-, jaw muscle-, and neural crest cells) as well as an eye-specific enhancer; and c) the -2937/-291 segment contained notochord- and eye-specific elements, while the -290/-1 segment was responsible for basal transcription and somite-specific expression