熱休克對TG(HSP70:BMP4)基因轉殖班馬魚(Danio rerio)之ntl基因在原腸期表現之影響

Abstract

過去的實驗所製備的TG(HSP70:BMP4)基因轉殖魚品系，可以利用熱緊迫蛋白質(HSP70)之啟動子來驅動BMP4基因Mrna的表現，來探討BMP4基因與頭部軟骨、心臟與消化道器官發育之間的關係。而由文獻中已知BMP4基因在早期胚胎發育背、腹軸形態形成扮演著重要的角色，因此本實驗的目的主要在測試是否經熱休克處理所產生過多的BMP4能影響基因轉殖斑馬魚早期胚胎背、腹軸組織的發育。本實驗以no tail (ntl)脊索標示基因為探討，利用全株原位雜交的方法，來比較不同發育時期的基因轉殖斑馬魚斗胚胎經過37℃，2小時之加熱處理後，對表現在脊索ntl基因的影響。結果顯示：32-64；64-128；128-256細胞期胚胎經熱處理可造成100%bud期胚胎其ntl基因在脊索表現不正常；在256-512細胞期則造成19%bud期胚胎ntl在脊索的兵現在細化(thinner)以及81%有不連續的情形，而在未經熱處理之基因轉殖魚胚胎 ，與不論是否加熱之野生型斑馬魚胚胎ntl在脊索的表現皆是100%正常。因此本實驗結果顯示TG(HSP70:BMP4)基因轉殖魚早期胚胎經過熱處理處，的確可誘發BMP4基因的過度表現，造成ntl的不正常表現進而影響脊索的發育。

A TG((HSP70:BMP4) transgenic fish line was generated previously. One can conditionally activate BMP4 expression by heat shock treatment to study the relationship between BMP4 and the development of pharyngeal arch, heart, and digestive tract. Previous studies have shown the BMP4 plays important role in dorsal-ventral patterning during gastrulation. The aim of this report is to investigate whether BMP4 expression can be induced by heat treatment and subsequently affects dorsal-ventral patterning of early transgenic fish embryos. Whole-mount in situ hybridization using ntl notochord marker gene as a probe was conducted to compare the effect on ntl expression pattern after heat treatment of early transgenic embryos from different developmental stages at 37℃ for 2 h. Our results demonstrated that respective heat treatment of 32-64 cells, 64-128 cells, and 128-256 cells caused 100% bud embryos showing abnormal ntl expression pattern in the notochord. Heat treatment of 256-512 cells caused 19% bud embryos showing thinner ntl distribution and 81% embryos showing discontinuous ntl expression in the notochord. However, heat treatment of different stages embryos from either transgenic lines or wild type zebrafish does not affect ntl expression in the notochord. Overall our results indicate that heat treatment of early TG((HSP70:BMP4) transgenic fish embryos can induce BMP4 over expression to have an effect on ntl expression and following notochord development.