Du S J, Dienhart M
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore 21202, USA.
Dev Dyn. 2001 Dec;222(4):655-66. doi: 10.1002/dvdy.1219.
Zebrafish tiggy-winkle hedgehog (twhh) is a member of the hedgehog gene family that plays an important role in patterning brain, neural tube, somites, and eyes. To better understand the regulation of its tissue-specific expression, the activity of the twhh promoter was determined in zebrafish embryos by transient and transgenic expression analysis. Transient expression studies revealed that the 5.2-kb twhh promoter drove green fluorescence protein (GFP) expression in the notochord, floor plate, and branchial arches. Deletion analysis showed that distinct regions of the twhh promoter regulated the respective notochord or floor plate specific expression. To confirm the tissue specificity of the twhh promoter, transgenic zebrafish containing the twhh-GFP transgene were generated. GFP expression was analyzed in the F1, F2, and F3 generations of the transgenic embryos. The results confirmed the tissue-specific expression of the transgene in the notochord, floor plate, and branchial arches. In addition, GFP expression was also found in the pectoral fin buds, retina, and epithelial lining cells of the Kupffer's vesicle in the transgenic fish embryos. The expression pattern of the twhh-GFP transgene mimicked the expression of the endogenous twhh mRNAs in the floor plate, fin buds, branchial arches, retina, and epithelial lining cells of the Kupffer's vesicle. The expression in the notochord, however, did not mimic the pattern of the endogenous twhh expression. To determine whether no tail (ntl) or floating head (flh) mutants that have developmental defect in the notochord or the Kupffer's vesicle may affect the GFP expression in these regions, GFP expression was analyzed in ntl or flh transgenic embryos. No GFP expression could be detected in the midline region of the ntl transgenic embryos. However, in flh transgenic embryos, although GFP expression was affected in the midline region, its expression in the Kupffer's vesicle appeared normal. Together, these data indicated that the 5.2-kb twhh promoter contains regulatory elements for tissue-specific expression of twhh in the floor plate, pectoral fin bud, branchial arches, retina, and Kupffer's vesicle.
斑马鱼刺猬基因（twhh）是刺猬基因家族的成员，在大脑、神经管、体节和眼睛的模式形成中发挥重要作用。为了更好地理解其组织特异性表达的调控机制，通过瞬时和转基因表达分析，在斑马鱼胚胎中测定了twhh启动子的活性。瞬时表达研究表明，5.2kb的twhh启动子驱动绿色荧光蛋白（GFP）在脊索、底板和鳃弓中表达。缺失分析表明，twhh启动子的不同区域分别调控脊索或底板的特异性表达。为了证实twhh启动子的组织特异性，构建了含有twhh-GFP转基因的斑马鱼。对转基因胚胎的F1、F2和F3代进行了GFP表达分析。结果证实了转基因在脊索、底板和鳃弓中的组织特异性表达。此外，在转基因鱼胚胎的胸鳍芽、视网膜和库普弗囊的上皮衬里细胞中也发现了GFP表达。twhh-GFP转基因的表达模式与内源性twhh mRNA在底板、鳍芽、鳃弓、视网膜和库普弗囊上皮衬里细胞中的表达模式相似。然而，在脊索中的表达并不模拟内源性twhh的表达模式。为了确定在脊索或库普弗囊中存在发育缺陷的无尾（ntl）或浮头（flh）突变体是否会影响这些区域的GFP表达，对ntl或flh转基因胚胎进行了GFP表达分析。在ntl转基因胚胎的中线区域未检测到GFP表达。然而，在flh转基因胚胎中，虽然中线区域的GFP表达受到影响，但其在库普弗囊中的表达似乎正常。总之，这些数据表明，5.2kb的twhh启动子包含在底板、胸鳍芽、鳃弓、视网膜和库普弗囊中twhh组织特异性表达的调控元件。
