Enhancer trap lines with GFP driven by smad6b and frizzled1 regulatory sequences for the study of epithelial morphogenesis in the developing zebrafish inner ear.
Enhancer trap lines with GFP driven by smad6b and frizzled1 regulatory sequences for the study of epithelial morphogenesis in the developing zebrafish inner ear.
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使用荧光蛋白的组织特异性表达在斑马鱼胚胎中进行实时成像可以对驱动感觉器官形态发生和细胞分化的机制产生重要的见解。脊椎动物内耳半规管的形态发生需要上皮细胞的复杂重排，包括上皮突起的生长、粘附、融合和穿孔，以产生形成每个管的枢纽的组织柱。我们报告的插入位点和表达模式的两个增强子陷阱线在发展中的斑马鱼胚胎，其中每个突出的不同方面的上皮细胞的形态发生在内耳。由smad6b调控序列驱动的膜连接EGFP在整个耳上皮中表达，在耳的外侧和感觉嵴中表达最强烈。第二个增强子陷阱线，与细胞质的绿色荧光蛋白驱动的frizzled1（fzd1）调控序列，特别标志着细胞的腹侧投影和支柱在发展中的耳朵，和边缘细胞的感觉嵴，连同可变的表达在视网膜和骨骺，和神经元在其他地方的发展中的中枢神经系统。我们已经使用了一种组合的方法来确定这两个转基因的插入位点，这是通过随机插入产生的，并表明，靶向基因座扩增是一种快速和可靠的方法，用于随机插入的转基因的插入位点的鉴定。Baldera，Mendale等人描述了两种增强子捕获系，其显示GFP在发育中的斑马鱼内耳中的表达。这些线是由smad6b和fzd1基因座的调控元件驱动的，将有助于成像上皮形态发生的动态事件，产生这种复杂的感觉器官系统。
Live imaging in the zebrafish embryo using tissue‐specific expression of fluorescent proteins can yield important insights into the mechanisms that drive sensory organ morphogenesis and cell differentiation. Morphogenesis of the semicircular canal ducts of the vertebrate inner ear requires a complex rearrangement of epithelial cells, including outgrowth, adhesion, fusion and perforation of epithelial projections to generate pillars of tissue that form the hubs of each canal. We report the insertion sites and expression patterns of two enhancer trap lines in the developing zebrafish embryo, each of which highlight different aspects of epithelial cell morphogenesis in the inner ear. A membrane‐linked EGFP driven by smad6b regulatory sequences is expressed throughout the otic epithelium, most strongly on the lateral side of the ear and in the sensory cristae. A second enhancer trap line, with cytoplasmic EGFP driven by frizzled1 (fzd1) regulatory sequences, specifically marks cells of the ventral projection and pillar in the developing ear, and marginal cells in the sensory cristae, together with variable expression in the retina and epiphysis, and neurons elsewhere in the developing central nervous system. We have used a combination of methods to identify the insertion sites of these two transgenes, which were generated through random insertion, and show that Targeted Locus Amplification is a rapid and reliable method for the identification of insertion sites of randomly inserted transgenes. Baldera, Baxendale, et al. describe two enhancer trap lines that show the expression of GFP in the developing zebrafish inner ear. These lines are driven by regulatory elements at the smad6b and fzd1 loci, and will be useful for imaging the dynamic events of epithelial morphogenesis that generate this intricate sensory organ system.
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