Müller F, Chang B, Albert S, Fischer N, Tora L, Strähle U
Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, CU de Strasbourg, France.
Development. 1999 May;126(10):2103-16. doi: 10.1242/dev.126.10.2103.
The signalling molecule Sonic hedgehog (Shh) controls a wide range of differentiation processes during vertebrate development. Numerous studies have suggested that the absolute levels as well as correct spatial and temporal expression of shh are critical for its function. To investigate the regulation of shh expression, we have studied the mechanism controlling its spatial expression in the zebrafish. We employed an enhancer screening strategy in zebrafish embryos based on co-injection of putative enhancer sequences with a reporter construct and analysis of mosaic expression in accumulated expression maps. Enhancers were identified in intron 1 and 2 that mediate floor plate and notochord expression. These enhancers also drive notochord and floor plate expression in the mouse embryo strongly suggesting that the mechanisms controlling shh expression in the midline are conserved between zebrafish and mouse. Functional analysis in the zebrafish embryo revealed that the intronic enhancers have a complex organisation. Two activator regions, ar-A and ar-C, were identified in intron 1 and 2, respectively, which mediate mostly notochord and floor plate expression. In contrast, another activating region, ar-B, in intron 1 drives expression in the floor plate. Deletion fine mapping of ar-C delineated three regions of 40 bp to be essential for activity. These regions do not contain binding sites for HNF3beta, the winged helix transcription factor previously implicated in the regulation of shh expression, indicating the presence of novel regulatory mechanisms. A T-box transcription factor-binding site was found in a functionally important region that forms specific complexes with protein extracts from wild-type but not from notochord-deficient mutant embryos.
信号分子音猬因子（Shh）在脊椎动物发育过程中控制着广泛的分化过程。大量研究表明，Shh的绝对水平以及其在空间和时间上的正确表达对其功能至关重要。为了研究Shh表达的调控机制，我们研究了斑马鱼中控制其空间表达的机制。我们在斑马鱼胚胎中采用了增强子筛选策略，该策略基于将推定的增强子序列与报告构建体共注射，并在累积表达图谱中分析镶嵌表达。在内含子1和2中鉴定出介导底板和脊索表达的增强子。这些增强子在小鼠胚胎中也驱动脊索和底板表达，这强烈表明斑马鱼和小鼠中控制中线Shh表达的机制是保守的。对斑马鱼胚胎的功能分析表明，内含子增强子具有复杂的结构。分别在内含子1和2中鉴定出两个激活区域，即ar-A和ar-C，它们主要介导脊索和底板表达。相比之下，内含子1中的另一个激活区域ar-B驱动底板中的表达。ar-C的缺失精细定位确定了三个40bp的区域对活性至关重要。这些区域不包含HNF3β的结合位点，HNF3β是先前涉及Shh表达调控的翼状螺旋转录因子，这表明存在新的调控机制。在一个功能重要区域中发现了一个T盒转录因子结合位点，该位点与野生型而非脊索缺陷型突变胚胎的蛋白提取物形成特异性复合物。
