A functional screen for sonic hedgehog regulatory elements across a 1 Mb interval identifies long-range ventral forebrain enhancers
A functional screen for sonic hedgehog regulatory elements across a 1 Mb interval identifies long-range ventral forebrain enhancers
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DOI：
10.1242/dev.02239
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发表时间：
2006-02-01
期刊：
影响因子：
4.6
通讯作者：
Epstein, DJ
中科院分区：
文献类型：
--
作者：
Jeong, YS;El-Jaick, K;Epstein, DJ
分泌蛋白音猬因子（Shh）在脊椎动物中枢神经系统（CNS）腹侧中线的形成中起着不可或缺的作用。在缺乏Shh功能的情况下，腹侧中线发育受到干扰，导致前脑无裂畸形（HPE），这是一种脑部结构畸形，以及沿前后神经轴长度方向的神经元模式形成和路径寻找缺陷。理解腹侧神经管发育的核心是Shh转录在中枢神经系统中是如何被调控的。为了解决这个问题，我们设计了一种增强子捕获实验，以系统地筛选Shh基因座周围1Mb的DNA，检测其将报告基因表达靶向转基因小鼠胚胎中Shh转录位点的能力。这项分析发现了分布在400kb范围内的6个增强子，它们的联合活性覆盖了小鼠胚胎中枢神经系统中从腹侧前脑到脊髓后端的所有Shh表达位点。为了评估这些增强子对Shh表达整体模式的相对贡献，我们在转基因细菌人工染色体（Bac）报告基因实验中删除了单个元件。研究发现，在腹侧脊髓、后脑和端脑部分区域存在冗余机制控制类Shh报告基因的活性，而独特的元件调控腹侧中脑、大部分腹侧间脑和部分端脑的类Shh表达。三个人类前脑无裂畸形病例中，在Shh上游发生易位断点的远端存在三个腹侧前脑增强子，这表明这些调控元件从Shh启动子上移位可能是这些患者发生前脑无裂畸形的原因。
The secreted protein sonic hedgehog (Shh) plays an integral role in forming the ventral midline of the vertebrate central nervous system (CNS). In the absence of Shh function, ventral midline development is perturbed resulting in holoprosencephaly (HPE), a structural malformation of the brain, as well as in neuronal patterning and path finding defects along the length of the anteroposterior neuraxis. Central to the understanding of ventral neural tube development is how Shh transcription is regulated in the CNS. To address this issue, we devised an enhancer trap assay to systematically screen 1 Mb of DNA surrounding the Shh locus for the ability to target reporter gene expression to sites of Shh transcription in transgenic mouse embryos. This analysis uncovered six enhancers distributed over 400 kb, the combined activity of which covered all sites of Shh expression in the mouse embryonic CNS from the ventral forebrain to the posterior extent of the spinal cord. To evaluate the relative contribution of these enhancers to the overall pattern of Shh expression, individual elements were deleted in the context of a transgenic Bac reporter assay. Redundant mechanisms were found to control Shh-like reporter activity in the ventral spinal cord, hindbrain and regions of the telencephalon, whereas unique elements regulated Shh-like expression in the ventral midbrain, the majority of the ventral diencephalon and parts of the telencephalon. Three ventral forebrain enhancers locate on the distal side of translocation breakpoints that occurred upstream of Shh in human cases of HPE, suggesting that displacement of these regulatory elements from the Shh promoter is a likely cause of HPE in these individuals.