Limb-type specific distinct upstream regulation of a common Hand2-Shh pathway

描述（由申请人提供）：脊椎动物前肢（臂）和后肢（腿）是同源组织，并共享许多模式程序的发展。然而，令人惊讶的是，最近的研究表明，控制共同遗传程序的上游调控程序似乎在遗传上是不同的。确定肢体类型特异性的、不同的上游调控机制控制共同的过程，并揭示这些程序如何不同，是发育生物学中重要的未解之谜。本研究将通过阐明Sonic hedgehog （Shh）基因在后肢芽中的空间激活机制来解决这些问题。在前肢和后肢芽发育的早期阶段，Shh的表达是通过HAND2和HOX蛋白直接调控的顺式元件在后间质中诱导的。然而，Hand2的上游调控在前肢和后肢芽中似乎有所不同。据报道，在Hox9四重敲除（Hox-a9, b9, c9, d9）中，前肢芽中Hand2和Shh的表达未被激活，而后肢芽中Hand2和Shh的表达正常。据设想，在类似的情况下，涉及不同的Hox类似物可以控制后肢早期芽中的Hand2和Shh。然而，没有发现在后肢芽中调控Hand2和Shh早期表达的Hox基因。因此，Hand2-Shh通路上游的后肢芽特异性机制仍然难以捉摸。通过条件敲除Islet1 （Isl1）和Gata6获得的初步数据表明，这两个基因位于hand2依赖性Shh诱导的上游，特别是在后肢芽中。通过观察和进一步分析，本课题的具体假设为：Gata6将Isl1空间限制在后肢形成区后区，触发后肢芽后区Hand2-Shh通路。Aim 1将确定Gata6的负调控是否会限制Isl1对Hand2-Shh通路的后肢特异性激活。病毒介导的Isl1和Gata6异位表达分析，随后检测标记基因/蛋白。我们将对Gata6和Isl1进行双KO，以确定它们在后肢芽Shh激活中的关系。目的2将确定Gata6通过其顺式调控元件负调控Isl1的机制。将进行体外DNA结合试验、荧光素酶报告基因试验检测转录调控、体内染色质免疫沉淀试验检测体内结合和小鼠胚胎LacZ报告基因试验。综上所述，本研究将确定不同的转录因子（前肢芽中的Hox9s和后肢芽中的Isl1）在两种同源组织中作为共同的Hand2-Shh通路的调节因子。此外，该提案将确定Gata6的一个新的上游调控系统，该系统以后肢特定的方式导致Shh的正确空间激活。

DESCRIPTION (provided by applicant): Vertebrate forelimbs (arms) and hindlimbs (legs) are homologous tissues, and share many patterning programs for development. Surprisingly, however, recent studies suggest that the upstream regulatory programs controlling common genetic programs for their development seem to be genetically distinct. Identifying limb-type specific, distinct upstream regulatory mechanisms that control common processes, and unveiling how these programs differ, are important unanswered questions in developmental biology. This proposal will address these questions by elucidating the mechanisms of spatial activation of the Sonic hedgehog (Shh) gene specifically in hindlimb buds. During early stages of the development of both fore- and hindlimb buds, Shh expression is induced in the posterior mesenchyme through a cis-element directly regulated by HAND2 and HOX proteins. However, upstream regulations of Hand2 seem to be different in fore- and hindlimb buds. It is reported that in Hox9 quadruple knockout (Hox-a9, b9, c9, d9), Hand2 and Shh expression were not activated in forelimb buds, while expression of Hand2 and Shh in hindlimb buds was normal. It was envisaged that a similar scenario involving different Hox paralogs could control Hand2 and Shh in early hindlimb buds. However, no Hox genes that regulate the early expression of Hand2 and Shh in hindlimb buds have been identified. Thus, the hindlimb bud- specific mechanism acting upstream of the Hand2-Shh pathway remains elusive. Preliminary data for this proposal, obtained by conditional knockout (cKO) of Islet1 (Isl1) and Gata6, suggests that these two genes are upstream of Hand2-dependent Shh induction specifically in hindlimb buds. Based on observation and further analysis, the specific hypothesis of the proposed project is that spatial restriction of Isl1 to the posterior region of the hindlimb-forming area by Gata6 triggers the Hand2-Shh pathway in the posterior region of the hindlimb bud. Aim 1 will determine whether negative regulation by Gata6 restricts Isl1 for hindlimb-specific activation of the Hand2-Shh pathway. Virus-mediated ectopic expression analyses of Isl1 and Gata6, followed by examination of marker genes/proteins, will be carried out. Double KO of Gata6 and Isl1 will be performed to determine their relationship in Shh activation in hindlimb buds. Aim 2 will determine the mechanism by which Gata6 negatively regulates Isl1 through its cis-regulatory element. In vitro DNA binding assay, luciferase reporter assay to test transcriptional regulation, in vivo chromatin immunoprecipitation assay to test in vivo binding and mouse embryo LacZ reporter assay will be performed. Combined, this proposal will determine that different transcription factors (Hox9s in fore- and Isl1 in hindlimb buds) act as regulators of a common Hand2-Shh pathway in two homologous tissues. Moreover, the proposal will identify a novel upstream regulation system by Gata6 that leads to correct spatial activation of Shh in a hindlimb specific manner. PUBLIC HEALTH RELEVANCE: Congenital limb malformations are common birth defects occurring 1 in every 500 to 1,000 human live births, and abnormal genetic programming is a major cause. Identification of genes and mechanisms that specifically regulate critical processes of limb development, such as spatially regulated induction of Shh, is important to understanding human health as well as developing appropriate information required for genetic counseling.