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，并共享许多发育模式程序。然而，令人惊讶的是，最近的研究表明，控制共同遗传程序发育的上游调节程序似乎在遗传上是不同的。确定肢体类型的具体，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，是重要的发育生物学未回答的问题。该提案将通过阐明特别是在后肢芽中的Sonic hedgehog（Shh）基因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的表达通过HAND 2和HOX蛋白直接调控的顺式元件在后间充质中诱导。然而，Hand 2的上游调控似乎是不同的，在前，后肢芽。据报道，在Hox 9四重敲除（Hox-a9，b 9，c9，d9）中，Hand 2和Shh在前肢芽中的表达不被激活，而Hand 2和Shh在后肢芽中的表达正常。据设想，涉及不同Hox旁系同源物的类似情况可以控制早期后肢芽中的Hand 2和Shh。然而，没有Hox基因，调节早期表达的手2和Shh在后肢芽已被确定。因此，作用于Hand 2-Shh通路上游的后肢芽特异性机制仍然难以捉摸.通过条件性敲除（cKO）Islet 1（Isl 1）和Gata 6获得的初步数据表明，这两个基因是Hand 2依赖性Shh诱导的上游，特别是在后肢芽中。基于观察和进一步分析，该项目的具体假设是，Gata 6将Isl 1空间限制在后肢形成区的后部区域，触发了后肢芽后部区域的Hand 2-Shh通路。目的1将确定Gata 6的负调控是否限制了Isl 1对Hand 2-Shh通路的后肢特异性激活。将进行Isl 1和Gata 6的病毒介导的异位表达分析，然后检查标记基因/蛋白质。将进行Gata 6和Isl 1的双KO以确定它们在后肢芽中Shh激活中的关系。目的2将确定Gata 6通过其顺式调节元件负调控Isl 1的机制。将进行体外DNA结合试验、检测转录调控的荧光素酶报告基因试验、检测体内结合的体内染色质免疫沉淀试验和小鼠胚胎LacZ报告基因试验。结合，这一建议将确定不同的转录因子（Hox 9 s在前肢芽和Isl 1在后肢芽）作为一个共同的Hand 2-Shh通路在两个同源组织的调节器。此外，该提案将通过Gata 6确定一种新型上游调节系统，该系统可以以后肢特异性方式正确激活Shh。

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.