Limb-Type Specific Distinct Upstream Regulation of a Common Hand2-Shh Pathway

描述(申请人提供)：脊椎动物前肢(手臂)和后肢(腿)是同源组织，具有许多共同的发育图案化程序。然而，令人惊讶的是，最近的研究表明，控制它们发育的共同遗传程序的上游调控程序似乎在基因上是不同的。识别控制共同过程的特定肢体类型的独特上游调控机制，并揭示这些程序的不同之处，是发育生物学中重要的悬而未决的问题。这项建议将通过阐明Sonic Hedgehog(Shh)基因在后肢芽中特异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部间充质中诱导的。然而，Hand2的上游调控在前肢和后肢芽中似乎不同。据报道，在Hox9四重基因敲除(Hox-A9、b9、c9、D9)中，Hand2和Shh在前肢芽中不表达，而在后肢芽中表达正常。据设想，类似的情况涉及不同的HOX近亲动物可以控制早期后肢芽中的Hand2和Shh。然而，目前还没有发现调控Hand2和Shh在后肢芽早期表达的Hox基因。因此，作用于Hand2-Shh通路上游的后肢芽特异性机制仍然难以捉摸。通过对Islet1(Isl1)和GATA6进行条件性敲除(CKO)获得的初步数据表明，这两个基因是依赖Hand2的Shh诱导的上游，特异地在后肢芽中诱导。根据观察和进一步的分析，该项目的具体假设是，GATA6将Isl1的空间限制在后肢形成区的后部，触发了后肢芽后部的Hand2-Shh通路。目的1将确定GATA6的负调控是否限制Isl1的后肢特异性激活Hand2-Shh通路。将进行病毒介导的Isl1和GATA6的异位表达分析，随后将进行标记基因/蛋白质的检测。将进行GATA6和Isl1的双重KO，以确定它们在后肢芽Shh激活中的关系。目的2将确定GATA6通过其顺式调节元件负性调节ISL1的机制。体外DNA结合实验，荧光素酶报告基因转录调控实验，体内染色质免疫沉淀实验，体内结合实验和小鼠胚胎LacZ报告基因实验。结合这一建议，这一建议将确定不同的转录因子(前肢芽中的Hox9S和后肢芽中的Isl1)在两个同源组织中作为共同的Hand2-Shh途径的调节因子。此外，该提案将通过GATA6确定一个新的上游调控系统，该系统以后肢特异的方式导致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.