CELL FATE DETERMINATION IN THE MESODERM OF DROSOPHILA

我们的研究结果表明，作为一种基本的螺旋-环-螺旋(BHLH)转录因子，Twist在中胚层的指定、其向不同组织的细分、躯体肌肉的图案化以及成年肌祖细胞的分化中起着核心作用。我们将专注于Twist如何能够执行这些不同的角色。根据我们的发现，在体内和体外，Twist可以与bHLH蛋白形成同源二聚体和异源二聚体，无子(Da)，我们预测Twist的活性受到与中胚层中不同发育阶段的其他HLH蛋白相互作用的严格调控。我们已经确定了四个可能与Twist相互作用的HLH候选蛋白。基于功能数据的表达和丢失，我们认为Twist、Da和大毛虫之间的相互作用对于早期的中胚层事件是关键的，而Twist、L的Scut、Da和毛发之间的相互作用对后来的事件是决定性的。为了验证这一假说，我们将使用基因技术来控制Twist的剂量和这些潜在的伙伴。如果这些蛋白质相互作用，我们将看到中胚层发育的缺陷。然后，我们将结合使用体外[例如，凝胶移动]和体内[例如，用拴系二聚体过度表达]技术来研究Twist和候选HLH蛋白之间的关系，以评估这些蛋白是否直接相互作用来激活或抑制目标基因。我们的初步发现表明，Twist同源二聚体激活了肌源性通路，而Twist/Da异源二聚体抑制了肌源性命运。我们将使用体内和体外技术来绘制激活所需的Twist结构域。我们将测试这些结构域是否通过改变Twist同源二聚体和异二聚体的比例来影响中胚层的发育，以及脊椎动物的Twist、Wingless、Decapentaplecic、Notch和RAS信号通路是否至少部分地影响中胚层的发育。我们将在体内测试，在突变的胚胎中过度表达特定二聚体的信号通路是否可以挽救中胚层缺陷。由于与已知的HLH蛋白和Twist的相互作用不能完全解释Twist的功能范围，我们还将筛选未知的HLH蛋白和在Twist依赖的增强子上与Twist相互作用的共同调节因子。随着果蝇和其他物种之间基因和机制的高度保守，我们的数据将有助于理解所有物种在肌肉发育过程中做出的类似决定。事实上，小鼠和人类Twist、Wg和N同源物的突变会导致发育异常，我们的工作将对理解这些缺陷和疾病产生直接影响。

The goal of this study is to understand the underlying mechanisms by which a uniform population of early mesoderm cells gives rise to a variety of different tissues Our findings indicate that Twist, a basic helix-loop-helix (bHLH) transcription factor, plays a central role in specification of the mesoderm, its subdivision into different tissues, patterning of the somatic muscles, and differentiation of the adult muscle progenitors. We will focus on how Twist is able to execute these different roles. Based on our findings that, in vivo and in vitro, Twist can form homodimers and heterodimers with the bHLH protein, Daughterless (Da), we predict that Twist activity is tightly regulated by interactions with other HLH proteins in the mesoderm at different points in development. We have defined four HLH candidate proteins that may interact with Twist. Based on expression and loss of function data, we propose that interactions between Twist, Da, and Extramachrochaete are critical for early mesodermal events, whereas interactions between Twist, L'Scute,Da and Hairy are decisive for later events.To test this hypothesis, we will use genetic techniques to manipulate the dosage of Twist and each of these potential partners. If these proteins interact, we will see defects in mesodermal development. We will then examine the relationship between Twist and the candidate HLH protein, using a combination of in vitro [e.g., gel shift] and in vivo [e.g., overexpresssion with tethered dimers] techniques to assess if the proteins interact directly to activate or repress target genes. Our preliminary findings indicate that Twist homodimers activate the myogenic pathway, whereas Twist/Da heterodimers repress the myogenic fate. We will use both in vivo and in vitro techniques to map the domains of Twist required for activation. We will test whether these domains transform Da and vertebrate Twist, Wingless, Decapentaplegic, Notch and Ras signaling pathways affect mesoderm development, at least in part, by altering ratios of Twist homodimers and heterodimers. We will test in vivo, if overexpression of particular dimers in embryos mutant for a signaling pathway rescues the mesodermal defects. Since interactions with the known HLH proteins and Twist will not completely explain the scope of Twist's functions, we will also screen for unknown HLH proteins and co-regulators that interact with Twist on Twist-dependent enhancers. With the strong conservation of genes and mechanisms between drosophila and other species, our data will help understand similar decisions that occur in muscle development in all species. Indeed, mutations in homologues of Twist, Wg, and N in mouse and human lead to developmental abnormalities and our work will have a direct impact on understanding these defects and diseases.