CELL FATE DETERMINATION IN THE MESODERM OF DROSOPHILA

本研究的目的是了解早期中胚层细胞统一群体产生多种不同组织的潜在机制。我们的研究结果表明，Twist，一个基本螺旋-环-螺旋（bHLH）转录因子，在中胚层的特化，其细分为不同组织，成体肌肉的模式和成年肌肉祖先的分化中起着核心作用。我们将重点关注Twist如何能够执行这些不同的角色。根据我们的研究结果，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，Daughterless (Da)，我们预测在发育的不同阶段，Twist的活性受到与中胚层其他HLH蛋白相互作用的严格调节。我们已经确定了四种可能与Twist相互作用的HLH候选蛋白。基于表达和功能丧失数据，我们提出Twist、Da和Extramachrochaete之间的相互作用对早期中胚层事件至关重要，而Twist、L’sccute、Da和Hairy之间的相互作用对后期事件起决定性作用。为了验证这一假设，我们将使用基因技术来控制Twist和这些潜在合作伙伴的剂量。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，我们将检查Twist和候选HLH蛋白之间的关系，使用体外[例如，凝胶转移]和体内[例如，用系留二聚体过表达]技术的组合来评估蛋白质是否直接相互作用以激活或抑制靶基因。我们的初步研究结果表明，Twist同型二聚体激活了肌生成途径，而Twist/Da异源二聚体抑制了肌生成命运。我们将使用体内和体外技术来绘制Twist激活所需的结构域。我们将测试这些结构域是否转化Da和脊椎动物Twist、Wingless、Decapentaplegic、Notch和Ras信号通路，至少在一定程度上通过改变Twist同二聚体和异二聚体的比例来影响中胚层发育。我们将在体内测试，如果过度表达特定的二聚体在胚胎突变的信号通路挽救中胚层缺陷。由于与已知的HLH蛋白和Twist的相互作用并不能完全解释Twist的功能范围，我们还将筛选未知的HLH蛋白和在Twist依赖增强子上与Twist相互作用的共同调节因子。由于果蝇和其他物种之间的基因和机制具有很强的保守性，我们的数据将有助于理解所有物种肌肉发育中发生的类似决定。事实上，小鼠和人类中Twist、Wg和N的同源基因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有直接的影响。

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.