Mechanisms of signal integration in developmental control of organ size and tissue patterning

项目总结 我们的工作旨在为理解信号机制提供新的见解 器官生长的发育控制。我们的初步数据表明，Capicua(CIC)是增长的目标- 控制信号通路，并表明CIC整合了几个上游信号来控制器官的大小。 CIC是一种转录抑制蛋白，受细胞外受体酪氨酸激酶(RTK)的调节 信号调节激酶(ERK)通路是控制器官大小和组织结构的关键系统之一 在人类和模型实验动物中的模式。我们的初步研究已经确定了一种激酶迷你脑 (MNB)作为一种新型的CIC调节因子，与RTK/ERK信号通路并行作用。ERK信令组件，MNB (人类DYRK1A)和CIC是高度保守的蛋白质，在人类中它们水平的变化导致 几种疾病，如风湿病、神经退行性疾病和癌症。DYRK1A一直积极 作为唐氏综合征的致病因素之一被调查，并被认为是正确的 中枢神经系统的发育和生长。我们在此应用程序中追求的一个令人兴奋的假设 ERK和MNB的信号汇聚在CIC上，调节其作为生长抑制因子的活性。考虑到 人类病理可由ERK和DYRK1A信号的定量变化引起，因此应用 量化的方法，以了解潜在的机制。果蝇提供了一种独特的 有机会在体内进行这样的研究。我们建议利用这个强大的实验平台来进行 对控制增长的发展信号进行定量和系统层面的分析。我们将使用状态- 最先进的蛋白质组学方法，如亲和纯化质谱学(AP-MS)，以鉴定结合 并分析上游信号如何改变这些相互作用，进而导致 中投公司活动的变化。然后，我们将应用定量成像分析来系统地测试已识别的 参与三个分子成果的互动伙伴：CIC降解、核出口和 转录抑制物活性的抑制。此外，我们将调查CIC如何整合来自 ERK和MNB激酶通过识别和功能验证这些靶向的磷酸化位点 两个激酶，并通过测试这两个激酶在CIC调节和生长控制中的相对贡献 独立的体内检测。总而言之，我们的多层次实验计划旨在为 CIC解释RTK/ERK信号的分子机制 机制与MNB的输入相交，MNB是一种新型CIC调节器。从长远来看，这项工作将推动我们的 了解参与器官大小控制的途径之间的复杂调控关系，以及 可能为开发ERK、CIC和DYRK1A相关疗法提供新的靶点。

PROJECT SUMMARY Our work is designed to provide new insights into understanding of signaling mechanisms responsible for the developmental control of organ growth. Our preliminary data have implicated Capicua (Cic) as a target of growth- controlling signaling pathways and suggest that Cic integrates several upstream signals to control organ size. Cic is a transcriptional repressor protein that is regulated by the receptor tyrosine kinase (RTK)-extracellular signal regulated kinase (ERK) pathway, which is one of the key systems involved in organ size control and tissue patterning in humans and model experimental animals. Our preliminary studies have identified a kinase Minibrain (Mnb) as a novel Cic regulator which acts in parallel to RTK/ERK signaling. ERK signaling components, Mnb (human DYRK1A), and Cic are highly conserved proteins, and alterations in their levels in humans result in several diseases, such as Rasopathies, neurodegenerative disorders, and cancer. DYRK1A has been actively investigated as one of the causative factors in Down syndrome, and is thought to be important for proper development and growth of the central nervous system. An exciting hypothesis that we pursue in this application is that signals from ERK and Mnb converge on Cic to regulate its activity as a growth suppressor. Given that human pathologies can result from quantitative changes in ERK and DYRK1A signaling, it is essential to apply quantitative approaches in order to understand the underlying mechanisms. Drosophila offers a unique opportunity to carry out such research in vivo. We propose to use this powerful experimental platform to carry out a quantitative and systems-level analysis of developmental signals controlling growth. We will use state-of- the-art proteomic methods, such as affinity purification mass spectrometry (AP-MS), to identify the binding partners of Cic in the embryo and analyze how upstream signals alter these interactions, which in turn leads to changes in Cic activity. We will then apply quantitative imaging assays to systematically test the identified interacting partners for their involvement in three molecular outcomes: Cic degradation, nuclear export, and inhibition of transcriptional repressor activity. Furthermore, we will investigate how Cic integrates signals from the ERK and Mnb kinases by identifying and functionally validating the phosphorylation sites targeted by these two kinases, and by testing the relative contributions of these kinases to Cic regulation and growth control in four independent in vivo assays. In summary, our multi-level experimental plan is designed to provide insights into the molecular mechanisms of RTK/ERK signal interpretation by Cic, as well as to determine how these mechanisms intersect with the input from Mnb, a novel Cic regulator. In the long term, this work will advance our understanding of the complex regulatory relationships between the pathways involved in organ size control, and may suggest new targets for developing ERK, Cic, and DYRK1A-related therapies.