The response of primary mesenchyme cells to VEGF

原代间充质细胞对VEGF的反应

• 批准号: 1456837
• 负责人: Derk Joester
• 金额: $ 69万
• 依托单位: Northwestern University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1456837&HistoricalAwards=false
• 关键词: response; primary; mesenchyme; cells; VEGF

The skeleton of the sea urchin embryo is a powerful model system for how genetic information is transformed into anatomical features. An intricate network of more than 100 regulatory genes is known to be involved at the top level. The role of this "managerial" network, however, is primarily to make decisions, not to do any of the actual work. It was recently discovered that proper development requires that a small group of cells in the embryo send a message to another group, the primary mesenchyme cells (PMCs), using a protein called vascular endothelial growth factor (VEGF). PMCs are the cells that lay down the skeleton, and once given the word, they do this pretty much on their own. The research team hopes to learn how PMCs respond to the "go"-signal they receive from other cells, what genetic programs they run that affect the shape of the skeleton, and what kind of behavioral and structural changes result from running these programs. In the big picture, this would help understand how top-level decisions in the cell lead to actual changes in cell behavior and the emergence of a skeleton with complex architecture, and will hopefully contribute to an understanding of how such mechanisms evolve. An important part of the proposed activities is the development of lab modules for undergraduate engineering students, the integration of undergraduate researchers in the project, and outreach activities at local high schools using a mobile sea urchin lab.Spiculogenesis in the sea urchin embryo, i.e. formation of the endoskeleton by primary mesenchyme cells (PMC), is the result of a stereotypical sequence of morphogenetic events. Key steps include an epithelial-mesenchymal transition, patterning, and cell-cell fusion. In the late gastrula stage, PMC syncytia begin secreting the endoskeleton. While the current model of the skeletogenic gene regulatory network (GRN) is arguably one of the best understood in any organism, there is currently a gap in our understanding of how the high-level GRN circuitry is integrated with regulation of complex cellular behavior. Recent work identified the importance of ectoderm-derived factors, including vascular endothelial growth factor (VEGF). The Principal Investigator and collaborators discovered that a recombinant VEGF has a dramatic concentration-dependent effect on the shape of spicules deposited by PMC in vitro. Triradiates, i.e. branching spicules that closely resemble those initially deposited in the embryo, require a threshold concentration of rVEGF. Below this concentration, markedly different spicule shapes are formed. VEGF is, thus, an important extrinsic regulator of morphogenetic events in PMC. The research team will therefore perform a series of in vitro experiments that aim to unravel the role of VEGF signaling in PMCs. Specifically, they will: a) determine the influence of rVEGF concentration on the skeletogenic GRN and its downstream circuitry by a combination of quantitative PCR and deep sequencing; b) investigate the effect of VEGF on cell behavior before the onset of spiculogenesis, including proliferation, survival, motility, and patterning, and c) study the effect of VEGF on ultrastructure, specifically cytoskeletal rearrangement, in the PMC syncytium. The proposed research will not only help integrate a mechanistic view of PMC skeletal morphogenesis by connecting top-level regulatory circuits with more proximal control over cellular behavior, but also provide a basis for understanding how such regulatory networks evolve to give rise to different morphological features. Since VEGF is also a key player in the development of vascular, nervous, and tracheal networks, this research may help discern functions of this ligand during evolution. An important part of the proposed activities is the development of lab modules that cast sea urchin skeletal morphogenesis in the context of an entry-level chemical and biological engineering/materials science class, the integration of undergraduate researchers in the project, and outreach activities at local high schools using a mobile sea urchin lab.

海胆胚胎的骨骼是一个强大的模型系统，可以解释遗传信息如何转化为解剖特征。一个由100多个调控基因组成的复杂网络被认为是在最高水平上参与的。然而，这个“管理”网络的作用主要是做决策，而不是做任何实际工作。最近发现，正常的发育需要胚胎中的一小群细胞使用一种称为血管内皮生长因子（VEGF）的蛋白质向另一组细胞（初级间充质细胞（PMC））发送信息。后内侧皮层细胞是奠定骨骼的细胞，一旦有命令，它们几乎是靠自己完成的。研究小组希望了解PMC如何响应它们从其他细胞接收的“go”信号，它们运行哪些影响骨骼形状的遗传程序，以及运行这些程序会导致什么样的行为和结构变化。从大局来看，这将有助于理解细胞中的顶层决策如何导致细胞行为的实际变化以及具有复杂结构的骨架的出现，并有望有助于理解这些机制如何进化。拟议的活动的一个重要组成部分是本科工程专业学生的实验室模块的开发，本科研究人员在项目中的整合，并在当地高中使用移动的海胆labor.Spiculogenesis在海胆胚胎，即形成的内骨骼的初级间充质细胞（PMC），外展活动的结果是一个刻板的序列的形态发生事件。关键步骤包括上皮-间充质转化、图案化和细胞-细胞融合。在原肠胚晚期，PMC合胞体开始分泌内骨骼。虽然目前的骨骼生成基因调控网络（GRN）模型可以说是任何生物体中最好的理解之一，但目前我们对高水平GRN电路如何与复杂细胞行为的调控相结合的理解存在差距。最近的工作确定了外胚层衍生因子的重要性，包括血管内皮生长因子（VEGF）。主要研究者和合作者发现，重组VEGF在体外对PMC沉积的针状体形状具有显著的浓度依赖性作用。三辐射体，即与最初沉积在胚胎中的那些非常相似的分支针状体，需要rVEGF的阈值浓度。低于此浓度，形成明显不同的骨针形状。因此，VEGF是PMC中形态发生事件的重要外在调节因子。因此，研究小组将进行一系列体外实验，旨在揭示VEGF信号在PMC中的作用。具体而言，它们将：a）通过定量PCR和深度测序的组合，确定rVEGF浓度对成骨GRN及其下游回路的影响; B）研究VEGF对毛刺发生之前的细胞行为的影响，包括增殖、存活、运动性和模式化，和c）研究VEGF对PMC合胞体中的超微结构，特别是细胞骨架重排的影响。拟议的研究不仅有助于通过连接顶层调控回路与更近端的细胞行为控制来整合PMC骨骼形态发生的机制观点，而且还为理解这种调控网络如何演变以产生不同的形态特征提供了基础。 由于VEGF在血管、神经和气管网络的发育中也是一个关键角色，这项研究可能有助于识别这种配体在进化过程中的功能。拟议活动的一个重要组成部分是开发实验室模块，在入门级化学和生物工程/材料科学课程的背景下铸造海胆骨骼形态发生，将本科研究人员纳入该项目，并使用移动的海胆实验室在当地高中开展推广活动。