The response of primary mesenchyme cells to VEGF

海胆胚胎的骨骼是一个强大的模型系统，可以用来研究遗传信息如何转化为解剖特征。已知顶层涉及一个由100多个调控基因组成的复杂网络。然而，这个“管理”网络的作用主要是做决定，而不是做任何实际工作。最近发现，胚胎的正常发育需要胚胎中的一小部分细胞使用一种名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。PMC是奠定骨骼的细胞，一旦被赋予这个词，它们几乎就会自己完成这一任务。研究小组希望了解PMC如何对它们从其他细胞接收到的“GO”信号做出反应，它们运行的哪些遗传程序会影响骨骼的形状，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顶层决策如何导致细胞行为的实际变化，以及具有复杂架构的骨架的出现，并有望有助于理解此类机制是如何演变的。拟议活动的一个重要部分是为工科本科生开发实验模块，将本科生研究人员纳入该项目，以及在当地高中使用流动海胆实验室开展推广活动。海胆胚胎的刺激发生，即初级间充质细胞(PMC)内骨骼的形成，是一系列定型形态发生事件的结果。关键步骤包括上皮-间充质转化、图案化和细胞-细胞融合。原肠胚后期，PMC合胞体开始分泌内骨骼。虽然目前的骨骼发育基因调控网络(GRN)模型可以说是所有生物体中理解得最好的模型之一，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。最近的工作确认了外胚层衍生因子的重要性，包括血管内皮生长因子(VEGF)。主要研究人员和合作者发现，重组血管内皮生长因子在体外对PMC沉积的针状物的形状具有显著的浓度依赖效应。三次辐射，即与最初沉积在胚胎中的分支针状体非常相似的分枝针状体，需要一个阈值浓度的rVEGF。低于这个浓度，就会形成明显不同的针叶形状。因此，血管内皮生长因子是PMC中重要的形态发生事件的外在调节因子。因此，研究小组将进行一系列体外实验，旨在揭示血管内皮生长因子信号在PMC中的作用。具体地说，他们将：a)通过定量PCR和深度测序相结合的方法来确定rVEGF浓度对骨源性GRN及其下游电路的影响；b)研究VEGF在毛刺发生开始之前对细胞行为的影响，包括增殖、存活、运动和构型；以及c)研究VEGF对PMC合胞体内超微结构的影响，特别是细胞骨架重排。这项拟议的研究不仅将顶层调控电路与对细胞行为的更近端控制联系在一起，有助于整合PMC骨骼形态发生的机制观点，而且还为理解此类调控网络如何演变而产生不同的形态特征提供了基础。由于血管内皮生长因子在血管、神经和气管网络的发展中也是一个关键角色，这项研究可能有助于辨别该配体在进化过程中的功能。拟议活动的一个重要部分是开发实验单元，在初级化学和生物工程/材料科学课程的背景下预测海胆的骨骼形态发生，将本科生研究人员纳入该项目，并利用流动海胆实验室在当地高中开展外联活动。

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.