The response of primary mesenchyme cells to VEGF

Northwestern University, Evanston IL

Investigators

Abstract

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.