A major challenge in developmental biology is to understand the intricate cellular interactions between tissues that underlie morphogenesis of complex organs. To dissect the molecular basis of morphogenesis it s advantageous to study simple model tissues or organs. The long term objective of this proposal is a molecular and cellular explanation of embryonic morphogenesis of epidermis of the nematode C. elegans. In embryonic development the epidermis spreads over substrate cells, the ventral neuroblasts. Our previous work showed that signaling via the Eph receptor tyrosine kinase and its ephrin ligands is required for movements of substrate ventral neuroblasts. A specific form of Eph signaling involving both receptor and ligand activation (bidirectional signaling) likely promotes adhesion or attraction among ventral neuroblasts. Here we propose to investigate how Eph signaling and parallel pathways regulate neuroblast adhesion and movements. 1. We will analyze the cellular basis of ventral neuroblast motility by a combination of confocal timelapse analysis, genetics, pharmacological interventions, and laser microsurgery. The results will allow us to interpret how the signaling pathways affect motility. 2. Reverse signaling by GPI-linked ephrins is poorly understood. In RNAi enhancer screens we identified a G protein subunit as a potential component of ephrin reverse signaling. We will define the role of this G protein in ephrin signaling using genetic and biochemical tests. 3. Eph signaling functions partly redundantly with several other pathways that provide cell adhesion in the embryo and in axon guidance. To address the nature of this redundancy we will analyze one such parallel pathway involving KAL-1, the C. elegans ortholog of anosmin-1. We have shown KAL-1 interacts with the cell surface heparan sulfated proteoglycans syndecan and glypican. We will define the regulatory pathway of KAL-1 and HSPGs and whether they are required in the same cells. HSPGs are essential for C. elegans embryonic morphogenesis. We will identify the other core proteins that account for this essential function by a combination of candidate gene testing and biochemical purification. 4. Epidermal cells migrate over the ventral neuroblast substrate. The molecules involved in epidermal- substrate adhesion are not known. Our preliminary data suggest netrin signaling has functions both in neuroblast migration and in epidermal substrate attachment. We will define the roles of netrin signaling in morphogenesis and test the hypothesis that netrin acts over short range in epidermal substrate adhesion. Relevance: An understanding of morphogenesis is relevant to treatment of human birth defects, to the development of artificial organs and tissue repair, and to tumor development. Eph signaling in particular has been implicated in human genetic disease, cancer, and in maintenance of neural stem cell niches. ? ? ?
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