Masters Thesis

The Role of SLIT/ROBO in contact inhibition of locomotion in Neural Crest cells

Neural Crest cells (NCC) are a stem cell population originating from the dorsal-most region of the neural tube, delaminating from the neural tube in an epithelial to mesenchymal transition (EMT). These cells are highly motile and migrate extensively throughout the developing embryo and generate a variety of different cell types. This migration process has a precise pattern of cell-cell interaction, which allows the neural crest cells collide with each other, has been called Contact Inhibition of Locomotion (CIL). This process enables cells to change it current direction after contacting another cell during migration; while the loss of this activity has been linked to metastasis. We believe a key player in CIL is Slit/Robo signaling; Robo receptors are expressed in trunk, while Slit, a ligand, are found around the gut. The objective of my research is to determine the role of Slit/Robo in CIL and the effects they have on Neural crest cell motility and cytoskeleton. Previous research (De Bellard, 2003), has shown Slit/Robo signaling prevents trunk NCC (TNCC) from entering the gut but allows NCC from the vagal region. 1) Cultured NCC in media conditioned by HEK/HEK-SLIT2 to determine if soluble Slit2 can enhance CIL behavior. I will quantify this by measuring the time that cells remain in contact after collision and their acceleration rates after they move away from each other. This is a hallmark of CIL. 2) In order to examine how Slit/Robo affects neural crest precursor proliferation, I will Electroporate chick embryos after introducing a plasmid, Robo -/- ∆ 2 (RD2) which silences Robo signaling. Then observe whether Robo signaling decreases cell proliferation within the neural crest; includes NCC and neural crest derived cell lines (i.e. SpL201). NCC cell-cell interaction and measure the amount of time which NCC remain in contact after the initial collision; In addition, the acceleration and directionality will be measured also to determine how their migration behavior patterns are affected.3) By performing RNA sequencing, initially on SpL201; then NCC, key molecules effected once the cell line is transfected with RD2. This will provide a visual analysis on how RD2 changes cell migration, and allow us to examine how Slit/Robo signaling affects neural crest mechanisms. The importance of this project is to pioneer the molecular effects on the physiological behaviors these pre-cursor cells' achieve. This will lead to advancement in knowledge of these migratory variations, which uncover a pathway to understanding mechanisms linking cell receptor interaction to the behavior of the cell within its environment.

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