Theses and Dissertations at Montana State University (MSU)
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Item The role of FGF signaling in neural crest development(Montana State University - Bozeman, College of Letters & Science, 2014) Dunkel, Haley Ann Arthun; Chairperson, Graduate Committee: Frances LefcortThe mechanisms that stimulate Neural Crest Cell (NCC) migration and cessation into discrete sympathetic ganglia (SG) and dorsal root ganglia (DRG) are incompletely understood. In this study we investigated the role of Endothelial Cells (ECs) and the shared growth factors of the nervous and vascular system: fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) in the development of the peripheral nervous system. We hypothesized that ECs impact neural crest patterning. Using live time-lapse microscopy, we found that NCCs and ECs interact extensively during neural crest migration and DRG development, including stimulating proliferation of Pax3+ neural progenitors. These studies also revealed that ECs of the intersomitic vessel perhaps act as a substrate for migrating NCCs while the perisomitic vessel acted as a boundary keeping NCCs in the forming DRG. In order to determine the role of ECs in PNS development we focally eliminated them with FGF receptor (FGFR) blocker Su5402. Su5402 focally reduced ECs and caused NCCs to remain dorsal. Given that FGFs effects could be direct or indirect on NCC, we sought to reduce FGF signaling solely in NCe. To this end we overexpressed a dominant negative FGFRl (DNFGFR1) and FGFR3 in ovo in NCCs. Our studies demonstrated that while blocking FGFR3 signaling did not impair NCC migration, NCCs expressing the DNFGFRl behaved similarly as did the Su4502 treated NCCs: they stayed dorsally at the expense of the SG and DRG and exhibited distinct morphologies. Stimulating FGFR signaling with the ligand FGF8 promoted migration in vitro and increased ventral migration of NCCs in vivo. To determine if the alterations in DNFGFRl + NCCs was due to changes in cell adhesion molecule function, we transfected NCC with DNFGFRl and gain and loss of function Ncadherin constructs. Surprisingly the fate of DNFGFR1+ NCCs was rescued by increasing expression of N-cadherin, while double transfections of DNFGFRl and DN-N-cadherin further impaired migration. While it is possible that N-cadherin and FGFRl directly interact, the exact interactions of FGFRl and N-cadherin need to be further investigated. FGFR3 was not found to function in NCC migration, but preliminary findings show it is required for TrkA+ neural differentiation.Item Analysis of the expression and function of chicken protocadherin 1 in neural crest cell migration and peripheral nervous system formation(Montana State University - Bozeman, College of Letters & Science, 2007) Bononi, Judy; Chairperson, Graduate Committee: Roger Bradley.The necessary steps of development from a single cell to a multi-celled functional organism are complex. Many molecules have been identified and their roles characterized in this process. One interesting population of cells includes the highly migratory neural crest cells (NCCs) unique to the vertebrate embryo and existing transiently during early embryonic development. The NCCs migrate along specific pathways at specific timepoints, stop at target locations, differentiate and give rise to a variety of cell types and tissues. Trunk NCCs must choose between two different migratory pathways: the ventral route, giving rise to neurons and glia of the dorsal root ganglia (DRG), sympathetic ganglia (SG), Schwann cells of the ventral root (VR); or the dorsolateral pathway, giving rise to melanocytes. Although many aspects of neural crest migration have been elucidated, cessation of migration and subsequent differentiation at target structures is not clearly defined. One family of molecules involved in various steps of NCC migration is the cell-cell adhesion molecules, the cadherins. To investigate the involvement of cadherins in NCC migration and differentiation during development using the avian model system, a combination of experiments and techniques including a library screen, in situ hybridization, in ovo electroporation, immunohistochemical and immunofluorescence staining as well as live time-lapse confocal imaging were performed. Results from these experiments produced the discovery and isolation of a novel molecule in the family of cadherin adhesion molecules, chicken protocadherin-1 (cPcdh1). Expression analysis showed cPcdh1 expressed in migrating NCCs, the DRG, SG and Schwann cells along the VR. A distinct expression pattern showed cPcdh1 along the periphery of the DRG, where crest cells are in an undifferentiated and mitotically active state. Further testing with deletion constructs and siRNA demonstrated when cPcdh1 function is inhibited, a greater percentage of cells migrate to the SG and VR at the expense of the DRG. Time-lapse confocal imaging showed cPcdh1 cells having an elongated cell shape with contact primarily being formed with neighboring cells along the periphery and longer cell-cell contact than observed in the control. Collectively, the results provide evidence for cPcdh1 involvement in NCC migration arrest and DRG formation.