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    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.
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    Sympathetic ganglia formation in the chick peripheral nervous system
    (Montana State University - Bozeman, College of Letters & Science, 2005) Kasemeier-Kulesa, Jennifer Caroline; Chairperson, Graduate Committee: Frances Lefcort.
    The neural crest is a unique population of pluripotent cells that are crucial in vertebrate embryogenesis. In the trunk, NCCs migrate along a ventromedial pathway give rise to the dorsal root ganglia or the sympathetic ganglia (SG), or follow a dorsolateral pathway and give rise to melanocytes. Intriguingly, NCCs following the ventromedial pathway migrate in a metameric pattern through the rostral half somite and avoid the caudal somite and this is thought to dictate the metameric pattern of the SG. Static analyses have characterized the development of these structures, but timelapse imaging of NCCs in their normal environment could potentially reveal unidentified cellular and molecular interactions integral to SG development. However, because NCCs migrate deep within the embryo, it is challenging to track NCCs little is known about the cellular mechanisms mediating their migration, aggregation and differentiation. Here, we follow fluorescently labeled trunk NCCs using a novel sagittal explant culture system and timelapse confocal microscopy. We show trunk NCCs migrate in chain-like formations, and restriction to the rostral somite is not maintained once these cells arrive at the dorsal aorta. Instead, discrete SG only arise after an intermixing of cells along the ventral border of the somite followed by segregation into ganglia. The diverse cell migratory behaviors and active reorganization at the target sites suggest that cell-cell and cell-environment interactions are coordinated with dynamic molecular processes. In a screen for molecules expressed on NCCs during SG formation, we identified the cell adhesion molecule N-cadherin expressed once NCCs arrive adjacent to the dorsal aorta and form ganglia. Additionally, altering cadherin function drastically alters ganglia size. Additionally, we found EphrinB1 expression was absent adjacent to the dorsal aorta as NCCs had dispersed in this corridor, but strikingly up-regulated in the inter-ganglionic regions after discrete ganglia formation. The ephrinB1 receptor, EphB2, is also expressed on NCCs distributed adjacent to the dorsal aorta when discrete ganglia form. Altering ephrinB1/EphB2 signaling interferes with formation of the primary SG chain and blocks the formation of discrete ganglia. Taken together, these results indicate the importance of adhesive and inhibitory mechanisms in the formation of SG.
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