The Involvement Of Myosin X In The Motility Of Immortalized GnRH Neuronal Cells

Gonadotropin-releasing hormone (GnRH) neurons are critical for the reproductive competence in mammalian, which have an important role in regulating the pituitary-gonadai axis。During development, the GnRH-secreting neurons originate in or around the olfactory placode and migrate along vomeronasal nerve fibers through the nasal septum, cross the cribriform plate, and travel associated with the caudal branch of the vomeronasal nerve until they reach the preoptic area and hypothalamus. The molecular cues regulating their migratory process from the olfactory placode into the brain are not well known, however. Netrin-1 and its receptor, DCC, were reported regulating the migration of GnRH neurons. Loss of netrin-1 or DCC function resulted in the migration of many GnRH neurons to inappropriate destinations. However, the mechanism of GnRH migration is still not well known. Recently, Myosin X (Myo X), an actin-based motor protein in vertebrate was reported to interact with DCC by the FERM domain. In addition to the full-length Myo X (Myo X FL), a headless form was expressed in the brain which is specifically expressed in the neuronal cells. So far, little is known about the function of this motor-less Myo X during the development of nervous system. The evidence from immunohistochemistry assay demonstrated Myo X expressed in the whole mouse embryonic brain including the migrating pathway of GnRH neuroblasts. Thus, experiments were performed to investigate the role of Myo X in the motility of GnRH cells.In this study, immortalized GnRH NLT cells, retaining the phenotypic characteristics of GnRH neurons and showing a high ability of migration, were employed to investigate the role of headless Myo X in the GnRH neuron migration. The immortalized GnRH cells are common models to investigate the biological characteristics of the GnRH neurons in vitro. DCC and two forms of Myo X were expressed in NLT cells and the distributions of DCC and Myo X were co-localized in the NLT cells. Myo X regulated the filopidial initiation and extention in NLT cells and filopodia were motile structures associated with the cell motility. To study the role of Myo X in the migration, the expressing level of Myo X was decreased by Myo X siRNA in NLT cells. In the collagen gel assay, the migratory distance of NLT cells was decreased and the migratory direction was disordered by the decreased expressing level of Myo X. The stably transfected cells overexpressing the headless Myo X were established to investigate the role of headless Myo X in the migration of GnRH cells. In the chemomigration assays, the cells overexpressing the headless Myo X migrated shorter distance and showed a significant decrease in number of migratory cells as compared with the control, but the migratory direction had not been changed. The cells overexpressing the headless Myo X formed less focal adhesion and spread more slowly than the wild-type NLT cells and the GFP-expressing cells. The NLT cells overexpressing headless Myo X were difficult to aggregation due to the decreased level of NCAM which was essential to morphogenesis of nervous system and the migration of GnRH neurons in vivo. It suggested that the headless Myo X has a role in the migration of GnRH neurons independent of Myo X FL...