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Molecular mechanisms of ventral tangential migration of lot cells, the guide post neurons in the lateral olfactory tract Molecular mechanisms of ventral tangential migration of lot cells, the guide post neurons in the lateral olfactory tract 伊藤, 圭祐イトウ, ケイスケ ITO, Keisuke The brain functions are accomplished via communications between vast numbers of neurons. Thus, the construction of elaborated neural networks is indispensable for exerting a normal brain functions. Brain development is categorized into several steps such as neurogenesis, neuronal migration, axon projection, synaptogenesis, etc. Among them, neuronal migration is one of the most fundamental processes. If neurons do not migrate directionally, the consequences wi11 be a chaotic brain because numerous subtypes of neurons will be randomly intermixed. Neuronal migration helps different neuronal populations to segregate into distinctive compartments, whereas it also contributes to the dispersion of one neurohal population to occupy a large domain. In this way, distinct neuronal populations are appropriately arranged in the brain, which enables the complicated brain functions. Neurons migrate basically in two types of streams, radial and tangential. In the radial migration, neurons migrate verdcally to the surface of a brain, whereas in the tangential migration, neurons migrate parallel to the surface. The radial migration is a main migratory mode for constructing the six-layer cerebral cortex, and thus this mode have been well studied by many Laboratories for several decades. However, it has been only recently revealed that the tangential migration is also a critical migratory stream for the brain development. The most famous example of the tangential migration is provided by GABA interneurons, which migrate from the ventral telencephalon towards the dorsal neocortex thorough the so-called "dorsal tangential migration stream". However, there is also a stream in the opposite direction, "ventral tangential migration". Although this migration has been far less studied compared with the dorsal tangential migration, there is a good model system for analyzing the ventral tangential migration, which is lot cells. Lot cells are a neuronal population recognized by monoclonal antibody (mAb) lot1. It has been revealed that these neurons are involved in the formation of the lateral olfactory tract (LOT), the fascicle of olfactory bulb axons extending on the surface of the telencephalon. Our group previously reproted that newborn lot cells migrate through the ventral tangential pathway. During the early developmental phase at E9-10, lot cells differentiate from the ventricular zone of the dorsal neocortex region, and migrate on the surface of the neocortex ventrally and tangentially. After finishing the migration, the cells accumulate at the presumptive LOT region and make a cellular array, which guides or allows olfactory bulb axons to form the accurate LOT. This migration pattern is quite unique from the viewpoint that the cell migration controls the following axon projection, but molecular mechanisms of the lot cell migration still remain unknown. Our group previously performed combinational culture of early telencephalic explants, and suggested that the lot cell migration is non-cell autonomously controlled by multiple guidance cues; the neocortex region contains gradually distributed guidance cues to orient the migrating cells into the ventral direction, whereas the ventral part of the telencephalon has some mechanisms to exclude lot cells, probably mediated by short-range repulsive cues. An axon guidance molecule, Netrin-1 has an attractive effect on the migration of lot cells in vitro. However, the expression of netrin-1 is only restricted in the ventral part of the telencephalon, .thus Netrin-1 knockout mice exhibit only weak defects in the migration of lot cells. These results suggest that some other guidance molecules probably attract the lot cell migration. Also the repulsive cues for lot cells, which should be essential for the final arrangement of the cells, were virtually unidentified. In order to understand molecular mechanisms of the lot cell migration, I took two types of approaches; candidate screening and pharmacological perturbation. First, I screened candidate guidance molecules. cDNAs for various guidance molecules were transfected into HEK293T line cells, and the cell aggregates expressing the candidate molecules were made. Subsequently, the effects of candidate molecules were investigated by co-culturing these cell aggregates with telencephalic slices, after labeling the cells in the ventral tangential migration stream with a fluorescent dye, DiI. Among many candidate molecules, I found that a repulsive axon guidance molecule, SemaphOrin3F had a repulsive effect on the lot cell migration. Sema3F receptor, Neuropilin-2, was expressed in lot cells, and Sema3F was expressed in the region surrounding the presumptive LOT region. The cells in Neuropilin-2 knockout mice did not respond to Sema3F in the culture system. I examined the distribution pattern of lot cells in Nrp2 knockout mice and found that some lot cells were ectopically distributed in the medial region of the telencephalon. The majority of lot cells, however, normally aligned at the presumptive LOT region and they did not cross over the presumptive LOT region ventrally. These results indicate that Sema3F functions in confinement of lot cells on the surface of the neocortex, but not exclusion of cells from the ventral telencephalon Second, I tested various pharmacological drugs in culture, and found that a protein kinase inhibitor, K252a inhibits the migration of lot cells but does not inhibit the extension of leading processes. This result is interesting because it may provide a new insight into the mechanisms of neuronal migration. Neurons usually migrate long distances by the locomotion mode in which the leading processes and the cell body migrate in a coordinated manner. However, K252a seemed to convert this migration mode into the neurite extension mode such as the axon projection or dendrite extension. Thus, I hoped that the effect of K252a would give an important clue for understanding the switch of the migratory modes, and further analyzed this interesting phenomenon in the time-lapse video microscopy to detail the kinetics of the effect. This analysis showed that K252a robustly decreased the migration speed of cell bodies but not the extension of leading processes. K252a also converted the locomotion mode of the cerebellar granule cells into the neurite extension mode, suggesting that the switch of the migratory modes by K252a is rather a general phenomenon observable in various neuronal populations. I found one drug, roscovitine had a similar effect with K252a. Roscovitine is an inhibitor for cycling dependent kinases (CDK), and thus I overexpressed a dominant negative form of neuronal CDK, CDK5 in migrating neurons. Overexpression of the dominant negative CDK5 induced the extension of leading processes and slowdowned the migration speed of cell bodies. Therefore, CDK5 activity may be one of the critical components for the switching of the migratory modes from the locomotion to the neurite extension. 総研大甲第1059号 eng thesis https://ir.soken.ac.jp/records/1030 博士（理学） 2007-03-23 総合研究大学院大学 https://ir.soken.ac.jp/record/1030/files/甲1059_要旨.pdf application/pdf 369.0 kB 2016-02-17