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