During development of the neocortex, the marginal zone (layer I) and the subplate (layer VII) are the first layers to form from a primordial plexiform neoropil. The cortical plate (layers II-VI) is subsequently established between these superficial and deep components of the primordial plexiform neuropil. Neurons in the early zones are thought to play important roles in the formation of the cortex: the Cajal-Retzius cells of the marginal zone are instrumental in neuronal migration and laminar formation, and cells of the subplate are involved in the formation of cortical connections. Using the fluorescent tracer 1,1'-dioctodecyl-3,3,3', 3'-tetramethylindocarbocyanine (DiI), we have shown here that a substantial proportion of neurons of the marginal zone, including cells with features of Cajal-Retzius cells, and of the subplate and lower intermediate zone are not born in the ventricular neuroepithelium but instead originate in the medial ganglionic eminence (MGE), the pallidal primordium. These neurons follow a tangential migratory route to their positions in the developing cortex. They express the neurotransmitter GABA but seem to lack the calcium binding protein calretinin; some migrating cells found in the marginal zone express reelin. In addition, migrating cells express the LIM-homeobox gene Lhx6, a characteristic marker of the MGE. It is suggested that this gene uniquely or in combination with other transcription factors may be involved in the decision of MGE cells to differentiate in situ or migrate to the neocortex.

The conventional scheme of cortical formation shows that postmitotic neurons migrate away from the germinal ventricular zone to their positions in the developing cortex, guided by the processes of radial glial cells. However, recent studies indicate that different neuronal types adopt distinct modes of migration in the developing cortex. Here, we review evidence for two modes of radial movement: somal translocation, which is adopted by the early-generated neurons; and glia-guided locomotion, which is used predominantly by pyramidal cells. Cortical interneurons, which originate in the ventral telencephalon, use a third mode of migration. They migrate tangentially into the cortex, then seek the ventricular zone before moving radially to take up their positions in the cortical anlage.

Large numbers of dying cells are found in proliferating tissues, suggesting a link between cell death and cell division. We detected and quantified dying cells during pre-and early postnatal development of the rat cerebral cortex using in situ end labeling of DNA fragmentation [terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)] and electron microscopy. The proliferative zones that give rise to the neuronal and glial cell types of the cortex, the ventricular and, to a larger extent, the subventricular zones showed higher incidence of cell death than other regions of the developing cortex during the period of neurogenesis. Gel electrophoresis of DNA isolated from the subventricular zone of newborn animals showed a ladder pattern that is characteristic of apoptosis. The number of apoptotic cells remained high in this zone for at least 2 weeks, during which period cells continued to divide. The correlation between cell division and cell death was studied in the subventricular zone of newborn rats; cumulative labeling with bromodeoxyuridine showed that 71% of TUNEL-labeled cells had taken up this S-phase marker before undergoing cell death. Using bromodeoxyuridine and [ 3 H]-thymidine in succession to identify a cohort of proliferating cells, we found that the clearance time of TUNEL-positive nuclei was 2 hr and 20 min. A comparison between the number of mitotic figures and that of TUNEL-positive nuclei showed that cell death affects one in every 14 cells produced by dividing ventricular zone cells at embryonic day 16 and one in every 1.5 cells produced in the subventricular zone of newborn rats. In addition, we found that most of TUNEL-positive cells were in the G1 phase of their cell cycle. We conclude that apoptosis is prominent in the proliferating neuroepithelium of the developing rat cerebral cortex and that it is related to the progression of the cell cycle.

The Slit genes encode secreted ligands that regulate axon branching, commissural axon pathfinding and neuronal migration. The principal identified receptor for Slit is Robo (Roundabout in Drosophila). To investigate Slit signalling in forebrain development, we generated Robo1 knockout mice by targeted deletion of exon 5 of the Robo1 gene. Homozygote knockout mice died at birth, but prenatally displayed major defects in axon pathfinding and cortical interneuron migration. Axon pathfinding defects included dysgenesis of the corpus callosum and hippocampal commissure, and abnormalities in corticothalamic and thalamocortical targeting. Slit2 and Slit1/2 double mutants display malformations in callosal development, and in corticothalamic and thalamocortical targeting, as well as optic tract defects. In these animals, corticothalamic axons form large fasciculated bundles that aberrantly cross the midline at the level of the hippocampal and anterior commissures, and more caudally at the medial preoptic area. Such phenotypes of corticothalamic targeting were not observed in Robo1 knockout mice but, instead, both corticothalamic and thalamocortical axons aberrantly arrived at their respective targets at least 1 day earlier than controls. By contrast, in Slit mutants, fewer thalamic axons actually arrive in the cortex during development. Finally, significantly more interneurons (up to twice as many at E12.5 and E15.5) migrated into the cortex of Robo1 knockout mice, particularly in both rostral and parietal regions, but not caudal cortex. These results indicate that Robo1 mutants have distinct phenotypes, some of which are different from those described in Slit mutants, suggesting that additional ligands, receptors or receptor partners are likely to be involved in Slit/Robo signalling.

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