Neuropilin is a neuronal cell surface protein and has been shown to function as a receptor for a secreted protein, semaphorin III/D, that can induce neuronal growth cone collapse and repulsion of neurites in vitro. The roles of neuropilin in vivo, however, are unknown. Here, we report that neuropilin-deficient mutant mice produced by targeted disruption of the neuropilin gene show severe abnormalities in the trajectory of efferent fibers of the PNS. We also describe that neuropilin-deprived dorsal root ganglion neurons are perfectly protected from growth cone collapse elicited by semaphorin III/D. Our results indicate that neuropilin-semaphorin III/D-mediated chemorepulsive signals play a major role in guidance of PNS efferents.

The six-layered neocortex permits complex information processing in all mammalian species. Because its homologous region (the pallium) in nonmammalian amniotes has a different architecture, the ability of neocortical progenitors to generate an orderly sequence of distinct cell types was thought to have arisen in the mammalian lineage. This study, however, shows that layer-specific neuron subtypes do exist in the chick pallium. Deep- and upper-layer neurons are not layered but are segregated in distinct mediolateral domains in vivo. Surprisingly, cultured chick neural progenitors produce multiple layer-specific neuronal subtypes in the same chronological sequence as seen in mammals. These results suggest that the temporal sequence of the neocortical neurogenetic program was already inherent in the last common ancestor of mammals and birds and that mammals use this conserved program to generate a uniformly layered neocortex, whereas birds impose spatial constraints on the sequence to pattern the pallium.

During development, mitral cells, the main output neurons of the olfactory bulb, project axons into a very narrow part of the telencephalon and form an axonal bundle called the lateral olfactory tract (LOT). The present study shows that before the first mitral cell axons elongate, the LOT position is already marked with a subset of early-generated neurons that are recognized by monoclonal antibody lot1 (lot cells). Mitral cell axons choose the lot cell position for their growth pathway and maintain a close contact with the cells until LOT formation is completed. Ablation of lot cells prevented LOT formation in organotypic culture. These results suggest that lot cells are "guidepost cells" for mitral cell axons.

No abstract

Neural circuitry formation depends on the molecular control of axonal projection during development. By screening with fluorophore-assisted light inactivation in the developing mouse brain, we identified cartilage acidic protein–1B as a key molecule for lateral olfactory tract (LOT) formation and named it LOT usher substance (LOTUS). We further identified Nogo receptor–1 (NgR1) as a LOTUS-binding protein. NgR1 is a receptor of myelin-derived axon growth inhibitors, such as Nogo, which prevent neural regeneration in the adult. LOTUS suppressed Nogo-NgR1 binding and Nogo-induced growth cone collapse. A defasciculated LOT was present in lotus-deficient mice but not in mice lacking both lotus- and ngr1. These findings suggest that endogenous antagonism of NgR1 by LOTUS is crucial for normal LOT formation.

The early-generated neurons designated as lot cells specifically mark the future site of the lateral olfactory tract (LOT) and guide LOT axons. We investigated the mechanism of how lot cells develop and get localized in the LOT position. Lot cells differentiated from neuroepithelial cells in all regions of the neocortex but not from those in the ganglionic eminence in culture. Cell tracing analyses demonstrated that lot cells generated from the neocortex subsequently followed a tangential migration stream ventrally toward the LOT position. Mutant mouse embryos lacking the function of transcription factor Gli3 showed disturbances of the migration stream and translocation of lot cells in the dorsal telencephalon. These results reveal a new type of neuronal migration in the telencephalon and introduce an unexpected dramatic feature of the earliest regionalization of the telencephalon.

The metabotropic glutamate receptor subtype 1 (mGluR1, Grm1) in cerebellar Purkinje cells (PCs) is essential for motor coordination and motor learning. At the synaptic level, mGluR1 has a critical role in long-term synaptic depression (LTD) at parallel fiber (PF)-PC synapses, and in developmental elimination of climbing fiber (CF)-PC synapses. mGluR1a, a predominant splice variant in PCs, has a long carboxyl (C)-terminal domain that interacts with Homer scaffolding proteins. Cerebellar roles of the C-terminal domain at both synaptic and behavior levels remain poorly understood. To address this question, we introduced a short variant, mGluR1b, which lacks this domain into PCs of mGluR1-knock-out (KO) mice (mGluR1b-rescue mice). In mGluR1b-rescue mice, mGluR1b showed dispersed perisynaptic distribution in PC spines. Importantly, mGluR1b-rescue mice exhibited impairments in inositol 1,4,5-trisphosphate receptor (IP 3 R)-mediated Ca 2ϩ release, CF synapse elimination, LTD induction, and delay eyeblink conditioning: they showed normal transient receptor potential canonical (TRPC) currents and normal motor coordination. In contrast, PC-specific rescue of mGluR1a restored all cerebellar defects of mGluR1-KO mice. We conclude that the long C-terminal domain of mGluR1a is required for the proper perisynaptic targeting of mGluR1, IP 3 R-mediated Ca 2ϩ release, CF synapse elimination, LTD, and motor learning, but not for TRPC currents and motor coordination.

MiceTime-pregnant ICR wild-type mice were purchased from SLC (Shizuoka, Japan). Mice expressing green fluorescent protein (GFP; green mice) were provided by Okabe et al. (Okabe et al., 1997). Xt J /Xt J mutant embryos, which have deletions in the Gli3 gene, were generated as described previously (Tomioka et al., 2000). When the Xt J mutation was introduced into green mice, green mice were crossed with Xt J /+ mice and the resulting Xt J /+ mice expressing GFP were intercrossed to obtain Xt J /Xt J green mouse embryos. Heterozygous netrin 1 mutant mice (Serafini et al., 1996) and Dcc mutant mice (Fazeli et al., 1997) were crossed to obtain homozygous embryos. The day on which a vaginal plug was found was designated embryonic day 0.5 (E0.5). All experimental protocols were approved by the Animal Committee of National Institute of Genetics. Organotypic culture of telencephalon stripsEach telencephalic hemisphere of the E10.5 mouse embryo was dissected along the dorsoventral axis into a strip ~1.5 mm wide, including the neocortex, the presumptive LOT area and the lateral and medial ganglionic eminences (GE) (see Fig. 1A). In the strip, the presumptive LOT area was located around the middle along the dorsoventral axis. In a standard culture, the strip was labeled by injection of dextran tetramethylrhodamine In the developing nervous system, functional neural networks are constructed with intricate coordination of neuronal migrations and axonal projections. We have previously reported a ventral tangential migration of a special type of cortical neurons, lot cells, in the mouse embryo. These neurons originate from the ventricular zone of the entire neocortex, tangentially migrate in the surface layer of the neocortex into the ventral direction, align in the future pathway of the lateral olfactory tract (LOT) and eventually guide the projection of LOT axons. In this study, we developed an organotypic culture system to investigate the regulation of this cell migration in the developing telencephalon. Our data show that the neocortex contains the signals that direct lot cells ventrally, that the ganglionic eminence excludes lot cells by repelling the migration and that lot cells are attracted to netrin 1, an axon guidance factor. Furthermore, we demonstrate that mutations in the genes encoding netrin 1 and its functional receptor Dcc lead to inappropriate distribution of lot cells and subsequent partial disruption of LOT projection. These results suggest that netrin 1 regulates the migration of lot cells and LOT projections, possibly by ensuring the correct distribution of these guidepost neurons. Molecular Probes, Eugene, OR) or 1,1-dioctadecyl-3,3,3Ј3Ј-tetrametylindocarbacyanine perchlorate (DiI; Molecular Probes) into a small area of the dorsal neocortex, unfolded on a collagen-coated membrane filter (Transwell-COL inserts #3492, Corning, Acton, MA) and cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 (Sigma-Aldrich, St Louis, MO) containing 10% fetal bovine serum (Cansera, Rexdale, ONT, Canada) and 5% hor...