During their migration, cerebellar granule cells switch from a tangential to a radial mode of migration. We have previously demonstrated that this involves the transmembrane semaphorin Sema6A. We show here that plexin-A2 is the receptor that controls Sema6A function in migrating granule cells. In plexin-A2-deficient (Plxna2(-/-)) mice, which were generated by homologous recombination, many granule cells remained in the molecular layer, as we saw in Sema6a mutants. A similar phenotype was observed in mutant mice that were generated by mutagenesis with N-ethyl-N-nitrosourea and had a single amino-acid substitution in the semaphorin domain of plexin-A2. We found that this mutation abolished the ability of Sema6A to bind to plexin-A2. Mouse chimera studies further suggested that plexin-A2 acts in a cell-autonomous manner. We also provide genetic evidence for a ligand-receptor relationship between Sema6A and plexin-A2 in this system. Using time-lapse video microscopy, we found that centrosome-nucleus coupling and coordinated motility were strongly perturbed in Sema6a(-/-) and Plxna2(-/-) granule cells. This suggests that semaphorin-plexin signaling modulates cell migration by controlling centrosome positioning.

Slits, semaphorins and netrins are three families of proteins that can attract or repel growing axons and migrating neurons in the developing nervous system of vertebrates and invertebrates. Recent studies have shown that they are widely expressed outside the nervous system and that they may play important roles in cancers. Several of the genes encoding these proteins are localized on chromosomal region associated with frequent loss-of-heterozygosity in tumors and cancer cell lines and there is also significant hypermethylation of their promoter suggesting that they may act as tumor suppressors. In addition, proteins in all these families and their receptors appear to control the vascularization of the tumors. Last, many axon guidance molecules also regulate cell migration and apoptosis in normal and tumorigenic tissues. Overall, this suggests that molecules that could mimick or block the activity of axon guidance molecules may be used as therapeutic agents for the treatment of malignancy.

The transmembrane semaphorin protein Sema6A is broadly expressed in the developing nervous system. Sema6A repels several classes of developing axons in vitro and contributes to thalamocortical axon guidance in vivo. Here we show that during cerebellum development, Sema6A is selectively expressed by postmitotic granule cells during their tangential migration in the deep external granule cell layer, but not during their radial migration. In Sema6A-deficient mice, many granule cells remain ectopic in the molecular layer where they differentiate and are contacted by mossy fibers. The analysis of ectopic granule cell morphology in Sema6a-/- mice, and of granule cell migration and neurite outgrowth in cerebellar explants, suggests that Sema6A controls the initiation of granule cell radial migration, probably through a modulation of nuclear and/or soma translocation. Finally, the analysis of mouse chimeras suggests that this function of Sema6A is primarily non-cell-autonomous.

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Cerebellar granule cell progenitors proliferate postnatally in the upper part of the external granule cell layer (EGL) of the cerebellum. Postmitotic granule cells differentiate and migrate, tangentially in the EGL and then radially through the molecular and Purkinje cell layers. The molecular control of the transition between proliferation and differentiation in cerebellar granule cells is poorly understood. We show here that the transmembrane receptor Plexin-B2 is expressed by proliferating granule cell progenitors. To study Plexin-B2 function, we generated a targeted mutation of mouse Plexin-B2. Most Plexin-B2 Ϫ/Ϫ mutants die at birth as a result of neural tube closure defects. Some mutants survive but their cerebellum cytoarchitecture is profoundly altered. This is correlated with a disorganization of the timing of granule cell proliferation and differentiation in the EGL. Many differentiated granule cells migrate inside the cerebellum and keep proliferating. These results reveal that Plexin-B2 controls the balance between proliferation and differentiation in granule cells.

Mutations in doublecortin (DCX) are associated with intractable epilepsy in humans, due to a severe disorganization of the neocortex and hippocampus known as classical lissencephaly. However, the basis of the epilepsy in lissencephaly remains unclear. To address potential functional redundancy with murin Dcx, we targeted one of the closest homologues, doublecortin-like kinase 2 (Dclk2). Here, we report that Dcx; Dclk2-null mice display frequent spontaneous seizures that originate in the hippocampus, with most animals dying in the first few months of life. Elevated hippocampal expression of c-fos and loss of somatostatin-positive interneurons were identified, both known to correlate with epilepsy. Dcx and Dclk2 are coexpressed in developing hippocampus, and, in their absence, there is dosagedependent disrupted hippocampal lamination associated with a cellautonomous simplification of pyramidal dendritic arborizations leading to reduced inhibitory synaptic tone. These data suggest that hippocampal dysmaturation and insufficient receptive field for inhibitory input may underlie the epilepsy in lissencephaly, and suggest potential therapeutic strategies for controlling epilepsy in these patients.epilepsy ͉ receptive field ͉ pyramidal neuron ͉ dendrites ͉ delamination A pproximately 1 in 10 people will have a seizure sometime during their life, and the prevalence of epilepsy in the general population is Ϸ3%. Cortical dysplasia, which includes defects in both neocortex and hippocampus, can result from disordered neuronal cell proliferation, migration, or differentiation, and is identified in Ͼ25% of children with intractable epilepsy (1), the type of epilepsy associated with the highest morbidity and mortality.Classical lissencephaly (defined as smooth brain, with simplified or absent gyri and sulci) is due to alterations in neuronal migration and differentiation. The frequency of epilepsy in lissencephaly is probably 100%, and is typically associated with lethality in the first or second decade of life. One of the major causative genes in humans is doublecortin (DCX), which encodes a microtubule binding and stabilizing protein (2).DCX has 2 close homologues in mice, doublecortin-like kinase 1 (Dclk1, also known as Dcamkl1) and doublecortin-like kinase 2 (Dclk2, also known as Dck2), both with broad nervous system expression, to include both mitotic neuroblasts and adult neurons, whereas Dcx is mostly expressed in postmitotic immature neurons, and also transiently in adult neuroblasts (3). Previous analysis of Dcx; Dclk1 double knockouts demonstrated functional redundancy during cortical and hippocampal lamination (4, 5). To further uncover functional redundancy in the DCX gene family, we targeted Dclk2, a gene encoding a highly conserved N-terminal doublecortin domain and a C-terminal kinase domain-containing protein (6). Here, we report that mice deficient in both Dcx and Dclk2 display frequent and spontaneous epileptic seizures, bearing some resemblance to the phenotype observed in humans. Results Dclk2 Knockout Leav...

SUMMARY Acute gene inactivation using short hairpin RNA (shRNA, knockdown) in developing brain is a powerful technique to study genetic function, however, discrepancies between knockdown and knockout murine phenotypes have left unanswered questions. For example, doublecortin (Dcx) knockdown but not knockout shows a neocortical neuronal migration phenotype. Here we report that in utero electroporation of shRNA, but not siRNA or miRNA to Dcx demonstrates a migration phenotype in Dcx knockouts akin to the effect in wildtype mice, suggesting shRNA-mediated off-target toxicity. This effect was not limited to Dcx, as it was observed in Dclk1 knockouts, as well as with a fraction of scrambled shRNAs, suggesting a sequence-dependent but not sequence-specific effect. Profiling RNAs from electroporated cells showed a defect in endogenous let7 miRNA levels, and disruption of let7 or Dicer recapitulated the migration defect. The results suggest that shRNA-mediated knockdown can produce untoward migration effects by altering endogenous miRNA pathways.

Plexin-domain containing 2 (Plxdc2) is a relatively uncharacterised transmembrane protein with an area of nidogen homology and a plexin repeat (PSI domain) in its extracellular region. Here, we describe Plxdc2 expression in the embryonic mouse, with particular emphasis on the developing central nervous system. Using light microscopy and optical projection tomography (OPT), we analyse RNA in situ hybridization patterns and expression of two reporter genes, -geo (a fusion of -galactosidase to neomycin phosphotransferase) and placental alkaline phosphatase (PLAP) in a Plxdc2 gene trap mouse line (KST37; [Leighton, P.A., Mitchell, K.J., Goodrich, L.V., Lu, X., Pinson, K., Scherz, P., Skarnes, W.C., Tessier-Lavigne, M., 2001. DeWning brain wiring patterns and mechanisms through gene trapping in mice. Nature 410, 174-179]). At mid-embryonic stages (E9.5-E11.5) Plxdc2-geo expression is prominent in a number of patterning centres of the brain, including the cortical hem, midbrain-hindbrain boundary and the midbrain Xoorplate. Plxdc2 is expressed in other tissues, most notably the limbs, lung buds and developing heart, as well as the spinal cord and dorsal root ganglia. At E15.5, expression is apparent in a large number of discrete nuclei and structures throughout the brain, including the glial wedge and derivatives of the cortical hem. Plxdc2-geo expression is particularly strong in the developing Purkinje cell layer, especially in the posterior half of the cerebellum. The PLAP marker is expressed in a number of axonal tracts, including the posterior commissure, mammillotegmental tract and cerebellar peduncle. We compare Plxdc2-geo expression in the embryonic brain with the much more restricted expression of the related gene Plxdc1 and with members of the Wnt family (Wnt3a, Wnt5a and Wnt8b) that show a striking overlap with Plxdc2 expression in certain areas.