The migration of cerebellar rhombic lip derivatives

We have used cell labelling, co-culture and time-lapse confocal microscopy to investigate tangential neuronal migration from the rhombic lip. Cerebellar rhombic lip derivatives demonstrate a temporal organisation with respect to their morphology and response to migration cues. Early born cells, which migrate into ventral rhombomere 1, have a single long leading process that turns at the midline and becomes an axon. Later born granule cell precursors also migrate ventrally but halt at the lateral edge of the cerebellum, correlating with a loss of sensitivity to netrin 1 and expression of Robo2. The rhombic lip and ventral midline express Slit2 and both early and late migrants are repelled by sources of Slit2 in co-culture. These studies reveal an intimate relationship between birthdate, response to migration cues and neuronal fate in an identified population of migratory cells. The use of axons in navigating cell movement suggests that tangential migration is an elaboration of the normal process of axon extension.     

Cellular and molecular guidance of GABAergic neuronal migration from an extracortical origin to the neocortex.

Formation of the normal mammalian cerebral cortex requires the migration of GABAergic inhibitory interneurons from an extracortical origin, the lateral ganglionic eminence (LGE). Mechanisms guiding the migratory direction of these neurons, or other neurons in the neocortex, are not well understood. We have used an explant assay to study GABAergic neuronal migration and found that the ventricular zone (VZ) of the LGE is repulsive to GABAergic neurons. Furthermore, the secreted protein Slit is a chemorepellent guiding the migratory direction of GABAergic neurons, and blockade of endogenous Slit signaling inhibits the repulsive activity in the VZ. These results have revealed a cellular source of guidance for GABAergic neurons, demonstrated a molecular cue important for cortical development, and suggested a guidance mechanism for the migration of extracortical neurons into the neocortex.     

Hes genes and neurogenin regulate non-neural versus neural fate specification in the dorsal telencephalic midline

The choroid plexus in the brain is unique because it is a non-neural secretory tissue. It secretes the cerebrospinal fluid and functions as a blood-brain barrier, but the precise mechanism of specification of this non-neural tissue has not yet been determined. Using mouse embryos and lineage-tracing analysis, we found that the prospective choroid plexus region initially gives rise to Cajal-Retzius cells, specialized neurons that guide neuronal migration. Inactivation of the bHLH repressor genes Hes1, Hes3 and Hes5 upregulated expression of the proneural gene neurogenin 2 (Ngn2) and prematurely depleted Bmp-expressing progenitor cells, leading to enhanced formation of Cajal-Retzius cells and complete loss of choroid plexus epithelial cells. Overexpression of Ngn2 had similar effects. These data indicate that Hes genes promote specification of the fate of choroid plexus epithelial cells rather than the fate of Cajal-Retzius cells by antagonizing Ngn2 in the dorsal telencephalic midline region, and thus this study has identified a novel role for bHLH genes in the process of deciding which cells will have a non-neural versus a neural fate.     

Dab2ip Regulates Neuronal Migration and Neurite Outgrowth in the Developing Neocortex

Dab2ip (DOC-2/DAB2 interacting protein) is a member of the Ras GTPase-activating protein (GAP) family that has been previously shown to function as a tumor suppressor in several systems. Dab2ip is also highly expressed in the brain where it interacts with Dab1, a key mediator of the Reelin pathway that controls several aspects of brain development and function. We found that Dab2ip is highly expressed in the developing cerebral cortex, but that mutations in the Reelin signaling pathway do not affect its expression. To determine whether Dab2ip plays a role in brain development, we knocked down or over expressed it in neuronal progenitor cells of the embryonic mouse neocortex using in utero electroporation. Dab2ip down-regulation severely disrupts neuronal migration, affecting preferentially late-born principal cortical neurons. Dab2ip overexpression also leads to migration defects. Structure-function experiments in vivo further show that both PH and GRD domains of Dab2ip are important for neuronal migration. A detailed analysis of transfected neurons reveals that Dab2ip down- or up-regulation disrupts the transition from a multipolar to a bipolar neuronal morphology in the intermediate zone. Knock down of Dab2ip in neurons ex-vivo indicates that this protein is necessary for proper neurite development and for the expression of several major neuronal microtubule associated proteins (MAPs), which are important for neurite growth and stabilization. Thus, our study identifies, for the first time, a critical role for Dab2ip in mammalian cortical development and begins to reveal molecular mechanisms that underlie this function.     

Roundabout signalling, cell contact and trophic support confine longitudinal glia and axons in the Drosophila CNS

Contrary to our knowledge of the genetic control of midline crossing, the mechanisms that generate and maintain the longitudinal axon pathways of the Drosophila CNS are largely unknown. The longitudinal pathways are formed by ipsilateral pioneer axons and the longitudinal glia. The longitudinal glia dictate these axonal trajectories and provide trophic support to later projecting follower neurons. Follower interneuron axons cross the midline once and join these pathways to form the longitudinal connectives. Once on the contralateral side, longitudinal axons are repelled from recrossing the midline by the midline repulsive signal Slit and its axonal receptor Roundabout. We show that longitudinal glia also transiently express roundabout, which halts their ventral migration short of the midline. Once in contact with axons, glia cease to express roundabout and become dependent on neurons for their survival. Trophic support and cell-cell contact restrict glial movement and axonal trajectories. The significance of this relationship is revealed when neuron-glia interactions are disrupted by neuronal ablation or mutation in the glial cells missing gene, which eliminates glia, when axons and glia cross the midline despite continued midline repellent signalling.     

Expression of Kv1.3 potassium channels regulates density of cortical interneurons

The Kv1.3 protein is a member of the large family of voltage‐dependent K⁺ subunits (Kv channels), which assemble to form tetrameric membrane‐spanning channels that provide a selective pore for the conductance of K⁺ across the cell membrane. Kv1.3 differs from most other Kv channels in that deletion of Kv1.3 gene produces very striking changes in development and structure of the olfactory bulb, where Kv1.3 is expressed at high levels, resulting in a lower threshold for detection of odors, an increased number of synaptic glomeruli and alterations in the levels of a variety of neuronal signaling molecules. Because Kv1.3 is also expressed in the cerebral cortex, we have now examined the effects of deletion of the Kv1.3 gene on the expression of interneuron populations of the cerebral cortex. Using unbiased stereology we found an increase in the number of parvalbumin (PV) cells in whole cerebral cortex of Kv1.3^?/? mice relative to that in wild‐type mice, and a decrease in the number of calbindin (CB), calretinin (CR), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and somatostatin (SOM) interneurons. These changes are accompanied by a decrease in the cortical volume such that the cell density of PV interneurons is significantly increased and that of SOM neurons is decreased in Kv1.3^?/? animals. Our studies suggest that, as in the olfactory bulb, Kv1.3 plays a unique role in neuronal differentiation and/or survival of interneuron populations and that expression of Kv1.3 is required for normal cortical function. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73:841–855, 2013     

Directional guidance of interneuron migration to the cerebral cortex relies on subcortical Slit1/2-independent repulsion and cortical attraction

Tangential migration from the basal telencephalon to the cortex is a highly directional process, yet the mechanisms involved are poorly understood. Here we show that the basal telencephalon contains a repulsive activity for tangentially migrating cells, whereas the cerebral cortex contains an attractive activity. In vitro experiments demonstrate that the repulsive activity found in the basal telencephalon is maintained in mice deficient in both Slit1 and Slit2, suggesting that factors other than these are responsible for this activity. Correspondingly, in vivo analysis demonstrates that interneurons migrate to the cortex in the absence of Slit1 and Slit2, or even in mice simultaneously lacking Slit1, Slit2 and netrin 1. Nevertheless, loss of Slit2 and, even more so, Slit1 and Slit2 results in defects in the position of other specific neuronal populations within the basal telencephalon, such as the cholinergic neurons of the basal magnocellular complex. These results demonstrate that whereas Slit1 and Slit2 are not necessary for tangential migration of interneurons to the cortex, these proteins regulate neuronal migration within the basal telencephalon by controlling cell positioning close to the midline.     

Dapper Antagonist of Catenin-1 Cooperates with Dishevelled-1 during Postsynaptic Development in Mouse Forebrain GABAergic Interneurons

Synaptogenesis has been extensively studied along with dendritic spine development in glutamatergic pyramidal neurons, however synapse development in cortical interneurons, which are largely aspiny, is comparatively less well understood. Dact1, one of 3 paralogous Dact (Dapper/Frodo) family members in mammals, is a scaffold protein implicated in both the Wnt/β-catenin and the Wnt/Planar Cell Polarity pathways. We show here that Dact1 is expressed in immature cortical interneurons. Although Dact1 is first expressed in interneuron precursors during proliferative and migratory stages, constitutive Dact1 mutant mice have no major defects in numbers or migration of these neurons. However, cultured cortical interneurons derived from these mice have reduced numbers of excitatory synapses on their dendrites. We selectively eliminated Dact1 from mouse cortical interneurons using a conditional knock-out strategy with a Dlx-I12b enhancer-Cre allele, and thereby demonstrate a cell-autonomous role for Dact1 during postsynaptic development. Confirming this cell-autonomous role, we show that synapse numbers in Dact1 deficient cortical interneurons are rescued by virally-mediated re-expression of Dact1 specifically targeted to these cells. Synapse numbers in these neurons are also rescued by similarly targeted expression of the Dact1 binding partner Dishevelled-1, and partially rescued by expression of Disrupted in Schizophrenia-1, a synaptic protein genetically implicated in susceptibility to several major mental illnesses. In sum, our results support a novel cell-autonomous postsynaptic role for Dact1, in cooperation with Dishevelled-1 and possibly Disrupted in Schizophrenia-1, in the formation of synapses on cortical interneuron dendrites.     

Expression and function of Slit1alpha, a novel alternative splicing product for slit1

Slits are large molecular and extracellular glycoproteins that may function as chemorepellents in axon guidance and neural cell migration. The heterogeneity of the mRNA for slit has been described. Its variants indicate considerable potential for alternative splicing, resulting in the generation of multiple protein isoforms. We examined the regions in which these isoforms are expressed, and identified the highest expression of a splicing product for slit1 in rat brain rather than in other organs. The splicing product, Slit1alpha, arises through alternative splicing at the C-terminus of Slit1, causing defects in the cysteine knot domain. We show that slit1alpha exists in the hippocampus and cerebral cortex in rat brain by in situ hybridization, and that it acts as a chemorepellent in olfactory bulb axon guidance in vitro. These findings suggest that Slit1alpha is an active Slit1 protein specific in the vertebrate nervous system.     

Vertebrate slit, a secreted ligand for the transmembrane protein roundabout, is a repellent for olfactory bulb axons

The olfactory bulb plays a central role in olfactory information processing through its connections with both peripheral and cortical structures. Axons projecting from the olfactory bulb to the telencephalon are guided by a repulsive activity in the septum. The molecular nature of the repellent is not known. We report here the isolation of vertebrate homologs of the Drosophila slit gene and show that Slit protein binds to the transmembrane protein Roundabout (Robo). Slit is expressed in the septum whereas Robo is expressed in the olfactory bulb. Functionally, Slit acts as a chemorepellent for olfactory bulb axons. These results establish a ligand-receptor relationship between two molecules important for neural development, suggest a role for Slit in olfactory bulb axon guidance, and reveal the existence of a new family of axon guidance molecules.     

Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons.

Doublecortin (DCX) is required for normal migration of neurons into the cerebral cortex, since mutations in the human gene cause a disruption of cortical neuronal migration. To date, little is known about the distribution of DCX protein or its function. Here, we demonstrate that DCX is expressed in migrating neurons throughout the central and peripheral nervous system during embryonic and postnatal development. DCX protein localization overlaps with microtubules in cultured primary cortical neurons, and this overlapping expression is disrupted by microtubule depolymerization. DCX coassembles with brain microtubules, and recombinant DCX stimulates the polymerization of purified tubulin. Finally, overexpression of DCX in heterologous cells leads to a dramatic microtubule phenotype that is resistant to depolymerization. Therefore, DCX likely directs neuronal migration by regulating the organization and stability of microtubules.     

Localization of type I insulin-like growth factor receptor messenger RNA in the adult rat brain by in situ hybridization.

Using multiple 35S-labeled oligonucleotide probes concurrently, the type I insulin-like growth factor receptor (IGF-I-R) mRNA was demonstrated by Northern blot hybridization in newborn and adult rat brain as a single species of approximately 11 kilobases. The probes were used to localize IGF-I-R mRNA by in situ hybridization in slices of adult rat brain. The highest levels of IGF-I-R mRNA expression were found in the glomerular and mitral cell body layers of the olfactory bulb, the granule cell body layers of the dentate gyrus and cerebellum, the pyramidal cell body layers of the piriform cortex and Ammon's horn, and the choroid plexus. The lowest levels of IGF-I-R mRNA expression were found in white matter. At the cellular level, IGF-I-R mRNA was expressed by a variety of neurons, by epithelial cells of the choroid plexus, and by ependymal cells of the third ventricle. Of the neuron types studied, the highest levels of IGF-I-R mRNA were consistently found in perikarya of mitral and tufted cells in the olfactory bulb, in pyramidal cells of the piriform cortex and Ammon's horn, and in granule cells of the dentate gyrus. There was a close congruency between the distribution of IGF-I binding and IGF-I-R mRNA at the regional level. Neuropil layers in the cerebral cortex, olfactory bulb, hippocampus, and cerebellum contained a high level of IGF-I binding, whereas the adjacent cell body layers contained a high level of the IGF-I-R mRNA. We conclude that in these regions, IGF-I-R mRNA is synthesized in neuronal cell bodies, and the receptors are transported to axons and dendrites in adjacent synapse-rich layers, where appropriate IGF effects are achieved.     

Slit proteins are not dominant chemorepellents for olfactory tract and spinal motor axons.

Members of the Slit family are large extracellular glycoproteins that may function as chemorepellents in axon guidance and neuronal cell migration. Their actions are mediated through members of the Robo family that act as their receptors. In vertebrates, Slit causes chemorepulsion of embryonic olfactory tract, spinal motor, hippocampal and retinal ganglion cell axons. Since Slits are expressed in the septum and floor plate during the period when these tissues cause chemorepulsion of olfactory tract and spinal motor axons respectively, it has been proposed that Slits function as guidance cues. We have tested this hypothesis in collagen gel co-cultures using soluble Robo/Fc chimeras, as competitive inhibitors, to disrupt Slit interactions. We find that the addition of soluble Robo/Fc has no effect on chemorepulsion of olfactory tract and spinal motor axons when co-cultured with septum or floor plate respectively. Thus, we conclude that although Slits are expressed in the septum and floor plate, their proteins do not contribute to the major chemorepulsive activities emanating from these tissues which cause repulsion of olfactory tract and spinal motor axons.     

Prosaposin Overexpression following Kainic Acid-Induced Neurotoxicity

Because excessive glutamate release is believed to play a pivotal role in numerous neuropathological disorders, such as ischemia or seizure, we aimed to investigate whether intrinsic prosaposin (PS), a neuroprotective factor when supplied exogenously in vivo or in vitro, is up-regulated after the excitotoxicity induced by kainic acid (KA), a glutamate analog. In the present study, PS immunoreactivity and its mRNA expression in the hippocampal and cortical neurons showed significant increases on day 3 after KA injection, and high PS levels were maintained even after 3 weeks. The increase in PS, but not saposins, detected by immunoblot analysis suggests that the increase in PS-like immunoreactivity after KA injection was not due to an increase in saposins as lysosomal enzymes after neuronal damage, but rather to an increase in PS as a neurotrophic factor to improve neuronal survival. Furthermore, several neurons with slender nuclei inside/outside of the pyramidal layer showed more intense PS mRNA expression than other pyramidal neurons. Based on the results from double immunostaining using anti-PS and anti-GABA antibodies, these neurons were shown to be GABAergic interneurons in the extra- and intra-pyramidal layers. In the cerebral cortex, several large neurons in the V layer showed very intense PS mRNA expression 3 days after KA injection. The choroid plexus showed intense PS mRNA expression even in the normal rat, and the intensity increased significantly after KA injection. The present study indicates that inhibitory interneurons as well as stimulated hippocampal pyramidal and cortical neurons synthesize PS for neuronal survival, and the choroid plexus is highly activated to synthesize PS, which may prevent neurons from excitotoxic neuronal damage. To the best of our knowledge, this is the first study that demonstrates axonal transport and increased production of neurotrophic factor PS after KA injection.     

Arx homeobox gene is essential for development of mouse olfactory system

The olfactory system provides an excellent model in which to study cell proliferation, migration, differentiation, axon guidance, dendritic morphogenesis, and synapse formation. We report here crucial roles of the Arx homeobox gene in the developing olfactory system by analyzing its mutant phenotypes. Arx protein was expressed strongly in the interneurons and weakly in the radial glia of the olfactory bulb, but in neither the olfactory sensory neurons nor bulbar projection neurons. Arx-deficient mice showed severe anatomical abnormalities in the developing olfactory system: (1) size reduction of the olfactory bulb, (2) reduced proliferation and impaired entry into the olfactory bulb of interneuron progenitors, (3) loss of tyrosine hydroxylase-positive periglomerular cells, (4) disorganization of the layer structure of the olfactory bulb, and (5) abnormal axonal termination of olfactory sensory neurons in an unusual axon-tangled structure, the fibrocellular mass. Thus, Arx is required for not only the proper developmental processes of Arx-expressing interneurons, but also the establishment of functional olfactory neural circuitry by affecting Arx-non-expressing sensory neurons and projection neurons. These findings suggest a likely role of Arx in regulating the expression of putative instructive signals produced in the olfactory bulb for the proper innervation of olfactory sensory axons.     

Subcellular Localization and Differentiation-Induced Redistribution of the Protein Tyrosine Phosphatase PTP-BL in Neuroblastoma Cells

1.In cells of epithelial origin the protein tyrosine phosphatase PTP-BL is predominantly localized at the apical membrane of polarized cells. This large submembranous multidomain PTP is also expressed in cells of neuronal origin. We studied the localization of PTP-BL in mouse neuroblastoma cells utilizing EGFP-tagged versions of the protein. 2. In proliferating Neuro-2a cells, immunofluorescence and immuno-electron microscopy revealed a submembranous FERM domain-dependent localization at cell-cell boundaries for EGFP-PTP-BL. Additionally, significant amounts of EGFP-PTP-BL are located in the cytoplasm as well as in nuclei. Upon serum depletion-induced differentiation of Neuro-2a cells, a partial shift of EGFP-PTP-BL from a cortical localization to cytoskeleton-like F-actin-positive structures is observed. Parallel biochemical studies corroborate this finding and reveal a serum depletion-induced shift of EFGP-PTP-BL from a membrane(-associated) fraction to an NP40-soluble cytoskeletal fraction. 3. Different pools of PTP-BL-containing protein complexes can be discerned in neuronal cells, reflecting distinct molecular microenvironments in which PTP-BL may exert its function.     

Localization of organic anion transporting polypeptide 3 (oatp3) in mouse brain parenchymal and capillary endothelial cells

Organic anion transporting polypeptide 3 (oatp3) transports various CNS-acting endogenous compounds, including thyroid hormones and prostaglandin E2, between extra- and intracellular spaces, suggesting a possible role in CNS function. The purpose of this study was to clarify the expression and localization of oatp3 in the mouse brain. RT-PCR analysis revealed that oatp3 mRNA is expressed in brain capillary-rich fraction, conditionally immortalized brain capillary endothelial cells, choroid plexus, brain and lung, but not in liver or kidney, where oatp1, 2 and 5 mRNAs were detected. Immunohistochemical analysis with anti-oatp3 antibody suggests that oatp3 protein is localized at the brush-border membrane of mouse choroid plexus epithelial cells. Furthermore, intense immunoreactivity was detected in neural cells in the border region between hypothalamus and thalamus, and in the olfactory bulb. Immunoreactivity was also detected in brain capillary endothelial cells in the cerebral cortex. These localizations in the mouse brain suggest that oatp3 plays roles in blood-brain and -cerebrospinal fluid barrier transport of organic anions and signal mediators, and in hormone uptake by neural cells.