Cntnap4 differentially contributes to GABAergic and dopaminergic synaptic transmission.

Although considerable evidence suggests that the chemical synapse is a lynchpin underlying affective disorders, how molecular insults differentially affect specific synaptic connections remains poorly understood. For instance, Neurexin 1a and 2 (NRXN1 and NRXN2) and CNTNAP2 (also known as CASPR2), all members of the neurexin superfamily of transmembrane molecules, have been implicated in neuropsychiatric disorders. However, their loss leads to deficits that have been best characterized with regard to their effect on excitatory cells. Notably, other disease-associated genes such as BDNF and ERBB4 implicate specific interneuron synapses in psychiatric disorders. Consistent with this, cortical interneuron dysfunction has been linked to epilepsy, schizophrenia and autism. Using a microarray screen that focused upon synapse-associated molecules, we identified Cntnap4 (contactin associated protein-like 4, also known as Caspr4) as highly enriched in developing murine interneurons. In this study we show that Cntnap4 is localized presynaptically and its loss leads to a reduction in the output of cortical parvalbumin (PV)-positive GABAergic (γ-aminobutyric acid producing) basket cells. Paradoxically, the loss of Cntnap4 augments midbrain dopaminergic release in the nucleus accumbens. In Cntnap4 mutant mice, synaptic defects in these disease-relevant neuronal populations are mirrored by sensory-motor gating and grooming endophenotypes; these symptoms could be pharmacologically reversed, providing promise for therapeutic intervention in psychiatric disorders.

Genes expressed in Atoh1 neuronal lineages arising from the r1/isthmus rhombic lip.

During embryogenesis, the rhombic lip of the fourth ventricle is the germinal origin of a diverse collection of neuronal populations that ultimately reside in the brainstem and cerebellum. Rhombic lip neurogenesis requires the bHLH transcription factor Atoh1 (Math1), and commences shortly after neural tube closure (E9.5). Within the rhombomere 1-isthmus region, the rhombic lip first produces brainstem and deep cerebellar neurons (E9.5-E12), followed by granule cell precursors after E12. While Atoh1 function is essential for all of these populations to be specified, the downstream genetic programs that confer specific properties to early and late born Atoh1 lineages are not well characterized. We have performed a comparative microarray analysis of gene expression within early and later born cohorts of Atoh1 expressing neural precursors purified from E14.5 embryos using a transgenic labeling strategy. We identify novel transcription factors, cell surface molecules, and cell cycle regulators within each pool of Atoh1 lineages that likely contribute to their distinct developmental trajectories and cell fates. In particular, our analysis reveals new insights into the genetic programs that regulate the specification and proliferation of granule cell precursors, the putative cell of origin for the majority of medulloblastomas.

Telencephalic cells take a tangent: non-radial migration in the mammalian forebrain.

During development of the mammalian telencephalon, cells migrate via diverse pathways to reach their final destinations. In the developing neocortex, projection neurons are generated from cells that migrate radially from the underlying ventricular zone. In contrast, subsets of cells that populate the ventral piriform cortex and olfactory bulb reach these sites by migrating long distances. Additionally, it has been recently established that cells migrate tangentially from the ventral ganglionic eminences to the developing cortex. These tangentially migrating cells are a significant source of cortical interneurons and possibly other cell types such as oligodendrocytes. Here we summarize the known routes of migration in the developing telencephalon, with a particular focus on tangential migration. We also review recent genetic and transplantation studies that have given greater insight into the understanding of these processes and the molecular cues that may guide these migrating cells.

In utero fate mapping reveals distinct migratory pathways and fates of neurons born in the mammalian basal forebrain.

Recent studies suggest that neurons born in the developing basal forebrain migrate long distances perpendicularly to radial glia and that many of these cells reach the developing neocortex. This form of tangential migration, however, has not been demonstrated in vivo, and the sites of origin, pathways of migration and final destinations of these neurons in the postnatal brain are not fully understood. Using ultrasound-guided transplantation in utero, we have mapped the migratory pathways and fates of cells born in the lateral and medial ganglionic eminences (LGE and MGE) in 13.5-day-old mouse embryos. We demonstrate that LGE and MGE cells migrate along different routes to populate distinct regions in the developing brain. We show that LGE cells migrate ventrally and anteriorly, and give rise to the projecting medium spiny neurons in the striatum, nucleus accumbens and olfactory tubercle, and to granule and periglomerular cells in the olfactory bulb. By contrast, we show that the MGE is a major source of neurons migrating dorsally and invading the developing neocortex. MGE cells migrate into the neocortex via the neocortical subventricular zone and differentiate into the transient subpial granule neurons in the marginal zone and into a stable population of GABA-, parvalbumin- or somatostatin-expressing interneurons throughout the cortical plate.

N-terminal fatty-acylation of sonic hedgehog enhances the induction of rodent ventral forebrain neurons.

The adult basal ganglia arise from the medial and lateral ganglionic eminences, morphologically distinct structures found in the embryonic telencephalon. We have previously shown that temporal changes in sonic hedgehog (Shh) responsiveness determine the sequential induction of embryonic neurons that populate the medial and lateral ganglionic eminences. In this report, we show that Shh-mediated differentiation of neurons that populate the lateral ganglionic eminence express different combinations of the homeobox-containing transcription factors Dlx, Mash1 and Islet 1/2. Furthermore, we show that N-terminal fatty-acylation of Shh significantly enhances its ability to induce the differentiation of rat E11 telencephalic neurons expressing Dlx, Islet 1/2 or Mash1. Recent evidence indicates that in utero injection of the E9.5 mouse forebrain with retroviruses encoding wild-type Shh induces the ectopic expression of Dlx2 and severe deformities in the brain. In this report, we show that Shh containing a mutation at the site of acylation prevents either of these phenotypes. These results suggest that N-terminal fatty-acylation of Shh may play an important role in Shh-dependent signaling during rodent ventral forebrain formation.

Telencephalic neural progenitors appear to be restricted to regional and glial fates before the onset of neurogenesis.

The contribution of early cell lineage to regional fate in the mammalian forebrain remains poorly understood. Previous lineage-tracing studies using retroviral methods were only begun at mid-neurogenesis and have suffered from region-specific retroviral silencing. We have been able to study cell lineage in the telencephalon from the onset of neurogenesis by using ultrasound backscatter microscopy to label the forebrain neuroepithelium and a modified retroviral lineage library to overcome regional silencing. Our studies suggest that by embryonic day 9.5, forebrain clones are primarily restricted to territories within anatomically demarcated regional boundaries, such as the cortex, striatum and hypothalamus. In addition, we observed a subset of clones that appeared to be composed entirely of glia. These observations suggest that both regional and cell-type restrictions exist within progenitor populations before the first forebrain cells become postmitotic.

Calcium-dependent adhesion is necessary for the maintenance of prosomeres.

Cell adhesion has been suggested to function in the establishment and maintenance of the segmental organization of the central nervous system. Here we tested the role of different classes of adhesion molecules in prosencephalic segmentation. Specifically, we examined the ability of progenitors from different prosomeres to reintegrate and differentiate within various brain regions after selective maintenance or removal of different classes of calcium-dependent versus -independent surface molecules. This analysis implicates calcium-dependent adhesion molecules as central to the maintenance of prosomeres. Only conditions that spared calcium-dependent adhesion systems but ablated more general (calcium-independent) adhesion systems resulted in prosomere-specific integration after transplantation. Among the members of this class of adhesion molecules, R-cadherin shows a striking pattern of prosomeric expression during development. To test whether expression of this molecule was sufficient to direct progenitor integration to prosomeres expressing R-cadherin, we used a retroviral-mediated gain-of-function approach. We found that progenitors originally isolated from prosomere P2 (a region which does not express R-cadherin), when forced to express this molecule, can now integrate more readily into R-cadherin-expressing regions, such as the cortex, the ventral thalamus, and the hypothalamus. Nonetheless, our analysis suggests that while calcium-dependent molecules are able to direct prosomere-specific integration, they are not sufficient to induce progenitors to change their regional identity. While diencephalic progenitors from R-cadherin-expressing regions of prosomere 5 could integrate into R-cadherin-expressing regions of the cortex, they did not express the cortex-specific gene Emx1 or the telencephalic-specific gene Bf-1. Furthermore, diencephalic progenitors that integrate heterotopically into the cortex do not persist postnatally, whereas the same progenitors survive and differentiate when they integrate homotopically into the diencephalon. Together our results implicate calcium-dependent adhesion molecules as key mediators of prosomeric organization but suggest that they are not sufficient to bestow regional identities.

An acylatable residue of Hedgehog is differentially required in Drosophila and mouse limb development.

The Drosophila Hedgehog protein and its vertebrate counterpart Sonic hedgehog are required for a wide variety of patterning events throughout development. Hedgehog proteins are secreted from cells and undergo autocatalytic cleavage and cholesterol modification to produce a mature signaling domain. This domain of Sonic hedgehog has recently been shown to acquire an N-terminal acyl group in cell culture. We have investigated the in vivo role that such acylation might play in appendage patterning in mouse and Drosophila; in both species Hedgehog proteins define a posterior domain of the limb or wing. A mutant form of Sonic hedgehog that cannot undergo acylation retains significant ability to repattern the mouse limb. However, the corresponding mutation in Drosophila Hedgehog renders it inactive in vivo, although it is normally processed. Furthermore, overexpression of the mutant form has dominant negative effects on Hedgehog signaling. These data suggest that the importance of the N-terminal cysteine of mature Hedgehog in patterning appendages differs between species.

Radial glial cell line C6-R integrates preferentially in adult white matter and facilitates migration of coimplanted neurons in vivo.

C6-R is a cell line derived from C6 glioma cells that exhibits key properties of radial glia including the ability to support neuronal migration in culture. To explore its potential use in promoting neuronal migration in vivo, we analyzed the behavior of C6-R cells in the intact and injured adult rat CNS. At 6-11 days postimplantation at the splenium of the corpus callosum, green fluorescent protein-labeled C6-R cells were observed primarily in either the corpus callosum or the hippocampus in the brain, and in the spinal cord they migrated more extensively in the white matter than in the grey matter. To determine whether C6-R cells retain their ability to promote neuronal migration in vivo, they were coinjected with labeled neurons into adult brain. When rat embryonic neurons were coimplanted with C6-R cells, the neurons and C6-R cells comigrated through a much larger volume than neurons alone or neurons coimplanted with fibroblasts. In brains preinjured with ibotenic acid, C6-R cells as well as coimplanted neurons distributed widely within the lesion site and migrated into adjacent brain tissue, while transplants with neurons alone were restricted primarily to the lesion site. The results suggest that radial glial cell lines can serve as a scaffold for neuronal migration that may facilitate development of experimental models for neural transplantation and regeneration.

Sonic hedgehog contributes to oligodendrocyte specification in the mammalian forebrain.

This study addresses the role of Sonic hedgehog (Shh) in promoting the generation of oligodendrocytes in the mouse telencephalon. We show that in the forebrain, expression of the early oligodendrocyte markers Olig2, plp/dm20 and PDGFR(alpha) corresponds to regions of Shh expression. To directly test if Shh can induce the development of oligodendrocytes within the telencephalon, we use retroviral vectors to ectopically express Shh within the mouse embryonic telencephalon. We find that infections with Shh-expressing retrovirus at embryonic day 9.5, result in ectopic Olig2 and PDGFR(alpha) expression by mid-embryogenesis. By postnatal day 21, cells expressing ectopic Shh overwhelmingly adopt an oligodendrocyte identity. To determine if the loss of telencephalic Shh correspondingly results in the loss of oligodendrocyte production, we studied Nkx2.1 mutant mice in which telencephalic expression of Shh is selectively lost. In accordance with Shh playing a role in oligodendrogenesis, within the medial ganglionic eminence of Nkx2.1 mutants, the early expression of PDGFR(alpha) is absent and the level of Olig2 expression is diminished in this region. In addition, in these same mutants, expression of both Shh and plp/dm20 is lost in the hypothalamus. Notably, in the prospective amygdala region where Shh expression persists in the Nkx2.1 mutant, the presence of plp/dm20 is unperturbed. Further supporting the idea that Shh is required for the in vivo establishment of early oligodendrocyte populations, expression of PDGFR(alpha) can be partially rescued by virally mediated expression of Shh in the Nkx2.1 mutant telencephalon. Interestingly, despite the apparent requirement for Shh for oligodendrocyte specification in vivo, all regions of either wild-type or Nkx2.1 mutant telencephalon are competent to produce oligodendrocytes in vitro. Furthermore, analysis of CNS tissue from Shh null animals definitively shows that, in vitro, Shh is not required for the generation of oligodendrocytes. We propose that oligodendrocyte specification is negatively regulated in vivo and that Shh generates oligodendrocytes by overcoming this inhibition. Furthermore, it appears that a Shh-independent pathway for generating oligodendrocytes exists.

Spatiotemporal selectivity of response to Notch1 signals in mammalian forebrain precursors.

The olfactory bulb, neocortex and archicortex arise from a common pool of progenitors in the dorsal telencephalon. We studied the consequences of supplying excess Notch1 signal in vivo on the cellular and regional destinies of telencephalic precursors using bicistronic replication defective retroviruses. After ventricular injections mid-neurogenesis (E14.5), activated Notch1 retrovirus markedly inhibited the generation of neurons from telencephalic precursors, delayed the emergence of cells from the subventricular zone (SVZ), and produced an augmentation of glial progeny in the neo- and archicortex. However, activated Notch1 had a distinct effect on the progenitors of the olfactory bulb, markedly reducing the numbers of cells of any type that migrated there. To elucidate the mechanism of the cell fate changes elicited by Notch1 signals in the cortical regions, short- and long-term cultures of E14.5 telencephalic progenitors were examined. These studies reveal that activated Notch1 elicits a cessation of proliferation that coincides with an inhibition of the generation of neurons. Later, during gliogenesis, activated Notch1 triggers a rapid cellular proliferation with a significant increase in the generation of cells expressing GFAP. To examine the generation of cells destined for the olfactory bulb, we used stereotaxic injections into the early postnatal anterior subventricular zone (SVZa). We observed that precursors of the olfactory bulb responded to Notch signals by remaining quiescent and failing to give rise to differentiated progeny of any type, unlike cortical precursor cells, which generated glia instead of neurons. These data show that forebrain precursors vary in their response to Notch signals according to spatial and temporal cues, and that Notch signals influence the composition of forebrain regions by modulating the rate of proliferation of neural precursor cells.

The Gsh2 homeodomain gene controls multiple aspects of telencephalic development.

Homeobox genes have recently been demonstrated to be important for the proper patterning of the mammalian telencephalon. One of these genes is Gsh2, whose expression in the forebrain is restricted to the ventral domain. In this study, we demonstrate that Gsh2 is a downstream target of sonic hedgehog and that lack of Gsh2 results in profound defects in telencephalic development. Gsh2 mutants have a significant decrease in the expression of numerous genes that mark early development of the lateral ganglionic eminence, the striatal anlage. Accompanying this early loss of patterning genes is an initial expansion of dorsal telencephalic markers across the cortical-striatal boundary into the lateral ganglionic eminence. Interestingly, as development proceeds, there is compensation for this early loss of markers that is coincident with a molecular re-establishment of the cortical-striatal boundary. Despite this compensation, there is a defect in the development of distinct subpopulations of striatal neurons. Moreover, while our analysis suggests that the migration of the ventrally derived interneurons to the developing cerebral cortex is not significantly affected in Gsh2 mutants, there is a distinct delay in the appearance of GABAergic interneurons in the olfactory bulb. Taken together, our data support a model in which Gsh2, in response to sonic hedgehog signaling, plays a crucial role in multiple aspects of telencephalic development.

Radial glial identity is promoted by Notch1 signaling in the murine forebrain.

In vertebrates, Notch signaling is generally thought to inhibit neural differentiation. However, whether Notch can also promote specific early cell fates in this context is unknown. We introduced activated Notch1 (NIC) into the mouse forebrain, before the onset of neurogenesis, using a retroviral vector and ultrasound imaging. During embryogenesis, NIC-infected cells became radial glia, the first specialized cell type evident in the forebrain. Thus, rather than simply inhibiting differentiation, Notch1 signaling promoted the acquisition of an early cellular phenotype. Postnatally, many NIC-infected cells became periventricular astrocytes, cells previously shown to be neural stem cells in the adult. These results suggest that Notch1 promotes radial glial identity during embryogenesis, and that radial glia may be lineally related to stem cells in the adult nervous system.

A method for rapid gain-of-function studies in the mouse embryonic nervous system.

We used ultrasound image-guided injections of high-titer retroviral vectors to obtain widespread introduction of genes into the mouse nervous system in utero as early as embryonic day 8.5 (E8.5). The vectors used included internal promoters that substantially improved proviral gene expression in the ventricular zone of the brain. To demonstrate the utility of this system, we extended our previous work in vitro by infecting the telencephalon in vivo as early as E8. 5 with a virus expressing Sonic Hedgehog. Infected embryos showed gross morphological brain defects, as well as ectopic expression of ventral telencephalic markers characteristic of either the medial or lateral ganglionic eminences.

Generation of a radial-like glial cell line.

Rat C6 glioma is a cell line that has been used extensively as a model of astroglia. Although this cell line retains many of the properties of developing glia, it does not resemble morphologically the specialized form of glia found embryonically, the radial glia. In experiments designed to study a mutant form of receptor protein tyrosine phosphatase beta, we isolated a subclone of C6 called C6-R which, like radial glia, assumes a highly polarized radial-like morphology in culture. C6-R cells and, to a somewhat lesser extent, C6 cells, express cytoskeletal proteins found in developing astroglia including glial fibrillary acidic protein and RC1. As seen with radial glia, cerebellar granule cell bodies and neurites migrated along radial processes of C6-R cells in culture. Morphological analysis of dye-labeled cells injected into the developing forebrain revealed that a large fraction (approximately 60%) of the C6-R cells in the cortex assumed a radial orientation and about half of these (approximately 30%) made contact with the pial surface. In contrast, the parental C6 cells generally formed aggregates and only displayed a radial alignment when associated with blood vessels. These results suggest that we have generated a stable cell line from C6 glioma which has adopted certain key features of radial glia, including the ability to promote neuronal migration in culture and integrate radially in vivo in response to local cues. This cell line may be particularly useful for studying receptors on radial glia that mediate neuronal migration.

Regionalization within the mammalian telencephalon is mediated by changes in responsiveness to Sonic Hedgehog.

The cortex and basal ganglia are the major structures of the adult brain derived from the embryonic telencephalon. Two morphologically distinct regions of the basal ganglia are evident within the mature ventral telencephalon, the globus pallidus medially, and the striatum, which is positioned between the globus pallidus and the cortex. Deletion of the Sonic Hedgehog gene in mice indicates that this secreted signaling molecule is vital for the generation of both these ventral telencephalic regions. Previous experiments showed that Sonic Hedgehog induces differentiation of ventral neurons characteristic of the medial ganglionic eminence, the embryonic structure which gives rise to the globus pallidus. In this paper, we show that later in development, Sonic Hedgehog induces ventral neurons with patterns of gene expression characteristic of the lateral ganglionic eminence. This is the embryonic structure from which the striatum is derived. These results suggest that temporally regulated changes in Sonic Hedgehog responsiveness are integral in the sequential induction of basal telencephalic structures.

Telencephalic progenitors maintain anteroposterior identities cell autonomously.

Grafting experiments have demonstrated that determination of anteroposterior (AP) identity is an early step in neural patterning that precedes dorsoventral (DV) specification [1,2]. These studies used pieces of tissue, however, rather than individual cells to address this question. It thus remains unclear whether the maintenance of AP identity is a cell-autonomous property or a result of signaling between cells within the grafted tissue. Previously, we and others [3-5] have used transplants of dissociated brain cells to show that individual telencephalic precursor cells can adopt host-specific DV identities when they integrate within novel regions of the telencephalon. We have now undertaken a set of transplantations during the same mid-neurogenic period used in the previous studies to assess the ability of telencephalic progenitors to integrate and differentiate into more posterior regions of the neuraxis. We observed that telencephalic progenitors were capable of integrating and migrating within different AP levels of the central nervous system (CNS). Despite this, we found that telencephalic progenitors that integrated within the diencephalon and the mesencephalon continued to express a telencephalic marker until adulthood. We speculate that during neurogenesis individual progenitors are determined in terms of their AP but not their DV identity. Hence, AP identity is maintained cell autonomously within individual progenitors.

Transplantation as a tool to study progenitors within the vertebrate nervous system.

In recent years, many studies have focused on the fate and potential of neural progenitors in vertebrates. While much progress has been made, many questions remain about the mechanisms which lead to neural diversity, in terms of both the regionalization of the nervous system and specification of cell fates within those regions. Studies aimed at addressing these questions have fallen into three main categories: in vivo lineage tracings, in vitro differentiation analyses, and in vivo cell transplantation studies. This body of work has pointed to the existence of both pluripotent and unipotent neural progenitors, and has suggested that both cell intrinsic and extrinsic cues play a role in the determination of neural cell fate. In addition, the existence of neural "stem cells" maintained into adulthood has been suggested. This review will focus on transplantation studies in mammals, and will emphasize how this method has been useful as a means of determining the changing potential of neural precursors and their environments within the developing nervous system.

Cooperation of intrinsic and extrinsic signals in the elaboration of regional identity in the posterior cerebral cortex.

Understanding the compartmentalization of the neocortex (isocortex) of the mammalian brain into functional areas is a challenging problem [1-3] . Unlike pattern formation in the spinal cord and hindbrain, it does not involve the specification of distinct cells types: distinct areas differ in their patterns of connectivity and cytoarchitecture. It has been suggested that signals intrinsic to the neocortical neuroepithelium specify regional fate [3]. Alternatively, spatial patterning might be imposed by extrinsic cues such as thalamocortical projections [4-6]. Recent results highlight the ability of early precursor cells of the telencephalic neuroepithelium to 'remember' their spatial position from times before thalamic innervation [7,8] [9-12]. An influence from the thalamus, however, cannot be ruled out as there is a precise invasion of the correct cortical areas by the corresponding projections [13,14]. Furthermore, cortical neuronal progenitors have been proposed to adopt new connection patterns after transplantation [6,7], as well as when the thalamic input is rerouted [15,16]. Here, we describe the transient expression of the homeobox gene Otx2 in the posterior, prospective visual, neocortex and use it to analyze the establishment of posterior cortical fate. The results suggest that whereas intrinsic cortical information is sufficient to specify regional fate, extrinsic signals from the thalamus are involved in the expansion or maintenance of the population of cells expressing Otx2 but not in regionalization.