Sequential phases of cortical specification involve Neurogenin-dependent and -independent pathways

Neocortical projection neurons, which segregate into six cortical layers according to their birthdate, have diverse morphologies, axonal projections and molecular profiles, yet they share a common cortical regional identity and glutamatergic neurotransmission phenotype. Here we demonstrate that distinct genetic programs operate at different stages of corticogenesis to specify the properties shared by all neocortical neurons. Ngn1 and Ngn2 are required to specify the cortical (regional), glutamatergic (neurotransmitter) and laminar (temporal) characters of early-born (lower-layer) neurons, while simultaneously repressing an alternative subcortical, GABAergic neuronal phenotype. Subsequently, later-born (upper-layer) cortical neurons are specified in an Ngn-independent manner, requiring instead the synergistic activities of Pax6 and Tlx, which also control a binary choice between cortical/glutamatergic and subcortical/GABAergic fates. Our study thus reveals an unanticipated heterogeneity in the genetic mechanisms specifying the identity of neocortical projection neurons.     

In Vitro Differentiation of Mouse Embryonic Stem Cells into Neurons of the Dorsal Forebrain

Pluripotent embryonic stem cells (ESCs) are able to differentiate into all cell types in the organism including cortical neurons. To follow the dynamic generation of progenitors of the dorsal forebrain in vitro, we generated ESCs from D6-GFP mice in which GFP marks neocortical progenitors and neurons after embryonic day (E) 10.5. We used several cell culture protocols for differentiation of ESCs into progenitors and neurons of the dorsal forebrain. In cell culture, GFP-positive cells were induced under differentiation conditions in quickly formed embryoid bodies (qEBs) after 10–12 day incubation. Activation of Wnt signaling during ESC differentiation further stimulated generation of D6-GFP-positive cortical cells. In contrast, differentiation protocols using normal embryoid bodies (nEBs) yielded only a few D6-GFP-positive cells. Gene expression analysis revealed that multiple components of the canonical Wnt signaling pathway were expressed during the development of embryoid bodies. As shown by immunohistochemistry and quantitative qRT-PCR, D6-GFP-positive cells from qEBs expressed genes that are characteristic for the dorsal forebrain such as Pax6, Dach1, Tbr1, Tbr2, or Sox5. qEBs culture allowed the formation of a D6-GFP positive pseudo-polarized neuroepithelium with the characteristic presence of N-cadherin at the apical pole resembling the structure of the developing neocortex.     

The early topography of thalamocortical projections is shifted in Ebf1 and Dlx1/2 mutant mice

The prevailing model to explain the formation of topographic projections in the nervous system stipulates that this process is governed by information located within the projecting and targeted structures. In mammals, different thalamic nuclei establish highly ordered projections with specific neocortical domains and the mechanisms controlling the initial topography of these projections remain to be characterized. To address this issue, we examined Ebf1(-/-) embryos in which a subset of thalamic axons does not reach the neocortex. We show that the projections that do form between thalamic nuclei and neocortical domains have a shifted topography, in the absence of regionalization defects in the thalamus or neocortex. This shift is first detected inside the basal ganglia, a structure on the path of thalamic axons, and which develops abnormally in Ebf1(-/-) embryos. A similar shift in the topography of thalamocortical axons inside the basal ganglia and neocortex was observed in Dlx1/2(-/-) embryos, which also have an abnormal basal ganglia development. Furthermore, Dlx1 and Dlx2 are not expressed in the dorsal thalamus or in cortical projections neurons. Thus, our study shows that: (1) different thalamic nuclei do not establish projections independently of each other; (2) a shift in thalamocortical topography can occur in the absence of major regionalization defects in the dorsal thalamus and neocortex; and (3) the basal ganglia may contain decision points for thalamic axons' pathfinding and topographic organization. These observations suggest that the topography of thalamocortical projections is not strictly determined by cues located within the neocortex and may be regulated by the relative positioning of thalamic axons inside the basal ganglia.     

Interdigitated paralemniscal and lemniscal pathways in the mouse barrel cortex

Primary sensory cortical areas receive information through multiple thalamic channels. In the rodent whisker system, lemniscal and paralemniscal thalamocortical projections, from the ventral posteromedial nucleus (VPM) and posterior medial nucleus (POm) respectively, carry distinct types of sensory information to cortex. Little is known about how these separate streams of activity are parsed and integrated within the neocortical microcircuit. We used quantitative laser scanning photostimulation to probe the organization of functional thalamocortical and ascending intracortical projections in the mouse barrel cortex. To map the thalamocortical projections, we recorded from neocortical excitatory neurons while stimulating VPM or POm. Neurons in layers (L)4, L5, and L6A received dense input from thalamus (L4, L5B, and L6A from VPM; and L5A from POm), whereas L2/3 neurons rarely received thalamic input. We further mapped the lemniscal and paralemniscal circuits from L4 and L5A to L2/3. Lemniscal L4 neurons targeted L3 within a column. Paralemniscal L5A neurons targeted a superficial band (thickness, 60 μm) of neurons immediately below L1, defining a functionally distinct L2 in the mouse barrel cortex. L2 neurons received input from lemniscal L3 cells and paralemniscal L5A cells spread over multiple columns. Our data indicate that lemniscal and paralemniscal information is segregated into interdigitated cortical layers.     

The microcephaly gene Donson is essential for progenitors of cortical glutamatergic and GABAergic neurons

Biallelic mutations in DONSON, an essential gene encoding for a replication fork protection factor, were linked to skeletal abnormalities and microcephaly. To better understand DONSON function in corticogenesis, we characterized Donson expression and consequences of conditional Donson deletion in the mouse telencephalon. Donson was widely expressed in the proliferation and differentiation zones of the embryonic dorsal and ventral telencephalon, which was followed by a postnatal expression decrease. Emx1-Cre-mediated Donson deletion in progenitors of cortical glutamatergic neurons caused extensive apoptosis in the early dorsomedial neuroepithelium, thus preventing formation of the neocortex and hippocampus. At the place of the missing lateral neocortex, these mutants exhibited a dorsal extension of an early-generated paleocortex. Targeting cortical neurons at the intermediate progenitor stage using Tbr2-Cre evoked no apparent malformations, whereas Nkx2.1-Cre-mediated Donson deletion in subpallial progenitors ablated 75% of Nkx2.1-derived cortical GABAergic neurons. Thus, the early telencephalic neuroepithelium depends critically on Donson function. Our findings help explain why the neocortex is most severely affected in individuals with DONSON mutations and suggest that DONSON-dependent microcephaly might be associated with so far unrecognized defects in cortical GABAergic neurons. Targeting Donson using an appropriate recombinase is proposed as a feasible strategy to ablate proliferating and nascent cells in experimental research.     

Multiple roles of ephrins during the formation of thalamocortical projections: maps and more

The functional architecture of the cerebral cortex is based on intrinsic connections that precisely link neurons from distinct cortical laminae as well as layer-specific afferent and efferent projections. Experimental strategies using in vitro assays originally developed by Friedrich Bonhoeffer have suggested that positional cues confined to individual layers regulate the assembly of local cortical circuits and the formation of thalamocortical projections. One of these wiring molecules is ephrinA5, a ligand for Eph receptor tyrosine kinases. EphrinA5 and Eph receptors exhibit highly dynamic expression patterns in distinct regions of the cortex and thalamus during early and late stages of thalamocortical and cortical circuit formation. In vitro assays suggest that ephrinA5 is a multifunctional wiring molecule for different populations of cortical and thalamic axons. Additionally, the expression patterns of ephrinA5 during cortical development are consistent with this molecule regulating, in alternative ways, specific components of thalamic and cortical connectivity. To test this directly, the organization of thalamocortical projections was examined in mice lacking ephrinA5 gene expression. The anatomical studies in ephrinA5 knockout animals revealed a miswiring of limbic thalamic projections and changes in neocortical circuits that were predicted from the expression pattern and the in vitro analysis of ephrinA5 function.     

Induction of excitatory and inhibitory presynaptic differentiation by GluD1

The δ subfamily of ionotropic glutamate receptor subunits consists of GluD1 and GluD2. GluD2, which is selectively expressed in cerebellar Purkinje neurons, has been shown to contribute to the formation of synapses between granule neurons and Purkinje neurons through interaction with Cbln1 (cerebellin precursor protein1) and presynaptic Neurexin. On the other hand, the synaptogenic activity of GluD1, which is expressed not in the cerebellum but in the hippocampus, remains to be characterized. Here, we report that GluD1 expressed in non-neuronal HEK cells, induced presynaptic differentiation of granule neurons through its N-terminal domain in co-cultures with cerebellar neurons, similarly to GluD2. We also show that GluD1 rescued the defect of synapse formation in GluD2-knockout Purkinje neurons, indicating the functional similarity of GluD1 and GluD2. In contrast, GluD1 expression alone did not induce presynaptic differentiation in co-cultures of HEK cells with hippocampal neurons. However, when Cbln1 was exogenously added to the culture medium, GluD1 induced presynaptic differentiation of not only glutamatergic presynaptic terminals but also GABAergic ones. Cbln1 is not expressed in hippocampal neurons but is expressed in entorhinal cortical neurons projecting to the hippocampus. In co-cultures of HEK cells expressing GluD1 and entorhinal cortical neurons, both glutamatergic and GABAergic presynaptic terminals were formed on the HEK cells without exogenous application of Cbln1. These results suggest that GluD1 might contribute to the formation of specific synapses in the hippocampus such as those formed by the projecting neurons of the entorhinal cortex.     

A cell-autonomous requirement for the cell cycle regulatory protein, Rb, in neuronal migration

Precise cell cycle regulation is critical for nervous system development. To assess the role of the cell cycle regulator, retinoblastoma (Rb) protein, in forebrain development, we studied mice with telencephalon-specific Rb deletions. We examined the role of Rb in neuronal specification and migration of diverse neuronal populations. Although layer specification occurred at the appropriate time in Rb mutants, migration of early-born cortical neurons was perturbed. Consistent with defects in radial migration, neuronal cell death in Rb mutants specifically affected Cajal-Retzius neurons. In the ventral telencephalon, although calbindin- and Lhx6-expressing cortical neurons were generated at embryonic day 12.5, their tangential migration into the neocortex was dramatically and specifically reduced in the mutant marginal zone. Cell transplantation assays revealed that defects in tangential migration arose owing to a cell-autonomous loss of Rb in migrating interneurons and not because of a defective cortical environment. These results revealed a cell-autonomous role for Rb in regulating the tangential migration of cortical interneurons. Taken together, we reveal a novel requirement for the cell cycle protein, Rb, in the regulation of neuronal migration.     

Role of presenilin-1 in cortical lamination and survival of Cajal-Retzius neurons

Presenilin-1 (PS1), the major causative gene of familial Alzheimer disease, regulates neuronal differentiation and Notch signaling during early neural development. To investigate the role of PS1 in neuronal migration and cortical lamination of the postnatal brain, we circumvented the perinatal lethality of PS1-null mice by generating a conditional knockout (cKO) mouse in which PS1 inactivation is restricted to neural progenitor cells (NPCs) and NPC-derived neurons and glia. BrdU birthdating analysis revealed that many late-born neurons fail to migrate beyond the early-born neurons to arrive at their appropriate positions in the superficial layer, while the migration of the early-born neurons is largely normal. The migration defect of late-born neurons coincides with the progressive reduction of radial glia in PS1 cKO mice. In contrast to the premature loss of Cajal-Retzius (CR) neurons in PS1-null mice, generation and survival of CR neurons are unaffected in PS1 cKO mice. Furthermore, the number of proliferating meningeal cells, which have been shown to be important for the survival of CR neurons, is increased in PS1-null mice but not in PS1 cKO mice. These findings show a cell-autonomous role for PS1 in cortical lamination and radial glial development, and a non-cell-autonomous role for PS1 in CR neuron survival.     

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.     

Dpy19l1, a multi-transmembrane protein, regulates the radial migration of glutamatergic neurons in the developing cerebral cortex

During corticogenesis, the regulation of neuronal migration is crucial for the functional organization of the neocortex. Glutamatergic neurons are major excitatory components of the mammalian neocortex. In order to elucidate the specific molecular mechanisms underlying their development, we used single-cell microarray analysis to screen for mouse genes that are highly expressed in developing glutamatergic neurons. We identified dpy-19-like 1 (Dpy19l1), a homolog of C. elegans dpy-19, which encodes a putative multi-transmembrane protein shown to regulate directed migration of Q neuroblasts in C. elegans. At embryonic stages Dpy19l1 is highly expressed in glutamatergic neurons in the mouse cerebral cortex, whereas in the subpallium, where GABAergic neurons are generated, expression was below detectable levels. Downregulation of Dpy19l1 mediated by shRNA resulted in defective radial migration of glutamatergic neurons in vivo, which was restored by the expression of shRNA-insensitive Dpy19l1. Many Dpy19l1-knockdown cells were aberrantly arrested in the intermediate zone and the deep layer and, additionally, some extended single long processes towards the pial surface. Furthermore, we observed defective radial migration of bipolar cells in Dpy19l1-knockdown brains. Despite these migration defects, these cells correctly expressed Cux1, which is a marker for upper layer neurons, suggesting that Dpy19l1 knockdown results in migration defects but does not affect cell type specification. These results indicate that Dpy19l1 is required for the proper radial migration of glutamatergic neurons, and suggest an evolutionarily conserved role for the Dpy19 family in neuronal migration.     

Cultured subventricular zone progenitor cells transduced with neurogenin-2 become mature glutamatergic neurons and integrate into the dentate gyrus

We have previously shown that transplantation of immature DCX+/NeuN+/Prox1+ neurons (found in the neonatal DG), but not undifferentiated neuronal progenitor cells (NPCs) from ventral subventricular zone (SVZ), results in neuronal maturation in vivo within the dentate niche. Here we investigated whether we could enhance the integration of SVZ NPCs by forced expression of the proneural gene Neurogenin 2 (NEUROG2). NPCs cultured from neonatal GFP-transgenic rat SVZ for 7 days in a non-differentiating medium were transduced with a retrovirus encoding NEUROG2 and DsRed or the DsRed reporter gene alone (control). By 3 days post-transduction, the NEUROG2-transduced cells maintained in culture contained mostly immature neurons (91% DCX+; 76% NeuN+), whereas the control virus-transduced cells remained largely undifferentiated (30% DCX+; <1% NeuN+). At 6 weeks following transplantation into the DG of adult male rats, there were no neurons among the transplanted cells treated with the control virus but the majority of the NEUROG2-transduced DsRed+ SVZ cells became mature neurons (92% NeuN+; DCX-negative). Although the NEUROG2-transduced SVZ cells did not express the dentate granule neuron marker Prox1, most of the NEUROG2-transduced SVZ cells (78%) expressed the glutamatergic marker Tbr1, suggesting the acquisition of a glutamatergic phenotype. Moreover, some neurons extended dendrites into the molecular layer, grew axons containing Ankyrin G+ axonal initial segments, and projected into the CA3 region, thus resembling mature DG granule neurons. A proportion of NEUROG2 transduced cells also expressed c-Fos and P-CREB, two markers of neuronal activation. We conclude that NEUROG2-transduction is sufficient to promote neuronal maturation and integration of transplanted NPCs from SVZ into the DG.     

Ventral migration of early-born neurons requires Dcc and is essential for the projections of primary afferents in the spinal cord

Neuronal migration and lamina-specific primary afferent projections are crucial for establishing spinal cord circuits, but the underlying mechanisms are poorly understood. Here, we report that in mice lacking Dcc (deleted in colorectal cancer), some early-born neurons could not migrate ventrally in the spinal cord. Conversely, forced expression of Dcc caused ventral migration and prevented dorsolateral migration of late-born spinal neurons. In the superficial layer of the spinal cord of Dcc-/- mutants, mislocalized neurons are followed by proprioceptive afferents, while their presence repels nociceptive afferents through Sema3a. Thus, our study has shown that Dcc is a key molecule required for ventral migration of early-born neurons, and that appropriate neuronal migration is a prerequisite for, and coupled to, normal projections of primary afferents in the developing spinal cord.     

Pallial origin of basal forebrain cholinergic neurons in the nucleus basalis of Meynert and horizontal limb of the diagonal band nucleus

The majority of the cortical cholinergic innervation implicated in attention and memory originates in the nucleus basalis of Meynert and in the horizontal limb of the diagonal band nucleus of the basal prosencephalon. Functional alterations in this system give rise to neuropsychiatric disorders as well as to the cognitive alterations described in Parkinson and Alzheimer's diseases. Despite the functional importance of these basal forebrain cholinergic neurons very little is known about their origin and development. Previous studies suggest that they originate in the medial ganglionic eminence of the telencephalic subpallium; however, our results identified Tbr1-expressing, reelin-positive neurons migrating from the ventral pallium to the subpallium that differentiate into cholinergic neurons in the basal forebrain nuclei projecting to the cortex. Experiments with Tbr1 knockout mice, which lack ventropallial structures, confirmed the pallial origin of cholinergic neurons in Meynert and horizontal diagonal band nuclei. Also, we demonstrate that Fgf8 signaling in the telencephalic midline attracts these neurons from the pallium to follow a tangential migratory route towards the basal forebrain.