C3G regulates cortical neuron migration, preplate splitting and radial glial cell attachment

Neuronal migration is integral to the development of the cerebral cortex and higher brain function. Cortical neuron migration defects lead to mental disorders such as lissencephaly and epilepsy. Interaction of neurons with their extracellular environment regulates cortical neuron migration through cell surface receptors. However, it is unclear how the signals from extracellular matrix proteins are transduced intracellularly. We report here that mouse embryos lacking the Ras family guanine nucleotide exchange factor, C3G (Rapgef1, Grf2), exhibit a cortical neuron migration defect resulting in a failure to split the preplate into marginal zone and subplate and a failure to form a cortical plate. C3G-deficient cortical neurons fail to migrate. Instead, they arrest in a multipolar state and accumulate below the preplate. The basement membrane is disrupted and radial glial processes are disorganised and lack attachment in C3G-deficient brains. C3G is activated in response to reelin in cortical neurons, which, in turn, leads to activation of the small GTPase Rap1. In C3G-deficient cells, Rap1 GTP loading in response to reelin stimulation is reduced. In conclusion, the Ras family regulator C3G is essential for two aspects of cortex development, namely radial glial attachment and neuronal migration.     

ADAM17 Is Critical for Multipolar Exit and Radial Migration of Neuronal Intermediate Progenitor Cells in Mice Cerebral Cortex

The radial migration of neuronal progenitor cells is critical for the development of cerebral cortex layers. They go through a critical step transforming from multipolar to bipolar before outward migration. A Disintegrin and Metalloprotease 17 (ADAM17) is a transmembrane protease which can process many substrates involved in cell-cell interaction, including Notch, ligands of EGFR, and some cell adhesion molecules. In this study, we used in utero electroporation to knock down or overexpress ADAM17 at embryonic day 14.5 (E14.5) in neuronal progenitor cells to examine the role of ADAM17 in cortical embryonic neurogenesis. Our results showed that the radial migration of ADAM17-knocked down cells were normal till E16.5 and reached the intermediate zone (IZ). Then most transfected cells stopped migration and stayed at the IZ to inner cortical plate (CP) layer at E18.5, and there was higher percentage of multipolar cells at IZ layer in the ADAM17-knocked down group compared to the cells in control group. Marker staining revealed that those ADAM17-knocked down cells differentiated normally from neural stem cells (NSCs) to neuronal intermediate progenitor cells (nIPCs) but did not differentiate into mature neurons. The migration and multipolar exit defects caused by ADAM17 knockdown could be partially rescued by over-expressing an shRNA resistant ADAM17, while overexpressing ADAM17 alone did not affect the radial migration. Taken together, our results showed for the first time that, ADAM17 is critical in regulating the multipolar-stage exit and radial migration of the nIPCs during telencephalon cortex development in mice.     

Multimodal tangential migration of neocortical GABAergic neurons independent of GPI-anchored proteins

Neuronal migration is crucial for the construction of neuronal architecture such as layers and nuclei. Most inhibitory interneurons in the neocortex derive from the basal forebrain and migrate tangentially; however, little is known about the mode of migration of these neurons in the cortex. We used glutamate decarboxylase (Gad)67-green fluorescent protein (GFP) knock-in embryonic mice with expression of GFP in gamma-aminobutyric acid (GABA)-ergic neurons and performed time-lapse analysis. In coronal slices, many GFP-positive neurons in the lower intermediate zone (IZ) and subventricular zone (SVZ) showed robust tangential migration from lateral to medial cortex, while others showed radial and non-radial migration mostly towards the pial surface. In flat-mount preparations, GFP-positive neurons of the marginal zone (MZ) showed multidirectional tangential migration. Some of these neurons descended toward the cortical plate (CP). Intracortical migration of these neurons was largely unaffected by a treatment that cleaves glycosylphosphatidylinositol (GPI) anchors. These findings suggest that tangential migration of cortical interneurons from lateral to medial cortex predominantly occurs in the IZ/SVZ and raise the possibility that a part of the pial surface-directed neurons in the IZ/SVZ reach the MZ, whereby they spread into the whole area of the cortex. At least a part of these neurons may descend toward the CP. Our results also suggest that intracortical migration of GABAergic neurons occurs independent of GPI-anchored proteins.     

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.     

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.     

Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex

The majority of neurons in the adult neocortex are produced embryonically during a brief but intense period of neuronal proliferation. The radial glial cell, a transient embryonic cell type known for its crucial role in neuronal migration, has recently been shown to function as a neuronal progenitor cell and appears to produce most cortical pyramidal neurons. Radial glial cell modulation could thus affect neuron production, neuronal migration, and overall cortical architecture; however, signaling mechanisms among radial glia have not been studied directly. We demonstrate here that calcium waves propagate through radial glial cells in the proliferative cortical ventricular zone (VZ). Radial glial calcium waves occur spontaneously and require connexin hemichannels, P2Y1 ATP receptors, and intracellular IP3-mediated calcium release. Furthermore, we show that wave disruption decreases VZ proliferation during the peak of embryonic neurogenesis. Taken together, these results demonstrate a radial glial signaling mechanism that may regulate cortical neuronal production.     

Netrin 1 regulates ventral tangential migration of guidepost neurons in the lateral olfactory tract

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.     

Ubiquitination of the GTPase Rap1B by the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity

The development of a polarised morphology with multiple dendrites and a single axon is an essential step in the differentiation of neurons. The establishment of neuronal polarity is directed by the sequential activity of the GTPases Rap1B and Cdc42. Rap1B is initially present in all neurites of unpolarised neurons, but becomes restricted to the tip of a single process during the establishment of neuronal polarity where it specifies axonal identity. Here, we show that the ubiquitin ligases Smad ubiquitination regulatory factor-1 (Smurf1) and Smurf2 are essential for neurite growth and neuronal polarity, respectively, and regulate the GTPases Rho and Rap1B in hippocampal neurons. Smurf2 is required for the restriction of Rap1B to a single neurite. Smurf2 ubiquitinates inactive Rap1B and initiates its degradation through the ubiquitin/proteasome pathway (UPS). Degradation of Rap1B restricts it to a single neurite and thereby ensures that neurons extend a single axon.     

Place- and time-dependent expression of mouse sFRP-1 during development of the cerebral neocortex.

Wnts are a family of secreted proteins involved in multiple developmental mechanisms during nervous system development, including cell proliferation, cell migration, axon guidance and specification of cell positional information. We report here the expression of sFRP-1 mRNA, encoding a putative inhibitor of Wnt, in the developing mouse neocortex during the entire period when neurons for the neocortex are born. We show that sFRP-1 mRNA expression is spatially restricted to the proliferative zones during the period, when neurons are known to be generated in large numbers for the enlarging cortical plate.     

Oversulfated chondroitin sulfate plays critical roles in the neuronal migration in the cerebral cortex

Chondroitin sulfate (CS) proteoglycans bind with various proteins through CS chains in a CS structure-dependent manner, in which oversulfated structures, such as iB (IdoA(2-O-sulfate)alpha1-3GalNAc(4-O-sulfate)), D (GlcA(2-O-sulfate)beta1-3GalNAc(6-O-sulfate)), and E (GlcAbeta1-3GalNAc(4,6-O-disulfate)) units constitute the critical functional module. In this study, we examined the expression and function of three CS sulfotransferases in the developing neocortex: uronyl 2-O-sulfotransferase (UST), N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (4,6-ST) and dermatan 4-O-sulfotransferase-1 (D4-ST), which are responsible for the synthesis of oversulfated structures. The CS chains of the neocortex of mouse embryos contained significant amounts of D and E units that are generated by UST and 4,6-ST, respectively. UST and 4,6-ST mRNAs were expressed in the ventricular and subventricular zones, and their expression increased during late embryonic development. In utero electroporation experiments indicated that knockdown of UST and 4,6-ST resulted in the disturbed migration of cortical neurons. The neurons electroporated with the short hairpin RNA constructs of UST and 4,6-ST accumulated in the lower intermediate zone and in the subventricular zone, showing a multipolar morphology. The cDNA constructs of UST and 4,6-ST rescued the defects caused by the RNA interference, and the neurons were able to migrate radially. On the other hand, knockdown of D4-ST, which is involved in the biosynthesis of the iB unit, caused no migratory defects. These results revealed that specific oversulfated structures in CS chains play critical roles in the migration of neuronal precursors during cortical development.     

Tangential migration and proliferation of intermediate progenitors of GABAergic neurons in the mouse telencephalon

In the embryonic neocortex, neuronal precursors are generated in the ventricular zone (VZ) and accumulate in the cortical plate. Recently, the subventricular zone (SVZ) of the embryonic neocortex was recognized as an additional neurogenic site for both principal excitatory neurons and GABAergic inhibitory neurons. To gain insight into the neurogenesis of GABAergic neurons in the SVZ, we investigated the characteristics of intermediate progenitors of GABAergic neurons (IPGNs) in mouse neocortex by immunohistochemistry, immunocytochemistry, single-cell RT-PCR and single-cell array analysis. IPGNs were identified by their expression of some neuronal and cell cycle markers. Moreover, we investigated the origins of the neocortical IPGNs by Cre-loxP fate mapping in transgenic mice and the transduction of part of the telencephalic VZ by Cre-reporter plasmids, and found them in the medial and lateral ganglionic eminence. Therefore, they must migrate tangentially within the telencephalon to reach the neocortex. Cell-lineage analysis by simple-retrovirus transduction revealed that the neocortical IPGNs self-renew and give rise to a small number of neocortical GABAergic neurons and to a large number of granule and periglomerular cells in the olfactory bulb. IPGNs are maintained in the neocortex and may act as progenitors for adult neurogenesis.     

Sox11基因对发育期小鼠大脑皮层神经元迁移的影响

目的: 以小鼠为实验动物研究精神分裂症易感基因Sox11对皮层神经元迁移的影响。方法: 应用实时荧光定量PCR、原位杂交等技术明确发育期 (E14.5, P0, P7, P14) Sox11于大脑皮层的表达模式;应用质粒构建、转染、胚胎电转、免疫荧光染色等技术,对不同时期 (E17.5, P0, P4, P7) 的小鼠分别转染对照shRNA质粒、mSox11 shRNA质粒和mSox11 shRNA干扰恢复后质粒,研究Sox11在神经元放射性迁移中的作用。结果: 与对照组神经元相比,转染mSox11 shRNA的神经元迁移明显延迟。当对照组神经元有一部分已经到达新皮层的表层时,大部分转染mSox11 shRNA的神经元仍停留在新皮层中间区;使用大鼠Sox11基因 (rSox11) 过表达载体对小鼠Sox11基因的干扰进行恢复后,神经元迁移完成后的分布情况与对照基本一致。小鼠Sox11干扰后和干扰恢复后,室管膜下区 (SVZ)、中间区 (IZ) 和皮层板 (CP) 内迁移神经元分布具有显著性差异 (P＜0.01)。结论: Sox11可以促进皮层神经元的迁移,提示Sox11在小鼠皮层神经元迁移过程中发挥重要功能。     

Guidance of cortical radial migration by gradient of diffusible factors

During the development of cerebral cortex, newborn pyramidal neurons originated from the ventricle wall migrate outwardly to the superficial layer of cortex under the guidance of radial glial filaments. Whether this radial migration of young neurons is guided by gradient of diffusible factors or simply driven by a mass action of newly generated neurons at the ventricular zone is entirely unknown, a potential guidance mechanism that has long been overlooked. Our recent study showed that a guidance molecule semaphorin-3A, which is expressed in descending gradient across cortical layers, may serve as a chemoattractive guidance signal for radial migration of newborn cortical neurons toward upper layers. We hypothesize the existence of four groups of extracellular factors that can guide the radial migration of young neurons: (1) attractive factors expressing in superficial layers of cortex, (2) repulsive factors enriched in the ventricular zone, (3) pro-migratory factors uniformly expressed in all cortical layers and (4) stop signals locally expressed in the outmost layer of cortex.Key words: radial migration, cortex, guidance, semaphorin, diffusible factors, growth coneThe mammalian cerebral cortex has the typical laminar structure, the formation of which is essential for neurons in each cortical layer to establish the specific input and output connections with other brain regions. The development of the cortical laminar structure is known to involve the well-coordinated radial migration of newborn pyramidal neurons during development.¹ After young neurons are generated from the ventricular zone (VZ) and subventricular zone (SVZ), they leave their birthplace and migrate along radial glial filaments toward the surface of cortical plate (CP), crossing existing cortical layers composed of earlier born neurons and eventually settling down beneath the marginal zone (MZ, layer I).^1–3 It is generally accepted that the adhesion between neurons and radial glial filaments provides the directionality for these young neurons, and the targeting of neurons to specific lamina was controlled by the selective detachment of migrating neurons from radial glial fibers upon reaching the designated cortical layer.^2,3 However, we believe that the radial glial fibers can only serve as the adhesive scaffold for migrating neurons and constrain their migration in the radial dimension; it remains an open question regarding the nature of the signals that cause newborn neurons to migrate consistently outward along the fiber rather than inward. Whether the radial migration of cortical neurons is guided by gradient of diffusible factors or simply driven by a mass action of newly generated neurons at the VZ is entirely unknown, a potential guidance mechanism that has long been overlooked.Recently we found that the radial migration of layer II/III cortical neurons during development is guided by an extracellular guidance molecule semaphorin-3A (Sema3A).⁴ We observed that Sema3A is expressed in a descending gradient across the cortical layers, whereas its receptor neuropilin-1 (NP1) is expressed at a high level in migrating neurons. By in utero electroporation, we were able to monitor the migration of a subpopulation of cortical neurons in their native environment and examine the effect of perturbing Sema3A signaling. We found that downregulation or conditional knockout of NP1 in young neurons impeded their radial migration with severe misorientation of affected neurons during their migration without altering their cell fate. Studies in cultured cortical slices further showed the requirement of the endogenous gradient of Sema3A for the proper migration of newborn neurons. Results from transwell chemotaxis assays in dissociated culture of newborn cortical neurons also supported the notion that Sema3A attracts the migration of these neurons through the receptor NP1. Thus, Sema3A may serve as a chemoattractive guidance signal for the radial migration of newborn cortical neurons toward upper layers. This is the first demonstration that radial migration of cortical neurons is guided by gradient of extracellular guidance factors. This study also suggests that guidance factors may guide the radial migration by their actions on the growth cone of the leading process of migrating neurons, via mechanisms similar to that found for their actions on axon guidance and dendritic orientation, followed by long-range cytoplasmic signaling that coordinates the forward motility of the entire neuron.⁵In this study, we have only observed an attractive effect of Sema3A in the radial migration of the layer II/III cortical neurons. However, to form the highly ordered laminar structure of the cortex, the entire process of neuronal migration is likely to depend on coordinated actions of multiple factors in the developing cortex, including other semaphorin family members and other guidance molecules, e.g., slits⁶ and ephrins,⁷ which are also expressed in the CP. We hypothesize that four groups of extracellular factors orchestrate to promote the proper radial migration and cortical lamination: (1) factors that are expressed in superficial layers of cortex and in a descending gradient, like Sema3A, may attract the upward migration of newborn neurons (attractive factors), (2) factors enriched in the VZ may exert repulsive action and help to “push” newborn neurons out of their birthplace (repulsive factors), (3) those factors widely expressed in all cortical layers may promote the motility of migrating neurons (pro-migratory factors) and (4) Some repulsive cues may be locally expressed in the superficial layer of cortex to prevent the over migration of neurons when they have arrived at the outmost layer (stop signal). Under the guidance of these four groups of factors, newborn neurons migrate all the way from VZ to the outmost layer of CP and then settle down. One of our recent tasks is to try to identify these four groups of factors.If the radial migration and cortical lamination are guided by diffusible factors, why is radial glial system necessary for this migration process? In other words, why earlier-born neurons in different layers cannot provide the supportive adhesion to young neurons during their radial migration? A potential explanation is that neurons in cortex undergo maturation after terminating their migration, accompanying with changes in their expression profiles of adhesion ligands, and become less and less supportive to the neuronal migration. In contrast, as a kind of cortical progenitor cells, radial glial cells maintain a relatively ‘young’ state and continue to express supportive adhesion ligands over a very long developmental stage. Thus, only the radial glial filament is capable of providing a bridge for newborn neurons to migrate over a very long distance across the non-permissive cell layers. In summary, we believe that during the cortical radial migration, signals from diffusible factors override the adhesive signal from radial glial fibers to promote the appropriate migration and placement of newborn neurons.? Open in a separate windowFigure 1A schematic diagram for the guidance of cortical radial migration by diffusible factors. (A) A model for the distribution of four groups of guidance factors in developing cortex. Radial glial filaments are shown in red, young neurons are in green. There may exist a descending gradient of attractive factors in upper cortical layers (yellow) and an ascending gradient of repulsive factors (blue) near the ventricular zone (VZ). Stop signals (purple) may come from the surface of cortex, and pro-migratory factors (dots) may be widely distributed. (B) Representative image of EGFP-labeled neurons migrating along radial glial filaments in the cortical tissue of E20 mouse. Sections were counterstained with DAPI (Red). Scale bar, 100 µm.     

Ena/VASP proteins regulate cortical neuronal positioning

Development of the multilayered cerebral cortex involves extensive regulated migration of neurons arising from the deeper germinative layers of the mammalian brain. The anatomy and formation of the cortical layers has been well characterized; however, the underlying molecular mechanisms that control the migration and the final positioning of neurons within the cortex remain poorly understood. Here, we report evidence for a key role of Ena/VASP proteins, a protein family implicated in the spatial control of actin assembly and previously shown to negatively regulate fibroblast cell speeds, in cortical development. Ena/VASP proteins are highly expressed in the developing cortical plate in cells bordering Reelin-expressing Cajal-Retzius cells and in the intermediate zone through which newly born cells migrate. Inhibition of Ena/VASP function through retroviral injections in utero led to aberrant placement of early-born pyramidal neurons in the superficial layers of both the embryonic and the postnatal cortex in a cell-autonomous fashion. The abnormally placed pyramidal neurons exhibited grossly normal morphology and polarity. Our results are consistent with a model in which Ena/VASP proteins function in vivo to control the position of neurons in the mouse neocortex.     

C3G regulates the size of the cerebral cortex neural precursor population

The mechanisms regulating the size of the cerebral cortex are poorly understood. Here, we demonstrate that the Rap1 guanine nucleotide exchange factor, C3G (Grf2, Rapgef1), controls the size of the cerebral precursor population. Mice lacking C3G show overproliferation of the cortical neuroepithelium. C3G-deficient neuroepithelial cells accumulate nuclear beta-catenin and fail to exit the cell cycle in vivo. C3G mutant neural precursor cells fail to activate Rap1, exhibit activation of Akt/PKB, inhibition of the beta-catenin-degrading enzyme, Gsk3beta and accumulation of cytosolic and nuclear beta-catenin when exposed to growth factors, in vitro. Our results show that the size of the cortical neural precursor population is controlled by C3G-mediated inhibition of the Ras signalling pathway.     

The interaction of mPar3 with the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity

The Par polarity complex consisting of the evolutionarily conserved proteins mPar3, mPar6, and aPKC regulates cell polarity in many cell types including neurons. Here we show that mPar3 is required for the establishment of neuronal polarity and links the Smurf2 to Kinesin-2. The HECT domain E3 ubiquitin ligase Smurf2 ensures that neurons extend only a single axon by initiating the degradation of inactive Rap1B through the ubiquitin/proteasome system. Its interaction with mPar3 is required to localize Smurf2 to growth cones and restrict Rap1B to the axon. Interfering with the binding of mPar3 to Kinesin-2 or Smurf2 to mPar3 and knockdown of mPar3 by RNAi disrupt the establishment of neuronal polarity through the failure to restrict Rap1B to a single neurite.