Cdk5 is required for multipolar-to-bipolar transition during radial neuronal migration and proper dendrite development of pyramidal neurons in the cerebral cortex

The mammalian cerebral cortex consists of six layers that are generated via coordinated neuronal migration during the embryonic period. Recent studies identified specific phases of radial migration of cortical neurons. After the final division, neurons transform from a multipolar to a bipolar shape within the subventricular zone-intermediate zone (SVZ-IZ) and then migrate along radial glial fibres. Mice lacking Cdk5 exhibit abnormal corticogenesis owing to neuronal migration defects. When we introduced GFP into migrating neurons at E14.5 by in utero electroporation, we observed migrating neurons in wild-type but not in Cdk5(-/-) embryos after 3-4 days. Introduction of the dominant-negative form of Cdk5 into the wild-type migrating neurons confirmed specific impairment of the multipolar-to-bipolar transition within the SVZ-IZ in a cell-autonomous manner. Cortex-specific Cdk5 conditional knockout mice showed inverted layering of the cerebral cortex and the layer V and callosal neurons, but not layer VI neurons, had severely impaired dendritic morphology. The amount of the dendritic protein Map2 was decreased in the cerebral cortex of Cdk5-deficient mice, and the axonal trajectory of cortical neurons within the cortex was also abnormal. These results indicate that Cdk5 is required for proper multipolar-to-bipolar transition, and a deficiency of Cdk5 results in abnormal morphology of pyramidal neurons. In addition, proper radial neuronal migration generates an inside-out pattern of cerebral cortex formation and normal axonal trajectories of cortical pyramidal neurons.     

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.     

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.     

Radial glial cell development and transformation are disturbed in reeler forebrain

Radial glia are among the earliest cell types to differentiate in the developing mammalian forebrain. Glial fibers span the early cortical wall, forming a dense scaffold; this persists throughout corticogenesis, providing a cellular substrate which supports and directs the migration of young neurons. Although the mechanisms regulating radial glial cell development are poorly understood, a secreted cortical radial glial differentiation signal was recently identified in the embryonic mouse forebrain. This signal is abundant at the time radial glia function to support neuronal migration, and down-regulated perinatally, when radial glia are known to undergo transformation into astrocytes. Therefore, it seems that this signal functions as a radial glial maintenance factor, the availability of which regulates the phenotype of cortical astroglia. Here the differentiation signal is further characterized as RF60, a protein with a molecular weight of approximately 60 kD. In addition, the neurologic mutant mouse reeler provides a genetic model for analysis of RF60 function. Radial glia in reeler cortex are shown to be poorly differentiated and the radial scaffold is shown to be maintained for a shorter time than normal. Furthermore, although astroglial cells from normal cortex are induced to elaborate a radial phenotype by RF60, reeler astroglia show an impaired differentiation response to this. These findings suggest that an intrinsic defect in glial differentiation contributes to the phenotype of abnormal cortical lamination seen in reeler mouse, and indicate that RF60 may play a critical role in normal cortical patterning. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 459–472, 1997     

Characterization and identification of Sox2+ radial glia cells derived from rat embryonic cerebral cortex

During the central nervous system (CNS) development, radial glia cells (RGCs) play at least two essential roles, they contribute to neuronal production and the subsequent guidance of neuronal migration, whereas its precise distribution and contribution to cerebral cortex remains less understood. In this research, we used Vimentin as an astroglial marker and Sox2 as a neural progenitor marker to identify and investigate RGCs in rat cerebral cortex at embryonic day (E) 16.5. We found that the Sox2+ progenitor cells localized in the germinal zone (GZ) of E16.5 cerebral cortex, ~95% Sox2+ cells co-localized with Vimentin+ or Nestin+ radial processes which extended to the pial surface across the cortical plate (CP). In vitro, we obtained RG-like cells from E16.5 cerebral cortex on adherent conditions, these Sox2+ Radial glia (RG)-like cells shared some properties with RGCs in vivo, and these Sox2+ RG-like cells could differentiate into astrocytes, oligodendrocytes and presented the radial glia—neuron lineage differentiation ability. Taken together, we identified and investigated some characterizations and properties of Sox2+ RGCs derived from E16.5 cerebral cortex, we suggested that the embryonic Sox2+ progenitor cells which located in the cortical GZ were mainly composed of Sox2+ RGCs, and the cortex-derived Sox2+ RG-like cells displayed the radial glia—neuron lineage differentiation ability as neuronal progenitors in vitro.     

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.     

Formation and preservation of cortical layers in slice cultures.

During cortical development, neurons generated at the same time in the ventricular zone migrate out into the cortical plate and form a cortical layer (Berry and Eayrs, 1963, Nature 197:984-985; Berry and Rogers, 1965, J. Anat. 99:691-709). We have been studying both the formation and maintenance of cortical layers in slice cultures from rat cortex. The bromodeoxyuridine (BrdU) method was used to label cortical neurons on their birthday in vivo. When slice cultures were prepared from animals at different embryonic and postnatal ages, all cortical layers that have already been established in vivo remained preserved for several weeks in vitro. In slice cultures prepared during migration in the cortex, cells continued to migrate towards the pial side of the cortical slice, however, migration ceased after about 1 week in culture. Thus, cortical cells reached their final laminar position only in slice cultures from postnatal animals, whereas in embryonic slice, migrating cells became scattered throughout the cortex. Previous studies demonstrated that radial glia fibers are the major substrate for migrating neurons (Rakic, 1972, J. Comp. Neurol. 145:61-84; Hatten and Mason, 1990, Experientia 46:907-916). Using antibodies directed against the intermediate filament Vimentin, radial glial cells were detected in all slice cultures where cell migration did occur. Comparable to the glia development in vivo, radial glial fibers disappeared and astrocytes containing the glia fibrillary-associated protein (GFAP) differentiated in slice cultures from postnatal cortex, after the neurons have completed their migration. In contrast, radial glial cells were detected over the whole culture period, and very few astrocytes differentiated in embryonic slices, where cortical neurons failed to finish their migration. The results of this study indicate that the local environment is sufficient to sustain the layered organization of the cortex and support the migration of cortical neurons. In addition, our results reveal a close relationship between cell migration and the developmental status of glial cells.     

In vivo knockdown of ckit impairs neuronal migration and axonal extension in the cerebral cortex

The tyrosine kinase receptor cKit and its ligand stem cell factor (SCF) are well known mediators in proliferation, survival, and positive chemotaxis of different cell types in the hematopoietic system. However, and in spite of previous reports showing robust expression of cKit and SCF in the brain during development, their possible function in the cerebral cortex has not been clarified. In this study, embryonic knockdown expression of cKit in the rat cortex by in utero electroporation of specific RNAi resulted in delayed radial migration of cortical neurons. In conditional Nestin‐cKit KO homozygous mutants, radial migration in the cortex was also delayed. The opposite phenotype was observed after overexpressing cKit in the cortex: radial migration was accelerated. Callosal fibers electroporated with cKit RNAi were also delayed in their extension within the contralateral cortex and eventually failed to innervate their target area. In vitro experiments showed that, whereas SCF was able to promote migration of cortical neurons, it had no effect on cortical neurite outgrowth. In summary, our results demonstrate that (1) cKit is necessary for radial migration of cortical neurons, probably through SCF binding and (2) cKit is necessary for the correct formation of the callosal projection, most likely by a mechanism not involving SCF. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 871–887, 2013     

N-WASP通过polyPro和VCA结构域调控大脑皮层神经元迁移

在大脑皮层发育过程中,神经元迁移是一个动态的复杂过程,与细胞骨架构建和重塑的调控息息相关。N-WASP蛋白是Wiskott-Aldrich综合征蛋白家族(WASP-WAVE family)的一个重要成员,又名WAS-like蛋白(WASL),直接参与细胞骨架中肌动蛋白丝状分支的动态调控。本研究通过蛋白免疫印迹检测发现N-WASP表达于小鼠胚胎发育时期(E12.5~E18.5)的大脑皮层中,并且其表达水平随着发育逐渐降低。利用在体子宫内胚胎电转实验,结果发现过表达或者敲低N-WASP均会造成不同程度的大脑皮层神经元迁移障碍,说明N-WASP在大脑皮层神经元迁移中起到关键作用。N-WASP蛋白主要包含4个结构域：WH1、GBD、polyPro和VCA。为进一步研究N-WASP各结构域在神经元迁移中的调控功能,设计了一系列的显性负性突变实验。通过过表达结构域删除的N-WASP蛋白,发现ΔpolyPro、ΔVCA和ΔWH1均能造成神经元迁移障碍。但是,过表达不能结合Cdc42的N-WASP蛋白(H208D突变体)却不能造成明显的神经元迁移障碍。另外,单独过表达N-WASP的结构域polyPro或VCA能够造成神经元迁移障碍,而过表达WH1结构域却不能影响迁移。最后,通过过表达polyPro和VCA结构域同时删除的N-WASP (WH1-GBD),发现WH1-GBD结构域对神经元迁移没有明显影响。上述结果表明N-WASP蛋白主要是通过polyPro和VCA两个结构域调控大脑皮层神经元的迁移过程。     

alpha3beta1 integrin modulates neuronal migration and placement during early stages of cerebral cortical development

We show that alpha3 integrin mutation disrupts distinct aspects of neuronal migration and placement in the cerebral cortex. The preplate develops normally in alpha3 integrin mutant mice. However, time lapse imaging of migrating neurons in embryonic cortical slices indicates retarded radial and tangential migration of neurons, but not ventricular zone-directed migration. Examination of the actin cytoskeleton of alpha3 integrin mutant cortical cells reveals aberrant actin cytoskeletal dynamics at the leading edges. Deficits are also evident in the ability of developing neurons to probe their cellular environment with filopodial and lamellipodial activity. Calbindin or calretinin positive upper layer neurons as well as the deep layer neurons of alpha3 integrin mutant mice expressing EGFP were misplaced. These results suggest that alpha3beta1 integrin deficiency impairs distinct patterns of neuronal migration and placement through dysregulated actin dynamics and defective ability to search and respond to migration modulating cues in the developing cortex.     

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.     

Cellular migration patterns in the developing mouse cerebral cortex

The migration patterns of embryonic mouse cortical cells were investigated using a replication-incompetent retrovirus vector (BAG). The lateral ventricles of embryonic day 12 mouse embryos were infected with BAG and brains were harvested 2, 3, 4 and 6 days after infection. The location and morphology of all infected cortical cells were recorded from serial sections of entire brains, which were then reconstructed in three dimensions. Examination of the distribution of labelled cells revealed that there were migration patterns characteristic of each medial-lateral domain of the cortex. In the medial and dorsal areas, migration was often radial, although tangential spread increased with survival time, in large part due to ramification of cells in the intermediate zone. In the dorsolateral and lateral areas of the cortex, radial migration was generally not observed. Rather, variable extents of tangential migration occurred, and often resulted in wide separation of cells in the cortical plate. Almost all of the cellular dispersion occurred in the intermediate zone, although a modest degree of dispersion also occurred within the cortical plate itself. Most dispersion occurred in the mediolateral plane, with relatively little dispersion along the anteroposterior axis. Though characteristic migration patterns could be defined, wide variability in the extents of radial migration and tangential separation of cells was seen. The patterns of migration paralleled the distribution of radial glial fibers in all areas, and are most likely a reflection of the role of this network in supporting the migration of cortical neurons. The extent and variability of cellular dispersion supports a lineage-independent mechanism of cortical column ontogenesis.