Radial Glial Scaffold Research Articles

Spatiotemporal tissue maturation of thalamocortical pathways in the human fetal brain.

The development of connectivity between the thalamus and maturing cortex is a fundamental process in the second half of human gestation, establishing the neural circuits that are the basis for several important brain functions. In this study, we acquired high-resolution in utero diffusion magnetic resonance imaging (MRI) from 140 fetuses as part of the Developing Human Connectome Project, to examine the emergence of thalamocortical white matter over the second to third trimester. We delineate developing thalamocortical pathways and parcellate the fetal thalamus according to its cortical connectivity using diffusion tractography. We then quantify microstructural tissue components along the tracts in fetal compartments that are critical substrates for white matter maturation, such as the subplate and intermediate zone. We identify patterns of change in the diffusion metrics that reflect critical neurobiological transitions occurring in the second to third trimester, such as the disassembly of radial glial scaffolding and the lamination of the cortical plate. These maturational trajectories of MR signal in transient fetal compartments provide a normative reference to complement histological knowledge, facilitating future studies to establish how developmental disruptions in these regions contribute to pathophysiology.

TMEM161B modulates radial glial scaffolding in neocortical development

TMEM161B encodes an evolutionarily conserved widely expressed novel 8-pass transmembrane protein of unknown function in human. Here we identify TMEM161B homozygous hypomorphic missense variants in our recessive polymicrogyria(PMG) cohort. Patients carrying TMEM161B mutations exhibit striking neocortical PMG and intellectual disability. Tmem161b knockout mice fail to develop midline hemispheric cleavage, whereas knock-in of patient mutations and patient-derived brain organoids show defects in apical cell polarity and radial glial scaffolding. We found that TMEM161B modulates actin filopodia, functioning upstream of the Rho-GTPase CDC42. Our data link TMEM161B with human PMG, likely regulating radial glia apical polarity during neocortical development.

Embryonic mouse medial neocortex as a model system for studying the radial glial scaffold in fetal human neocortex

Neocortex is the evolutionarily newest region in the brain, and is a structure with diversified size and morphology among mammalian species. Humans have the biggest neocortex compared to the body size, and their neocortex has many foldings, that is, gyri and sulci. Despite the recent methodological advances in in vitro models such as cerebral organoids, mice have been continuously used as a model system for studying human neocortical development because of the accessibility and practicality of in vivo gene manipulation. The commonly studied neocortical region, the lateral neocortex, generally recapitulates the developmental process of the human neocortex, however, there are several important factors missing in the lateral neocortex. First, basal (outer) radial glia (bRG), which are the main cell type providing the radial scaffold to the migrating neurons in the fetal human neocortex, are very few in the mouse lateral neocortex, thus the radial glial scaffold is different from the fetal human neocortex. Second, as a consequence of the difference in the radial glial scaffold, migrating neurons might exhibit different migratory behavior and thus distribution. To overcome those problems, we propose the mouse medial neocortex, where we have earlier revealed an abundance of bRG similar to the fetal human neocortex, as an alternative model system. We found that similar to the fetal human neocortex, the radial glial scaffold, neuronal migration and neuronal distribution are tangentially scattered in the mouse medial neocortex. Taken together, the embryonic mouse medial neocortex could be a suitable and accessible in vivo model system to study human neocortical development and its pathogenesis.

Cell-Scale Modeling to Probe Mechanobiology during Early Cortical Development

Background/Objective: During early cerebral cortex development, neurons form from proliferative glial cells near the ventricular (apical) surface, then migrate along radial glial scaffolds to the cortical surface. In species with wrinkled brains, the presence of basal radial glial cells (bRGCs), radial glial cells which have detached from the ventricular surface, is correlated to the process of gyrification. While mechanical forces are also involved in gyrus creation, the link between the mechanical and biological aspects of this process remains unelucidated. In this study, we hypothesized that radial tension may lead to the production of gyri via the intermediary creation of bRGCs. 
 Methods: To test this hypothesis, the cell-level modeling software CX3D was used to simulate a system in which radial tension acts on radial glial cells (RGCs), facilitating the semi-stochastic production of bRGCs during the process of neocortex development. The outcome of this model was contrasted with a control case in which bRGCs were not allowed to form, and the two models were compared based upon the presence of neurons on the basal surface. 
 Results: The production of bRGCs via tension corresponded to a significant increase in the presence of neurons on the pial surface, even if the total number of glial cells—and thus total number of neurons generated—remained constant. Additionally, the likelihood of neurons moving more basally was found to be significantly greater in the presence of bRGCs. 
 Conclusion and Potential Impact: These results were interpreted to be indications of early gyrus formation. Thus, this study showed that bRGCs—and, ultimately, gyri—may arise from mechanical tension, indicating a possible link between the biological and mechanical explanations of gyrus formation. By providing an alternative lens through which to understand cortical folding, this may have implications for future lines of inquiry, which may expand our understanding of neuro-pathologies associated with misfolding, such as autism and epilepsy.

Neurotransmitters and Endothelins Acting on Radial Glial G-Protein-Coupled Receptors Are, Through Proteolytic NRG/ErbB4 Activation, Able to Modify the Migratory Behavior of Neocortical Cells and Mediate Bipolar-to-Multipolar Transition.

Cell-cell communication plays a central role in the guidance of migrating neurons during the development of the cerebral cortex. Neuregulins (NRGs) are essential mediators for migration and maintenance of the radial glial scaffold. We show, in this study that soluble NRG reduces neuronal motility, causes transition of bipolar cells to multipolar ones, and induces neuronal mitosis. Blocking the NRG receptor, ErbB4, results in reduction of neuron-neuron and neuron-radial glial contacts and causes an increase in neuronal motility. Blocking the radial glial metabotropic glutamate receptor 5 (mGluR5), the nonselective cation channel transient receptor potential 3 (TRPC3), or matrix metalloproteinases (MMPs) results in similar effects as ErbB4 blockade. Soluble NRG counteract the changes in motility pattern. Stimulation of other radial glial G-protein-coupled receptors (GPCRs), such as muscarinic acetylcholine receptors or endothelin receptors counteract all the effect of mGluR5 blockade, but not that of ErbB4, TRPC3, and MMP blockade. The results indicate that neurotransmitters and endothelins acting on radial glial GPCRs are, through proteolytic NRG/ErbB4 activation, able to modify the migratory behavior of neurons.

Lgl1 deficiency disrupts hippocampal development and impairs cognitive performance in mice.

Cellular polarity is crucial for brain development and morphogenesis. Lethal giant larvae 1 (Lgl1) plays a crucial role in the establishment of cell polarity from Drosophila to mammalian cells. Previous studies have found the importance of Lgl1 in the development of cerebellar, olfactory bulb, and cerebral cortex. However, the role of Lgl1 in hippocampal development during the embryonic stage and function in adult mice is still unknown. In our study, we created Lgl1-deficient hippocampus mice by using Emx1-Cre mice. Histological analysis showed that the Emx1-Lgl1-/- mice exhibited reduced size of the hippocampus with severe malformations of hippocampal cytoarchitecture. These defects mainly originated from the disrupted hippocampal neuroepithelium, including increased cell proliferation, abnormal interkinetic nuclear migration, reduced differentiation, increased apoptosis, gradual disruption of adherens junctions, and abnormal neuronal migration. The radial glial scaffold was disorganized in the Lgl1-deficient hippocampus. Thus, Lgl1 plays a distinct role in hippocampal neurogenesis. In addition, the Emx1-Lgl1-/- mice displayed impaired behavioral performance in the Morris water maze and fear conditioning test.

Expression of the RNA methyltransferase Nsun5 is essential for developing cerebral cortex

Nsun5 gene, encoding a cytosine-5 RNA methyltransferase, is deleted in about 95% patients with Williams-Beuren syndrome (WBS). WBS is a neurodevelopmental disorder and characterized by cognitive disorder. We generated single-gene Nsun5 knockout (Nsun5-KO) mice and reported that the Nsun5 deletion leads to deficit in spatial cognition. This study focused on investigating the influence of Nsun5 deficiency in the development of cerebral cortex. In comparison with wild-type littermates, the cortical thickness in postnatal day 10 Nsun5-KO mice was obviously reduced with an abnormal laminar organization, and the processes of pyramidal cells were shorter and finer. Nsun5 was selectively expressed in radial glial cells (RGCs) of cerebral cortex from embryonic day (E) 12.5 to E16.5, but not in intermediate progenitor cells (IPCs) or neocortical neurons. The Nsun5 deletion did not alter proliferation of RGCs or differentiation of RGCs into IPCs. Notably, the ablation of Nsun5 disrupted the growth of radial glial scaffolds, thus numerous basal processes of RGCs failed to reach pial basement membrane. Level of cell polarity regulator Cdc42 protein in radial glial scaffolds of E14.5 Nsun5-KO mice was reduced, but the level of Cdc42 mRNA was unchanged. The dysfunction of glial scaffolds impeded the radial migration of upper-layer and deeper-layer neurons to cause their subcortical accumulation and apoptosis, resulting in an obvious thinness of the cortical plate in E18.5 Nsun5-KO mice. These findings establish a critical role of Nsun5 in development of cerebral cortex through regulating radial glial scaffolds of RGCs to control migration of neocortical neurons.

Derepression of sonic hedgehog signaling upon Gpr161 deletion unravels forebrain and ventricular abnormalities

Inverse gradients of transcriptional repressors antagonize the transcriptional effector response to morphogens. However, the role of such inverse regulation might not manifest solely from lack of repressors. Sonic hedgehog (Shh) patterns the forebrain by being expressed ventrally; however, absence of antagonizing Gli3 repressor paradoxically cause insufficient pathway activation. Interestingly, lack of the primary cilia-localized G-protein-coupled receptor, Gpr161 increases Shh signaling in the mouse neural tube from coordinated lack of Gli3 repressor and Smoothened-independent activation. Here, by deleting Gpr161 in mouse neuroepithelial cells and radial glia at early mid-gestation we detected derepression of Shh signaling throughout forebrain, allowing determination of the pathophysiological consequences. Accumulation of cerebrospinal fluid (hydrocephalus) was apparent by birth, although usual causative defects in multiciliated ependymal cells or aqueduct were not seen. Rather, the ventricular surface was expanded (ventriculomegaly) during embryogenesis from radial glial overproliferation. Cortical phenotypes included polymicrogyria in the medial cingulate cortex, increased proliferation of intermediate progenitors and basal radial glia, and altered neocortical cytoarchitectonic structure with increased upper layer and decreased deep layer neurons. Finally, periventricular nodular heterotopia resulted from disrupted neuronal migration, while the radial glial scaffold was unaffected. Overall, suppression of Shh pathway during early mid-gestation prevents ventricular overgrowth, and regulates cortical gyration and neocortical/periventricular cytoarchitecture.

Multimodal Single-Cell Analysis Reveals Physiological Maturation in the Developing Human Neocortex

In the developing human neocortex, progenitor cells generate diverse cell types prenatally. Progenitor cells and newborn neurons respond to signaling cues, including neurotransmitters. While single-cell RNA sequencing has revealed cellular diversity, physiological heterogeneity has yet to be mapped onto these developing and diverse cell types. By combining measurements of intracellular Ca2+ elevations in response to neurotransmitter receptor agonists and RNA sequencing of the same single cells, we show that Ca2+ responses are cell-type-specific and change dynamically with lineage progression. Physiological response properties predict molecular cell identity and additionally reveal diversitynot captured by single-cell transcriptomics. We find that the serotonin receptor HTR2A selectively activates radial glia cells in the developing human, butnot mouse, neocortex, and inhibiting HTR2A receptorsin human radial glia disrupts the radial glial scaffold. We show highly specific neurotransmittersignaling during neurogenesis in the developing human neocortex and highlight evolutionarilydivergent mechanisms of physiological signaling.

Loss of Lgl1 Disrupts the Radial Glial Fiber-guided Cortical Neuronal Migration and Causes Subcortical Band Heterotopia in Mice

Radial glial cells (RGCs) are neuronal progenitors and function as scaffolds for neuronal radial migration in the developing cerebral cortex. These functions depend on a polarized radial glial scaffold, which is of fundamental importance for brain development. Lethal giant larvae 1 (Lgl1), a key regulator for cell polarity from Drosophila to mammals, plays a key role in tumorigenesis and brain development. To overcome neonatal lethality in Lgl1-null mice and clarify the role of Lgl1 in mouse cerebral cortex development and function, we created Lgl1 dorsal telencephalon-specific knockout mice mediated by Emx1-Cre. Lgl1Emx1 conditional knockout (CKO) mice had normal life spans and could be used for function research. Histology results revealed that the mutant mice displayed an ectopic cortical mass in the dorsolateral hemispheric region between the normotopic cortex and the subcortical white matter, resembling human subcortical band heterotopia (SBH). The Lgl1Emx1 CKO cortex showed disrupted adherens junctions (AJs), which were accompanied by ectopic RGCs and intermediate progenitors, and disorganization of the radial glial fiber system. The early- and late-born neurons failed to reach the destined position along the disrupted radial glial fiber scaffold and instead accumulated in ectopic positions and formed SBH. Additionally, the absence of Lgl1 led to severe abnormalities in RGCs, including hyperproliferation, impaired differentiation, and increased apoptosis. Lgl1Emx1 CKO mice also displayed deficiencies in anxiety-related behaviors. We concluded that Lgl1 is essential for RGC development and neural migration during cerebral cortex development.

Microglia: An Intrinsic Component of the Proliferative Zones in the Fetal Rhesus Monkey (Macaca mulatta) Cerebral Cortex.

Microglial cells are increasingly recognized as modulators of brain development. We previously showed that microglia colonize the cortical proliferative zones in the prenatal brain and regulate the number of precursor cells through phagocytosis. To better define cellular interactions between microglia and proliferative cells, we performed lentiviral vector-mediated intraventricular gene transfer to induce enhanced green fluorescent protein expression in fetal cerebrocortical cells. Tissues were collected and counterstained with cell-specific markers to label microglial cells and identify other cortical cell types. We found that microglial cells intimately interact with the radial glial scaffold and make extensive contacts with neural precursor cells throughout the proliferative zones, particularly in the rhesus monkey fetus when compared to rodents. We also identify a subtype of microglia, which we term 'periventricular microglia', that interact closely with mitotic precursor cells in the ventricular zone. Our data suggest that microglia are structural modulators that facilitate remodeling of the proliferative zones as precursor cells migrate away from the ventricle and may facilitate the delamination of precursor cells. Taken together, these results indicate that microglial cells are an integral component of cortical proliferative zones and contribute to the interactive milieu in which cortical precursor cells function.

PDK1 Deficit Impairs the Development of the Dentate Gyrus in Mice.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) is crucial for the development of the dentate gyrus (DG), the first gateway receiving afferent inputs from the entorhinal cortex. However, the role of PDK1 in DG development is unclear. In the present study, by crossing Pdk1fl/fl mice with the Emx1-cre line, we identified that the ablation of PDK1 disrupted the development of DG via decreasing the proliferation, and increasing the differentiation of dentate neural progenitor cells, downregulating AKT activity and upregulating GSK3β signaling. Moreover, PDK1 deletion disrupted the distribution of Reelin+ cells and decreased the level of Reelin mRNA which may contribute to the defective migration of progenitor cells and the disrupted radial glial scaffolds. Furthermore, the inhibition of GSK3β activity partially restored the decreased proliferation of primary neural stem cells in vitro. Taken together, our data indicated that the ablation of PDK1 affected the proliferation and differentiation of dentate neural progenitor cells in mice.

Trajectory Analysis Unveils Reelin's Role in the Directed Migration of Granule Cells in the Dentate Gyrus.

Reelin controls neuronal migration and layer formation. Previous studies in reeler mice deficient in Reelin focused on the result of the developmental process in fixed tissue sections. It has remained unclear whether Reelin affects the migratory process, migration directionality, or migrating neurons guided by the radial glial scaffold. Moreover, Reelin has been regarded as an attractive signal because newly generated neurons migrate toward the Reelin-containing marginal zone. Conversely, Reelin might be a stop signal because migrating neurons in reeler, but not in wild-type mice, invade the marginal zone. Here, we monitored the migration of newly generated proopiomelanocortin-EGFP-expressing dentate granule cells in slice cultures from reeler, reeler-like mutants and wild-type mice of either sex using real-time microscopy. We discovered that not the actual migratory process and migratory speed, but migration directionality of the granule cells is controlled by Reelin. While wild-type granule cells migrated toward the marginal zone of the dentate gyrus, neurons in cultures from reeler and reeler-like mutants migrated randomly in all directions as revealed by vector analyses of migratory trajectories. Moreover, live imaging of granule cells in reeler slices cocultured to wild-type dentate gyrus showed that the reeler neurons changed their directions and migrated toward the Reelin-containing marginal zone of the wild-type culture, thus forming a compact granule cell layer. In contrast, directed migration was not observed when Reelin was ubiquitously present in the medium of reeler slices. These results indicate that topographically administered Reelin controls the formation of a granule cell layer.SIGNIFICANCE STATEMENT Neuronal migration and the various factors controlling its onset, speed, directionality, and arrest are poorly understood. Slice cultures offer a unique model to study the migration of individual neurons in an almost natural environment. In the present study, we took advantage of the expression of proopiomelanocortin-EGFP by newly generated, migrating granule cells to analyze their migratory trajectories in hippocampal slice cultures from wild-type mice and mutants deficient in Reelin signaling. We show that the compartmentalized presence of Reelin is essential for the directionality, but not the actual migratory process or speed, of migrating granule cells leading to their characteristic lamination in the dentate gyrus.

Regulation of radial glial process growth by glutamate via mGluR5/TRPC3 and neuregulin/ErbB4.

Radial glial cells play an essential role through their function as guides for neuronal migration during development. Disruption of metabotropic glutamate receptor 5 (mGluR5) function retards the growth of radial glial processes in vitro. Neuregulins (NRG) are activated by proteolytic cleavage and regulate (radial) glial maintenance via ErbB3/ErbB4 receptors. We show here that blocking ErbB4 disrupts radial process extension. Soluble NRG acting on ErbB4 receptors is able to promote radial process extension in particular where process elongation has been impeded by blockade of mGluR5, the nonselective cation channel canonical transient receptor potential 3 (TRPC3), or matrix metalloproteases (MMP). NRG does not restore retarded process growth caused by ErbB4 blockade. Stimulation of muscarinic receptors restores process elongation due to mGluR5 blockade but not that caused by TRPC3, MMP or ErbB4 blockade suggesting that muscarinic receptors can replace mGluR5 with respect to radial process extension. Additionally, NRG/ErbB4 causes Ca2+ mobilization in a population of cells through cooperation with ErbB1 receptors. Our results indicate that mGluR5 promotes radial process growth via NRG activation by a mechanism involving TRPC3 channels and MMPs. Thus neurotransmitters acting on G-protein coupled receptors could play a central role in the maintenance of the radial glial scaffold through activation of NRG/ErbB4 signaling.

Intersectin 1 is a component of the Reelin pathway to regulate neuronal migration and synaptic plasticity in the hippocampus

Brain development and function depend on the directed and coordinated migration of neurons from proliferative zones to their final position. The secreted glycoprotein Reelin is an important factor directing neuronal migration. Loss of Reelin function results in the severe developmental disorder lissencephaly and is associated with neurological diseases in humans. Reelin signals via the lipoprotein receptors very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), but the exact mechanism by which these receptors control cellular function is poorly understood. We report that loss of the signaling scaffold intersectin 1 (ITSN1) in mice leads to defective neuronal migration and ablates Reelin stimulation of hippocampal long-term potentiation (LTP). Knockout (KO) mice lacking ITSN1 suffer from dispersion of pyramidal neurons and malformation of the radial glial scaffold, akin to the hippocampal lamination defects observed in VLDLR or ApoER2 mutants. ITSN1 genetically interacts with Reelin receptors, as evidenced by the prominent neuronal migration and radial glial defects in hippocampus and cortex seen in double-KO mice lacking ITSN1 and ApoER2. These defects were similar to, albeit less severe than, those observed in Reelin-deficient or VLDLR/ ApoER2 double-KO mice. Molecularly, ITSN1 associates with the VLDLR and its downstream signaling adaptor Dab1 to facilitate Reelin signaling. Collectively, these data identify ITSN1 as a component of Reelin signaling that acts predominantly by facilitating the VLDLR-Dab1 axis to direct neuronal migration in the cortex and hippocampus and to augment synaptic plasticity.

Evidence of early defects in Cajal-Retzius cell localization during brain development in a mouse model of dystroglycanopathy.

The secondary dystroglycanopathies represent a heterogeneous group of congenital muscular dystrophies characterized by the defective glycosylation of alpha dystroglycan. These disorders are associated with mutations in at least 17 genes, including Fukutin-related protein (FKRP). At the severe end of the clinical spectrum there is substantial brain involvement, and cobblestone lissencephaly is highly suggestive of these disorders. The precise pathogenesis of this phenotype has, however, remained unclear with most attention focused on the disruption to the radial glial scaffold. Here, we set out to investigate whether lesions are apparent prior to the differentiation of the radial glia. A detailed investigation of the structural brain defects from embryonic day 10.5 (E10.5) up until the time of birth (P0) was undertaken in the Fkrp-deficient mice (FKRPKD ). Reelin, and downstream PI3K/Akt signalling pathways were analysed using Western blot. We show that early basement membrane defects and neuroglial ectopia precede radial glial cell differentiation. Furthermore, we identify mislocalization of Cajal-Retzius cells which nonetheless is not associated with any apparent disruption to the reelin, and downstream PI3K/Akt signalling pathways. These observations identify Cajal-Retzius cell mislocalization as an early event during the development of cortical defects thereby identifying an earlier onset and more complex pathogenesis than originally reported for the secondary dystroglycanopathies. Overall this study provides new insight into central nervous system involvement in this group of diseases.

DNA Methyltransferase 1 Is Indispensable for Development of the Hippocampal Dentate Gyrus.

Development of the hippocampal dentate gyrus (DG) in the mammalian brain is achieved through multiple processes during late embryonic and postnatal stages, with each developmental step being strictly governed by extracellular cues and intracellular mechanisms. Here, we show that the maintenance DNA methyltransferase 1 (Dnmt1) is critical for development of the DG in the mouse. Deletion of Dnmt1 in neural stem cells (NSCs) at the beginning of DG development led to a smaller size of the granule cell layer in the DG. NSCs lacking Dnmt1 failed to establish proper radial processes or to migrate into the subgranular zone, resulting in aberrant neuronal production in the molecular layer of the DG and a reduction of integrated neurons in the granule cell layer. Interestingly, prenatal deletion of Dnmt1 in NSCs affected not only the developmental progression of the DG but also the properties of NSCs maintained into adulthood: Dnmt1-deficient NSCs displayed impaired neurogenic ability and proliferation. We also found that Dnmt1 deficiency in NSCs decreased the expression of Reelin signaling components in the developing DG and increased that of the cell cycle inhibitors p21 and p57 in the adult DG. Together, these findings led us to propose that Dnmt1 functions as a key regulator to ensure the proper development of the DG, as well as the proper status of NSCs maintained into adulthood, by modulating extracellular signaling and intracellular mechanisms. Here, we provide evidence that Dnmt1 is required for the proper development of the hippocampal dentate gyrus (DG). Deletion of Dnmt1 in neural stem cells (NSCs) at an early stage of DG development impaired the ability of NSCs to establish secondary radial glial scaffolds and to migrate into the subgranular zone of the DG, leading to aberrant neuronal production in the molecular layer, increased cell death, and decreased granule neuron production. Prenatal deletion of Dnmt1 in NSCs also induced defects in the proliferation and neurogenic ability of adult NSCs. Furthermore, we found that Dnmt1 regulates the expression of key extracellular signaling components during developmental stages while modulating intracellular mechanisms for proliferation and neuronal production of NSCs in the adult.

Fstl1 is involved in the regulation of radial glial scaffold development.

BackgroundRadial glial cells (RGCs), the instructive scaffolds for neuronal migration, are well characterized by their unique morphology and polarization; these cells extend elongated basal processes to the pial basement membrane (BM) and parallel to one another. However, little is known about the mechanisms that underlie the developmental regulation and maintenance of this unique morphology.ResultsHere, by crossing Fstl1fl/fl mice with an EIIa-Cre line, we identified a new role for the secreted glycoprotein Follistatin like-1 (FSTL1). The ablation of Fstl1 in both of its cortical expression domains, the ventricular zone (VZ) and the pia mater, resulted in RGC morphologic disruption; basal processes were not parallel to each other, and endfeet exhibited greater density and branching. However, Fstl1 deletion in only the VZ in the Emx1IREScre; Fstl1fl/fl line did not affect RGC morphology, indicating that FSTL1 derived from the pia mater might be more important for RGC morphology. In addition, upper-layer projection neurons, not deeper-layer projection neurons, failed to reach their appropriate positions. We also found that BMP, AKT/PKB, Cdc42, GSK3β, integrin and reelin signals, which have previously been reported to regulate RGC development, were unchanged, indicating that Fstl1 may function through a unique mechanism.ConclusionsIn the present study, we identified a new role for FSTL1 in the development of radial glial scaffolds and the neuronal migration of upper-layer projection neurons. Our findings will improve understanding of the regulation of RGC development and neuronal migration.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0144-8) contains supplementary material, which is available to authorized users.

Chemokine Signaling Controls Integrity of Radial Glial Scaffold in Developing Spinal Cord and Consequential Proper Position of Boundary Cap Cells.

Radial glial cells are the neural progenitors of the developing CNS and have long radial processes that guide radially migrating neurons. The integrity of the radial glial scaffold, in particular proper adhesion between the endfeet of radial processes and the pial basement membrane (BM), is important for the cellular organization of the CNS, as indicated by evidence emerging from the developing cortex. However, the mechanisms underlying the maintenance of radial glial scaffold integrity during development, when the neuroepithelium rapidly expands, are still poorly understood. Here, we addressed this issue in the developing mouse spinal cord. We show that CXCR4, a receptor of chemokine CXCL12, is expressed in spinal cord radial glia. Conditional knock-out of Cxcr4 in radial glia caused disrupted radial glial scaffold with gaps at the pial endfeet layer and consequentially led to an invasion of boundary cap (BC) cells into the spinal cord. Because BC cells are PNS cells normally positioned at the incoming and outgoing axonal roots, their invasion into the spinal cord suggests a compromised CNS/PNS boundary in the absence of CXCL12/CXCR4 signaling. Both disrupted radial glial scaffold and invasion of BC cells into the CNS were also present in mice deficient in CXCR7, a second receptor of CXCL12. We further show that CXCL12 signaling promotes the radial glia adhesion to BM components and activates integrin β1 avidity. Our study unravels a novel molecular mechanism that deploys CXCL12/CXCR4/CXCR7 for the maintenance of radial glial scaffold integrity, which in turn safeguards the CNS/PNS boundary during spinal cord development.

Impairment of radial glial scaffold-dependent neuronal migration and formation of double cortex by genetic ablation of afadin

Studies of human brain malformations, such as lissencephaly and double cortex, have revealed the importance of neuronal migration during cortical development. Afadin, a membrane scaffolding protein, regulates the formation of adherens junctions (AJs) and cell migration to form and maintain tissue structures. Here, we report that mice with dorsal telencephalon-specific ablation of afadin gene exhibited defects similar to human double cortex, in which the heterotopic cortex was located underneath the normotopic cortex. The normotopic cortex of the mutant mice was arranged in the pattern similar to the cortex of the control mice, while the heterotopic cortex was disorganized. As seen in human patients, double cortex in the mutant mice was formed by impaired neuronal migration during cortical development. Genetic ablation of afadin in the embryonic cerebral cortex disrupted AJs of radial glial cells, likely resulting in the retraction of the apical endfeet from the ventricular surface and the dispersion of radial glial cells from the ventricular zone to the subventricular and intermediate zones. These results indicate that afadin is required for the maintenance of AJs of radial glial cells and that the disruption of AJs might cause an abnormal radial scaffold for neuronal migration. In contrast, the proliferation or differentiation of radial glial cells was not significantly affected. Taken together, these findings indicate that afadin is required for the maintenance of the radial glial scaffold for neuronal migration and that the genetic ablation of afadin leads to the formation of double cortex.