Phosphorylation of Focal Adhesion Kinase at Tyr925: Role in Glia-Dependent and Independent Migration Through Regulating Cofilin and N-Cadherin

The adult neocortex is a six-layered structure, consisting of nearly continuous layers of neurons that are generated with large temporal diversity. During development, cortical neurons originating from the ventricular zone migrate towards the Reelin-containing marginal zone in an inside-out arrangement. Focal adhesion kinase (FAK), one tyrosine kinase localizing to focal adhesions, has been shown to be activated by Src, an important downstream molecule of Reelin signaling, at tyrosine 925 (Y925). Up to date, the precise molecular mechanisms of FAK and its phosphorylation at Y925 during neuronal migration are still unclear. Combining in utero electroporation with immunohistochemistry and live imaging, we examined the function of FAK in regulating neuronal migration. We show that phosphorylated FAK is colocalized with Reelin positive cells in the developing neocortex and hippocampus. Phosphorylation of FAK at Y925 is significantly reduced in reeler mice. Overexpression and dephosphorylation of FAK impair locomotion and translocation, resulting in migration inhibition and dislocation of both late-born and early-born neurons. These migration defects are highly correlated to the function of FAK in regulating cofilin phosphorylation and N-Cadherin expression, both are involved in Reelin signaling pathway. Thus, phosphorylation of focal adhesion kinase at Y925 is crucial for both glia-dependent and independent neuronal migration.

Regulation of neuronal progenitor delamination by dynein-driven post-Golgi apical transport

Radial glial (RG) cells are the neural stem cells of the developing neocortex. Apical RG (aRG) cells can delaminate to generate basal RG (bRG) cells, a cell type associated with human brain expansion. Here, we report that this delamination is regulated by the post-Golgi secretory pathway. Using in situ subcellular live imaging, we show that post-Golgi transport of RAB6+ vesicles occurs toward the minus ends of microtubules and depends on dynein. We demonstrate that the apical determinant Crumbs3 (CRB3) is also transported by dynein. Double knockout of RAB6A/A' and RAB6B impairs apical localization of CRB3, and induces a retraction of aRG cell apical process, leading to delamination and ectopic division. These defects are phenocopied by knock-out of the dynein activator LIS1. Overall, our results identify a RAB6-dynein-LIS1 complex for Golgi to apical surface transport in aRG cells, and highlights the role of this pathway in the maintenance of neuroepithelial integrity.

Developmental stage-specific patterned activity contributes to callosal axon projections

The developing neocortex exhibits patterned spontaneous network activity with various synchrony levels. However, the role of such activity in the formation of cortical circuits remains unclear. We previously reported that the development of callosal axon projections, one of the major long-range axonal projections in the brain, is activity dependent. Here, using a genetic method to manipulate network activity in a stage-specific manner, we demonstrated that spontaneous cortical network activity contributes to the region- and lamina-specific projections of callosal axons in the mouse visual cortex and that this process has a critical period: restoring neuronal activity during that period resumed the projections, whereas restoration after the period failed. Furthermore, in vivo imaging revealed that less correlated network activity was critical. Together, our findings suggest that a distinct pattern of spontaneous network activity in a specific developmental stage underlies the formation of long-range axonal projections in the developing neocortex.

Integrative genomic analysis of early neurogenesis reveals a temporal genetic program for differentiation and specification of preplate and Cajal-Retzius neurons

Neurogenesis in the developing neocortex begins with the generation of the preplate, which consists of early-born neurons including Cajal-Retzius (CR) cells and subplate neurons. Here, utilizing the Ebf2-EGFP transgenic mouse in which EGFP initially labels the preplate neurons then persists in CR cells, we reveal the dynamic transcriptome profiles of early neurogenesis and CR cell differentiation. Genome-wide RNA-seq and ChIP-seq analyses at multiple early neurogenic stages have revealed the temporal gene expression dynamics of early neurogenesis and distinct histone modification patterns in early differentiating neurons. We have identified a new set of coding genes and lncRNAs involved in early neuronal differentiation and validated with functional assays in vitro and in vivo. In addition, at E15.5 when Ebf2-EGFP+ cells are mostly CR neurons, single-cell sequencing analysis of purified Ebf2-EGFP+ cells uncovers molecular heterogeneities in CR neurons, but without apparent clustering of cells with distinct regional origins. Along a pseudotemporal trajectory these cells are classified into three different developing states, revealing genetic cascades from early generic neuronal differentiation to late fate specification during the establishment of CR neuron identity and function. Our findings shed light on the molecular mechanisms governing the early differentiation steps during cortical development, especially CR neuron differentiation.

Neural crest cell-derived pericytes act as pro-angiogenic cells in human neocortex development and gliomas

Central nervous system diseases involving the parenchymal microvessels are frequently associated with a ‘microvasculopathy’, which includes different levels of neurovascular unit (NVU) dysfunction, including blood–brain barrier alterations. To contribute to the understanding of NVU responses to pathological noxae, we have focused on one of its cellular components, the microvascular pericytes, highlighting unique features of brain pericytes with the aid of the analyses carried out during vascularization of human developing neocortex and in human gliomas. Thanks to their position, centred within the endothelial/glial partition of the vessel basal lamina and therefore inserted between endothelial cells and the perivascular and vessel-associated components (astrocytes, oligodendrocyte precursor cells (OPCs)/NG2-glia, microglia, macrophages, nerve terminals), pericytes fulfil a central role within the microvessel NVU. Indeed, at this critical site, pericytes have a number of direct and extracellular matrix molecule- and soluble factor-mediated functions, displaying marked phenotypical and functional heterogeneity and carrying out multitasking services. This pericytes heterogeneity is primarily linked to their position in specific tissue and organ microenvironments and, most importantly, to their ontogeny. During ontogenesis, pericyte subtypes belong to two main embryonic germ layers, mesoderm and (neuro)ectoderm, and are therefore expected to be found in organs ontogenetically different, nonetheless, pericytes of different origin may converge and colonize neighbouring areas of the same organ/apparatus. Here, we provide a brief overview of the unusual roles played by forebrain pericytes in the processes of angiogenesis and barriergenesis by virtue of their origin from midbrain neural crest stem cells. A better knowledge of the ontogenetic subpopulations may support the understanding of specific interactions and mechanisms involved in pericyte function/dysfunction, including normal and pathological angiogenesis, thereby offering an alternative perspective on cell subtype-specific therapeutic approaches.

Semaphorin 3C attracts MGE-derived cortical interneurons in the deep migratory stream guiding them into the developing neocortex

While it is known that Semaphorin 3C acts as a guidance cue for axons during brain development, their potential role during interneuron migration is largely unknown. One striking observation is that Sema3C demarcates the pallial/subpallial border and the intracortical pathway of cortical interneurons in the dorsal telencephalon. Moreover, migrating cortical interneurons express Neuropilin1 and Neuropilin2, described receptors for Semaphorin 3A, 3F and 3C. All these reasons prompt us to examine possible roles for Sema3C on cortical interneuron migration. Using several in vitro approaches, we showed that Nrp1-expressing MGE-derived interneurons from the deep migratory stream migrate towards the increasing Sema3C gradients. In contrast, inhibitory neurons from the superficial migratory stream that express Nrp2, do not respond to this guidance cue. In the present study, we proposed that diffusible Sema3C expressed in the Pallium provides a permissive corridor that attracts the Nrp1- expressing interneurons from the DMS into the dorsal telencephalon.

PAX6: a transcriptional guardian for glutamatergic fate in the developing neocortex

During forebrain development, the transcription factor PAX6 is highly expressed by progenitors in the dorsal telencephalon (dTel) i.e. the primitive cerebral cortex with a sharp boundary at the pallial-subpallial boundary, thereby establishing the dorso-ventral patterning of the forebrain and regulating the generation of cortical glutamatergic neurons. Strikingly, removal of Pax6led to a diversion away from the glutamatergic identity in a subset of cortical progenitors indicated by ectopic gene expression. We postulate that PAX6 confers glutamatergic fate in progenitors by preventing them from responding to signaling cues such as SHH that can induce abberant fates. In the present study, we used the transgenic mouse model with Pax6conditionally deleted in the cortex using a tamoxifen-inducible Emx1-CreER T2 transgene combined with a floxed Pax6 and an EGFP constructs. Single-cell transcriptome revealed multiple ectopic leanages in cortical progenitors with morphogen-regulated transcriptional signatures upon Pax6deletion. We also undertook a candidate approach to investigate how attenuation of signaling cues by surgical and pharmacological means using in vitroslice culture affect the magnitude of ectopic gene expression in the cortical progenitors. We demonstrated that attenuation of interneuron migration into the cortex and inhibition of SHH signaling pathway in slice culture substatially reduced aberrant gene expression in the cortical progenitors. Our findings suggest that ventral cues from vTel such as SHH possess ventralizing effect on cortical progenitors, this is consistent with a requirement for PAX6 to resist such effects in order to safeguard glutamatergic fate.