Integrative mechanisms of oriented neuronal migration in the developing brain.

The emergence of functional neuronal connectivity in the developing cerebral cortex depends on neuronal migration. This process enables appropriate positioning of neurons and the emergence of neuronal identity so that the correct patterns of functional synaptic connectivity between the right types and numbers of neurons can emerge. Delineating the complexities of neuronal migration is critical to our understanding of normal cerebral cortical formation and neurodevelopmental disorders resulting from neuronal migration defects. For the most part, the integrated cell biological basis of the complex behavior of oriented neuronal migration within the developing mammalian cerebral cortex remains an enigma. This review aims to analyze the integrative mechanisms that enable neurons to sense environmental guidance cues and translate them into oriented patterns of migration toward defined areas of the cerebral cortex. We discuss how signals emanating from different domains of neurons get integrated to control distinct aspects of migratory behavior and how different types of cortical neurons coordinate their migratory activities within the developing cerebral cortex to produce functionally critical laminar organization.

APC sets the Wnt tone necessary for cerebral cortical progenitor development.

Adenomatous polyposis coli (APC) regulates the activity of ß-catenin, an integral component of Wnt signaling. However, the selective role of the APC-ß-catenin pathway in cerebral cortical development is unknown. Here we genetically dissected the relative contributions of APC-regulated ß-catenin signaling in cortical progenitor development, a necessary early step in cerebral cortical formation. Radial progenitor-specific inactivation of the APC-ß-catenin pathway indicates that the maintenance of appropriate ß-catenin-mediated Wnt tone is necessary for the orderly differentiation of cortical progenitors and the resultant formation of the cerebral cortex. APC deletion deregulates ß-catenin, leads to high Wnt tone, and disrupts Notch1 signaling and primary cilium maintenance necessary for radial progenitor functions. ß-Catenin deregulation directly disrupts cilium maintenance and signaling via Tulp3, essential for intraflagellar transport of ciliary signaling receptors. Surprisingly, deletion of ß-catenin or inhibition of ß-catenin activity in APC-null progenitors rescues the APC-null phenotype. These results reveal that APC-regulated ß-catenin activity in cortical progenitors sets the appropriate Wnt tone necessary for normal cerebral cortical development.

Adenomatous polyposis coli regulates radial axonal sorting and myelination in the PNS.

The tumor suppressor protein adenomatous polyposis coli (APC) is multifunctional - it participates in the canonical Wnt/ß-catenin signal transduction pathway as well as modulating cytoskeleton function. Although APC is expressed by Schwann cells, the role that it plays in these cells and in the myelination of the peripheral nervous system (PNS) is unknown. Therefore, we used the Cre-lox approach to generate a mouse model in which APC expression is specifically eliminated from Schwann cells. These mice display hindlimb weakness and impaired axonal conduction in sciatic nerves. Detailed morphological analyses revealed that APC loss delays radial axonal sorting and PNS myelination. Furthermore, APC loss delays Schwann cell differentiation in vivo, which correlates with persistent activation of the Wnt signaling pathway and results in perturbed extension of Schwann cell processes and disrupted lamellipodia formation. In addition, APC-deficient Schwann cells display a transient diminution of proliferative capacity. Our data indicate that APC is required by Schwann cells for their timely differentiation to mature, myelinating cells and plays a crucial role in radial axonal sorting and PNS myelination.

New insights into the development of the human cerebral cortex.

The cerebral cortex constitutes more than half the volume of the human brain and is presumed to be responsible for the neuronal computations underlying complex phenomena, such as perception, thought, language, attention, episodic memory and voluntary movement. Rodent models are extremely valuable for the investigation of brain development, but cannot provide insight into aspects that are unique or highly derived in humans. Many human psychiatric and neurological conditions have developmental origins but cannot be studied adequately in animal models. The human cerebral cortex has some unique genetic, molecular, cellular and anatomical features, which need to be further explored. The Anatomical Society devoted its summer meeting to the topic of Human Brain Development in June 2018 to tackle these important issues. The meeting was organized by Gavin Clowry (Newcastle University) and Zoltán Molnár (University of Oxford), and held at St John's College, Oxford. The participants provided a broad overview of the structure of the human brain in the context of scaling relationships across the brains of mammals, conserved principles and recent changes in the human lineage. Speakers considered how neuronal progenitors diversified in human to generate an increasing variety of cortical neurons. The formation of the earliest cortical circuits of the earliest generated neurons in the subplate was discussed together with their involvement in neurodevelopmental pathologies. Gene expression networks and susceptibility genes associated to neurodevelopmental diseases were discussed and compared with the networks that can be identified in organoids developed from induced pluripotent stem cells that recapitulate some aspects of in vivo development. New views were discussed on the specification of glutamatergic pyramidal and γ-aminobutyric acid (GABA)ergic interneurons. With the advancement of various in vivo imaging methods, the histopathological observations can be now linked to in vivo normal conditions and to various diseases. Our review gives a general evaluation of the exciting new developments in these areas. The human cortex has a much enlarged association cortex with greater interconnectivity of cortical areas with each other and with an expanded thalamus. The human cortex has relative enlargement of the upper layers, enhanced diversity and function of inhibitory interneurons and a highly expanded transient subplate layer during development. Here we highlight recent studies that address how these differences emerge during development focusing on diverse facets of our evolution.

Bcl-xL Is Essential for the Survival and Function of Differentiated Neurons in the Cortex That Control Complex Behaviors.

UNLABELLED: Apoptosis plays an essential role during brain development, yet the precise mechanism by which this pathway is regulated in the brain remains unknown. In particular, mammalian cells are known to express multiple anti-apoptotic Bcl-2 family proteins. However, the cells of the developing brain could also exist in a primed state in which the loss of a single anti-apoptotic Bcl-2 family protein is sufficient to trigger apoptosis. Here, we examined the critical role of Bcl-xL, an anti-apoptotic protein, during brain development. Using conditional knock-out mice in which Bcl-xL is deleted in neural progenitor cells (Bcl-xL(Emx1-Cre)), we show that the loss of Bcl-xL is not sufficient to trigger apoptosis in these proliferating progenitors. In contrast, specific populations of postmitotic neurons derived from these progenitors, including upper layer cortical neurons and the CA1-CA3 regions of the hippocampus, were acutely dependent on Bcl-xL. Consistent with this finding, deletion of Bcl-xL selectively in the postmitotic neurons in the brain (Bcl-xL(Nex-Cre)) also resulted in similar patterns of apoptosis. This Bcl-xL deficiency-induced neuronal death was a consequence of activation of the apoptotic pathway, because the cell death was rescued with codeletion of the proapoptotic proteins Bax and Bak. Importantly, the loss of these Bcl-xL-dependent neurons led to severe neurobehavioral abnormalities, including deficits in motor learning, hyperactivity, and increased risk-taking and self-injurious behaviors. Together, our results identify a population of neurons in the developing brain that are acutely dependent on Bcl-xL during the peak period of synaptic connectivity that are important for the establishment of higher-order complex behaviors. SIGNIFICANCE STATEMENT: Although Bcl-xL is known to inhibit apoptosis, exactly which cells in the brain are dependent on Bcl-xL has remained unclear because of the embryonic lethality of mice globally deleted for Bcl-xL. Here, we conditionally deleted Bcl-xL in the brain and found that this did not result in widespread apoptosis in the proliferating progenitors. Instead, Bcl-xL deficiency induced apoptosis in a select population of differentiated neurons predominantly in the early postnatal stages. Importantly, these Bcl-xL-dependent neurons are not essential for survival of the organism but instead regulate complex behaviors. Our results show that the selective loss of these Bcl-xL-dependent neurons results in mice exhibiting severe neurobehavioral abnormalities, including self-injurious and risk-taking behaviors, hyperactivity, and learning and memory defects.

Transcriptional co-regulation of neuronal migration and laminar identity in the neocortex.

The cerebral neocortex is segregated into six horizontal layers, each containing unique populations of molecularly and functionally distinct excitatory projection (pyramidal) neurons and inhibitory interneurons. Development of the neocortex requires the orchestrated execution of a series of crucial processes, including the migration of young neurons into appropriate positions within the nascent neocortex, and the acquisition of layer-specific neuronal identities and axonal projections. Here, we discuss emerging evidence supporting the notion that the migration and final laminar positioning of cortical neurons are also co-regulated by cell type- and layer-specific transcription factors that play concomitant roles in determining the molecular identity and axonal connectivity of these neurons. These transcriptional programs thus provide direct links between the mechanisms controlling the laminar position and identity of cortical neurons.

Mapping of neuronal and glial primary cilia contactome and connectome in the human cerebral cortex.

Primary cilia act as antenna receivers of environmental signals and enable effective neuronal or glial responses. Disruption of their function is associated with circuit disorders. To understand the signals these cilia receive, we comprehensively mapped cilia's contacts within the human cortical connectome using serial-section EM reconstruction of a 1 mm3 cortical volume, spanning the entire cortical thickness. We mapped the "contactome" of cilia emerging from neurons and astrocytes in every cortical layer. Depending on the layer and cell type, cilia make distinct patterns of contact. Primary cilia display cell-type- and layer-specific variations in size, shape, and microtubule axoneme core, which may affect their signaling competencies. Neuronal cilia are intrinsic components of a subset of cortical synapses and thus a part of the connectome. This diversity in the structure, contactome, and connectome of primary cilia endows each neuron or glial cell with a unique barcode of access to the surrounding neural circuitry.

Cdc42 and Gsk3 modulate the dynamics of radial glial growth, inter-radial glial interactions and polarity in the developing cerebral cortex.

Polarized radial glia are crucial to the formation of the cerebral cortex. They serve as neural progenitors and as guides for neuronal placement in the developing cerebral cortex. The maintenance of polarized morphology is essential for radial glial functions, but the extent to which the polarized radial glial scaffold is static or dynamic during corticogenesis remains an open question. The developmental dynamics of radial glial morphology, inter-radial glial interactions during corticogenesis, and the role of the cell polarity complexes in these activities remain undefined. Here, using real-time imaging of cohorts of mouse radial glia cells, we show that the radial glial scaffold, upon which the cortex is constructed, is highly dynamic. Radial glial cells within the scaffold constantly interact with one another. These interactions are mediated by growth cone-like endfeet and filopodia-like protrusions. Polarized expression of the cell polarity regulator Cdc42 in radial glia regulates glial endfeet activities and inter-radial glial interactions. Furthermore, appropriate regulation of Gsk3 activity is required to maintain the overall polarity of the radial glia scaffold. These findings reveal dynamism and interactions among radial glia that appear to be crucial contributors to the formation of the cerebral cortex. Related cell polarity determinants (Cdc42, Gsk3) differentially influence radial glial activities within the evolving radial glia scaffold to coordinate the formation of cerebral cortex.

Disrupted-in-Schizophrenia-1 expression is regulated by beta-site amyloid precursor protein cleaving enzyme-1-neuregulin cascade.

Neuregulin-1 (NRG1) and Disrupted-in-Schizophrenia-1 (DISC1) are promising susceptibility factors for schizophrenia. Both are multifunctional proteins with roles in a variety of neurodevelopmental processes, including progenitor cell proliferation, migration, and differentiation. Here, we provide evidence linking these factors together in a single pathway, which is mediated by ErbB receptors and PI3K/Akt. We show that signaling by NRG1 and NRG2, but not NRG3, increase expression of an isoform of DISC1 in vitro. Receptors ErbB2 and ErbB3, but not ErbB4, are responsible for transducing this effect, and PI3K/Akt signaling is also required. In NRG1 knockout mice, this DISC1 isoform is selectively reduced during neurodevelopment. Furthermore, a similar decrease in DISC1 expression is seen in beta-site amyloid precursor protein cleaving enzyme-1 (BACE1) knockout mice, in which NRG1/Akt signaling is reportedly impaired. In contrast to neuronal DISC1 that was reported and characterized, expression of DISC1 in other types of cells in the brain has not been addressed. Here we demonstrate that DISC1, like NRG and ErbB proteins, is expressed in neurons, astrocytes, oligodendrocytes, microglia, and radial progenitors. These findings may connect NRG1, ErbBs, Akt, and DISC1 in a common pathway, which may regulate neurodevelopment and contribute to susceptibility to schizophrenia.

Radial glia promote microglial development through integrin αVß8 -TGFß1 signaling.

Microglia diversity emerges from interactions between intrinsic genetic programs and environment-derived signals, but how these processes unfold and interact in the developing brain remains unclear. Here, we show that radial glia-expressed integrin beta 8 (ITGB8) expressed in radial glia progenitors activates microglia-expressed TGFß1, permitting microglial development. Domain-restricted deletion of Itgb8 in these progenitors establishes complementary regions with developmentally arrested "dysmature" microglia that persist into adulthood. In the absence of autocrine TGFß1 signaling, we find that microglia adopt a similar dysmature phenotype, leading to neuromotor symptoms almost identical to Itgb8 mutant mice. In contrast, microglia lacking the TGFß signal transducers Smad2 and Smad3 have a less polarized dysmature phenotype and correspondingly less severe neuromotor dysfunction. Finally, we show that non-canonical (Smad-independent) signaling partially suppresses disease and development associated gene expression, providing compelling evidence for the adoption of microglial developmental signaling pathways in the context of injury or disease.

MARCKS modulates radial progenitor placement, proliferation and organization in the developing cerebral cortex.

The radial glial cells serve as neural progenitors and as a migratory guide for newborn neurons in the developing cerebral cortex. These functions require appropriate organization and proliferation of the polarized radial glial scaffold. Here, we demonstrate in mice that the myristoylated alanine-rich C-kinase substrate protein (MARCKS), a prominent cellular substrate for PKC, modulates radial glial placement and expansion. Loss of MARCKS results in ectopic collection of mitotically active radial progenitors away from the ventricular zone (VZ) in the upper cerebral wall. Apical restriction of key polarity complexes [CDC42, beta-catenin (CTNNB1), N-cadherin (CDH2), myosin IIB (MYOIIB), aPKCzeta, LGL, PAR3, pericentrin, PROM1] is lost. Furthermore, the radial glial scaffold in Marcks null cortex is compromised, with discontinuous, non-radial processes apparent throughout the cerebral wall and deformed, bulbous, unbranched end-feet at the basal ends. Further, the density of radial processes within the cerebral cortex is reduced. These deficits in radial glial development culminate in aberrant positioning of neurons and disrupted cortical lamination. Genetic rescue experiments demonstrate, surprisingly, that phosphorylation of MARCKS by PKC is not essential for the role of MARCKS in radial glial cell development. By contrast, the myristoylation domain of MARCKS needed for membrane association is essential for MARCKS function in radial glia. The membrane-associated targeting of MARCKS and the resultant polarized distribution of signaling complexes essential for apicobasal polarity may constitute a critical event in the appropriate placement, proliferation and organization of polarized radial glial scaffold in the developing cerebral cortex.

Strategies for analyzing neuronal progenitor development and neuronal migration in the developing cerebral cortex.

The emergence of functional neuronal connectivity in the developing cerebral cortex depends on 1) neural progenitor differentiation, which leads to the generation of appropriate number and types of neurons, and 2) neuronal migration, which enables the appropriate positioning of neurons so that the correct patterns of functional synaptic connectivity between neurons can emerge. In this review, we discuss 1) currently available methods to study neural progenitor development and differentiation in the developing cerebral cortex and emerging technologies in this regard, 2) assays to study the migration of descendents of progenitors (i.e., neurons) in vitro and in vivo, and 3) the use of these assays to probe the molecular control of these events in the developing brain and evaluation of gene functions disrupted in human neurodevelopmental disorders.

Netrin-1-alpha3beta1 integrin interactions regulate the migration of interneurons through the cortical marginal zone.

Cortical GABAergic interneurons, most of which originate in the ganglionic eminences, take distinct tangential migratory trajectories into the developing cerebral cortex. However, the ligand-receptor systems that modulate the tangential migration of distinct groups of interneurons into the emerging cerebral wall remain unclear. Here, we show that netrin-1, a diffusible guidance cue expressed along the migratory routes traversed by GABAergic interneurons, interacts with alpha3beta1 integrin to promote interneuronal migration. In vivo analysis of interneuron-specific alpha3beta1 integrin, netrin-1-deficient mice (alpha3(lox/-)Dlx5/6-CIE, netrin-1(-/-)) reveals specific deficits in the patterns of interneuronal migration along the top of the developing cortical plate, resulting in aberrant interneuronal positioning throughout the cerebral cortex and hippocampus of conditional alpha3(lox/-)Dlx5/6-CIE, netrin-1(-/-) mice. These results indicate that specific guidance mechanisms, such as netrin-1-alpha3beta1 integrin interactions, modulate distinct routes of interneuronal migration and the consequent positioning of groups of cortical interneurons in the developing cerebral cortex.

Coordinating cerebral cortical construction and connectivity: Unifying influence of radial progenitors.

Radial progenitor development and function lay the foundation for the construction of the cerebral cortex. Radial glial scaffold, through its functions as a source of neurogenic progenitors and neuronal migration guide, is thought to provide a template for the formation of the cerebral cortex. Emerging evidence is challenging this limited view. Intriguingly, radial glial scaffold may also play a role in axonal growth, guidance, and neuronal connectivity. Radial glial cells not only facilitate the generation, placement, and allocation of neurons in the cortex but also regulate how they wire up. The organization and function of radial glial cells may thus be a unifying feature of the developing cortex that helps to precisely coordinate the right patterns of neurogenesis, neuronal placement, and connectivity necessary for the emergence of a functional cerebral cortex. This perspective critically explores this emerging view and its impact in the context of human brain development and disorders.

Navigating the ventricles: Novel insights into the pathogenesis of hydrocephalus.

Congenital hydrocephalus occurs in one in 500-1000 babies born in the United States and acquired hydrocephalus may occur as the consequence of stroke, intraventricular and subarachnoid hemorrhage, traumatic brain injuries, brain tumors, craniectomy or may be idiopathic, as in the case of normal pressure hydrocephalus. Irrespective of its prevalence and significant impact on quality of life, neurosurgeons still rely on invasive cerebrospinal fluid shunt systems for the treatment of hydrocephalus that are exceptionally prone to failure and/or infection. Further understanding of this process at a molecular level, therefore, may have profound implications for improving treatment and quality of life for millions of individuals worldwide. The purpose of this article is to review the current research landscape on hydrocephalus with a focus on recent advances in our understanding of cerebrospinal fluid pathways from an evolutionary, genetics and molecular perspective.

Nap1-regulated neuronal cytoskeletal dynamics is essential for the final differentiation of neurons in cerebral cortex.

The cytoskeletal regulators that mediate the change in the neuronal cytoskeletal machinery from one that promotes oriented motility to one that facilitates differentiation at the appropriate locations in the developing neocortex remain unknown. We found that Nck-associated protein 1 (Nap1), an adaptor protein thought to modulate actin nucleation, is selectively expressed in the developing cortical plate, where neurons terminate their migration and initiate laminar-specific differentiation. Loss of Nap1 function disrupts neuronal differentiation. Premature expression of Nap1 in migrating neurons retards migration and promotes postmigratory differentiation. Nap1 gene mutation in mice leads to neural tube and neuronal differentiation defects. Disruption of Nap1 retards the ability to localize key actin cytoskeletal regulators such as WAVE1 to the protrusive edges where they are needed to elaborate process outgrowth. Thus, Nap1 plays an essential role in facilitating neuronal cytoskeletal changes underlying the postmigratory differentiation of cortical neurons, a critical step in functional wiring of the cortex.

The essential role of primary cilia in cerebral cortical development and disorders.

Primary cilium, first described in the 19th century in different cell types and organisms by Alexander Ecker, Albert Kolliker, Aleksandr Kowalevsky, Paul Langerhans, and Karl Zimmermann (Ecker, 1844; Kolliker, 1854; Kowalevsky, 1867; Langerhans, 1876; Zimmermann, 1898), play an essential modulatory role in diverse aspects of nervous system development and function. The primary cilium, sometimes referred to as the cell's 'antennae', can receive wide ranging inputs from cellular milieu, including morphogens, growth factors, neuromodulators, and neurotransmitters. Its unique structural and functional organization bequeaths it the capacity to hyper-concentrate signaling machinery in a restricted cellular domain approximately one-thousandth the volume of cell soma. Thus enabling it to act as a signaling hub that integrates diverse developmental and homestatic information from cellular milieu to regulate the development and function of neural cells. Dysfunction of primary cilia contributes to the pathophysiology of several brain malformations, intellectual disabilities, epilepsy, and psychiatric disorders. This review focuses on the most essential contributions of primary cilia to cerebral cortical development and function, in the context of neurodevelopmental disorders and malformations. It highlights the recent progress made in identifying the mechanisms underlying primary cilia's role in cortical progenitors, neurons and glia, in health and disease. A future challenge will be to translate these insights and advances into effective clinical treatments for ciliopathies.

Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor sema 3A.

Variants in the high confident autism spectrum disorder (ASD) gene ANK2 target both ubiquitously expressed 220 kDa ankyrin-B and neurospecific 440 kDa ankyrin-B (AnkB440) isoforms. Previous work showed that knock-in mice expressing an ASD-linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities. Expression of this variant or loss of AnkB440 caused axonal hyperbranching in vitro, which implicated AnkB440 microtubule bundling activity in suppressing collateral branch formation. Leveraging multiple mouse models, cellular assays, and live microscopy, we show that AnkB440 also modulates axon collateral branching stochastically by reducing the number of F-actin-rich branch initiation points. Additionally, we show that AnkB440 enables growth cone (GC) collapse in response to chemorepellent factor semaphorin 3 A (Sema 3 A) by stabilizing its receptor complex L1 cell adhesion molecule/neuropilin-1. ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse. We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal targeting and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.

MicroRNA-29 is an essential regulator of brain maturation through regulation of CH methylation.

Although embryonic brain development and neurodegeneration have received considerable attention, the events that govern postnatal brain maturation are less understood. Here, we identify the miR-29 family to be strikingly induced during the late stages of brain maturation. Brain maturation is associated with a transient, postnatal period of de novo non-CG (CH) DNA methylation mediated by DNMT3A. We examine whether an important function of miR-29 during brain maturation is to restrict the period of CH methylation via its targeting of Dnmt3a. Deletion of miR-29 in the brain, or knockin mutations preventing miR-29 to specifically target Dnmt3a, result in increased DNMT3A expression, higher CH methylation, and repression of genes associated with neuronal activity and neuropsychiatric disorders. These mouse models also develop neurological deficits and premature lethality. Our results identify an essential role for miR-29 in restricting CH methylation in the brain and illustrate the importance of CH methylation regulation for normal brain maturation.

Doubling up on microtubule stabilizers: synergistic functions of doublecortin-like kinase and doublecortin in the developing cerebral cortex.

Dynamic regulation of neuronal cytoskeletal machinery in response to extracellular cues enables distinct changes in neuronal development in the cerebral cortex. In this issue of Neuron, three related studies on doublecortin-like kinase, a microtubule-associated protein related to doublecortin, by Shu et al., Koizumi et al., and Deuel et al., provide evidence that doublecortin-like kinase is essential for proper neurogenesis, neuronal migration, and axonal wiring.