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Loss-of-function mutations in chromatin remodeler gene ARID1A are a cause of Coffin-Siris syndrome, a developmental disorder characterized by dysgenesis of corpus callosum. Here, we characterize Arid1a function during cortical development and find unexpectedly selective roles for Arid1a in subplate neurons (SPNs). SPNs, strategically positioned at the interface of cortical gray and white matter, orchestrate multiple developmental processes indispensable for neural circuit wiring. We find that pancortical deletion of Arid1a leads to extensive mistargeting of intracortical axons and agenesis of corpus callosum. Sparse Arid1a deletion, however, does not autonomously misroute callosal axons, implicating noncell-autonomous Arid1a functions in axon guidance. Supporting this possibility, the ascending axons of thalamocortical neurons, which are not autonomously affected by cortical Arid1a deletion, are also disrupted in their pathfinding into cortex and innervation of whisker barrels. Coincident with these miswiring phenotypes, which are reminiscent of subplate ablation, we unbiasedly find a selective loss of SPN gene expression following Arid1a deletion. In addition, multiple characteristics of SPNs crucial to their wiring functions, including subplate organization, subplate axon-thalamocortical axon cofasciculation ("handshake"), and extracellular matrix, are severely disrupted. To empirically test Arid1a sufficiency in subplate, we generate a cortical plate deletion of Arid1a that spares SPNs. In this model, subplate Arid1a expression is sufficient for subplate organization, subplate axon-thalamocortical axon cofasciculation, and subplate extracellular matrix. Consistent with these wiring functions, subplate Arid1a sufficiently enables normal callosum formation, thalamocortical axon targeting, and whisker barrel development. Thus, Arid1a is a multifunctional regulator of subplate-dependent guidance mechanisms essential to cortical circuit wiring.

Loss-of-function mutations in chromatin remodeler geneARID1Aare a cause of Coffin-Siris syndrome, a developmental disorder characterized by dysgenesis of corpus callosum. Here, we characterizeArid1afunction during cortical development and find unexpectedly selective roles forArid1ain subplate neurons. Subplate neurons (SPNs), strategically positioned at the interface of cortical grey and white matter, orchestrate multiple developmental processes indispensable for neural circuit wiring. We find that pan-cortical deletion ofArid1aleads to extensive mistargeting of intracortical axons and agenesis of corpus callosum. SparseArid1adeletion, however, does not autonomously misroute callosal axons, implicating non-cell autonomousArid1afunctions in axon guidance. Supporting this possibility, the ascending axons of thalamocortical neurons, which are not autonomously affected by corticalArid1adeletion, are also disrupted in their pathfinding into cortex and innervation of whisker barrels. Coincident with these miswiring phenotypes, which are reminiscent of subplate ablation, we unbiasedly find a selective loss of SPN gene expression followingArid1adeletion. In addition, multiple characteristics of SPNs crucial to their wiring functions, including subplate organization, subplate-thalamocortical axon co-fasciculation (“handshake”), and extracellular matrix, are severely disrupted. To empirically testArid1asufficiency in subplate, we generate a cortical plate deletion ofArid1athat spares SPNs. In this model, subplateArid1aexpression is sufficient for subplate-thalamocortical axon co-fasciculation and extracellular matrix assembly. Consistent with these wiring functions, subplateArid1asufficiently enables normal callosum formation, thalamocortical axon targeting, and whisker barrel development. Thus,Arid1ais a multifunctional regulator of subplate-dependent guidance mechanisms essential to cortical circuit wiring.SignificanceThe cognitive, perceptive, and motor capabilities of the mammalian cerebral cortex depend on assembly of circuit connectivity during development. Subplate neurons, strategically located at the junction of grey and white matter, orchestrate the wiring of cortical circuits. Using a new approach to study gene necessity and sufficiency in subplate neurons, we uncover an essential role for chromatin remodelerArid1ain subplate neuron gene expression and axon guidance functions. Cortical deletion ofArid1adisrupts subplate-dependent formation of corpus callosum, targeting of thalamocortical axons, and development of sensory maps. Together, our study identifiesArid1aas a central regulator of subplate-dependent axon pathfinding, establishes subplate function as essential to callosum development, and highlights non-cell autonomous mechanisms in neural circuit formation and disorders thereof.

In early brain development, ascending thalamocortical axons (TCAs) navigate through the ventral telencephalon (VTel) to reach their target regions in the young cerebral cortex. Descending, deep-layer cortical axons subsequently target appropriate thalamic and subcortical target regions. However, precisely how and when corticothalamic axons (CTAs) identify their appropriate, reciprocal thalamic targets remains unclear. We show here that EphB1 and EphB2 receptors control proper navigation of a subset of TCA and CTA projections through the VTel. We show in vivo that EphB receptor forward signaling and the ephrinB1 ligand are required during the early navigation of L1-CAM(+) thalamic fibers in the VTel, and that the misguided thalamic fibers in EphB1/2 KO mice appear to interact with cortical subregion-specific axon populations during reciprocal cortical axon guidance. As such, our findings suggest that descending cortical axons identify specific TCA subpopulations in the dorsal VTel to coordinate reciprocal cortical-thalamic connectivity in the early developing brain.

International audience; TSHZ3, which encodes a zinc-finger transcription factor, was recently positioned as a hub gene in a module of the genes with the highest expression in the developing human neocortex, but its functions remained unknown. Here we identify TSHZ3 as the critical region for a syndrome associated with heterozygous deletions at 19q12-q13.11, which includes autism spectrum disorder (ASD). In Tshz3-null mice, differentially expressed genes include layer-specific markers of cerebral cortical projection neurons (CPNs), and the human orthologs of these genes are strongly associated with ASD. Furthermore, mice heterozygous for Tshz3 show functional changes at synapses established by CPNs and exhibit core ASD-like behavioral abnormalities. These findings highlight essential roles for Tshz3 in CPN development and function, whose alterations can account for ASD in the newly defined TSHZ3 deletion syndrome.

The co-emergence of a six-layered cerebral neocortex and its corticospinal output system is one of the evolutionary hallmarks of mammals. However, the genetic programs that underlie their development and evolution remain poorly understood. Here we identify a conserved non-exonic element (E4) that acts as a cortex-specific enhancer for the nearby gene Fezf2 (also known as Fezl and Zfp312), which is required for the specification of corticospinal neuron identity and connectivity. We find that SOX4 and SOX11 functionally compete with the repressor SOX5 in the transactivation of E4. Cortex-specific double deletion of Sox4 and Sox11 leads to the loss of Fezf2 expression, failed specification of corticospinal neurons and, independent of Fezf2, a reeler-like inversion of layers. We show evidence supporting the emergence of functional SOX-binding sites in E4 during tetrapod evolution, and their subsequent stabilization in mammals and possibly amniotes. These findings reveal that SOX transcription factors converge onto a cis-acting element of Fezf2 and form critical components of a regulatory network controlling the identity and connectivity of corticospinal neurons.

Fragile X syndrome (FXS), the leading monogenic cause of intellectual disability and autism, results from loss of function of the RNA-binding protein FMRP. Here we show that FMRP regulates the translation of neuronal nitric oxide synthase 1 (NOS1) in the developing human neocortex. Whereas NOS1 mRNA is ubiquitously expressed, NOS1 protein is transiently co-expressed with FMRP during early synaptogenesis in layer- and region-specific subpopulations of pyramidal neurons. These include mid-fetal layer 5 subcortically projecting neurons arranged into alternating columns in the prospective Broca’s area and orofacial motor cortex. Human NOS1 translation is activated by FMRP via interactions with coding region binding motifs absent from mouse Nos1 mRNA, which is expressed in mouse pyramidal neurons, but not efficiently translated. Correspondingly, neocortical NOS1 protein levels are severely reduced in developing human FXS cases but not FMRP-deficient mice. Thus, alterations in FMRP post-transcriptional regulation of NOS1 in developing neocortical circuits may contribute to cognitive dysfunction in FXS.

The corticospinal (CS) tract is involved in controlling discrete voluntary skilled movements in mammals. The CS tract arises exclusively from layer (L) 5 projection neurons of the cerebral cortex, and its formation requires L5 activity of Fezf2 ( Fezl , Zfp312 ). How this L5-specific pattern of Fezf2 expression and CS axonal connectivity is established with such remarkable fidelity had remained elusive. Here we show that the transcription factor TBR1 directly binds the Fezf2 locus and represses its activity in L6 corticothalamic projection neurons to restrict the origin of the CS tract to L5. In Tbr1 null mutants, CS axons ectopically originate from L6 neurons in a Fezf2 -dependent manner. Consistently, misexpression of Tbr1 in L5 CS neurons suppresses Fezf2 expression and effectively abolishes the CS tract. Taken together, our findings show that TBR1 is a direct transcriptional repressor of Fezf2 and a negative regulator of CS tract formation that restricts the laminar origin of CS axons specifically to L5.

Local mRNA translation in growing axons allows for rapid and precise regulation of protein expression in response to extrinsic stimuli. However, the role of local translation in mature CNS axons is unknown. Such a mechanism requires the presence of translational machinery and associated mRNAs in circuit-integrated brain axons. Here we use a combination of genetic, quantitative imaging and super-resolution microscopy approaches to show that mature axons in the mammalian brain contain ribosomes, the translational regulator FMRP and a subset of FMRP mRNA targets. This axonal translational machinery is associated with Fragile X granules (FXGs), which are restricted to axons in a stereotyped subset of brain circuits. FXGs and associated axonal translational machinery are present in hippocampus in humans as old as 57 years. This FXG-associated axonal translational machinery is present in adult rats, even when adult neurogenesis is blocked. In contrast, in mouse this machinery is only observed in juvenile hippocampal axons. This differential developmental expression was specific to the hippocampus, as both mice and rats exhibit FXGs in mature axons in the adult olfactory system. Experiments in Fmr1 null mice show that FMRP regulates axonal protein expression but is not required for axonal transport of ribosomes or its target mRNAs. Axonal translational machinery is thus a feature of adult CNS neurons. Regulation of this machinery by FMRP could support complex behaviours in humans throughout life.

A mechanistic understanding of the pathophysiology underpinning psychiatric disorders is essential for the development of targeted molecular therapies. For fragile X syndrome (FXS), recent mechanistic studies have been focused on the metabotropic glutamate receptor (mGluR) signaling pathway. This line of research has led to the discovery of promising candidate drugs currently undergoing various phases of clinical trial, and represents a model of how biological insights can inform therapeutic strategies in neurodevelopmental disorders. Although mGluR signaling is a key mechanism at which targeted treatments can be directed, it is likely to be one of many mechanisms contributing to FXS. A more complete understanding of the molecular and neural underpinnings of the disorder is expected to inform additional therapeutic strategies. Alterations in the assembly of neural circuits in the neocortex have been recently implicated in genetic studies of autism and schizophrenia, and may also contribute to FXS. In this review, we explore dysregulated nitric oxide signaling in the developing neocortex as a novel candidate mechanism of FXS. This possibility stems from our previous work demonstrating that neuronal nitric oxide synthase 1 (NOS1 or nNOS) is regulated by the FXS protein FMRP in the mid-fetal human neocortex. Remarkably, in the mid-late fetal and early postnatal neocortex of human FXS patients, NOS1 expression is severely diminished. Given the role of nitric oxide in diverse neural processes, including synaptic development and plasticity, the loss of NOS1 in FXS may contribute to the etiology of the disorder. Here, we outline the genetic and neurobiological data that implicate neocortical dysfunction in FXS, review the evidence supporting dysregulated nitric oxide signaling in the developing FXS neocortex and its contribution to the disorder, and discuss the implications for targeting nitric oxide signaling in the treatment of FXS and other psychiatric illnesses.

The neocortex underlies not only remarkable motor and sensory capabilities, but also some of our most distinctly human cognitive functions. The emergence of these higher functions during evolution was accompanied by structural changes in the neocortex, including the acquisition of areal specializations such as Broca's speech and language area. The study of these evolutionary mechanisms, which likely involve species-dependent gene expression and function, represents a substantial challenge. These species differences, however, may represent valuable opportunities to understand the molecular underpinnings of neocortical evolution. Here, we discuss nitric oxide signaling as a candidate mechanism in the assembly of neocortical circuits underlying language and higher cognitive functions. This hypothesis was based on the highly specific mid-fetal pattern of nitric oxide synthase 1 (NOS1, previously nNOS) expression in the pyramidal (projection) neurons of two human neocortical areas respectively involved in speech and language, and higher cognition; the frontal operculum (FOp) and the anterior cingulate cortex (ACC). This expression is transiently present during mid-gestation, suggesting that NOS1 may be involved in the development of these areas and the assembly of their neural circuits. As no other gene product is known to exhibit such exquisite spatiotemporal expression, NOS1 represents a remarkable candidate for these functions.

Autism spectrum disorders (ASD) impair social cognition and communication, key higher order functions centered in the human neocortex. The assembly of neocortical circuitry is a precisely regulated developmental process susceptible to genetic alterations that can ultimately affect cognitive abilities. Because ASD is an early onset neurodevelopmental disorder that disrupts functions executed by the neocortex, miswiring of neocortical circuits has been hypothesized to be an underlying mechanism of ASD. This possibility is supported by emerging genetic findings and data from imaging studies. Recent research on neocortical development has identified transcription factors (TFs) as key determinants of neocortical circuit assembly, mediating diverse processes including neuronal specification, migration, and wiring. Many of these TFs (TBR1, SOX5, FEZF2, and SATB2) have been implicated in ASD. Here, I will discuss the functional roles of these transcriptional programs in neocortical circuit development and their neurobiological implications for the emerging etiology of ASD.

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.

Cataloged from PDF version of article. The biological basis for regional and inter-species differences in cerebral cortical morphology is poorly understood. We focused on consanguineous Turkish families with a single affected member with complex bilateral occipital cortical gyration abnormalities. By using whole-exome sequencing, we initially identified a homozygous 2-bp deletion in LAMC3, the laminin 33 gene, leading to an immediate premature termination codon. In two other affected individuals with nearly identical phenotypes, we identified a homozygous nonsense mutation and a compound heterozygous mutation. In human but not mouse fetal brain, LAMC3 is enriched in postmitotic cortical plate neurons, localizing primarily to the somatodendritic compartment. LAMC3 expression peaks between late gestation and late infancy, paralleling the expression of molecules that are important in dendritogenesis and synapse formation. The discovery of the molecular basis of this unusual occipital malformation furthers our understanding of the complex biology underlying the formation of cortical gyrations. © 2011 Nature America, Inc. All rights reserved.

WOS: 000281616300034, PubMed ID: 20729831, The development of the human cerebral cortex is an orchestrated process involving the generation of neural progenitors in the periventricular germinal zones, cell proliferation characterized by symmetric and asymmetric mitoses, followed by migration of post-mitotic neurons to their final destinations in six highly ordered, functionally specialized layers(1,2). An understanding of the molecular mechanisms guiding these intricate processes is in its infancy, substantially driven by the discovery of rare mutations that cause malformations of cortical development(3-6). Mapping of disease loci in putative Mendelian forms of malformations of cortical development has been hindered by marked locus heterogeneity, small kindred sizes and diagnostic classifications that may not reflect molecular pathogenesis. Here we demonstrate the use of whole-exome sequencing to overcome these obstacles by identifying recessive mutations in WD repeat domain 62 (WDR62) as the cause of a wide spectrum of severe cerebral cortical malformations including microcephaly, pachygyria with cortical thickening as well as hypoplasia of the corpus callosum. Some patients with mutations in WDR62 had evidence of additional abnormalities including lissencephaly, schizencephaly, polymicrogyria and, in one instance, cerebellar hypoplasia, all traits traditionally regarded as distinct entities. In mice and humans, WDR62 transcripts and protein are enriched in neural progenitors within the ventricular and subventricular zones. Expression of WDR62 in the neocortex is transient, spanning the period of embryonic neurogenesis. Unlike other known microcephaly genes, WDR62 does not apparently associate with centrosomes and is predominantly nuclear in localization. These findings unify previously disparate aspects of cerebral cortical development and highlight the use of whole-exome sequencing to identify disease loci in settings in which traditional methods have proved challenging., Yale Program on Neurogenetics; Yale Center for Human Genetics and Genomics; National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [RC2 NS070477, UL1 RR024139NIH, UO1MH081896]; National Institutes of Health Neuroscience Microarray ConsortiumUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [U24 NS051869-02S1], We are indebted to the patients and families who have contributed to this study. We thank J. Noonan for expert advice and C. Camputaro for her help with three-dimensional reconstruction of the magnetic resonance images. This study was supported by the Yale Program on Neurogenetics, the Yale Center for Human Genetics and Genomics, and National Institutes of Health grants RC2 NS070477 (to M.G.), UL1 RR024139NIH (Yale Clinical and Translational Science Award) and UO1MH081896 (to N.S.). SNP genotyping was supported in part by a National Institutes of Health Neuroscience Microarray Consortium award U24 NS051869-02S1 (to S.M.). R.P.L. is an investigator of the Howard Hughes Medical Institute.

Cortical excitatory glutamatergic projection neurons and inhibitory GABAergic interneurons follow substantially different developmental programs. In rodents, projection neurons originate from progenitors within the dorsal forebrain, whereas interneurons arise from progenitors in the ventral forebrain. In contrast, it has been proposed that in humans, the majority of cortical interneurons arise from progenitors within the dorsal forebrain, suggesting that their origin and migration is complex and evolutionarily divergent. However, whether molecularly defined human cortical interneuron subtypes originate from distinct progenitors, including those in the ventral forebrain, remains unknown. Furthermore, abnormalities in cortical interneurons have been linked to human disorders, yet no distinct cell population selective loss has been reported. Here we show that cortical interneurons expressing nitric oxide synthase 1, neuropeptide Y, and somatostatin, are either absent or substantially reduced in fetal and infant cases of human holoprosencephaly (HPE) with severe ventral forebrain hypoplasia. Notably, another interneuron subtype normally abundant from the early fetal period, marked by calretinin expression, and different subtypes of projection neuron were present in the cortex of control and HPE brains. These findings have important implications for the understanding of neuronal pathogenesis underlying the clinical manifestations associated with HPE and the developmental origins of human cortical interneuron diversity.

Neocortical projection neurons exhibit layer-specific molecular profiles and axonal connections. Here we show that the molecular identities of early-born subplate and deep-layer neurons are not acquired solely during generation or shortly thereafter but undergo progressive postmitotic refinement mediated by SOX5. Fezf2 and Bcl11b , transiently expressed in all subtypes of newly postmigratory early-born neurons, are subsequently downregulated in layer 6 and subplate neurons, thereby establishing their layer 5-enriched postnatal patterns. In Sox5 -null mice, this downregulation is disrupted, and layer 6 and subplate neurons maintain an immature differentiation state, abnormally expressing these genes postnatally. Consistent with this disruption, SOX5 binds and represses a conserved enhancer near Fezf2 . The Sox5 -null neocortex exhibits failed preplate partition and laminar inversion of early-born neurons, loss of layer 5 subcerebral axons, and misrouting of subplate and layer 6 corticothalamic axons to the hypothalamus. Thus, SOX5 postmitotically regulates the migration, postmigratory differentiation, and subcortical projections of subplate and deep-layer neurons.

SummaryPyramidal neurons of the neocortex can be subdivided into two major groups: deep- (DL) and upper-layer (UL) neurons. Here we report that the expression of the AT-rich DNA-binding protein Satb2 defines two subclasses of UL neurons: UL1 (Satb2 positive) and UL2 (Satb2 negative). In the absence of Satb2, UL1 neurons lose their identity and activate DL- and UL2-specific genetic programs. UL1 neurons in Satb2 mutants fail to migrate to superficial layers and do not contribute to the corpus callosum but to the corticospinal tract, which is normally populated by DL axons. Ctip2, a gene required for the formation of the corticospinal tract, is ectopically expressed in all UL1 neurons in the absence of Satb2. Satb2 protein interacts with the Ctip2 genomic region and controls chromatin remodeling at this locus. Satb2 therefore is required for the initiation of the UL1-specific genetic program and for the inactivation of DL- and UL2-specific genes.

The polarity and adhesion of radial glial cells (RGCs), which function as progenitors and migrational guides for neurons, are critical for morphogenesis of the cerebral cortex. These characteristics largely depend on cadherin-based adherens junctions, which anchor apical end-feet of adjacent RGCs to each other at the ventricular surface. Here, we show that mouse numb and numb-like are required for maintaining radial glial adherens junctions. Numb accumulates in the apical end-feet, where it localizes to adherens junction–associated vesicles and interacts with cadherins. Numb and Numbl inactivation in RGCs decreases proper basolateral insertion of cadherins and disrupts adherens junctions and polarity, leading to progenitor dispersion and disorganized cortical lamination. Conversely, overexpression of Numb prolongs RGC polarization, in a cadherin-dependent manner, beyond the normal neurogenic period. Thus, by regulating RGC adhesion and polarity, Numb and Numbl are required for the tissue architecture of neurogenic niches and the cerebral cortex.

Pyramidal neurons of the cerebral cortex display marked layer- and subtype-specific differences in their axonal projections and dendritic morphologies. Here we show that transcription factor Zfp312 is selectively expressed by layer V and VI subcortical projection pyramidal neurons and their progenitor cells. Knocking down Zfp312 with small interfering RNAs dramatically reduced the number of subcortical axonal projections from deep-layer pyramidal neurons and altered their dendritic morphology. In contrast, misexpression of Zfp312 in cortically projecting pyramidal neurons of layers II and III induced the expression of Tbr1, a transcription factor enriched in deep-layer neurons, and the formation of ectopic subcortical axonal projections. Thus, our results indicate that transcription factor Zfp312 plays a critical role in layer- and neuronal subtype-specific patterning of cortical axonal projections and dendritic morphologies.