Protein Synthesis in the Developing Neocortex at Near-Atomic Resolution Reveals Ebp1-Mediated Neuronal Proteostasis at the 60S Tunnel Exit.

Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development.

Species-dependent posttranscriptional regulation of NOS1 by FMRP in the developing cerebral cortex.

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 translation of neuronal nitric oxide synthase 1 (NOS1) in the developing human neocortex. Whereas NOS1 mRNA is widely expressed, NOS1 protein is transiently coexpressed with FMRP during early synaptogenesis in layer- and region-specific pyramidal neurons. These include midfetal 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 posttranscriptional regulation of NOS1 in developing neocortical circuits may contribute to cognitive dysfunction in FXS.

mRNA-Decapping Associated DcpS Enzyme Controls Critical Steps of Neuronal Development.

Homozygous mutations in the gene encoding the scavenger mRNA-decapping enzyme, DcpS, have been shown to underlie developmental delay and intellectual disability. Intellectual disability is associated with both abnormal neocortical development and mRNA metabolism. However, the role of DcpS and its scavenger decapping activity in neuronal development is unknown. Here, we show that human neurons derived from patients with a DcpS mutation have compromised differentiation and neurite outgrowth. Moreover, in the developing mouse neocortex, DcpS is required for the radial migration, polarity, neurite outgrowth, and identity of developing glutamatergic neurons. Collectively, these findings demonstrate that the scavenger mRNA decapping activity contributes to multiple pivotal roles in neural development and further corroborate that mRNA metabolism and neocortical pathologies are associated with intellectual disability.

Somato-dendritic morphology and axon origin site specify von Economo neurons as a subclass of modified pyramidal neurons in the human anterior cingulate cortex.

Von Economo neurons (VENs) are modified pyramidal neurons characterized by an extremely elongated rod-shaped soma. They are abundant in layer V of the anterior cingulate cortex (ACC) and fronto-insular cortex (FI) of the human brain, and have long been described as a human-specific neuron type. Recently, VENs have been reported in the ACC of apes and the FI of macaque monkeys. The first description of the somato-dendritic morphology of VENs in the FI by Cajal in 1899 (Textura del Sistema Nervioso del Hombre y de los Vertebrados, Tomo II. Madrid: Nicolas Moya) strongly suggested that they were a unique neuron subtype with specific morphological features. It is surprising that a clarification of this extremely important observation has not yet been attempted, especially as possible misidentification of other oval or fusiform cells as VENs has become relevant in many recently published studies. Here, we analyzed sections of Brodmann area 24 (ACC) stained with rapid Golgi and Golgi-Cox in five adult human specimens, and confirmed Cajal's observations. In addition, we established a comprehensive morphological description of VENs. VENs have a distinct somato-dendritic morphology that allows their clear distinction from other modified pyramidal neurons. We established that VENs have a perpendicularly oriented, stick-shaped core part consisting of the cell body and two thick extensions - an apical and basal stem. The perpendicular length of the core part was 150-250 µm and the thickness was 10-21 µm. The core part was characterized by a lack of clear demarcation between the cell body and the two extensions. Numerous thin, spiny and horizontally oriented side dendrites arose from the cell body. The basal extension of the core part typically ended by giving numerous smaller dendrites with a brush-like branching pattern. The apical extension had a topology typical for apical dendrites of pyramidal neurons. The dendrites arising from the core part had a high dendritic spine density. The most distinct feature of VENs was the distant origin site of the axon, which arose from the ending of the basal extension, often having a common origin with a dendrite. Quantitative analysis found that VENs could be divided into two groups based on total dendritic length - small VENs with a peak total dendritic length of 1500-2500 µm and large VENs with a peak total dendritic length of 5000-6000 µm. Comparative morphological analysis of VENs and other oval and fusiform modified pyramidal neurons showed that on Nissl sections small VENs might be difficult to identify, and that oval and fusiform neurons could be misidentified as VENs. Our analysis of Golgi slides of Brodmann area 9 from a total of 32 adult human subjects revealed only one cell resembling VEN morphology. Thus, our data show that the numerous recent reports on the presence of VENs in non-primates in other layers and regions of the cortex need further confirmation by showing the dendritic and axonal morphology of these cells. In conclusion, our study provides a foundation for further comprehensive morphological and functional studies on VENs between different species.

Temporally defined neocortical translation and polysome assembly are determined by the RNA-binding protein Hu antigen R.

Precise spatiotemporal control of mRNA translation machinery is essential to the development of highly complex systems like the neocortex. However, spatiotemporal regulation of translation machinery in the developing neocortex remains poorly understood. Here, we show that an RNA-binding protein, Hu antigen R (HuR), regulates both neocorticogenesis and specificity of neocortical translation machinery in a developmental stage-dependent manner in mice. Neocortical absence of HuR alters the phosphorylation states of initiation and elongation factors in the core translation machinery. In addition, HuR regulates the temporally specific positioning of functionally related mRNAs into the active translation sites, the polysomes. HuR also determines the specificity of neocortical polysomes by defining their combinatorial composition of ribosomal proteins and initiation and elongation factors. For some HuR-dependent proteins, the association with polysomes likewise depends on the eukaryotic initiation factor 2 alpha kinase 4, which associates with HuR in prenatal developing neocortices. Finally, we found that deletion of HuR before embryonic day 10 disrupts both neocortical lamination and formation of the main neocortical commissure, the corpus callosum. Our study identifies a crucial role for HuR in neocortical development as a translational gatekeeper for functionally related mRNA subgroups and polysomal protein specificity.

Thalamic WNT3 Secretion Spatiotemporally Regulates the Neocortical Ribosome Signature and mRNA Translation to Specify Neocortical Cell Subtypes.

Neocortical development requires tightly controlled spatiotemporal gene expression. However, the mechanisms regulating ribosomal complexes and the timed specificity of neocortical mRNA translation are poorly understood. We show that active mRNA translation complexes (polysomes) contain ribosomal protein subsets that undergo dynamic spatiotemporal rearrangements during mouse neocortical development. Ribosomal protein specificity within polysome complexes is regulated by the arrival of in-growing thalamic axons, which secrete the morphogen Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3). Thalamic WNT3 release during midneurogenesis promotes a change in the levels of Ribosomal protein L7 in polysomes, thereby regulating neocortical translation machinery specificity. Furthermore, we present an RNA sequencing dataset analyzing mRNAs that dynamically associate with polysome complexes as neocortical development progresses, and thus may be regulated spatiotemporally at the level of translation. Thalamic WNT3 regulates neocortical translation of two such mRNAs, Foxp2 and Apc, to promote FOXP2 expression while inhibiting APC expression, thereby driving neocortical neuronal differentiation and suppressing oligodendrocyte maturation, respectively. This mechanism may enable targeted and rapid spatiotemporal control of ribosome composition and selective mRNA translation in complex developing systems like the neocortex. SIGNIFICANCE STATEMENT: The neocortex is a highly complex circuit generating the most evolutionarily advanced complex cognitive and sensorimotor functions. An intricate progression of molecular and cellular steps during neocortical development determines its structure and function. Our goal is to study the steps regulating spatiotemporal specificity of mRNA translation that govern neocortical development. In this work, we show that the timed secretion of Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3) by ingrowing axons from the thalamus regulates the combinatorial composition of ribosomal proteins in developing neocortex, which we term the "neocortical ribosome signature." Thalamic WNT3 further regulates the specificity of mRNA translation and development of neurons and oligodendrocytes in the neocortex. This study advances our overall understanding of WNT signaling and the spatiotemporal regulation of mRNA translation in highly complex developing systems.

Prenatal deletion of the RNA-binding protein HuD disrupts postnatal cortical circuit maturation and behavior.

The proper functions of cortical circuits are dependent upon both appropriate neuronal subtype specification and their maturation to receive appropriate signaling. These events establish a balanced circuit that is important for learning, memory, emotion, and complex motor behaviors. Recent research points to mRNA metabolism as a key regulator of this development and maturation process. Hu antigen D (HuD), an RNA-binding protein, has been implicated in the establishment of neuronal identity and neurite outgrowth in vitro. Therefore, we investigated the role of HuD loss of function on neuron specification and dendritogenesis in vivo using a mouse model. We found that loss of HuD early in development results in a defective early dendritic overgrowth phase and pervasive deficits in neuron specification in the lower neocortical layers and defects in dendritogenesis in the CA3 region of the hippocampus. Subsequent behavioral analysis revealed a deficit in performance of a hippocampus-dependent task: the Morris water maze. Further, HuD knock-out (KO) mice exhibited lower levels of anxiety than their wild-type counterparts and were overall less active. Last, we found that HuD KO mice are more susceptible to auditory-induced seizures, often resulting in death. Our findings suggest that HuD is necessary for the establishment of neocortical and hippocampal circuitry and is critical for their function.

Extraordinary neoteny of synaptic spines in the human prefrontal cortex.

The major mechanism for generating diversity of neuronal connections beyond their genetic determination is the activity-dependent stabilization and selective elimination of the initially overproduced synapses [Changeux JP, Danchin A (1976) Nature 264:705-712]. The largest number of supranumerary synapses has been recorded in the cerebral cortex of human and nonhuman primates. It is generally accepted that synaptic pruning in the cerebral cortex, including prefrontal areas, occurs at puberty and is completed during early adolescence [Huttenlocher PR, et al. (1979) Brain Res 163:195-205]. In the present study we analyzed synaptic spine density on the dendrites of layer IIIC cortico-cortical and layer V cortico-subcortical projecting pyramidal neurons in a large sample of human prefrontal cortices in subjects ranging in age from newborn to 91 y. We confirm that dendritic spine density in childhood exceeds adult values by two- to threefold and begins to decrease during puberty. However, we also obtained evidence that overproduction and developmental remodeling, including substantial elimination of synaptic spines, continues beyond adolescence and throughout the third decade of life before stabilizing at the adult level. Such an extraordinarily long phase of developmental reorganization of cortical neuronal circuitry has implications for understanding the effect of environmental impact on the development of human cognitive and emotional capacities as well as the late onset of human-specific neuropsychiatric disorders.

Early Regional Patterning in the Human Prefrontal Cortex Revealed by Laminar Dynamics of Deep Projection Neuron Markers.

Early regional patterning and laminar position of cortical projection neurons is determined by activation and deactivation of transcriptional factors (TFs) and RNA binding proteins (RBPs) that regulate spatiotemporal framework of neurogenetic processes (proliferation, migration, aggregation, postmigratory differentiation, molecular identity acquisition, axonal growth, dendritic development, and synaptogenesis) within transient cellular compartments. Deep-layer projection neurons (DPN), subplate (SPN), and Cajal-Retzius neurons (CRN) are early-born cells involved in the establishment of basic laminar and regional cortical architecture; nonetheless, laminar dynamics of their molecular transcriptional markers remain underexplored. Here we aimed to analyze laminar dynamics of DPN markers, i.e., transcription factors TBR1, CTIP2, TLE4, SOX5, and RBP CELF1 on histological serial sections of the human frontal cortex between 7.5-15 postconceptional weeks (PCW) in reference to transient proliferative, migratory, and postmigratory compartments. The subtle signs of regional patterning were seen during the late preplate phase in the pattern of sublaminar organization of TBR1+/Reelin+ CRN and TBR1+ pioneering SPN. During the cortical plate (CP)-formation phase, TBR1+ neurons became radially aligned, forming continuity from a well-developed subventricular zone to CP showing clear lateral to medial regional gradients. The most prominent regional patterning was seen during the subplate formation phase (around 13 PCW) when a unique feature of the orbitobasal frontal cortex displays a "double plate" pattern. In other portions of the frontal cortex (lateral, dorsal, medial) deep portion of CP becomes loose and composed of TBR1+, CTIP2+, TLE4+, and CELF1+ neurons of layer six and later-born SPN, which later become constituents of the expanded SP (around 15 PCW). Overall, TFs and RBPs mark characteristic regional laminar dynamics of DPN, SPN, and CRN subpopulations during remarkably early fetal phases of the highly ordered association cortex development.

Integrin-KCNB1 potassium channel complexes regulate neocortical neuronal development and are implicated in epilepsy.

Potassium (K+) channels are robustly expressed during prenatal brain development, including in progenitor cells and migrating neurons, but their function is poorly understood. Here, we investigate the role of voltage-gated K+ channel KCNB1 (Kv2.1) in neocortical development. Neuronal migration of glutamatergic neurons was impaired in the neocortices of KCNB1 null mice. Migratory defects persisted into the adult brains, along with disrupted morphology and synaptic connectivity. Mice developed seizure phenotype, anxiety, and compulsive behavior. To determine whether defective KCNB1 can give rise to developmental channelopathy, we constructed Knock In (KI) mice, harboring the gene variant Kcnb1R312H (R312H mice) found in children with developmental and epileptic encephalopathies (DEEs). The R312H mice exhibited a similar phenotype to the null mice. Wild type (WT) and R312H KCNB1 channels made complexes with integrins α5ß5 (Integrin_K+ channel_Complexes, IKCs), whose biochemical signaling was impaired in R312H brains. Treatment with Angiotensin II in vitro, an agonist of Focal Adhesion kinase, a key component of IKC signaling machinery, corrected the neuronal abnormalities. Thus, a genetic mutation in a K+ channel induces severe neuromorphological abnormalities through non-conducting mechanisms, that can be rescued by pharmacological intervention. This underscores a previously unknown role of IKCs as key players in neuronal development, and implicate developmental channelopathies in the etiology of DEEs.

Celf4 controls mRNA translation underlying synaptic development in the prenatal mammalian neocortex.

Abnormalities in neocortical and synaptic development are linked to neurodevelopmental disorders. However, the molecular and cellular mechanisms governing initial synapse formation in the prenatal neocortex remain poorly understood. Using polysome profiling coupled with snRNAseq on human cortical samples at various fetal phases, we identify human mRNAs, including those encoding synaptic proteins, with finely controlled translation in distinct cell populations of developing frontal neocortices. Examination of murine and human neocortex reveals that the RNA binding protein and translational regulator, CELF4, is expressed in compartments enriched in initial synaptogenesis: the marginal zone and the subplate. We also find that Celf4/CELF4-target mRNAs are encoded by risk genes for adverse neurodevelopmental outcomes translating into synaptic proteins. Surprisingly, deleting Celf4 in the forebrain disrupts the balance of subplate synapses in a sex-specific fashion. This highlights the significance of RNA binding proteins and mRNA translation in evolutionarily advanced synaptic development, potentially contributing to sex differences.

Making sense of mRNA landscapes: Translation control in neurodevelopment.

Like all other parts of the central nervous system, the mammalian neocortex undergoes temporally ordered set of developmental events, including proliferation, differentiation, migration, cellular identity, synaptogenesis, connectivity formation, and plasticity changes. These neurodevelopmental mechanisms have been characterized by studies focused on transcriptional control. Recent findings, however, have shown that the spatiotemporal regulation of post-transcriptional steps like alternative splicing, mRNA traffic/localization, mRNA stability/decay, and finally repression/derepression of protein synthesis (mRNA translation) have become just as central to the neurodevelopment as transcriptional control. A number of dynamic players act post-transcriptionally in the neocortex to regulate these steps, as RNA binding proteins (RBPs), ribosomal proteins (RPs), long non-coding RNAs, and/or microRNA. Remarkably, mutations in these post-transcriptional regulators have been associated with neurodevelopmental, neurodegenerative, inherited, or often co-morbid disorders, such as microcephaly, autism, epilepsy, intellectual disability, white matter diseases, Rett-syndrome like phenotype, spinocerebellar ataxia, and amyotrophic lateral sclerosis. Here, we focus on the current state, advanced methodologies and pitfalls of this exciting and upcoming field of RNA metabolism with vast potential in understanding fundamental neurodevelopmental processes and pathologies. This article is categorized under: Translation > Translation Regulation RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Molecular and cellular evolution of the primate dorsolateral prefrontal cortex.

The granular dorsolateral prefrontal cortex (dlPFC) is an evolutionary specialization of primates that is centrally involved in cognition. We assessed more than 600,000 single-nucleus transcriptomes from adult human, chimpanzee, macaque, and marmoset dlPFC. Although most cell subtypes defined transcriptomically are conserved, we detected several that exist only in a subset of species as well as substantial species-specific molecular differences across homologous neuronal, glial, and non-neural subtypes. The latter are exemplified by human-specific switching between expression of the neuropeptide somatostatin and tyrosine hydroxylase, the rate-limiting enzyme in dopamine production in certain interneurons. The above molecular differences are also illustrated by expression of the neuropsychiatric risk gene FOXP2, which is human-specific in microglia and primate-specific in layer 4 granular neurons. We generated a comprehensive survey of the dlPFC cellular repertoire and its shared and divergent features in anthropoid primates.

Numb and Numbl are required for maintenance of cadherin-based adhesion and polarity of neural progenitors.

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.

Effect of environmental enrichment on morphology of deep layer III and layer V pyramidal cells of occipital cortex in oldest-old rat - A quantitative golgi cox study.

Dendrites and dendritic spine density regress extensively during aging in rats housed under standard conditions (SC), which can be ameliorated by housing in the enriched environment (EE). This event is particularly pronounced on neurons where high rates of plasticity are conceivable, such as on projection neurons of archicortical regions of dentate gyrus'. However, effects of EE on neocortical projection neurons are still poorly understood. Therefore, we investigated the effect of EE housing on a deep layer III (L3) and layer V pyramidal cell (L5) morphology in the associative occipital neocortex of male Sprague-Dawley rats at 24 months of age. Rats were randomly distributed in two groups and reared under either SC (n=5) or EE conditions (n=6) for 26 days. In depth quantitative analysis of dendritic tree morphology and spine density on occipital projection neurons, from Golgi-Cox stained sections, showed similar trend in both EE occipital layers L3 and L5. Significant increase was found in total number of dendritic segments (L3 - 37.5 %, L5 - 33 %) and in dendritic diameter of intermediate segments (for more than 20 %), while increase in total spine number was around the level of significance (p>0.55; L3 - 30 %, L5 - 64 %). These findings suggest an outgrowth of new dendritic segments, When compared to archicortical region of dentate gyrus, effects of aging in the associative occipital cortex were less pronounced. Taken together, these findings suggest that structures being more affected by the aging process are more susceptible to the environmental enrichment in old age.

Evolution of the Neocortex Through RNA-Binding Proteins and Post-transcriptional Regulation.

The human neocortex is undoubtedly considered a supreme accomplishment in mammalian evolution. It features a prenatally established six-layered structure which remains plastic to the myriad of changes throughout an organism's lifetime. A fundamental feature of neocortical evolution and development is the abundance and diversity of the progenitor cell population and their neuronal and glial progeny. These evolutionary upgrades are partially enabled due to the progenitors' higher proliferative capacity, compartmentalization of proliferative regions, and specification of neuronal temporal identities. The driving force of these processes may be explained by temporal molecular patterning, by which progenitors have intrinsic capacity to change their competence as neocortical neurogenesis proceeds. Thus, neurogenesis can be conceptualized along two timescales of progenitors' capacity to (1) self-renew or differentiate into basal progenitors (BPs) or neurons or (2) specify their fate into distinct neuronal and glial subtypes which participate in the formation of six-layers. Neocortical development then proceeds through sequential phases of proliferation, differentiation, neuronal migration, and maturation. Temporal molecular patterning, therefore, relies on the precise regulation of spatiotemporal gene expression. An extensive transcriptional regulatory network is accompanied by post-transcriptional regulation that is frequently mediated by the regulatory interplay between RNA-binding proteins (RBPs). RBPs exhibit important roles in every step of mRNA life cycle in any system, from splicing, polyadenylation, editing, transport, stability, localization, to translation (protein synthesis). Here, we underscore the importance of RBP functions at multiple time-restricted steps of early neurogenesis, starting from the cell fate transition of transcriptionally primed cortical progenitors. A particular emphasis will be placed on RBPs with mostly conserved but also divergent evolutionary functions in neural progenitors across different species. RBPs, when considered in the context of the fascinating process of neocortical development, deserve to be main protagonists in the story of the evolution and development of the neocortex.

Adult Upper Cortical Layer Specific Transcription Factor CUX2 Is Expressed in Transient Subplate and Marginal Zone Neurons of the Developing Human Brain.

Cut-Like Homeobox 2 (Cux2) is a transcription factor involved in dendrite and spine development, and synapse formation of projection neurons placed in mouse upper neocortical layers. Therefore, Cux2 is often used as an upper layer marker in the mouse brain. However, expression of its orthologue CUX2 remains unexplored in the human fetal neocortex. Here, we show that CUX2 protein is expressed in transient compartments of developing neocortical anlage during the main fetal phases of neocortical laminar development in human brain. During the early fetal phase when neurons of the upper cortical layers are still radially migrating to reach their final place in the cortical anlage, CUX2 was expressed in the marginal zone (MZ), deep cortical plate, and pre-subplate. During midgestation, CUX2 was still expressed in the migrating upper cortical neurons as well as in the subplate (SP) and MZ neurons. At the term age, CUX2 was expressed in the gyral white matter along with its expected expression in the upper layer neurons. In sum, CUX2 was expressed in migratory neurons of prospective superficial layers and in the diverse subpopulation of transient postmigratory SP and MZ neurons. Therefore, our findings indicate that CUX2 is a novel marker of distinct transient, but critical histogenetic events during corticogenesis. Given the Cux2 functions reported in animal models, our data further suggest that the expression of CUX2 in postmigratory SP and MZ neurons is associated with their unique dendritic and synaptogenesis characteristics.

Extrinsic Regulators of mRNA Translation in Developing Brain: Story of WNTs.

Extrinsic molecules such as morphogens can regulate timed mRNA translation events in developing neurons. In particular, Wingless-type MMTV integration site family, member 3 (Wnt3), was shown to regulate the translation of Foxp2 mRNA encoding a Forkhead transcription factor P2 in the neocortex. However, the Wnt receptor that possibly mediates these translation events remains unknown. Here, we report Frizzled member 7 (Fzd7) as the Wnt3 receptor that lays downstream in Wnt3-regulated mRNA translation. Fzd7 proteins co-localize with Wnt3 ligands in developing neocortices. In addition, the Fzd7 proteins overlap in layer-specific neuronal subpopulations expressing different transcription factors, Foxp1 and Foxp2. When Fzd7 was silenced, we found decreased Foxp2 protein expression and increased Foxp1 protein expression, respectively. The Fzd7 silencing also disrupted the migration of neocortical glutamatergic neurons. In contrast, Fzd7 overexpression reversed the pattern of migratory defects and Foxp protein expression that we found in the Fzd7 silencing. We further discovered that Fzd7 is required for Wnt3-induced Foxp2 mRNA translation. Surprisingly, we also determined that the Fzd7 suppression of Foxp1 protein expression is not Wnt3 dependent. In conclusion, it is exhibited that the interaction between Wnt3 and Fzd7 regulates neuronal identity and the Fzd7 receptor functions as a downstream factor in ligand Wnt3 signaling for mRNA translation. In particular, the Wnt3-Fzd7 signaling axis determines the deep layer Foxp2-expressing neurons of developing neocortices. Our findings also suggest that Fzd7 controls the balance of the expression for Foxp transcription factors in developing neocortical neurons. These discoveries are presented in our manuscript within a larger framework of this review on the role of extrinsic factors in regulating mRNA translation.

Developmental and Behavioral Phenotypes in a Mouse Model of DDX3X Syndrome.

BACKGROUND: Mutations in the X-linked gene DDX3X account for approximately 2% of intellectual disability in females, often comorbid with behavioral problems, motor deficits, and brain malformations. DDX3X encodes an RNA helicase with emerging functions in corticogenesis and synaptogenesis. METHODS: We generated a Ddx3x haploinsufficient mouse (Ddx3x+/- females) with construct validity for DDX3X loss-of-function mutations. We used standardized batteries to assess developmental milestones and adult behaviors, as well as magnetic resonance imaging and immunostaining of cortical projection neurons to capture early postnatal changes in brain development. RESULTS: Ddx3x+/- females showed physical, sensory, and motor delays that evolved into behavioral anomalies in adulthood, including hyperactivity, anxiety-like behaviors, cognitive impairments in specific tasks (e.g., contextual fear memory but not novel object recognition memory), and motor deficits. Motor function declined with age but not if mice were previously exposed to behavioral training. Developmental and behavioral changes were associated with a reduction in brain volume, with some regions (e.g., cortex and amygdala) disproportionally affected. Cortical thinning was accompanied by defective cortical lamination, indicating that Ddx3x regulates the balance of glutamatergic neurons in the developing cortex. CONCLUSIONS: These data shed new light on the developmental mechanisms driving DDX3X syndrome and support construct and face validity of this novel preclinical mouse model.

Selective depletion of molecularly defined cortical interneurons in human holoprosencephaly with severe striatal hypoplasia.

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.