miR-409-3p represses Cited2 to refine neocortical layer V projection neuron identity.

The evolutionary emergence of the corticospinal tract and corpus callosum are thought to underpin the expansion of complex motor and cognitive abilities in mammals. Molecular mechanisms regulating development of the neurons whose axons comprise these tracts, the corticospinal and callosal projection neurons, remain incompletely understood. Our previous work identified a genomic cluster of microRNAs (miRNAs), Mirg/12qF1, that is unique to placental mammals and specifically expressed by corticospinal neurons, and excluded from callosal projection neurons, during development. We found that one of these, miR-409-3p, can convert layer V callosal into corticospinal projection neurons, acting in part through repression of the transcriptional regulator Lmo4. Here we show that miR-409-3p also directly represses the transcriptional co-regulator Cited2, which is highly expressed by callosal projection neurons from the earliest stages of neurogenesis. Cited2 is highly expressed by intermediate progenitor cells (IPCs) in the embryonic neocortex while Mirg, which encodes miR-409-3p, is excluded from these progenitors. miR-409-3p gain-of-function (GOF) in IPCs results in a phenocopy of established Cited2 loss-of-function (LOF). At later developmental stages, both miR-409-3p GOF and Cited2 LOF promote the expression of corticospinal at the expense of callosal projection neuron markers in layer V. Taken together, this work identifies previously undescribed roles for miR-409-3p in controlling IPC numbers and for Cited2 in controlling callosal fate. Thus, miR-409-3p, possibly in cooperation with other Mirg/12qF1 miRNAs, represses Cited2 as part of the multifaceted regulation of the refinement of neuronal cell fate within layer V, combining molecular regulation at multiple levels in both progenitors and post-mitotic neurons.

Vitamin D modulates cortical transcriptome and behavioral phenotypes in an Mecp2 heterozygous Rett syndrome mouse model.

Rett syndrome (RTT) is an X-linked neurological disorder caused by mutations in the transcriptional regulator MECP2. Mecp2 loss-of-function leads to the disruption of many cellular pathways, including aberrant activation of the NF-κB pathway. Genetically attenuating the NF-κB pathway in Mecp2-null mice ameliorates hallmark phenotypes of RTT, including reduced dendritic complexity, raising the question of whether NF-κB pathway inhibitors could provide a therapeutic avenue for RTT. Vitamin D is a known inhibitor of NF-κB signaling; further, vitamin D deficiency is prevalent in RTT patients and male Mecp2-null mice. We previously demonstrated that vitamin D rescues the aberrant NF-κB activity and reduced neurite outgrowth of Mecp2-knockdown cortical neurons in vitro, and that dietary vitamin D supplementation rescues decreased dendritic complexity and soma size of neocortical projection neurons in both male hemizygous Mecp2-null and female heterozygous mice in vivo. Here, we have identified over 200 genes whose dysregulated expression in the Mecp2+/- cortex is modulated by dietary vitamin D. Genes normalized with vitamin D supplementation are involved in dendritic complexity, synapses, and neuronal projections, suggesting that the rescue of their expression could underpin the rescue of neuronal morphology. Further, there is a disruption in the homeostasis of the vitamin D synthesis pathway in Mecp2+/- mice, and motor and anxiety-like behavioral phenotypes in Mecp2+/- mice correlate with circulating vitamin D levels. Thus, our data indicate that vitamin D modulates RTT pathology and its supplementation could provide a simple and cost-effective partial therapeutic for RTT.

Epigenetic regulation of nervous system development and function.

Building a brain is complicated but maintaining one may be an even greater challenge. Epigenetic mechanisms, including DNA methylation, histone and chromatin modifications, and the actions of non-coding RNAs, play an indispensable role in both. They orchestrate long-term changes in gene expression that underpin establishment of cellular identity as well as the distinct functionality of each cell type, while providing the needed plasticity for the brain to respond to a changing environment. The rapid expansion of studies on these epigenetic mechanisms over the last few decades has brought an evolving definition of the term epigenetics, including in the specialized context of the nervous system. The goal of this special issue is thus not only to bring a greater understanding of the myriad ways in which epigenetic mechanisms regulate nervous system development and function, but also to provide a platform for discussion of what is and what is not epigenetics. To this end, the editors have compiled a collection of review articles highlighting some of the remarkable breadth of epigenetic mechanisms that act at all stages of neuronal development and function, spanning from neurodevelopment, through learning and memory, and neurodegeneration.

Proteomic and transcriptional changes associated with MeCP2 dysfunction reveal nodes for therapeutic intervention in Rett syndrome.

Mutations in the methyl-CpG binding protein 2 (MECP2) gene cause Rett syndrome (RTT), an X-linked neurodevelopmental disorder predominantly impacting females. MECP2 is an epigenetic transcriptional regulator acting mainly to repress gene expression, though it plays multiple gene regulatory roles and has distinct molecular targets across different cell types and specific developmental stages. In this review, we summarize MECP2 loss-of-function associated transcriptome and proteome disruptions, delving deeper into the latter which have been comparatively severely understudied. These disruptions converge on multiple biochemical and cellular pathways, including those involved in synaptic function and neurodevelopment, NF-κB signaling and inflammation, and the vitamin D pathway. RTT is a complex neurological disorder characterized by myriad physiological disruptions, in both the central nervous system and peripheral systems. Thus, treating RTT will likely require a combinatorial approach, targeting multiple nodes within the interactomes of these cellular pathways. To this end, we discuss the use of dietary supplements and factors, namely, vitamin D and polyunsaturated fatty acids (PUFAs), as possible partial therapeutic agents given their demonstrated benefit in RTT and their ability to restore homeostasis to multiple disrupted cellular pathways simultaneously. Further unravelling the complex molecular alterations induced by MECP2 loss-of-function, and contextualizing them at the level of proteome homeostasis, will identify new therapeutic avenues for this complex disorder.

Atypical Neocortical Development in the Cited2 Conditional Knockout Leads to Behavioral Deficits Associated with Neurodevelopmental Disorders.

The mammalian neocortex develops from a single layer of neuroepithelial cells to form a six-layer heterogeneous mosaic of differentiated neurons and glial cells. This process requires a complex choreography of temporally and spatially restricted transcription factors and epigenetic regulators. Even subtle disruptions in this regulation can alter the way the neocortex forms and functions, leading to a neurodevelopmental disorder. One epigenetic regulator that is essential for the precise development of the neocortex is CITED2 (CBP/p300 Interacting Transactivator with ED-rich termini). Cited2 is highly expressed by intermediate progenitor cells in the subventricular zone during the generation of the superficial layers of the neocortex. A forebrain-specific conditional knockout of Cited2 (cKO) exhibits reduced proliferation of intermediate progenitor cells embryonically, leading to reduced thickness of the superficial layers and reduced corpus callosum (CC) volume postnatally. Further, the Cited2 cKO display disruptions in balanced neocortical arealization, with a specific reduction in the somatosensory neocortical length, and dysregulation of precise, area-specific neuronal connectivity. Here, we explore the behavioral consequences resulting from this aberrant neocortical development. We demonstrate that Cited2 cKO mice display decreased maternal separation-induced ultrasonic vocalizations (USVs) as neonates, and an increase in rearing behavior and lack of habituation following repeated acoustic startle as adults. They do not display alterations in anxiety-like behavior, overall locomotor activity, or social interactions. Together with the morphological, molecular, and connectivity disruptions, these results identify the Cited2 cKO neocortex as an ideal system to study mechanisms underlying neurodevelopmental and neuroanatomical disruptions with relevance to human neurodevelopmental disorders.

An evolutionarily acquired microRNA shapes development of mammalian cortical projections.

The corticospinal tract is unique to mammals and the corpus callosum is unique to placental mammals (eutherians). The emergence of these structures is thought to underpin the evolutionary acquisition of complex motor and cognitive skills. Corticospinal motor neurons (CSMN) and callosal projection neurons (CPN) are the archetypal projection neurons of the corticospinal tract and corpus callosum, respectively. Although a number of conserved transcriptional regulators of CSMN and CPN development have been identified in vertebrates, none are unique to mammals and most are coexpressed across multiple projection neuron subtypes. Here, we discover 17 CSMN-enriched microRNAs (miRNAs), 15 of which map to a single genomic cluster that is exclusive to eutherians. One of these, miR-409-3p, promotes CSMN subtype identity in part via repression of LMO4, a key transcriptional regulator of CPN development. In vivo, miR-409-3p is sufficient to convert deep-layer CPN into CSMN. This is a demonstration of an evolutionarily acquired miRNA in eutherians that refines cortical projection neuron subtype development. Our findings implicate miRNAs in the eutherians' increase in neuronal subtype and projection diversity, the anatomic underpinnings of their complex behavior.

Vitamin D Supplementation Rescues Aberrant NF-κB Pathway Activation and Partially Ameliorates Rett Syndrome Phenotypes in Mecp2 Mutant Mice.

Rett syndrome (RTT) is a severe, progressive X-linked neurodevelopmental disorder caused by mutations in the transcriptional regulator MECP2 We previously identified aberrant NF-κB pathway upregulation in brains of Mecp2-null mice and demonstrated that genetically attenuating NF-κB rescues some characteristic neuronal RTT phenotypes. These results raised the intriguing question of whether NF-κB pathway inhibitors might provide a therapeutic avenue in RTT. Here, we investigate whether the known NF-κB pathway inhibitor vitamin D ameliorates neuronal phenotypes in Mecp2-mutant mice. Vitamin D deficiency is prevalent among RTT patients, and we find that Mecp2-null mice similarly have significantly reduced 25(OH)D serum levels compared with wild-type littermates. We identify that vitamin D rescues aberrant NF-κB pathway activation and reduced neurite outgrowth of Mecp2 knock-down cortical neurons in vitro Further, dietary supplementation with vitamin D in early symptomatic male Mecp2 hemizygous null and female Mecp2 heterozygous mice ameliorates reduced neocortical dendritic morphology and soma size phenotypes and modestly improves reduced lifespan of Mecp2-nulls. These results elucidate fundamental neurobiology of RTT and provide foundation that NF-κB pathway inhibition might be a therapeutic target for RTT.

Sex differences in Mecp2-mutant Rett syndrome model mice and the impact of cellular mosaicism in phenotype development.

There is currently no effective treatment for Rett syndrome (RTT), a severe X-linked progressive neurodevelopmental disorder caused by mutations in the transcriptional regulator MECP2. Because MECP2 is subjected to X-inactivation, most affected individuals are female heterozygotes who display cellular mosaicism for normal and mutant MECP2. Males who are hemizygous for mutant MECP2 are more severely affected than heterozygous females and rarely survive. Mecp2 loss-of-function is less severe in mice, however, and male hemizygous null mice not only survive until adulthood, they have been the most commonly studied model system. Although heterozygous female mice better recapitulate human RTT, they have not been as thoroughly characterized. This is likely because of the added experimental challenges that they present, including delayed and more variable phenotypic progression and cellular mosaicism due to X-inactivation. In this review, we compare phenotypes of Mecp2 heterozygous female mice and male hemizygous null mouse models. Further, we discuss the complexities that arise from the many cell-type and tissue-type specific roles of MeCP2, as well as the combination of cell-autonomous and non-cell-autonomous disruptions that result from Mecp2 loss-of-function. This is of particular importance in the context of the female heterozygous brain, composed of a mixture of MeCP2+ and MeCP2- cells, the ratio of which can alter RTT phenotypes in the case of skewed X-inactivation. The goal of this review is to provide a clearer understanding of the pathophysiological differences between the mouse models, which is an essential consideration in the design of future pre-clinical studies.

Caveolin1 Identifies a Specific Subpopulation of Cerebral Cortex Callosal Projection Neurons (CPN) Including Dual Projecting Cortical Callosal/Frontal Projection Neurons (CPN/FPN).

The neocortex is composed of many distinct subtypes of neurons that must form precise subtype-specific connections to enable the cortex to perform complex functions. Callosal projection neurons (CPN) are the broad population of commissural neurons that connect the cerebral hemispheres via the corpus callosum (CC). Currently, how the remarkable diversity of CPN subtypes and connectivity is specified, and how they differentiate to form highly precise and specific circuits, are largely unknown. We identify in mouse that the lipid-bound scaffolding domain protein Caveolin 1 (CAV1) is specifically expressed by a unique subpopulation of Layer V CPN that maintain dual ipsilateral frontal projections to premotor cortex. CAV1 is expressed by over 80% of these dual projecting callosal/frontal projection neurons (CPN/FPN), with expression peaking early postnatally as axonal and dendritic targets are being reached and refined. CAV1 is localized to the soma and dendrites of CPN/FPN, a unique population of neurons that shares information both between hemispheres and with premotor cortex, suggesting function during postmitotic development and refinement of these neurons, rather than in their specification. Consistent with this, we find that Cav1 function is not necessary for the early specification of CPN/FPN, or for projecting to their dual axonal targets. CPN subtype-specific expression of Cav1 identifies and characterizes a first molecular component that distinguishes this functionally unique projection neuron population, a population that expands in primates, and is prototypical of additional dual and higher-order projection neuron subtypes.

Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity.

UNLABELLED: The neocortex contains hundreds to thousands of distinct subtypes of precisely connected neurons, allowing it to perform remarkably complex tasks of high-level cognition. Callosal projection neurons (CPN) connect the cerebral hemispheres via the corpus callosum, integrating cortical information and playing key roles in associative cognition. CPN are a strikingly diverse set of neuronal subpopulations, and development of this diversity requires precise control by a complex, interactive set of molecular effectors. We have found that the transcriptional coregulator Cited2 regulates and refines two stages of CPN development. Cited2 is expressed broadly by progenitors in the embryonic day 15.5 subventricular zone, during the peak of superficial layer CPN birth, with a progressive postmitotic refinement in expression, becoming restricted to CPN of the somatosensory cortex postnatally. We generated progenitor-stage and postmitotic forebrain-specific Cited2 conditional knock-out mice, using the Emx1-Cre and NEX-Cre mouse lines, respectively. We demonstrate that Cited2 functions in progenitors, but is not necessary postmitotically, to regulate both (1) broad generation of layer II/III CPN and (2) acquisition of precise area-specific molecular identity and axonal/dendritic connectivity of somatosensory CPN. This novel CPN subtype-specific and area-specific control from progenitor action of Cited2 adds yet another layer of complexity to the multistage developmental regulation of neocortical development. SIGNIFICANCE STATEMENT: This study identifies Cited2 as a novel subtype-specific and area-specific control over development of distinct subpopulations within the broad population of callosal projection neurons (CPN), whose axons connect the two cerebral hemispheres via the corpus callosum (CC). Currently, how the remarkable diversity of CPN subtypes is specified, and how they differentiate to form highly precise and specific circuits, are largely unknown. We found that Cited2 functions within subventricular zone progenitors to both broadly regulate generation of superficial layer CPN throughout the neocortex, and to refine precise area-specific development and connectivity of somatosensory CPN. Gaining insight into molecular development and heterogeneity of CPN will advance understanding of both diverse functions of CPN and of the remarkable range of neurodevelopmental deficits correlated with CPN/CC development.

Reduction of aberrant NF-κB signalling ameliorates Rett syndrome phenotypes in Mecp2-null mice.

Mutations in the transcriptional regulator Mecp2 cause the severe X-linked neurodevelopmental disorder Rett syndrome (RTT). In this study, we investigate genes that function downstream of MeCP2 in cerebral cortex circuitry, and identify upregulation of Irak1, a central component of the NF-κB pathway. We show that overexpression of Irak1 mimics the reduced dendritic complexity of Mecp2-null cortical callosal projection neurons (CPN), and that NF-κB signalling is upregulated in the cortex with Mecp2 loss-of-function. Strikingly, we find that genetically reducing NF-κB signalling in Mecp2-null mice not only ameliorates CPN dendritic complexity but also substantially extends their normally shortened lifespan, indicating broader roles for NF-κB signalling in RTT pathogenesis. These results provide new insight into both the fundamental neurobiology of RTT, and potential therapeutic strategies via NF-κB pathway modulation.

Development, specification, and diversity of callosal projection neurons.

Callosal projection neurons (CPN) are a diverse population of neocortical projection neurons that connect the two hemispheres of the cerebral cortex via the corpus callosum. They play key roles in high-level associative connectivity, and have been implicated in cognitive syndromes of high-level associative dysfunction, such as autism spectrum disorders. CPN evolved relatively recently compared to other cortical neuron populations, and have undergone disproportionately large expansion from mouse to human. While much is known about the anatomical trajectory of developing CPN axons, and progress has been made in identifying cellular and molecular controls over midline crossing, only recently have molecular-genetic controls been identified that specify CPN populations, and help define CPN subpopulations. In this review, we discuss the development, diversity and evolution of CPN.

MBD2 and MeCP2 regulate distinct transitions in the stage-specific differentiation of olfactory receptor neurons.

DNA methylation-dependent gene silencing is initiated by DNA methyltransferases (DNMTs) and mediated by methyl-binding domain proteins (MBDs), which recruit histone deacetylases (HDACs) to silence DNA, a process that is essential for normal development. Here, we show that the MBD proteins MBD2 and MeCP2 regulate distinct transitional stages of olfactory receptor neuron (ORN) differentiation in vivo. Mbd2 null progenitors display enhanced proliferation, recapitulated by HDAC inhibition, and Mbd2 null ORNs have a decreased lifespan. Mecp2 null ORNs, on the other hand, temporarily stall at the stage of terminal differentiation, retaining expression of the immature neuronal protein GAP43 after initiating expression of mature neuronal genes. The Gap43 promoter is highly methylated in the mature, but not embryonic olfactory epithelium (OE), suggesting that Gap43 may be regulated by DNA methylation during ORN differentiation. Thus, MBD2 and MeCP2 may mediate distinct, sequential transitions of ORN differentiation-an epigenetic mechanism that may be relevant to developmental regulation throughout the nervous system.

Novel subtype-specific genes identify distinct subpopulations of callosal projection neurons.

Little is known about the molecular development and heterogeneity of callosal projection neurons (CPN), cortical commissural neurons that connect homotopic regions of the two cerebral hemispheres via the corpus callosum and that are critical for bilateral integration of cortical information. Here we report on the identification of a series of genes that individually and in combination define CPN and novel CPN subpopulations during embryonic and postnatal development. We used in situ hybridization analysis, immunocytochemistry, and retrograde labeling to define the layer-specific and neuron-type-specific distribution of these newly identified CPN genes across different stages of maturation. We demonstrate that a subset of these genes (e.g., Hspb3 and Lpl) appear specific to all CPN (in layers II/III and V-VI), whereas others (e.g., Nectin-3, Plexin-D1, and Dkk3) discriminate between CPN of the deep layers and those of the upper layers. Furthermore, the data show that several genes finely subdivide CPN within individual layers and appear to label CPN subpopulations that have not been described previously using anatomical or morphological criteria. The genes identified here likely reflect the existence of distinct programs of gene expression governing the development, maturation, and function of the newly identified subpopulations of CPN. Together, these data define the first set of genes that identify and molecularly subcategorize distinct populations of callosal projection neurons, often located in distinct subdivisions of the canonical cortical laminae.

Epigenetic regulation of nervous system development by DNA methylation and histone deacetylation.

Alterations in the epigenetic modulation of gene expression have been implicated in several developmental disorders, cancer, and recently, in a variety of mental retardation and complex psychiatric disorders. A great deal of effort is now being focused on why the nervous system may be susceptible to shifts in activity of epigenetic modifiers. The answer may simply be that the mammalian nervous system must first produce the most complex degree of developmental patterning in biology and hardwire cells functionally in place postnatally, while still allowing for significant plasticity in order for the brain to respond to a rapidly changing environment. DNA methylation and histone deacetylation are two major epigenetic modifications that contribute to the stability of gene expression states. Perturbing DNA methylation, or disrupting the downstream response to DNA methylation - methyl-CpG-binding domain proteins (MBDs) and histone deacetylases (HDACs) - by genetic or pharmacological means, has revealed a critical requirement for epigenetic regulation in brain development, learning, and mature nervous system stability, and has identified the first distinct gene sets that are epigenetically regulated within the nervous system. Epigenetically modifying chromatin structure in response to different stimuli appears to be an ideal mechanism to generate continuous cellular diversity and coordinate shifts in gene expression at successive stages of brain development - all the way from deciding which kind of a neuron to generate, through to how many synapses a neuron can support. Here, we review the evidence supporting a role for DNA methylation and histone deacetylation in nervous system development and mature function, and present a basis from which to understand how the clinical use of HDAC inhibitors may impact nervous system function.

Histone deacetylases 1 and 2 are expressed at distinct stages of neuro-glial development.

The deacetylation of histone proteins, catalyzed by histone deacetylases (HDACs), is a common epigenetic modification of chromatin, associated with gene silencing. Although HDAC inhibitors are used clinically to treat nervous system disorders, such as epilepsy, very little is known about the expression pattern of the HDACs in the central nervous system. Identifying the cell types and developmental stages that express HDAC1 and HDAC2 within the brain is important for determining the therapeutic mode of action of HDAC inhibitors, and evaluating potential side effects. Here, we examined the expression of HDAC1 and HDAC2 in the murine brain at multiple developmental ages. HDAC1 is expressed in neural stem cells/progenitors and glia. In contrast, HDAC2 is initiated in neural progenitors and is up-regulated in post-mitotic neuroblasts and neurons, but not in fully differentiated glia. These results identify key developmental stages of HDAC expression and suggest transitions of neural development that may utilize HDAC1 and/or HDAC2.

Stage-specific induction of DNA methyltransferases in olfactory receptor neuron development.

DNA methylation-dependent gene silencing, mediated by DNA methyltransferases (DNMTs), is essential for normal mammalian development and its dysregulation has been implicated in neurodevelopmental disorders. Despite this, little is known about DNMTs in the developing or mature nervous system. Here, we show that DNMT1, 3a and 3b are expressed at discrete developmental stages in the olfactory neuron lineage, coincident with key shifts in developmental gene expression. DNMT1 is induced in cycling progenitors and is retained in post-mitotic olfactory receptor neurons (ORNs). DNMT3b is restricted to mitotic olfactory progenitors, whereas DNMT3a is expressed only in post-mitotic immature neurons prior to ORN terminal maturation, coincident with histone deacetylase 2 (HDAC2), a key downstream effector of methylation-dependent chromatin condensation. Similar stage-specific expression of DNMT3b and 3a was also found in other developing sensory and CNS neurons. This suggests that progressive lineage restriction regulated by methylation-dependent silencing could be a highly conserved mechanism shared by multiple lineages in the developing nervous system.

Olfactory horizontal basal cells demonstrate a conserved multipotent progenitor phenotype.

Stem cells of adult regenerative organs share a common goal but few established conserved mechanisms. Within the neural stem cell niche of the mouse olfactory epithelium, we identified a combination of extracellular matrix (ECM) receptors that regulate adhesion and mitosis in non-neural stem cells [intercellular adhesion molecule-1 (ICAM-1), beta1, beta4, and alpha-1, -3, and -6 integrins] and on horizontal basal cells (HBCs), candidate olfactory neuro-epithelial progenitors. Using ECM receptors as our guide, we recreated a defined microenvironment in vitro that mimics olfactory basal lamina and, when supplemented with epidermal growth factor, transforming growth factor alpha, and leukemia inhibitory factor, allows us to preferentially expand multiple clonal adherent colony phenotypes from individual ICAM-1+ and ICAM-1+/beta1 integrin+-selected HBCs. The most highly mitotic colony-forming HBCs demonstrate multipotency, spontaneously generating more ICAM-positive presumptive HBCs, a combination of olfactory neuroglial progenitors, and neurons of olfactory and potentially nonolfactory phenotypes. HBCs thus possess a conserved adhesion receptor expression profile similar to non-neural stem cells, preferential self-replication in an in vitro environment mimicking their in vivo niche, and contain subpopulations of cells that can produce multiple differentiated neuronal and glial progeny from within and beyond the olfactory system in vitro.