The embryonic basal ganglia generates multiple projection neurons and interneuron subtypes from distinct progenitor domains. Combinatorial interactions of transcription factors and chromatin are thought to regulate gene expression. In the medial ganglionic eminence, the NKX2-1 transcription factor controls regional identity and, with LHX6, is necessary to specify pallidal projection neurons and forebrain interneurons. Here, we dissected the molecular functions of NKX2-1 by defining its chromosomal binding, regulation of gene expression, and epigenetic state. NKX2-1 binding at distal regulatory elements led to a repressed epigenetic state and transcriptional repression in the ventricular zone. Conversely, NKX2-1 is required to establish a permissive chromatin state and transcriptional activation in the sub-ventricular and mantle zones. Moreover, combinatorial binding of NKX2-1 and LHX6 promotes transcriptionally permissive chromatin and activates genes expressed in cortical migrating interneurons. Our integrated approach provides a foundation for elucidating transcriptional networks guiding the development of the MGE and its descendants.

Little is known about genetic mechanisms that regulate the ratio of cortical excitatory and inhibitory neurons. We show that NPAS1 and NPAS3 transcription factors (TFs) are expressed in progenitor domains of the mouse basal ganglia (subpallium, MGE, and CGE). NPAS1(-/-) mutants had increased proliferation, ERK signaling, and expression of Arx in the MGE and CGE. NPAS1(-/-) mutants also had increased neocortical inhibition (sIPSC and mIPSC) and generated an excess of somatostatin(+) (SST) (MGE-derived) and vasoactive intestinal polypeptide(+) (VIP) (CGE-derived) neocortical interneurons, but had a normal density of parvalbumin(+) (PV) (MGE-derived) interneurons. In contrast, NPAS3(-/-) mutants showed decreased proliferation and ERK signaling in progenitors of the ganglionic eminences and had fewer SST(+) and VIP(+) interneurons. NPAS1 repressed activity of an Arx enhancer, and Arx overexpression resulted in increased proliferation of CGE progenitors. These results provide insights into genetic regulation of cortical interneuron numbers and cortical inhibitory tone.

Short non-coding transcripts can be transcribed from distant-acting transcriptional enhancer loci, but the prevalence of such enhancer RNAs (eRNAs) within the transcriptome, and the association of eRNA expression with tissue-specific enhancer activity in vivo remain poorly understood. Here, we investigated the expression dynamics of tissue-specific non-coding RNAs in embryonic mouse tissues via deep RNA sequencing. Overall, approximately 80% of validated in vivo enhancers show tissue-specific RNA expression that correlates with tissue-specific enhancer activity. Globally, we identified thousands of tissue-specifically transcribed non-coding regions (TSTRs) displaying various genomic hallmarks of bona fide enhancers. In transgenic mouse reporter assays, over half of tested TSTRs functioned as enhancers with reproducible activity in the predicted tissue. Together, our results demonstrate that tissue-specific eRNA expression is a common feature of in vivo enhancers, as well as a major source of extragenic transcription, and that eRNA expression signatures can be used to predict tissue-specific enhancers independent of known epigenomic enhancer marks.

The accurate and comprehensive identification of functional regulatory sequences in mammalian genomes remains a major challenge. Here we describe site-specific integration fluorescence-activated cell sorting followed by sequencing (SIF-seq), an unbiased, medium-throughput functional assay for the discovery of distant-acting enhancers. Targeted single-copy genomic integration into pluripotent cells, reporter assays and flow cytometry are coupled with high-throughput DNA sequencing to enable parallel screening of large numbers of DNA sequences. By functionally interrogating >500 kilobases (kb) of mouse and human sequence in mouse embryonic stem cells for enhancer activity we identified enhancers at pluripotency loci including NANOG. In in vitro-differentiated cardiomyocytes and neural progenitor cells, we identified cardiac enhancers and neuronal enhancers, respectively. SIF-seq is a powerful and flexible method for de novo functional identification of mammalian enhancers in a potentially wide variety of cell types.

Massively parallel sequencing of barcoded DNA samples significantly increases screening efficiency for clinically important genes. Short read aligners are well suited to single nucleotide and indel detection. However, methods for CNV detection from targeted enrichment are lacking. We present a method combining coverage with map information for the identification of deletions and duplications in targeted sequence data.

The mammalian telencephalon plays critical roles in cognition, motor function, and emotion. Though many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here, we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified more than 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising more than 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders.

Several methods to identify tagging single-nucleotide polymorphisms (SNPs) are in common use for genetic epidemiologic studies; however, there may be loss of information when using only a subset of SNPs. We sought to compare the ability of commonly used pairwise, multimarker, and haplotype-based tagging SNP selection methods to detect known associations with quantitative expression phenotypes. Using data from HapMap release 21 on unrelated Utah residents with ancestors from northern and western Europe (CEPH-Utah, CEU), we selected tagging SNPs in five chromosomal regions using ldSelect, Tagger, and TagSNPs. We found that SNP subsets did not substantially overlap, and that the use of trio data did not greatly impact SNP selection. We then tested associations between HapMap genotypes and expression phenotypes on 28 CEU individuals as part of Genetic Analysis Workshop 15. Relative to the use of all SNPs (n = 210 SNPs across all regions), most subset methods were able to detect single-SNP and haplotype associations. Generally, pairwise selection approaches worked extremely well, relative to use of all SNPs, with marked reductions in the number of SNPs required. Haplotype-based approaches, which had identified smaller SNP subsets, missed associations in some regions. We conclude that the optimal tagging SNP method depends on the true model of the genetic association (i.e., whether a SNP or haplotype is responsible); unfortunately, this is often unknown at the time of SNP selection. Additional evaluations using empirical and simulated data are needed.

Medial ganglionic eminence (MGE)-derived somatostatin (SST)+ and parvalbumin (PV)+ cortical interneurons (CINs), have characteristic molecular, anatomical and physiological properties. However, mechanisms regulating their diversity remain poorly understood. Here, we show that conditional loss of the Tuberous Sclerosis Complex (TSC) gene, Tsc1, which inhibits the mammalian target of rapamycin (MTOR), causes a subset of SST+ CINs, to express PV and adopt fast-spiking (FS) properties, characteristic of PV+ CINs. Milder intermediate phenotypes also occur when only one allele of Tsc1 is deleted. Notably, treatment of adult mice with rapamycin, which inhibits MTOR, reverses the phenotypes. These data reveal novel functions of MTOR signaling in regulating PV expression and FS properties, which may contribute to TSC neuropsychiatric symptoms. Moreover, they suggest that CINs can exhibit properties intermediate between those classically associated with PV+ or SST+ CINs, which may be dynamically regulated by the MTOR signaling.

The TSC1 and TSC2 genes are connected to multiple syndromes from Tuberous Sclerosis Complex (TSC) to autism spectrum disorder (ASD), with uncertainty if genetic variants cause all or subsets of phenotypes based on the location and type of change. For TSC1, few have addressed if non-TSC associated genetic variants have direct contributions to changes in neurological genotype-to-phenotype impacts, including elevated rates of ASD and seizures. Dominant variants cause TSC, yet TSC1 has many heritable variants not dominant for TSC that are poorly understood in neurological function, with some associated with ASD. Herein, we examined how missense variants in TSC1, R336W, T360N, T393I, S403L, and H732Y, impacted the development of cortical inhibitory interneurons, cell-types whose molecular, cellular, and physiological properties are altered after the loss of mouse TSC1. We found these variants complemented a known phenotype caused by loss of TSC1, increased cell size. However, distinct variants, particularly S403L showed deficits in complementing an increase in parvalbumin levels and exhibited smaller amplitude after hyperpolarizations. Overall, these data show that subtle phenotypes can be induced by some TSC1 missense variants and provide an in vivo system to assess TSC1 variants' neurological impact better.

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants1,2. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.

Genetic studies of autism have revealed causal roles for chromatin remodeling gene mutations. Chromodomain helicase DNA binding protein 8 (CHD8) encodes a chromatin remodeler with significant de novo mutation rates in sporadic autism. However, relationships between CHD8 genomic function and autism-relevant biology remain poorly elucidated. Published studies utilizing ChIP-seq to map CHD8 protein-DNA interactions have high variability, consistent with technical challenges and limitations associated with this method. Thus, complementary approaches are needed to establish CHD8 genomic targets and regulatory functions in developing brain. We used in utero CHD8 Targeted DamID followed by sequencing (TaDa-seq) to characterize CHD8 binding in embryonic mouse cortex. CHD8 TaDa-seq reproduced interaction patterns observed from ChIP-seq and further highlighted CHD8 distal interactions associated with neuronal loci. This study establishes TaDa-seq as a useful alternative for mapping protein-DNA interactions in vivo and provides insights into the regulatory targets of CHD8 and autism-relevant pathophysiology associated with CHD8 mutations.

In utero exposure to maternal immune activation (MIA) is an environmental risk factor for neurodevelopmental and neuropsychiatric disorders. Animal models provide an opportunity to identify mechanisms driving neuropathology associated with MIA. We performed time-course transcriptional profiling of mouse cortical development following induced MIA via poly(I:C) injection at E12.5. MIA-driven transcriptional changes were validated via protein analysis, and parallel perturbations to cortical neuroanatomy were identified via imaging. MIA-induced acute upregulation of genes associated with hypoxia, immune signaling, and angiogenesis, by 6 hr following exposure. This acute response was followed by changes in proliferation, neuronal and glial specification, and cortical lamination that emerged at E14.5 and peaked at E17.5. Decreased numbers of proliferative cells in germinal zones and alterations in neuronal and glial populations were identified in the MIA-exposed cortex. Overall, paired transcriptomic and neuroanatomical characterization revealed a sequence of perturbations to corticogenesis driven by mid-gestational MIA.

RAS/MAPK gene dysfunction underlies various cancers and neurocognitive disorders. Although the roles of RAS/MAPK genes have been well studied in cancer, less is known about their function during neurodevelopment. There are many genes that work in concert to regulate RAS/MAPK signaling, suggesting that if common brain phenotypes could be discovered they could have a broad impact on the many other disorders caused by distinct RAS/MAPK genes. We assessed the cellular and molecular consequences of hyperactivating the RAS/MAPK pathway using two distinct genes in a cell type previously implicated in RAS/MAPK-mediated cognitive changes, cortical GABAergic interneurons. We uncovered some GABAergic core programs that are commonly altered in each of the mutants. Notably, hyperactive RAS/MAPK mutants bias developing cortical interneurons towards those that are somatostatin positive. The increase in somatostatin-positive interneurons could also be prevented by pharmacological inhibition of the core RAS/MAPK signaling pathway. Overall, these findings present new insights into how different RAS/MAPK mutations can converge on GABAergic interneurons, which may be important for other RAS/MAPK genes and related disorders.

The forebrain is the seat of higher-order brain functions, and many human neuropsychiatric disorders are due to genetic defects affecting forebrain development, making it imperative to understand the underlying genetic circuitry. Recent progress now makes it possible to begin fully elucidating the genomic regulatory mechanisms that control forebrain gene expression. Herein, we discuss the current knowledge of how transcription factors drive gene expression programs through their interactions with cis-acting genomic elements, such as enhancers; how analyses of chromatin and DNA modifications provide insights into gene expression states; and how these approaches yield insights into the evolution of the human brain.

Chimeric genes can be caused by structural genomic rearrangements that fuse together portions of two different genes to create a novel gene. We hypothesize that brain-expressed chimeras may contribute to schizophrenia. Individuals with schizophrenia and control individuals were screened genome wide for copy-number variants (CNVs) that disrupted two genes on the same DNA strand. Candidate events were filtered for predicted brain expression and for frequency < 0.001 in an independent series of 20,000 controls. Four of 124 affected individuals and zero of 290 control individuals harbored such events (p = 0.002); a 47 kb duplication disrupted MATK and ZFR2, a 58 kb duplication disrupted PLEKHD1 and SLC39A9, a 121 kb duplication disrupted DNAJA2 and NETO2, and a 150 kb deletion disrupted MAP3K3 and DDX42. Each fusion produced a stable protein when exogenously expressed in cultured cells. We examined whether these chimeras differed from their parent genes in localization, regulation, or function. Subcellular localizations of DNAJA2-NETO2 and MAP3K3-DDX42 differed from their parent genes. On the basis of the expression profile of the MATK promoter, MATK-ZFR2 is likely to be far more highly expressed in the brain during development than the ZFR2 parent gene. MATK-ZFR2 includes a ZFR2-derived isoform that we demonstrate localizes preferentially to neuronal dendritic branch sites. These results suggest that the formation of chimeric genes is a mechanism by which CNVs contribute to schizophrenia and that, by interfering with parent gene function, chimeras may disrupt critical brain processes, including neurogenesis, neuronal differentiation, and dendritic arborization.

Enhancers are distal regulatory elements that can activate tissue-specific gene expression and are abundant throughout mammalian genomes. Although substantial progress has been made toward genome-wide annotation of mammalian enhancers, their temporal activity patterns and global contributions in the context of developmental in vivo processes remain poorly explored. Here we used epigenomic profiling for H3K27ac, a mark of active enhancers, coupled to transgenic mouse assays to examine the genome-wide utilization of enhancers in three different mouse tissues across seven developmental stages. The majority of the ∼90,000 enhancers identified exhibited tightly temporally restricted predicted activity windows and were associated with stage-specific biological functions and regulatory pathways in individual tissues. Comparative genomic analysis revealed that evolutionary conservation of enhancers decreases following midgestation across all tissues examined. The dynamic enhancer activities uncovered in this study illuminate rapid and pervasive temporal in vivo changes in enhancer usage that underlie processes central to development and disease.

Synaptogenesis has been extensively studied along with dendritic spine development in glutamatergic pyramidal neurons, however synapse development in cortical interneurons, which are largely aspiny, is comparatively less well understood. Dact1, one of 3 paralogous Dact (Dapper/Frodo) family members in mammals, is a scaffold protein implicated in both the Wnt/β-catenin and the Wnt/Planar Cell Polarity pathways. We show here that Dact1 is expressed in immature cortical interneurons. Although Dact1 is first expressed in interneuron precursors during proliferative and migratory stages, constitutive Dact1 mutant mice have no major defects in numbers or migration of these neurons. However, cultured cortical interneurons derived from these mice have reduced numbers of excitatory synapses on their dendrites. We selectively eliminated Dact1 from mouse cortical interneurons using a conditional knock-out strategy with a Dlx-I12b enhancer-Cre allele, and thereby demonstrate a cell-autonomous role for Dact1 during postsynaptic development. Confirming this cell-autonomous role, we show that synapse numbers in Dact1 deficient cortical interneurons are rescued by virally-mediated re-expression of Dact1 specifically targeted to these cells. Synapse numbers in these neurons are also rescued by similarly targeted expression of the Dact1 binding partner Dishevelled-1, and partially rescued by expression of Disrupted in Schizophrenia-1, a synaptic protein genetically implicated in susceptibility to several major mental illnesses. In sum, our results support a novel cell-autonomous postsynaptic role for Dact1, in cooperation with Dishevelled-1 and possibly Disrupted in Schizophrenia-1, in the formation of synapses on cortical interneuron dendrites.

The SMARCA4 (also known as BRG1 in humans) chromatin remodeling factor is critical for establishing lineage-specific chromatin states during early mammalian development. However, the role of SMARCA4 in tissue-specific gene regulation during embryogenesis remains poorly defined. To investigate the genome-wide binding landscape of SMARCA4 in differentiating tissues, we engineered a Smarca4(FLAG) knock-in mouse line. Using ChIP-seq, we identified ∼51,000 SMARCA4-associated regions across six embryonic mouse tissues (forebrain, hindbrain, neural tube, heart, limb, and face) at mid-gestation (E11.5). The majority of these regions was distal from promoters and showed dynamic occupancy, with most distal SMARCA4 sites (73%) confined to a single or limited subset of tissues. To further characterize these regions, we profiled active and repressive histone marks in the same tissues and examined the intersection of informative chromatin states and SMARCA4 binding. This revealed distinct classes of distal SMARCA4-associated elements characterized by activating and repressive chromatin signatures that were associated with tissue-specific up- or down-regulation of gene expression and relevant active/repressed biological pathways. We further demonstrate the predicted active regulatory properties of SMARCA4-associated elements by retrospective analysis of tissue-specific enhancers and direct testing of SMARCA4-bound regions in transgenic mouse assays. Our results indicate a dual active/repressive function of SMARCA4 at distal regulatory sequences in vivo and support its role in tissue-specific gene regulation during embryonic development.

Epidemiological and clinical evidence points to cancer as a comorbidity in people with autism spectrum disorders (ASD). A significant overlap of genes and biological processes between both diseases has also been reported.

Mafb and c-Maf transcription factor (TF) expression is enriched in medial ganglionic eminence (MGE) lineages, beginning in late-secondary progenitors and continuing into mature parvalbumin (PV+) and somatostatin (SST+) interneurons. However, the functions of Maf TFs in MGE development remain to be elucidated. Herein, Mafb and c-Maf were conditionally deleted, alone and together, in the MGE and its lineages. Analyses of Maf mutant mice revealed redundant functions of Mafb and c-Maf in secondary MGE progenitors, where they repress the generation of SST+ cortical and hippocampal interneurons. By contrast, Mafb and c-Maf have distinct roles in postnatal cortical interneuron (CIN) morphological maturation, synaptogenesis, and cortical circuit integration. Thus, Mafb and c-Maf have redundant and opposing functions at different steps in CIN development.