Targeted blockade of aberrant sodium current in a stem cell-derived neuron model of SCN3A encephalopathy.

Missense variants in SCN3A encoding the voltage-gated sodium (Na+) channel α subunit Nav1.3 are associated with SCN3A-related neurodevelopmental disorder (SCN3A-NDD), a spectrum of disease that includes epilepsy and malformation of cortical development. How genetic variation in SCN3A leads to pathology remains unclear, as prior electrophysiological work on disease-associated variants has been performed exclusively in heterologous cell systems. To further investigate the mechanisms of SCN3A-NDD pathogenesis, we used CRISPR/Cas9 gene editing to modify a control human induced pluripotent stem cell (iPSC) line to express the recurrent de novo missense variant SCN3A c.2624T>C (p.Ile875Thr). With the established Ngn2 rapid induction protocol, we generated glutamatergic forebrain-like neurons (iNeurons), which we showed to express SCN3A mRNA and Nav1.3-mediated Na+ currents. We performed detailed whole-cell patch clamp recordings to determine the effect of the SCN3A-p.Ile875Thr variant on endogenous Na+ currents in, and intrinsic excitability of, human neurons. Compared to control iNeurons, variant-expressing iNeurons exhibit markedly increased slowly-inactivating/persistent Na+ current, abnormal firing patterns with paroxysmal bursting and plateau-like potentials with action potential failure, and a hyperpolarized voltage threshold for action potential generation. We then validated these findings using a separate iPSC line generated from a patient harbouring the SCN3A-p.Ile875Thr variant compared to a corresponding CRISPR-corrected isogenic control line. Finally, we found that application of the Nav1.3-selective blocker ICA-121431 normalizes action potential threshold and aberrant firing patterns in SCN3A-p.Ile1875Thr iNeurons; in contrast, consistent with action as a Na+ channel blocker, ICA-121431 decreases excitability of control iNeurons. Our findings demonstrate that iNeurons can model the effects of genetic variation in SCN3A yet reveal a complex relationship between gain-of-function at the level of the ion channel versus impact on neuronal excitability. Given the transient expression of SCN3A in the developing human nervous system, selective blockade or suppression of Nav1.3-containing Na+ channels could represent a therapeutic approach towards SCN3A-NDD.

The NANCI-Nkx2.1 gene duplex buffers Nkx2.1 expression to maintain lung development and homeostasis.

A subset of long noncoding RNAs (lncRNAs) is spatially correlated with transcription factors (TFs) across the genome, but how these lncRNA-TF gene duplexes regulate tissue development and homeostasis is unclear. We identified a feedback loop within the NANCI (Nkx2.1-associated noncoding intergenic RNA)-Nkx2.1 gene duplex that is essential for buffering Nkx2.1 expression, lung epithelial cell identity, and tissue homeostasis. Within this locus, Nkx2.1 directly inhibits NANCI, while NANCI acts in cis to promote Nkx2.1 transcription. Although loss of NANCI alone does not adversely affect lung development, concurrent heterozygous mutations in both NANCI and Nkx2.1 leads to persistent Nkx2.1 deficiency and reprogramming of lung epithelial cells to a posterior endoderm fate. This disruption in the NANCI-Nkx2.1 gene duplex results in a defective perinatal innate immune response, tissue damage, and progressive degeneration of the adult lung. These data point to a mechanism in which lncRNAs act as rheostats within lncRNA-TF gene duplex loci that buffer TF expression, thereby maintaining tissue-specific cellular identity during development and postnatal homeostasis.

22q11.2 Deletion-Associated Blood-Brain Barrier Permeability Potentiates Systemic Capillary Leak Syndrome Neurologic Features.

We present a case study of a young male with a history of 22q11.2 deletion syndrome (22qDS), diagnosed with systemic capillary leak syndrome (SCLS) who presented with acute onset of diffuse anasarca and sub-comatose obtundation. We hypothesized that his co-presentation of neurological sequelae might be due to blood-brain barrier (BBB) susceptibility conferred by the 22q11.2 deletion, a phenotype that we have previously identified in 22qDS. Using pre- and post-intravenous immunoglobulins (IVIG) patient serum, we studied circulating biomarkers of inflammation and assessed the potential susceptibility of the 22qDS BBB. We employed in vitro cultures of differentiated BBB-like endothelial cells derived from a 22qDS patient and a healthy control. We found evidence of peripheral inflammation and increased serum lipopolysaccharide (LPS) alongside endothelial cells in circulation. We report that the patient's serum significantly impairs barrier function of the 22qDS BBB compared to control. Only two other cases of pediatric SCLS with neurologic symptoms have been reported, and genetic risk factors have been suggested in both instances. As the third case to be reported, our findings are consistent with the hypothesis that genetic susceptibility of the BBB conferred by genes such as claudin-5 deleted in the 22q11.2 region promoted neurologic involvement during SCLS in this patient.

Immune status of the murine 22q11.2 deletion syndrome model.

Mice modeling the hemizygous deletion of chromosome 22q11.2 (22qMc) have been utilized to address various clinical phenotypes associated with the disease, including cardiac malformations, altered neural circuitry, and behavioral deficits. Yet, the status of the T cell compartment, an important clinical concern among 22q11.2 deletion syndrome (22qDS) patients, has not been addressed. While infancy and early childhood in 22qDS are associated with deficient T cell numbers and thymic hypoplasia, which can be severe in a small subset of patients, studies suggest normalization of the T cell counts by adulthood. We found that adult 22qMc do not exhibit thymic hypoplasia or altered thymic T cell development. Our findings that immune cell counts and inflammatory T cell activation are unaffected in 22qMc lend support to the hypothesis that human 22qDS immunodeficiencies are secondary to thymic hypoplasia, rather than intrinsic effects due to the deletion. Furthermore, the 22q11.2 deletion does not impact the differentiation capacity of T cells, nor their activity and response during inflammatory activation. Thus, 22qMc reflects the T cell compartment in adult 22qDS patients, and our findings suggest that 22qMc may serve as a novel model to address experimental and translational aspects of immunity in 22qDS.

Deficits in Seizure Threshold and Other Behaviors in Adult Mice without Gross Neuroanatomic Injury after Late Gestation Transient Prenatal Hypoxia.

Intrauterine hypoxia is a common cause of brain injury in children resulting in a broad spectrum of long-term neurodevelopmental sequela, including life-long disabilities that can occur even in the absence of severe neuroanatomic damage. Postnatal hypoxia-ischemia rodent models are commonly used to understand the effects of ischemia and transient hypoxia on the developing brain. Postnatal models, however, have some limitations. First, they do not test the impact of placental pathologies on outcomes from hypoxia. Second, they primarily recapitulate severe injury because they provoke substantial cell death, which is not seen in children with mild hypoxic injury. Lastly, they do not model preterm hypoxic injury. Prenatal models of hypoxia in mice may allow us to address some of these limitations to expand our understanding of developmental brain injury. The published rodent models of prenatal hypoxia employ multiple days of hypoxic exposure or complicated surgical procedures, making these models challenging to perform consistently in mice. Furthermore, large animal models suggest that transient prenatal hypoxia without ischemia is sufficient to lead to significant functional impairment to the developing brain. However, these large animal studies are resource-intensive and not readily amenable to mechanistic molecular studies. Therefore, here we characterized the effect of late gestation (embryonic day 17.5) transient prenatal hypoxia (5% inspired oxygen) on long-term anatomical and neurodevelopmental outcomes in mice. Late gestation transient prenatal hypoxia increased hypoxia-inducible factor 1 alpha protein levels (a marker of hypoxic exposure) in the fetal brain. Hypoxia exposure predisposed animals to decreased weight at postnatal day 2, which normalized by day 8. However, hypoxia did not affect gestational age at birth, litter size at birth, or pup survival. No differences in fetal brain cell death or long-term gray or white matter changes resulted from hypoxia. Animals exposed to prenatal hypoxia did have several long-term functional consequences, including sex-dichotomous changes. Hypoxia exposure was associated with a decreased seizure threshold and abnormalities in hindlimb strength and repetitive behaviors in males and females. Males exposed to hypoxia had increased anxiety-related deficits, whereas females had deficits in social interaction. Neither sex developed any motor or visual learning deficits. This study demonstrates that late gestation transient prenatal hypoxia in mice is a simple, clinically relevant paradigm for studying putative environmental and genetic modulators of the long-term effects of hypoxia on the developing brain.

MitoScape: A big-data, machine-learning platform for obtaining mitochondrial DNA from next-generation sequencing data.

The growing number of next-generation sequencing (NGS) data presents a unique opportunity to study the combined impact of mitochondrial and nuclear-encoded genetic variation in complex disease. Mitochondrial DNA variants and in particular, heteroplasmic variants, are critical for determining human disease severity. While there are approaches for obtaining mitochondrial DNA variants from NGS data, these software do not account for the unique characteristics of mitochondrial genetics and can be inaccurate even for homoplasmic variants. We introduce MitoScape, a novel, big-data, software for extracting mitochondrial DNA sequences from NGS. MitoScape adopts a novel departure from other algorithms by using machine learning to model the unique characteristics of mitochondrial genetics. We also employ a novel approach of using rho-zero (mitochondrial DNA-depleted) data to model nuclear-encoded mitochondrial sequences. We showed that MitoScape produces accurate heteroplasmy estimates using gold-standard mitochondrial DNA data. We provide a comprehensive comparison of the most common tools for obtaining mtDNA variants from NGS and showed that MitoScape had superior performance to compared tools in every statistically category we compared, including false positives and false negatives. By applying MitoScape to common disease examples, we illustrate how MitoScape facilitates important heteroplasmy-disease association discoveries by expanding upon a reported association between hypertrophic cardiomyopathy and mitochondrial haplogroup T in men (adjusted p-value = 0.003). The improved accuracy of mitochondrial DNA variants produced by MitoScape will be instrumental in diagnosing disease in the context of personalized medicine and clinical diagnostics.

Disruption of the blood-brain barrier in 22q11.2 deletion syndrome.

Neuroimmune dysregulation is implicated in neuropsychiatric disorders including schizophrenia. As the blood-brain barrier is the immunological interface between the brain and the periphery, we investigated whether this vascular phenotype is intrinsically compromised in the most common genetic risk factor for schizophrenia, the 22q11.2 deletion syndrome (22qDS). Blood-brain barrier like endothelium differentiated from human 22qDS+schizophrenia-induced pluripotent stem cells exhibited impaired barrier integrity, a phenotype substantiated in a mouse model of 22qDS. The proinflammatory intercellular adhesion molecule-1 was upregulated in 22qDS+schizophrenia-induced blood-brain barrier and in 22qDS mice, indicating compromise of the blood-brain barrier immune privilege. This immune imbalance resulted in increased migration/activation of leucocytes crossing the 22qDS+schizophrenia blood-brain barrier. We also found heightened astrocyte activation in murine 22qDS, suggesting that the blood-brain barrier promotes astrocyte-mediated neuroinflammation. Finally, we substantiated these findings in post-mortem 22qDS brain tissue. Overall, the barrier-promoting and immune privilege properties of the 22qDS blood-brain barrier are compromised, and this might increase the risk for neuropsychiatric disease.

Generation of cerebral cortical GABAergic interneurons from pluripotent stem cells.

The cerebral cortex functions by the complex interactions of intrinsic and extrinsic neuronal activities, glial actions, and the effects of humoral factors. The intrinsic neuronal influences are mediated by two major subclasses: excitatory glutamatergic neurons that generally have axonal projections extending beyond the neuron's locality and inhibitory GABAergic neurons that generally project locally. These interneurons can be grouped based on morphological, neurochemical, electrophysiological, axonal targeting, and circuit influence characteristics. Cortical interneurons (CIns) can also be grouped based on their origins within the subcortical telencephalon. Interneuron subtypes, of which a dozen or more are thought to exist, are characterized by combinations of these subgrouping features. Due to their well-documented relevance to the causes of and treatments for neuropsychiatric disorders, and to their remarkable capacity to migrate extensively following transplantation, there has been tremendous interest in generating cortical GABAergic interneurons from human pluripotent stem cells. In this concise review, we discuss recent progress in understanding how interneuron subtypes are generated in vivo, and how that progress is being applied to the generation of rodent and human CIns in vitro. In addition, we will discuss approaches for the rigorous designation of interneuron subgroups or subtypes in transplantation studies, and challenges to this field, including the protracted maturation of human interneurons.

Duration of culture and sonic hedgehog signaling differentially specify PV versus SST cortical interneuron fates from embryonic stem cells.

Medial ganglionic eminence (MGE)-derived GABAergic cortical interneurons (cINs) consist of multiple subtypes that are involved in many cortical functions. They also have a remarkable capacity to migrate, survive and integrate into cortical circuitry after transplantation into postnatal cortex. These features have engendered considerable interest in generating distinct subgroups of interneurons from pluripotent stem cells (PSCs) for the study of interneuron fate and function, and for the development of cell-based therapies. Although advances have been made, the capacity to generate highly enriched pools of subgroup fate-committed interneuron progenitors from PSCs has remained elusive. Previous studies have suggested that the two main MGE-derived interneuron subgroups--those expressing somatostatin (SST) and those expressing parvalbumin (PV)--are specified in the MGE from Nkx2.1-expressing progenitors at higher or lower levels of sonic hedgehog (Shh) signaling, respectively. To further explore the role of Shh and other factors in cIN fate determination, we generated a reporter line such that Nkx2.1-expressing progenitors express mCherry and postmitotic Lhx6-expressing MGE-derived interneurons express GFP. Manipulations of Shh exposure and time in culture influenced the subgroup fates of ESC-derived interneurons. Exposure to higher Shh levels, and collecting GFP-expressing precursors at 12 days in culture, resulted in the strongest enrichment for SST interneurons over those expressing PV, whereas the strongest enrichment for PV interneurons was produced by lower Shh and by collecting mCherry-expressing cells after 17 days in culture. These findings confirm that fate determination of cIN subgroups is crucially influenced by Shh signaling, and provide a system for the further study of interneuron fate and function.

Loss of the neurodevelopmental gene Zswim6 alters striatal morphology and motor regulation.

The zinc-finger SWIM domain-containing protein 6 (ZSWIM6) is a protein of unknown function that has been associated with schizophrenia and limited educational attainment by three independent genome-wide association studies. Additionally, a putatively causal point mutation in ZSWIM6 has been identified in several cases of acromelic frontonasal dysostosis with severe intellectual disability. Despite the growing number of studies implicating ZSWIM6 as an important regulator of brain development, its role in this process has never been examined. Here, we report the generation of Zswim6 knockout mice and provide a detailed anatomical and behavioral characterization of the resulting phenotype. We show that Zswim6 is initially expressed widely during embryonic brain development but becomes restricted to the striatum postnatally. Loss of Zswim6 causes a reduction in striatal volume and changes in medium spiny neuron morphology. These changes are associated with alterations in motor control, including hyperactivity, impaired rotarod performance, repetitive movements, and behavioral hyperresponsiveness to amphetamine. Together, our results show that Zswim6 is indispensable to normal brain function and support the notion that Zswim6 might serve as an important contributor to the pathogenesis of schizophrenia and other neurodevelopmental disorders.

Interneuron precursor transplants in adult hippocampus reverse psychosis-relevant features in a mouse model of hippocampal disinhibition.

GABAergic interneuron hypofunction is hypothesized to underlie hippocampal dysfunction in schizophrenia. Here, we use the cyclin D2 knockout (Ccnd2(-/-)) mouse model to test potential links between hippocampal interneuron deficits and psychosis-relevant neurobehavioral phenotypes. Ccnd2(-/-) mice show cortical PV(+) interneuron reductions, prominently in hippocampus, associated with deficits in synaptic inhibition, increased in vivo spike activity of projection neurons, and increased in vivo basal metabolic activity (assessed with fMRI) in hippocampus. Ccnd2(-/-) mice show several neurophysiological and behavioral phenotypes that would be predicted to be produced by hippocampal disinhibition, including increased ventral tegmental area dopamine neuron population activity, behavioral hyperresponsiveness to amphetamine, and impairments in hippocampus-dependent cognition. Remarkably, transplantation of cells from the embryonic medial ganglionic eminence (the major origin of cerebral cortical interneurons) into the adult Ccnd2(-/-) caudoventral hippocampus reverses these psychosis-relevant phenotypes. Surviving neurons from these transplants are 97% GABAergic and widely distributed within the hippocampus. Up to 6 mo after the transplants, in vivo hippocampal metabolic activity is lowered, context-dependent learning and memory is improved, and dopamine neuron activity and the behavioral response to amphetamine are normalized. These findings establish functional links between hippocampal GABA interneuron deficits and psychosis-relevant dopaminergic and cognitive phenotypes, and support a rationale for targeting limbic cortical interneuron function in the prevention and treatment of schizophrenia.

Cortical parvalbumin GABAergic deficits with α7 nicotinic acetylcholine receptor deletion: implications for schizophrenia.

Dysfunction of cortical parvalbumin (PV)-containing GABAergic interneurons has been implicated in cognitive deficits of schizophrenia. In humans microdeletion of the CHRNA7 (α7 nicotinic acetylcholine receptor, nAChR) gene is associated with cortical dysfunction in a broad spectrum of neurodevelopmental and neuropsychiatric disorders including schizophrenia while in mice similar deletion causes analogous abnormalities including impaired attention, working-memory and learning. However, the pathophysiological roles of α7 nAChRs in cortical PV GABAergic development remain largely uncharacterized. In both in vivo and in vitro models, we identify here that deletion of the α7 nAChR gene in mice impairs cortical PV GABAergic development and recapitulates many of the characteristic neurochemical deficits in PV-positive GABAergic interneurons found in schizophrenia. α7 nAChR null mice had decreased cortical levels of GABAergic markers including PV, glutamic acid decarboxylase 65/67 (GAD65/67) and the α1 subunit of GABAA receptors, particularly reductions of PV and GAD67 levels in cortical PV-positive interneurons during late postnatal life and adulthood. Cortical GABAergic synaptic deficits were identified in the prefrontal cortex of α7 nAChR null mice and α7 nAChR null cortical cultures. Similar disruptions in development of PV-positive GABAergic interneurons and perisomatic synapses were found in cortical cultures lacking α7 nAChRs. Moreover, NMDA receptor expression was reduced in GABAergic interneurons, implicating NMDA receptor hypofunction in GABAergic deficits in α7 nAChR null mice. Our findings thus demonstrate impaired cortical PV GABAergic development and multiple characteristic neurochemical deficits reminiscent of schizophrenia in cortical PV-positive interneurons in α7 nAChR gene deletion models. This implicates crucial roles of α7 nAChRs in cortical PV GABAergic development and dysfunction in schizophrenia and other neuropsychiatric disorders.

Dense and overlapping innervation of pyramidal neurons by chandelier cells.

Chandelier (or axo-axonic) cells are a distinct group of GABAergic interneurons that innervate the axon initial segments of pyramidal cells and thus could have an important role controlling the activity of cortical circuits. To understand their connectivity, we labeled upper layers chandelier cells (ChCs) from mouse neocortex with a genetic strategy and studied how their axons contact local populations of pyramidal neurons, using immunohistochemical detection of axon initial segments. We studied ChCs located in the border of layers 1 and 2 from primary somatosensory cortex and found that practically all ChC axon terminals contact axon initial segments, with an average of three to five boutons per cartridge. By measuring the number of putative GABAergic synapses in initial segments, we estimate that each pyramidal neuron is innervated, on average, by four ChCs. Additionally, each individual ChC contacts 35-50% of pyramidal neurons within the areas traversed by its axonal arbor, with pockets of very high innervation density. Finally, ChCs have similar innervation patterns at different postnatal ages (P18-P90), with only relatively small lateral expansions of their arbor and increases in the total number of their cartridges during the developmental period analyzed. We conclude that ChCs innervate neighboring pyramidal neurons in a dense and overlapping manner, a connectivity pattern that could enable ChCs to exert a widespread influence on their local circuits.

Variant-to-function analysis of the childhood obesity chr12q13 locus implicates rs7132908 as a causal variant within the 3' UTR of FAIM2.

The ch12q13 locus is among the most significant childhood obesity loci identified in genome-wide association studies. This locus resides in a non-coding region within FAIM2; thus, the underlying causal variant(s) presumably influence disease susceptibility via cis-regulation. We implicated rs7132908 as a putative causal variant by leveraging our in-house 3D genomic data and public domain datasets. Using a luciferase reporter assay, we observed allele-specific cis-regulatory activity of the immediate region harboring rs7132908. We generated isogenic human embryonic stem cell lines homozygous for either rs7132908 allele to assess changes in gene expression and chromatin accessibility throughout a differentiation to hypothalamic neurons, a key cell type known to regulate feeding behavior. The rs7132908 obesity risk allele influenced expression of FAIM2 and other genes and decreased the proportion of neurons produced by differentiation. We have functionally validated rs7132908 as a causal obesity variant that temporally regulates nearby effector genes and influences neurodevelopment and survival.

3D genomic features across >50 diverse cell types reveal insights into the genomic architecture of childhood obesity.

The prevalence of childhood obesity is increasing worldwide, along with the associated common comorbidities of type 2 diabetes and cardiovascular disease in later life. Motivated by evidence for a strong genetic component, our prior genome-wide association study (GWAS) efforts for childhood obesity revealed 19 independent signals for the trait; however, the mechanism of action of these loci remains to be elucidated. To molecularly characterize these childhood obesity loci we sought to determine the underlying causal variants and the corresponding effector genes within diverse cellular contexts. Integrating childhood obesity GWAS summary statistics with our existing 3D genomic datasets for 57 human cell types, consisting of high-resolution promoter-focused Capture-C/Hi-C, ATAC-seq, and RNA-seq, we applied stratified LD score regression and calculated the proportion of genome-wide SNP heritability attributable to cell type-specific features, revealing pancreatic alpha cell enrichment as the most statistically significant. Subsequent chromatin contact-based fine-mapping was carried out for genome-wide significant childhood obesity loci and their linkage disequilibrium proxies to implicate effector genes, yielded the most abundant number of candidate variants and target genes at the BDNF, ADCY3, TMEM18 and FTO loci in skeletal muscle myotubes and the pancreatic beta-cell line, EndoC-BH1. One novel implicated effector gene, ALKAL2 - an inflammation-responsive gene in nerve nociceptors - was observed at the key TMEM18 locus across multiple immune cell types. Interestingly, this observation was also supported through colocalization analysis using expression quantitative trait loci (eQTL) derived from the Genotype-Tissue Expression (GTEx) dataset, supporting an inflammatory and neurologic component to the pathogenesis of childhood obesity. Our comprehensive appraisal of 3D genomic datasets generated in a myriad of different cell types provides genomic insights into pediatric obesity pathogenesis.

Losing your inhibition: linking cortical GABAergic interneurons to schizophrenia.

GABAergic interneurons of the cerebral cortex (cINs) play crucial roles in many aspects of cortical function. The diverse types of cINs are classified into subgroups according to their morphology, intrinsic physiology, neurochemical markers and synaptic targeting. Recent advances in mouse genetics, imaging and electrophysiology techniques have greatly advanced our efforts to understand the role of normal cIN function and its dysfunction in neuropsychiatric disorders. In schizophrenia (SCZ), a wealth of data suggests that cIN function is perturbed, and that interneuron dysfunction may underlie key symptoms of the disease. In this review, we discuss the link between cINs and SCZ, focusing on the evidence for GABAergic signaling deficits from both SCZ patients and mouse models.

Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex.

Neuroscience produces a vast amount of data from an enormous diversity of neurons. A neuronal classification system is essential to organize such data and the knowledge that is derived from them. Classification depends on the unequivocal identification of the features that distinguish one type of neuron from another. The problems inherent in this are particularly acute when studying cortical interneurons. To tackle this, we convened a representative group of researchers to agree on a set of terms to describe the anatomical, physiological and molecular features of GABAergic interneurons of the cerebral cortex. The resulting terminology might provide a stepping stone towards a future classification of these complex and heterogeneous cells. Consistent adoption will be important for the success of such an initiative, and we also encourage the active involvement of the broader scientific community in the dynamic evolution of this project.

Spatial and temporal bias in the mitotic origins of somatostatin- and parvalbumin-expressing interneuron subgroups and the chandelier subtype in the medial ganglionic eminence.

GABAergic interneurons modulate cortical activity through the actions of distinct subgroups. Recent studies using interneuron transplants have shown tremendous promise as cell-based therapies for seizure disorders, Parkinson's disease, and in the study of neocortical plasticity. Previous reports identified a spatial bias for the origins of parvalbumin (PV)- and somatostatin (SST)-expressing interneuron subgroups within the medial ganglionic eminence (MGE). In the current study, the mitotic origins of these interneurons are examined by harvesting MGE cells at 2 time points and evaluating their neurochemical profiles after transplantation into neonatal mouse cortex. Although the dorsal MGE (dMGE)-SST and ventral MGE (vMGE)-PV bias were confirmed, both subgroups originate from progenitors located throughout the MGE. The dMGE bias was also found for SST subgroups that coexpress calretinin or reelin. In contrast, another major subgroup of SST interneuron, neuropeptide Y-expressing, does not appear to originate within the MGE. Finally, novel evidence is provided that a clinically important subtype of PV-expressing interneuron, the chandelier (axo-axonic) cell, is greatly enriched in transplants from the vMGE at embryonic day 15. These findings have important implications both for the study of interneuron fate determination and for studies that use interneuron precursor transplantation to alter cortical activity.

CHEX-seq detects single-cell genomic single-stranded DNA with catalytical potential.

Genomic DNA (gDNA) undergoes structural interconversion between single- and double-stranded states during transcription, DNA repair and replication, which is critical for cellular homeostasis. We describe "CHEX-seq" which identifies the single-stranded DNA (ssDNA) in situ in individual cells. CHEX-seq uses 3'-terminal blocked, light-activatable probes to prime the copying of ssDNA into complementary DNA that is sequenced, thereby reporting the genome-wide single-stranded chromatin landscape. CHEX-seq is benchmarked in human K562 cells, and its utilities are demonstrated in cultures of mouse and human brain cells as well as immunostained spatially localized neurons in brain sections. The amount of ssDNA is dynamically regulated in response to perturbation. CHEX-seq also identifies single-stranded regions of mitochondrial DNA in single cells. Surprisingly, CHEX-seq identifies single-stranded loci in mouse and human gDNA that catalyze porphyrin metalation in vitro, suggesting a catalytic activity for genomic ssDNA. We posit that endogenous DNA enzymatic activity is a function of genomic ssDNA.

Variant-to-function analysis of the childhood obesity chr12q13 locus implicates rs7132908 as a causal variant within the 3' UTR of FAIM2.

The ch12q13 obesity locus is among the most significant childhood obesity loci identified in genome-wide association studies. This locus resides in a non-coding region within FAIM2; thus, the underlying causal variant(s) presumably influence disease susceptibility via an influence on cis-regulation within the genomic region. We implicated rs7132908 as a putative causal variant at this locus leveraging a combination of our inhouse 3D genomic data, public domain datasets, and several computational approaches. Using a luciferase reporter assay in human primary astrocytes, we observed allele-specific cis-regulatory activity of the immediate region harboring rs7132908. Motivated by this finding, we went on to generate isogenic human embryonic stem cell lines homozygous for either rs7132908 allele with CRISPR-Cas9 homology-directed repair to assess changes in gene expression due to genotype and chromatin accessibility throughout a differentiation to hypothalamic neurons, a key cell type known to regulate feeding behavior. We observed that the rs7132908 obesity risk allele influenced the expression of FAIM2 along with other genes, decreased the proportion of neurons produced during differentiation, up-regulated cell death gene sets, and conversely down-regulated neuron differentiation gene sets. We have therefore functionally validated rs7132908 as a causal obesity variant which temporally regulates nearby effector genes at the ch12q13 locus and influences neurodevelopment and survival.