Publications by authors named "John L Rubenstein"

221 Publications

Developmental dynamics of voltage-gated sodium channel isoform expression in the human and mouse brain.

Genome Med 2021 Aug 23;13(1):135. Epub 2021 Aug 23.

Department of Psychiatry and Behavioral Sciences, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA, 94158, USA.

Background: Genetic variants in the voltage-gated sodium channels SCN1A, SCN2A, SCN3A, and SCN8A are leading causes of epilepsy, developmental delay, and autism spectrum disorder. The mRNA splicing patterns of all four genes vary across development in the rodent brain, including mutually exclusive copies of the fifth protein-coding exon detected in the neonate (5N) and adult (5A). A second pair of mutually exclusive exons is reported in SCN8A only (18N and 18A). We aimed to quantify the expression of individual exons in the developing human brain.

Methods: RNA-seq data from 783 human brain samples across development were analyzed to estimate exon-level expression. Developmental changes in exon utilization were validated by assessing intron splicing. Exon expression was also estimated in RNA-seq data from 58 developing mouse neocortical samples.

Results: In the mature human neocortex, exon 5A is consistently expressed at least 4-fold higher than exon 5N in all four genes. For SCN2A, SCN3A, and SCN8A, a brain-wide synchronized 5N to 5A transition occurs between 24 post-conceptual weeks (2nd trimester) and 6 years of age. In mice, the equivalent 5N to 5A transition begins at or before embryonic day 15.5. In SCN8A, over 90% of transcripts in the mature human cortex include exon 18A. Early in fetal development, most transcripts include 18N or skip both 18N and 18A, with a transition to 18A inclusion occurring from 13 post-conceptual weeks to 6 months of age. No other protein-coding exons showed comparably dynamic developmental trajectories.

Conclusions: Exon usage in SCN1A, SCN2A, SCN3A, and SCN8A changes dramatically during human brain development. These splice isoforms, which alter the biophysical properties of the encoded channels, may account for some of the observed phenotypic differences across development and between specific variants. Manipulation of the proportion of splicing isoforms at appropriate stages of development may act as a therapeutic strategy for specific mutations or even epilepsy in general.
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http://dx.doi.org/10.1186/s13073-021-00949-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8383430PMC
August 2021

COUP-TFI specifies the medial entorhinal cortex identity and induces differential cell adhesion to determine the integrity of its boundary with neocortex.

Sci Adv 2021 Jul 2;7(27). Epub 2021 Jul 2.

Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.

Development of cortical regions with precise, sharp, and regular boundaries is essential for physiological function. However, little is known of the mechanisms ensuring these features. Here, we show that determination of the boundary between neocortex and medial entorhinal cortex (MEC), two abutting cortical regions generated from the same progenitor lineage, relies on COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I), a patterning transcription factor with graded expression in cortical progenitors. In contrast with the classical paradigm, we found that increased COUP-TFI expression expands MEC, creating protrusions and disconnected ectopic tissue. We further developed a mathematical model that predicts that neuronal specification and differential cell affinity contribute to the emergence of an instability region and boundary sharpness. Correspondingly, we demonstrated that high expression of COUP-TFI induces MEC cell fate and protocadherin 19 expression. Thus, we conclude that a sharp boundary requires a subtle interplay between patterning transcription factors and differential cell affinity.
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http://dx.doi.org/10.1126/sciadv.abf6808DOI Listing
July 2021

Cracking the Codes of Cortical Glial Progenitors: Evidence for the Common Lineage of Astrocytes and Oligodendrocytes.

Neurosci Bull 2021 Apr 13;37(6):437-439. Epub 2021 Apr 13.

Institute of Life Sciences, College of Life and Environmental Sciences, College of Basic Medical Science, Hangzhou Normal University, Hangzhou, 311121, China.

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http://dx.doi.org/10.1007/s12264-021-00675-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055804PMC
April 2021

Ultraconserved enhancer function does not require perfect sequence conservation.

Nat Genet 2021 04 29;53(4):521-528. Epub 2021 Mar 29.

Environmental Genomics & System Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.

Ultraconserved enhancer sequences show perfect conservation between human and rodent genomes, suggesting that their functions are highly sensitive to mutation. However, current models of enhancer function do not sufficiently explain this extreme evolutionary constraint. We subjected 23 ultraconserved enhancers to different levels of mutagenesis, collectively introducing 1,547 mutations, and examined their activities in transgenic mouse reporter assays. Overall, we find that the regulatory properties of ultraconserved enhancers are robust to mutation. Upon mutagenesis, nearly all (19/23, 83%) still functioned as enhancers at one developmental stage, as did most of those tested again later in development (5/9, 56%). Replacement of endogenous enhancers with mutated alleles in mice corroborated results of transgenic assays, including the functional resilience of ultraconserved enhancers to mutation. Our findings show that the currently known activities of ultraconserved enhancers do not necessarily require the perfect conservation observed in evolution and suggest that additional regulatory or other functions contribute to their sequence constraint.
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http://dx.doi.org/10.1038/s41588-021-00812-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038972PMC
April 2021

Gene regulatory networks controlling differentiation, survival, and diversification of hypothalamic Lhx6-expressing GABAergic neurons.

Commun Biol 2021 01 21;4(1):95. Epub 2021 Jan 21.

Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.

GABAergic neurons of the hypothalamus regulate many innate behaviors, but little is known about the mechanisms that control their development. We previously identified hypothalamic neurons that express the LIM homeodomain transcription factor Lhx6, a master regulator of cortical interneuron development, as sleep-promoting. In contrast to telencephalic interneurons, hypothalamic Lhx6 neurons do not undergo long-distance tangential migration and do not express cortical interneuronal markers such as Pvalb. Here, we show that Lhx6 is necessary for the survival of hypothalamic neurons. Dlx1/2, Nkx2-2, and Nkx2-1 are each required for specification of spatially distinct subsets of hypothalamic Lhx6 neurons, and that Nkx2-2+/Lhx6+ neurons of the zona incerta are responsive to sleep pressure. We further identify multiple neuropeptides that are enriched in spatially segregated subsets of hypothalamic Lhx6 neurons, and that are distinct from those seen in cortical neurons. These findings identify common and divergent molecular mechanisms by which Lhx6 controls the development of GABAergic neurons in the hypothalamus.
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http://dx.doi.org/10.1038/s42003-020-01616-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820013PMC
January 2021

Regulatory Elements Inserted into AAVs Confer Preferential Activity in Cortical Interneurons.

eNeuro 2020 Nov/Dec;7(6). Epub 2020 Dec 17.

Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, University of California San Francisco Weill Institute for Neurosciences, University of California San Francisco, San Francisco, CA 94158

Cortical interneuron (CIN) dysfunction is thought to play a major role in neuropsychiatric conditions like epilepsy, schizophrenia and autism. It is therefore essential to understand how the development, physiology, and functions of CINs influence cortical circuit activity and behavior in model organisms such as mice and primates. While transgenic driver lines are powerful tools for studying CINs in mice, this technology is limited in other species. An alternative approach is to use viral vectors such as AAV, which can be used in multiple species including primates and also have potential for therapeutic use in humans. Thus, we sought to discover gene regulatory enhancer elements (REs) that can be used in viral vectors to drive expression in specific cell types. The present study describes the systematic genome-wide identification of putative REs (pREs) that are preferentially active in immature CINs by histone modification chromatin immunoprecipitation and sequencing (ChIP-seq). We evaluated two novel pREs in AAV vectors, alongside the well-established enhancer, and found that they drove CIN-specific reporter expression in adult mice. We also showed that the identified pRE could drive sufficient expression of channelrhodopsin for optogenetic rescue of behavioral deficits in the mouse model of fast-spiking CIN dysfunction.
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http://dx.doi.org/10.1523/ENEURO.0211-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768279PMC
June 2021

A Chromatin Accessibility Atlas of the Developing Human Telencephalon.

Cell 2020 08 30;182(3):754-769.e18. Epub 2020 Jun 30.

Department of Psychiatry, Langley Porter Psychiatric Institute, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA, USA. Electronic address:

To discover regulatory elements driving the specificity of gene expression in different cell types and regions of the developing human brain, we generated an atlas of open chromatin from nine dissected regions of the mid-gestation human telencephalon, as well as microdissected upper and deep layers of the prefrontal cortex. We identified a subset of open chromatin regions (OCRs), termed predicted regulatory elements (pREs), that are likely to function as developmental brain enhancers. pREs showed temporal, regional, and laminar differences in chromatin accessibility and were correlated with gene expression differences across regions and gestational ages. We identified two functional de novo variants in a pRE for autism risk gene SLC6A1, and using CRISPRa, demonstrated that this pRE regulates SCL6A1. Additionally, mouse transgenic experiments validated enhancer activity for pREs proximal to FEZF2 and BCL11A. Thus, this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease.
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http://dx.doi.org/10.1016/j.cell.2020.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415678PMC
August 2020

LacZ-reporter mapping of Dlx5/6 expression and genoarchitectural analysis of the postnatal mouse prethalamus.

J Comp Neurol 2021 02 18;529(2):367-420. Epub 2020 Jun 18.

Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, UCSF Medical School, San Francisco, California, USA.

We present here a thorough and complete analysis of mouse P0-P140 prethalamic histogenetic subdivisions and corresponding nuclear derivatives, in the context of local tract landmarks. The study used as fundamental material brains from a transgenic mouse line that expresses LacZ under the control of an intragenic enhancer of Dlx5 and Dlx6 (Dlx5/6-LacZ). Subtle shadings of LacZ signal, jointly with pan-DLX immunoreaction, and several other ancillary protein or RNA markers, including Calb2 and Nkx2.2 ISH (for the prethalamic eminence, and derivatives of the rostral zona limitans shell domain, respectively) were mapped across the prethalamus. The resulting model of the prethalamic region postulates tetrapartite rostrocaudal and dorsoventral subdivisions, as well as a tripartite radial stratification, each cell population showing a characteristic molecular profile. Some novel nuclei are proposed, and some instances of potential tangential cell migration were noted.
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http://dx.doi.org/10.1002/cne.24952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671952PMC
February 2021

Enhancing WNT Signaling Restores Cortical Neuronal Spine Maturation and Synaptogenesis in Tbr1 Mutants.

Cell Rep 2020 04;31(2):107495

Department of Psychiatry and UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

Tbr1 is a high-confidence autism spectrum disorder (ASD) gene encoding a transcription factor with distinct pre- and postnatal functions. Postnatally, Tbr1 conditional knockout (CKO) mutants and constitutive heterozygotes have immature dendritic spines and reduced synaptic density. Tbr1 regulates expression of several genes that underlie synaptic defects, including a kinesin (Kif1a) and a WNT-signaling ligand (Wnt7b). Furthermore, Tbr1 mutant corticothalamic neurons have reduced thalamic axonal arborization. LiCl and a GSK3β inhibitor, two WNT-signaling agonists, robustly rescue the dendritic spines and the synaptic and axonal defects, suggesting that this could have relevance for therapeutic approaches in some forms of ASD.
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http://dx.doi.org/10.1016/j.celrep.2020.03.059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473600PMC
April 2020

Cortical Neural Stem Cell Lineage Progression Is Regulated by Extrinsic Signaling Molecule Sonic Hedgehog.

Cell Rep 2020 03;30(13):4490-4504.e4

Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA 95064, USA. Electronic address:

Neural stem cells (NSCs) in the prenatal neocortex progressively generate different subtypes of glutamatergic projection neurons. Following that, NSCs have a major switch in their progenitor properties and produce γ-aminobutyric acid (GABAergic) interneurons for the olfactory bulb (OB), cortical oligodendrocytes, and astrocytes. Herein, we provide evidence for the molecular mechanism that underlies this switch in the state of neocortical NSCs. We show that, at around E16.5, mouse neocortical NSCs start to generate GSX2-expressing (GSX2) intermediate progenitor cells (IPCs). In vivo lineage-tracing study revealed that GSX2 IPC population gives rise not only to OB interneurons but also to cortical oligodendrocytes and astrocytes, suggesting that they are a tri-potential population. We demonstrated that Sonic hedgehog signaling is both necessary and sufficient for the generation of GSX2 IPCs by reducing GLI3R protein levels. Using single-cell RNA sequencing, we identify the transcriptional profile of GSX2 IPCs and the process of the lineage switch of cortical NSCs.
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http://dx.doi.org/10.1016/j.celrep.2020.03.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197103PMC
March 2020

deletion results in depletion of the transcription factor and a specific loss of parvalbumin cortical interneurons.

Proc Natl Acad Sci U S A 2020 03 2;117(11):6189-6195. Epub 2020 Mar 2.

Department of Pediatrics and Human Development, Michigan State University, Grand Rapids, MI 49503;

Neurofibromatosis 1 (NF1) is caused by mutations in the gene, which encodes the protein, neurofibromin, an inhibitor of Ras activity. Cortical GABAergic interneurons (CINs) are implicated in NF1 pathology, but the cellular and molecular changes to CINs are unknown. We deleted mouse from the medial ganglionic eminence, which gives rise to both oligodendrocytes and CINs that express somatostatin and parvalbumin. loss led to a persistence of immature oligodendrocytes that prevented later-generated oligodendrocytes from occupying the cortex. Moreover, molecular and cellular properties of parvalbumin (PV)-positive CINs were altered by the loss of , without changes in somatostatin (SST)-positive CINs. We discovered that loss of results in a dose-dependent decrease in expression, the transcription factor necessary to establish SST and PV CINs, which was rescued by the MEK inhibitor SL327, revealing a mechanism whereby a neurofibromin/Ras/MEK pathway regulates a critical CIN developmental milestone.
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http://dx.doi.org/10.1073/pnas.1915458117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084085PMC
March 2020

Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors.

Neuron 2020 04 5;106(1):37-65.e5. Epub 2020 Feb 5.

Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities.
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http://dx.doi.org/10.1016/j.neuron.2020.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7377387PMC
April 2020

Interneuron Transplantation Rescues Social Behavior Deficits without Restoring Wild-Type Physiology in a Mouse Model of Autism with Excessive Synaptic Inhibition.

J Neurosci 2020 03 27;40(11):2215-2227. Epub 2020 Jan 27.

Weill Institute for Neurosciences,

Manipulations that enhance GABAergic inhibition have been associated with improved behavioral phenotypes in autism models, suggesting that autism may be treated by correcting underlying deficits of inhibition. Interneuron transplantation is a method for increasing recipient synaptic inhibition, and it has been considered a prospective therapy for conditions marked by deficient inhibition, including neuropsychiatric disorders. It is unknown, however, whether interneuron transplantation may be therapeutically effective only for conditions marked by reduced inhibition, and it is also unclear whether transplantation improves behavioral phenotypes solely by normalizing underlying circuit defects. To address these questions, we studied the effects of interneuron transplantation in male and female mice lacking the autism-associated gene, , in GABAergic interneurons. mutant mice exhibit social behavior deficits, elevated synaptic inhibition in prefrontal cortex, abnormal baseline and social interaction-evoked electroencephalogram (EEG) signals, and an altered composition of cortical interneuron subtypes. Transplantation of wild-type embryonic interneurons from the medial ganglionic eminence into the prefrontal cortex of neonatal mutants rescued social behavior despite exacerbating excessive levels of synaptic inhibition. Furthermore, transplantation did not normalize recipient EEG signals measured during baseline states. Interneuron transplantation can thus correct behavioral deficits even when those deficits are associated with elevated synaptic inhibition. Moreover, transplantation does not exert therapeutic effects solely by restoring wild-type circuit states. Our findings indicate that interneuron transplantation could offer a novel cell-based approach to autism treatment while challenging assumptions that effective therapies must reverse underlying circuit defects. Imbalances between neural excitation and inhibition are hypothesized to contribute to the pathophysiology of autism. Interneuron transplantation is a method for altering recipient inhibition, and it has been considered a prospective therapy for neuropsychiatric disorders, including autism. Here we examined the behavioral and physiological effects of interneuron transplantation in a mouse genetic model of autism. They demonstrate that transplantation rescues recipient social interaction deficits without correcting a common measure of recipient inhibition, or circuit-level physiological measures. These findings demonstrate that interneuron transplantation can exert therapeutic behavioral effects without necessarily restoring wild-type circuit states, while highlighting the potential of interneuron transplantation as an autism therapy.
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http://dx.doi.org/10.1523/JNEUROSCI.1063-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7083289PMC
March 2020

Functionally Distinct Connectivity of Developmentally Targeted Striosome Neurons.

Cell Rep 2019 11;29(6):1419-1428.e5

Neuroscience Graduate Program, UCSF, San Francisco, CA 94158, USA; Weill Institute for Neurosciences, UCSF, San Francisco, CA 94158, USA; Kavli Institute for Fundamental Neuroscience, UCSF, San Francisco, CA 94158, USA; Department of Neurology, UCSF, San Francisco, CA 94158, USA. Electronic address:

One long-standing model of striatal function divides the striatum into compartments called striosome and matrix. While some anatomical evidence suggests that these populations represent distinct striatal pathways with differing inputs and outputs, functional investigation has been limited by the methods for identifying and manipulating these populations. Here, we utilize hs599 mice as a new tool for targeting striosome projection neurons and testing their functional connectivity. Extending anatomical work, we demonstrate that striosome neurons receive greater synaptic input from prelimbic cortex, whereas matrix neurons receive greater input from primary motor cortex. We also identify functional differences in how striosome and matrix neurons process excitatory input, providing the first electrophysiological method for delineating striatal output neuron subtypes. Lastly, we provide the first functional demonstration that striosome neurons are the predominant striatal output to substantia nigra pars compacta dopamine neurons. These results identify striosome and matrix as functionally distinct striatal pathways.
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http://dx.doi.org/10.1016/j.celrep.2019.09.076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6866662PMC
November 2019

Tsc1 represses parvalbumin expression and fast-spiking properties in somatostatin lineage cortical interneurons.

Nat Commun 2019 11 1;10(1):4994. Epub 2019 Nov 1.

Department of Pediatrics and Human Development, 400 Monroe Ave. NW, Grand Rapids, MI, 49503, USA.

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.
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http://dx.doi.org/10.1038/s41467-019-12962-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825152PMC
November 2019

Genomic Resolution of DLX-Orchestrated Transcriptional Circuits Driving Development of Forebrain GABAergic Neurons.

Cell Rep 2019 08;28(8):2048-2063.e8

Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94158, USA; Development and Stem Cell Biology Program, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

DLX transcription factors (TFs) are master regulators of the developing vertebrate brain, driving forebrain GABAergic neuronal differentiation. Ablation of Dlx1&2 alters expression of genes that are critical for forebrain GABAergic development. We integrated epigenomic and transcriptomic analyses, complemented with in situ hybridization (ISH), and in vivo and in vitro studies of regulatory element (RE) function. This revealed the DLX-organized gene regulatory network at genomic, cellular, and spatial levels in mouse embryonic basal ganglia. DLX TFs perform dual activating and repressing functions; the consequences of their binding were determined by the sequence and genomic context of target loci. Our results reveal and, in part, explain the paradox of widespread DLX binding contrasted with a limited subset of target loci that are sensitive at the epigenomic and transcriptomic level to Dlx1&2 ablation. The regulatory properties identified here for DLX TFs suggest general mechanisms by which TFs orchestrate dynamic expression programs underlying neurodevelopment.
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http://dx.doi.org/10.1016/j.celrep.2019.07.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6750766PMC
August 2019

Immature excitatory neurons develop during adolescence in the human amygdala.

Nat Commun 2019 06 21;10(1):2748. Epub 2019 Jun 21.

Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA, 94143, USA.

The human amygdala grows during childhood, and its abnormal development is linked to mood disorders. The primate amygdala contains a large population of immature neurons in the paralaminar nuclei (PL), suggesting protracted development and possibly neurogenesis. Here we studied human PL development from embryonic stages to adulthood. The PL develops next to the caudal ganglionic eminence, which generates inhibitory interneurons, yet most PL neurons express excitatory markers. In children, most PL cells are immature (DCX+PSA-NCAM+), and during adolescence many transition into mature (TBR1+VGLUT2+) neurons. Immature PL neurons persist into old age, yet local progenitor proliferation sharply decreases in infants. Using single nuclei RNA sequencing, we identify the transcriptional profile of immature excitatory neurons in the human amygdala between 4-15 years. We conclude that the human PL contains excitatory neurons that remain immature for decades, a possible substrate for persistent plasticity at the interface of the hippocampus and amygdala.
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http://dx.doi.org/10.1038/s41467-019-10765-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588589PMC
June 2019

Excitation-inhibition balance as a framework for investigating mechanisms in neuropsychiatric disorders.

Mol Psychiatry 2019 09 14;24(9):1248-1257. Epub 2019 May 14.

Department of Psychiatry, Weill Institute for Neurosciences, and Kavli Institute for Fundamental Neuroscience, University of California San Francisco, San Francisco, CA, 94143, USA.

In 2003 Rubenstein and Merzenich hypothesized that some forms of Autism (ASD) might be caused by a reduction in signal-to-noise in key neural circuits, which could be the result of changes in excitatory-inhibitory (E-I) balance. Here, we have clarified the concept of E-I balance, and updated the original hypothesis in light of the field's increasingly sophisticated understanding of neuronal circuits. We discuss how specific developmental mechanisms, which reduce inhibition, affect cortical and hippocampal functions. After describing how mutations of some ASD genes disrupt inhibition in mice, we close by suggesting that E-I balance represents an organizing framework for understanding findings related to pathophysiology and for identifying appropriate treatments.
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http://dx.doi.org/10.1038/s41380-019-0426-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742424PMC
September 2019

Transcriptomic metaanalyses of autistic brains reveals shared gene expression and biological pathway abnormalities with cancer.

Mol Autism 2019 8;10:17. Epub 2019 Apr 8.

1Biomedical Research Networking Center of Mental Health (CIBERSAM), Madrid, Spain.

Background: 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.

Methods: Here, for the first time, we compared the gene expression profiles of ASD frontal cortex tissues and 22 cancer types obtained by differential expression meta-analysis and report gene, pathway, and drug set-based overlaps between them.

Results: Four cancer types (brain, thyroid, kidney, and pancreatic cancers) presented a significant overlap in gene expression deregulations in the same direction as ASD whereas two cancer types (lung and prostate cancers) showed differential expression profiles significantly deregulated in the opposite direction from ASD. Functional enrichment and LINCS L1000 based drug set enrichment analyses revealed the implication of several biological processes and pathways that were affected jointly in both diseases, including impairments of the immune system, and impairments in oxidative phosphorylation and ATP synthesis among others. Our data also suggest that brain and kidney cancer have patterns of transcriptomic dysregulation in the PI3K/AKT/MTOR axis that are similar to those found in ASD.

Conclusions: Comparisons of ASD and cancer differential gene expression meta-analysis results suggest that brain, kidney, thyroid, and pancreatic cancers are candidates for direct comorbid associations with ASD. On the other hand, lung and prostate cancers are candidates for inverse comorbid associations with ASD. Joint perturbations in a set of specific biological processes underlie these associations which include several pathways previously implicated in both cancer and ASD encompassing immune system alterations, impairments of energy metabolism, cell cycle, and signaling through PI3K and G protein-coupled receptors among others. These findings could help to explain epidemiological observations pointing towards direct and inverse comorbid associations between ASD and specific cancer types and depict a complex scenario regarding the molecular patterns of association between ASD and cancer.
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http://dx.doi.org/10.1186/s13229-019-0262-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6454734PMC
May 2019

Dlx1/2 are Central and Essential Components in the Transcriptional Code for Generating Olfactory Bulb Interneurons.

Cereb Cortex 2019 12;29(11):4831-4849

State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, MOE Frontier Research Center for Brain Science, Department of Neurology, Zhongshan Hospital, Fudan University, Shanghai 200032, PR China.

Generation of olfactory bulb (OB) interneurons requires neural stem/progenitor cell specification, proliferation, differentiation, and young interneuron migration and maturation. Here, we show that the homeobox transcription factors Dlx1/2 are central and essential components in the transcriptional code for generating OB interneurons. In Dlx1/2 constitutive null mutants, the differentiation of GSX2+ and ASCL1+ neural stem/progenitor cells in the dorsal lateral ganglionic eminence is blocked, resulting in a failure of OB interneuron generation. In Dlx1/2 conditional mutants (hGFAP-Cre; Dlx1/2F/- mice), GSX2+ and ASCL1+ neural stem/progenitor cells in the postnatal subventricular zone also fail to differentiate into OB interneurons. In contrast, overexpression of Dlx1&2 in embryonic mouse cortex led to ectopic production of OB-like interneurons that expressed Gad1, Sp8, Sp9, Arx, Pbx3, Etv1, Tshz1, and Prokr2. Pax6 mutants generate cortical ectopia with OB-like interneurons, but do not do so in compound Pax6; Dlx1/2 mutants. We propose that DLX1/2 promote OB interneuron development mainly through activating the expression of Sp8/9, which further promote Tshz1 and Prokr2 expression. Based on this study, in combination with earlier ones, we propose a transcriptional network for the process of OB interneuron development.
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http://dx.doi.org/10.1093/cercor/bhz018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917526PMC
December 2019

Mafb and c-Maf Have Prenatal Compensatory and Postnatal Antagonistic Roles in Cortical Interneuron Fate and Function.

Cell Rep 2019 01;26(5):1157-1173.e5

Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94158, USA; Neuroscience Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2019.01.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602795PMC
January 2019

Genome-wide de novo risk score implicates promoter variation in autism spectrum disorder.

Science 2018 12;362(6420)

Department of Psychiatry, UCSF Weill Institute for Neurosciences, University of California, San Francisco, CA, USA.

Whole-genome sequencing (WGS) has facilitated the first genome-wide evaluations of the contribution of de novo noncoding mutations to complex disorders. Using WGS, we identified 255,106 de novo mutations among sample genomes from members of 1902 quartet families in which one child, but not a sibling or their parents, was affected by autism spectrum disorder (ASD). In contrast to coding mutations, no noncoding functional annotation category, analyzed in isolation, was significantly associated with ASD. Casting noncoding variation in the context of a de novo risk score across multiple annotation categories, however, did demonstrate association with mutations localized to promoter regions. We found that the strongest driver of this promoter signal emanates from evolutionarily conserved transcription factor binding sites distal to the transcription start site. These data suggest that de novo mutations in promoter regions, characterized by evolutionary and functional signatures, contribute to ASD.
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http://dx.doi.org/10.1126/science.aat6576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6432922PMC
December 2018

Neonatal Tbr1 Dosage Controls Cortical Layer 6 Connectivity.

Neuron 2018 11 11;100(4):831-845.e7. Epub 2018 Oct 11.

Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, and Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Psychiatry, Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

An understanding of how heterozygous loss-of-function mutations in autism spectrum disorder (ASD) risk genes, such as TBR1, contribute to ASD remains elusive. Conditional Tbr1 deletion during late mouse gestation in cortical layer 6 neurons (Tbr1 mutants) provides novel insights into its function, including dendritic patterning, synaptogenesis, and cell-intrinsic physiology. These phenotypes occur in heterozygotes, providing insights into mechanisms that may underlie ASD pathophysiology. Restoring expression of Wnt7b largely rescues the synaptic deficit in Tbr1 mutant neurons. Furthermore, Tbr1 heterozygotes have increased anxiety-like behavior, a phenotype seen ASD. Integrating TBR1 chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) data from layer 6 neurons and activity of TBR1-bound candidate enhancers provides evidence for how TBR1 regulates layer 6 properties. Moreover, several putative TBR1 targets are ASD risk genes, placing TBR1 in a central position both for ASD risk and for regulating transcriptional circuits that control multiple steps in layer 6 development essential for the assembly of neural circuits.
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http://dx.doi.org/10.1016/j.neuron.2018.09.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6250594PMC
November 2018

Genomic analysis of transcriptional networks directing progression of cell states during MGE development.

Neural Dev 2018 09 14;13(1):21. Epub 2018 Sep 14.

Department of Psychiatry, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA, 94143, USA.

Background: Homeodomain (HD) transcription factor (TF) NKX2-1 critical for the regional specification of the medial ganglionic eminence (MGE) as well as promoting the GABAergic and cholinergic neuron fates via the induction of TFs such as LHX6 and LHX8. NKX2-1 defines MGE regional identity in large part through transcriptional repression, while specification and maturation of GABAergic and cholinergic fates is mediated in part by transcriptional activation via TFs such as LHX6 and LHX8. Here we analyze the signaling and TF pathways, downstream of NKX2-1, required for GABAergic and cholinergic neuron fate maturation.

Methods: Differential ChIP-seq analysis was used to identify regulatory elements (REs) where chromatin state was sensitive to change in the Nkx2-1cKO MGE at embryonic day (E) 13.5. TF motifs in the REs were identified using RSAT. CRISPR-mediated genome editing was used to generate enhancer knockouts. Differential gene expression in these knockouts was analyzed through RT-qPCR and in situ hybridization. Functional analysis of motifs within hs623 was analyzed via site directed mutagenesis and reporter assays in primary MGE cultures.

Results: We identified 4782 activating REs (aREs) and 6391 repressing REs (rREs) in the Nkx2-1 conditional knockout (Nkx2-1cKO) MGE. aREs are associated with basic-Helix-Loop-Helix (bHLH) TFs. Deletion of hs623, an intragenic Tcf12 aRE, caused a reduction of Tcf12 expression in the sub-ventricular zone (SVZ) and mantle zone (MZ) of the MGE. Mutation of LHX, SOX and octamers, within hs623, caused a reduction of hs623 activity in MGE primary cultures.

Conclusions: Tcf12 expression in the SVZ of the MGE is mediated through aRE hs623. The activity of hs623 is dependent on LHX6, SOX and octamers. Thus, maintaining the expression of Tcf12 in the SVZ involves on TF pathways parallel and genetically downstream of NKX2-1.
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http://dx.doi.org/10.1186/s13064-018-0119-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138899PMC
September 2018

The Epigenetic Factor Landscape of Developing Neocortex Is Regulated by Transcription Factors Pax6→ Tbr2→ Tbr1.

Front Neurosci 2018 22;12:571. Epub 2018 Aug 22.

Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, WA, United States.

Epigenetic factors (EFs) regulate multiple aspects of cerebral cortex development, including proliferation, differentiation, laminar fate, and regional identity. The same neurodevelopmental processes are also regulated by transcription factors (TFs), notably the Pax6→ Tbr2→ Tbr1 cascade expressed sequentially in radial glial progenitors (RGPs), intermediate progenitors, and postmitotic projection neurons, respectively. Here, we studied the EF landscape and its regulation in embryonic mouse neocortex. Microarray and hybridization assays revealed that many EF genes are expressed in specific cortical cell types, such as intermediate progenitors, or in rostrocaudal gradients. Furthermore, many EF genes are directly bound and transcriptionally regulated by Pax6, Tbr2, or Tbr1, as determined by chromatin immunoprecipitation-sequencing and gene expression analysis of TF mutant cortices. Our analysis demonstrated that Pax6, Tbr2, and Tbr1 form a direct feedforward genetic cascade, with direct feedback repression. Results also revealed that each TF regulates multiple EF genes that control DNA methylation, histone marks, chromatin remodeling, and non-coding RNA. For example, Tbr1 activates and to promote the formation of non-canonical Polycomb repressive complex 1 (PRC1). Also, Pax6, Tbr2, and Tbr1 collectively drive massive changes in the subunit isoform composition of BAF chromatin remodeling complexes during differentiation: for example, a novel switch from (Baf40c) to (Baf40a), the latter directly activated by Tbr2. Of 11 subunits predominantly in neuronal BAF, 7 were transcriptionally activated by Pax6, Tbr2, or Tbr1. Using EFs, Pax6→ Tbr2→ Tbr1 effect persistent changes of gene expression in cell lineages, to propagate features such as regional and laminar identity from progenitors to neurons.
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http://dx.doi.org/10.3389/fnins.2018.00571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113890PMC
August 2018

SP8 and SP9 coordinately promote D2-type medium spiny neuron production by activating expression.

Development 2018 07 23;145(14). Epub 2018 Jul 23.

State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Department of Neurology, Zhongshan Hospital, Fudan University, Shanghai 200032, China

Dopamine receptor DRD1-expressing medium spiny neurons (D1 MSNs) and dopamine receptor DRD2-expressing medium spiny neurons (D2 MSNs) are the principal projection neurons in the striatum, which is divided into dorsal striatum (caudate nucleus and putamen) and ventral striatum (nucleus accumbens and olfactory tubercle). Progenitors of these neurons arise in the lateral ganglionic eminence (LGE). Using conditional deletion, we show that mice lacking the transcription factor genes and lose virtually all D2 MSNs as a result of reduced neurogenesis in the LGE, whereas D1 MSNs are largely unaffected. SP8 and SP9 together drive expression of the transcription factor in a spatially restricted domain of the LGE subventricular zone. Conditional deletion of also prevents the formation of most D2 MSNs, phenocopying the mutants. Finally, ChIP-Seq reveals that SP9 directly binds to the promoter and a putative enhancer of Thus, this study defines components of a transcription pathway in a regionally restricted LGE progenitor domain that selectively drives the generation of D2 MSNs.
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http://dx.doi.org/10.1242/dev.165456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6078334PMC
July 2018

Sp9 Regulates Medial Ganglionic Eminence-Derived Cortical Interneuron Development.

Cereb Cortex 2019 06;29(6):2653-2667

State Key Laboratory of Medical Neurobiology, Department of Neurology, Institutes of Brain Science, Zhongshan Hospital, Fudan University, Shanghai, China.

Immature neurons generated by the subpallial MGE tangentially migrate to the cortex where they become parvalbumin-expressing (PV+) and somatostatin (SST+) interneurons. Here, we show that the Sp9 transcription factor controls the development of MGE-derived cortical interneurons. SP9 is expressed in the MGE subventricular zone and in MGE-derived migrating interneurons. Sp9 null and conditional mutant mice have approximately 50% reduction of MGE-derived cortical interneurons, an ectopic aggregation of MGE-derived neurons in the embryonic ventral telencephalon, and an increased ratio of SST+/PV+ cortical interneurons. RNA-Seq and SP9 ChIP-Seq reveal that SP9 regulates MGE-derived cortical interneuron development through controlling the expression of key transcription factors Arx, Lhx6, Lhx8, Nkx2-1, and Zeb2 involved in interneuron development, as well as genes implicated in regulating interneuron migration Ackr3, Epha3, and St18. Thus, Sp9 has a central transcriptional role in MGE-derived cortical interneuron development.
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http://dx.doi.org/10.1093/cercor/bhy133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519701PMC
June 2019

An analytical framework for whole-genome sequence association studies and its implications for autism spectrum disorder.

Nat Genet 2018 04 26;50(5):727-736. Epub 2018 Apr 26.

Department of Biology, Eastern Nazarene College, Quincy, MA, USA.

Genomic association studies of common or rare protein-coding variation have established robust statistical approaches to account for multiple testing. Here we present a comparable framework to evaluate rare and de novo noncoding single-nucleotide variants, insertion/deletions, and all classes of structural variation from whole-genome sequencing (WGS). Integrating genomic annotations at the level of nucleotides, genes, and regulatory regions, we define 51,801 annotation categories. Analyses of 519 autism spectrum disorder families did not identify association with any categories after correction for 4,123 effective tests. Without appropriate correction, biologically plausible associations are observed in both cases and controls. Despite excluding previously identified gene-disrupting mutations, coding regions still exhibited the strongest associations. Thus, in autism, the contribution of de novo noncoding variation is probably modest in comparison to that of de novo coding variants. Robust results from future WGS studies will require large cohorts and comprehensive analytical strategies that consider the substantial multiple-testing burden.
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http://dx.doi.org/10.1038/s41588-018-0107-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5961723PMC
April 2018

Tbr1 instructs laminar patterning of retinal ganglion cell dendrites.

Nat Neurosci 2018 05 9;21(5):659-670. Epub 2018 Apr 9.

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA.

Visual information is delivered to the brain by >40 types of retinal ganglion cells (RGCs). Diversity in this representation arises within the inner plexiform layer (IPL), where dendrites of each RGC type are restricted to specific sublaminae, limiting the interneuronal types that can innervate them. How such dendritic restriction arises is unclear. We show that the transcription factor Tbr1 is expressed by four mouse RGC types with dendrites in the outer IPL and is required for their laminar specification. Loss of Tbr1 results in elaboration of dendrites within the inner IPL, while misexpression in other cells retargets their neurites to the outer IPL. Two transmembrane molecules, Sorcs3 and Cdh8, act as effectors of the Tbr1-controlled lamination program. However, they are expressed in just one Tbr1 RGC type, supporting a model in which a single transcription factor implements similar laminar choices in distinct cell types by recruiting partially non-overlapping effectors.
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http://dx.doi.org/10.1038/s41593-018-0127-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5920715PMC
May 2018

Ultraconserved Enhancers Are Required for Normal Development.

Cell 2018 01 18;172(3):491-499.e15. Epub 2018 Jan 18.

Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA; U.S. Department of Energy Joint Genome Institute, Walnut Creek, CA 94598, USA; School of Natural Sciences, University of California Merced, Merced, CA 95343, USA. Electronic address:

Non-coding "ultraconserved" regions containing hundreds of consecutive bases of perfect sequence conservation across mammalian genomes can function as distant-acting enhancers. However, initial deletion studies in mice revealed that loss of such extraordinarily constrained sequences had no immediate impact on viability. Here, we show that ultraconserved enhancers are required for normal development. Focusing on some of the longest ultraconserved sites genome wide, located near the essential neuronal transcription factor Arx, we used genome editing to create an expanded series of knockout mice lacking individual or combinations of ultraconserved enhancers. Mice with single or pairwise deletions of ultraconserved enhancers were viable and fertile but in nearly all cases showed neurological or growth abnormalities, including substantial alterations of neuron populations and structural brain defects. Our results demonstrate the functional importance of ultraconserved enhancers and indicate that remarkably strong sequence conservation likely results from fitness deficits that appear subtle in a laboratory setting.
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http://dx.doi.org/10.1016/j.cell.2017.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5786478PMC
January 2018
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