Regulatory elements with preferential activity in cortical interneurons identified by histone modification ChIP-seq
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE158428 | GEO | 2020/09/24
REPOSITORIES: GEO
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Project description:Viral genetictools that target specificbraincell types could transform basicneuroscienceand targeted gene therapy. Here, we use comparative open chromatin analysis to identify thousands of human-neocortical-subclass-specific putative enhancers from across the genome tocontrol gene expressionin adeno-associated virus (AAV) vectors. The cellular specificity of reporter expression from enhancer-AAVs is established by molecular profiling after systemic AAV delivery in mouse. Over 30% of enhancer-AAVs produce specific expression in the targeted subclass, including both excitatory and inhibitory subclasses. We present a collection ofParvalbumin(PVALB) enhancer-AAVs that show highly enriched expression not only in cortical PVALB cells but also in some subcortical PVALB populations. Five vectors maintain PVALB-enriched expression in primateneocortex. These results demonstrate how genome-wide open chromatin data mining and cross-species AAV validation can be used to create the next generation of non-species-restricted viral genetic tools.
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Project description:Malformations of cortical development are the underlying eitiology of many cases of Mental Retardation and Epilepsy. Subtle, below the resolution of current MRI, cortical dysplasias are probably involved in many cases of MR, Epilepsy and Autism for which no diagnosis can currently be made. Therefore, understanding the process of cortical development will be vital in diagnosing and eventual treatment of many patients with these conditions. More specifically, the cortex forms from two major populations of neuroblasts which reach their final destination in the cortex by differerent mechanisms. One is radial migration from ventricular neuroblasts to the cortical plate. These cells are excititory projection neurons and glia. The second pathway is from the ventral ganglionic eminences and tangential migration of the interneuronal population of primarily inhibitory neurons. Much less is known about the control of the latter process, and many of these currently undiagnosed subtle malformations may stem from abnormalities of this tangential migration. This project focuses on the understanding the control of the tangentially migrating inhibitory interneurons. We aim to uncover the transcriptional differences between two distinct neuronal populations, GFP positive cells in the ganglionic emience and GFP positive cells within the cortical plate, in order to understand the genetic control of tangential migration. We will acomplish this aim by targeting two different labeled transcripts againts the affymetrix mouse genome arrays. We hypothesis that there will be distinct differences in mRNA transcription profiles between the ganglionic eminence and cortical plate within developing interneurons. The differences in transcript profiles will be genes that regulate the migration and differentiation of these developing neurons. Transgenic mice have been generated which express green flourscent protien (GFP) based on the activity of the Dlx 5-6 promotor. Dlx 5-6 are highly regulated genes, and are expressed only in developing interneurons with in the embryonic brain. The expression of this gene begins at around E8 within the medial and lateral ganglionic eminence (GE) and then remain present as cells born within these regions migrate out into the developing cortex. Therefore, we can take advantage of these mice by harvesting embryos at a midpoint in their development (E13.5-14.5) and dissect out GFP positive cells from the ganglionic eminence and from the cortical plate. In order to obtain enough cells to extract sufficent mRNA, we have choosen to perform microdisction of the entire GE and entire cortical plate then break up the tissue into single cell suspension and Florescent activated cell sort (FACs) the populations of GFP + and GFP - cells. We then will extract the total RNA from the GFP postive cell populations using a Trizol based method and purify the RNA with a Quiagen column purification method. The purified RNA will then be amplified using the Affymetrix 1 or 2 round T7 based amplification procedure in order to generate the microgram quantities of labelled cRNA. The labelled biotin cRNA will be sent to the microarray consortium for hybridization againt the Affy mouse genome chip. Three embryos will be pooled in order to obtain sufficent RNA and 6 GFP positive GE and cortical plate samples will be used. The six replicates will be obtained from at least 3 different pregnant mice.
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Project description:Recent success in identifying gene regulatory elements in the context of recombinant adeno-associated virus vectors have enabled cell type-restricted gene expression. However, within the cerebral cortex these tools are presently limited to broad classes of neurons. To overcome this limitation, we developed a strategy that led to the identification of multiple novel enhancers to target functionally distinct neuronal subtypes. By investigating the regulatory landscape of the disease gene Scn1a, we identified enhancers that target the breadth of its expression, including two selective for parvalbumin and vasoactive intestinal peptide cortical interneurons. Demonstrating the functional utility of these elements, we found that the PV-specific enhancer allowed for the selective targeting and manipulation of fast-spiking cortical interneurons across species, from mice to humans.
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Project description:Human embryonic stem cells (hESCs) were specified as ventral telencephalic neuroectoderm (day 4) and then into medial ganglionic emininence (MGE)-like progenitors (day 15) and were subsequently differentiated into cortical interneuron (cIN)-like cells (day 25-35), by modification of previously published protocols. RNA-seq analysis at days 0, 4, 15, 25, and 35 defined transcriptome signatures for MGE and cIN cell identity. Further integration of these gene expression signatures with ChIP-seq for the NKX2-1 transcription factor in MGE-like progenitors defined NKX2-1 putative direct targets, including genes involved in both MGE specification and in several aspects of later cIN differentiation (migration, synaptic function). Among the NKX2-1 direct targets with MGE and cIN enriched expression was CHD2, a chromatin remodeling protein. Since CHD2 haploinsufficiency can cause epilepsy and/or autism, which can involve altered cIN development or function, we evaluated CHD2 requirements in these processes. Transcriptome changes were evaluated in CHD2 knockdown MGE-like progenitors at day 15, revealing diminished expression of genes involved in MGE specification and cIN differentiation including channel and synaptic genes implicated in epilepsy, while later cIN electrophysiological properties were also altered. We defined some shared cis-regulatory elements bound by both NKX2-1 and CHD2 and characterized their ability to cooperatively regulate cIN gene transcription through these elements. We used these data to construct regulatory networks underlying MGE specification and cIN differentiation and to define requirements for CHD2 and its ability to cofunction with NKX2-1 in this process.
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