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Transcriptomic and anatomic parcellation of 5-HT3AR expressing cortical interneuron subtypes revealed by single-cell RNA sequencing

ABSTRACT: Cortical GABAergic interneurons constitute a highly diverse population of inhibitory neurons that are key regulators of cortical microcircuit function. An important and heterogeneous group of cortical interneurons specifically expresses the serotonin receptor 3A (5-HT3AR) but how this diversity emerges during development is poorly understood. Here we use single-cell transcriptomics to identify gene expression patterns operating in Htr3a-GFP+ interneurons during early steps of cortical circuit assembly. We identify 3 main molecular types of Htr3a-GFP+ interneurons, each displaying distinct developmental dynamics of gene expression. The transcription factor Meis2 is specifically enriched in a type of Htr3a-GFP+ interneurons spatially confined to the cortical white matter. These MEIS2 expressing interneurons appear to originate from a restricted region located at the embryonic pallial-subpallial boundary. Overall, this study identifies MEIS2 as a subclass-specific marker for 5-HT3AR-containing interstitial interneurons and demonstrates that the transcriptional and anatomical parcellation of cortical interneurons is developmentally coupled.

ORGANISM(S): Mus musculus

PROVIDER: GSE90860 | GEO | 2017/03/08

SECONDARY ACCESSION(S): PRJNA356240

REPOSITORIES: GEO

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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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Transcriptomic and anatomic parcellation of 5-HT3AR expressing cortical interneuron subtypes revealed by single-cell RNA sequencing

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