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Individual Human Cortical Progenitors Can Produce Excitatory and Inhibitory Neurons

ABSTRACT: The cerebral cortex is a cellularly-complex structure comprised of a rich diversity of neuronal and glial cell types. Cortical neurons can be broadly categorized into two classes—glutamatergic excitatory neurons and GABAergic inhibitory interneurons. Previous developmental studies in rodents have led to the prevailing model that while excitatory neurons are born from progenitors located in the cortex, cortical interneurons are born from a separate population of progenitors located outside of the developing cortex in the ganglionic eminences1-5. However, the developmental potential of human cortical progenitors has not been thoroughly explored. Here we show that in addition to excitatory neurons and glia, human cortical progenitors are also capable of producing GABAergic neurons with the transcriptional characteristics and morphologies of cortical interneurons. By developing a cellular barcoding tool called “ScRNAseq-compatible Tracer for Identifying Clonal Relationships” (STICR), we were able to perform clonal lineage tracing of 1912 primary human cortical progenitors from six specimens and capture both the transcriptional identities and clonal relationships of their resulting progeny. A subpopulation of cortically-born GABAergic neurons were transcriptionally similar to cortical interneurons born from the caudal ganglionic eminence and these cells were frequently related to excitatory neurons and glia. Thus, our results demonstrate that individual human cortical progenitors can generate both excitatory neurons and cortical interneurons, providing a new framework for understanding the origins of neuronal diversity in the human cortex.

ORGANISM(S): Synthetic plasmid

PROVIDER: GSE187875 | GEO | 2021/11/06

REPOSITORIES: GEO

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Project description:GABAergic interneurons are lost in conditions including epilepsy and CNS injury, but there are few culture models available to study their function. Towards the goal of obtaining renewable sources of GABAergic neurons, we used the molecular profile of a functionally-incomplete GABAergic precursor clone to screen 17 new clones isolated from GFP+ rat E14.5 cortex and ganglionic eminence (GE) that were generated by viral introduction of v-myc. The clones grow as neurospheres in medium with FGF2, and after withdrawal of FGF2 they exhibit varying patterns of differentiation. Transcriptional profiling and qPCR indicated that one clone (GE6) expresses high levels of mRNAs encoding Dlx1, 2, 5 and 6, glutamate decarboxylases, and presynaptic proteins including neuropeptide Y and somatostatin. Protein expression confirmed that GE6 is a progenitor with restricted differentiation giving rise mostly to neurons with GABAergic markers. In co-cultures with hippocampal neurons, GE6 neurons became electrically excitable and received both inhibitory and excitatory synapses. After withdrawal of FGF2 in cultures of GE6 alone, neurons matured to express BetaIII-tubulin, and staining for synaptophysin and vesicular GABA transporter (VGAT) were robust after 1-2 weeks of differentiation. GE6 neurons also became electrically excitable and displayed synaptic activity, but synaptic currents were carried by chloride and were blocked by bicuculline. The results suggest that the GE6 clone, which is ventrally derived from the GE, resembles GABAergic interneuron progenitors that migrate into the developing forebrain. This is the first report of a relatively stable fetal clone that can be differentiated into GABAergic interneurons with functional synapses. The purpose was to compare differentiation patterns of several different immortalized rat neural progenitor clones to identify early stages in differentiation. The cell clones studies were: GE6 (GABAergic neuronal precursor), GE2 (non-neuronal precursor, CTX8 (multipotential precursor), L2.2 (interneuronal precursor), and L2.3 (multipotential precursor). Five rat neural precursor cell clones were compared at three different time points following FGF2 withdrawal, which triggers differentiation. Three sister culture replicates were performed for each cell clone and time point, yielding 45 samples. One microarray failed so we have 44 microarray results in the dataset.

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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Individual Human Cortical Progenitors Can Produce Excitatory and Inhibitory Neurons

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