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| Status |
Public on Jan 15, 2025 |
| Title |
Identification of a Subpopulation of Astrocyte Progenitor Cells in the Neonatal Subventricular Zone: Evidence that Migration is Regulated by Glutamate Signaling |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
In adult mice engineered to express enhanced green fluorescent protein (eGFP) under the control of the glutamate transporter 1 (GLT1) gene, eGFP is found in all cortical astrocytes. However, when 8.3 kilobases of the human GLT1/EAAT2 gene is used to control expression of tdTomato (tdT), tdT is only found in a subpopulation of cortical astrocytes. The eGFP mice have been used to define mechanisms of transcriptional regulation using astrocytes cultured from cortex of 1-3 day old mice. Using the same cultures, we were never able to induce tdT+ expression. We hypothesized that these cells might not have migrated into the cortex by this age. In this study, we characterized the ontogeny of tdT+ cells, performed single-cell RNA sequencing (scRNA-seq), and tracked their migration in organotypic slice cultures. At postnatal day (PND) 1, tdT+ cells were observed in the subventricular zone and striatum but not in the cortex, and they did not express eGFP. At PND7, tdT+ cells began to appear in the cortex with their numbers increasing with age. At PND1, scRNA-seq demonstrates that the tdT+ cells are molecularly heterogeneous, with a subpopulation expressing astrocytic markers, subsequently validated with immunofluorescence. In organotypic slices, tdT+ cells migrate into the cortex and express GLT1, NF1A, and eGFP after 7 days. Ionotropic glutamate receptor (iGluR) antagonists reduced (25 to 50%) the distance tdT+ cells migrate from the subventricular zone into cortex. The pan-glutamate transport inhibitor, TFB-TBOA, dramatically increased (10-fold) the number of tdT+ cells in the cortex. In conclusion, although tdT is expressed by non-glial cells at PND1, it is also expressed by glial progenitors that migrate into the cortex postnatally. With this fluorescent labelling, we provide novel evidence that glutamate signaling contributes to the control of glia precursor migration.
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| Overall design |
The dorsal anterior forebrain of three postnatal day 1 double reporter (BAC-GLT1-eGFP/8.3kb-EAAT2-tdTomato) mice were dissected and dissociated into a single cell suspension using enzymatic and mechanical dissociation. eGFP+ or tdT+ cells were isolated using FACS. Next-generation sequencing libraries were prepared using the 10x Genomics Chromium Single Cell 3’ Reagent kit v3. Libraries were uniquely indexed using the Chromium i7 Sample Index Kit. After preparing a library and performing quality control on a High Sensitivity D1000 ScreenTape station, 17,596 tdT+ and 16,956 eGFP+ cells were loaded and sequenced on an Illumina NovaSeq sequencer in a paired-end, single indexing run. Data were then processed using the cellranger pipeline (10x genomics, v6.0.0) for demultiplexing and alignment of sequencing reads to the mm10 transcriptome, with the tdT (GenBank Accession #AY678269.1) and eGFP (GenBank Accession #MK387175.1) transcripts added to produce the feature-barcode matrices. A pool of three mice was use, with no replicates.
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| Contributor(s) |
Martinez-Lozada Z, Guillem AM, Song I, Gonzalez MV, Takano H, Parikh E, Rothstein JD, Putt ME, Robinson MB |
| Citation(s) |
39789409 |
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| Submission date |
Jul 26, 2024 |
| Last update date |
Jan 16, 2025 |
| Contact name |
Michael Gonzalez |
| E-mail(s) |
michael.gonzalez1@pennmedicine.upenn.edu
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| Phone |
9098389935
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| Organization name |
University of Pennsylvania
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| Department |
Translational Medicine and Human Genetics
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| Lab |
Fajgenbaum
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| Street address |
3535 Market St
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| City |
Philadelphia |
| State/province |
Pennsylvania |
| ZIP/Postal code |
19122 |
| Country |
USA |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (2) |
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| Relations |
| BioProject |
PRJNA1140624 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE273223_RAW.tar |
152.3 Mb |
(http)(custom) |
TAR (of MTX, TSV) |
SRA Run Selector |
| Raw data are available in SRA |
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