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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 10, 2023 |
| Title |
Droplet-based genome-wide forward genetic screening of astrocyte-microglia communication mechanisms |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
SPEACC-seq is a novel high-throughput method which enables forward genetic screens to identify cell-cell interaction mechanisms that uncovered an astrocyte-microglia regulatory circuit mediated by amphiregulin and IL33-ST2. signaling. Cell-cell interactions in the central nervous system (CNS) play central roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. For example, several factors mediate microglia-astrocyte interactions that promote CNS pathology, but less is known about regulatory interactions that limit tissue pathology. Here we report the development of SPEACC-seq (Stimulation, Perturbation, and Encapsulation of interACting Cells followed by Sequencing), a forward genetic screening platform which combines genome-wide CRISPR/Cas9 perturbations, cell co-culture in picoliter droplets, and microfluidic-based fluorescence activated droplet sorting to identify mechanisms of cell-cell communication. Using SPEACC-seq in combination with an in vivo perturb-seq screen, we identified microglia-produced amphiregulin as a suppressor of disease promoting astrocyte responses in experimental autoimmune encephalomyelitis (EAE), a pre-clinical model of multiple sclerosis (MS). The production of microglial amphiregulin was induced via ST2 signaling by IL-33 released from astrocytes during EAE. Indeed, the genetic inactivation of ST2 or amphiregulin in microglia, or IL-33 or amphiregulin signaling in astrocytes resulted in the worsening of EAE, suggesting that IL-33-induced microglial amphiregulin limits disease-promoting astrocyte responses associated with CNS pathology. This regulatory loop was also detected in human astrocytes and microglia both in vitro and in MS patient CNS samples. In summary, we developed SPEACC-seq, a high-throughput, droplet-based forward genetic screening platform for the identification of cell-cell interaction mechanisms, which identified a novel microglia-astrocyte negative feedback loop that limits CNS pathology.
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| Overall design |
For samples with 4OTR Prefix the experiment is designed to study the effect of IL33 knockout and ST2 knockout in Astrocytes and Microglia (3 samples for each condition in each cell type); for samples with 4M67 prefix the experiment contain samples with Areg knockout and Egfr knockout in Astrocytes and Microglia (3 samples for each condition). In the end two single cell experiment is conducted on the CNS tissue in Naive Mouse and EAE induced Mouse.
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| Contributor(s) |
Lee H, Li Z, Clark I, Quintana F, Wheeler M |
| Citation(s) |
36893254 |
| Submission date |
Apr 07, 2022 |
| Last update date |
Mar 11, 2023 |
| Contact name |
Michael Wheeler |
| E-mail(s) |
mwheeler0@bwh.harvard.edu
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| Organization name |
Brigham and Women's Hospital
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| Department |
Neurology
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| Street address |
60 Fenwood Rd.
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (50)
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| GSM6034407 |
IL33, WT, Astrocytes (4OTR.TAAGGCGA_CTAGTCGA) |
| GSM6034408 |
IL33, WT, Astrocytes (4OTR.CGTACTAG_CTAGTCGA) |
| GSM6034409 |
IL33, WT, Astrocytes (4OTR.AGGCAGAA_CTAGTCGA) |
| GSM6034410 |
IL33, KO, Astrocytes (4OTR.TCCTGAGC_CTAGTCGA) |
| GSM6034411 |
IL33, KO, Astrocytes (4OTR.GGACTCCT_CTAGTCGA) |
| GSM6034412 |
IL33, KO, Astrocytes (4OTR.TAGGCATG_CTAGTCGA) |
| GSM6034413 |
IL33, WT, Microglia (4OTR.CTCTCTAC_CTAGTCGA) |
| GSM6034414 |
IL33, WT, Microglia (4OTR.CGAGGCTG_CTAGTCGA) |
| GSM6034415 |
IL33, WT, Microglia (4OTR.AAGAGGCA_CTAGTCGA) |
| GSM6034416 |
IL33, KO, Microglia (4OTR.GTAGAGGA_CTAGTCGA) |
| GSM6034417 |
IL33, KO, Microglia (4OTR.GCTCATGA_CTAGTCGA) |
| GSM6034418 |
IL33, KO, Microglia (4OTR.ATCTCAGG_CTAGTCGA) |
| GSM6034419 |
ST2, WT, Astrocytes (4OTR.TAAGGCGA_AGCTAGAA) |
| GSM6034420 |
ST2, WT, Astrocytes (4OTR.CGTACTAG_AGCTAGAA) |
| GSM6034421 |
ST2, WT, Astrocytes (4OTR.AGGCAGAA_AGCTAGAA) |
| GSM6034422 |
ST2, KO, Astrocytes (4OTR.TCCTGAGC_AGCTAGAA) |
| GSM6034423 |
ST2, KO, Astrocytes (4OTR.GGACTCCT_AGCTAGAA) |
| GSM6034424 |
ST2, KO, Astrocytes (4OTR.TAGGCATG_AGCTAGAA) |
| GSM6034425 |
ST2, WT, Microglia (4OTR.CTCTCTAC_AGCTAGAA) |
| GSM6034426 |
ST2, WT, Microglia (4OTR.CGAGGCTG_AGCTAGAA) |
| GSM6034427 |
ST2, WT, Microglia (4OTR.AAGAGGCA_AGCTAGAA) |
| GSM6034428 |
ST2, KO, Microglia (4OTR.GTAGAGGA_AGCTAGAA) |
| GSM6034429 |
ST2, KO, Microglia (4OTR.GCTCATGA_AGCTAGAA) |
| GSM6034430 |
ST2, KO, Microglia (4OTR.ATCTCAGG_AGCTAGAA) |
| GSM6034431 |
CD11b, Scramble-1, MG (4M67_ACTCGCTA_AGCTAGAA) |
| GSM6034432 |
CD11b, Scramble-1, AS (4M67_ACTCGCTA_CTAGTCGA) |
| GSM6034433 |
GFAP, Scramble-2, MG (4M67_ACTGAGCG_AGCTAGAA) |
| GSM6034434 |
GFAP, Scramble-2, AS (4M67_ACTGAGCG_CTAGTCGA) |
| GSM6034435 |
CD11b, Areg-3, MG (4M67_ATGCGCAG_AGCTAGAA) |
| GSM6034436 |
CD11b, Areg-3, AS (4M67_ATGCGCAG_CTAGTCGA) |
| GSM6034437 |
GFAP, Scramble-3, MG (4M67_CCTAAGAC_AGCTAGAA) |
| GSM6034438 |
GFAP, Scramble-3, AS (4M67_CCTAAGAC_CTAGTCGA) |
| GSM6034439 |
GFAP, EGFR-1, MG (4M67_CGATCAGT_AGCTAGAA) |
| GSM6034440 |
GFAP, EGFR-1, AS (4M67_CGATCAGT_CTAGTCGA) |
| GSM6034441 |
CD11b, Areg-1, MG (4M67_CGGAGCCT_AGCTAGAA) |
| GSM6034442 |
CD11b, Areg-1, AS (4M67_CGGAGCCT_CTAGTCGA) |
| GSM6034443 |
CD11b, Scramble-3, MG (4M67_GCGTAGTA_AGCTAGAA) |
| GSM6034444 |
CD11b, Scramble-3, AS (4M67_GCGTAGTA_CTAGTCGA) |
| GSM6034445 |
CD11b, Scramble-2, MG (4M67_GGAGCTAC_AGCTAGAA) |
| GSM6034446 |
CD11b, Scramble-2, AS (4M67_GGAGCTAC_CTAGTCGA) |
| GSM6034447 |
CD11b, Areg-2, MG (4M67_TACGCTGC_AGCTAGAA) |
| GSM6034448 |
CD11b, Areg-2, AS (4M67_TACGCTGC_CTAGTCGA) |
| GSM6034449 |
GFAP, Scramble-1, MG (4M67_TAGCGCTC_AGCTAGAA) |
| GSM6034450 |
GFAP, Scramble-1, AS (4M67_TAGCGCTC_CTAGTCGA) |
| GSM6034451 |
GFAP, EGFR-3, MG (4M67_TCGACGTC_AGCTAGAA) |
| GSM6034452 |
GFAP, EGFR-3, AS (4M67_TCGACGTC_CTAGTCGA) |
| GSM6034453 |
GFAP, EGFR-2, MG (4M67_TGCAGCTA_AGCTAGAA) |
| GSM6034454 |
GFAP, EGFR-2, AS (4M67_TGCAGCTA_CTAGTCGA) |
| GSM6034455 |
EAE.Single.Cells [10X] |
| GSM6034456 |
Naive.Single.Cells [10X] |
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| Relations |
| BioProject |
PRJNA824546 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE200457_RAW.tar |
185.4 Mb |
(http)(custom) |
TAR (of TAR) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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