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| Status |
Public on Jul 31, 2020 |
| Title |
Regulation of dynamic pigment cell states at single-cell resolution |
| Organism |
Strongylocentrotus purpuratus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Pigment cells bear molecules with diverse physiological roles across phylogeny and are often under strict evolutionary selection. Thus, the mechanisms of regulating these cells, and the pigments within them, are of critical importance. Here, we explore the regulation of pigment cells in the purple sea urchin Strongylocentrotus purpuratus, an emerging model for understudied pigments at a molecular level. Sea urchins display a variety of pigments in both embryos and adults, but how the pigment cells form during development has not been resolved, nor has their breadth of biological significance been revealed. Known genes expressed by embryonic pigment cells are the transcriptional factor glial cells missing (gcm), and the enzymes pks1 and fmo1,2 and 3 (polyketide synthase1, flavin-dependent monooxygenase 1, 2, 3 respectively). In this study, we took advantage of single cell RNA-seq technology to interrogate the cell state of emerging pigment cells, and to test their diversity in fate and function.
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| Overall design |
Eggs and sperm of S.purpuratus were spawned by injection of 0.5M KCl into the adult coelomic cavity. Embryos were cultured in filtered (0.2micron) sea water collected at the Marine Biological laboratories in Woods Hole MA, until the appropriate stage for dissociation (48 and 72 hours post fertilization). To test the function of gcm, eggs were dejelled before fertilization by washing in pH4.0 seawater, and injected post fertilization in presence of 1mM 3-AT with either a control morpholino or the gcm morpholino used at a concentration of 0.5mM. The resulting wild type controls and albino embryos were collected at 48 hpf. These 4 samples (non injected 48 and 72 hpf, control morpholino injected 48 hpf and gcm morpholino injected 48 hpf embryos) were used for single cell RNA seq. Briefly, the collected embryos were washed twice with calcium-free sea water, and then suspended hyalin-extraction media (HEM) for 10-15 minutes, depending on the stage of dissociation. When cells were beginning to dissociate, the embryos were collected and washed in 0.5M NaCl, gently sheared with a pipette, run through a 40micron Nitex mesh, counted on a hemocytometer, and diluted to reach the appropriate concentration for the single cell RNA-seq protocol using the 10X Chromium technology (v3 Chemistry).
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| Contributor(s) |
Perillo M, Oulhen N, Foster S, Spurrell M, Calestani C, Wessel G |
| Citation(s) |
32812865 |
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| Submission date |
Jul 30, 2020 |
| Last update date |
Sep 08, 2020 |
| Contact name |
nathalie oulhen |
| E-mail(s) |
nathalie_oulhen@brown.edu
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| Organization name |
Brown university
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| Street address |
185 meeting street
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| City |
Providence |
| State/province |
RI |
| ZIP/Postal code |
02912 |
| Country |
USA |
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| Platforms (1) |
| GPL28450 |
Illumina HiSeq 4000 (Strongylocentrotus purpuratus) |
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| Samples (4)
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| GSM4704280 |
48 hours post fertilization embryos |
| GSM4704281 |
72 hours post fertilization embryos |
| GSM4704282 |
48 hours post fertilization embryos, control MO injected |
| GSM4704283 |
48 hours post fertilization embryos, gcm MO injected |
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| Relations |
| BioProject |
PRJNA649738 |
| SRA |
SRP274329 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE155427_RAW.tar |
67.0 Mb |
(http)(custom) |
TAR (of MTX, TSV) |
| GSE155427_Sp48MO.rds.gz |
89.5 Mb |
(ftp)(http) |
RDS |
| GSE155427_Sp48and72.rds.gz |
307.2 Mb |
(ftp)(http) |
RDS |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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