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| Status |
Public on Jul 04, 2020 |
| Title |
A single cell RNA-seq resource for early sea urchin development |
| Organism |
Strongylocentrotus purpuratus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Here we leverage the sea urchin embryo for its rich history of developmental transitions, its well-established gene regulatory network, and the ability to readily dissociate embryos into single cells to interrogate the embryo by single cell RNA-seq. We tested eight developmental stages in S. purpuratus, from the eight-cell stage to late in gastrulation. The analysis revealed cell types derived from the abundant cells of the three germ layers as well as the rare cells of the germline. We used these datasets to parse out 22 major cell states of the embryo with focus on key transition stages for cell type specification of each germ layer. Sub-clustering of these major embryonic domains revealed over 50 cell states with distinct transcript profiles. Further, we identified the transcript profile of two cell states expressing germ cell factors, one we conclude are the primordial germ cells, and the other transiently present prior to gastrulation. We hypothesize that these cells of the veg2 tier of the early embryo may represent a lineage that converts to the germ line when the primordial germ cells are deleted (Voronina et al., 2008). This broad resource will hopefully enable the community to identify other cell states and genes of interest to expose the underpinning of developmental mechanisms.
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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. Fertilization was accomplished in sea water containing 10mM p-aminobenzoic acid to reduce cross-linking of the fertilization envelope, and which was washed out after 30 minutes. 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. Multiple fertilizations were initiated in this study and timed such that the appropriate stages of embryonic development were reached at a common endpoint. The embryos were then collected and 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 scmRNA-seq protocol. Equal numbers of embryos were used in each time point and at no time were cells or embryos pelleted in a centrifuge.
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| Contributor(s) |
Foster S, Oulhen N, Wessel G |
| Citation(s) |
32816969 |
| Submission date |
Apr 23, 2020 |
| Last update date |
Oct 05, 2020 |
| Contact name |
Gary Wessel |
| Organization name |
Brown University
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| Department |
Molecular Biology, Cell Biology & Biochemistry
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| Street address |
185 Meeting St
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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 (8)
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| Relations |
| BioProject |
PRJNA627693 |
| SRA |
SRP258127 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE149221_RAW.tar |
398.3 Mb |
(http)(custom) |
TAR (of MTX, TSV) |
| GSE149221_SpInteg.rds.gz |
1.9 Gb |
(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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