2019 Fiscal Year Annual Research Report
Cell type-specific mechanisms of desiccation tolerance in the anhydrobiotic insect
| Project/Area Number |
18H02217
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
グセフ オレグ 国立研究開発法人理化学研究所, 科技ハブ産連本部, ユニットリーダー (30711999)
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| Project Period (FY) |
2018-04-01 – 2021-03-31
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| Keywords | anhydrobiosis / Pv11 cells / scRNAseq / promoters / desiccation / enhancers / CAGE / transcriptome |
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| Outline of Annual Research Achievements |
In this year we have successfully achieved one of the main task of the study: creation of detailed libraries and obtaining sequence data for brains of anhydrobiotic chironomid on different states of anhydrobiosis. For analysis we used teh following design: we separately analyzed D0(wet), D48(dry larvae) and recovered larvae, as well as a single mix containing cells from 8 points of desiccations-rehydration cycle. The such "mix", we are going to utilize Velocity bioinformatics approach to analyze the "transitions" of expression responsein cell types along the "desiccation" trajectory.
Using combined methods of CAGE-Seq (bulk version) and single cell data we have identified key cell types in brain of the chironomid. We found that major cell type composition in normally hydrated larvae are similar to that of other diptera insects (compared to Drosophila). We observed strong transcriptional response to desiccation specific for different cell types in brains. Using obtained bulk tissues-specific and single cell data we identified key promising promoters and transcriptional factors hierarchy interaction in Pv11 cells upon anhydrobiosis.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Although we have some delay in sequencing results due to COVID-19,general progress of the study moves according to the schedule. We successfully profiled brain of ahnhydrobiotic midge on different stages of anhydrobiosis using 10xGenomics platform. More than 5000 cells were successfully identified based on the sequencing results and we could successfully discriminate main cell types. Now we analyze brain-cell type specific response to desiccation to recognize "common" and cell-specific types of transcriptional response and "minimal essential set" of protective genes required for desiccation tolerance. Also, in the parallel study we successfully developed an approach to identify transcription factor network hierarchy upon desiccation on Pv11 cells.
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| Strategy for Future Research Activity |
We plan to continue analyzing results of brain-derived single cell data, as one of the most interesting data-set obtained. The research paper on complexity of types of nervous cells and their reaction to desiccation is to be submitted. In collaboration with NARO (Prof. Kikawada group)to confirm contribution of identified genes in anhydrobiosis, we will conduct
experiments for knockout (in Pv11 cell line, using recently optimized for Pv CRISP-Cas9 technique) and knockdown (in vivo, using RNAi technique in the Pv larvae). Using markers identified by single cell analysis, "surviving" Pv11 sub-populating will be purified from the cell line, and then key anhydrobiotic genes will be knock-outed by CRISPR-Cas9. The effect of knockout will be assayed by desiccation-rehydration of the cells.
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