| 研究実績の概要 |
Hematopoiesis is the process by which the blood cells of an individual are formed. However, despite being a crucial physiological phenomenon of animals, most of our knowledge derives from studies using a handful of classic animal models, mostly representatives of jawed vertebrates. With this in mind, I set to study the lamprey, long-distant relative of jawed vertebrates that is understudied in terms of the blood system. During FY2019 I have mainly completed my analysis of RNA-seq data derived from different embryological samples of the Arctic lamprey, Lethenteron camtschaticum (syn. Lethenteron japonicum), from stage 8 to 28. These RNA-seq data was used to de novo assemble a transcriptome than, together with L. camtschaticum’s genome assembly (Mehta et al., 2013. PNAS. 110(40), 16044-16049), was utilized to screen for orthologous counterparts of genes known to have an important role in jawed vertebrates’ hematopoiesis: these include genes for Erg (2 paralogues), Jam (2 paralogues), Lmo1/2 (1 paralogue), Myb (3 paralogues), Runx (2 paralogues, previously described), SCL (2 paralogues), TIE receptors (2 paralogues) and VEGFR (4 paralogues). Surprisingly, I found that the lamprey genome contains more paralogs than jawed vertebrates typically have for the gene GATA1/2/3, for which lamprey has 6 paralogs, unlike the four present in jawed vertebrates. Last, I have already performed in situ hybridization revealing the spatio-temporal expression of many of these genes, revealing the presence of structures similar to the blood cell islands of jawed vertebrate embryos.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
I have been able to achieve the objectives I set for FY2019. However, during last FY our laboratory was not able to obtain fresh adult individuals of the lamprey, delaying the sampling for the necessary RNA-seq data, which was obtained from frozen samples from previous years.
Now, in FY2020, the recent outbreak of a new type of coronavirus has halted all research activity at RIKEN, completely overlapping with the lamprey breeding season. This might incur in certain future delays in the project.
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| 今後の研究の推進方策 |
For FY2020, I plan to carry out the originally proposed research plan, in which I will perform functional genomics experiments using some of the described genes from FY2019. Scl is a key transcription factor with important functions in the formation of both primitive erythropoiesis and myelopoiesis in both zebrafish and mouse, as well as in the generation of endothelial cells. Lamprey Scl genes are thus ideal candidate genes for this proof of principle experiments. First, during the lamprey breeding season I will inject CRISPR/Cas9 constructs aimed at knocking out both lamprey Scl together with mock injections as control. I will study the phenotype obtained from knock out embryos, paying especial attention to those that affect the circulatory system and the formation of blood. In order to do these, I will keep a pool of embryos for in situ hybridization assays, using some of the genes described in FY2019 as markers. Then, RNA-seq data will be obtained from both knock out and control pools of embryos, and a comprehensive analysis of the genes affected by Scl knock out will be determined by means of differential expression analysis. Finally, I will perform a GO enrichment analysis, which will tell me about the processes and function of genes affected, which I expect to be genes involved in the formation of blood and vessels.
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| 次年度使用額が生じた理由 |
The lack of fresh adult and therefore embryological samples of the lamprey on FY2019 precluded me to obtain as much material as I planned. However, I was able to sequence enough samples for the project to continue.
For the next fiscal year, I will use the incurring amount to carry out RNA-seq at a higher depth, increasing the sensibility of the experiments in terms of lowly expressed genes that might be affected by the knocked out embryos.
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