2019 Fiscal Year Annual Research Report
Study on metabolism/physiological functions of retinoic acid in metamorphic larvae of the Pleuronectiformes
| Project/Area Number |
18J20627
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| Research Institution | Tohoku University |
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Principal Investigator |
陳 其然 東北大学, 農学研究科, 特別研究員(DC1)
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| Project Period (FY) |
2018-04-25 – 2021-03-31
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| Keywords | retinoic acid / metabolism / RNA-Seq / in situ hybridization / metamorphosis / Japanese flounder |
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| Outline of Annual Research Achievements |
1. Exogenous RA induced negative feedback reaction in the RA metabolism pathway to decrease RA content by up-regulating RA degradation and conversion genes. ISH analysis confirmed the up-regulation of RA catabolic enzymes and bone-related genes in basal cells that envelope notochord. The results may provide new insights into the metamorphic bone formation and mechanism of RA regulation in metamorphosing Japanese flounder.
2. Transient exposure of metamorphosing larvae to retinoic acid (RA) induced progenitors on the blind-side to differentiate into gch2-positive chromatoblasts. In addition, the RAR antagonist, BMS493 treatment suppressed the gch2-positive signal on ocular side. Thus, we indicated that RA is essential for flounder chromatophore progenitors to differentiate into chromatoblasts. At the time of chromatoblast differentiation to melanophores, cyp26b1, which inactivates RA, was up-regulated on the blind-side skin compared to the ocular-side. Therefore, we suppose that ocular-side specific pigmentation is regulated by the inhibition of RA-signaling by cyp26b1 on the blind-side. The data were published in the Journal of Experimental Zoology Part B: Molecular and Developmental Evolution.
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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
This year, we further analyze the RNA-seq results. Obtained data suggest that negative feedback of RA-signaling is particularly induced in G-stage flounder. Several genes related to bone formation were checked by in situ hybridization, and most of them showed up-regulation in basal cells that surround notochord. By BMS493 treatment, the gch2 expression was suppressed, indicating the critical role of RA in the initiation of chromatophore progenitor cell differentiation. cyp26b1 in blind side of skin was significantly higher than the ocular side during metamorphosing stages. We suppose cyp26b1 that expressed in blind side of skin down-regulate the local RA concentration to cause the asymmetric pigmentation in Japanese flounder. The results together will contribute to an understanding of RA function and asymmetry in flounder.
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| Strategy for Future Research Activity |
Challenges 1: (1) Sox10 positive cells representing not only chromatophore progenitors but also glia precursors in the DRG and axons. There is no lineage tracing system established in Japanese flounder.
Solution: In zebrafish, Cre-lox system was used to trace the Sox10 positive cells from expression until the apoptosis. We have succeeded in the transfer of plasmid into flounder cells, thus, we can study the pigment cell lineage in flounder using this method.
Challenges 2: BMS493 suppressed the expression of gch2 positive cells in the skin, but little information exists talking about the mechanism of RA-regulation in the initiation of chromatophore progenitor differentiation in metamorphosing flounders.
Solution: Japanese flounders at mid-climax stage will be separated into two groups. The first group will be treated with DMSO as control and the second group will be treated with BMS493. The larvae from each group will be collected and send to RNA-seq to compare the transcriptomic difference.
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