2018 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 signaling / metabolism / RNA-Seq / in situ hybridization / metamorphosis / Japanese flounder |
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| Outline of Annual Research Achievements |
1. RNA-Seq for RA-treated and DMSO-treated flounder larvae
The RNA-Seq data will be used for further analysis for the understanding of RA function in flatfish. In this experiment, there are several genes related to bone formation were detected up- or down-regulated by RA treatment, which gives insights into RA function in bone formation. In the top 30 up-regulated genes, most of them are related to hematopoiesis, RA catabolism, immune system and collagenase. And in top 30 down-regulated genes, most of these genes are related with collagens and extracellular matrix. The results reveal the possibility of participation of RA in bone formation, hematopoiesis and immune system. These results were presented in The Japanese Society of Fisheries Science Spring Meeting.
2. Expression profile of RA synthases and catabolic enzymes
Obtained data suggests that RA-signaling is particularly active in the central nervous system. aldh1a2 was strongly expressed in the intestine and liver, which may function as the main sources of RA during metamorphic development. aldh1a3 was expressed in the larval pituitary, giving interesting implications for endocrine function. RA synthases and catabolic enzymes were detected in canals of spinal cord or regions close to brain ventricle, which suggests RA could function through cerebralspinal fluid. These results were published in the paper “Expression profiles of RA synthases and catabolic enzymes in newly hatched and metamorphosing larvae of Japanese flounder, Paralichthys olivaceus” in General and Comparative Endocrinology.
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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
In this year, we have understood the expression pattern of retinoic acid (RA) synthases and catabolic enzymes in flounder by in situ hybridizaiton. Obtained data suggests that RA-signaling is particularly active in the central nervous system. aldh1a2 is strongly expressed in the intestine, which may function as the main sources of RA during metamorphic development. aldh1a3 is expressed in the larval pituitary, giving interesting implications for endocrine function. RA synthases were detected in canals of spinal cord or regions close to brain ventricle, which suggests the RA synthesized at tectum, spinal cord, pituitary may secret into the cerebrospinal fluid to exert effects on neural cells expressing RA receptors. The results explain where and how RA is synthesized and degraded in flounder, which gives insights into the function of RA in different tissues.
By using RNA-Seq, genes that up- or down-regulated by excess RA were screened out. Most of these genes were related to collagen, hemoglobin, RA metabolism and complement system. The results reveal the possibility of participation of RA in bone formation, hematogenesis and immune system. The results will be used for further analysis of RA functions in fish development.
These results together establish a solid foundation for future research, which will contribute to an understanding of RA function and asymmetry in flounder.
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| Strategy for Future Research Activity |
Challenges 1: We have understood the expression pattern of retinoic acid (RA) synthases and catabolic enzymes in flounder, but where retinoic acid receptors (RARs) express is still unknown. In previous attempts, we proved RARs expression is very hard to be detected by in situ hybridization during metamorphosing stages.
Solution and plans: Flounder embryos during embryogenesis will be collected twice a day and fixed by 4%PFA. Whole mount in situ hybridization will be proceeded to detect the expression of RARs in different tissues. Since RA synthases and catabolic enzymes have very similar expression patterns between embryos and larvae, we assume that the RARs expression in larvae could also be similar to embryos.
Challenges 2: Excess RA treatment was proved to cause severe effects on bone and pigment formation to metamorphosing Japanese flounder. But it is difficult to know if it is by the function of endogenous retinoic acid that affects flounder development (especially at pigmentation and bone formation).
Materials and methods: Japanese flounders at mid-climax stage will be separated into six groups, incubated by different chemicals include DMSO, RA, DEAB (antagonist of RA synthases), R115866 (antagonist of RA catabolic enzymes), BMS493 (antagonist of RA receptors) and the combination of DEAB and BMS493. By comparing different expression patterns of genes and phenotypes of fish between these groups, it can help us to understand the endogenous RA function.
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Research Products
(2 results)
