| 研究実績の概要 |
I have made IGFBP5a knockout (KO) medaka and a heterologous KO (IGFBP5a+/-) line is maintained. By crossing the heterologous KO individuals, homologous KO (IGFBP5a-/-) were created, but the survival rate was terribly low (7 individuals after 298 genotyping [2.3 % survival rate to adult]). Furthermore, the fertility of the IGFBP5a-/- individuals are poor, thus maintaining a stable KO line is challenging.
Using a newly developed method known as in situ hybridization chain reaction (ISHCR), I have localized the expression of IGFBP5a with other key transporters in the gill of medaka and chum salmon under different salinities. IGFBP5a is localized in the accessory cells, co-expressing TRPV6 (a calcium channel).
With ISHCR, I have analyzed the distribution of NKA isoform expression in chum salmon gill from different salinities and performed morphometrics analysis on the ionocytes expressing different degree of NKA isoforms (doi: 10.1007/s10695-023-01212-6.)
I have developed two new antibodies against the IGFBP5a proteins in medaka and chum salmon, and initial observation suggested that the IGFBP5a proteins are located on the major ionocytes, but not on accessory cells, indicating that the IGFBP5a could be produced in the accessory cells and transferred to the ionocytes.
To disseminate the research result, I have attended a domestic conference organized by the Japanese Society of Comparative Endocrinology in November 2023, and presented the progress of the project, which has attracted many domestic researchers.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
The major setback of the functional analysis of IGFBP5a is the low survival rate of the IGFBP5a-/- medaka individuals. Due to the low survival rate as described before, it did not allow the planned salinity challenge experiment and other in vivo experiment. The reason for the low survival remains unidentified and I am investigating the histology of the IGFBP5a-/- individuals, to look for any hint of organ under-development or potential defects. The results will be important basic information for the role of IGFBP5a in the development process.
Another reason for the hindered progress is that the initial custom-made antibodies for IGFBP5a were not specific, thus I have re-designed the epitopes, and remake the specific antibodies. The newly made antibodies were now under investigation.
Salinity challenges to the IGFBP5a-/- individuals are pending because of the lack of individual numbers, but salinity challenges to heterogenous KO (IGFBP5a+/-) individuals have been performed, and so far no defects in seawater adaptability were observed.
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| 今後の研究の推進方策 |
In the final year of the project, I will focus on finding the reason of the low survival rate of the IGFBP5a-/- medaka. By using the ISHCR and IHC combination, I can study the transcription, translation and translocation of the IGFBP5a in fish ionocytes. With the limited number of IGFBP5a-/- individuals, I aim to study the histology of the KO medaka to search the possible roles of IGFBP5a on normal development in general. Since the IGFBP5a was confirmed to be present on accessory cells instead of ionocytes, it sheds lights on the functional role of accessory cells, which is mostly not studied. Each ionocyte is clearly coupled with an accessory cell to complete the ion-transporting apparatus, thus IGFBP5a is involved in the osmoregulatory process. Moreover, the possible translocation of IGFBP5a proteins from accessory cells to ionocytes are novel to our knowledge, and this may intrigue many fellow researchers.
I aim to present the project data in an international conference to promote the research topic and gather collaborators. I have planned to visit Prof. Cunming Duan of University of Michigan to discuss the progress and future collaboration. Prof. Duan is interested in the methods ISHCR and IHC of IGFBP5a and I will visit his laboratory in Michigan to transfer the technique.
I aim to summarize the current results and compose publications that report the findings in peer-reviewed journals. I would like to establish myself as one of the experts in this topics, and attract many researchers and collaborators to carry out further investigation on IGFBP5a in fish.
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