The roles of insulin-like growth factor 5 (IGFBP5) on fish osmoregulation

2022 Fiscal Year Research-status Report

• Project/Area Number: 22K06291
• Research Institution: Toho University
• Principal Investigator: 黄 國成 東邦大学, 理学部, 博士研究員 (40526901)
• Project Period (FY): 2022-04-01 – 2025-03-31
• Keywords: osmoregulation / IGFBP5 / medaka / chum salmon / ISH chain reaction / ionocyte

Outline of Annual Research Achievements

I have made IGFBP5a knockout (KO) medaka using CRISPR-Cas9 system. I have introduced a 5-nucleotide deletion in the IGFBP5a gene in drR medaka and established a heterologous KO line. With crossing between the heterologous line, it is in the process to breed homologous IGFBP5a KO medaka line.
The salinity tolerance and transporter expression in the ionocytes in heterologous KO medaka was examined and no apparent difference was observed compared with the WT drR medaka.
I have made two custom antibodies for the IGFBP5a in medaka and chum salmon respectively, but the antibodies for IGFBP5a were not desirable as the immunohistochemistry and Western blot results showed non-specific signals at the mucus.
I have established a novel method to detect the transcript localization using in situ hybridization chain reaction (ISHCR). The method can distinguish closely resemble gene isoforms and I have demonstrated the distinct localization of Na-K-ATPase (NKA) alpha-1a and alpha-1b in chum salmon gill, which cannot be distinguished by conventional in situ hybridization method using cRNA probes. I have used the same method to study the localization of IGFBP5a in chum salmon ionocytes, and initial result suggested that the IGFBP5a is not colocalized with NKA, an observation different from that of zebrafish.
I found a sequential increase in IGFBP5a expression during embryonic stages, in which the smoltification occurs in chum salmon. However, seawater treatment downregulated the IGFBP5a expression significantly in smolting chum salmon, an observation that is different from that of medaka.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

The making of the IGFBP5a-KO line in medaka was more challenging than expected as it is likely that the loss of IGFBP5a may have impacted the the growth and reproduction of the mutants. So far, few homologous individuals could be obtain and their fertility was poor, which contributing to the major obstacle in the progress of the project.

Strategy for Future Research Activity

I will continue to establish 5a-KO medaka and study the physiological characteristics of the 5a-KO on the osmoregulatory performance.
After the establishment of the KO-line, I will perform RNA-seq analysis on the gills of wild-type and KO medaka in freshwater and seawater to examine the global influence of losing of IGFBP5a in the body system. I will perform STRING analysis to examine the protein-protein interaction to deduce what protein networks are influenced most by the 5a-KO during salinity acclimation.
I consider remaking the chum salmon IGFBP5a antibody using a different peptide region or to purify the antibodies using affinity purification.
I will extend the use of ISHCR to examine the localization and expression levels of IGFBP5a and other ionocyte markers in medaka and chum salmon.