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Gene duplication of C-type natriuretic peptide-4 (CNP4) in teleost lineage elicits subfunctionalization of ancestral CNP

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Abstract

The diversified natriuretic peptide (NP) family, consisting of four CNPs (CNP1-4), ANP, BNP, and VNP, has been identified in the eel. Here, we successfully cloned additional cnp genes from the brain of eel (a basal teleost) and zebrafish (a later branching teleost). The genes were identified as paralogues of cnp4 generated by the third round of whole genome duplication (3R) in the teleost lineage, thereby being named eel cnp4b and zebrafish cnp4-like, respectively. To examine the histological patterns of their expressions, we employed a newly developed in situ hybridization (ISH) chain reaction using short hairpin DNAs, in addition to conventional ISH. Eel cnp4b was expressed in the medulla oblongata, while mRNAs of eel cnp4a (former cnp4) were localized in the preoptic area. In the zebrafish brain, cnp4-like mRNA was undetectable, while the known cnp4 was expressed in both the preoptic area and medulla oblongata. Together with the different mRNA distribution of cnp4a and cnp4b in eel peripheral tissues determined by RT-PCR and ISH, it is suggested that subfunctionalization by duplicated cnp4s in ancestral teleosts has been retained only in basal teleosts. Intriguingly, cnp4b-expressing neurons in the glossopharyngeal-vagal motor complex of the medulla oblongata were co-localized with choline acetyltransferase, suggesting an involvement of Cnp4b in swallowing and respiration functions that are modulated by the vagus. Since teleost Cnp4 is an ortholog of mammalian CNP, the identified localization of teleost Cnp4 will contribute to future studies aimed at deciphering the physiological functions of CNP.

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Acknowledgements

We thank Dr. Andre P. Seale of the University of Hawaii for critical reading of the manuscript and Dr. Wataru Iwasaki of Atmosphere and Ocean Research Institute, the University of Tokyo, for providing an eel genome database. We also thank Ms. Masako Ino of Nikon Solutions for the technical assistance of confocal laser microscopy.

Funding

This work was supported in part by JSPS KAKENHI Grants (nos. 20K06242 to TT and 20K15604 to YK), the Scientific research funds to YK from Okayama University.

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Authors and Affiliations

  1. Faculty of Science, Ushimado Marine Institute, Okayama University, 130-17 Kashino, Setouchi, Okayama, 701-4303, Japan

    Yukitoshi Katayama

  2. Department of Biomolecular Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan

    Yukitoshi Katayama, Ami Saito & Takehiro Tsukada

  3. Graduate School of Natural Science and Technology, Okayama University, 1-1-1 Tsushima-Naka, Kita, Okayama, Okayama, 700-8530, Japan

    Maho Ogoshi

  4. Department of Anatomy, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota, Tokyo, 143-8540, Japan

    Yousuke Tsuneoka

  5. Department of Anatomy, Faculty of Medicine, Tottori University, 86 Nishicho, Yonago, Tottori, 683-8503, Japan

    Takao Mukuda

  6. Division of Molecular Pharmacology, Department of Pharmacology, Jichi Medical University School of Medicine, 3311–1 Yakushiji, Shimotsuke, Tochigi, 329-0498, Japan

    Morio Azuma

  7. Faculty of Science, Shizuoka University, 836 Otani, Suruga, Shizuoka, Shizuoka, 422-8529, Japan

    Makoto Kusakabe

  8. Laboratory of Physiology, Atmosphere and Ocean Research Institute, the University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba, 277-8564, Japan

    Yoshio Takei

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  1. Yukitoshi Katayama
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Contributions

YK wrote the manuscript and analyzed data; AS performed in situ hybridization for cnp4s and immunohistochemistry; MO performed synteny and phylogenetic analyses; YT established and designed in situ hybridization chain reaction in eel brain; TM analyzed eel brain anatomy; MA established signal enhancement method of in situ hybridization for cnp4s; MK sampled and processed eel tissues for reverse-transcription PCR; TY performed in silico analyses; TT coordinated the project. All authors read, edited, and approved the final manuscript.

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Correspondence to Takehiro Tsukada.

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Supplementary Information

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441_2022_3596_MOESM1_ESM.eps

Supplementary Figure 1. Principle of in situ hybridization chain reaction. A Short hairpin DNA (H1, H2) and initiator. Hairpin DNA is composed of toehold, stem, and loop domains, and is conjugated to a fluorophore. Complementary sequences of a, b, and c are a’, b’ and c’, respectively. Initiator first hybridizes with H1 to open the hairpin structure, which promotes hybridization to H2, followed by continuous H1-H2 hybridization. B Design of split-initiator probes. Each probe has 25 nucleotide (nt) complementary to the target mRNA, 2 nt spacer sequence, and 9 nt partial initiator sequence of b’. Only when a set of split-initiator probes hybridizes to the target mRNA, H1 recognizes the initiator sequence and chain reaction starts. C A schematic diagram of in situ HCR. In this experiments, 4 and 2 sets of split-initiator probes against eCNP4a and eCNP4b mRNAs were designed, respectively. (EPS 1786 KB)

441_2022_3596_MOESM2_ESM.eps

Supplementary Figure 2. Nucleotide and amino acid sequences of eel Cnp4b. A Nucleotide sequence of eel cnp4b mRNA. Capital letters indicate coding sequence. B Comparison of amino acid sequence among eel Cnps. Signal peptide, furin cleavage site (RXXR) and endopeptidase cleavage site (KK: dibasic amino acid), and disulfide bond are shown. Gray fillings indicate mature Cnp sequences. Eel cnp2 transcripts has not been found. (EPS 2165 KB)

441_2022_3596_MOESM3_ESM.eps

Supplementary Figure 3. Nucleotide and amino acid sequences of zebrafish Cnp4-like. A Nucleotide sequence of zebrafish cnp4-like mRNA. Capital letters indicate coding sequence. B Amino acid sequence of zebrafish Cnp4s. Signal peptide, furin cleavage site (RXXR) and endopeptidase cleavage site (KK: dibasic amino acid), and disulfide bond are shown. Gray fillings indicate mature Cnp sequences (EPS 1873 KB)

441_2022_3596_MOESM4_ESM.eps

Supplementary Figure 4. Original agarose gel image of Fig. 3A. A cnp4a, B cnp4b, and C Elongation Factor 1 α (ef1a) (EPS 4739 KB)

441_2022_3596_MOESM5_ESM.eps

Supplementary Figure 5. Brain atlas of the Japanese eel (Sagittal view). A Ob: olfactory bulb, Tel: telencephalon, TeO: optic tectum, Ce: cerebellum, MO: medulla oblongata, AP: area postrema, and P: pituitary. B in situ hybridization using eel cnp4a/4b sense probe in the eel brain as control. P: pituitary, II: optic nerve. Scale bars: 1 mm. C in situ hybridization using cnp4b sense probe in the eel peripheral tissues as control. Scale bars: 50 µm (EPS 124781 KB)

441_2022_3596_MOESM6_ESM.eps

Supplementary Figure 6. Characterization of medaka cnp4-expressing cells in medaka brain. In situ hybridization for medaka cnp4. Medaka cnp4-expressing neurons were localized in the preoptic area a and the medulla oblongata b. Magnified views of red box a, b are shown in the bottom panels. Scale bars: 1mm (upper panels), 100 µm (bottom panels)(EPS 47204 KB)

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Katayama, Y., Saito, A., Ogoshi, M. et al. Gene duplication of C-type natriuretic peptide-4 (CNP4) in teleost lineage elicits subfunctionalization of ancestral CNP. Cell Tissue Res 388, 225–238 (2022). https://doi.org/10.1007/s00441-022-03596-y

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