Haruhito Koda*, Runa Sekine*, Sae Watanabe*, Tsubaki Yamada*, Keita Tanaka*,1*Biology Club of Niigata Meikun High School, Niigata 950-0116, JapanRunning title: Mutation of mpv17 in iridophore reduced medakaKeywords: medaka, iridophore, mpv17 , MDDS (mitochondrial DNA depletion syndrome) 1Correspondence: Keita Tanaka, Biology Club of Niigata Meikun High School, Niigata 950-0116, Japan. Tel.: +81-25-257-2131. Email: keita_sc@yahoo.co.jpABSTRACTWe discovered a novel iridophore mutant of medaka (Oryzias latipes ), designated as panda (pa ) due to its black eye trait. Throughout the juvenile to adult stages, this mutant shows reduced iridophores. Using linkage analysis and whole genome sequencing, we identified mpv17 as the strong candidate causal gene of pa . Knocking out mpv17 in d-rR embryos showed that this gene was indeed causal. The gene encodes MPV17, a membrane transport protein localized on the inner mitochondrial membrane, which is thought to supply the matrix with deoxynucleotide phosphates and/or nucleotide precursors for mitochondrial DNA synthesis. No mutants caused by this gene have been reported in medaka. However, a similar mutant called transparent exists in zebrafish, caused by the same gene. Developmental observations revealed that iridophores in the mutants initially formed but subsequently disappeared during growth, suggesting that MPV17 is involved in maintaining rather than forming iridophores. Although mutations in this gene are lethal in mammals such as mice and humans, no such symptoms were observed in medaka. Therefore, our findings could lead to the discovery of new therapeutic strategy for mitochondrial DNA depletion syndrome, an intractable disease caused by mutations in MPV17.INTRODUCTIONThe body color of animal results from skin pigment cells derived from neural crest cells. While mammals and birds have one type of pigment cell, melanocytes, fish have multiple types: melanophores (black), xanthophores (yellow), iridophores (iridescent), erythrophores (red), cyanophores (blue), and leucophores (white) (Fujii, 1993). Because all these pigment cells are derived from neural crest cells and are easily distinguished by their color, fish have been studied as model organisms to characterize the mechanisms underlying the regulation of cell fate decisions in pluripotent cells. The medaka (O. latipes ) has four types of chromatophores: melanophores, xanthophores, iridophores, and leucophores (Takeuchi, 1976; Kelsh et al., 1996). In Medaka, body color mutants have been collected and lineages maintained by Tomita (Kelsh et al., 2004). One of these, an orange-red mutant, is homozygous for the b allele encoding a transporter that mediates melanin synthesis (Fukumachi et al., 2001). Medaka many leucophores-3 (ml-3) mutant embryos exhibit excessive formation of leucophores and absence of xanthophores. Previous research revealed that ml-3 encodes sox5, which has a cell-autonomous role in the xanthophore lineage. pax7a is expressed in neural crest cells and is required for both xanthophore and leucophore lineages (Nagao et al., 2014; Kimura et al., 2014). The few melanophore (fm) mutant of medaka is characterized by reduced numbers of melanophores and leucophores. kit-ligand a (kitlga) was identified as the gene whose mutation gives rise to the fm phenotype (Otsuki, 2019). Iridophores contain guanine crystals with a high refractive index, which efficiently reflect light at the interface with the cytoplasm, resulting in Tyndall phenomena and structural color. pnp4a has already been identified as the causal gene for gu: guanineless, an iridophore mutant of medaka (Kimura et al., 2017). Here, we report the discovery of a novel iridophore mutant medaka and identify its causal gene.MATERIALS AND METHODSMedaka Strains and Rearing ConditionsMedaka were reared at 26°C under a 14-h light/10-h dark cycle. We collected wild O. latipes from an irrigation canal in Kitagata, Akiha-ward, Niigata City (northern Japanese population) in 2020-2021 (Fig. 1A). We caught two iridophore-deficient individuals out of 1,605 fish and named this mutation panda (pa) due to its black eye trait (Fig. 1B). The T5 strain, which is homozygous for the gu mutation, has been previously described (Tomita, 1992; Shimada and Shima, 2001) and was used as the gu mutant in this study. The gu mutant has a similar phenotype to pa . The d-rR strain (Hyodo, 1990) is a closed colony derived from a southern Japanese population. The HNI strain was used as the wild-type of  northern Japanese population O. latipes for reference genome. All experiments were conducted in accordance with the Guidelines for the Proper Conduct of Animal Experiments (Science Council of Japan) and the institutional guidelines for animal care and use. Complementation Test with T5 StrainA complementation test was performed to determine whether the causal genes for pa and gu are allelic. The T5 strain carries five pigment mutations (b , gu , lf , I ,wl ). Among these, the gu mutant exhibits a strong reduction in visible iridophores from larval to adult stages, like pa . The causal gene is located on an autosomal chromosome (chromosome 5), and has been identified as pnp4a (Kimura et al., 2017).Mode of Inheritance TestAn inheritance pattern test was performed using d-rR as the wild-type strain.Genomic PCR Analysis and Linkage AnalysisGenomic DNA was extracted from fin clips. Samples were suspended in 100 µl lysis buffer containing 2 µl proteinase K (10 mg/ml) and incubated at 65°C for 1 hour. Subsequently, 100 µl of chloroform-isopropyl alcohol was added and the mixture was inverted. From this, 60 µl of supernatant was collected and mixed with an equal volume of isopropyl alcohol. The mixture was centrifuged at 15,000 × g for 10 min. After discarding the supernatant and drying, 200 µl of 70% ethanol was added and centrifuged again under the same conditions. Then, it was dissolved in 50 µl TE. All extracted genomic DNA samples were stored at 4°C until use. PCR conditions were as follows: one cycle at 95°C for 30 sec; 30 cycles at 95°C for 5 sec, 55°C for 5 sec, and 72°C for 20 sec; followed by 72°C for 1 min. The products were electrophoresed on 12% polyacrylamide gels (Davis, 1964). Linkage analysis was performed on 32 F2 individuals obtained from crosses with d-rR, using M-markers (Kimura & Naruse, 2010) from chromosomes 1-24 for rough mapping (Table 1). Additional genetic markers were then developed for fine mapping, and linkage analysis was conducted using the same 96 F2 individuals. Primers used in the linkage analysis were designed by selecting sequences with In-dels between HNI and Hd-rR to facilitate band differentiation during electrophoresis. The primers and genetic markers used for additional linkage analysis are shown in Table 1. The recombination map was constructed using MAPL98 for Windows (Ukai et al., 1995).Whole Genome Sequencing  pa DNA was extracted from fin clips using the same method as for PCR and submitted to Novogene for library preparation. Sequencing was performed using Illumina’s NovaSeq6000 NGS with 150 bp paired-end reads, generating 28 Gbp (approximately 35× coverage of the 0.8 Gbp medaka genome). The sequence was compared with the HNI reference genome (Genome assembly ASM223471v1) to identify nucleotide substitutions and mutations. We detected SNPs and InDels using SAMtools(Li et al., 2009) with the following parameter: 'mpileup -m 2 -F 0.002 -d 1000'. The structure of the protein encoded by the identified causal gene was estimated using AlphaFold3 (Abramson et al., 2024) and PPM 3.0 (Lomize et al., 2022).Knockout of the Candidate Causal GeneGene targeting was performed using the CRISPR-Cas system-based RNA-guided endonuclease, which has emerged as a simple and efficient tool for targeted genome editing in medaka (Ansai and Kinoshita, 2014). Single guide RNA (sgRNA) was designed targeting exon 4 of mpv17 (Fig. 3A). Microinjection was performed using the d-rR strain. DNA was extracted from fin clips of pa-like individuals and PCR products were directly sequenced.Data availability All strains in this study are available from the NBRP, Medaka (www.shigen.nig.ac.jp/medaka/). Whole Genome Sequence of panda(pa) have been deposited with links to BioProject accession number PRJDB19582 in the DDBJ BioProject database.