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Taste recognition mediated by taste receptors is critical for the survival of animals in nature and is an important determinant of nutritional status and quality of life in humans. However, many factors including aging, diabetes, zinc deficiency, infection with influenza or cold viruses, and chemotherapy can trigger dysgeusia, for which a standard treatment has not been established. We here established an engineered strain of medaka (Oryzias latipes) that expresses green fluorescent protein (GFP) from the endogenous taste 1 receptor 3 (T1R3) gene locus with the use of the CRISPR-Cas9 system. This T1R3-GFP knock-in (KI) strain allows direct visualization of expression from this locus by monitoring of GFP fluorescence. The pattern of GFP expression in the T1R3-GFP KI fish thus mimicked that of endogenous T1R3 gene expression. Furthermore, exposure of T1R3-GFP KI medaka to water containing monosodium glutamate or the anticancer agent 5-fluorouracil resulted in an increase or decrease, respectively, in GFP fluorescence intensity, effects that also recapitulated those on T1R3 mRNA abundance. Finally, screening for agents that affect GFP fluorescence intensity in T1R3-GFP KI medaka identified tryptophan as an amino acid that increases T1R3 gene expression. The establishment of this screening system for taste receptor expression in medaka provides a new tool for the development of potential therapeutic agents for dysgeusia.

Additional file 10. Review history.

Additional file 8: Figure S6. Circos plots. Circos plots for structural variants (DEL, INS, DUP, INV, TRA) discovered by the reference-anchored approach.

Additional file 10. Review history.

Additional file 8: Figure S9. Copenhagen plot. Plot showing genomic positions of significant eQTL SNPs.

Additional file 2: Figures S1-S5. Supplementary figures. Various supplementary figures.

Additional file 8: Figure S6. Circos plots. Circos plots for structural variants (DEL, INS, DUP, INV, TRA) discovered by the reference-anchored approach.

Additional file 4: Figures S1-S8. Supplementary figures. Various supplementary figures.

Additional file 9: Figure S10. Haploview plots. Linkage disequilibrium (LD) plots generated with Haploview for each of the 24 medaka chromosomes, showing r2 values in the MIKK panel for each pair of SNPs within 1 Mb of one another.

To cope with seasonal environmental changes, animals adapt their physiology and behaviour in response to photoperiod. However, the molecular mechanisms underlying these adaptive changes are not completely understood. Here, using genome-wide expression analysis, we show that an uncharacterized long noncoding RNA (lncRNA), LDAIR, is strongly regulated by photoperiod in Japanese medaka fish (Oryzias latipes). Numerous transcripts and signalling pathways are activated during the transition from short- to long-day conditions; however, LDAIR is one of the first genes to be induced and its expression shows a robust daily rhythm under long-day conditions. Transcriptome analysis of LDAIR knockout fish reveals that the LDAIR locus regulates a gene neighbourhood, including corticotropin releasing hormone receptor 2, which is involved in the stress response. Behavioural analysis of LDAIR knockout fish demonstrates that LDAIR affects self-protective behaviours under long-day conditions. Therefore, we propose that photoperiodic regulation of corticotropin releasing hormone receptor 2 by LDAIR modulates adaptive behaviours to seasonal environmental changes., ファイル公開：2019/11/01

While lower vertebrates contain adult stem cells (aSCs) that maintain homeostasis and drive un-exhaustive organismal growth, mammalian aSCs display mainly the homeostatic function. Understanding aSC-driven growth is of paramount importance to promote organ regeneration and prevent tumor formation in mammals. Here we present a clonal approach to address common or dedicated populations of aSCs for homeostasis and growth. Our functional assays on medaka gills demonstrate the existence of separate homeostatic and growth aSCs, which are clonal but differ in their topology. While homeostatic aSCs are fixed, embedded in the tissue, growth aSCs locate at the expanding peripheral zone. Modifications in tissue architecture can convert the homeostatic zone into a growth zone, indicating a leading role for the physical niche defining stem cell output. We hypothesize that physical niches are main players to restrict aSCs to a homeostatic function in animals with a fixed adult size.

Genomic location of potential off-target sites for sgT (sgRNA for Tbait) (DOCX 14 kb)

Nucleotide sequences of the joint region of the insertions for the sox5 and pax7a transgenic fish The PCR products that span the genomic DNA and the donor DNA (see Additional file 2: Table S2) were sequenced. The top two lines for each panel show the sequence of the genome and the donor. Underlined sequences correspond to sgRNA targets. The PAM sequences are indicated in green. The predicted digestion sites by CRISPR/Cas9 are indicated with arrowheads. Sequences in bold letters are expected to be present after the integrations. The predicted nucleotide sequence without an indel is shown in the third line as bold letters. (A) The 5′ side of the sox5 transgenic fish with the forward integration (#1, #3, and #4 strains; Additional file 2:Table S2). (B) The 5′ side of the sox5 transgenic fish with the reverse integration (#2 strains; Additional file 2: Table S2). (C) The 3′ side of the sox5 transgenic fish with the forward integration (#1, #2, #3, and #7 strains; Additional file 2: Table S2). (D) The 3′ side of the sox5 transgenic fish with the reverse integration (#6 strain; Additional file 2: Table S2). (E) The 5′ side of the pax7a transgenic fish with the forward integration (#1, #3, #4, and #6 strains; Additional file 2: Table S2). (F) The 5′ side of the pax7a transgenic fish with the reverse integration (#2 strains; Additional file 2: Table S2). (G) The 3′ side of the pax7a transgenic fish with the forward integration (#1and #6 strains; Additional file 2: Table S2). (H) The 3′ side of the pax7a transgenic fish with the reverse integration (#2, #3, #4, and #5 strains; Additional file 2: Table S2). (PDF 31 kb)

Nucleotide sequences of the joint region of the insertions for the sox5 and pax7a transgenic fish The PCR products that span the genomic DNA and the donor DNA (see Additional file 2: Table S2) were sequenced. The top two lines for each panel show the sequence of the genome and the donor. Underlined sequences correspond to sgRNA targets. The PAM sequences are indicated in green. The predicted digestion sites by CRISPR/Cas9 are indicated with arrowheads. Sequences in bold letters are expected to be present after the integrations. The predicted nucleotide sequence without an indel is shown in the third line as bold letters. (A) The 5′ side of the sox5 transgenic fish with the forward integration (#1, #3, and #4 strains; Additional file 2:Table S2). (B) The 5′ side of the sox5 transgenic fish with the reverse integration (#2 strains; Additional file 2: Table S2). (C) The 3′ side of the sox5 transgenic fish with the forward integration (#1, #2, #3, and #7 strains; Additional file 2: Table S2). (D) The 3′ side of the sox5 transgenic fish with the reverse integration (#6 strain; Additional file 2: Table S2). (E) The 5′ side of the pax7a transgenic fish with the forward integration (#1, #3, #4, and #6 strains; Additional file 2: Table S2). (F) The 5′ side of the pax7a transgenic fish with the reverse integration (#2 strains; Additional file 2: Table S2). (G) The 3′ side of the pax7a transgenic fish with the forward integration (#1and #6 strains; Additional file 2: Table S2). (H) The 3′ side of the pax7a transgenic fish with the reverse integration (#2, #3, #4, and #5 strains; Additional file 2: Table S2). (PDF 31 kb)

Status of the insertions in the transgenic fish generated in this study (DOCX 16 kb)

Status of the insertions in the transgenic fish generated in this study (DOCX 16 kb)

No significant effect of the presence of a far male to the female receptivity. (a) A Separation condition for the female mating receptivity test in triadic relationships. “Far: WT, Near: WT (focal), Wall: transparent”: two WT males were placed in the far and near zones, respectively. The female could see them and mated with the near male in the next morning. (b) The presence of a far male didn’t affect the female receptivity to the near male. Mean ± SEM. (c) The existence of the far male didn’t significantly affect the motivation to mate of the near male. Mean ± SEM. Mann–Whitney U test: U = 39, N 1 = N 2 = 11, P = 0.161. (PDF 109 kb)

Research & Knowledge, 2, 1

Low motivation for courtship behavior in V1a2 KO males. V1a2 KO males exhibited courtship behavior less frequently than WT males. Mean ± SEM. Mann–Whitney U test: U = 32.5, N 1 = N 2 = 11, P = 0.02. (PDF 62 kb)

No significant defect in visual capacity or locomotion in V1a2 KO males. (a) Apparatus for analysis of the optomotor response described previously [40]. (b) The moving velocity for 1 min of free-swimming V1a2 KO males did not differ significantly from that of WT males. Mean ± SEM. Mann–Whitney U test: U = 7, N 1 = N 2 = 5, P = 0.310. (c-d) Integrated angular velocity during 1 min of free-swimming in (c) WT and (d) V1a2 KO males. Each line indicates the raw data of five individual fish. (e) Ratio of the mean fish angular speed to mean stripe speed. Mean ± SEM. Mann–Whitney U test: U = 9, N 1 = N 2 = 5, P = 0.532. (PDF 197 kb)

The normal male mating activity in the female mating receptivity test. (a) The visual familiarization didn’t significantly affect the motivation to mate of males. Mean ± SEM. Kruskal-Wallis: chi-squared = 6.686, df = 2, P = 0.0353. post-hoc Steel test: “Near: WT, Wall: opaque” VS “Near: WT, Wall: transparent”, P = 0.115; “Near: WT, Wall: opaque” VS “Far: WT, Wall: transparent”, P = 0.689. (b) The existence of the near male didn’t significantly affect the motivation to mate of the far male. Mean ± SEM. Kruskal-Wallis: chi-squared = 1.0216, df = 2, P = 0.6. (PDF 106 kb)

Distance between the female and the near male under the separation condition. There was no significant difference in the distance between the female and the WT near male or the V1a2 KO near males in mate-guarding test. Meanâ Âąâ SEM. (PDF 58 kb)

A genetic screen for mutations affecting embryogenesis in the medaka, Oryzias latipes, identified a mutant, whiteout (who), that exhibited hypochromic anemia. The who mutant initially had the normal number of blood cells, but it then gradually decreased during the embryonic and larval stages. The blood cells in the who mutants show an elongated morphology and little hemoglobin activity. Genetic mappinng localized who to the vicinity of a LG12 marker, olgcl. By utilizing the highly conserved synteny between medaka and pufferfish, we identified a gene for delta-aminolevulinic acid dehydratase(ALAD), which is the second enzyme in the heme synthetic pathway, as a candidate for who. We found a missense mutation in the alad gene that was tightly linked to the who phenotype, strongly suggesting that the hypochromic anemia phenotype in the who mutant is caused by a loss of the alad function. Thus, who mutants represent a model for the human disease ALAD-deficiency porphyria.

A cDNA clone encoding the soluble guanylyl cyclase α2 subunit was isolated from medaka fish (Oryzias latipes) and designated as OIGCS-α2. The OIGCS-α2 cDNA was 3192 bp in length and the open reading frame (ORF) encodes a protein of 805 amino acids. The deduced amino acid sequence has high similarity to that of the mammalianα2 subunit gene except for the N-terminal regulatory domain. The C-terminal 5 amino acids, "RETSL", which have been reported to interact with the post synaptic density protein (PSD)-95 were conserved. An RNase protection assay with adult fish organs showed that O/GCS-α2 was expressed mainly in the brain and testis. The complete nucleotide sequence (about 41 kbp) of the OIGCS-α2 genomic DNA clone isolated from a medaka fish BAC library indicated that the OIGCS-α2 gene consisted of 9 exons and 8 introns. The 5'-flanking region and larger introns, such as introns 1, 4, and 7, contained the several fragments conserved in the nucleotide sequences of Rex6 (non-long terminal repeat retrotransposon), MHC class I genomic region, and OIGC1, the medaka fish homolog of the mammalian guanylyl cyclase B gene. Linkage analysis on the medaka fish chromosome demonstrated that the OIGCS-α2 gene was mapped to LG13; this mapping position was different from those for the OIGCS-α1 and OIGCS-β1 genes (LG1).

Review articles for this special issue

Background Oryzias latipes (Medaka) has been established as a vertebrate genetic model for over a century, and has recently been rediscovered outside its native Japan. The power of new sequencing methods now makes it possible to reinvigorate Medaka genetics, in particular by establishing a near-isogenic panel derived from a single wild population. Results Here we characterise the genomes of wild Medaka catches obtained from a single Southern Japanese population in Kiyosu as a precursor for the establishment of a near isogenic panel of wild lines. The population is free of significant detrimental population structure, and has advantageous linkage disequilibrium properties suitable for establishment of the proposed panel. Analysis of morphometric traits in five representative inbred strains suggests phenotypic mapping will be feasible in the panel. In addition high throughput genome sequencing of these Medaka strains confirms their evolutionary relationships on lines of geographic separation and provides further evidence that there has been little significant interbreeding between the Southern and Northern Medaka population since the Southern/Northern population split. The sequence data suggest that the Southern Japanese Medaka existed as a larger older population which went through a relatively recent bottleneck around 10,000 years ago. In addition we detect patterns of recent positive selection in the Southern population. Conclusions These data indicate that the genetic structure of the Kiyosu Medaka samples are suitable for the establishment of a vertebrate near isogenic panel and therefore inbreeding of 200 lines based on this population has commenced. Progress of this project can be tracked at http://www.ebi.ac.uk/birney-srv/medaka-ref-panel, Comment: 5 figures, 30 pages

The medaka, Oryzias latipes, has been widely used as an experimental animal in Japan, because of its relatively short life cycle (2 to 3 months), high fecundity (laying over 10 eggs daily) and small size (saving space for large-scale breeding). In this review, we will describe the development of the medaka as an experimental animal based on its characteristics, availability of several experimental methodologies, mutant and inbred strains, and availability of other species of the genus Oryzias and related genera. We also describe advantages and disadvantages of the medaka compared with other small freshwater fish such as zebrafish, platyfish and swordtail.