(A) Phylogenetic tree showing the relationship of medaka Gal to other known GAL family proteins. The number at each node indicates bootstrap values for 1000 replicates. Scale bar represents 0.1 …
(A) Nucleotide and deduced amino acid sequences of the medaka gal cDNA. The predicted signal peptide is underlined and the mature Gal polypeptide is boxed. Asterisk indicates stop codon. Nucleotide …
(A) Levels of gal expression in the whole brain of sex-reversed adult XX males and XY females versus typical adult XY males and XX females (n = 8 per group). ***p<0.001 (Bonferroni’s post hoc test). …
Representative micrographs showing the expression of androgen receptor (AR) and estrogen receptor (ER) genes in pPMp, where sexually dimorphic gal-expressing neurons reside, in males (A) and females …
(A, B) Sum of each aggressive behavioral act (chase, fin display, circle, strike, and bite) performed by wild-type (+/+), heterozygous (+/Δ2 and +/Δ10), and homozygous (Δ2/Δ2 and Δ10/Δ10) males from …
Two different lines of gal knockout medaka (Δ2 and Δ10) were generated by CRISPR/Cas9-mediated genome editing. (A) Gene structure of gal showing the location of the CRISPR target site, which is …
Various parameters in the mating behavior of males (A, B) and females (C, D) from Δ2 (A, C) and Δ10 (B, D) gal knockout lines. Results were compared among wild-type (+/+), heterozygous (+/Δ2 and …
(A, B) Sum of each aggressive behavioral act (chase, fin display, circle, strike, and bite) performed by KT-treated females from Δ2 (A) and Δ10 (B) gal knockout lines. Asterisks indicate significant …
(A, B) Line drawings of lateral (A) and ventral (B) views (anterior to the left) of the medaka brain showing the approximate levels of sections in panels C and D, respectively. (C) Representative …
(A) Structure of the transgene in gal-GFP transgenic medaka. A 7 bp sequence containing the translation initiation site of gal in a medaka BAC clone (clone ID: ola1-108J05) was replaced by a 2136 bp …
(A) Phylogenetic tree showing the relationship of medaka Galr1 and Galr2 to other known GAL receptors. The number at each node indicates bootstrap values for 1000 replicates. Scale bar represents …
(A) Nucleotide and deduced amino acid sequences of the medaka galr2 cDNA. Asterisk indicates stop codon. Nucleotide numbers are shown at the right of each sequence line. (B) Sequence comparison of …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Oryzias latipes) | gal | this paper | GenBank:LC532140 | |
Gene (O. latipes) | galr2 | this paper | GenBank:LC532141 | |
Gene (O. latipes) | actb | GenBank | GenBank:NM_001104808 | |
Strain, strain background (O. latipes) | d-rR | NBRP Medaka | strain ID:MT837 | maintained in a closed colony over 10 years in Okubo lab |
Genetic reagent (O. latipes) | gal knockout Δ2 line | this paper | N/A | generated and maintained in Okubo lab |
Genetic reagent (O. latipes) | gal knockout Δ10 line | this paper | N/A | generated and maintained in Okubo lab |
Genetic reagent (O. latipes) | gal-GFP transgenic | this paper | N/A | generated and maintained in Okubo lab |
Cell line (Homo sapiens) | HEK293T | Riken BRC Cell Bank | cell number:RCB2202; RRID:CVCL_0063 | |
Cell line (Escherichia coli) | DY380 | DOI:10.1038/35093556; DOI:10.1006/geno.2000.6451 | N/A | |
Transfected construct (H. sapiens) | pcDNA3.1/V5-His-TOPO | Thermo Fisher Scientific | cat#:K480001 | |
Transfected construct (H. sapiens) | pGL4.29 | Promega | cat#:E8471 | |
Transfected construct (H. sapiens) | pGL4.33 | Promega | cat#:E1340 | |
Transfected construct (H. sapiens) | pGL4.74 | Promega | cat#:E6921 | |
Antibody | alkaline phosphatase-conjugated anti-DIG antibody (sheep polyclonal) | Roche Diagnostics | cat#:11093274910; RRID:AB_514497 | (1:500 or 1:2000) |
Antibody | horseradish peroxidase-conjugated anti-fluorescein antibody (sheep polyclonal) | PerkinElmer | cat#:NEF710001EA; RRID:AB_2737388 | (1:1000) |
Antibody | anti-GAL antibody (rabbit polyclonal) | Enzo Life Sciences | cat#:BML-GA1161-0025; RRID:AB_2051473 | (1:200 or 1:500) |
Antibody | Alexa Fluor 555-conjugated goat anti-rabbit IgG (goat polyclonal) | Thermo Fisher Scientific | cat#:A-21428; RRID:AB_2535849 | (1:1000) |
Antibody | Alexa Fluor 488-conjugated goat anti-rabbit IgG (goat polyclonal) | Thermo Fisher Scientific | cat#:A-11070; RRID:AB_2534114 | (1:1000) |
Recombinant DNA reagent | full-length cDNA clone for medaka gal | this paper | clone ID:56_B03 | |
Recombinant DNA reagent | full-length cDNA clone for medaka galr2 | this paper | clone ID:39_L19 | |
Recombinant DNA reagent | pGEM-Teasy vector | Promega | cat#:A1360 | |
Recombinant DNA reagent | pCS2+hSpCas9 plasmid | Addgene | RRID:Addgene_51815 | |
Recombinant DNA reagent | medaka bacterial artificial chromosome (BAC) clone containing the gal locus | NBRP Medaka | clone ID:108_J05 | |
Recombinant DNA reagent | phrGFP II-1 mammalian expression vector | Agilent Technologies | cat#:240143 | |
Sequence-based reagent | CRISPR RNA (crRNA) for medaka gal | Fasmac | N/A | GAGCATCGGGCTGGTTATCGCGG |
Sequence-based reagent | trans-activating CRISPR RNA (tracrRNA) | Fasmac | cat#:GE-002 | |
Peptide, recombinant protein | medaka Gal peptide | Scrum | this paper | GWTLNSAGYLLGPHGIDGHRTLGDKQGLA-NH2 |
Commercial assay or kit | Isogen Poly(A)+Isolation Pack | Fujifilm Wako Pure Chemical Corporation | cat#:314–05651 | |
Commercial assay or kit | nylon membrane | Roche Diagnostics | cat#:11209272001 | |
Commercial assay or kit | DIG RNA Labeling Mix | Roche Diagnostics | cat#:11277073910 | |
Commercial assay or kit | T7 RNA polymerase | Roche Diagnostics | cat#:10881775001 | |
Commercial assay or kit | DIG Easy Hyb | Roche Diagnostics | cat#:11603558001 | |
Commercial assay or kit | CDP-Star | Roche Diagnostics | cat#:12041677001 | |
Commercial assay or kit | RNeasy Lipid Tissue Mini Kit | Qiagen | cat#:74804 | |
Commercial assay or kit | RNeasy Plus Universal Mini Kit | Qiagen | cat#:73404 | |
Commercial assay or kit | Omniscript RT Kit | Qiagen | cat#:205111 | |
Commercial assay or kit | SuperScript VILO cDNA Synthesis Kit | Thermo Fisher Scientific | cat#:11754050 | |
Commercial assay or kit | Power SYBR Green PCR Master Mix | Thermo Fisher Scientific | cat#:4367659 | |
Commercial assay or kit | LightCycler 480 SYBR Green I Master | Roche Diagnostics | cat#:04887352001 | |
Commercial assay or kit | TSA Plus Fluorescein System | PerkinElmer | cat#:NEL741001KT | |
Commercial assay or kit | mMessage mMachine SP6 Kit | Thermo Fisher Scientific | cat#:AM1340 | |
Commercial assay or kit | Dual-Luciferase Reporter Assay System | Promega | cat#:E1910 | |
Chemical compound, drug | methyltestosterone | Fujifilm Wako Pure Chemical Corporation | cat#:136–09931 | |
Chemical compound, drug | estradiol-17β (E2) | Fujifilm Wako Pure Chemical Corporation | cat#:058–04043 | |
Chemical compound, drug | 11-ketotestosterone (KT) | Cosmo Bio | cat#:117 ST | |
Chemical compound, drug | tricaine methane sulfonate | Sigma-Aldrich | cat#:E10521 | |
Chemical compound, drug | 5-bromo-4-chloro-3-indolyl phosphate | Roche Diagnostics | cat#:11383221001 | |
Chemical compound, drug | nitro blue tetrazolium | Roche Diagnostics | cat#:11383213001 | |
Chemical compound, drug | agarose, type IX-A | Sigma-Aldrich | cat#:A2576 | |
Chemical compound, drug | Fast Red | Roche Diagnostics | cat#:11496549001 | |
Chemical compound, drug | 4′,6-diamidino-2-phenylindole (DAPI) | Thermo Fisher Scientific | cat#:D1306 | |
Chemical compound, drug | blocking reagent | Roche Diagnostics | cat#:11096176001 | |
Chemical compound, drug | Lipofectamine LTX | Thermo Fisher Scientific | cat#:15338100 | |
Software, algorithm | InterPro | https://www.ebi.ac.uk/interpro/ | RRID:SCR_006695 | |
Software, algorithm | SignalP | http://www.cbs.dtu.dk/services/SignalP/ | RRID:SCR_015644 | |
Software, algorithm | ClustalW | http://clustalw.ddbj.nig.ac.jp/index.php | RRID:SCR_017277 | |
Software, algorithm | GraphPad Prism | GraphPad Software | RRID:SCR_002798 |
Abbreviations of medaka brain regions and nuclei.
Species names and GenBank accession numbers of the protein sequences used in this study.
Primers used in this study.