Male-predominant galanin mediates androgen-dependent aggressive chases in medaka
- Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan
- Division of Reproductive Biology, National Institute for Basic Biology, Japan
Figures
Figure 1 with 1 supplement
Male-biased sexual dimorphism exists in gal expression in the MPOA.
(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 …
Figure 1—figure supplement 1
Sequence information for medaka gal.
(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 …
Figure 2 with 1 supplement
Sexually dimorphic gal expression is dependent on adult sex steroids.
(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). …
Figure 2—figure supplement 1
Expression of sex steroid receptors in sexually dimorphic gal neurons in pPMp.
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 …
Figure 3 with 2 supplements
Genetic ablation of gal specifically suppresses male–male chases.
(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 …
Figure 3—figure supplement 1
Generation and verification of gal knockout medaka.
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 …
Figure 3—figure supplement 2
Mating behavior of gal knockout medaka.
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 …
Figure 4
Genetic ablation of gal attenuates androgen-induced chases in females.
(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 …
Figure 5 with 1 supplement
Gal peptide produced male-predominantly is transported to various brain regions.
(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 …
Figure 5—figure supplement 1
Generation and fluorescence imaging of gal-GFP transgenic medaka.
(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 …
Figure 6 with 1 supplement
Gal receptors coupled to different signaling pathways are expressed widely in the brain.
(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 …
Figure 6—figure supplement 1
Sequence information for medaka galr2.
(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 …
Author response image 1
Tables
Key resources table
| 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 |
Additional files
-
Supplementary file 1
Abbreviations of medaka brain regions and nuclei.
- https://cdn.elifesciences.org/articles/59470/elife-59470-supp1-v1.docx
-
Supplementary file 2
Species names and GenBank accession numbers of the protein sequences used in this study.
- https://cdn.elifesciences.org/articles/59470/elife-59470-supp2-v1.docx
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Supplementary file 3
Primers used in this study.
- https://cdn.elifesciences.org/articles/59470/elife-59470-supp3-v1.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/59470/elife-59470-transrepform-v1.docx
