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. 2019 Aug 6;8:e39495. doi: 10.7554/eLife.39495

Figure 4. Medaka possess an additional NPB gene, designated npbb.

(A) Phylogenetic tree of NPB and NPW. The number at each node indicates bootstrap values for 1000 replicates. Scale bar represents 0.1 substitution per site. For species names and GenBank accession numbers, see Supplementary file 3. (B) Ability of medaka Npba and Npbb to activate medaka Npbwr2. Receptor activation was assessed by measuring cAMP-responsive element-driven luciferase activity in cells transfected with Npbwr2. The x-axis shows the concentration of Npba and Npbb, and the y-axis shows luciferase activity as a percentage of that observed in the absence of Npba/Npbb. (C) Overall levels of npbb expression in the male (blue columns) and female (beige columns) brain dissected into three parts as determined by real-time PCR (n = 6 per sex). *, p<0.05 (unpaired t-test). For abbreviations of brain regions, see Supplementary file 1. (D) Distribution of npbb-expressing neurons in the male (blue columns) and female (beige columns) brain (n = 5 per sex). The data are split into three graphs for clarity. *, p<0.05; **, p<0.01; ***, p<0.001 (unpaired t-test). For abbreviations of brain nuclei, see Supplementary file 1. (E) Total area of npbb expression in PMm/PMg/PPp in males (blue column) and females (beige column) (n = 5 per sex). ***, p<0.001 (unpaired t-test). (F) Representative micrographs showing npbb-expressing neurons in different brain nuclei. Micrographs of both sexes are shown for Vs/Vp and PMm/PMg/PPp, where npbb expression is confined or almost confined to females. Micrographs of males only are shown for other nuclei, where sex differences were not detected or, if present, were not sufficiently demonstrated by photographs. (G) Coexpression of npba and npbb in the same neurons in Vs/Vp and PMm/PMg, but not in other brain nuclei. In each set of panels, the left and middle ones show images of npba (green) and npbb (magenta) expression, respectively, in the same sections; the right ones show the merged images with nuclear counterstaining (blue). Arrowheads indicate representative neurons coexpressing npba and npbb. Scale bars represent 50 μm. For abbreviations of brain nuclei, see Supplementary file 1. See also Figure 4—figure supplement 1 and Figure 4—figure supplement 2.

Figure 4.

Figure 4—figure supplement 1. Sequence information for medaka npbb.

Figure 4—figure supplement 1.

(A) Nucleotide and deduced amino acid sequences of the medaka npbb cDNA. The predicted signal peptide is underlined and the mature Npbb polypeptide is boxed. Asterisk indicates the stop codon. Nucleotide numbers are shown at the right of each sequence line. (B) Comparison of mature NPB polypeptide sequences from medaka and other vertebrate species. Identical amino acids in all sequences are shaded in beige. For species names and GenBank accession numbers, see Supplementary file 3.
Figure 4—figure supplement 2. Comparison of syntenic relationships of genes in the vicinity of gar, arowana, catfish, and medaka NPB genes.

Figure 4—figure supplement 2.

Genes are represented by arrows denoting the direction of transcription. gnav1, guanine nucleotide-binding protein (G protein) α v1; mafg, MAF bZIP transcription factor G; pcyt2, phosphate cytidylyltransferase 2, ethanolamine; rnf213, ring finger protein 213; sgsh, N-sulfoglucosamine sulfohydrolase; sirt7, sirtuin 7; slc26a11, solute carrier family 26 member 11; sox9, SRY (sex determining region Y)-box 9; sstr2, somatostatin receptor 2.