Hirooka A , Hamada M , Fujiyama D , Takanami K , Kobayashi Y , Oti T , Katayama Y , Sakamoto T , Sakamoto H .

21H00428 Grant-in-Aid for Scientific Research on Innovative Areas "Singularity Biology (No.8007)", 15K15202 Japan Society for the Promotion of Science , 15KK0257 Japan Society for the Promotion of Science , 15H05724 Japan Society for the Promotion of Science , 16H06280 Japan Society for the Promotion of Science , 961149 Japan Agency for Medical Research and Development

	Figure 1. Molecular phylogeny of precursors of gastrin-releasing peptide (GRP)/neuromedin B (NMB)/bombesin family peptides. GRP/NMB/bombesin family peptides are classified into a GRP clade (pink box) and an NMB/bombesin clade (gray frame). While GRP was found in all the animals examined, genes included in NMB clade were not found in frogs (yellow box). All bombesin-like peptides in frogs appeared to form a single bombesin clade (blue box). Species name in magenta: amphibians; bold: frogs. IDs for the protein sequences used in this analysis are shown in Supplementary Table S1.
	Figure 2. Schematic representation of gene synteny around the neuromedin B (NMB)/bombesin gene and gastrin-releasing peptide (GRP) genes in vertebrates. Horizontal lines (gray) indicate chromosome fragments. Genes are represented as arrows (yellow: NMB gene, blue: bombesin genes, pink: GRP genes, white: other genes) according to their transcriptional orientation. Protein IDs are shown below the genes. Broken lines indicate correspondences of orthologous genes. Gene synteny around the NMB/bombesin gene in vertebrates is highly conserved, suggesting an orthologous relationship between bombesin in frogs and NMB in other vertebrates.
	Figure 3. Molecular phylogeny of gastrin-releasing peptide-preferring receptor (GRPR)/neuromedin B-preferring receptor (NMB)/bombesin receptor subtype-3 (BRS-3). The GRPR/NMBR/BRS-3 family gene diverged into GRPR (pink box), NMBR (yellow box) and BRS-3 (blue box) in the ancestor of vertebrates. All the three receptors are widely conserved among vertebrates including frogs, although BRS-3 genes are not found in teleost and cartilaginous fish. Species name in magenta: amphibians; bold: frogs; cyan: cartilaginous fish. IDs for the protein sequences used in this analysis are shown in Supplementary Table S1.
	Figure 4. The expression of gastrin-releasing peptide (GRP) and GRP receptor (GRPR) mRNA in Xenopus tropicalis. (a) Reverse transcription (RT)-PCR analysis of GRP and GRPR mRNA expression. Gene expression of GRP (upper panel) and GRPR (middle panel) in the brain, spinal cord, heart, lung and stomach were examine by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as the internal control (bottom panel). Cropped gel images of electrophoresis are shown. Original images are shown in Supplementary Fig. S4. (b, c) The expression levels of GRP and GRPR mRNA in Xenopus central nervous system measured by the real-time quantitative PCR. Relative expression levels of GRP (b) and GRPR (c) in the telencephalon, the diencephalon/mesencephalon/pons/cerebellum, the medulla oblongata, and the spinal cord of males and females were analyzed. P values indicate statistical tests using linear mixed model with Bonferroni correction for multiple comparisons (vs. telencephalon, *P < 0.01; vs. diencephalon/mesencephalon/pons/cerebellum, †P < 0.01; vs. medulla oblongata, ‡P < 0.01; vs. spinal cord, §P < 0.05). No sex differences and no interactions between sex and tissue were detected. Dots and bars indicate values of each sample and means of the samples, respectively. Black: males, magenta: females.
	Figure 5. Cell bodies and fibers in Xenopus brain immunohistochemically labeled with the anti-gastrin-releasing peptide (GRP) antiserum. Schematic cross sections illustrating the distribution of GRP-like immunoreactivity (left panels; a–j) and representative photomicrographs (right panels; a″–j″) of transverse sections are shown. (k) Letters (a–j) in the schematic lateral profile of the brain indicate the rostrocaudal level of each transverse section. In the schematic sections, immunoreactive cell bodies and fibers in Xenopus brain are shown by magenta circles and magenta lines, respectively. The density of the symbols shown in the right hemispheres is roughly proportional to the relative density of the immunoreactive elements. The anatomical structures are indicated on the left hemispheres according to the nomenclature of ten Donkelaar (1998)70–72. Dorsal is up and lateral is right. The histology of the brain regions where cell bodies are densely distributed was shown by the Nissl staining (middle panels; a′–c′, e′, f′, i′). In the micrographs of immunohistochemical staining for GRP, relative weakly immunoreactive cell bodies and fibers are observed throughout the ventral telencephalic area, e.g. in the nucleus accumbens (Acc; a″ − 1) and in the striatum, ventral part (Strv; a″ − 2). Immunoreactive cell bodies are abundant in the amygdala, e.g. in the amygdala pars medialis (Apm; b″), and in the amygdala pars lateralis (Apl; c″ − 1). GRP-immunoreactive fibers are also observed in the preoptic area (Poa; c″ − 2) and in the nucleus preopticus magnocellularis (Mg; d″). In the diencephalon, GRP-immunoreactive somata are detected in the nucleus ventromedialis thalami (VM; e″) and in the tuberculum posterius (TP; f″). A weak distribution of GRP-immunoreactive fibers is also found in the area near the tractus opticus basalis (optb; g″), and the nucleus reticularis isthmi (Ris; h″). In the brainstem, GRP-immunoreactive cell bodies and fibers are observed in the nucleus raphes (Ra; I″). In the posterior part of the medulla oblongata, GRP-immunoreactive fibers and cell bodies are observed in the nucleus descendens nervi trigemini (Vds), the tractus descendens nervi trigemini (trVds; j″ − 1), the nucleus motorius nervi vagi (Xm; j” −  2), and the nucleus reticularis inferior (Ri; j″ − 3). Scale bars = 50 μm, 10 μm in enlarged images. Arrows indicate representative GRP-immunoreactive cell bodies. Arrowheads indicate representative GRP-immunoreactive fibers. For abbreviations, see Table 1.
	Figure 6. Western immunoblotting of gastrin-releasing peptide receptor (GRPR). The number on the left indicates the molecular weight (kDa). Extracts of protein from Xenopus brain and spinal cord were transferred onto polyvinylidene difluoride membranes and probed with the rabbit polyclonal antiserum against Xenopus GRPR (1:100,000). The antiserum recognized a single band at the expected molecular weight of GRPR (~ 43 kDa) on a Western blot of the brain and spinal cord. Preabsorption of the antiserum with an excess of antigen peptides (50 μg/ml) eliminated the staining of the ~ 43-kDa protein band. The gel image was processed by cropping to remove the irrelevant area. Original images are shown in Supplementary Fig. S5.
	Figure 7. The evolutionary lineage of the gastrin-releasing peptide (GRP)/bombesin. (a) Previous studies suggested that GRP (pink box) and neuromedin B (NMB, yellow box) should be considered as the mammalian equivalents of bombesin (blue box). The peptide like bombesin (black box) was thought to be an ancestral form of the bombesin-like family peptides. (b) Our current study demonstrates that GRP is not a mammalian orthologue of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frog species. The ancestral peptide (black box) is considered to be similar to GRP from the research of the basal chordate amphioxus36.

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