In terms of receptors for bombesin-like peptides, hydrophobicity analysis showed that Xenopus GRPR, NMBR, and BRS-3 contain seven transmembrane domains (TM1–7) as well as those in other vertebrates (e.g. human, rat, chicken, and zebrafish) (Fig. S2). Homology was highest in the hydrophobic domains, and the [Asp85] in the second hydrophobic domain that has possibly been linked to ligand binding is well conserved in vertebrates 35 (Fig. S2).
Phylogenetic analyses of GRP/NMB/bombesin and GRPR/NMBR/BRS-3
To clarify the relation among GRP/NMB/bombesin family, phylogenetic analysis of GRP/NMB/bombesin precursors (preprohormone) was performed (Fig. 1). Homologous genes for GRP/NMB/bombesin family peptides were found in gnathostomes (vertebrates except cyclostomes/agnathans). For the analysis, we used the deduced amino acid sequence in Mammalia Homo sapiens and Rattus norvegicus, Aves Gallus gallus, Reptilia Gekko japonicus, Amphibia X. laevis, X. tropicalis, Nanorana parkeri (frogs), Rhinatrema bivittatum and Microcaecilia unicolor (Caecilians), teleost fish Danio rerio and Oryzias latipes, cartilaginous fish Callorhinchus milii and Rhincodon typus, because their genomes have been decoded. In addition, the sequences of prepro-bombesin-like peptides, which have been reported for other frog species were also included (Fig. 1).
The prepro-GRP/NMB/bombesin sequences were divided into two major clades: GRP and NMB/bombesin clades (Fig. 1). A single GRP gene was found in almost all animals examined, although X. laevis which is allotetraploid has one gene on each of chromosome L and S (Fig. 1, pink box). The NMB/bombesin clade was further divided into the sub-clades: the NMB clade (Fig. 1, yellow box) and the bombesin clade (Fig. 1, blue box); the bombesin clade and NMB clade were found only in frogs; and all the other animals including caecilians, respectively. X. tropicalis and Nanorana parkeri have single bombesin gene, and X. laevis has the gene on each of chromosome L and S, but these frogs do not possess any genes of the NMB group (Fig. 1). In addition, all the precursors of bombesin-like peptides which have previously been identified in other frogs: one in Rana catesbeiana, R. pipiens, and Alytes maurus; two in Phyllomedusa sauvagii; three in Bombina orientalis; and four in Bombina variegate 34 were also included in the bombesin clade (Fig. 1).
These results indicate two possibilities for the evolution of NMB/bombesin: one is the specialization of NMB into bombesin in the frog lineage; the other is the divergence into NMB and bombesin clades resulting, respectively, in the undetectability of frog NMB and the disappearance of bombesin in vertebrates other than frogs. We therefore examined synteny in genes surrounding the amphibian NMB/bombesin locus (Fig. 2). Comparison of the genome of X. tropicalis, Nanorana parkeri, Microcaecilia unicolor and Rhinatrema bivittatum, indicates that the order of genes around the caecilian NMB genes and the frog bombesin genes were highly conserved, although an inversion of the ZNF11 - SEC592A – bombesin - KTI12 region of X. tropicalis genome has occurred. Thus, it can be concluded that bombesin and NMB are respective orthologues and that specialization of the NMB sequence in the frog lineage resulted in bombesin (Fig. 2).
We also performed phylogenetic analysis of the receptors for the GRP/NMB/bombesin family; GRPR, NMBR and BRS-3 (Supplemental Fig. 3). The results suggest that Gnathostomata basically have orthologues in each of the three groups with no specialization in the frog lineage as seen in NMB/bombesin. The BB4 receptor in Bombina also belongs to the mammalian BRS-3 group. In addition, the conservation of GRPR and NMB, BRS3 was not found in teleosts (e.g. Danio rerio and Oryzias latipes) and cartilaginous fish (e.g. Callorhinchus milii and Rhincodon typus), but only in archaic fish such as Latimeria chalumnae (coelacanth), Erpetoichthys calabaricus (reedfish), Lepisosteus oculatus (gar), and Acipenser ruthenus (sturgeon) (Supplemental Table 1).
Reverse transcription (RT)-PCR of GRP and GRPR mRNA in Xenopus
In contrast to the diversification of NMB/bombesin in the frog lineage and the loss of the BRS-3 gene in some fish lineages, GRP and the GRPR are widely conserved in vertebrates. In this study, we used frogs to investigate the principal (conserved, original) role of these bombesin-family systems. We confirmed the expression of GRP and GRPR mRNA in a variety of Xenopus tissues (brain, spinal cord, heart, lung, and stomach) by RT-PCR. Bands were detected at the expected sizes for GRP and GRPR genes in the brain (Fig. 3). GRP mRNA was highly expressed in the brain, spinal cord, stomach, and weakly expressed in the lung (Fig. 3; upper panel). Although GRPR mRNA was detected in all tissues, the expression level was low in the heart (Fig. 3; middle panel). As the internal control in Xenopus, nearly equivalent amounts of GAPDH cDNA were amplified from RNA preparations among these tissues, which showed that no significant RNA degradation had occurred and a proper RT was obtained (Fig. 3; bottom panel).
Real-time quantitative PCR (qPCR) of GRP and GRPR mRNA in Xenopus CNS
To quantify the GRP and GRPR expression at the transcription level, we performed real-time qPCR analyses for four parts of the CNS of males and females: the telencephalon; the diencephalon/mesencephalon/pons/cerebellum; the medulla oblongata; and the spinal cord. Although GRP and GRPR mRNA expression was detectable in these all tissues of both sexes, no sex differences and no interactions between sex and tissue were detected in any of the tissues we examined (black bars indicate means of males, magenta bars indicate means of females) (Fig. 4). Thus, in the comparisons of expression level between tissues, sexes were combined. The expression of GRP mRNA was higher in the diencephalon/mesencephalon/pons/cerebellum and the medulla oblongata, than in the telencephalon and the spinal cord (Fig. 4a). In contrast, the expression of GRPR mRNA was the highest in the spinal cord, and the lowest in the telencephalon (Fig. 4b).
Distribution of GRP in Xenopus CNS
The expression of GRP was next localized in Xenopus CNS. The examination of transverse (from rostral to caudal) brain and spinal cord sections revealed the presence of many cell bodies and fibers of GRP-immunoreactive (+) neurons in Xenopus CNS. The overall neuroanatomical distribution of GRP+ neuronal cell bodies and their fiber projections is schematically summarized in Table 1. The specificity of the GRP antiserum reactivity was confirmed by control absorption experiments in which the primary rabbit antiserum against Xenopus GRP20 − 29 was preabsorbed with an excess of Xenopus GRP20 − 29 antigen peptide ( [Ser2] form-NMC); in these experiments no immunostaining was seen (Supplemental Fig. 4). In the spinal cord, GRP+ fibers and numerous varicosities were found throughout the spinal grey matter area (Supplemental Fig. 4a, b, d, and e). A cluster of GRP+ cell bodies was located mainly in the dorsal field of spinal grey in the cervical spinal cord (df; Supplemental Fig. 4a, b, and e). In the thoracic and lumbosacral spinal cord, similar GRP+ fibers were frequently observed, but few GRP+ cell bodies could be detected.
Table 1
Localization of gastrin-releasing peptide (GRP)-immunoreactive cell bodies and fibers in Xenopus central nervous system.
Brain area
|
Cell bodies
|
Fibers
|
Acc
|
nucleus accumbens
|
++
|
+
|
Apl
|
amygdala pars laterails
|
++
|
+
|
Apm
|
amygdala pars medialis
|
++
|
+
|
Av
|
anteroventral tegmental nucleus
|
-
|
+
|
ca
|
commissura anterior
|
-
|
-
|
Cb
|
nucleus cerebelli
|
-
|
++
|
df
|
dorsal field of spinal grey
|
++
|
+++
|
Ea
|
nucleus entopeduncularis anterior
|
-
|
+
|
fr
|
fasciculus retroflexus
|
-
|
+
|
ftg
|
fasciculi tegmentales
|
-
|
++
|
Hbv
|
nucleus habenularis ventralis
|
-
|
+
|
Iflm
|
nucleus interstitialis of flm
|
-
|
+
|
lf
|
lateral field of spinal grey
|
-
|
++
|
lfb
|
lateral forebrain bundle
|
-
|
++
|
ll
|
lemniscus lateralis
|
-
|
++
|
mfb
|
medial forebrain bundle
|
-
|
++
|
Mg
|
nucleus preopticus magnocellularis
|
-
|
++
|
mmf
|
medial motor field of spinal grey
|
-
|
++
|
Mp
|
Medial pallium
|
-
|
-
|
ola
|
tractus olfactorius accessorius
|
+
|
+
|
optb
|
tractus opticus basalis
|
-
|
++
|
optl
|
tractus opticus lateralis
|
-
|
++
|
Pb
|
nucleus parabrachialis
|
-
|
+
|
Poa
|
nucleus preopticus anterior
|
-
|
++
|
Pv
|
posteroventral tegmental nucleus
|
-
|
+
|
Ra
|
nucleus raphes
|
+
|
++
|
Ri
|
nucleus reticularis inferior
|
+
|
-
|
Ris
|
nucleus reticularis isthmi
|
-
|
++
|
Rm
|
nucleus reticularis medius
|
-
|
++
|
SC
|
nucleus suprachiasmaticus
|
-
|
+
|
Strd
|
striatum, dorsal part
|
-
|
-
|
Strv
|
striatum, ventral part
|
-
|
+
|
TP
|
tuberculum posterius
|
+++
|
+
|
trVds
|
tractus descendens nervi trigemini
|
-
|
+
|
VH
|
nucleus hypothalamicus ventralis
|
-
|
-
|
VL
|
nucleus ventrolateralis thalami
|
-
|
++
|
vlf
|
ventrolateral field of spinal grey
|
-
|
++
|
VLs
|
superficial ventral nucleus
|
-
|
+
|
VM
|
nucleus ventromedialis thalami
|
++
|
++
|
vmf
|
ventromedial field of spinal grey
|
-
|
++
|
vt
|
ventriculus tertius
|
-
|
+
|
Vds
|
nucleus descendens nervi trigemini
|
-
|
++
|
VIIIv
|
nucleus ventralis nervi vestibulocochlearis
|
-
|
+
|
IXm
|
nucleus motorius nervi glossopharyngei
|
-
|
+
|
Xm
|
nucleus motorius nervi vagi
|
++
|
+
|
XII
|
nucleus motorius nervi hypoglossi
|
-
|
+
|
The relative density of labeling was classified as weak (+), moderate (++), and strong (+++). cc: central canal; LV: lateral ventricle; 3V: third ventricle.
|
In the telencephalon, abundant but weakly GRP-immunoreactive cell bodies and fibers were located throughout the ventral telencephalic area, e.g. cell bodies and fibers in the nucleus accumbens (Acc) and thin fibers in the tractus olfactorius accessorius (ola) (Fig. 5b). Immunoreactive cell bodies were abundant in the amygdala pars medialis (Apm; Fig. 5c), and in the amygdala pars lateralis (Apl; Fig. 5d). In the diencephalon, a small number of labeled cells were located in the nucleus ventromedialis thalami (VM; Fig. 5e). In the hypothalamus, many intensely labeled cell bodies and fibers were found in the tuberculum posterius (TP; Fig. 5f). In the brainstem, large but only weakly immunoreactive cell bodies and fibers were found in the nucleus raphes (Ra; Fig. 5g).
GRP+ fibers were widely distributed throughout the CNS (Table 1). A fine network of GRP+ fibers was also observed in the Apm, in the Apl, and prominently in the striatum, pars ventralis (Strv; Fig. 6b). GRP+ fibers were also found in some diencephalic nuclei surrounding the third ventricle; e.g. VM (Fig. 5e), nucleus preopticus anterior (Poa; Fig. 6c), and nucleus preopticus magnocellularis (Mg; Fig. 6d). A weak distribution of GRP+ fibers was also found in the area near the tractus opticus lateralis (optl), the tractus opticus basalis (optb; Fig. 6e), and the nucleus reticularis isthmi (Ris; Fig. 6f). In the posterior part of the medulla oblongata, the network of GRP+ fibers spreading in the nucleus descendens nervi trigemini (Vds) was intensely immunoreactive and such fibers were also scattered in the tractus descendens nervi trigemini (trVds; Fig. 6g).
Expression of GRPR protein in Xenopus CNS
Western immunoblot analysis with the polyclonal antiserum against Xenopus GRPR was performed to determine the presence of GRPR protein in homogenates derived from the brain and spinal cord of adult male Xenopus. An intense protein band was observed in the brain and spinal cord extracts, and its electrophoretic mobility was located at ~ 43 kDa, which is the expected molecular weight of Xenopus GRPR (Fig. 7). Preabsorption of the antiserum with an excess of antigen peptides (50 µg/mL) prevented the immunostaining of the ~ 43-kDa protein band in the brain and spinal cord (Fig. 7). Immunoblot analyses were repeated independently three times by using different three frogs and gave similar results.