Edited by: Sylvie Dufour, Muséum National d’Histoire Naturelle, France
Reviewed by: Gregoy Y. Bedecarrats, University of Guelph, Canada; Alexander S. Kauffman, University of California San Diego, USA
This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology.
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RFamides (RFa) are neuropeptides involved in many different physiological processes in vertebrates, such as reproductive behavior, pubertal activation of the reproductive endocrine axis, control of feeding behavior, and pain modulation. As research has focused mostly on their role in adult vertebrates, the possible roles of these peptides during development are poorly understood. However, the few studies that exist show that RFa are expressed early in development in different vertebrate classes, perhaps mostly associated with the central nervous system. Interestingly, the related peptide family of FMRFa has been shown to be important for brain development in invertebrates. In a teleost, the Japanese medaka, knockdown of genes in the Kiss system indicates that Kiss ligands and receptors are vital for brain development, but few other functional studies exist. Here, we review the literature of RFa in early vertebrate development, including the possible functional roles these peptides may play.
Neuropeptides with an arginine (R) and an amidated phenylalanine (F)-motif at its C-end (called RFamides or RFa) were first described in mollusks in the 70s [FMRFamide (FMRFa)] (
Interestingly, FMRFa are expressed in the nervous system at very early developmental stages in several phyla of metazoans, as mollusks (cephalopods and gastropods) (
The first RFa to be identified in vertebrates was NPFF (also known as F8F-amide) and NPAF (
Few studies have investigated the expression, location, or function of NPFF during development. However, one study in teleosts (
RFa and/or receptors | Species | Method | Antibody (or radioligand) | Embryonic stages | Location of peptide/mRNA in early developing central nervous system | Putative functions in early development | Reference |
---|---|---|---|---|---|---|---|
NPFF | Zebrafish ( |
ISH | – | 24, 30, 36 hpf, 2, 3, 4, 7 dpf, adult | Exclusively in large cells of the developing terminal nerve | – | ( |
FMRFa (NPFF + ?) | Zebrafish and sterlet ( |
ir | Pol 1:1000-1:20000 rabbit anti-FMRFa (Phoenix/Incstar) | 24–60 hpf and 5 dpf zebrafish, juvenile sterling | Developing terminal nerve, hyp | Involvement in brain functions | ( |
FMRFa (NPFF + ?) | Brown trout ( |
ir | Pol 1:500 rabbit anti-FMRFa (Chemicon/Incstar) | Embryos, alevins, fry | Developing terminal nerve, hyp (NAPv, NPPv) | Regulation of neural centers related to analgesia, feeding | ( |
FMRFa (NPFF + ?) | Lungfish ( |
ir | Pol 1:10000 anti-FMRFa, Phoenix | Just before hatching to juvenile stages | Paraventricular organ in hyp, terminal nerve at hatching | – | ( |
FMRFa (NPFF + ?) | Frog ( |
ir | Pol FMRFa antiserum (Peninsula labs) | Posthatching | tel and diencephalon (newly hatched) | Modulation of GnRH-neurons? | ( |
NPFF | African clawed frog ( |
ir | Pol 1:1000 rabbit anti-NPFF serum (from Dr. H.Y.T. Yang, Elisabeth’s Hospital, Washington, DC, USA) | E30–45, and through metamorphosis | Olfactory bulbs and ventral tel, hyp, NTS, and spinal cord in embryo | Regulation of α-MSH release? Spinal embryogenesis? | ( |
FMRFa (NPFF + ?) | Toad ( |
ir | Pol 1:30000 rabbit anti-FMRFa (Phoenix) | Embryonic and larval stages | Suprachiasmatic area in embryo (stage III6). Olfactory bulb, tel, suprachiasmatic hyp in early larvae | Neuromodulator/neurohormone during development | ( |
FMRFa (NPFF + ?) | Skink ( |
ir | Pol 1:10000, 1:30000 anti-FMRF (Phoenix) | 7 –70 dpf (birth)-neonatal | Fore- and hindbrain (terminal nerve, OB, hyp lateral preoptic area, suprachiasmatic area, and NAPv), MRF (35 dpf), plus NTS and vagus nerve close to birth | Regulation of blood pressure? Control of pituitary? | ( |
FMRFa (NPFF + ?) | Chicken ( |
ir | Pol 1:4000 FMRFa antiserum (Peninsula labs) | E11–19 | TN | – | ( |
FMRFa (NPFF + ?) | Japanese quail ( |
ir | 1:5000 Anti-FMRFa ( |
E2.5–12 | Fibers in diencephalon (hyp), brain stem, olfactory nerve, and cell bodies in septum at early stages. OB at later stages | – | ( |
FMRFa (NPFF + ?) | African clawed frog | ir | Pol 1:1000 rabbit anti-FMRFa (Diasorin, Stillwater, MN) | Through metamorphosis | Olfactory nerve, tel, suprachiasmatic hyp (prometamorphic stage 56) | – | ( |
NPFF and receptors | Mouse | Quantitive autoradiography | Radioligand: [125 I](1DME)Y8Famide | Post-natal | Almost all brain areas at P14 | Pro-opioid (P14) and anti-opioid effect (P21) of NPFF | ( |
NPFF | Rat | ISH, qPCR | – | E14-birth | Spinal cord, medulla (caudal NTS; E14), MRF (P0), pituitary | Sensory projection development in MRF, lactrotrope differentiation? | ( |
NPFF | Rat | ir | Pol rabbit anti rat F8Fa (FLFQPQRF) | E16, E18, E20, and post-natal | Fibers in median eminence (E20), cells in medulla (P1) | Role in homeostatic mechanisms, food intake in neonatals? | ( |
FMRFa (NPFF + ?) | Tree shrew ( |
ir on pituitary | Pol 1:1000 rabbit anti-FMRFa (Incstar, Stillwater, MN, USA) | E20–E41 | Pars intermedia of pituitary from E27 | Involved in early hormone secretion and releasing factor regulation? | ( |
FMRFa (NPFF + ?) | Tree shrew | ir | Pol 1:1000 rabbit anti-FMRFa (Incstar, Stillwater, MN, USA) | E19–E43 | Developing TN from E23 | – | ( |
In adult agnathans, NPFF RFa has been found expressed in the hypothalamus. Furthermore, it has been shown that NPFF stimulates the expression of the gonadotropin-β gene in the pituitary of hagfish, which suggests that NPFF can have a role in control of reproduction in lower vertebrates (
In zebrafish (
Similar to teleosts and lungfish, FMRFa-immunohistochemistry labels neurons of the TN of African clawed frog (
In contrast to the apparent situation in teleosts, the developing and the adult brains of amphibians and birds show presence of NPFF mRNA and protein also in brain areas other than the TN (
Interestingly, neurons in the nucleus of the solitary tract in the medulla show NPFFir at an early stage in
In the Japanese quail, the first NPFFir (F8F) fibers appear in the diencephalon (later hypothalamus) and the brain stem at embryonic stage (E) 6 (
Using an antibody against rat NPFF (F8F-amide), Kivipelto et al. showed the presence of fibers and terminal-like structures as early as E20 in the rat (
In an
Using an NPFF radioligand, Desprat et al. showed the presence of receptors for NPFF in regions of the developing mouse brain and spinal cord involved in the analgesic effects of opiates (
Interestingly, the TN of the mammal tree shrew (
In summary, NPFF is detected early in embryonic development in all vertebrates studied, see overview in Table
The PrRP group includes the peptides PrRP31 and PrRP20. A new member of this family, C-RFa is found in Japanese crucian carp (
It is believed that PrRP is involved in the control of pituitary function in fishes. Firstly, in many species of adult fishes, PrRP fibers project to and terminate on prolactin-producing cells of the pituitary. Secondly, C-RFa injections in rainbow trout and tilapia cause release of prolactin and somatostatin (
In mammals, PrRP was thought to act on the pituitary, because of the high expression of its receptor GPR10 in the anterior pituitary (
Very few studies have looked at the possible role of PrRP in development. However, the few that exist show that this peptide is expressed at an early stage in
In the teleost guppy, PrRPir cells were detected in the nucleus lateralis tuberis pars posterioris in the hypothalamus already at the day of birth (
In
In rat, PrRP mRNA and PrRPir cells are found in the nucleus of the solitary tract at E18, and in the ventral and lateral reticular nucleus of the caudal medulla oblongata at E20 (
The presence of mRNA of PrRP and its receptor GPR10 has also been investigated with
Studies of PrRP in vertebrate development are summarized in Table
RFa and/or receptors | Species | Method | Antibody | Embryonic stages | Location of peptide/mRNA in early developing CNS | Putative functions in early development | Reference |
---|---|---|---|---|---|---|---|
PrRP | Guppy ( |
ir | Pol rabbit anti-salmon PrRP ( |
0-P14 | Hyp, pituitary pars distalis at birth | Developmental role? | ( |
PrRP | qPCR | – | Premetamorphosis- climax (54–65) | Transiently increased expression in brain at prometamorphosis | – | ( |
|
PrRP | Chicken pituitary | RT-PCR | – | E8–20 | Expressed in pituitary at all stages studied | – | ( |
PrRP | Rat | ISH, RT-PCR, ir | M 40 μl/ml P2L-1C (mature PrRP)/P2L-1T (prepro-PrRP) mouse anti human PrRP ( |
E15, E18, E20, and post-natal | NTS (E18), MRF (E20), hyp (P13) | Role in embryonic brain development? | ( |
PrRP + GPR10 | Rat | ISH, qPCR | – | E14-birth | PrRP: MRF, pituitary (E19), GPR10: pallidum, hippocampus, and MRF (E15–17) | Lactrotrope differentiation? | ( |
GnIH was first described by Tsutsui et al. (
In adult vertebrates, GnIH positive cells are found in different regions of the brain, notably in hypothalamic regions like the avian paraventricular nucleus, from where they send their projections to GnRH1 neurons in the preoptic region or to gonadotrope cells in the pituitary. GnIH terminals and GnIH receptors have also been identified on GnRH2 neurons in birds and mammals, e.g., Ref. (
The spatial expression pattern should indicate potential functions, although much remains to be discovered when it comes to GnIH functions in general and during development in particular. Similar to in birds, GnIH in mammals have been shown to inhibit gonadotropin synthesis and release, either directly in the pituitary or via inhibition of hypothalamic GnRH-neurons. The situation seems different in frogs and teleost fish where GnIH can either inhibit or stimulate gonadotropin (and also growth hormone and prolactin) release, depending on reproductive stage, species, and sex, e.g., Ref. (
As most interest has focused on its role as an inhibitor of GnRH and gonadotropin release during reproduction, very little is known about GnIH during vertebrate development. Apart from some studies looking at pre-pubertal stages, the information we have is mostly limited to studies on the spatio-temporal expression pattern in mammalian and avian (post-natal) development, and some very few in teleost early development.
A recent article from Biswas and colleagues (
In birds, where GnIH was first characterized, the existing literature focuses on the function of the GnIH system during sexual development, especially during the pre-pubertal period. For instance, circulating gonadotropin levels have been found to be negatively correlated with hypothalamic GnIH content (
As mammalian model systems are less suited for studies of early embryogenesis, the few papers dealing with GnIH in mammalian development starts from late gestational stages. Yano et al. (
In summary, the expression of a seemingly functional GnIH system in fish, birds, and mammals already from early development suggests important developmental function(s) of this RFa in vertebrates. If these include more than the above mentioned regulatory (inhibitory/modulatory) effects on sexual development, remains to be seen. See Table
RFa (and/or receptors) | Species | Method | Antibody (or radioligand) | Embryonic stages | Location of peptide/mRNA in early developing CNS | Putative functions in early development | Reference |
---|---|---|---|---|---|---|---|
GnIH | Indian major carp ( |
ir | ? | Hatchling-fry-juvenile | Cells in olfactory system, NPP, NPPv, and fibers in optic tectum, PPD in pituitary, and MRF (P0) | – | ( |
GnIH + receptors | Zebrafish ( |
RT-PCR | - | Blastula-juvenile | GnIH first detected at 5-prime stage, receptors at all stages | Role in early development? | ( |
GnIH | Rat | ISH, RT-PCR, ir | M 10 μg/ml 1F3 anti-RFRP-1, P 16 μg/ml antisera anti – FRP-1 ( |
E15, E18, E20, and post-natal | Caudal portion of hyp (E16), many areas at E18 and E20 | Modulation of pain, response to stress during development? | ( |
GnIH and GPR147 | Rat | qPCR,ELISA | Pol rabbit anti-avian GnIH ( |
Pre-pubertal (P4–20) and peripubertal | GnIH and receptor mRNA and peptide present in hyp from P4 | – | ( |
GnIH | Rat | ISH + BrdU | – | Cell bodies generated at E13/E14 in tuberal hyp | – | ( |
|
GnIH | Mouse | ISH | – | P1, P10, P20 | mRNA and protein in dorsal-medial nucleus of hyp from P1 | – | ( |
Kisspeptins are RFa encoded by the
The product of the
Despite the accumulating data of the role of kisspeptins in adult vertebrates, less is known about kisspeptins during post-natal/pre-pubertal development, and very little is known regarding the potential expression and function of the kiss system during embryogenesis/early development. This could at least partly be due to the lack of a suitable model system. Because
The few existing data on kisspeptins during early development come from studies in medaka and zebrafish. We recently performed a study of kisspeptin ligand and receptor expression pattern and function during early development in medaka, exploiting the advantages of the teleost model system (
In Hodne et al., we performed a series of knockdown experiments that indicated several independent kiss systems during medaka embryonic development (
Knockdown of maternal
Zygotic knockdown of
Contrary to the observed phenotypes following zygotic knockdown of
A recent work by Zhao et al. has investigated the role of kiss on GnRH neuron development in zebrafish (
The existence of a functional kisspeptin system in birds is not clarified [see in Ref. (
In mammals, the kisspeptin system has been intensively investigated during the last decade. Whereas, most literature covers the key role of Kiss in regulating GnRH neuron around and after puberty, pre- and early neonatal stages have been looked into more closely during recent years [see reviews in Ref. (
It seems that the early kisspeptin systems are functional in rodents in that Kiss neurons already are in close contact with GnRH-neurons prenatally, and that GnRH-neurons are able to respond to kisspeptins by enhanced GnRH secretion during prenatal life (
In line with the more severe phenotypes observed in medaka following receptor knockdown (
Although more data are available regarding the role of kisspeptins during vertebrate development compared to the role of other RFa, there are still much work to be done. One important aspect probably will be to elucidate their role in neuronal migration/development, where they seemingly play a major role, at least in fish. See Table
RFa (and/or receptors) | Species | Method | Antibody | Embryonic stages | Location of peptide/mRNA in early developing CNS | Putative functions in early development | Reference |
---|---|---|---|---|---|---|---|
Kiss | Zebrafish ( |
qPCR, kiss treatment, electrophysiology | – | 1–7 dpf | Kiss1 and 2 mRNA detectable in brain from 1 dpf, increasing during development | Kiss1 stimulates GnRH neuron development, Kiss2 involved in development of trigeminal neurons | ( |
Kiss | Zebrafish | qPCR | – | 1, 3, 7, 30, 45 dpf, adult | – | ( |
|
GPR54 | Cobia ( |
qPCR | – | Post hatching-adult | – | ( |
|
Kiss and receptors | Medaka ( |
qPCR+ knockdown | – | From fertilization to newly hatched | – | Essential for brain and eye development | ( |
Kiss | Rat | qPCR, ISH ir, BrdU birth dating | Pol sheep anti-kiss (N-ter) AC067 | Embryonic rats from E11.5 to E21.5 | Kiss1 neurons in arcuate nucleus born from E12.5 | Involved in embryonic activation of the hypothalamic–hypophyseal–gonadal axis | ( |
Kiss | Rat | ISH | – | Post-natal (neonate to adult) | Anteroventral periventricular nucleus (P7 in males, P21 in females), arcuate nucleus (P3) | – | ( |
Kiss | Rat | ISH | – | Post-natal (P0–P19) | Anterior hyp (P11), arcuate nucleus (P0) | Role in sexual differentiation of neonatal brain | ( |
Kiss | Rat | Kiss stimulation ( |
– | Post-natal | – | Stimulating GnRH release in neonatals (5P) | ( |
Kiss + GPR54 | Rat | qPCR on hyp | – | Post-natal (P1–75) + adults | – | ( |
|
Kiss + GPR54 | Mouse | Transgenic mice | – | E12.5, E13.5, E14.5, and E16.5 | Regulating fetal GnRH activity? | ( |
|
Kiss + GPR54 | Mouse | RT-PCR, kiss treatment++ | E12.5, E13.5, E14.5, and E15.5 | Stimulates GnRH neurite growth | ( |
||
Kiss + GPR54 | Mouse | ISH, qPCR, ir | Pol 1:10000 rabbit anti-rodent-kiss 1 ( |
E13, E15, E17 to P35 | Involved in sexual differentiation of the brain during embryonic development? | ( |
|
GPR54 | Mouse | ISH, single cell qPCR, Ca2+ imaging | – | E12.5, E13.5, E14.5, E17.5, and adult | In GnRH-cells in nasal region and nasal forebrain junction (E13.5) | – | ( |
Kiss | Mouse | ir | Pol 1:5000 rabbit anti-kisspeptin-10 (no. 566) ( |
Post-natal-adults (P10–P61) | Anteroventral periventricular nucleus, preoptic periventricular nucleus in hyp (P25) and arcuate nucleus in hyp at all stages | Kiss neurons in anteroventral periventricular nucleus and preoptic periventricular nucleus in hyp involved in the sexually differentiated functioning of GnRH-neurons | ( |
Kiss | Mouse | ISH | – | Post-natal (P1–P16) | Anteroventral periventricular nucleus and preoptic periventricular nucleus in hyp from P10 | Involved in the sexually differentiated functioning of GnRH-neurons | ( |
Kiss | Mouse | ir | Pol 1:10000 rabbit anti-kisspeptin-10 (no. 566) ( |
Post-natal (P15–P30) + adults | Preoptic periventricular nucleus in hyp from P15 | – | ( |
The 26RF/QRFP group is the newest member of the RFa family, first described in 2003 in the brain of European green frog (
In adult goldfish,
The developmental expression of 26RFa/QRFP and GPR103 has only been studied in human adrenal gland and rat adrenals (
The RFa form a complex family with many different members acting in various physiological processes, with one peptide seemingly having several functions in the same animal in some cases. Common to all the peptides seems to be that they could have a role in appetite regulation, pain modulation, and reproduction. With the newest member of the RFa family found only a little over 10 years ago, the field of RFa is relatively new and requires much more research. Especially, how these peptides can influence development is poorly understood. However, the few studies of RFa in developing vertebrates show interesting results that may indicate that many of the RFa could have a separate role in development. Interestingly, it seems that all RFa are expressed early in development in many different groups of vertebrates. However, most of our knowledge of RFa comes from
The cellular pathways of RFa are poorly understood, and more research is required to find out how RFa can act on developmental processes. However, some RFa (26RFa and Kiss) have been shown to affect migration in cancer cells (
The field of RFa in vertebrates is exiting and rapidly expanding. The few developmental studies that have been done show promising and important results. Taken together, these studies indicate that RFa may have a role in development of the nervous system not yet identified. More research is needed, especially functional studies that can give insight into the role these peptides play in development.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by the Research Council of Norway, Grants Nos. 184851 (to Finn-Arne Weltzien) and 231767/F20 (to Guro Katrine Sandvik).