Involvement of Calcitonin and Its Receptor in the Control of Calcium-Regulating Genes and Calcium Homeostasis in Zebrafish (Danio rerio)

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A novel function of calcitonin gene-related peptide in body fluid Cl − homeostasis

International audienceVertebrates need to maintain extracellular chloride (Cl 2) concentrations to ensure the normal operation of physiological processes; the transition from aquatic to terrestrial environments necessitated the development of sophisticated mechanisms to ensure Cl 2 homeostasis in the face of fluctuating Cl 2 levels. Zebrafish calcitonin gene-related peptide (CGRP), unlike its splice variant calcitonin, does not respond to environmental Ca 2þ levels. This study aimed to test the hypothesis that CGRP is involved in the control of body fluid Cl 2 homeostasis. Acclimation to high-Cl 2 artificial water stimulated the mRNA expression of cgrp and the receptor (crlr1) when compared with low-Cl 2. CGRP knockdown induced upregulation of the Na þ-Cl 2 co-transporter (ncc2b), while overexpression of CGRP resulted in the downregulation of ncc2b mRNA synthesis and a simultaneous decrease in Cl 2 uptake in embryos. Consistent with these findings, knockdown of either cgrp or crlr1 was found to increase the density of NCC2b-expressing cells in embryos. This is the first demonstration that CGRP acts as a hypochloremic hormone through suppressing NCC2b expression and the differentiation of NCC-expressing ionocytes. Eluci-dation of this novel function of CGRP in fish body fluid Cl 2 homeostasis promises to enhance our understanding of the related physiology in vertebrates

MOLECULAR EVOLUTION OF GPCRS: Kisspeptin/kisspeptin receptors

International audienceFollowing the discovery of kisspeptin (Kiss) and its receptor (GPR54 or KissR) in mammals, phylogenetic studies revealed up to three Kiss and four KissR paralogous genes in other vertebrates. The multiplicity of Kiss and KissR types in vertebrates probably originated from the two rounds of whole-genome duplication (1R and 2R) that occurred in early vertebrates. This review examines compelling recent advances on molecular diversity and phylogenetic evolution of vertebrate Kiss and KissR. It also addresses, from an evolutionary point of view, the issues of the structure-activity relationships and interaction of Kiss with KissR and of their signaling pathways. Independent gene losses, during vertebrate evolution, have shaped the repertoire of Kiss and KissR in the extant vertebrate species. In particular, there is no conserved combination of a given Kiss type with a KissR type, across vertebrate evolution. The striking conservation of the biologically active ten-amino-acid C-terminal sequence of all vertebrate kisspeptins, probably allowed this evolutionary flexibility of Kiss/KissR pairs. KissR mutations, responsible for hypogonadotropic hypogonadism in humans, mostly occurred at highly conserved amino acid positions among vertebrate KissR. This further highlights the key role of these amino acids in KissR function. In contrast, less conserved KissR regions, notably in the intracellular C-terminal domain, may account for differential intracellular signaling pathways between vertebrate KissR. Cross talk between evolutionary and biomedical studies should contribute to further understanding of the Kiss/KissR structure-activity relationships and biological functions

Possible role of calcitonin gene-related peptide in osmoregulation via the endocrine control of the gill in a teleost, the eel, Anguilla anguilla

Osmoregulation is a major challenge in aquatic animals involving a complex endocrine control. We investigated the potential role of calcitonin gene-related peptide (CGRP, a neuromediator in mammals) in the endocrine control of the gill in a teleost, the eel. Transfer from freshwater to seawater induced an hyperosmolality and a concomitant large increase in plasma CGRP levels. Specific CGRP binding sites were characterized in the gill and their number was up-regulated after seawater transfer. This study suggests that the endocrine control of gill function during osmoregulation may represent an ancient role of CGRP in vertebrates

Recurrent DCC gene losses during bird evolution

Accepted for publication in Scientific ReportsDuring development, midline crossing by axons brings into play highly conserved families of receptors and ligands. The interaction between the secreted ligand Netrin-1 and its receptor Deleted in Colorectal Carcinoma (DCC) is thought to control midline attraction of crossing axons. Here, we studied the evolution of this ligand/receptor couple in birds taking advantage of a wealth of newly sequencedgenomes. From phylogeny and synteny analyses we can infer that the DCC gene has been conserved in most extant bird species, while two independent events haveled to its loss in two avian groups, passeriformes and galliformes. These convergent accidental gene loss events are likely related to chromosome Z rearrangement. Weshow, using whole-mount immunostaining and 3Disco clearing, that in the nervous system of all birds that have a DCC gene, DCC protein expression pattern is similar to other vertebrates. Surprisingly, we show that the early developmental pattern of commissural tracts is comparable in all birds, whether or not they have a DCC receptor. Interestingly, only 4 of the 5 genes encoding secreted netrins, the DCC ligands in vertebrates, were found in birds, but Netrin-5 was absent. Together, these results support a remarkable plasticity of commissural axon guidance mechanisms in birds

First identification of dopamine receptors in pikeperch, Sander lucioperca, during the pre-ovulatory period

International audienceDopamine (DA) is a ubiquitous neurotransmitter exerting a range of pleiotropic actions through two DA receptor families, the D1 and the D2. To date in vertebrates, a maximum of four receptor subtypes have been identified within the D1 family, D1 (former D1A), D5 (former D1B), D6 (former D1C and D1D) and D7 (former D1E), while the D2 family encloses five subtypes, D2, D3, D4, D8 (former D2like or D2l) and D9 (former D4-related sequence or D4-rs). In teleosts, no study has investigated in parallel all the DA receptors to identify and localize the whole receptor repertoire from both families. In pikeperch, Sander lucioperca, a species of interest for aquaculture development, the existence, number and location of the DA receptors are totally unknown. To address these questions, RNA-seq with de novo transcriptome reconstruction, functional annotation and phylogenetic analysis were performed to characterize the transcript repertoire of DA receptors in the brain of female pikeperch at the pre-ovulatory period. Ten different cDNA were identified and showed to belong to the D1 family: two D1, one D5a, one D6a and one D6b and to the D2 family: two spliced variants of D2, one D3, one D8 and one D9. Unlike zebrafish, the subtypes D4 and D7 have not yet been isolated in pikeperch. As expected D1, D3, D8 and D9 are mostly expressed in brain parts except for the cerebellum (D1 and D3). The inter-species differences in the number of DA receptors and the inter-organ differences in the gene expression of all receptors support the complexity of the dopaminergic actions in vertebrate

Basal teleosts provide new insights into the evolutionary history of teleost-duplicated aromatase

International audienceDuplicated cyp19a1 genes (cyp19a1a encoding aromatase a and cyp19a1b encoding aromatase b) have been identified in an increasing number of teleost species. Cyp19a1a is mainly expressed in the gonads, while cyp19a1b is mainly expressed in the brain, specifically in radial glial cells, as largely investigated by Kah and collaborators. The third round of whole-genome duplication that specifically occurred in the teleost lineage (TWGD or 3R) is likely at the origin of the duplicated cyp19a1 paralogs. In contrast to the situation in other teleosts, our previous studies identified a single cyp19a1 in eels (Anguilla), which are representative species of a basal group of teleosts, Elopomorpha. In the present study, using genome data mining and phylogenetic and synteny analyses, we confirmed that the whole aromatase genomic region was duplicated in eels, with most aromatase-neighboring genes being conserved in duplicate in eels, as in other teleosts. These findings suggest that specific gene loss of one of the 3R-duplicated cyp19a1 paralogs occurred in Elopomorpha after TWGD. Similarly, a single cyp19a1 gene was found in the arowana, which is a representative species of another basal group of teleosts, Osteoglossomorpha. In eels, the single cyp19a1 is expressed in both the brain and the gonads, as observed for the single CYP19A1 gene present in other vertebrates. The results of phylogenetic, synteny, closest neighboring gene, and promoter structure analyses showed that the single cyp19a1 of the basal teleosts shared conserved properties with both teleost cyp19a1a and cyp19a1b paralogs, which did not allow us to conclude which of the 3R-duplicated paralogs (cyp19a1a or cyp19a1b) was lost in Elopomorpha. Elopomorpha and Osteoglossomorpha cyp19a1 genes exhibited preserved ancestral functions, including expression in both the gonad and brain. We propose that the subfunctionalization of the 3R-duplicated cyp19a1 paralogs expressed specifically in the gonad or brain occurred in Clupeocephala, after the split of Clupeocephala from Elopomorpha and Osteoglossomorpha, which represented a driving force for the conservation of both 3R-duplicated paralogs in all extant Clupeocephala. In contrast, the functional redundancy of the undifferentiated 3R-duplicated cyp19a1 paralogs in elopomorphs and osteoglossomorphs would have favored the loss of one 3R paralog in basal teleosts

Gonadotropin-inhibitory hormone in teleosts: New insights from a basal representative, the eel

International audienceSince its discovery in birds, gonadotropin-inhibitory hormone (GnIH) has triggered investigation in the other groups of vertebrates. In the present study, we have identified a single gnih gene in the European eel (Anguilla anguilla), a representative species of a basal group of teleosts (Elopomorphs). We have also retrieved a single gnih gene in Osteoglossomorphs, as well as in more recently emerged teleosts, Clupeocephala. Phylogeny and synteny analyses allowed us to infer that one of the two gnih paralogs emerged from the teleost-specific whole genome duplication (TWGD or 3R), would have been lost shortly after the 3R, before the emergence of the basal groups of teleosts. This led to the presence of a single gnih in extant teleosts as in other vertebrates. Two gnih paralogs were still found in some teleost species, such as in salmonids, but resulting from the additional whole genome duplication that specifically occurred in this lineage (4R). Eel gnih was mostly expressed in the diencephalon part of the brain, as analyzed by quantitative real-time PCR. Cloning of eel gnih cDNA confirmed that the sequence of the GnIH precursor encoded three putative mature GnIH peptides (aaGnIH-1, aaGnIH-2 and aaGnIH-3), which were synthesized and tested for their direct effects on eel pituitary cells in vitro. Eel GnIH peptides inhibited the expression of gonadotropin subunits (lhβ, fshβ, and common a-subunit) as well as of GnRH receptor (gnrh-r2), with no effect on tshβ and gh expression. The inhibitory effect of GnIH peptides on gonadotropic function in a basal teleost is in agreement with an ancestral inhibitory role of GnIH in the neuroendocrine control of reproduction in vertebrates

Crustacean cardioactive peptides Expression, localization, structure, and a possible involvement in regulation of egg-laying in the cuttlefish Sepia officinalis

International audienceThe cuttlefish (Sepia officinalis) is a cephalopod mollusk distributed on the western European coast, in the West African Ocean and in the Mediterranean Sea. On the Normandy coast (France), cuttlefish is a target species of professional fishermen, so its reproduction strategy is of particular interest in the context of stock management. Egg-laying, which is coastal, is controlled by several types of regulators among which neuropeptides. The cuttlefish neuropeptidome was recently identified by Zatylny-Gaudin et al. (2016). Among the 38 neuropeptide families identified, some were significantly overexpressed in egg-laying females as compared to mature males. This study is focused on crustacean cardioactive peptides (CCAPs), a highly expressed neuropeptide family strongly suspected of being involved in the control of egg-laying. We investigated the functional and structural characterization and tissue mapping of CCAPs, as well as the expression patterns of their receptors. CCAPs appeared to be involved in oocyte transport through the oviduct and in mechanical secretion of capsular products. Immunocytochemistry revealed that the neuropeptides were localized throughout the central nervous system (CNS) and in the nerve endings of the glands involved in egg-capsule synthesis and secretion, i.e. the oviduct gland and the main nidamental glands. The CCAP receptor was expressed in these glands and in the subesophageal mass of the CNS. Multiple sequence alignments revealed a high level of conservation of CCAP protein precursors in Sepia officinalis and Loligo pealei, two cephalopod decapods. Primary sequences of CCAPs from the two species were fully conserved, and cryptic peptides detected in the nerve endings were also partially conserved, suggesting biological activity that remains unknown for the time being