New Insights Into the Evolutionary History of Melatonin Receptors in Vertebrates, With Particular Focus on Teleosts

Abstract

In order to improve our understanding of melatonin signaling, we have reviewed and revised the evolutionary history of melatonin receptor genes (mtnr) in vertebrates. All gnathostome mtnr genes have a conserved gene organization with two exons, except for mtnr1b paralogs of some teleosts that show intron gains. Phylogeny and synteny analyses demonstrate the presence of four mtnr subtypes, MTNR1A, MTNR1B, MTNR1C, MTNR1D that arose from duplication of an ancestral mtnr during the vertebrate tetraploidizations (1R and 2R). In tetrapods, mtnr1d was lost, independently, in mammals, in archosaurs and in caecilian amphibians. All four mtnr subtypes were found in two non-teleost actinopterygian species, the spotted gar and the reedfish. As a result of teleost tetraploidization (3R), up to seven functional mtnr genes could be identified in teleosts. Conservation of the mtnr 3R-duplicated paralogs differs among the teleost lineages. Synteny analysis showed that the mtnr1d was conserved as a singleton in all teleosts resulting from an early loss after tetraploidization of one of the teleost 3R and salmonid 4R paralogs. Several teleosts including the eels and the piranha have conserved both 3R-paralogs of mtnr1a, mtnr1b, and mtnr1c. Loss of one of the 3R-paralogs depends on the lineage: mtnr1ca was lost in euteleosts whereas mtnr1cb was lost in osteoglossomorphs and several ostariophysians including the zebrafish. We investigated the tissue distribution of mtnr expression in a large range of tissues in medaka. The medaka has conserved the four vertebrate paralogs, and these are expressed in brain and retina, and, differentially, in peripheral tissues. Photoperiod affects mtnr expression levels in a gene-specific and tissue-specific manner. This study provides new insights into the repertoire diversification and functional evolution of the mtnr gene family in vertebrates.

Keywords: functional evolution; gene duplication; medaka; melatonin receptors; phylogeny; synteny; teleosts; vertebrates.

Figure 1

Evolution of the gene structure of mtnr in vertebrates. Representation of the gene structure of the lamprey mtnr1-like, the four gnathostome mtnr subtypes represented by the four coelacanth mtnr, and gene of mtnr1b duplicates in teleosts. The gene representation includes the coding sequence and its features. Genomic DNA in 5′-3′ orientation is represented by black lines, and the coding sequences with boxes. To save place, long intronic sequences have been cut and length is indicated in kb on the corresponding intron sequence. Within the coding sequences, the different domains of mtnr1b are represented by different colors; blue represents extracellular domains and loops, yellow for the transmembrane domain, and red for the intracellular loops and cytosolic tail. Predicted N-glycosylation sites are indicated in green, and conserved cysteine residues in red. Gene references are given in the Supplementary Table 1.

Figure 2

Maximum-likelihood phylogeny tree of melatonin receptors of vertebrate representatives. Melatonin receptor phylogeny was inferred from alignment of the deduced amino-acid sequences of melatonin receptor using the PhyML algorithm with the AIC selection criteria of the Smart Model Selection and the tree Subtree Pruning and Regrafting (SPR) improvement algorithm. The four gnathostome monophyletic clades are indicated with different background colors. The blue line indicates teleost melatonin receptor clades. Branch nodes are supported by bootstrap analysis with 100 replicates and only nodes with bootstrap values above 50% were considered as supported.

Figure 3

Conserved syntenic regions of melatonin receptors in gnathostomes. Locations are given in Mb. Gene order follows that of the human chromosomes 4, 5, 11, and X. Each neighboring gene family is displayed with a specific color. Red crosses indicate mtnr gene loss. In the spotted gar, the mtnr1a was found as pseudogene in the genome assembly but a transcript encoding a functional receptor was found in the PhyloFish database. We assumed that the mtnr1a pseudogene form results from an assembly error, and therefore the mtnr1a is represented by the mtnr1a blue box, but with the perimeter edges dotted. The new gene locations in the genome assembly are indicated under the gene position. Full names and references of mtnr and neighboring genes are given in Supplementary Table 2.

Figure 4

Conserved syntenic region of melatonin receptors in actinopterygians. Locations are given in Mb. Gene order follows that of the spotted gar chromosomes LG4, LG3, LG7, and LG6. Each neighboring gene family is displayed with a specific color. Red crosses indicate mtnr gene loss. In the spotted gar, the mtnr1a was found as pseudogene, in the genome assembly but a transcript encoding a functional receptor was found in the PhyloFish database. We assumed that the mtnr1a pseudogene form results from an assembly error, and therefore the mtnr1a is represented by the mtnr1a blue box, but with the edges dotted. The new gene locations in the genome assembly are indicated under the gene position. Full names and references of mtnr and neighboring genes are given in Supplementary Table 2.

Figure 5

Mtnr gene neighborhood in the pre-vertebrate genome. Mtnr and its syntenic genes were mapped from the gene list of the pre-1R ancestral genome reconstruction (Genomicus 69.10). The gene pre-1R gene list is available for download on the Genomics webserver and (115). The gene order is relative.

Figure 6

Tissue distributions of the four melatonin receptors in reproductively active medaka females. Gene expression profiles of the four melatonin receptor paralogs: (A) mtnr1a, (B) mtnr1b, (C) mtnr1c, and (D) mtnr1d in brain (Br), pituitary (Pit), eye (Ey), gill (Gi), heart (He), spleen (Sp), adipose tissue (AT), liver (Li), intestine (Int), muscle (Mu), skin (Sk), kidney (Ki), and ovary (Ov) of reproductively active medaka females. The bar plots represent the relative expression calculated as the percentage of the mean of transcript levels (mean ± se) per tissue. The letter inside the parentheses indicates the 3R paralog identity.

Figure 7

Melatonin receptor expression profiles in mature female medaka during the day and night. Gene expression of the four melatonin receptor paralogs: (A) mtnr1a, (B) mtnr1b, (C) mtnr1c, and (D) mtnr1d in brain (Br), pituitary (Pit), eye (Ey), gill (Gi), heart (He), spleen (Sp), adipose tissue (AT), liver (Li), intestine (Int), muscle (Mu), skin (Sk), kidney (Ki), and ovary (Ov) of reproductively active medaka females. Fish were maintained under a prolonged photoperiod cycle of L14:D10 (light from 08:00 to 22:00) for one month prior to sampling and sampled either during the day (around 13:00) or the night (around 01:00) (n = 5 per group). Bar plots represent the mean ± se of the relative transcript levels for the 5 fish and the dots show the individual data points depending on the sampling time. Night sampling is indicated with blue dots and day sampling with pink dots. Bars along the x-axes represent the light phase (open bars) and dark phase (solid bars). Statistically significant changes in gene expression between day and night (Wilcoxon rank sum test) are indicated by an asterisk (*). Triangles (▴) indicate groups for which gene expression was detected in less than 3 out of 5 fish. Statistical comparisons were not done for these groups. The letter inside the parentheses indicates the 3R paralog identity.

Figure 8

Tissue distribution profiles of melatonin receptors in different ray-finned fish in the PhyloFish database. The bar plots represent the relative expression calculated as the percentage of the maximum rpkm (number of reads per kilo base per million reads) value per species. The mtnr transcripts were found by exploring the PhyloFish database (17) and transcript identity was established by phylogenetic analysis (data not shown). Subtype identity is given at the top of each graph. ND indicates a tissue for which data were not available.

Figure 9

Evolutionary scenario of the melatonin receptors in vertebrates. This evolutionary scenario was developed on the basis of the phylogeny and synteny analyses (Figures 2–4 and Supplementary Figures 2–5). The four receptor genes are derived from duplication of an ancient mtnr1 gene through vertebrate tetraploidization (1R and 2R). The teleost 3R event generated duplicates of the four mtnr subtypes. Multiple and selective gene losses occurred, leading to mtnr gene repertoires differing between the main gnathostome lineages. Genome tetraploidization events (1R, 2R, 3R, and 4R) are indicated in purple. Major gene gain and loss events as well as chromosome rearrangements are indicated in red boxes. Genes located on the same linkage group are represented by clusters on the genomic DNA line. Red crosses indicate gene loss. The red arc indicates genomic region fusion events. Colored tree branches represent mtnr gene lineages. Red star * on a tree branch indicates gene loss. The receptor identities are indicated in or beside the boxes. Paralogs originating from teleost 3R are designated by a and b suffixes, and salmonid 4R paralogs are designated by α and β suffixes.