Drastic neofunctionalization associated with evolution of the timezyme AANAT 500 Mya.

Melatonin (N-acetyl-5-methoxytrypamine) is the vertebrate hormone of the night: circulating levels at night are markedly higher than day levels. This increase is driven by precisely regulated increases in acetylation of serotonin in the pineal gland by arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the synthesis of melatonin. This unique essential role of AANAT in vertebrate timekeeping is recognized by the moniker the timezyme. AANAT is also found in the retina, where melatonin is thought to play a paracrine role. Here, we focused on the evolution of AANAT in early vertebrates. AANATs from Agnathans (lamprey) and Chondrichthyes (catshark and elephant shark) were cloned, and it was found that pineal glands and retinas from these groups express a form of AANAT that is compositionally, biochemically, and kinetically similar to AANATs found in bony vertebrates (VT-AANAT). Examination of the available genomes indicates that VT-AANAT is absent from other forms of life, including the Cephalochordate amphioxus. Phylogenetic analysis and evolutionary rate estimation indicate that VT-AANAT evolved from the nonvertebrate form of AANAT after the Cephalochordate-Vertebrate split over one-half billion years ago. The emergence of VT-AANAT apparently involved a dramatic acceleration of evolution that accompanied neofunctionalization after a duplication of the nonvertebrate AANAT gene. This scenario is consistent with the hypotheses that the advent of VT-AANAT contributed to the evolution of the pineal gland and lateral eyes from a common ancestral photodetector and that it was not a posthoc recruitment.

Somatotropic axis genes are expressed before pituitary onset during zebrafish and sea bass development.

The somatotropic axis, or growth hormone-insulin-like growth factor-1 (GH-IGF-1) axis, of fish is involved in numerous physiological process including regulation of ionic and osmotic balance, lipid, carbohydrate and protein metabolism, growth, reproduction, immune function and behavior. It is thought that GH plays a role in fish development but conflicting results have been obtained concerning the ontogeny of the somatotropic axis. Here we investigated the developmental expression of GH, GH-receptor (GHR) and IGF-1 genes and of a GH-like protein from fertilization until early stages of larval development in two Teleosts species, Danio rerio and Dicentrarchus labrax, by PCR, in situ hybridization and Western blotting. GH, GHR and IGF-1 mRNA were present in unfertilized eggs and at all stages of embryonic development, all three displaying a similar distribution in the two species. First located in the whole embryo (until 12 hpf in zebrafish and 76 hpf in sea bass), the mRNAs appeared then distributed in the head and tail, from where they disappeared progressively to concentrate in the forming pituitary gland. Proteins immunoreactive with a specific sea bass anti-GH antibody were also detected at all stages in this species. Differences in intensity and number of bands suggest that protein processing varies from early to later stages of development. The data show that all actors of the somatotropic axis are present from fertilization in these two species, suggesting they plays a role in early development, perhaps in an autocrine/paracrine mode as all three elements displayed a similar distribution at each stage investigated.

Evolution of AANAT: expansion of the gene family in the cephalochordate amphioxus.

BACKGROUND: The arylalkylamine N-acetyltransferase (AANAT) family is divided into structurally distinct vertebrate and non-vertebrate groups. Expression of vertebrate AANATs is limited primarily to the pineal gland and retina, where it plays a role in controlling the circadian rhythm in melatonin synthesis. Based on the role melatonin plays in biological timing, AANAT has been given the moniker "the Timezyme". Non-vertebrate AANATs, which occur in fungi and protists, are thought to play a role in detoxification and are not known to be associated with a specific tissue. RESULTS: We have found that the amphioxus genome contains seven AANATs, all having non-vertebrate type features. This and the absence of AANATs from the genomes of Hemichordates and Urochordates support the view that a major transition in the evolution of the AANATs may have occurred at the onset of vertebrate evolution. Analysis of the expression pattern of the two most structurally divergent AANATs in Branchiostoma lanceolatum (bl) revealed that they are expressed early in development and also in the adult at low levels throughout the body, possibly associated with the neural tube. Expression is clearly not exclusively associated with the proposed analogs of the pineal gland and retina. blAANAT activity is influenced by environmental lighting, but light/dark differences do not persist under constant light or constant dark conditions, indicating they are not circadian in nature. bfAANAT alpha and bfAANAT delta' have unusually alkaline (> 9.0) optimal pH, more than two pH units higher than that of vertebrate AANATs. CONCLUSIONS: The substrate selectivity profiles of bfAANAT alpha and delta' are relatively broad, including alkylamines, arylalkylamines and diamines, in contrast to vertebrate forms, which selectively acetylate serotonin and other arylalkylamines. Based on these features, it appears that amphioxus AANATs could play several roles, including detoxification and biogenic amine inactivation. The presence of seven AANATs in amphioxus genome supports the view that arylalkylamine and polyamine acetylation is important to the biology of this organism and that these genes evolved in response to specific pressures related to requirements for amine acetylation.

Melatonin effects on the hypothalamo-pituitary axis in fish.

Melatonin, a hormonal output signal of vertebrate circadian clocks, contributes to synchronizing behaviors and neuroendocrine regulations with the daily and annual variations of the photoperiod. Conservation and diversity characterize the melatonin system: conservation because its pattern of production and synchronizing properties are a constant among vertebrates; and diversity because regulation of both its synthesis and modes of action have been profoundly modified during vertebrate evolution. Studies of the targets and modes of action of melatonin in fish, and their parallels in mammals, are of interest to our understanding of time-related neuroendocrine regulation and its evolution from fish to mammals, as well as for aquacultural purposes.

Cloning and retinal expression of melatonin receptors in the European sea bass, Dicentrarchus labrax.

Melatonin contributes to synchronizing behaviors and physiological functions to daily and seasonal rhythm in fish. However, no coherent vision emerges because the effects vary with the species, sex, age, moment of the year or sexual cycle. And, scarce information is available concerning the melatonin receptors, which is crucial to our understanding of the role melatonin plays. We report here the full length cloning of three different melatonin receptor subtypes in the sea bass Dicentrarchus labrax, belonging, respectively, to the MT1, MT2 and Mel1c subtypes. MT1, the most abundantly expressed, was detected in the central nervous system, retina, and gills. MT2 was detected in the pituitary gland, blood cells and, to a lesser extend, in the optic tectum, diencephalon, liver and retina. Mel1c was mainly expressed in the skin; traces were found in the retina. The cellular sites of MT1 and MT2 expressions were investigated by in situ hybridization in the retina of pigmented and albino fish. The strongest signals were obtained with the MT1 riboprobes. Expression was seen in cells also known to express the enzymes of the melatonin biosynthesis, i.e., in the photoreceptor, inner nuclear and ganglion cell layers. MT1 receptor mRNAs were also abundant in the retinal pigment epithelium. The results are consistent with the idea that melatonin is an autocrine (neural retina) and paracrine (retinal pigment epithelium) regulator of retinal function. The molecular tools provided here will be of valuable interest to further investigate the targets and role of melatonin in nervous and peripheral tissues of fish.

Are We Reaching the Limits of Homo sapiens?

Echoing scientific and industrial progress, the Twentieth century was an unprecedented period of improvement for human capabilities and performances, with a significant increase in lifespan, adult height, and maximal physiological performance. Analyses of historical data show a major slow down occurring in the most recent years. This triggered large and passionate debates in the academic scene within multiple disciplines; as such an observation could be interpreted as our upper biological limits. Such a new phase of human history may be related to structural and functional limits determined by long term evolutionary constraints, and the interaction between complex systems and their environment. In this interdisciplinary approach, we call into question the validity of subsequent forecasts and projections through innovative and related biomarkers such as sport, lifespan, and height indicators. We set a theoretical framework based on biological and environmental relevance rather than using a typical single-variable forecasting approach. As demonstrated within the article, these new views will have major social, economical, and political implications.

Starting the zebrafish pineal circadian clock with a single photic transition.

The issue of what starts the circadian clock ticking was addressed by studying the developmental appearance of the daily rhythm in the expression of two genes in the zebrafish pineal gland that are part of the circadian clock system. One encodes the photopigment exorhodopsin and the other the melatonin synthesizing enzyme arylalkylamine N-acetyltransferase (AANAT2). Significant daily rhythms in AANAT2 mRNA abundance were detectable for several days after fertilization in animals maintained in a normal or reversed lighting cycle providing 12 h of light and 12 h of dark. In contrast, these rhythms do not develop if animals are maintained in constant lighting or constant darkness from fertilization. In contrast to exorhodopsin, rhythmicity of AANAT2 can be initiated by a pulse of light against a background of constant darkness, by a pulse of darkness against a background of constant lighting, or by single light-to-dark or dark-to-light transitions. Accordingly, these studies indicate that circadian clock function in the zebrafish pineal gland can be initiated by minimal photic cues, and that single photic transitions can be used as an experimental tool to dissect the mechanism that starts the circadian clock in the pineal gland.

Retinal, pineal and diencephalic expression of frog arylalkylamine N-acetyltransferase-1.

The arylalkylamine N-acetyltransferase (AANAT) is a key enzyme in the rhythmic production of melatonin. Two Aanats are expressed in Teleost fish (Aanat1 in the retina and Aanat2 in the pineal organ) but only Aanat1 is found in tetrapods. This study reports the cloning of Aanat1 from R. perezi. Transcripts were mainly expressed in the retina, diencephalon, intestine and testis. In the retina and pineal organ, Aanat1 expression was in the photoreceptor cells. Expression was also seen in ependymal cells of the 3rd ventricle and discrete cells of the suprachiasmatic area. The expression of Aanat1 in both the retina and pineal organ, and the absence of Aanat2 suggests that green frog resembles more to birds and mammals than to Teleost fish, as far as Aanat is concerned. The significance of Aanat1 in extra-pineal and extra-retinal tissues remains to be elucidated; in the diencephalon, it might be associated to the so-called deep brain photoreceptor cells.

Marine biodiversity characteristics.

Oceans contain the largest living volume of the "blue" planet, inhabited by approximately 235-250,000 described species, all groups included. They only represent some 13% of the known species on the Earth, but the marine biomasses are really huge. Marine phytoplankton alone represents half the production of organic matter on Earth while marine bacteria represent more than 10%. Life first appeared in the oceans more than 3.8 billion years ago and several determining events took place that changed the course of life, ranging from the development of the cell nucleus to sexual reproduction going through multi-cellular organisms and the capture of organelles. Of the 31 animal phyla currently listed, 12 are exclusively marine phyla and have never left the ocean. An interesting question is to try to understand why there are so few marine species versus land species? This pattern of distribution seems pretty recent in the course of Evolution. From an exclusively marine world, since the beginning until 440 million years ago, land number of species much increased 110 million years ago. Specific diversity and ancestral roles, in addition to organizational models and original behaviors, have made marine organisms excellent reservoirs for identifying and extracting molecules (>15,000 today) with pharmacological potential. They also make particularly relevant models for both fundamental and applied research. Some marine models have been the source of essential discoveries in life sciences. From this diversity, the ocean provides humankind with renewable resources, which are highly threatened today and need more adequate management to preserve ocean habitats, stocks and biodiversity.

Structural and functional evolution of the pineal melatonin system in vertebrates.

In most species daily rhythms are synchronized by the photoperiodic cycle. They are generated by the circadian system, which is made of a pacemaker, an entrainment pathway to this clock, and one or more output signals. In vertebrates, melatonin produced by the pineal organ is one of these outputs. The production of this time-keeping hormone is high at night and low during the day. Despite the fact that this is a well-preserved pattern, the pathways through which the photoperiodic information controls the rhythm have been profoundly modified from early vertebrates to mammals. The photoperiodic control is direct in fish and frogs and indirect in mammals. In the former, full circadian systems are found in photoreceptor cells of the pineal organ, retina, and possibly brain, thus forming a network where melatonin could be a hormonal synchronizer. In the latter, the three elements of a circadian system are scattered: the photoreceptive units are in the eyes, the clocks are in the suprachiasmatic nuclei of the hypothalamus, and the melatonin-producing units are in the pineal cells. Intermediate situations are observed in sauropsids. Differences are also seen at the level of the arylalkylamine N-acetyltransferase (AANAT), the enzyme responsible for the daily variations in melatonin production. In contrast to tetrapods, teleost fish AANATs are duplicated and display tissue-specific expression; also, pineal AANAT is special--it responds to temperature in a species-specific manner, which reflects the fish ecophysiological preferences. This review summarizes anatomical, structural, and molecular aspects of the evolution of the melatonin-producing system in vertebrates.

The timing of the shrew: continuous melatonin treatment maintains youthful rhythmic activity in aging Crocidura russula.

BACKGROUND: Laboratory conditions nullify the extrinsic factors that determine the wild expected lifespan and release the intrinsic or potential lifespan. Thus, wild animals reared in a laboratory often show an increased lifespan, and consequently an increased senescence phase. Senescence is associated with a broad suite of physiological changes, including a decreased responsiveness of the circadian system. The time-keeping hormone melatonin, an important chemical player in this system, is suspected to have an anti-aging role. The Greater White-toothed shrew Crocidura russula is an ideal study model to address questions related to aging and associated changes in biological functions: its lifespan is short and is substantially increased in captivity; daily and seasonal rhythms, while very marked the first year of life, are dramatically altered during the senescence process which starts during the second year. Here we report on an investigation of the effects of melatonin administration on locomotor activity of aging shrews. METHODOLOGY/PRINCIPAL FINDINGS: 1) The diel fluctuations of melatonin levels in young, adult and aging shrews were quantified in the pineal gland and plasma. In both, a marked diel rhythm (low diurnal concentration; high nocturnal concentration) was present in young animals but then decreased in adults, and, as a result of a loss in the nocturnal production, was absent in old animals. 2) Daily locomotor activity rhythm was monitored in pre-senescent animals that had received either a subcutaneous melatonin implant, an empty implant or no implant at all. In non-implanted and sham-implanted shrews, the rhythm was well marked in adults. A marked degradation in both period and amplitude, however, started after the age of 14-16 months. This pattern was considerably delayed in melatonin-implanted shrews who maintained the daily rhythm for significantly longer. CONCLUSIONS: This is the first long term study (>500 days observation of the same individuals) that investigates the effects of continuous melatonin delivery. As such, it sheds new light on the putative anti-aging role of melatonin by demonstrating that continuous melatonin administration delays the onset of senescence. In addition, the shrew appears to be a promising mammalian model for elucidating the precise relationships between melatonin and aging.

[Photoperiodic control of melatonin synthesis in fish pineal and retina]. / Contrôle photopériodique de la synthèse de mélatonine par la rétine et l'épiphyse de poisson.

Melatonin is the time-keeping molecule of vertebrates. The daily and annual variations of its rhythmic production allow synchronizing physiological functions and behaviours to the variations of the environment. In fish, melatonin is produced by the photoreceptor cells of the retina and pineal organ. It is also synthesized by other retinal cell types of the inner nuclear and ganglion cell layers. In most of the species investigated, the melatonin rhythm displays a high-at-night profile, resulting from the circadian control of the arylalkylamine N-acetyltranferase (AANAT) activity; AANAT is the penultimate enzyme in the melatonin biosynthesis pathway. Some fish species escape the high-at-night rule in the retina, and the rhythm displays a high-at-day profile, intermediate situations being sometimes observed. This review summarizes our current knowledge on the molecular and cellular mechanisms of the rhythmic control of production of an important circadian clock messenger, underlying their plasticity.

[Melatonin and neuroendocrine regulations in fish]. / Mélatonine et régulations neuroendocrines chez le poisson.

Melatonin is the time-keeping molecule of the organism. The production by the pineal organ is responsible for the diurnal and annual rhythms of plasma melatonin content. This contributes to synchronizing behavioural, biochemical and physiological processes to the environmental variations in photoperiod and temperature. Conservation and diversity characterize the melatonin system in vertebrates: conservation because its nocturnal pattern of production as well as its synchronizing properties are a constant; diversity because the modalities of its biosynthesis and modes of action have been profoundly modified in the course of evolution. This review summarizes our current knowledge on the targets and modes of action of melatonin in fish and comparisons are made with mammals.

Cloning and early expression pattern of two melatonin biosynthesis enzymes in the turbot (Scophthalmus maximus).

Melatonin biosynthesis from serotonin involves the sequential activation of the arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT). Photoperiod synchronizes a daily rhythm in pineal and retinal melatonin secretion through controlling AANAT activity. Teleost fish possess two Aanat, one expressed in the retina (AANAT1) and the other expressed in the pineal gland (AANAT2). We report here the full-length cloning of Aanat1, Aanat2, SmHiomt and Otx5 (orthodenticle homeobox homolog 5) in the turbot (Scophthalmus maximus, Sm), a flatfish belonging to an evolutionary recent group of Teleost. The temporal expression pattern of the genes investigated is consistent with the idea that OTX5 is needed for photoreceptor specification, and that the pineal gland differentiates before the retina. SmAanat2 expression remained pineal specific during the period of time investigated, whereas SmOtx5 and SmHiomt expressions were seen in both the retina and pineal gland. Our results do not support the existence of a second SmHiomt, as is the case for SmAanat. Neither SmAanat2 nor SmHiomt mRNAs displayed cyclic accumulation in the pineal organ of embryos and larvae maintained under a light-dark cycle from fertilization onward. This is in marked contrast with the situation observed with zebrafish Aanat2, indicating that the molecular mechanisms controlling the development of the pineal melatonin system have been modified during the evolution of Teleost.

Melatonin pathway: breaking the 'high-at-night' rule in trout retina.

Pineal melatonin synthesis increases at night in all vertebrates, due to an increase in the activity of arylalkylamine N-acetyltransferase (AANAT). Melatonin is also synthesized in the retina of some vertebrates and it is generally assumed that patterns of pineal and retinal AANAT activity and melatonin production are similar, i.e. they exhibit a high-at-night pattern. However, the situation in fish is atypical because in some cases retinal melatonin increases during the day, not the night. Consistent with this, we now report that light increases the activity and abundance of the AANAT expressed in trout retina, AANAT1, at a time when the activity and abundance of pineal AANAT, AANAT2, decreases. Likewise, exposure to darkness causes retinal AANAT protein and activity to decrease coincident with increases in the pineal gland. Rhythmic changes in retinal AANAT protein and activity are 180 degrees out of phase with those of retinal AANAT1 mRNA; all appear to be driven by environmental lighting, not by a circadian oscillator. The atypical high-during-the-day pattern of retinal AANAT1 activity may reflect an evolutionary adaptation that optimizes an autocrine/paracrine signaling role of melatonin in photoadaptation and phototransduction; alternatively, it might reflect an adaptation that broadens and enhances aromatic amine detoxification in the retina.

Preliminary observations on the spawning conditions of the European amphioxus (Branchiostoma lanceolatum) in captivity.

Members of the subphylum Cephalochordata, which include the genus Branchiostoma (i.e. amphioxus), represent the closest living invertebrate relatives of the vertebrates. To date, developmental studies have been carried out on three amphioxus species (the European Branchiostoma lanceolatum, the East Asian B. belcheri, and Floridian-Caribbean B. floridae). In most instances, adult animals have been collected from the field during their ripe season and allowed (or stimulated) to spawn in the laboratory. In any given year, dates of laboratory pawning have been limited by two factors. First, natural populations of these three most studied species of amphioxus are ripe, at most, for only a couple of months each year and, second, even when apparently ripe, animals spawn only at unpredictable intervals of every several days. This limited supply of living material hinders the development of amphioxus as a model system because this limitation makes it more difficult to work out protocols for new laboratory techniques. Therefore we are developing laboratory methods for increasing the number of amphioxus spawning dates per year. The present study found that a Mediterranean population of B. lanceolatum living near the Franco-Spanish border spawned naturally at the end of May and again at the end of June in 2003. Re-feeding experiments in the laboratory demonstrated that the gonads emptied at the end of May refilled with gametes by the end of June. We also found that animals with large gonads (both, obtained from the field and kept and fed at the laboratory during several weeks) could be induced to spawn in the laboratory out of phase with the field population if they were temperature shocked (spawning occurred 36 hours after a sustained increase in water temperature from 19 degrees C to 25 degrees C).