Functional or vestigial? The genomics of the pineal gland in Xenarthra

Vestigial organs are historical echoes of past phenotypes. Determining whether a specific organ constitutes a functional or vestigial structure can be a challenging task, given that distinct levels of atrophy may arise between and within lineages. The mammalian pineal gland, an endocrine organ involved in melatonin biorhythmicity, represents a classic example, often yielding contradicting anatomical observations. In Xenarthra (sloths, anteaters and armadillos), a peculiar mammalian order, the presence of a distinct pineal organ was clearly observed in some species (i.e. Linnaeus's two-toed sloth) but undetected in other closely related species (i.e. brown-throated sloth). In the nine-banded armadillo, contradicting evidence supports either functional or vestigial scenarios. Thus, to untangle the physiological status of the pineal gland in Xenarthra, we used a comparative genomics approach to investigate the evolution of the gene hub responsible for melatonin synthesis and signaling. We show that both synthesis and signaling compartments are eroded and were lost independently. Additionally, by expanding our analysis to 157 mammal genomes we offer a comprehensive view showing that species with very distinctive habitats and lifestyles have convergently evolved a similar phenotype: Cetacea, Pholidota, Dermoptera, Sirenia and Xenarthra. Our findings suggest that the recurrent inactivation of melatonin genes correlates with pineal atrophy, and endorse the use of genomic analyses to ascertain the physiological status of suspected vestigial structures.

Differential role of cAMP, cGMP and Ca2+ and involvement of kinases and CBP-CREB-CRE pathway in regulation of arylalkylamine N-acetyltransferase 2 gene in the pineal organ of air-breathing catfish, Clarias gariepinus

Transcription of arylalkylamine N-acetyltransferase 2 (aanat2) gene leads to formation of AANAT2 - the rate-limiting enzyme in melatonin synthesis pathway in photosensitive fish pineal organ. However, unlike in avian and mammalian pineal gland, there is practically no information on signal transduction pathway(s) involved in regulation of aanat2 gene transcription in the fish pineal organ. Therefore, we investigated the role of important molecular components of signalling via cAMP, cGMP, Ca2+ involving PKA, PKG, PKC, MeK and p38 MAP kinase as well as possible role of serine/threonine phosphatases, CREB and CBP using their specific inhibitors and/or activators in aanat2 gene transcription in the fish pineal organ maintained under in vitro culture-conditions. db-cAMP and db-cGMP stimulated the expression of aanat2 gene. db-cAMP- and cGMP-induced aanat2 gene expression was significantly reduced in the presence of H-89 (specific inhibitor of PKA), KT5823 (specific inhibitor of PKG), chelerythrine chloride (specific inhibitor of PKC), U0126 ethanolate (specific inhibitor of MeK) and SB 202190 monohydrochloride hydrate (specific inhibitor of p38 MAP kinase). Inhibitors of PP1 and PP2A significantly increased aanat2 gene expression as well as significantly reduced cAMP- and cGMP-induced gene transcription, while inhibitor of PP2B had no effect on aanat2 gene expression. Inhibitors of both CREB and CBP-CREB interaction completely blocked cAMP-induced aanat2 gene transcription. Based on these findings, we suggest that cAMP, cGMP and Ca2+ stimulate aanat2 gene transcription via PKA, PKG and PKC, respectively. Further, protein phosphatases and CBP-CREB-CRE pathway are actively involved in regulation of on aanat2 gene expression in the fish pineal organ.

Metabolism of Melatonin Synthesis-Related Indoles in the Turkey Pineal Organ and Its Modification by Monochromatic Light

The metabolism of pineal indoles is closely related to alterations in the light and dark phases of a daily cycle. Recent research showed important interspecies differences in the pineal biochemistry, and a strong impact of monochromatic light on many physiological processes in birds. Therefore, the aims of study were to characterize the metabolism of melatonin-synthesis indoles in the pineal organ of the domestic turkey, and to determine the changes occurring in this metabolism under the influence of different wavelengths and intensities of light. For this purpose, 3-week-old turkeys were kept under 16 lx white light, or under blue, green, and red light with intensities of 16, 32, and 64 lx during the photophase, and after 7 d were sacrificed at 4 h intervals. The activities of melatonin-synthesizing enzymes and the contents of indoles were measured in the same pineal organ. The results revealed that the activities of tryptophan hydroxylase and arylalkylamine N-acetyltransferase, and the levels of all tryptophan derivatives had significant daily changes in birds kept under each light condition used. The profile of pineal indole metabolism in 4-week-old turkeys was characterized by high-amplitude rhythms in the activity of arylalkylamine N-acetyltransferase and the contents of N-acetylserotonin and melatonin, equal relative amounts of serotonin and 5-hydroxyindoleacetic acid, and higher content of melatonin than N-acetylserotonin. The monochromatic light significantly modified the pineal indole metabolism, and its effects were dependent on the color and intensity of light. Pronounced changes occurred in the level of serotonin synthesis and the daily rhythm course of melatonin synthesis.

Roles of Direct Photoreception and the Internal Circadian Oscillator in the Regulation of Melatonin Secretion in the Pineal Organ of the Domestic Turkey: A Novel In Vitro Clock and Calendar Model

The regulation of melatonin secretion in the avian pineal organ is highly complex and shows prominent interspecies differences. The aim of this study was to determine the roles of direct photoreception and the internal oscillator in the regulation of melatonin secretion in the pineal organ of the domestic turkey. The pineal organs were collected from 12-, 13- and 14-week-old female turkeys reared under a 12 L:12 D cycle with the photophase from 07.00 to 19.00, and were incubated in superfusion culture for 3–6 days. The cultures were subjected to different light conditions including 12 L:12 D cycles with photophases between 07.00 and 19.00, 13.00 and 01.00 or 01.00 and 13.00, a reversed cycle 12 D:12 L, cycles with long (16 L:8 D) and short (8 L:16 D) photophases, and continuous darkness or illumination. The pineal organs were also exposed to light pulses of variable duration during incubation in darkness or to periods of darkness during the photophase. The secretion of melatonin was determined by direct radioimmunoassay. The turkey pineal organs secreted melatonin in a well-entrained diurnal rhythm with a very high amplitude. Direct photoreception as an independently acting mechanism was able to ensure quick and precise adaptation of the melatonin secretion rhythm to changes in light-dark conditions. The pineal organs secreted melatonin in circadian rhythms during incubation in continuous darkness or illumination. The endogenous oscillator of turkey pinealocytes was able to acquire and store information about the light-dark cycle and then to generate the circadian rhythm of melatonin secretion in continuous darkness according to the stored data. The obtained data suggest that the turkey pineal gland is highly autonomous in the generation and regulation of the melatonin secretion rhythm. They also demonstrate that the turkey pineal organ in superfusion culture is a valuable model for chronobiological studies, providing a highly precise clock and calendar. This system has several features which make it an attractive alternative to other avian pineal glands for circadian studies.