RESUMO
Light cues vary along the axis of periodicity, intensity and spectrum and perception of light is dependent on the photoreceptive capacity encoded within the genome and the opsins expressed. A global approach was taken to analyze the photoreceptive capacity and the effect of differing light conditions on a developing teleost prior to first feeding. The transcriptomes of embryos and alevins of Atlantic salmon (Salmo salar) exposed to different light conditions were analyzed, including a developmental series and a circadian profile. The results showed that genes mediating nonvisual photoreception are present prior to hatching when the retina is poorly differentiated. The clock genes were expressed early, but the circadian profile showed that only two clock genes were significantly cycling before first feeding. Few genes were differentially expressed between day and night within a light condition; however, many genes were significantly different between light conditions, indicating that light environment has an impact on the transcriptome during early development. Comparing the transcriptome data from constant conditions to periodicity of white light or different colors revealed overrepresentation of genes related to photoreception, eye development, muscle contraction, degradation of metabolites and cell cycle among others, and in constant light, several clock genes were upregulated. In constant white light and periodicity of green light, genes associated with DNA replication, chromatin remodeling, cell division and DNA repair were downregulated. The study implies a direct influence of light conditions on the transcriptome profile at early developmental stages, by a complex photoreceptive system where few clock genes are cycling.
Assuntos
Relógios Circadianos , Animais , Relógios Circadianos/genética , Fotoperíodo , Perfilação da Expressão Gênica , Transcriptoma/genética , Estágios do Ciclo de Vida , Ritmo Circadiano/genéticaRESUMO
In Atlantic salmon (Salmo salar), seasonal photoperiod is shown to regulate the onset of sexual maturation, yet which brain region(s) is involved, and how light information impacts the neuroendocrine system are still not fully understood in teleosts. Detailed knowledge about the photoperiodic regulation of maturation in fish is still missing. In birds, it is shown that gonadotropin-releasing hormone (Gnrh) is located in the same neurons as vertebrate ancient (VA) opsin, suggesting a direct photoreceptive regulation for the onset of sexual maturity. This study presents a comprehensive topographic mapping of gnrh2, gnrh3, kisspeptin 2 (kiss2), gonadotropin-inhibiting hormone (gnih), and VA opsin using in situ hybridization on mature Atlantic salmon brains. Neurons positive for gnrh3 are expressed in the olfactory bulb and ventral telencephalon, while gnrh2-positive neurons are located dorsally in the midbrain tegmentum. Gonadotropin-inhibiting hormone (Gnih)-expressing cell bodies are present in the ventral thalamus and extend caudally to the hypothalamus with kiss2-expressing cells appearing in a lateral position. VA opsin-positive cells are present in the telencephalon, the rostro-dorsal ring of the left habenula, the ventral thalamus, and the midbrain tegmentum. The results show no similar co-location as found in birds, hypothesizing that the photoreceptive modulation of Gnrh in salmon may interact through neuronal networks. The topography analyses of the essential neuroendocrine cells related to sexual maturation in the Atlantic salmon brain show that diencephalic (thalamus, hypothalamus) and midbrain (tegmentum) regions seem central for controlling sexual maturation.
Assuntos
Células Neuroendócrinas , Salmo salar , Animais , Opsinas/metabolismo , Salmo salar/metabolismo , Células Neuroendócrinas/metabolismo , Maturidade Sexual , Hormônio Liberador de Gonadotropina/metabolismo , Encéfalo/metabolismo , Gonadotropinas/metabolismoRESUMO
In nonmammalian vertebrates, photoreception takes place in the deep brain already early in development, but knowledge is lacking about the functions of these nonvisual photoreceptive systems. Prior to hatching, Atlantic halibut has a transient bilateral cluster of photoreceptive cells in the hindbrain. The cluster is imbedded in a neuronal network projecting to the narrow belt of hatching glands in the yolk sac. In halibut, hatching is inhibited in light and activated by transfer to darkness and c-fos analysis during hatching shows that the hindbrain cluster and hatching glands have neural activation. Unexpectedly, the hindbrain cluster expresses dual photopigments, vertebrate ancient opsin and melanopsin. Evolutionarily, these opsins are believed to belong to different classes of photopigments found in rhabdomeric and ciliary photoreceptors. The concept that an organism develops transient light sensitivity to target critical aspects of life history transitions as hatching provides a fascinating landscape to investigate the timing of other biological events.
Assuntos
Opsinas/metabolismo , Células Fotorreceptoras de Vertebrados/citologia , Opsinas de Bastonetes/metabolismo , Animais , Encéfalo/metabolismo , Linguado/embriologia , Larva/metabolismo , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras de Vertebrados/fisiologia , Retina/metabolismo , Rombencéfalo/metabolismo , Rombencéfalo/fisiologia , Vertebrados/metabolismoRESUMO
Unlike in mammals, persistent postembryonic retinal growth is a characteristic feature of fish, which includes major remodeling events that affect all cell types including photoreceptors. Consequently, visual capabilities change during development, where retinal sensitivity to different wavelengths of light (photopic vision), -and to limited photons (scotopic vision) are central capabilities for survival. Differently from well-established model fish, Atlantic cod has a prolonged larval stage where only cone photoreceptors are present. Rods do not appear until juvenile transition (metamorphosis), a hallmark of indirect developing species. Previously we showed that whole gene families of lws (red-sensitive) and sws1 (UV-sensitive) opsins have been lost in cod, while rh2a (green-sensitive) and sws2 (blue-sensitive) genes have tandem duplicated. Here, we provide a comprehensive characterization of a two-step developing duplex retina in Atlantic cod. The study focuses on cone subtype dynamics and delayed rod neurogenesis and differentiation in all cod life stages. Using transcriptomic and histological approaches we show that different opsins disappear in a topographic manner during development where central to peripheral retina is a key axis of expressional change. Early cone differentiation was initiated in dorso-temporal retina different from previously described in fish. Rods first appeared during initiation of metamorphosis and expression of the nuclear receptor transcription factor nr2e3-1, suggest involvement in rod specification. The indirect developmental strategy thus allows for separate studies of cones and rods development, which in nature correlates with visual changes linked to habitat shifts. The clustering of key retinal genes according to life stage, suggests that Atlantic cod with its sequenced genome may be an important resource for identification of underlying factors required for development and function of photopic and scotopic vision.
Assuntos
Gadus morhua/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese , Retina/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Bastonetes/citologia , Animais , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Gadus morhua/embriologia , Gadus morhua/genética , Duplicação Gênica , Larva , Estágios do Ciclo de Vida , Metamorfose Biológica , Opsinas/genética , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Retina/citologia , Retina/embriologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcriptoma , Visão OcularRESUMO
The melanocortin system is a key regulator of appetite and food intake in vertebrates. This system includes the neuropeptides neuropeptide y (NPY), agouti-related peptide (AGRP), cocaine- and amphetamine-regulated transcript (CART), and pro-opiomelanocortin (POMC). An important center for appetite control in mammals is the hypothalamic arcuate nucleus, with neurons that coexpress either the orexigenic NPY/AGRP or the anorexigenic CART/POMC neuropeptides. In ray-finned fishes, such a center is less characterized. The Atlantic salmon (Salmo salar) has multiple genes of these neuropeptides due to whole-genome duplication events. To better understand the potential involvement of the melanocortin system in appetite and food intake control, we have mapped the mRNA expression of npy, agrp, cart, and pomc in the brain of Atlantic salmon parr using in situ hybridization. After identifying hypothalamic mRNA expression, we investigated the possible intracellular coexpression of npy/agrp and cart/pomc in the tuberal hypothalamus by fluorescent in situ hybridization. The results showed that the neuropeptides were widely distributed, especially in sensory and neuroendocrine brain regions. In the hypothalamic lateral tuberal nucleus, the putative homolog to the mammalian arcuate nucleus, npya, agrp1, cart2b, and pomca were predominantly localized in distinct neurons; however, some neurons coexpressed cart2b/pomca. This is the first demonstration of coexpression of cart2b/pomca in the tuberal hypothalamus of a teleost. Collectively, our data suggest that the lateral tuberal nucleus is the center for appetite control in salmon, similar to that of mammals. Extrahypothalamic brain regions might also be involved in regulating food intake, including the olfactory bulb, telencephalon, midbrain, and hindbrain.
Assuntos
Neuropeptídeos , Salmo salar , Animais , Proteína Relacionada com Agouti/genética , Proteína Relacionada com Agouti/metabolismo , Pró-Opiomelanocortina/metabolismo , Salmo salar/genética , Salmo salar/metabolismo , Melanocortinas/genética , Melanocortinas/metabolismo , Hibridização in Situ Fluorescente , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Neuropeptídeo Y/genética , Neuropeptídeo Y/metabolismo , Hipotálamo/metabolismo , Encéfalo/metabolismo , RNA Mensageiro/metabolismo , MamíferosRESUMO
During the first feeding period, fish will adapt to exogenous feeding as their endogenous source of nutrients is depleted. This requires the development of a functional physiological system to control active search for food, appetite, and food intake. The Atlantic salmon (Salmo salar) melanocortin system, a key player in appetite control, includes neuronal circuits expressing neuropeptide y (npya), agouti-related peptide (agrp1), cocaine- and amphetamine-regulated transcript (cart), and proopiomelanocortin (pomca). Little is known about the ontogeny and function of the melanocortin system during early developmental stages. Atlantic salmon [0-730 day degrees (dd)] were reared under three different light conditions (DD, continuous darkness; LD, 14:10 Light: Dark; LL, continuous light) before the light was switched to LD and the fish fed twice a day. We examined the effects of different light conditions (DD LD , LD LD , and LL LD ) on salmon growth, yolk utilization, and periprandial responses of the neuropeptides npya1, npya2, agrp1, cart2a, cart2b, cart4, pomca1, and pomca2. Fish were collected 1 week (alevins, 830 dd, still containing yolk sac) and 3 weeks (fry, 991 dd, yolk sac fully consumed) into the first feeding period and sampled before (-1 h) and after (0.5, 1.5, 3, and 6 h) the first meal of the day. Atlantic salmon reared under DD LD , LD LD , and LL LD had similar standard lengths and myotome heights at the onset of first feeding. However, salmon kept under a constant light condition during endogenous feeding (DD LD and LL LD ) had less yolk at first feeding. At 830 dd none of the neuropeptides analyzed displayed a periprandial response. But 2 weeks later, and with no yolk remaining, significant periprandial changes were observed for npya1, pomca1, and pomca2, but only in the LD LD fish. This suggests that these key neuropeptides serve an important role in controlling feeding once Atlantic salmon need to rely entirely on active search and ingestion of exogenous food. Moreover, light conditions during early development did not affect the size of salmon at first feeding but did affect the mRNA levels of npya1, pomca1, and pomca2 in the brain indicating that mimicking natural light conditions (LD LD ) better stimulates appetite control.
RESUMO
Salmonids are ideal models as many species follow a distinct developmental program from demersal eggs and a large yolk sac to hatching at an advanced developmental stage. Further, these economically important teleosts inhabit both marine- and freshwaters and experience diverse light environments during their life histories. At a genome level, salmonids have undergone a salmonid-specific fourth whole genome duplication event (Ss4R) compared to other teleosts that are already more genetically diverse compared to many non-teleost vertebrates. Thus, salmonids display phenotypically plastic visual systems that appear to be closely related to their anadromous migration patterns. This is most likely due to a complex interplay between their larger, more gene-rich genomes and broad spectrally enriched habitats; however, the molecular basis and functional consequences for such diversity is not fully understood. This study used advances in genome sequencing to identify the repertoire and genome organization of visual opsin genes (those primarily expressed in retinal photoreceptors) from six different salmonids [Atlantic salmon (Salmo salar), brown trout (Salmo trutta), Chinook salmon (Oncorhynchus tshawytcha), coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss), and sockeye salmon (Oncorhynchus nerka)] compared to the northern pike (Esox lucius), a closely related non-salmonid species. Results identified multiple orthologues for all five visual opsin classes, except for presence of a single short-wavelength-sensitive-2 opsin gene. Several visual opsin genes were not retained after the Ss4R duplication event, which is consistent with the concept of salmonid rediploidization. Developmentally, transcriptomic analyzes of Atlantic salmon revealed differential expression within each opsin class, with two of the long-wavelength-sensitive opsins not being expressed before first feeding. Also, early opsin expression in the retina was located centrally, expanding dorsally and ventrally as eye development progressed, with rod opsin being the dominant visual opsin post-hatching. Modeling by spectral tuning analysis and atomistic molecular simulation, predicted the greatest variation in the spectral peak of absorbance to be within the Rh2 class, with a â¼40 nm difference in λ max values between the four medium-wavelength-sensitive photopigments. Overall, it appears that opsin duplication and expression, and their respective spectral tuning profiles, evolved to maximize specialist color vision throughout an anadromous lifecycle, with some visual opsin genes being lost to tailor marine-based vision.
RESUMO
To better understand the complexity of clock genes in salmonids, a taxon with an additional whole genome duplication, an analysis was performed to identify and classify gene family members (clock, arntl, period, cryptochrome, nr1d, ror, and csnk1). The majority of clock genes, in zebrafish and Northern pike, appeared to be duplicated. In comparison to the 29 clock genes described in zebrafish, 48 clock genes were discovered in salmonid species. There was also evidence of species-specific reciprocal gene losses conserved to the Oncorhynchus sister clade. From the six period genes identified three were highly significantly rhythmic, and circadian in their expression patterns (per1a.1, per1a.2, per1b) and two was significantly rhythmically expressed (per2a, per2b). The transcriptomic study of juvenile Atlantic salmon (parr) brain tissues confirmed gene identification and revealed that there were 2,864 rhythmically expressed genes (p < 0.001), including 1,215 genes with a circadian expression pattern, of which 11 were clock genes. The majority of circadian expressed genes peaked 2 h before and after daylight. These findings provide a foundation for further research into the function of clock genes circadian rhythmicity and the role of an enriched number of clock genes relating to seasonal driven life history in salmonids.
RESUMO
Photoreceptive inputs to the teleost brain are perceived as image of the visual world and as photo-modulation of neuroendocrine and neuronal signals. The retina and pineal organ are major receptive organs with projections to various parts of the brain, but in the past decades deep brain photoreceptors have emerged as candidates for photoreceptive inputs, either independent or in combination with projections from light sensory organs. This study aimed to test the effects of narrow bandwidth light using light-emitting diodes technology on brain neural activity through putative opsin stimulation in Atlantic salmon. The expression of c-fos, a known marker of neural activity, was compared in situ between dark-adapted salmon parr and following light stimulation with different wavelengths. c-fos expression increased with duration of light stimulation and the strongest signal was obtained in fish exposed to light for 120 minutes. Distinct and specific brain regions were activated following dark to light stimulation, such as the habenula, suprachiasmatic nucleus, thalamus, and hypothalamus. The c-fos expression was overlapping with photoreceptors expressing melanopsin and/or vertebrate ancient opsin, suggesting a potential direct activation by light. Interestingly in the habenula, a distinct ring of vertebrate ancient opsin and melanopsin expressing cells is overlapping with c-fos expression after neural activation. Salmon exposed to different spectra had neural activation in similar brain regions. The most apparent difference was melanopsin expression in the lateral cells of the lateral tuberal nuclus in the hypothalamus, which appeared to be specifically activated by red light. Light-stimulated neuronal activity in the deep brain was limited to subpopulations of neurons, mainly in regions with neuronal modulation activity, retinal and pineal innervations and known presence of nonvisual photoreceptors. The overlapping expression patterns of c-fos and nonvisual opsins support direct light stimulation of deep brain photoreceptors and the importance of these systems in light induced brain activity.
Assuntos
Encéfalo/metabolismo , Neurônios/metabolismo , Células Fotorreceptoras/metabolismo , Animais , Estimulação Luminosa , Proteínas Proto-Oncogênicas c-fos/metabolismo , Opsinas de Bastonetes/metabolismo , Salmo salarRESUMO
While it is well known that the notochord of bony fishes changes over developmental time, less is known about how it varies across different body regions. In the development of the Atlantic salmon, Salmo salar L., cranial and caudal ends of the notochord are overlaid by the formation of the bony elements of the neurocranium and caudal fin, respectively. To investigate, we describe how the notochord of the cranium and caudal fin changes from embryo to spawning adult, using light microscopy, SEM, TEM, dissection, and CT scanning. The differences are dramatic. In contrast to the abdominal and caudal regions, at the ends of the notochord vertebrae never develop. While the cranial notochord builds a tapering, unsegmented cone of chordal bone, the urostylic notochordal sheath never ossifies: adjacent, irregular bony elements form from the endoskeleton of the caudal fin. As development progresses, two previously undescribed processes occur. First, the bony cone of the cranial notochord, and its internal chordocytes, are degraded by chordoclasts, an undescribed function of the clastic cell type. Second, the sheath of the urostylic notochord creates transverse septae that partly traverse the lumen in an irregular pattern. By the adult stage, the cranial notochord is gone. In contrast, the urostylic notochord in adults is robust, reinforced with septae, covered by irregularly shaped pieces of cellular bone, and capped with an opistural cartilage that develops from the sheath of the urostylic notochord. A previously undescribed muscle, with its origin on the opistural cartilage, inserts on the lepidotrich ventral to it.
Assuntos
Nadadeiras de Animais/embriologia , Notocorda/embriologia , Salmo salar/embriologia , Crânio/embriologia , Nadadeiras de Animais/crescimento & desenvolvimento , Animais , Notocorda/crescimento & desenvolvimento , Salmo salar/crescimento & desenvolvimento , Crânio/crescimento & desenvolvimentoRESUMO
The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that mediates the toxicity of dioxins and dioxin-like compounds (DLCs) in vertebrates. Two clades of the Ahr family exist in teleosts (Ahr1 and Ahr2), and it has been demonstrated that Ahr2 is the main protein involved in mediating the toxicity of dioxins and DLCs in most teleost species. Recently, we characterized the Atlantic cod (Gadus morhua) Ahr1a and Ahr2a receptors. To further explore a possible subfunction partitioning of Ahr1a and Ahr2a in Atlantic cod we have mapped the expression and localization of ahr1a and ahr2a in early developmental stages. Atlantic cod embryos were continuously exposed in a passive-dosing exposure system to the Ahr agonist, benzo[a]pyrene (B[a]P), from five days post fertilization (dpf) until three days post hatching (dph). Expression of ahr1a, ahr2a, and the Ahr-target genes, cyp1a and ahrrb, was assessed in embryos (8 dpf and 10 dpf) and larvae (3 dph) with quantitative real-time PCR analyses (qPCR), while in situ hybridization was used to assess the localization of expression of ahr1a, ahr2a and cyp1a. Quantitative measurements showed an increased cyp1a expression in B[a]P-exposed samples at all sampling points, and for ahr2a at 10 dpf, confirming the activation of the Ahr-signalling pathway. Furthermore, B[a]P strongly induced ahr2a and cyp1a expression in the cardiovascular system and skin, respectively, of embryos and larvae. Induced expression of both ahr2a and cyp1a was also revealed in the liver of B[a]P-exposed larvae. Our results suggest that Ahr2a is the major subtype involved in mediating responses to B[a]P in early developmental stages of Atlantic cod, which involves transcriptional regulation of biotransformation genes, such as cyp1a. The focused expression of ahr1a in the eye of embryos and larvae, and the presence of ahr2a transcripts in the jaws and fin nodes, further indicate evolved specialized roles of the two Ahrs in ontogenesis.
Assuntos
Citocromo P-450 CYP1A1/metabolismo , Gadus morhua/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Benzo(a)pireno/toxicidade , Citocromo P-450 CYP1A1/genética , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Olho/efeitos dos fármacos , Olho/embriologia , Olho/metabolismo , Gadus morhua/genética , Gadus morhua/metabolismo , Larva/efeitos dos fármacos , Larva/genética , Fígado/efeitos dos fármacos , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Poluentes Químicos da Água/toxicidadeRESUMO
The complexity of the nonvisual photoreception systems in teleosts has just started to be appreciated, with colocalization of multiple photoreceptor types with unresolved functions. Here we describe an intricate expression pattern of melanopsins in early life stages of the marine flat fish Atlantic halibut (Hippoglossus hippoglossus), a period when the unpigmented brain is directly exposed to environmental photons. We show a refined and extensive expression of melanopsins in the halibut brain already at the time of hatching, long before the eyes are functional. We detect melanopsin in the habenula, suprachiasmatic nucleus, dorsal thalamus, and lateral tubular nucleus of first feeding larvae, suggesting conserved functions of the melanopsins in marine teleosts. The complex expression of melanopsins already at larval stages indicates the importance of nonvisual photoreception early in development. Most strikingly, we detect expression of both exorhodopsin and melanopsin in the pineal complex of halibut larvae. Double-fluorescence labeling showed that two clusters of melanopsin-positive cells are located lateral to the central rosette of exorhodopsin-positive cells. The localization of different photopigments in the pineal complex suggests that two parallel photoreceptor systems may be active. Furthermore, the dispersed melanopsin-positive cells in the spinal cord of halibut larvae at the time of hatching may be primary sensory cells or interneurons representing the first example of dispersed high-order photoreceptor cells. The appearance of nonvisual opsins early in the development of halibut provides an alternative model for studying the evolution and functional significance of nonvisual opsins.