RESUMO
BACKGROUND: Scales are mineralised exoskeletal structures that are part of the dermal skeleton. Scales have been mostly lost during evolution of terrestrial vertebrates whilst bony fish have retained a mineralised dermal skeleton in the form of fin rays and scales. Each scale is a mineralised collagen plate that is decorated with both matrix-building and resorbing cells. When removed, an ontogenetic scale is quickly replaced following differentiation of the scale pocket-lining cells that regenerate a scale. Processes promoting de novo matrix formation and mineralisation initiated during scale regeneration are poorly understood. Therefore, we performed transcriptomic analysis to determine gene networks and their pathways involved in dermal scale regeneration. RESULTS: We defined the transcriptomic profiles of ontogenetic and regenerating scales of zebrafish and identified 604 differentially expressed genes (DEGs). These were enriched for extracellular matrix, ossification, and cell adhesion pathways, but not in enamel or dentin formation processes indicating that scales are reminiscent to bone. Hypergeometric tests involving monogenetic skeletal disorders showed that DEGs were strongly enriched for human orthologues that are mutated in low bone mass and abnormal bone mineralisation diseases (P< 2× 10-3). The DEGs were also enriched for human orthologues associated with polygenetic skeletal traits, including height (P< 6× 10-4), and estimated bone mineral density (eBMD, P< 2× 10-5). Zebrafish mutants of two human orthologues that were robustly associated with height (COL11A2, P=6× 10-24) or eBMD (SPP1, P=6× 10-20) showed both exo- and endo- skeletal abnormalities as predicted by our genetic association analyses; col11a2Y228X/Y228X mutants showed exoskeletal and endoskeletal features consistent with abnormal growth, whereas spp1P160X/P160X mutants predominantly showed mineralisation defects. CONCLUSION: We show that scales have a strong osteogenic expression profile comparable to other elements of the dermal skeleton, enriched in genes that favour collagen matrix growth. Despite the many differences between scale and endoskeletal developmental processes, we also show that zebrafish scales express an evolutionarily conserved sub-population of genes that are relevant to human skeletal disease.
Assuntos
Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Perfilação da Expressão Gênica , Humanos , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Sea bream (Sparus aurata Linneaus) was acclimated to three salinity concentrations, viz. 5 (LSW), 38 (SW) and 55psµ (HSW) and three water temperatures regimes (12, 19 and 26 °C) for five weeks. Osmoregulatory capacity parameters (plasma osmolality, sodium, chloride, cortisol, and branchial and renal Na+,K+-ATPase activities) were also assessed. Salinity and temperature affected all of the parameters tested. Our results indicate that environmental temperature modulates capacity in sea bream, independent of environmental salinity, and set points of plasma osmolality and ion concentrations depend on both ambient salinity and temperature. Acclimation to extreme salinity resulted in stress, indicated by elevated basal plasma cortisol levels. Response to salinity was affected by ambient temperature. A comparison between branchial and renal Na+,K+-ATPase activities appears instrumental in explaining salinity and temperature responses. Sea bream regulate branchial enzyme copy numbers (Vmax) in hyperosmotic media (SW and HSW) to deal with ambient temperature effects on activity; combinations of high temperatures and salinity may exceed the adaptive capacity of sea bream. Salinity compromises the branchial enzyme capacity (compared to basal activity at a set salinity) when temperature is elevated and the scope for temperature adaptation becomes smaller at increasing salinity. Renal Na+,K+-ATPase capacity appears fixed and activity appears to be determined by temperature.
Assuntos
Osmorregulação/fisiologia , Salinidade , Dourada/fisiologia , Temperatura , Animais , Cloretos , Brânquias/metabolismo , Hidrocortisona/sangue , Rim/metabolismo , Dourada/sangue , Sódio , ATPase Trocadora de Sódio-Potássio/metabolismo , ÁguaRESUMO
Ciliopathies are Mendelian disorders caused by dysfunction of cilia, ubiquitous organelles involved in fluid propulsion (motile cilia) or signal transduction (primary cilia). Retinal dystrophy is a common phenotypic characteristic of ciliopathies since photoreceptor outer segments are specialized primary cilia. These ciliary structures heavily rely on intracellular minus-end directed transport of cargo, mediated at least in part by the cytoplasmic dynein 1 motor complex, for their formation, maintenance and function. Ninein-like protein (NINL) is known to associate with this motor complex and is an important interaction partner of the ciliopathy-associated proteins lebercilin, USH2A and CC2D2A. Here, we scrutinize the function of NINL with combined proteomic and zebrafish in vivo approaches. We identify Double Zinc Ribbon and Ankyrin Repeat domains 1 (DZANK1) as a novel interaction partner of NINL and show that loss of Ninl, Dzank1 or both synergistically leads to dysmorphic photoreceptor outer segments, accumulation of trans-Golgi-derived vesicles and mislocalization of Rhodopsin and Ush2a in zebrafish. In addition, retrograde melanosome transport is severely impaired in zebrafish lacking Ninl or Dzank1. We further demonstrate that NINL and DZANK1 are essential for intracellular dynein-based transport by associating with complementary subunits of the cytoplasmic dynein 1 motor complex, thus shedding light on the structure and stoichiometry of this important motor complex. Altogether, our results support a model in which the NINL-DZANK1 protein module is involved in the proper assembly and folding of the cytoplasmic dynein 1 motor complex in photoreceptor cells, a process essential for outer segment formation and function.
Assuntos
Proteínas de Transporte/genética , Dineínas/genética , Larva/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Células Fotorreceptoras de Vertebrados , Retina/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Animais , Transporte Biológico/genética , Cílios/genética , Células HEK293 , Humanos , Larva/crescimento & desenvolvimento , Neurogênese/genética , Proteômica , Transdução de Sinais , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Despite the use of fish models to study human mental disorders and dysfunctions, knowledge of regional telencephalic responses in non-mammalian vertebrates expressing alternative stress coping styles is poor. As perception of salient stimuli associated with stress coping in mammals is mainly under forebrain limbic control, we tested region-specific forebrain neural (i.e. mRNA abundance and monoamine neurochemistry) and endocrine responses under basal and acute stress conditions for previously characterised proactive and reactive Atlantic salmon. Reactive fish showed a higher degree of the neurogenesis marker proliferating cell nuclear antigen (pcna) and dopamine activity under basal conditions in the proposed hippocampus homologue (Dl) and higher post-stress plasma cortisol levels. Proactive fish displayed higher post-stress serotonergic signalling (i.e. higher serotonergic activity and expression of the 5-HT1A receptor) in the proposed amygdala homologue (Dm), increased expression of the neuroplasticity marker brain-derived neurotropic factor (bdnf) in both Dl and the lateral septum homologue (Vv), as well as increased expression of the corticotropin releasing factor 1 (crf1 ) receptor in the Dl, in line with active coping neuro-profiles reported in the mammalian literature. We present novel evidence of proposed functional equivalences in the fish forebrain with mammalian limbic structures.
Assuntos
Prosencéfalo/fisiologia , Salmo salar/fisiologia , Estresse Fisiológico , Migração Animal , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Hidrocortisona/sangue , Neurogênese , Plasticidade Neuronal , Oxigênio/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Mensageiro/genética , Receptor 5-HT1A de Serotonina/genética , Receptor 5-HT1A de Serotonina/metabolismo , Salmo salar/sangue , Transcrição GênicaRESUMO
Intellectual disability and seizures are frequently associated with hypomagnesemia and have an important genetic component. However, to find the genetic origin of intellectual disability and seizures often remains challenging because of considerable genetic heterogeneity and clinical variability. In this study, we have identified new mutations in CNNM2 in five families suffering from mental retardation, seizures, and hypomagnesemia. For the first time, a recessive mode of inheritance of CNNM2 mutations was observed. Importantly, patients with recessive CNNM2 mutations suffer from brain malformations and severe intellectual disability. Additionally, three patients with moderate mental disability were shown to carry de novo heterozygous missense mutations in the CNNM2 gene. To elucidate the physiological role of CNNM2 and explain the pathomechanisms of disease, we studied CNNM2 function combining in vitro activity assays and the zebrafish knockdown model system. Using stable Mg(2+) isotopes, we demonstrated that CNNM2 increases cellular Mg2+ uptake in HEK293 cells and that this process occurs through regulation of the Mg(2+)-permeable cation channel TRPM7. In contrast, cells expressing mutated CNNM2 proteins did not show increased Mg(2+) uptake. Knockdown of cnnm2 isoforms in zebrafish resulted in disturbed brain development including neurodevelopmental impairments such as increased embryonic spontaneous contractions and weak touch-evoked escape behaviour, and reduced body Mg content, indicative of impaired renal Mg(2+) absorption. These phenotypes were rescued by injection of mammalian wild-type Cnnm2 cRNA, whereas mammalian mutant Cnnm2 cRNA did not improve the zebrafish knockdown phenotypes. We therefore concluded that CNNM2 is fundamental for brain development, neurological functioning and Mg(2+) homeostasis. By establishing the loss-of-function zebrafish model for CNNM2 genetic disease, we provide a unique system for testing therapeutic drugs targeting CNNM2 and for monitoring their effects on the brain and kidney phenotype.
Assuntos
Encéfalo/metabolismo , Ciclinas/genética , Deficiência Intelectual/genética , Magnésio/metabolismo , Mutação de Sentido Incorreto/genética , Convulsões/genética , Adolescente , Animais , Proteínas de Transporte de Cátions , Linhagem Celular , Feminino , Células HEK293 , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/metabolismo , Rim/metabolismo , Masculino , Fenótipo , Convulsões/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
When Atlantic salmon parr migrate from fresh water towards the sea, they undergo extensive morphological, neural, physiological and behavioural changes. Such changes have the potential to affect their responsiveness to various environmental factors that impose stress. In this study we compared the stress responses in parr and post-smolt salmon following exposure to repeated chasing stress (RCS) for three weeks. At the end of this period, all fish were challenged with a novel stressor and sampled before (T0) and after 1h (T1). Parr had a higher growth rate than post-smolts. Plasma cortisol declined in the RCS groups within the first week suggesting a rapid habituation/desensitisation of the endocrine stress axis. As a result of the desensitised HPI axis, RCS groups showed a reduced cortisol response when exposed to the novel stressor. In preoptic area (POA) crf mRNA levels were higher in all post-smolt groups compared to parr. 11ßhsd2 decreased by RCS and by the novel stressor in post-smolt controls (T1), whereas no effect of either stress was seen in parr. The grs were low in all groups except for parr controls. In pituitary, parr controls had higher levels of crf1r mRNA than the other parr and post-smolt groups, whilst pomcb was higher in post-smolt control groups. Overall, 11ßhsd2 transcript abundance in parr was lower than post-smolt groups; after the novel stressor pomcs, grs and mr were up-regulated in parr control (T1). In summary, we highlight differences in the central stress response between parr and post-smolt salmon following RCS.
Assuntos
Reação de Fuga/fisiologia , Estágios do Ciclo de Vida/fisiologia , Salmo salar/fisiologia , Estresse Fisiológico/fisiologia , Animais , Água Doce , Regulação da Expressão Gênica , Hidrocortisona/sangue , Comportamento Predatório/fisiologia , Área Pré-Óptica/metabolismo , Salmo salar/sangue , Salmo salar/crescimento & desenvolvimento , Água do Mar , Estresse Fisiológico/genética , Estresse Psicológico/sangue , Estresse Psicológico/genéticaRESUMO
Thyroid hormones, in particular 3,5,3'-triiodothyronine or T3, are involved in multiple physiological processes in mammals such as protein, fat and carbohydrate metabolism. However, the metabolic actions of T3 in fish are still not fully elucidated. We therefore tested the effects of T3 on Sparus aurata energy metabolism and osmoregulatory system, a hyperthyroid-induced model that was chosen. Fish were implanted with coconut oil depots (containing 0, 2.5, 5.0 and 10.0µg T3/g body weight) and sampled at day 3 and 6 post-implantation. Plasma levels of free T3 as well as glucose, lactate and triglyceride values increased with increasing doses of T3 at days 3 and 6 post-implantation. Changes in plasma and organ metabolite levels (glucose, glycogen, triglycerides, lactate and total α amino acid) and enzyme activities related to carbohydrate, lactate, amino acid and lipid pathways were detected in organs involved in metabolism (liver) and osmoregulation (gills and kidney). Our data implicate that the liver uses amino acids as an energy source in response to the T3 treatment, increasing protein catabolism and gluconeogenic pathways. The gills, the most important extruder of ammonia, are fuelled not only by amino acids, but also by lactate. The kidney differs significantly in its substrate preference from the gills, as it obtained metabolic energy from lactate but also from lipid oxidation processes. We conclude that in S. aurata lipid catabolism and protein turnover are increased as a consequence of experimentally induced hyperthyroidism, with secondary osmoregulatory effects.
Assuntos
Metabolismo Energético , Hipertireoidismo/metabolismo , Dourada/metabolismo , Análise de Variância , Animais , Brânquias/metabolismo , Hipertireoidismo/sangue , Rim/metabolismo , Modelos Lineares , Fígado/metabolismo , Metaboloma , Concentração Osmolar , Dourada/sangue , ATPase Trocadora de Sódio-Potássio/metabolismo , Análise de Sobrevida , Tri-Iodotironina/sangueRESUMO
Combinations of stressors occur regularly throughout an animal's life, especially in agriculture and aquaculture settings. If an animal fails to acclimate to these stressors, stress becomes chronic, and a condition of allostatic overload arises with negative results for animal welfare. In the current study, we describe effects of exposing Atlantic salmon parr to an unpredictable chronic stressor (UCS) paradigm for 3â weeks. The paradigm involves exposure of fish to seven unpredictable stressors three times a day. At the end of the trial, experimental and control fish were challenged with yet another novel stressor and sampled before and 1â h after that challenge. Plasma cortisol decreased steadily over time in stressed fish, indicative of exhaustion of the endocrine stress axis. This was confirmed by a lower cortisol response to the novel stressor at the end of the stress period in chronically stressed fish compared with the control group. In the preoptic area (POA) and pituitary gland, chronic stress resulted in decreased gene expression of 11ßhsd2, gr1 and gr2 in the POA and increased expression of those genes in the pituitary gland. POA crf expression and pituitary expression of pomcs and mr increased, whereas interrenal gene expression was unaffected. Exposure to the novel stressor had no effect on POA and interrenal gene expression. In the pituitary, crfr1, pomcs, 11ßhsd2, grs and mr were down-regulated. In summary, our results provide a novel overview of the dynamic changes that occur at every level of the hypothalamic-pituitary gland-interrenal gland (HPI) axis as a result of chronic stress in Atlantic salmon.
Assuntos
Salmo salar/fisiologia , Estresse Fisiológico , Alostase , Animais , Glândulas Endócrinas/metabolismo , Pesqueiros , Expressão Gênica , Hidrocortisona/sangue , Glândula Inter-Renal/fisiologia , Hipófise/metabolismo , Área Pré-Óptica/fisiologia , Salmo salar/crescimento & desenvolvimentoRESUMO
Zebrafish (Danio rerio Hamilton) are increasingly used as a model to study the effects of chronic stress on brain and behaviour. In rodents, unpredictable chronic stress (UCS) has a stronger effect on physiology and behaviour during the active phase than during the resting phase. Here, we applied UCS during the daytime (active phase) for 7 and 14 days or during the night-time (resting phase) for 7 nights in an in-house-reared Tuebingen long-fin (TLF) zebrafish strain. Following UCS, inhibitory avoidance learning was assessed using a 3 day protocol where fish learn to avoid swimming from a white to a black compartment where they will receive a 3 V shock. Latencies of entering the black compartment were recorded before training (day 1; first shock) and after training on day 2 (second shock) and day 3 (no shock, tissue sampling). Fish whole-body cortisol content and expression levels of genes related to stress, fear and anxiety in the telencephalon were quantified. Following 14 days of UCS during the day, inhibitory avoidance learning decreased (lower latencies on days 2 and 3); minor effects were found following 7 days of UCS. Following 7 nights of UCS, inhibitory avoidance learning decreased (lower latency on day 3). Whole-body cortisol levels showed a steady increase compared with controls (100%) from 7 days of UCS (139%), to 14 days of UCS (174%) to 7 nights of UCS (231%), suggestive of an increasing stress load. Only in the 7 nights of UCS group did expression levels of corticoid receptor genes (mr, grα, grß) and of bdnf increase. These changes are discussed as adaptive mechanisms to maintain neuronal integrity and prevent overload, and as being indicative of a state of high stress load. Overall, our data suggest that stressors during the resting phase have a stronger impact than during the active phase. Our data warrant further studies on the effect of UCS on stress axis-related genes, especially grß; in mammals this receptor has been implicated in glucocorticoid resistance and depression.
Assuntos
Aprendizagem da Esquiva/fisiologia , Modelos Animais , Tempo de Reação/genética , Estresse Fisiológico/fisiologia , Peixe-Zebra/fisiologia , Animais , Ritmo Circadiano/fisiologia , Estimulação Elétrica , Perfilação da Expressão Gênica , Hidrocortisona/metabolismo , Receptores de Glucocorticoides/metabolismo , Fatores de TempoRESUMO
Corticotropin-releasing factor-binding protein (CRF-BP) is considered a key determinant for CRF receptor (CRF-R) activation by CRF and several related peptides. Earlier studies have shown that the CRF system is highly conserved in gene structures throughout evolution, yet little is known about the evolutionary conservation of its biological functions. Therefore, we address the functional properties of CRF-BP and CRF-Rs in a teleost fish (common carp; Cyprinus carpio L.). We report the finding of two similar, yet distinct, genes for both CRF-R1 and CRF-R2 in this species. The four receptors are differentially responsive to CRF, urotensin-I (UI), sauvagine, and urocortin-2 (Ucn-2) and -3 (Ucn-3) as shown by luciferase assays. In vitro, carp CRF-BP inhibits CRF- and UI-mediated activation of the newfound CRF-Rs, but its potency to do so varies between receptor and peptide ligand. This is the first paper to establish the functionality and physiological interplay between CRF-BP, CRF-Rs and CRF-family peptides in a teleostean species.
Assuntos
Carpas/metabolismo , Proteínas de Transporte/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Urotensinas/metabolismo , Proteínas de Anfíbios/metabolismo , Animais , AMP Cíclico/farmacologia , Células HEK293 , Humanos , Luciferases/metabolismo , Hormônios Peptídicos/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de Hormônio Liberador da Corticotropina/genética , Proteínas Recombinantes/metabolismo , Urocortinas/metabolismoRESUMO
Of the many stressors in aquaculture, transportation of fish has remained poorly studied. The objective of this study was therefore to assess the effects of a (simulated) commercial transportation on stress physiology of market-size African catfish (Clarias gariepinus). Catfish weighing approximately 1.25 kg were returned to the farm after 3 h of truck-transportation, and stress-related parameters were measured for up to 72 h following return. Recovery from transportation was assessed through blood samples measuring plasma cortisol, glucose and non-esterified fatty acids (NEFA) and gill histology. Also, the number of skin lesions was compared before and after transport. Pre-transport handling and sorting elevated plasma cortisol levels compared to unhandled animals (before fasting). Plasma cortisol levels were further increased due to transportation. In control fish, plasma cortisol levels returned to baseline values within 6 h, whereas it took 48 h to reach baseline values in transported catfish. Plasma glucose and NEFA levels remained stable and were similar across all groups. Transported catfish did not, on average, have more skin lesions than the handling group, but the number of skin lesions had increased compared to unhandled animals. The macroscopic condition of the gills was similar in control, transported and unhandled catfish; however, light microscopy and immunohistochemistry revealed atypical morphology and chloride cell migration normally associated with adverse water conditions. From our data, we conclude that transportation may be considered a strong stressor to catfish that may add to other stressors and thus inflict upon the welfare of the fish.
Assuntos
Aquicultura , Peixes-Gato/fisiologia , Estresse Fisiológico , Meios de Transporte , Animais , Glicemia , Ácidos Graxos não Esterificados/sangue , Brânquias/fisiopatologia , Hidrocortisona/sangue , Pele/fisiopatologia , Qualidade da ÁguaRESUMO
Aquaculture practices bring several stressful events to fish. Stressors not only activate the hypothalamus-pituitary-interrenal-axis, but also evoke cellular stress responses. Up-regulation of heat shock proteins (HSPs) is among the best studied mechanisms of the cellular stress response. An extract of the prickly pear cactus (Opuntia ficus indica), Pro-Tex, a soluble variant of TEX-OE(®), may induce expression of HSPs and reduce negative effects of cellular stress. Pro-Tex therefore is used to ameliorate conditions during stressful aquaculture-related practices. We tested Pro-Tex in zebrafish (Danio rerio), common carp (Cyprinus carpio L.) and yellowtail kingfish (Seriola lalandi) exposed to aquaculture-relevant stressors (thermal stress, net confinement, transport) and assessed its effects on stress physiology. Heat shock produced a mild increase in hsp70 mRNA expression in 5-day-old zebrafish larvae. Pro-Tex increased basal hsp70 mRNA expression, but decreased heat-shock-induced expression of hsp70 mRNA. In carp, Pro-Tex increased plasma cortisol and glucose levels, while it did not affect the mild stress response (increased plasma cortisol and glucose) to net confinement. In gills, and proximal and distal intestine, stress increased hsp70 mRNA expression; in the distal intestine, an additive enhancement of hsp70 mRNA expression by Pro-Tex was seen under stress. In yellowtail kingfish, Pro-Tex reduced the negative physiological effects of transport more efficiently than when fish were sedated with AQUI-S(®). Overall, our data indicate that Pro-Tex has protective effects under high levels of stress only. As Pro-Tex has potential for use in aquaculture, its functioning and impact on health and welfare of fish should be further studied.
Assuntos
Carpas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Opuntia/química , Perciformes/metabolismo , Extratos Vegetais/farmacologia , Estresse Fisiológico/fisiologia , Peixe-Zebra/metabolismo , Animais , Aquicultura/métodos , Glicemia/análise , Primers do DNA/genética , Relação Dose-Resposta a Droga , Proteínas de Choque Térmico/metabolismo , Hidrocortisona/sangue , Ácido Láctico/sangue , Larva/efeitos dos fármacos , Larva/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Estatísticas não Paramétricas , Estresse Fisiológico/efeitos dos fármacos , TemperaturaRESUMO
Introduction of zebrafish as a model for human diseases with symptomatic hypomagnesemia urges to identify the regulatory transport genes involved in zebrafish Mg(2+) physiology. In humans, mutations related to hypomagnesemia are located in the genes TRPM6 and CNNM2, encoding for a Mg(2+) channel and transporter, respectively; EGF (epidermal growth factor); SLC12A3, which encodes for the Na(+)-Cl(-) co-transporter NCC; KCNA1 and KCNJ10, encoding for the K(+) channels Kv1.1 and Kir4.1, respectively; and FXYD2, which encodes for the γ-subunit of the Na(+),K(+)-ATPase. Orthologues of these genes were found in the zebrafish genome. For cnnm2, kcna1 and kcnj10, two conserved paralogues were retrieved. Except for fxyd2, kcna1b and kcnj10 duplicates, transcripts of orthologues were detected in ionoregulatory organs such as the gills, kidney and gut. Gene expression analyses in zebrafish acclimated to a Mg(2+)-deficient (0 mM Mg(2+)) or a Mg(2+)-enriched (2 mM Mg(2+)) water showed that branchial trpm6, gut cnnm2b and renal slc12a3 responded to ambient Mg(2+). When changing the Mg(2+) composition of the diet (the main source for Mg(2+) in fish) to a Mg(2+)-deficient (0.01 % (w/w) Mg) or a Mg(2+)-enriched diet (0.7 % (w/w) Mg), mRNA expression of branchial trpm6, gut trpm6 and cnnm2 duplicates, and renal trpm6, egf, cnnm2a and slc12a3 was the highest in fish fed the Mg(2+)-deficient diet. The gene regulation patterns were in line with compensatory mechanisms to cope with Mg(2+)-deficiency or surplus. Our findings suggest that trpm6, egf, cnnm2 paralogues and slc12a3 are involved in the in vivo regulation of Mg(2+) transport in ionoregulatory organs of the zebrafish model.
Assuntos
Deficiência de Magnésio/genética , Proteínas de Peixe-Zebra/metabolismo , Animais , Ciclinas/genética , Ciclinas/metabolismo , Regulação da Expressão Gênica , Brânquias/metabolismo , Mucosa Intestinal/metabolismo , Rim/metabolismo , Canal de Potássio Kv1.1/genética , Canal de Potássio Kv1.1/metabolismo , Magnésio/metabolismo , Deficiência de Magnésio/metabolismo , Especificidade de Órgãos , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
To systematically identify novel gene functions essential for osteogenesis and skeletal mineralization, we performed a forward genetic mutagenesis screen in zebrafish and isolated a mutant that showed delayed skeletal mineralization. Analysis of the mutant phenotype in an osterix:nuclear-GFP transgenic background demonstrated that mutants contain osterix-expressing osteoblasts comparable to wild-type embryos. Positional cloning revealed a premature stop mutation in the macrophage-stimulating protein (msp) gene, predicted to result in a biologically inactive protein. Analysis of the embryonic expression pattern for the receptor for Msp, Ron, shows specific expression in the corpuscles of Stannius, a teleost-specific organ that produces stanniocalcin, a pivotal hormone in fish calcium homeostasis. Knockdown of Ron resulted in identical phenotypes as observed in msp mutants. Msp mutant embryos could be rescued by excess calcium. Consistent with a role for Msp/Ron in calcium homeostasis, calcium-regulating factors, such as pth1, pth2, stc1l, and trpv5/6 were significantly affected in msp mutant larvae. While Msp and Ron have previously been shown to play a critical role in a wide variety of biological processes, we introduce here the Msp/Ron signaling axis as a previously unappreciated player in calcium homeostasis and embryonic skeletal mineralization.
Assuntos
Cálcio/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Glicoproteínas/metabolismo , Fator de Crescimento de Hepatócito/genética , Homeostase/genética , Homeostase/fisiologia , Osteogênese/genética , Osteogênese/fisiologia , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
To assess the hypothesis that cortisol release in rainbow trout is modulated by glucose levels, we first evaluated cortisol release [basal and adrenocorticotropic hormone (ACTH)-regulated] by head kidney tissue superfused with medium reflecting hypoglycaemic, normoglycaemic or hyperglycaemic conditions. Next, cortisol release from head kidney fragments in static incubations was assessed in parallel with changes in parameters related to cortisol synthesis (mRNA abundance of StAR, P450scc, 3ßHSD and 11ßH) and the GK-mediated glucosensing mechanism (levels of glycogen and glucose, activities of GK, GSase and PK, and mRNA levels of GK, GLUT-2, Kir6.x-like and SUR-like). We then evaluated the effects of two inhibitors of glucose transport, cytochalasin B and phlorizin, on cortisol production and glucosensing mechanisms. The ACTH-induced release of cortisol proved to be modulated by glucose concentration such that increased release occurs under high glucose levels, and decreased ACTH-stimulated cortisol release occurs when glucose transport is inhibited by cytochalasin B. The release of cortisol can be associated with increased synthesis as enhanced mRNA abundance of genes related to cortisol synthesis was also noted in high glucose medium. Specific GK immunoreactivity in the cortisol-producing cells (not in chromaffin cells) further substantiates GK-mediated glucosensing in cortisol production. In contrast, no changes compatible with those of glucose levels and cortisol release/synthesis in the presence of ACTH were noted for any other putative glucosensor mechanisms based on LXR, SGLT-1 or Gnat3. These combined results are the first evidence for a mechanism in fish linking the synthesis and release of a non-pancreatic hormone like cortisol with circulating glucose levels. The relationship was evident for the regulated (ACTH-dependent) pathway and this suggests that under acute stress conditions glucose is important for the regulation of cortisol synthesis and release.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Glucose/farmacologia , Rim Cefálico/metabolismo , Hidrocortisona/metabolismo , Oncorhynchus mykiss/metabolismo , Análise de Variância , Animais , Área Sob a Curva , Citocalasina B/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicogênio/metabolismo , Rim Cefálico/citologia , Rim Cefálico/efeitos dos fármacos , Rim Cefálico/enzimologia , Imuno-Histoquímica , Microscopia Confocal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
Background: Melatonin is a key hormone in regulation of circadian rhythms, and involved in many rhythmic functions, such as feeding and locomotor activity. Melatonin reportedly counteracts stress responses in many vertebrates, including fish. However, targets for this action of melatonin and underlying mechanisms remain unknown. Results: This study reports potential anti-stress properties of melatonin in goldfish (Carassius auratus), with a focus on its effect on plasma cortisol, food intake, and locomotor activity, all of them involved in the responses to stress exposure. Indeed, acute injection of melatonin counteracted stress-induced hypercortisolinemia and reduced food intake. The reduced locomotor activity following melatonin treatment suggests a possible sedative role in fish. To assess whether this anti-stress effects of melatonin involve direct actions on interrenal tissue, in vitro cultures of head kidney (containing the interrenal cortisol-producing tissue) were carried out in presence of ACTH, melatonin, and luzindole, an antagonist of melatonin receptors. Melatonin in vitro reduced ACTH-stimulated cortisol release, an effect attenuated by luzindole; this suggests the presence of specific melatonin receptors in interrenal tissue. Conclusions: Our data support a role for melatonin as an anti-stress signal in goldfish, and suggest that the interrenal tissue of teleosts may be a plausible target for melatonin action decreasing cortisol production.
Assuntos
Melatonina , Triptaminas , Animais , Melatonina/farmacologia , Carpa Dourada , Hidrocortisona , Receptores de Melatonina , Hormônio AdrenocorticotrópicoRESUMO
Calcium is an essential ion serving a multitude of physiological roles. Aside from its role as a second messenger, it is an essential component of the vertebrate bone matrix. Efficient uptake and storage of calcium are therefore indispensable for all vertebrates. Transient receptor potential family, vanilloid type (TRPV)5 and TRPV6 channels are known players in transcellular calcium uptake, but the exact contribution of this pathway is unclear. We used forward genetic screening in zebrafish (Danio rerio) to identify genes essential in bone formation and identified a lethal zebrafish mutant (matt-und-schlapp) with severe defects in bone formation, including lack of ossification of the vertebral column and craniofacial structures. Mutant embryos show a 68% reduction in calcium content, and systemic calcium homeostasis is disturbed when compared with siblings. The phenotype can be partially rescued by increasing ambient calcium levels to 25 mM. We identified the mutation as a loss-of-function mutation in the single orthologue of TRPV5 and 6, trpv5/6. Expression in HEK293 cells showed that Trpv5/6 is a calcium-selective channel capable of inward calcium transport at physiological concentrations whereas the mutant channel is not. Taken together, this study provides both genetic and functional evidence that transcellular epithelial calcium uptake is vital to sustain life and enable bone formation.
Assuntos
Desenvolvimento Ósseo/fisiologia , Cálcio/metabolismo , Epitélio/embriologia , Epitélio/metabolismo , Canais de Cátion TRPV/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Desenvolvimento Ósseo/genética , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células HEK293 , Humanos , Dados de Sequência Molecular , Mutação , Canais de Cátion TRPV/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Appetite suppression is a common response to hypoxia in fish that confers significant energy savings. Yet little is known about the endocrine signals involved in the regulation of food intake during chronic hypoxia. Thus, we assessed the impact of chronic hypoxia on food intake, the expression of the potent anorexigenic signal leptin and its receptor (lepr), the mRNA levels of key hypothalamic appetite-regulating genes, and the activity of the hypothalamic-pituitary-interrenal (HPI) axis in common carp, Cyprinus carpio. Fish exposed to 10% O(2) saturation for 8 days were chronically anorexic and consumed on average 79% less food than normoxic controls. Hypoxia also elicited gradual and parallel increases in the expression of liver leptin-a-I, leptin-a-II, lepr and erythropoietin, a known hypoxia-responsive gene. In contrast, the liver mRNA levels of all four genes remained unchanged in normoxic fish pair-fed to the hypoxia treatment. In the hypothalamus, expression of the appetite-regulating genes were consistent with an inhibition and stimulation of hunger in the hypoxic and pair-fed fish, respectively, and reduced feed intake led to a decrease in lepr. Although both treatments elicited similar delayed increases in plasma cortisol, they were characterized by distinct HPI axis effector transcript levels and a marked differential increase in pituitary lepr expression. Together, these results show that a reduction in O(2) availability, and not feed intake, stimulates liver leptin-a expression in common carp and suggest that this pleiotropic cytokine is involved in the regulation of appetite and the endocrine stress response during chronic hypoxia.
Assuntos
Regulação do Apetite , Carpas/fisiologia , Proteínas de Peixes/genética , Leptina/genética , Receptores para Leptina/genética , Animais , Carpas/genética , Carpas/metabolismo , Ingestão de Alimentos , Regulação da Expressão Gênica , Hipotálamo/metabolismo , Hipotálamo/fisiologia , Hipóxia/metabolismo , Oxigênio/metabolismo , Hipófise/metabolismo , Hipófise/fisiologia , RNA Mensageiro/genéticaRESUMO
Proper functioning of the endocrine stress axis requires communication between the stress axis and other regulatory mechanisms. We here describe an intimate interplay between the stress axis and recombinant human leptin (rhLeptin) in a teleostean fish, the common carp Cyprinus carpio. Restraint stress (by netting up to 96h) increased plasma cortisol but did not affect hepatic leptin expression. Perifusion of pituitary glands or head kidneys with rhLeptin revealed direct effects of rhLeptin on both tissues. RhLeptin suppresses basal and CRF-induced ACTH-secretion in a rapid and concentration-dependent manner. The rhLeptin effect persisted for over an hour after administration had been terminated. RhLeptin decreases basal interrenal cortisol secretion in vitro, and by doing so attenuates ACTH-stimulated cortisol production; rhLeptin does not affect interrenal ACTH-sensitivity. Our findings show that the endocrine stress axis activity and leptin are inseparably linked in a teleostean fish, a notion relevant to further our insights in the evolution of leptin physiology in vertebrates.
Assuntos
Carpas/metabolismo , Leptina/farmacologia , Proteínas Recombinantes/farmacologia , Animais , Receptor de Asialoglicoproteína/metabolismo , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Estresse FisiológicoRESUMO
The precursor protein proopiomelanocortin (POMC) gives rise to a variety of biologically active peptides through cell-specific posttranslational processing. Two transcripts of pomc were found in the flatfish Solea senegalensis (ssePOMC-A and ssePOMC-B), that most likely represent subfunctionalized paralogues: ssePOMC-A lacks the N-terminal cleavage site for ß-MSH, whereas ssePOMC-B cannot yield ACTH and completely lacks the opioid consensus sequence in the ß-END region. An analysis of nucleotide substitution rates shows that the POMC-derived peptides possess well-conserved regions under purifying selection, except the ß-END derived from POMC-B, which has undergone positive selection. The calculated K(s) values for ssePOMC-A versus ssePOMC-B and zebrafish POMCαversus zebrafish POMCß are 0.40 and 0.72, respectively, indicating that the zebrafish POMC paralogues started to evolve almost twice as early in evolution, and that the Solea POMC paralogues arose independently from the whole genome duplication event that gave rise to the zebrafish paralogues. This makes ssePOMC-B the first identified POMCα orthologue that lacks the opioid consensus. Furthermore, pomc-a expression is down-regulated in chronic stressed S. senegalensis juveniles, whereas pomc-b expression levels remain unaffected, indicating different physiological roles for both POMC paralogues. The distribution of functional POMC-derived peptide hormones over two pomc genes in S. senegalensis suggests subfunctionalization of the paralogues, a relevant notion when studying POMC function in endocrine responses.