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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.
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Proteínas de Pez Cebra , Pez Cebra , Animales , Perfilación de la Expresión Génica , Humanos , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
This study assessed the lasting impact of dexamethasone (DEX) exposure during early development on tissue repair capacity at later life stages (5, 14, and 24 days post fertilization [dpf]) in zebrafish larvae. Using the caudal fin amputation model, we show that prior exposure to DEX significantly delays but does not prevent wound healing at all life stages studied. DEX-induced impairments on wound healing were fully restored to normal levels with longer post amputation recovery time. Further analyses revealed that DEX mainly exerted its detrimental effects in the early phase (0-5 hours) of wound-healing process. Specifically, we observed the following events: (1) massive amount of cell death both by necrosis and apoptosis; (2) significant reduction in the number as well as misplacement of macrophages at the wound site; (3) aberrant migration and misplacement of neutrophils and macrophages at the wound site. These events were accompanied by significant (likely compensatory) changes in the expression of genes involved in tissue patterning, including up-regulation of FKBP5 6 hours post DEX exposure and that of Wnt3a and RARγ at 24 hours post amputation. Taken together, this study provides evidence that DEX exposure during early sensitive periods of development appears to cause permanent alterations in the cellular/molecular immune processes that are involved in the early phase of wound healing in zebrafish. These findings are consistent with previous studies showing that antenatal course of DEX is associated with immediate and lasting alterations of the immune system in rodent models and humans. Therefore, the current findings support the use of the larval zebrafish model to study the impact of stress and stress hormone exposure in immature organisms on health risks in later life.
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Amputación Traumática/patología , Aletas de Animales/patología , Dexametasona/farmacología , Regeneración , Cicatrización de Heridas , Animales , Antiinflamatorios/farmacología , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Larva , Macrófagos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Regulación hacia Arriba , Cicatrización de Heridas/efectos de los fármacos , Pez Cebra/genéticaRESUMEN
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.
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Carpas/metabolismo , Proteínas Portadoras/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Urotensinas/metabolismo , Proteínas Anfibias/metabolismo , Animales , AMP Cíclico/farmacología , Células HEK293 , Humanos , Luciferasas/metabolismo , Hormonas Peptídicas/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de Hormona Liberadora de Corticotropina/genética , Proteínas Recombinantes/metabolismo , Urocortinas/metabolismoRESUMEN
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.
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Melatonina , Triptaminas , Animales , Melatonina/farmacología , Carpa Dorada , Hidrocortisona , Receptores de Melatonina , Hormona AdrenocorticotrópicaRESUMEN
Cryo-correlative light and electron microscopy (cryoCLEM) is a powerful strategy to high resolution imaging in the unperturbed hydrated state. In this approach fluorescence microscopy aids localizing the area of interest, and cryogenic focused ion beam/scanning electron microscopy (cryoFIB/SEM) allows preparation of thin cryo-lamellae for cryoET. However, the current method cannot be accurately applied on bulky (3D) samples such as tissues and organoids. 3D cryo-correlative imaging of large volumes is needed to close the resolution gap between cryo-light microscopy and cryoET, placing sub-nanometer observations in a larger biological context. Currently technological hurdles render 3D cryoCLEM an unexplored approach. Here we demonstrate a cryoCLEM workflow for tissues, correlating cryo-Airyscan confocal microscopy with 3D cryoFIB/SEM volume imaging. Accurate correlation is achieved by imprinting a FinderTOP pattern in the sample surface during high pressure freezing, and allows precise targeting for cryoFIB/SEM volume imaging.
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Microscopía Electrónica , Microscopía Fluorescente/métodos , Microscopía por Crioelectrón/métodos , Microscopía Confocal , CongelaciónRESUMEN
Purpose: To study the prevalence, level, and nature of sleep problems and fatigue experienced by Usher syndrome type 2a (USH2a) patients. Design: Cross-sectional study. Participants: Fifty-six genetically confirmed Dutch patients with syndromic USH2a and 120 healthy controls. Methods: Sleep quality, prevalence, and type of sleep disorders, chronotype, fatigue, and daytime sleepiness were assessed using 5 questionnaires: (1) Pittsburgh Sleep Quality Index, (2) Holland Sleep Disorders Questionnaire, (3) Morningness-Eveningness Questionnaire, (4) Checklist Individual Strength, and (5) Epworth Sleepiness Scale. For a subset of patients, recent data on visual function were used to study the potential correlation between the outcomes of the questionnaires and disease progression. Main Outcome Measures: Results of all questionnaires were compared between USH2a and control cohorts, and the scores of the patients were compared with disease progression defined by age, visual field size, and visual acuity. Results: Compared with the control population, patients with USH2a experienced a poorer quality of sleep, a higher incidence of sleep disorders, and higher levels of fatigue and daytime sleepiness. Intriguingly, the sleep disturbances and high levels of fatigue were not correlated with the level of visual impairment. These results are in accordance with the patients' experiences that their sleep problems already existed before the onset of vision loss. Conclusions: This study demonstrates a high prevalence of fatigue and poor sleep quality experienced by patients with USH2a. Recognition of sleep problems as a comorbidity of Usher syndrome would be a first step toward improved patient care. The absence of a relationship between the level of visual impairment and the severity of reported sleep problems is suggestive of an extraretinal origin of the sleep disturbances. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.
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Introduction: Mutations in the FOXE1 gene are implicated in cleft palate and thyroid dysgenesis in humans. Methods: To investigate whether zebrafish could provide meaningful insights into the etiology of developmental defects in humans related to FOXE1, we generated a zebrafish mutant that has a disruption in the nuclear localization signal in the foxe1 gene, thereby restraining nuclear access of the transcription factor. We characterized skeletal development and thyroidogenesis in these mutants, focusing on embryonic and larval stages. Results: Mutant larvae showed aberrant skeletal phenotypes in the ceratohyal cartilage and had reduced whole body levels of Ca, Mg and P, indicating a critical role for foxe1 in early skeletal development. Markers of bone and cartilage (precursor) cells were differentially expressed in mutants in post-migratory cranial neural crest cells in the pharyngeal arch at 1 dpf, at induction of chondrogenesis at 3 dpf and at the start of endochondral bone formation at 6 dpf. Foxe1 protein was detected in differentiated thyroid follicles, suggesting a role for the transcription factor in thyroidogenesis, but thyroid follicle morphology or differentiation were unaffected in mutants. Discussion: Taken together, our findings highlight the conserved role of Foxe1 in skeletal development and thyroidogenesis, and show differential signaling of osteogenic and chondrogenic genes related to foxe1 mutation.
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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.
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Desarrollo Óseo/fisiología , Calcio/metabolismo , Epitelio/embriología , Epitelio/metabolismo , Canales Catiónicos TRPV/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Desarrollo Óseo/genética , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Células HEK293 , Humanos , Datos de Secuencia Molecular , Mutación , Canales Catiónicos TRPV/genética , Proteínas de Pez Cebra/genéticaRESUMEN
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.
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Sistema Endocrino/fisiología , Peces Planos/fisiología , Proopiomelanocortina/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Filogenia , Proopiomelanocortina/análisis , Proopiomelanocortina/genéticaRESUMEN
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.
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Carpas/metabolismo , Leptina/farmacología , Proteínas Recombinantes/farmacología , Animales , Receptor de Asialoglicoproteína/metabolismo , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Estrés FisiológicoRESUMEN
Background and Objective: A key pathway controlling skeletal development is fibroblast growth factor (FGF) and FGF receptor (FGFR) signaling. Major regulatory functions of FGF signaling are chondrogenesis, endochondral and intramembranous bone development. In this study we focus on fgfr2, as mutations in this gene are found in patients with craniofacial malformations. The high degree of conservation between FGF signaling of human and zebrafish (Danio rerio) tempted us to investigate effects of the mutated fgfr2 sa10729 allele in zebrafish on cartilage and bone formation. Methods: We stained cartilage and bone in 5 days post fertilization (dpf) zebrafish larvae and compared mutants with wildtypes. We also determined the expression of genes related to these processes. We further investigated whether pharmacological blocking of all FGFRs with the inhibitor BGJ398, during 0-12 and 24-36 h post fertilization (hpf), affected craniofacial structure development at 5 dpf. Results: We found only subtle differences in craniofacial morphology between wildtypes and mutants, likely because of receptor redundancy. After exposure to BGJ398, we found dose-dependent cartilage and bone malformations, with more severe defects in fish exposed during 0-12 hpf. These results suggest impairment of cranial neural crest cell survival and/or differentiation by FGFR inhibition. Compensatory reactions by upregulation of fgfr1a, fgfr1b, fgfr4, sp7 and dlx2a were found in the 0-12 hpf group, while in the 24-36 hpf group only upregulation of fgf3 was found together with downregulation of fgfr1a and fgfr2. Conclusions: Pharmacological targeting of FGFR1-4 kinase signaling causes severe craniofacial malformations, whereas abrogation of FGFR2 kinase signaling alone does not induce craniofacial skeletal abnormalities. These findings enhance our understanding of the role of FGFRs in the etiology of craniofacial malformations.
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Anomalías Craneofaciales , Pez Cebra , Animales , Humanos , Pez Cebra/genética , Larva/genética , Compuestos de Fenilurea , Factores de Crecimiento de Fibroblastos/genética , Anomalías Craneofaciales/inducido químicamente , Proteínas de Pez Cebra/genética , Receptor Tipo 4 de Factor de Crecimiento de FibroblastosRESUMEN
Corticotropin-releasing factor (CRF) plays a central role in the regulation of the stress axis. In mammals, CRF as well as its receptors and its CRF-binding protein (CRF-BP) are expressed in a variety of organs and tissues outside the central nervous system. One of these extrahypothalamic sites is the adrenal gland, where the paracrine actions of adrenal CRF influence cortical steroidogenesis and adrenal blood flow. Although the central role of CRF signaling in the initiation and regulation of the stress response has now been established throughout vertebrates, information about the possible peripheral presence of CRF in earlier vertebrate lineages is scant. We established the expression of CRF, CRF-BP, and the CRF receptor 1 in a panel of peripheral organs of common carp (Cyprinus carpio). Out of all the peripheral organs tested, CRF and CRF-BP are most abundantly expressed in the carp head kidney, the fish equivalent of the mammalian adrenal gland. This expression localizes to chromaffin cells. Furthermore, detectable quantities of CRF are released from the intact head kidney following in vitro stimulation with 8-bromo-cAMP in a superfusion setup. The presence of CRF and CRF-BP within the chromaffin compartment of the head kidney suggests that a pathway homologous to the mammalian intra-adrenal CRF system is present in the head kidney of fish. It follows that such a system to locally fine-tune the outcome of the centrally initiated stress response has been an integral part of the vertebrate endocrine system since the common ancestor of teleostean fishes and mammals.
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Glándulas Suprarrenales/química , Evolución Biológica , Carpas/metabolismo , Proteínas Portadoras/análisis , Hormona Liberadora de Corticotropina/análisis , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Glándulas Suprarrenales/efectos de los fármacos , Glándulas Suprarrenales/metabolismo , Animales , Secuencia de Bases , Proteínas Portadoras/metabolismo , Células Cromafines/química , Células Cromafines/efectos de los fármacos , Células Cromafines/metabolismo , Hormona Liberadora de Corticotropina/genética , Hormona Liberadora de Corticotropina/metabolismo , Cartilla de ADN/genética , Expresión Génica , Inmunohistoquímica , Microscopía Confocal , Datos de Secuencia Molecular , Técnicas de Cultivo de Órganos , ARN Mensajero/análisis , Radioinmunoensayo , Receptores de Hormona Liberadora de Corticotropina/análisis , Receptores de Hormona Liberadora de Corticotropina/genética , Estimulación QuímicaRESUMEN
In fish, the hypothalamus-pituitary-interrenal axis (HPI-axis), the equivalent of the hypothalamus-pituitary-adrenal axis (HPA-axis) in mammals, is activated during stress and leads to production and release of cortisol by the interregnal cells in the head kidney. In mammals, the cytokine interleukin-1beta (IL-1beta) takes a key position in the innate immune and inflammatory responses and influences the HPA-axis. In fish, studies that address the effects of cytokines on HPI-axis activation are limited. We quantitatively assessed expression of IL-1beta and its receptor, IL-1RI (the latter was cloned and sequenced), in an acute restraint stress paradigm in common carp, Cyprinus carpio. We also considered expression of the pituitary hormones prolactin (PRL) and GH that have been shown to be structurally related to cytokines and have immunomodulatory actions. Pituitary PRL expression increased fourfold during stress; GH mRNA levels were unaffected. Following restraint, hypothalamic IL-1beta expression was upregulated; in head kidney and pituitary pars intermedia, IL-1RI expression significantly increased. We suggest that during acute stress IL-1beta signalling in the HPI-axis becomes more sensitive, since both ligand and receptor expressions are enhanced. In vitro, recombinant carp IL-1beta stimulates release of alpha-MSH and N-Ac beta-endorphin from the pituitary gland. This observation concurs with increased in vivo plasma levels of alpha-MSH and N-Ac beta-endorphin following restraint. Our findings combined lead us to conclude that IL-1beta affects the activity of the HPI-axis and, in turn, expression profiles of genes encoding IL-1beta and its receptor are modified during acute stress. Our study provides convincing evidence for bi-directional communication of the HPI-axis and the immune system in fish.
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Carpas/metabolismo , Interleucina-1beta/genética , Receptores Tipo I de Interleucina-1/genética , Estrés Psicológico , Animales , Secuencia de Bases , Pollos , Expresión Génica/efectos de los fármacos , Hormona del Crecimiento/genética , Caballos , Humanos , Hipotálamo/metabolismo , Interleucina-1beta/farmacología , Riñón/metabolismo , Macaca , Ratones , Datos de Secuencia Molecular , Hipófisis/metabolismo , Prolactina/genética , ARN Mensajero/análisis , Ratas , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Salmón , Alineación de Secuencia , Takifugu , alfa-MSH/genética , betaendorfina/genéticaRESUMEN
Knowledge of the mechanisms by which fish excrete their metabolic nitrogenous waste and insights into nitrogen cycling in aquaculture systems is of utmost importance to improve the sustainable commercial production of fish. In fish, most nitrogenous waste is excreted via the gills as ammonia, a potentially toxic nitrogenous compound. In this study; activity assays, physiological experiments, molecular analysis and microscopy were used to show that the gills of fish harbor a unique combination of hitherto overlooked nitrogen-cycle microorganisms that can theoretically detoxify excreted ammonia by converting it into inert dinitrogen gas. By doing so, these microorganisms may benefit from the ammonia supply by the host and prevent the build-up of this compound to toxic concentrations. This novel relationship between vertebrates and microorganisms may shed new light on nitrogen handling by ammonotelic fish species.
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Iodothyronine deiodinases catalyze the conversion of the thyroid prohormone T(4) to T(3) by outer ring deiodination (ORD) of the iodothyronine molecule. The catalytic cycle of deiodinases is considered to be critically dependent on a reducing thiol cosubstrate that regenerates the selenoenzyme to its native state. The endogenous cosubstrate has still not been firmly identified; in studies in vitro the sulfhydryl reagent dithiothreitol (DTT) is commonly used to activate ORD. We now have characterized an ORD activity in the teleost gilthead seabream (Sparus auratus) that is inhibited by DTT. DTT inhibited reverse T(3) (rT(3)) ORD by 70 and 100% in kidney homogenates (IC(50) 0.4 mmol/liter) and microsomes (IC(50) 0.1 mmol/liter), respectively. The omission of DTT from the incubation medium restored renal ORD Michaelis-Menten kinetics with a Michaelis constant value of 5 mumol/liter rT(3) and unmasked the inhibition by 6-n-propyl-2-thiouracil. A putative seabream deiodinase type 1 (saD1), derived from kidney mRNA, showed high homology (> or = 41% amino acid identity) with vertebrate deiodinases type 1. Features of this putative saD1 include a selenocysteine encoded by an in-frame UGA codon, consensus sequences, and a predicted secondary structure for a selenocysteine insertion sequence and an amino acid composition of the catalytic center that is identical with reported consensus sequences for deiodinase type 1. Remarkably, three of six cysteines that are present in the deduced saD1 protein occur in the predicted amino terminal hydrophobic region. We suggest that the effects of DTT on rT(3) ORD can be explained by interactions with the cysteines unique to the putative saD1 protein.
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Ditiotreitol/farmacología , Yoduro Peroxidasa/antagonistas & inhibidores , Yoduro Peroxidasa/metabolismo , Dorada/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Codón , Secuencia de Consenso , ADN Complementario/genética , Yoduro Peroxidasa/genética , Yodo/metabolismo , Riñón/enzimología , Cinética , Microsomas/enzimología , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Selenocisteína/genética , Triyodotironina Inversa/antagonistas & inhibidores , Triyodotironina Inversa/metabolismoRESUMEN
The acute stress response is a key regulatory system for the maintenance of homeostatic equilibrium that is activated upon an imminent or ongoing disturbance of the "milieu interieur". In general, the stress response in bony fish is similar to that of mammals. The recent cloning and characterization of corticotropin-releasing hormone-binding protein (CRH-BP) in carp (Cyprinus carpio L.) reflects the conservation of the CRH signaling system throughout vertebrates. Now, we can start to investigate the processes that are mediated by the factors that make up this system. The stress response is only one of these processes.
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Carpas/metabolismo , Estrés Fisiológico/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Hormona Liberadora de Corticotropina/metabolismo , Hormona Liberadora de Corticotropina/fisiología , Humanos , Adenohipófisis/metabolismoRESUMEN
Tight interactions among different cell types contributing to bone formation are of key importance in the maintenance of bone homeostasis. Based on the high similarity in responses to (anti)osteogenic signals between zebrafish scales and mammalian bone, we developed and validated a model to screen large numbers of compounds using ex-vivo cultured scales of a sp7:luciferase transgenic zebrafish. This model combines the high predictive value of explant cultures with quick, sensitive, and quantifiable readout converging the effects via various pathways including WNT-signaling, to SP7/osterix promoter activity. Sp7 is pivotal in osteoblast differentiation and activity and its promoter activity provides an excellent surrogate for sp7 expression. Bmp-2a was shown to dose-dependently increase sp7-driven luciferase activity ex vivo. Next, we identified novel effects on bone for 51.7% of the compounds from a small library of WNT-signaling modulators, including a strong osteogenic effect for niclosamide. From all previously characterized compounds, the effect on bone was correctly predicted for 70% of compounds, resulting in a 7% false positive- and 21% false negative rate. The proposed sp7:luciferase zebrafish scale model is unique, powerful and efficient new tool to assess compounds with osteogenic effects, prior to further testing in rodents.
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Estructuras Animales/metabolismo , Bioensayo/métodos , Luciferasas/metabolismo , Osteogénesis , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Proteína Morfogenética Ósea 2 , Osteoblastos/metabolismo , Regiones Promotoras Genéticas/genética , Regeneración , Transducción de Señal , Factor de Transcripción Sp7 , Factores de Transcripción/genética , Proteínas Wnt/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Adrenocorticotropic hormone (ACTH) takes a central role in the hypothalamo-pituitary-interrenal axis (HPI axis), which is activated during stress. ACTH is produced by the corticotrope cells of the pituitary pars distalis (PD) and is under control of factors from the nucleus preopticus (NPO). The distribution of ACTH in the hypothalamo-pituitary system in common carp (Cyprinus carpio L.) was assessed by immunohistochemistry. ACTH and beta-endorphin immunoreactivity was observed in the ACTH cells in the PD and in the NPO. Nerve fibers, originating from the NPO and projecting to the pituitary gland, contain beta-endorphin, but not ACTH, and these fibers either control the pituitary pars intermedia (PI) through beta-endorphin or release it to the blood. The release of pituitary ACTH (studied in a superfusion setup) must in vivo be under predominant inhibitory control of dopamine. Release of ACTH is stimulated by corticotropin-releasing hormone, but only when ACTH cells experience dopaminergic inhibition. The expression of the precursor pro-opiomelanocortin in (POMC) NPO, PD and PI was studied in an acute restraint stress paradigm by real-time quantitative polymerase chain reaction (RQ-PCR). POMC gene expression is upregulated in these three key tissues of the hypothalamo-pituitary complex, revealing a hitherto unforeseen complex role for POMC-derived peptides in the regulation of responses to stress.
Asunto(s)
Hormona Adrenocorticotrópica/análisis , Carpas/metabolismo , Hipófisis/química , Área Preóptica/química , Hormona Adrenocorticotrópica/metabolismo , Animales , Hormona Liberadora de Corticotropina/farmacología , Dopamina/farmacología , Inmunohistoquímica/métodos , Masculino , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Área Preóptica/efectos de los fármacos , Área Preóptica/metabolismo , Proopiomelanocortina/análisis , Radioinmunoensayo/métodosRESUMEN
In fish nutrition, the ratio between omega-3 and omega-6 poly-unsaturated fatty acids influences skeletal development. Supplementation of fish oils with vegetable oils increases the content of omega-6 fatty acids, such as arachidonic acid in the diet. Arachidonic acid is metabolized by cyclooxygenases to prostaglandin E2, an eicosanoid with effects on bone formation and remodeling. To elucidate effects of poly-unsaturated fatty acids on developing and existing skeletal tissues, zebrafish (Danio rerio) were fed (micro-) diets low and high in arachidonic acid content. Elasmoid scales, dermal skeletal plates, are ideal to study skeletal metabolism in zebrafish and were exploited in the present study. The fatty acid profile resulting from a high arachidonic acid diet induced mild but significant increase in matrix resorption in ontogenetic scales of adult zebrafish. Arachidonic acid affected scale regeneration (following removal of ontogenetic scales): mineral deposition was altered and both gene expression and enzymatic matrix metalloproteinase activity changed towards enhanced osteoclastic activity. Arachidonic acid also clearly stimulates matrix metalloproteinase activity in vitro, which implies that resorptive effects of arachidonic acid are mediated by matrix metalloproteinases. The gene expression profile further suggests that arachidonic acid increases maturation rate of the regenerating scale; in other words, enhances turnover. The zebrafish scale is an excellent model to study how and which fatty acids affect skeletal formation.
Asunto(s)
Estructuras Animales/fisiología , Ácido Araquidónico/farmacología , Dermis/fisiología , Pez Cebra/fisiología , Estructuras Animales/efectos de los fármacos , Animales , Calcio/metabolismo , Dermis/efectos de los fármacos , Dinoprostona/metabolismo , Perfilación de la Expresión Génica , Metaloproteinasas de la Matriz/metabolismo , Fósforo/metabolismo , Regeneración/efectos de los fármacosRESUMEN
Matrix metalloproteinases (MMPs) are key enzymes in the turnover of extracellular matrix in health, disease, development and regeneration. We have studied zebrafish scale regeneration to ascertain the role of MMP-2 and MMP-9 in these processes. Scales were plucked from the surface of anaesthetised adult male zebrafish, and the scales that regenerated in the scale pocket were recovered at various time points after plucking. Analyses consisted of (i) mmp-9 in situ hybridisation; (ii) MMP-9+TRAcP double-staining; (iii) qRT-PCR for mmp-2 and mmp-9; (iv) zymography for gelatinolytic activity and (v) a hydroxyproline assay. We found that mmp-9 positive cells were confined to the episquamal side of the scales. Ontogenetic scales had irregular clusters of mono- and multinucleated mmp-9 expressing cells along their lateral margins and radii. During regeneration, mmp-9 positive cells were seen on the scale plate, but not along the lateral margins. Double staining for TRAcP and MMP-9 revealed the osteoclastic nature of these cells. During early scale regeneration, mmp-2 and mmp-9 transcripts increased in abundance in the scale, enzymatic MMP activity increased and collagen degradation was detected by means of hydroxyproline measurements. Near the end of regeneration, all of these parameters returned to the basal values seen in ontogenetic scales. These findings suggest that MMPs play an important role in remodelling of the scale plate during regeneration, and that this function resides in mononucleated and multinucleated osteoclasts which co-express TRAcP and mmp-9. Our findings suggest that the fish scale regeneration model may be a useful system in which to study the cells and mechanisms responsible for regeneration, development and skeletal remodelling.