RESUMEN
BACKGROUND: The identification of DNA methyltransferases (Dnmt) expression patterns during development and their regulation is important to understand the epigenetic mechanisms that modulate larval plasticity in marine fish. In this study, dnmt1 and dnmt3 paralogs were identified in the flatfish Solea senegalensis and expression patterns in early developmental stages and juveniles were determined. Additionally, the regulation of Dnmt transcription by a specific inhibitor (5-aza-2'-deoxycytidine) and temperature was evaluated. RESULTS: Five paralog genes of dnmt3, namely dnmt3aa, dnmt3ab, dnmt3ba, dnmt3bb.1 and dnmt3bb.2 and one gene for dnmt1 were identified. Phylogenetic analysis revealed that the dnmt gene family was highly conserved in teleosts and three fish-specific genes, dnmt3aa, dnmt3ba and dnmt3bb.2 have evolved. The spatio-temporal expression patterns of four dnmts (dnmt1, dnmt3aa, dnmt3ab and dnmt3bb.1) were different in early larval stages although all of them reduced expression with the age and were detected in neural organs and dnmt3aa appeared specific to somites. In juveniles, the four dnmt genes were expressed in brain and hematopoietic tissues such as kidney, spleen and gills. Treatment of sole embryos with 5-aza-2'-deoxycytidine down-regulated dntm1 and up-regulated dntm3aa. Moreover, in lecithotrophic larval stages, dnmt3aa and dnmt3ab were temperature sensitive and their expression was higher in larvae incubated at 16 °C relative to 20 °C. CONCLUSION: Five dnmt3 and one dnmt1 paralog were identified in sole and their distinct developmental and tissue-specific expression patterns indicate that they may have different roles during development. The inhibitor 5-aza-2'-deoxycytidine modified the transcript abundance of dntm1 and dntm3aa in embryos, which suggests that a regulatory feedback mechanism exists for these genes. The impact of thermal regime on expression levels of dnmt3aa and dnmt3ab in lecithotrophic larval stages suggests that these paralogs might be involved in thermal programing.
Asunto(s)
Proteínas de Peces/genética , Peces Planos/genética , Regulación del Desarrollo de la Expresión Génica/genética , Metiltransferasas/genética , Animales , Metilación de ADN , Inhibidores Enzimáticos/farmacología , Peces Planos/clasificación , Peces Planos/embriología , Peces Planos/crecimiento & desarrollo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/química , Filogenia , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
In marine fish, high epithelial bicarbonate secretion by the intestine generates luminal carbonate precipitates of divalent cations that play a key role in water and ion homeostasis. In vitro studies highlight the involvement of the calciotropic hormones PTHrP (parathyroid hormone-related protein) and stanniocalcin (STC) in the regulation of epithelial bicarbonate transport. The present study tested the hypothesis that calciotropic hormones have a regulatory role in carbonate precipitate formation in vivo. Sea bream (Sparus aurata) juveniles received single intraperitoneal injections of piscine PTHrP(1-34), the PTH/PTHrP receptor antagonist PTHrP(7-34) or purified sea bream STC, or were passively immunized with polyclonal rabbit antisera raised against sea bream STC (STC-Ab). Endocrine effects on the expression of the basolateral sodium bicarbonate co-transporter (Slc4a4.A), the apical anion exchangers Slc26a6.A and Slc26a3.B, and the V-type proton pump ß-subunit (Atp6v1b) in the anterior intestine were evaluated. In keeping with their calciotropic nature, the hypocalcaemic factors PTHrP(7-34) and STC up-regulated gene expression of all transporters. In contrast, the hypercalcaemic factor PTHrP(1-34) and STC antibodies down-regulated transporters involved in the bicarbonate secretion cascade. Changes in intestine luminal precipitate contents provoked by calcaemic endocrine factors validated these results: 24 h post-injection either PTHrP(1-34) or immunization with STC-Ab reduced the carbonate precipitate content in the sea bream intestine. In contrast, the PTH/PTHrP receptor antagonist PTHrP(7-34) increased not only the precipitated fraction but also the concentration of HCO3(-) equivalents in the intestinal fluid. These results confirm the hypothesis that calciotropic hormones have a regulatory role in carbonate precipitate formation in vivo in the intestine of marine fish. Furthermore, they illustrate for the first time in fish the counteracting effect of PTHrP and STC, and reveal an unexpected contribution of calcaemic factors to acid-base balance.
Asunto(s)
Glicoproteínas/metabolismo , Mucosa Intestinal/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Fragmentos de Péptidos/metabolismo , Dorada/metabolismo , Equilibrio Ácido-Base , Animales , Carbonatos/metabolismo , Precipitación Química , Expresión Génica , Glicoproteínas/fisiología , Transporte Iónico , Proteína Relacionada con la Hormona Paratiroidea/fisiología , Fragmentos de Péptidos/fisiologíaRESUMEN
Endocrine disruption results from exposure to chemicals that alter the function of the endocrine system in animals. Chronic 60 days of exposure to a low dose (0.1 µM) of ioxynil (IOX) or diethylstilbestrol (DES) via food was used to determine the effects of these chemicals on the physiology of the heart and thyroid follicles in juvenile zebrafish. Immunofluorescence analysis and subsequent 3D morphometric analysis of the zebrafish heart revealed that chronic exposure to IOX induced ventricle deformation and significant volume increase (p < 0.001). DES exposure caused a change in ventricle morphology, but volume was unaffected. Alongside, it was found that DES exposure upregulated endothelial related genes (angptl1b, mhc1lia, mybpc2a, ptgir, notch1b and vwf) involved in vascular homeostasis. Both IOX and DES exposure caused a change in thyroid follicle morphology. Notably, in IOX exposed juveniles, thyroid follicle hypertrophy was observed; and in DES-exposed fish, an enlarged thyroid field was present. In summary, chronic exposure of juvenile zebrafish to IOX and DES affected the heart and the thyroid. Given that both chemicals are able to change the morphology of the thyroid it indicates that they behave as endocrine disruptive chemicals (EDCs). Heart function dynamically changes thyroid morphology, and function and hence it is likely that the observed cardiac effects of IOX and DES are the source of altered thyroid status in these fish.
Asunto(s)
Disruptores Endocrinos , Pez Cebra , Animales , Dietilestilbestrol/toxicidad , Yodobencenos , Nitrilos , Glándula Tiroides , Pez Cebra/fisiologíaRESUMEN
BACKGROUND: Endocrine disruption is one of the consequences of industrialization and chemicals released into the environment have a profound impact on organisms. Waterborne micromolar concentrations of ioxynil (IOX) and diethylstilbestrol (DES) in fish affect the development of the heart, vasculature and thyroid gland. OBJECTIVES: The present study aimed to determine how IOX and DES disrupt the crosstalk between the developing thyroid gland and cardio-vascular system in zebrafish. METHODS: Twelve hours post fertilization (hpf) wild type, Tg(fli1:GFP) or Tg(cmalc2:GFPCaaX) zebrafish embryos were exposed to 0.1⯵M IOX or DES for 36â¯h (up until 48â¯hpf) or 60â¯h (up until 72â¯hpf). Embryos were used for vascular endothelial cell sorting, whole-mount immunohistochemistry, tissue selective transcriptomics, selected gene expression analysis by quantitative real-time polymerase chain reaction analysis and determination of heart rate by live imaging. RESULTS: Exposure of zebrafish embryos to IOX and DES (0.1⯵M) increased heart beat frequency and reduced ventricle volume and aorta diameter. The transcriptome of endothelial cells from blood vessels of hypertrophic, dilated and arrhythmogenic right ventricular cardiomyopathy was significantly changed and compound-specific toxic effects were found in IOX and DES exposed embryos. Both DES and IOX directly affected vascular and heart development and this indirectly impaired thyroid gland development in zebrafish. Even though the toxicity end-point of the two chemicals was similar, their action seemed to be via different gene regulatory pathways and physiological mechanisms. CONCLUSION: IOX and DES directly disrupt cardiovascular development and there is an associated disruption of thyroid tissue that most likely has long term consequences for this endocrine axis.
Asunto(s)
Sistema Cardiovascular/efectos de los fármacos , Sistema Cardiovascular/embriología , Dietilestilbestrol/toxicidad , Embrión no Mamífero/efectos de los fármacos , Disruptores Endocrinos/toxicidad , Nitrilos/toxicidad , Animales , Sistema Endocrino/efectos de los fármacos , Yodobencenos/toxicidad , Glándula Tiroides/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Pez Cebra/embriologíaRESUMEN
Flatfish metamorphosis is a unique post-embryonic developmental event in which thyroid hormones (THs) drive the development of symmetric pelagic larva into asymmetric benthic juveniles. One of the eyes migrates to join the other eye on the opposite side of the head. Developmental mechanisms at the basis of the acquisition of flatfish anatomical asymmetry remain an open question. Here we demonstrate that an TH responsive asymmetric centre, determined by deiodinase 2 expression, ventrally juxtaposed to the migrating eye in sole (Solea senegalensis) correlates with asymmetric cranial ossification that in turn drives eye migration. Besides skin pigmentation that is asymmetric between dorsal and ventral sides, only the most anterior head region delimited by the eyes becomes asymmetric whereas the remainder of the head and organs therein stay symmetric. Sub-ocular ossification is common to all flatfish analysed to date, so we propose that this newly discovered mechanism is universal and is associated with eye migration in all flatfish.
Asunto(s)
Ojo/crecimiento & desarrollo , Peces Planos/anatomía & histología , Peces Planos/crecimiento & desarrollo , Metamorfosis Biológica , Hormonas Tiroideas/metabolismo , Animales , Ojo/anatomía & histología , Peces Planos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Cráneo/anatomía & histologíaRESUMEN
BACKGROUND: Flatfish metamorphosis is a thyroid hormone (TH) driven process which leads to a dramatic change from a symmetrical larva to an asymmetrical juvenile. The effect of THs on muscle and in particular muscle sarcomer protein genes is largely unexplored in fish. The change in Troponin T (TnT), a pivotal protein in the assembly of skeletal muscles sarcomeres and a modulator of calcium driven muscle contraction, during flatfish metamophosis is studied. RESULTS: In the present study five cDNAs for halibut TnT genes were cloned; three were splice variants arising from a single fast TnT (fTnT) gene; a fourth encoded a novel teleost specific fTnT-like cDNA (AfTnT) expressed exclusively in slow muscle and the fifth encoded the teleost specific sTnT2. THs modified the expression of halibut fTnT isoforms which changed from predominantly basic to acidic isoforms during natural and T4 induced metamorphosis. In contrast, expression of red muscle specific genes, AfTnT and sTnT2, did not change during natural metamorphosis or after T4 treatment. Prior to and after metamorphosis no change in the dorso-ventral symmetry or temporal-spatial expression pattern of TnT genes and muscle fibre organization occurred in halibut musculature. CONCLUSION: Muscle organisation in halibut remains symmetrical even after metamorphosis suggesting TH driven changes are associated with molecular adaptations. We hypothesize that species specific differences in TnT gene expression in teleosts underlies different larval muscle developmental programs which better adapts them to the specific ecological constraints.
Asunto(s)
Lenguado/fisiología , Metamorfosis Biológica/fisiología , Isoformas de Proteínas/metabolismo , Troponina T/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Lenguado/anatomía & histología , Regulación del Desarrollo de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Músculo Esquelético/anatomía & histología , Músculo Esquelético/fisiología , Filogenia , Isoformas de Proteínas/genética , Alineación de Secuencia , Tiroxina/metabolismo , Distribución Tisular , Troponina T/clasificación , Troponina T/genéticaRESUMEN
Thyroid hormones (THs) are essential for embryonic brain development but the genetic mechanisms involved in the action of maternal THs (MTHs) are still largely unknown. As the basis for understanding the underlying genetic mechanisms of MTHs regulation we used an established zebrafish monocarboxylic acid transporter 8 (MCT8) knock-down model and characterised the transcriptome in 25hpf zebrafish embryos. Subsequent mapping of differentially expressed genes using Reactome pathway analysis together with in situ expression analysis and immunohistochemistry revealed the genetic networks and cells under MTHs regulation during zebrafish embryogenesis. We found 4,343 differentially expressed genes and the Reactome pathway analysis revealed that TH is involved in 1681 of these pathways. MTHs regulated the expression of core developmental pathways, such as NOTCH and WNT in a cell specific context. The cellular distribution of neural MTH-target genes demonstrated their cell specific action on neural stem cells and differentiated neuron classes. Taken together our data show that MTHs have a role in zebrafish neurogenesis and suggest they may be involved in cross talk between key pathways in neural development. Given that the observed MCT8 zebrafish knockdown phenotype resembles the symptoms in human patients with Allan-Herndon-Dudley syndrome our data open a window into understanding the genetics of this human congenital condition.
Asunto(s)
Desarrollo Embrionario/genética , Perfilación de la Expresión Génica , Exposición Materna , Hormonas Tiroideas/metabolismo , Transcriptoma , Animales , Desarrollo Embrionario/efectos de los fármacos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Fenotipo , Hormonas Tiroideas/farmacología , Pez CebraRESUMEN
Anuran and flatfish metamorphosis are tightly regulated by thyroid hormones that are the necessary and sufficient factors that drive this developmental event. In the present study whole mount in situ hybridization (WISH) and quantitative PCR in sole are used to explore the central regulation of flatfish metamorphosis. Central regulation of the thyroid in vertebrates is mediated by the hypothalamus-pituitary-thyroid (HPT) axis. Teleosts diverge from other vertebrates as hypothalamic regulation in the HPT axis is proposed to be through hypothalamic inhibition although the regulatory factor remains enigmatic. The dynamics of the HPT axis during sole metamorphosis revealed integration between the activity of the thyrotrophes in the pituitary and the thyroid follicles. No evidence was found supporting a role for thyroid releasing hormone (trh) or corticotrophin releasing hormone (crh) in hypothalamic control of TH production during sole metamorphosis. Intriguingly the results of the present study suggest that neither hypothalamic trh nor crh expression changes during sole metamorphosis and raises questions about the role of these factors and the hypothalamus in regulation of thyrotrophs.
Asunto(s)
Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Peces Planos/crecimiento & desarrollo , Hipotálamo/metabolismo , Glándula Tiroides/metabolismo , Animales , Hormona Liberadora de Corticotropina/genética , Hormona Liberadora de Corticotropina/metabolismo , Peces Planos/metabolismo , Hibridación in Situ , Metamorfosis Biológica , Hipófisis/metabolismo , Reacción en Cadena de la Polimerasa , Tiroglobulina/genética , Tiroglobulina/metabolismo , Hormona Liberadora de Tirotropina/genética , Hormona Liberadora de Tirotropina/metabolismoRESUMEN
Teleost eggs contain an abundant store of maternal thyroid hormones (THs), and early in zebrafish embryonic development, all the genes necessary for TH signaling are expressed. Nonetheless the function of THs in embryonic development remains elusive. To test the hypothesis that THs are fundamental for zebrafish embryonic development, an monocarboxilic transporter 8 (Mct8) knockdown strategy was deployed to prevent maternal TH uptake. Absence of maternal THs did not affect early specification of the neural epithelia but profoundly modified later dorsal specification of the brain and spinal cord as well as specific neuron differentiation. Maternal THs acted upstream of pax2a, pax7, and pax8 genes but downstream of shha and fgf8a signaling. The lack of inhibitory spinal cord interneurons and increased motoneurons in the mct8 morphants is consistent with their stiff axial body and impaired mobility. The mct8 mutations are associated with X-linked mental retardation in humans, and the cellular and molecular consequences of MCT8 knockdown during embryonic development in zebrafish provides new insight into the potential role of THs in this condition.
Asunto(s)
Transportadores de Ácidos Monocarboxílicos/genética , Neurogénesis/genética , Neuronas/citología , Hormonas Tiroideas/metabolismo , Pez Cebra/embriología , Animales , Encéfalo/embriología , Diferenciación Celular/genética , Morfolinos/genética , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box/genética , Transducción de Señal , Médula Espinal/embriología , Receptores alfa de Hormona Tiroidea/biosíntesis , Receptores alfa de Hormona Tiroidea/metabolismo , Receptores beta de Hormona Tiroidea/biosíntesis , Receptores beta de Hormona Tiroidea/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
In the present work, five genes encoding different Na(+),K(+) ATPase (NKA) α-isoforms in the teleost Solea senegalensis are described for the first time. Sequence analysis of predicted polypeptides revealed a high degree of conservation across teleosts and mammals. Phylogenetic analysis clustered the five genes into three main clades: α1 (designated atp1a1a and atp1a1b), α2 (designated atp1a2) and α3 (designated atp1a3a and atp1a3b) isoforms. Transcriptional analysis in larvae showed distinct expression profiles during development. In juvenile tissues, the atp1a1a gene was highly expressed in osmoregulatory organs, atp1a2 in skeletal muscle, atp1a1b in brain and heart and atp1a3a and atp1a3b mainly in brain. Quantification of mRNA abundance after a salinity challenge showed that atp1a1a transcript levels increased significantly in the gill of soles transferred to high salinity water (60 ppt). In contrast, atp1a3a transcripts increased at low salinity (5 ppt). In situ hybridization (ISH) analysis revealed that the number of ionocytes expressing atp1a1a transcripts in the primary gill filaments was higher at 35 and 60 ppt than at 5 ppt and remained undetectable or at very low levels in the lamellae at 5 and 35 ppt but increased at 60 ppt. Immunohistochemistry showed a higher number of positive cells in the lamellae. Whole-mount analysis of atp1a1a mRNA in young sole larvae revealed that it was localized in gut, pronephric tubule, gill, otic vesicle, yolk sac ionocytes and chordacentrum. Moreover, atp1a1a mRNAs increased at mouth opening (3 DPH) in larvae incubated at 36 ppt with a greater signal in gills.
Asunto(s)
Peces Planos/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Peces Planos/crecimiento & desarrollo , Branquias/metabolismo , Larva/crecimiento & desarrollo , Larva/metabolismo , Datos de Secuencia Molecular , Especificidad de Órganos , Presión Osmótica , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Salinidad , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Some environmental contaminants are thought to cause disruption of the thyroid system in vertebrates acting as endocrine disrupting chemicals (EDCs). Such chemicals may affect synthesis, transport and metabolism of thyroid hormones (THs). Ioxynil (IOX) and diethylstilbestrol (DES) are potential EDCs with strong affinity in vitro for sea bream transthyretin (TTR), a TH distributor protein (THDP). The aim of the present study was to establish how such chemicals influence the thyroid axis in sea bream (Sparus aurata). DES, IOX and propilthyouracil (PTU, a goitrogen) were administered in the diet to sea bream juveniles at 1 mg/kg fish (n = 14/treatment) for 21 days. After exposure plasma TH levels, quantified by RIA, were similar to those of control fish (p > 0.05) in all treatment groups. Analysis by quantitative PCR revealed that all treatments down-regulated TSH gene transcription (p < 0.05) in the brain and pituitary and deiodinase II and III transcription in the brain (p < 0.001). In contrast, PTU caused DII up-regulation in the liver (p < 0.05). Thyroid receptor beta (TRbeta) transcription was down-regulated in the pituitary by PTU (p < 0.001) and DES (p < 0.05). TTR plasma levels, quantified by ELISA, were elevated by all the chemicals including PTU (p < 0.001) which also increased TTR gene transcription in the liver (p < 0.05). Thyroid histology indicated follicular hyperstimulation in all treatments with marked hyperplasia, hypertrophy and colloid depletion in the PTU group. It appears therefore, that in vitro TTR-binders, IOX and DES, can strongly influence several components of the fish thyroid system in vivo but that the thyroid axis may have the ability to maintain or re-establish plasma TH homeostasis.
Asunto(s)
Dietilestilbestrol/toxicidad , Nitrilos/toxicidad , Dorada/fisiología , Glándula Tiroides/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Yodobencenos/toxicidad , Prealbúmina/análisis , Hormonas Tiroideas/sangreRESUMEN
Developmental models for skin exist in terrestrial and amphibious vertebrates but there is a lack of information in aquatic vertebrates. We have analysed skin epidermal development of a bony fish (teleost), the most successful group of extant vertebrates. A specific epidermal type I keratin cDNA (hhKer1), which may be a bony-fish-specific adaptation associated with the divergence of skin development (scale formation) compared with other vertebrates, has been cloned and characterized. The expression of hhKer1 and collagen 1alpha1 in skin taken together with the presence or absence of keratin bundle-like structures have made it possible to distinguish between larval and adult epidermal cells during skin development. The use of a flatfish with a well-defined larval to juvenile transition as a model of skin development has revealed that epidermal larval basal cells differentiate directly to epidermal adult basal cells at the climax of metamorphosis. Moreover, hhKer1 expression is downregulated at the climax of metamorphosis and is inversely correlated with increasing thyroxin levels. We suggest that, whereas early mechanisms of skin development between aquatic and terrestrial vertebrates are conserved, later mechanisms diverge.