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  • Molecular and cellular changes in skin and muscle during metamorphosis of Atlantic halibut (Hippoglossus hippoglossus) are accompanied by changes in deiodinases expression
    Publication . Campinho, Marco António; Galay-Burgos, M.; Silva, Nádia; Costa, R. A.; Alves, Ricardo N.; Sweeney, Glen E.; Power, Deborah
    Flatfish metamorphosis is the most dramatic postnatal developmental event in teleosts. Thyroid hormones (TH), thyroxine (T4) and 3,3′-5′-triiodothyronine (T3) are the necessary and sufficient factors that induce and regulate flatfish metamorphosis. Most of the cellular and molecular action of TH is directed through the binding of T3 to thyroid nuclear receptors bound to promoters with consequent changes in the expression of target genes. The conversion of T4 to T3 and nuclear availability of T3 depends on the expression and activity of a family of 3 selenocysteine deiodinases that activate T4 into T3 or degrade T4 and T3.
  • A thyroid hormone regulated asymmetric responsive centre is correlated with eye migration during flatfish metamorphosis
    Publication . Campinho, Marco António; Silva, Nádia; Martins, Gabriel G.; Anjos, Liliana; Florindo, Claudia; Roman-Padilla, Javier; Garcia-Cegarra, Ana; Louro, Bruno; Manchado, Manuel; Power, Deborah
    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.
  • Maternal thyroid hormones are essential for neural development in Zebrafish
    Publication . Campinho, Marco António; Saraiva, João; Florindo, Claudia; Power, Deborah M.
    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.
  • Endocrine regulation of carbonate precipitate formation in marine fish intestine by stanniocalcin and PTHrP
    Publication . Gregorio, Silvia F.; Carvalho, Edison S. M.; Campinho, Marco A.; Power, Deborah M.; Canario, Adelino V. M.; Fuentes, Juan
    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 cotransporter (Slc4a4.A), the apical anion exchangers Slc26a6.A and Slc26a3.B, and the V-type proton pump beta-subunit (Atp6v1b) in the anterior intestine were evaluated. In keeping with their calciotropic nature, the hypocalcaemic factors PTHrP(7-34) and STC upregulated gene expression of all transporters. In contrast, the hypercalcaemic factor PTHrP(1-34) and STC antibodies downregulated transporters involved in the bicarbonate secretion cascade. Changes in intestine luminal precipitate contents provoked by calcaemic endocrine factors validated these results: 24 h postinjection 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.
  • Biological characterization of Cynara cardunculus L. Methanolic extracts: antioxidant, anti-proliferative, anti-migratory and anti-angiogenic activities
    Publication . Velez, Zélia; Campinho, Marco António; Guerra, Ângela R.; García, Laura; Ramos, Patricia; Guerreiro, Olinda; Felício, Laura; Schmitt, Fernando; Duarte, Maria
    Cynara cardunculus (Cc) is a multipurpose species; beyond its use in southwestern European cuisine, it is also used for the production of solid biofuel, seed oil, biodiesel, paper pulp and cheese, as well as animal feed. In addition, Cc has a long tradition of use in folk medicine as a diuretic and liver protector. The value of this species as a source of bioactive compounds is known; however, pharmacological use would further increase its cultivation. The main goal of the current work was to evaluate the potential of Cc as source of anti-carcinogenic phytochemicals. Different methanolic extracts obtained from wild and cultivated plants were tested for antioxidant activity and effect on breast tumor cell viability. The most effective extract, both as antioxidant and inhibition of tumor cell viability, was tested for effects on angiogenesis and tumor cell migration capacity. All the extracts tested had high antioxidant activity; however, only green leaves and dry head extracts exhibit anti-proliferative activity. Green cultivated leaves (GCL) were the most effective extract both as antioxidant and inhibiting the proliferation of tumor cells; it is equally active inhibiting tumor cell migration and in vivo angiogenesis. GCL extract is an effective inhibitor of several key points in tumor development and thus a promising source of anti-carcinogenic phytochemicals.
  • The goitrogenic efficiency of thioamides in a marine teleost, sea bream (Sparus auratus)
    Publication . Campinho, Marco António; Morgado, Isabel; Pinto, Patricia IS; Silva, Nádia; Power, Deborah
    Studies on the role of thyroid hormones (THs) in teleost fish physiology have deployed the synthetic goitrogens, methimazol (MMI), propilthiouracil (PTU) and thiourea (TU) that are used to treat human hyperthyroidism. However, the action of the goitrogens, MMI, PTU and TU at different levels of the hypothalamic–pituitary–thyroid (HPT) axis in teleosts is largely unknown. The central importance of the hypothalamus and pituitary in a number of endocrine regulated systems and the cross-talk that occurs between different endocrine axes makes it pertinent to characterize the effects of MMI, PTU and TU, on several endpoints of the thyroid system. The marine teleost, sea bream (Sparus auratus) was exposed to MMI, PTU and TU (1 mg/kg wet weight per day), via the diet for 21 days. Radioimmunoassays (RIA) of plasma THs and ELISA of the TH carrier transthyretin (TTR) revealed that MMI was the only chemical that significantly reduced plasma TH levels (p < 0.05), although both MMI and PTU significantly (p < 0.05) reduced plasma levels of circulating TTR (p < 0.05). Histological analysis of the thyroid tissue revealed modifications in thyrocyte activity that explain the reduced circulating levels of THs. MMI also significantly (p < 0.05) up-regulated transcript abundance of liver deiodinase 1 and 2 while significantly (p < 0.05) decreasing TRb expression in the pituitary, all hallmarks of HPT axis action of goitrogens in vertebrates. The results indicate that in the sea bream MMI is the most effective goitrogen followed by PTU and that TU (1 mg/kg wet weight for 21 days) failed to have a goitrogenic effect. The study highlights the non-uniform effect of goitrogens on the thyroid axis of sea bream and provides the basis for future studies of thyroid disrupting pollutants.
  • Phylogeny, expression patterns and regulation of DNA Methyltransferases in early development of the flatfish, Solea senegalensis
    Publication . Firmino, Joana; Carballo, Carlos; Armesto, Paula; Campinho, Marco António; Power, Deborah M.; Manchado, Manuel
    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 degrees C relative to 20 degrees 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.
  • Ioxynil and diethylstilbestrol disrupt vascular and heart development in zebrafish
    Publication . Li, Yi-Feng; Canario, Adelino; Power, Deborah; Campinho, Marco António
    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.
  • Transcriptomics reveal an integrative role for maternal thyroid hormones during zebrafish embryogenesis
    Publication . Silva, Nadia; Louro, Bruno; Trindade, Marlene; Power, Deborah M.; Campinho, Marco A.
    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.
  • Teleost metamorphosis: the role of thyroid hormone
    Publication . Campinho, Marco António
    In most teleosts, metamorphosis encompasses a dramatic post-natal developmental process where the free-swimming larvae undergo a series of morphological, cellular and physiological changes that enable the larvae to become a fully formed, albeit sexually immature, juvenile fish. In all teleosts studied to date thyroid hormones (TH) drive metamorphosis, being the necessary and sufficient factors behind this developmental transition. During metamorphosis, negative regulation of thyrotropin by thyroxine (T4) is relaxed allowing higher whole-body levels of T4 that enable specific responses at the tissue/cellular level. Higher local thyroid cellular signaling leads to cell-specific responses that bring about localized developmental events. TH orchestrate in a spatial-temporal manner all local developmental changes so that in the end a fully functional organism arises. In bilateral teleost species, the most evident metamorphic morphological change underlies a transition to a more streamlined body. In the pleuronectiform lineage (flatfishes), these metamorphic morphological changes are more dramatic. The most evident is the migration of one eye to the opposite side of the head and the symmetric pelagic larva development into an asymmetric benthic juvenile. This transition encompasses a dramatic loss of the embryonic derived dorsal-ventral and left-right axis. The embryonic dorsal-ventral axis becomes the left-right axis, whereas the embryonic left-right axis becomes, irrespectively, the dorsal-ventral axis of the juvenile animal. This event is an unparalleled morphological change in vertebrate development and a remarkable display of the capacity of TH-signaling in shaping adaptation and evolution in teleosts. Notwithstanding all this knowledge, there are still fundamental questions in teleost metamorphosis left unanswered: how the central regulation of metamorphosis is achieved and the neuroendocrine network involved is unclear; the detailed cellular and molecular events that give rise to the developmental processes occurring during teleost metamorphosis are still mostly unknown. Also in flatfish, comparatively little is still known about the developmental processes behind asymmetric development. This review summarizes the current knowledge on teleost metamorphosis and explores the gaps that still need to be challenged.