Browsing by Author "Fernandez, Ignacio"
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- Comparative analysis of zebrafish bone morphogenetic proteins 2, 4 and 16: molecular and evolutionary perspectivesPublication . Marques, Cátia L.; Fernandez, Ignacio; Viegas, Michael; Cox, C. J.; Martel, Paulo; Rosa, Joana; Cancela, Leonor; Laizé, VincentBMP2, BMP4 and BMP16 form a subfamily of bone morphogenetic proteins acting as pleiotropic growth factors during development and as bone inducers during osteogenesis. BMP16 is the most recent member of this subfamily and basic data regarding protein structure and function, and spatio-temporal gene expression is still scarce. In this work, insights on BMP16 were provided through the comparative analysis of structural and functional data for zebrafish BMP2a, BMP2b, BMP4 and BMP16 genes and proteins, determined from three-dimensional models, patterns of gene expression during development and in adult tissues, regulation by retinoic acid and capacity to activate BMP-signaling pathway. Structures of Bmp2a, Bmp2b, Bmp4 and Bmp16 were found to be remarkably similar; with residues involved in receptor binding being highly conserved. All proteins could activate the BMP-signaling pathway, suggesting that they share a common function. On the contrary, stage-and tissue-specific expression of bmp2, bmp4 and bmp16 suggested the genes might be differentially regulated (e.g. different transcription factors, enhancers and/or regulatory modules) but also that they are involved in distinct physiological processes, although with the same function. Retinoic acid, a morphogen known to interact with BMP-signaling during bone formation, was shown to downregulate the expression of bmp2, bmp4 and bmp16, although to different extents. Taxonomic and phylogenetic analyses indicated that bmp16 diverged before bmp2 and bmp4, is not restricted to teleost fish lineage as previously reported, and that it probably arose from a whole genomic duplication event that occurred early in vertebrate evolution and disappeared in various tetrapod lineages through independent events.
- Coordinated regulation of chromatophore differentiation and melanogenesis during the ontogeny of skin pigmentation of Solea senegalensis (Kaup, 1858)Publication . Darias, Maria J.; Andree, Karl B.; Boglino, Anais; Fernandez, Ignacio; Estevez, Alicia; Gisbert, EnricAbnormal pigmentation of Senegalese sole has been described as one problem facing the full exploitation of its commercial production. To improve our understanding of flatfish pigmentation of this commercially important species we have evaluated eleven genes related to two different processes of pigmentation: melanophore differentiation, and melanin production. The temporal distribution of gene expression peaks corresponds well with changes in pigmentation patterns and the intensity of skin melanization. Several gene ratios were also examined to put in perspective possible genetic markers for the different stages of normal pigmentation development. Further, the phenotypic changes that occur during morphogenesis correspond well with the main transitions in gene expression that occur. Given the dramatic phenotypic alterations which flatfish undergo, including the asymmetric coloration that occurs between the ocular and the blind side, and the synchrony of the two processes of morphogenesis and pigmentation ontogenesis, these species constitute an interesting model for the study of pigmentation. In this study we present a first approximation towards explaining the genetic mechanisms for regulating pigmentation ontogeny in Senegalese sole, Solea senegalensis.
- Quantitative assessment of the regenerative and mineralogenic performances of the zebrafish caudal finPublication . Cardeira Da Silva, João; Gavaia, Paulo J.; Fernandez, Ignacio; Cengiz, Ibrahim Fatih; Moreira-Silva, Joana; Oliveira, Joaquim Miguel; Reis, Rui L.; Cancela, Leonor; Laizé, VincentThe ability of zebrafish to fully regenerate its caudal fin has been explored to better understand the mechanisms underlying de novo bone formation and to develop screening methods towards the discovery of compounds with therapeutic potential. Quantifying caudal fin regeneration largely depends on successfully measuring new tissue formation through methods that require optimization and standardization. Here, we present an improved methodology to characterize and analyse overall caudal fin and bone regeneration in adult zebrafish. First, regenerated and mineralized areas are evaluated through broad, rapid and specific chronological and morphometric analysis in alizarin red stained fins. Then, following a more refined strategy, the intensity of the staining within a 2D longitudinal plane is determined through pixel intensity analysis, as an indicator of density or thickness/volume. The applicability of this methodology on live specimens, to reduce animal experimentation and provide a tool for in vivo tracking of the regenerative process, was successfully demonstrated. Finally, the methodology was validated on retinoic acid-and warfarin-treated specimens, and further confirmed by micro-computed tomography. Because it is easily implementable, accurate and does not require sophisticated equipment, the present methodology will certainly provide valuable technical standardization for research in tissue engineering, regenerative medicine and skeletal biology.
- The xenobiotic sensor PXR in a marine flatfish species (Solea senegalensis): Gene expression patterns and its regulation under different physiological conditionsPublication . Marques, Carlos; Roberto, Vania Palma; Granadeiro, Luis; Trindade, Marlene; Gavaia, Paulo; Laizé, Vincent; Leonor Cancela, M.; Fernandez, IgnacioThe pregnane X receptor (PXR) is a nuclear receptor belonging to the NR1I sub-family and a known master regulator of xenobiotic metabolism. New roles have been recently proposed in mammals through its activation by vitamin K (VK) such as regulation of glucose metabolism, bone homeostasis, reproduction, neuronal development and cognitive capacities. In marine fish species little is known about PXR and its potential roles. Here, expression patterns of pxr transcripts and conservation of protein domains were determined in the Senegalese sole (Solea senegalensis), a marine flatfish model species in aquatic ecotoxicology. In addition to a full coding sequence transcript (sspxrl), two variants lacking DNA and/or ligand binding domains (sspxr2 and sspxr3) were also identified. The expression of sspxrl during early development and in adult tissues was ubiquitous, but highest levels were observed in liver, intestine and skin. Expression was also detected by in situ hybridization in chondrocytes and cells from the granular and inner nuclear layers in three month old fish. Finally, sspxrl expression was shown to be differentially regulated under physiological conditions related with fasting, VK and warfarin metabolism. The present work provides new and basic knowledge regarding pxr sequence and expression patterns in a marine flatfish species to unveil the potential impact of xenobiotics on marine fish physiology, and will allow a better and more ecosystemic environmental risk assessment of different pollutants over the marine environments with the development of reporter assays using PXR sequences from evolutionary distantly marine species (such as vertebrate and invertebrate marine species). (C) 2017 Elsevier Ltd. All rights reserved.
- Vitamin A affects flatfish development in a thyroid hormone signaling and metamorphic stage dependent mannerPublication . Fernandez, Ignacio; Ortiz-Delgado, Juan B.; Darias, Maria J.; Hontoria, Francisco; Andree, Karl B.; Manchado, Manuel; Sarasquete, Carmen; Gisbert, EnricVitamin A (VA) and retinoid derivatives are known morphogens controlling vertebrate development. Despite the research effort conducted during the last decade, the precise mechanism of how VA induces post-natal bone changes, and particularly those operating through crosstalk with the thyroid hormones (THs) remain to be fully understood. Since effects and mechanisms seem to be dose and time-dependent, flatfish are an interesting study model as they undergo a characteristic process of metamorphosis driven by THs that can be followed by external appearance. Here, we studied the effects of VA imbalance that might determine Senegalese sole (Solea senegalensis) skeletogenetic phenotype through development of thyroid follicles, THs homeostasis and signaling when a dietary VA excess was specifically provided during pre-, pro-or post-metamorphic stages using enriched rotifers and Artemia as carriers. The increased VA content in enriched live prey was associated to a higher VA content in fish at all developmental stages. Dietary VA content clearly affected thyroid follicle development, T3 and T4 immunoreactive staining, skeletogenesis and mineralization in a dose and time-dependent fashion. Gene expression analysis showed that VA levels modified the mRNA abundance of VA- and TH-specific nuclear receptors at specific developmental stages. Present results provide new and key knowledge to better understand how VA and TH pathways interact at tissue, cellular and nuclear level at different developmental periods in Senegalese sole, unveiling how dietary modulation might determine juvenile phenotype and physiology.