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Research Project
AQUATOX - Assessment of mineralogenic toxicity of aquatic pollutants
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Publications
The zebrafish operculum: a powerful system to assess osteogenic bioactivities of molecules with pharmacological and toxicological relevance
Publication . Tarasco, Marco; Laizé, Vincent; Cardeira, João; Leonor Cancela, M.; Gavaia, Paulo
Bone disorders affect millions of people worldwide and available therapeutics have a limited efficacy, often presenting undesirable side effects. As such, there is a need for novel molecules with bone anabolic properties. The aim of this work was to establish a rapid, reliable and reproducible method to screen for molecules with osteogenic activities, using the zebrafish operculum to assess bone formation. Exposure parameters were optimized through morphological analysis of the developing operculum of larvae exposed to calcitriol, a molecule with known pro-osteogenic properties. An exposure of 3 days initiated at 3 days post-fertilization was sufficient to stimulate operculum formation, while not affecting survival or development of the larvae. Dose dependent pro- and anti-osteogenic effects of calcitriol and cobalt chloride, respectively, demonstrated the sensitivity of the method and the suitability of the operculum system. A double transgenic reporter line expressing fluorescent markers for early and mature osteoblasts was used to gain insights into the effects of calcitriol and cobalt at the cellular level, with osteoblast maturation shown to be stimulated and inhibited, respectively, in the operculum of exposed fish. The zebrafish operculum represents a consistent, robust and rapid screening system for the discovery of novel molecules with osteogenic, anti-osteoporotic or osteotoxic activity.
Quantitative assessment of the regenerative and mineralogenic performances of the zebrafish caudal fin
Publication . Cardeira Da Silva, João; Gavaia, Paulo J.; Fernandez, Ignacio; Cengiz, Ibrahim Fatih; Moreira-Silva, Joana; Oliveira, Joaquim Miguel; Reis, Rui L.; Cancela, Leonor; Laizé, Vincent
The 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 conditions
Publication . Marques, Carlos; Roberto, Vania Palma; Granadeiro, Luis; Trindade, Marlene; Gavaia, Paulo; Laizé, Vincent; Leonor Cancela, M.; Fernandez, Ignacio
The 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.
Anti-osteogenic activity of cadmium in zebrafish
Publication . Tarasco, Marco; Cardeira Da Silva, João; Viegas, Michael; Caria, Joana; Martins, Gil; Gavaia, Paulo; Cancela, M. Leonor; Laizé, Vincent
Among the many anthropogenic chemicals that end up in the aquatic ecosystem,
heavy metals, in particular cadmium, are hazardous compounds that have been shown to affect
developmental, reproductive, hepatic, hematological, and immunological functions in teleost fish.
There is also evidence that cadmium disturbs bone formation and skeletal development, but data is
scarce. In this work, zebrafish was used to further characterize the anti-osteogenic/osteotoxic effects
of cadmium and gain insights into underlying mechanisms. Upon exposure to cadmium, a reduction
of the opercular bone growth was observed in 6-days post-fertilization (dpf) larvae and an increase in
the incidence of skeletal deformities was evidenced in 20-dpf post-larvae. The extent and stiffness
of newly formed bone was also affected in adult zebrafish exposed to cadmium while regenerating
their caudal fin. A pathway reporter assay revealed a possible role of the MTF-1 and cAMP/PKA
signaling pathways in mechanisms of cadmium osteotoxicity, while the expression of genes involved
in osteoblast differentiation and matrix production was strongly reduced in cadmium-exposed
post-larvae. This work not only confirmed cadmium anti-osteogenic activity and identified targeted
pathways and genes, but it also suggested that cadmium may affect biomechanical properties of bone.
Isolation, culture, and differentiation of Blastema cells from the regenerating caudal fin of zebrafish
Publication . Vijayakumar, Parameswaran; Cancela, M. Leonor; Laizé, Vincent
The caudal fin of teleost fish has become an excellent system for investigating the mechanisms
of epimorphic regeneration. Upon amputation of the caudal fin, a mass of undi erentiated cells,
called blastema, proliferate beneath the wound-epidermis and di erentiate into various cell types to
faithfully restore the missing fin structures. Here we describe a protocol that can be used to isolate
and culture blastema cells from zebrafish. Primary cultures were initiated from 36 h post-amputation
(hpa) blastema and optimal cell growth was achieved using L-15 medium supplemented with 5%
fetal bovine serum in plates either coated with fibronectin or uncoated. After seeding, zebrafish
blastema cells formed a uniform culture and exhibited polygonal shapes with prominent nucleus,
while various cell types were also observed after few days in culture indicating cell di erentiation.
Upon treatment with all-trans retinoic acid, zebrafish blastema cells di erentiated into neuron-like and
oligodendritic-like cells. Immunocytochemistry data also revealed the presence of mesenchymal and
neuronal cells. The availability of blastema cell cultures could contribute to a better understanding of
epimorphic regeneration by providing a mean to investigate the mechanisms underlying blastema
cell di erentiation. Furthermore, this protocol is simple, rapid, and cost-e cient, and can be virtually
applied to the development of any fish blastema cell culture.
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Funding agency
Fundação para a Ciência e a Tecnologia
Funding programme
3599-PPCDT
Funding Award Number
PTDC/MAR/112992/2009