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See detailTemperature preference of Nile tilapia (Oreochromis niloticus) juveniles induces spontaneous sex reversal
Nivelle, Renaud ULiege; Gennotte, Vincent ULiege; Muller, Marc ULiege et al

in PLoS ONE (2019)

Nile tilapia (Oreochromis niloticus) is an African freshwater fish that displays a genetic sex determination system (XX|XY) where high temperatures (above 32°C to 36.5°C) induce masculinization. In Nile ... [more ▼]

Nile tilapia (Oreochromis niloticus) is an African freshwater fish that displays a genetic sex determination system (XX|XY) where high temperatures (above 32°C to 36.5°C) induce masculinization. In Nile tilapia, the thermosensitive period was reported from 10 to 30 days post fertilization. In their natural environment, juveniles may encounter high temperatures that are above the optimal temperature for growth (27–30°C). The relevance of the thermal sex reversal mechanism in a natural context remains unclear. The main objective of our study is to determine whether sexually undifferentiated juveniles spontaneously prefer higher, unfavorable temperatures and whether this choice skews the sex ratio toward males. Five full-sib progenies (from 100% XX crosses) were subjected to (1) a horizontal three-compartment thermal step gradient (thermal continuum 28°C– 32°C– 36.5°C) during the thermosensitive period, (2) a control continuum (28°C– 28°C– 28°C) and (3) a thermal control tank (36.5°C). During the first days of the treatment, up to an average of 20% of the population preferred the masculinizing compartment of the thermal continuum (36.5°C) compared to the control continuum. During the second part of the treatment, juveniles preferred the lower, nonmasculinizing 32°C temperature. This short exposure to higher temperatures was sufficient to significantly skew the sex ratio toward males, compared to congeners raised at 28°C (from 5.0 ± 6.7% to 15.6 ± 16.5% of males). The proportion of males was significantly different in the thermal continuum, thermal control tank and control continuum, and it was positively correlated among populations. Our study shows for the first time that Nile tilapia juveniles can choose a masculinizing temperature during a short period of time. This preference is sufficient to induce sex reversal to males within a population. For the first time, behavior is reported as a potential player in the sex determination mechanism of this species. [less ▲]

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See detailIndividual identification and marking techniques for zebrafish
Delcourt, Johann ULiege; Ovidio, Michaël ULiege; Denoël, Mathieu ULiege et al

in Reviews in Fish Biology and Fisheries (2018), 28(4), 839-864

In laboratory fish research, the zebrafish Danio rerio (Cyprinidae) represents the equivalent of the mouse in mammalian research. This species has become a major model for studies in developmental and ... [more ▼]

In laboratory fish research, the zebrafish Danio rerio (Cyprinidae) represents the equivalent of the mouse in mammalian research. This species has become a major model for studies in developmental and behavioural genetics, neurophysiology, biomedicine, ecotoxicology, and behavioural and evolutionary ecology. To meet the need for accurate and reproducible data in both fundamental and applied sciences, it is of primary importance to be able to tag and/or recognize individual zebrafish. However, classic methods used in fish ecology and aquaculture are generally difficult to apply to such small fish. Recently, various new tagging methods have been developed. This paper presents a first review of current identification and marking methods applied to zebrafish, from external observation methods (such as skin pattern recognition, fin clipping, scale regeneration, colour and transgenic methods) to the most advanced technological developments in electronic (low- and high- radio-frequencies PIT tags, microchip) and image analysis methods (video tracking). This review aims to help researchers and zebrafish facility managers select the identification method (ID) best adapted to their needs. The main characteristics of each ID method are examined (including detection range, durability, speed and repetitiveness, ID code combination, size dependence and ethical considerations), and their pros and cons are summarized in a decision table to help select the most appropriate option for a research or management program. Finally, contextual applications of these ID methods and future developments are discussed. [less ▲]

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See detailA realistic mixture of Persistent Organic Pollutants (POPs) reveals possible synergism to inhibit the transactivation activities of the rat Aryl hydrocarbon Receptor (rAhR) in vitro
Doan Thi Que, ULiege; Muller, Marc ULiege; Berntsen, HF et al

in Toxicology Letters (2018, October 01)

While organisms are exposed to mixtures of persistent organic pollutants (POPs), scientific studies usually focus on the toxicity of a single compound at a time and few have addressed the mixture effect ... [more ▼]

While organisms are exposed to mixtures of persistent organic pollutants (POPs), scientific studies usually focus on the toxicity of a single compound at a time and few have addressed the mixture effect. This study aims to determine how a realistic mixture of POPs can affect transactivation of the rat Aryl hydrocarbon Receptor (rAhR) in vitro. Luciferase reporter Dioxin responsive rat hepatoma cell lines (DR-H4IIE) were used to screen both rAhR agonistic and antagonistic activities of 29 compounds: six perfluorinated (PFAA), seven brominated (Br), and 16 chlorinated (Cl) compounds (seven polychlorinated biphenyls (PCBs) and nine organochlorine pesticides) listed as POPs under the 2001 Stockholm Convention. Only 5 (2 Cl and 3 Br) out of the 29 compounds presented rAhR agonistic activities while 16 (13 Cl and 3 Br) were rAhR antagonists. No effect was observed for PFAAs. To test possible interactions between these compounds, a mixture of these 29 POPs and six sub-mixtures (PFAA, Br, Cl, Cl + Br, Cl + PFAA and Br + PFAA), prepared based on the respective concentrations found in Scandinavian human blood with a normal daily intake, were tested for the same activities. Not surprisingly, POP mixture also displayed a rAhR antagonistic activity (IC50 = 371 ± 52 times the blood level) with the lowest effective concentration found at 75-time blood level. This level could be plausibly reached in humans after a food contamination incident or in highly exposed sub-populations. Testing the sub-mixtures showed that the Cl mixture is responsible for the antagonism of the POP mixture, contributing to 80% of the POP response. When DR-H4IIE cells were exposed to the Cl + PFAA mixture, the antagonist level was the same as the response of the POP mixture. This indicates that PFAAs are probably non-specific rAhR antagonists as they did not induce any antagonist response when tested alone. The IC50 of the Cl mixture calculated from the measured IC50 of all 13 active chlorinated compounds, using an additive model, was about the same as the measured IC50, 1.9 M and 2.3 M, respectively. This suggests that these compounds act additively in the Cl mixture. In contrast, the calculated and measured IC50 for the total POP mixture were 22 M and 43.2 M, respectively, along with non-specific rAhR antagonism of PFAA mixture, indicating a possible synergistic effect. [less ▲]

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See detailLandmark detection in 2D bioimages for geometric morphometrics: a multi-resolution tree-based approach.
Vandaele, Rémy ULiege; Aceto, Jessica; Muller, Marc ULiege et al

in Scientific Reports (2018), 8(1), 538

The detection of anatomical landmarks in bioimages is a necessary but tedious step for geometric morphometrics studies in many research domains. We propose variants of a multi-resolution tree-based ... [more ▼]

The detection of anatomical landmarks in bioimages is a necessary but tedious step for geometric morphometrics studies in many research domains. We propose variants of a multi-resolution tree-based approach to speed-up the detection of landmarks in bioimages. We extensively evaluate our method variants on three different datasets (cephalometric, zebrafish, and drosophila images). We identify the key method parameters (notably the multi-resolution) and report results with respect to human ground truths and existing methods. Our method achieves recognition performances competitive with current existing approaches while being generic and fast. The algorithms are integrated in the open-source Cytomine software and we provide parameter configuration guidelines so that they can be easily exploited by end-users. Finally, datasets are readily available through a Cytomine server to foster future research. [less ▲]

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See detailMass spectrometry imaging of small xenobiotics on Danio rerio : influence of molecular profiles modification as potential localization asset
Tiquet, Mathieu ULiege; Muller, Marc ULiege; De Pauw, Edwin ULiege

Poster (2017, June 02)

MALDI Mass spectrometry often fail to locate small xenobiotics present in low concentration in tissues due to ion suppression effect. This new method compare tissues of contaminated zebrafish to controles ... [more ▼]

MALDI Mass spectrometry often fail to locate small xenobiotics present in low concentration in tissues due to ion suppression effect. This new method compare tissues of contaminated zebrafish to controles with the statistical tool called receiver operating characteristic. Results cannot directly locate the xenobiotic but can indicate which tissues are affected by the contamination and thus give a hint on the biolocalization. [less ▲]

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See detailBasic Helix loop Helix transcription factor twist1a and twist1b and their involvement in the skeletal development of zebrafish.
Zappia, Jérémie ULiege; Windhausen, Thomas ULiege; Renn, Jörg ULiege et al

Poster (2017, May)

1. Objective The twist1a and twist1b genes code for transcription factors involved during embryogenesis in the craniofacial development. The human ortholog of twist1a and twist1b, TWIST1 has been shown to ... [more ▼]

1. Objective The twist1a and twist1b genes code for transcription factors involved during embryogenesis in the craniofacial development. The human ortholog of twist1a and twist1b, TWIST1 has been shown to be mutated in cases of the Saethre–Chotzen syndrome (SCS). However, the roles of these genes in skeletal development in zebrafish remain to be clarified. The aim of our research is to characterize the effects of the knock-down of twist1a and twist1b in the zebrafish. 2. Methods Antisens morpholino are used for the knock-down of twist1a and twist1b (respectively MoTwist1a and MoTwist1b). The sox9a expression is investigated using whole amount in situ hybridization. 3. Results We show the spatio-temporal expression pattern of twist1a and twist1b during the first 6 days of zebrafish development and present the effects observed on cartilage and bone formation upon knock-down of these genes in zebrafish larvae. The results reveal a decrease of the cranial cartilage and bone formation in both MoTwist1a and MoTwist1b. Interestingly, when MoTwist1a and MoTwist1b are co-injected, the cranial cartilage formation is strongly reduced, while ectopic cartilage formation appeared in the front of the head. We also show an increase in expression of sox9a, a master regulator of chondrogenesis, in morphants. 4. Summary and Conclusions Even though it was shown that twist1a and twist1b maintain a pool of osteoprogenitor-like cells, we show that a knock-down of these genes induces a decrease of skeletal formation. Our hypothesis is that the morphants run out of progenitor cells to assure correct bone and cartilage formation. In the future, we plan to generate twist1a mutants in order to further characterize the effect of this mutation. The twist1a mutant will eventually serve for a rescue study using human TWIST1 mRNA, wild-type or mutant in order to have a better understanding of the human disorder SCS. [less ▲]

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See detailReplication Errors Made During Oogenesis Lead to Detectable De Novo mtDNA Mutations in Zebrafish Oocytes with a Low mtDNA Copy Number.
Otten, Auke B. C.; Stassen, Alphons P. M.; Adriaens, Michiel et al

in Genetics (2016), 204(4), 1423-1431

Of all pathogenic mitochondrial DNA (mtDNA) mutations in humans, ~25% is de novo, although the occurrence in oocytes has never been directly assessed. We used next generation sequencing to detect point ... [more ▼]

Of all pathogenic mitochondrial DNA (mtDNA) mutations in humans, ~25% is de novo, although the occurrence in oocytes has never been directly assessed. We used next generation sequencing to detect point mutations directly in the mtDNA of 3-15 individual mature oocytes and three somatic tissues from eight zebrafish females. Various statistical and biological filters allowed reliable detection of de novo variants with heteroplasmy >/=1.5%. In total, we detected 38 de novo base substitutions, but no insertions or deletions. These 38 de novo mutations were present in 19 of 103 mature oocytes, indicating that ~20% of the mature oocytes carry at least one de novo mutation with heteroplasmy >/=1.5%. This frequency of de novo mutations is close to that deducted from the reported error rate of polymerase gamma, the mitochondrial replication enzyme, implying that mtDNA replication errors made during oogenesis are a likely explanation. Substantial variation in the mutation prevalence among mature oocytes can be explained by the highly variable mtDNA copy number, since we previously reported that ~20% of the primordial germ cells have a mtDNA copy number of </=73 and would lead to detectable mutation loads. In conclusion, replication errors made during oogenesis are an important source of de novo mtDNA base substitutions and their location and heteroplasmy level determine their significance. [less ▲]

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See detailToxicity of organometal halide perovskite solar cells
Babayigit, Aslihan; Ethirajan, Anitha; Muller, Marc ULiege et al

in Nature Materials (2016), 15

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See detailAssessing the toxicity of Pb- and Sn-based perovskite solar cells in model organism Danio rerio
Babayigit, Aslihan; Dinh Duy Thanh, ULiege; Ethirajan, Anitha et al

in Scientific Reports (2016), 6

Intensive development of organometal halide perovskite solar cells has lead to a dramatic surge in power conversion efficiency up to 20%. Unfortunately, the most efficient perovskite solar cells all ... [more ▼]

Intensive development of organometal halide perovskite solar cells has lead to a dramatic surge in power conversion efficiency up to 20%. Unfortunately, the most efficient perovskite solar cells all contain lead (Pb), which is an unsettling flaw that leads to severe environmental concerns and is therefore a stumbling block envisioning their large-scale application. Aiming for the retention of favorable electro-optical properties, tin (Sn) has been considered the most likely substitute. Preliminary studies have however shown that Sn-based perovskites are highly unstable and, moreover, Sn is also enlisted as a harmful chemical, with similar concerns regarding environment and health. To bring more clarity into the appropriateness of both metals in perovskite solar cells, we provide a case study with systematic comparison regarding the environmental impact of Pb- and Sn-based perovskites, using zebrafish (Danio Rerio) as model organism. Uncovering an unexpected route of intoxication in the form of acidification, it is shown that Sn based perovskite may not be the ideal Pb surrogate. [less ▲]

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See detailDifferences in Strength and Timing of the mtDNA Bottleneck between Zebrafish Germline and Non-germline Cells.
Otten, Auke BC; Theunissen, Tom EJ; Derhaag, Josien G et al

in Cell Reports (2016)

We studied the mtDNA bottleneck in zebrafish to elucidate size, timing, and variation in germline and non-germline cells. Mature zebrafish oocytes contain, on average, 19.0 3 106 mtDNA molecules with high ... [more ▼]

We studied the mtDNA bottleneck in zebrafish to elucidate size, timing, and variation in germline and non-germline cells. Mature zebrafish oocytes contain, on average, 19.0 3 106 mtDNA molecules with high variation between oocytes. During embryo- genesis, the mtDNA copy number decreases to $170 mtDNA molecules per primordial germ cell (PGC), a number similar to that in mammals, and to $50 per non-PGC. These occur at the same developmental stage, implying considerable variation in mtDNA copy number in (non-)PGCs of the same female, dictated by variation in the mature oocyte. The pres- ence of oocytes with low mtDNA numbers, if similar in humans, could explain how (de novo) mutations can reach high mutation loads within a single gener- ation. High mtDNA copy numbers in mature oocytes are established by mtDNA replication during oocyte development. Bottleneck differences between germ- line and non-germline cells, due to early differentia- tion of PGCs, may account for different distribution patterns of familial mutations. [less ▲]

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See detailEffects of microgravity simulation on zebrafish transcriptomes and bone physiology; exposure starting at 5 days post-fertilization.
Aceto, Jessica ULiege; Nourizadeh-Lillabadi, Rasoul; Bradamante, Silvia et al

in NPJ Microgravity (2016), 2

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See detailEndocrine activity of mycotoxins and mycotoxin mixtures
Demaegdt; Daminet; Evrard et al

in Food and Chemical Toxicology (2016), 96

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See detailPb-based versus Sn-based perovskite solar cells: Toxicity and environmental burden
Babayigit, Aslihan; Dinh Duy Thanh, ULiege; Ethirajan, Anitha et al

Poster (2015, May)

Organometal halide perovskites have rapidly evolved into strong contenders to compete with silicon in the quest for low-cost photovoltaics, with their added value being solution-processability. Their ... [more ▼]

Organometal halide perovskites have rapidly evolved into strong contenders to compete with silicon in the quest for low-cost photovoltaics, with their added value being solution-processability. Their primary drawback, however, is that so far the presence of lead (Pb) is required to obtain the highly favorable electro-optical properties of the most successful perovskite crystals such as CH3NH3PbI3. Together with their tendency to degrade under the influence of humidity, and the corresponding disintegration of the unit cell, this implies that Pb compounds can be released into the environment upon failure of a perovskite module. As already known from literature, Pb is a rather toxic element causing irreversible neurological, nephrotic and hepatic damage. Hence, finding a non-harmful alternative metal, exhibiting similar electro-optical characteristics in the resulting perovskite crystal, could be the solution to improve and ultimately commercialize perovskite-based solar cells. Tin (Sn), also being a group IV metal, has been deemed the most appropriate alternative. However, Sn is also enlisted as a harmful chemical. Animal and human volunteer studies have shown that toxicity symptoms like fever, nausea, nephropathy, etc. emerge upon excessive uptake, raising question marks regarding the suitability of Sn as a more environmentally friendly alternative to Pb in perovskite solar cells. This contribution aims to make a first step towards the assessment of the environmental burden of both Pb and Sn based solar cells in the form of a toxicity study. Well-established aquatic model organisms are exposed to the appropriate degradation products, according to well-defined guidelines of the Organization for Economic Co-operation and Development (OECD). This allows a systematic comparison of Sn and Pb-containing decayed compounds regarding their potentially harmful effects on the environment, and sheds light onto the applicability of both corresponding perovskite families in large-scale photovoltaic systems. [less ▲]

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See detailEffects of some controversial food additives on zebrafish embryonic development
Dinh Duy Thanh, ULiege; Voncken, Audrey; Curé, Yoann et al

Poster (2015, January 27)

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See detailEstrogenic Evaluation and Organochlorine Identification in Blubber of North Sea Harbour Porpoise (Phocoena phocoena) Stranded on the North Sea Coast
Didimo Imazaki, Pedro Henrique ULiege; Brose, François ULiege; Jauniaux, Thierry ULiege et al

in BioMed Research International (2015), 2015(Article ID 438295), 13

Thirteen individual organochlorine compounds at 3 concentrations (80, 400, and 2000 ng/mL culture medium), as well as mixtures, were assayed for the estrogen receptor (ER) activation or inhibition, using ... [more ▼]

Thirteen individual organochlorine compounds at 3 concentrations (80, 400, and 2000 ng/mL culture medium), as well as mixtures, were assayed for the estrogen receptor (ER) activation or inhibition, using a luciferase reporter gene assay (RGA). None of the PCB 138, 153, or 180 or their mixture induced a response in the RGA. o,p'-DDT was the most potent xenoestrogen fromthe DDT group, inducing a response already at 80 ng/mL. From the HCH and HCB group, only 𝛽-HCH (at 400 and 2000 ng/mL) and 𝛿-HCH (at 2000 ng/mL) displayed estrogenic activities.These 13 organochlorines were determined by GC-MS in 12 samples of North Sea harbor porpoise blubber. The PCBs were the main contaminants. Within each group, PCB 153 (6.0 × 102∼4.2 × 104 𝜇g/kg), p,p'- DDE (5.1 × 102∼8.6 × 103 𝜇g/kg), and HCB (7.6 × 101∼1.5 × 103 𝜇g/kg) were the compounds found in highest concentrations.The hormonal activity of the porpoise blubber samples was also assayed in RGA, where two samples showed estrogenic activity, seven samples showed antiestrogenic activity, and one sample showed both estrogenic and antiestrogenic activity. Our results suggest that the 13 POPs measured by GC-MS in the samples cannot explain alone the estrogenicity of the extracts. [less ▲]

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See detailBMP Signaling Regulates Bone Morphogenesis in Zebrafish through Promoting Osteoblast Function as Assessed by Their Nitric Oxide Production
Windhausen, Thomas ULiege; Squifflet, Steeve; Renn, Jörg ULiege et al

in Molecules (2015), 20

Bone morphogenetic proteins (BMPs) control many developmental and physiological processes, including skeleton formation and homeostasis. Previous studies in zebrafish revealed the crucial importance of ... [more ▼]

Bone morphogenetic proteins (BMPs) control many developmental and physiological processes, including skeleton formation and homeostasis. Previous studies in zebrafish revealed the crucial importance of proper BMP signaling before 48 h post-fertilization (hpf) for cartilage formation in the skull. Here, we focus on the involvement of the BMP pathway between 48 and 96 hpf in bone formation after 96 hpf. Using BMP inhibitors and the expression of a dominant-negative BMP receptor, we analyze whether the loss of BMP signaling affects osteoblastogenesis, osteoblast function and bone mineralization. To this end, we used the transgenic zebrafish line Tg(osterix:mCherry), detection of nitric oxide (NO) production, and alizarin red staining, respectively. We observed that inhibition of BMP signaling between 48 and 72 hpf led to a reduction of NO production and bone mineralization. Osteoblast maturation and chondrogenesis, on the other hand, seemed unchanged. Osteoblast function and bone formation were less affected when BMP signaling was inhibited between 72 and 96 hpf. These results suggest that for the onset of bone formation, proper BMP signaling between 48 and 72 hpf is crucial to ensure osteoblast function and ossification. Furthermore, detection of NO in developing zebrafish larvae appears as an early indicator of bone calcification activity. [less ▲]

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See detailPhenotype Classification of Zebrafish Embryos by Supervised Learning
Jeanray, Nathalie ULiege; Marée, Raphaël ULiege; Pruvot, Benoist et al

in PLoS ONE (2015), 10(1), 01169891-20

Zebrafish is increasingly used to assess biological properties of chemical substances and thus is becoming a specific tool for toxicological and pharmacological studies. The effects of chemical substances ... [more ▼]

Zebrafish is increasingly used to assess biological properties of chemical substances and thus is becoming a specific tool for toxicological and pharmacological studies. The effects of chemical substances on embryo survival and development are generally evaluated manually through microscopic observation by an expert and documented by several typical photographs. Here, we present a methodology to automatically classify brightfield images of wildtype zebrafish embryos according to their defects by using an image analysis approach based on supervised machine learning. We show that, compared to manual classification, automatic classification results in 90 to 100 % agreement with consensus voting of biological experts in nine out of eleven considered defects in 3 days old zebrafish larvae. Automation of the analysis and classification of zebrafish embryo pictures reduces the workload and time required for the biological expert and increases the reproducibility and objectivity of this classification. [less ▲]

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See detailZebrafish bone and general physiology are differently affected by hormones or changes in gravity.
Aceto, Jessica ULiege; Nourizadeh-Lillabadi, Rasoul; Marée, Raphaël ULiege et al

in PLoS ONE (2015), 10(6), 1-42

Teleost fish such as zebrafish (Danio rerio) are increasingly used for physiological, genetic and developmental studies. Our understanding of the physiological consequences of altered gravity in an entire ... [more ▼]

Teleost fish such as zebrafish (Danio rerio) are increasingly used for physiological, genetic and developmental studies. Our understanding of the physiological consequences of altered gravity in an entire organism is still incomplete. We used altered gravity and drug treatment experiments to evaluate their effects specifically on bone formation and more generally on whole genome gene expression. By combining morphometric tools with an objective scoring system for the state of development for each element in the head skeleton and specific gene expression analysis, we confirmed and characterized in detail the decrease or increase of bone formation caused by a 5 day treatment (from 5dpf to 10 dpf) of, respectively parathyroid hormone (PTH) or vitamin D3 (VitD3). Microarray transcriptome analysis after 24 hours treatment reveals a general effect on physiology upon VitD3 treatment, while PTH causes more specifically developmental effects. Hypergravity (3g from 5dpf to 9 dpf) exposure results in a significantly larger head and a significant increase in bone formation for a subset of the cranial bones. Gene expression analysis after 24 hrs at 3g revealed differential expression of genes involved in the development and function of the skeletal, muscular, nervous, endocrine and cardiovascular systems. Finally, we propose a novel type of experimental approach, the "Reduced Gravity Paradigm", by keeping the developing larvae at 3g hypergravity for the first 5 days before returning them to 1g for one additional day. 5 days exposure to 3g during these early stages also caused increased bone formation, while gene expression analysis revealed a central network of regulatory genes (hes5, sox10, lgals3bp, egr1, edn1, fos, fosb, klf2, gadd45ba and socs3a) whose expression was consistently affected by the transition from hyper- to normal gravity. [less ▲]

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See detailEffects of some controversial food additives on zebrafish embryonic development
Dinh Duy Thanh, ULiege; Voncken, Audrey; Curé, Yoann et al

Poster (2014, December 04)

Background information: There are rising concerns about potential hazardous properties of food additives, forcing legislator to tighten management policy and requiring extensive, yet animal- minimized ... [more ▼]

Background information: There are rising concerns about potential hazardous properties of food additives, forcing legislator to tighten management policy and requiring extensive, yet animal- minimized, testing strategies. The zebrafish embryo is an emerging model system for chemical testing with many advantages that made it amenable to high-throughput assays at the in vivo level. In this study, we applied a panel of tests to evaluate toxicity, particularly neurobehavioral effects, of seven substances including standard compounds and controversial food additives. Methods: Zebrafish wildtype and transgenic fluorescent embryos were exposed to different concentrations of four food additives: Sodium benzoate (SB), Monosodium glutamate (MSG), Tartrazine (TTZ), and Quinoline yellow (QY). Method validation was carried out using three other substances: Ethanol (EtOH), Dimethyl sulfoxide (DMSO), 3,4-Dichloroaniline (DCA). Morphological and lethal effects were recorded and the data were analysed to determine median lethal concentration (LC50), median effective concentration (EC50), effective concentration 10% (EC10), and teratogenic index (TI) values as well as concentration-response equations. Delayed effects of substances on larval locomotion were inspected using the light/dark challenge. Gene expression analysis was carried out using transgenic fluorescent lines. Results: LC50 values of three standard compounds (EtOH, DMSO, and DCA) reveal a high correlation with previously validated data, proving the reliability of our method. Effects of each substance on zebrafish embryonic morphology and lethality were determined as well as the corresponding concentration-response curves. Calculated toxicological indexes revealed that SB belongs to Cat.3 aquatic toxicity class, while QY is the most teratogenic substance. At EC10, all additives exhibited a delayed effect on zebrafish larval locomotion in compound-specific patterns. Observation of transgenic fluorescent embryos and locomotion analysis of hatched larvae reveal that SB could decrease the zebrafish motoneuron differentiation rate, while TTZ exhibited anti-angiogenic effects. Conclusion: Our results demonstrate that our test panel is reliable as a means to assess and categorise chemical toxicity. Also, our data suggest the need to reconsider the safety of food additives SB, TTZ, and QY as well as other controversial food additives in further studies. [less ▲]

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