Fin ray branching is defined by TRAP+ osteolytic tubules in zebrafish.

The shaping of bone structures relies on various cell types and signaling pathways. Here, we use the zebrafish bifurcating fin rays during regeneration to investigate bone patterning. We found that the regenerating fin rays form via two mineralization fronts that undergo an osteoblast-dependent fusion/stitching until the branchpoint, and that bifurcation is not simply the splitting of one unit into two. We identified tartrate-resistant acid phosphatase-positive osteolytic tubular structures at the branchpoints, hereafter named osteolytic tubules (OLTs). Chemical inhibition of their bone-resorbing activity strongly impairs ray bifurcation, indicating that OLTs counteract the stitching process. Furthermore, by testing different osteoactive compounds, we show that the position of the branchpoint depends on the balance between bone mineralization and resorption activities. Overall, these findings provide a unique perspective on fin ray formation and bifurcation, and reveal a key role for OLTs in defining the proximo-distal position of the branchpoint.

Metabolic bone disorders and the promise of marine osteoactive compounds.

Metabolic bone disorders and associated fragility fractures are major causes of disability and mortality worldwide and place an important financial burden on the global health systems. These disorders result from an unbalance between bone anabolic and resorptive processes and are characterized by different pathophysiological mechanisms. Drugs are available to treat bone metabolic pathologies, but they are either poorly effective or associated with undesired side effects that limit their use. The molecular mechanism underlying the most common metabolic bone disorders, and the availability, efficacy, and limitations of therapeutic options currently available are discussed here. A source for the unmet need of novel drugs to treat metabolic bone disorders is marine organisms, which produce natural osteoactive compounds of high pharmaceutical potential. In this review, we have inventoried the marine osteoactive compounds (MOCs) currently identified and spotted the groups of marine organisms with potential for MOC production. Finally, we briefly examine the availability of in vivo screening and validation tools for the study of MOCs.

The osteogenic and mineralogenic potential of the microalgae Skeletonema costatum and Tetraselmis striata CTP4 in fish models.

Skeletal disorders are problematic aspects for the aquaculture industry as skeletal deformities, which affect most species of farmed fish, increase production costs and affect fish welfare. Following recent findings that show the presence of osteoactive compounds in marine organisms, we evaluated the osteogenic and mineralogenic potential of commercially available microalgae strains Skeletonema costatum and Tetraselmis striata CTP4 in several fish systems. Ethanolic extracts increased extracellular matrix mineralization in gilthead seabream (Sparus aurata) bone-derived cell cultures and promoted osteoblastic differentiation in zebrafish (Danio rerio) larvae. Long-term dietary exposure to both extracts increased bone mineralization in zebrafish and upregulated the expression of genes involved in bone formation (sp7, col1a1a, oc1, and oc2), bone remodeling (acp5a), and antioxidant defenses (cat, sod1). Extracts also improved the skeletal status of zebrafish juveniles by reducing the incidence of skeletal anomalies. Our results indicate that both strains of microalgae contain osteogenic and mineralogenic compounds, and that ethanolic extracts have the potential for an application in the aquaculture sector as dietary supplements to support fish bone health. Future studies should also identify osteoactive compounds and establish whether they can be used in human health to broaden the therapeutic options for bone erosive disorders such as osteoporosis.

Zebrafish Models to Study Ectopic Calcification and Calcium-Associated Pathologies.

Ectopic calcification refers to the pathological accumulation of calcium ions in soft tissues and is often the result of a dysregulated action or disrupted function of proteins involved in extracellular matrix mineralization. While the mouse has traditionally been the go-to model organism for the study of pathologies associated with abnormal calcium deposition, many mouse mutants often have exacerbated phenotypes and die prematurely, limiting the understanding of the disease and the development of effective therapies. Since the mechanisms underlying ectopic calcification share some analogy with those of bone formation, the zebrafish (Danio rerio)-a well-established model for studying osteogenesis and mineralogenesis-has recently gained momentum as a model to study ectopic calcification disorders. In this review, we outline the mechanisms of ectopic mineralization in zebrafish, provide insights into zebrafish mutants that share phenotypic similarities with human pathological mineralization disorders, list the compounds capable of rescuing mutant phenotypes, and describe current methods to induce and characterize ectopic calcification in zebrafish.

Transcriptional regulation of human DUSP4 gene by cancer-related transcription factors.

Dual specificity phosphatase 4 (DUSP4), a member of the dual specificity phosphatase family, is responsible for the dephosphorylation and inactivation of ERK, JNK and p38, which are mitogen-activated protein kinases involved in cell proliferation, differentiation and apoptosis, but also in inflammation processes. Given its importance for cellular signalling, DUSP4 is subjected to a tight regulation and there is growing evidence that its expression is dysregulated in several tumours. However, the mechanisms underlying DUSP4 transcriptional regulation remain poorly understood. Here, we analysed the regulation of the human DUSP4 promoters 1 and 2, located upstream of exons 1 and 2, respectively, by the cancer-related transcription factors (TFs) STAT3, FOXA1, CTCF and YY1. The presence of binding sites for these TFs was predicted in both promoters through the in silico analysis of DUSP4, and their functionality was assessed through luciferase activity assays. Regulatory activity of the TFs tested was found to be promoter-specific. While CTCF stimulated the activity of promoter 2 that controls the transcription of variants 2 and X1, STAT3 stimulated the activity of promoter 1 that controls the transcription of variant 1. YY1 positively regulated both promoters, although to different extents. Through site-directed mutagenesis, the functionality of YY1 binding sites present in promoter 2 was confirmed. This study provides novel insights into the transcriptional regulation of DUSP4, contributing to a better comprehension of the mechanisms of its dysregulation observed in several types of cancer.

New insights into benzo[⍺]pyrene osteotoxicity in zebrafish.

Persistent and ubiquitous organic pollutants, such as the polycyclic aromatic hydrocarbon benzo[⍺]pyrene (BaP), represent a major threat to aquatic organisms and human health. Beside some well-documented adverse effects on the development and reproduction of aquatic organisms, BaP was recently shown to affect fish bone formation and skeletal development through mechanisms that remain poorly understood. In this work, zebrafish bone-related in vivo assays were used to evaluate the osteotoxic effects of BaP during bone development and regeneration. Acute exposure of zebrafish larvae to BaP from 3 to 6 days post-fertilization (dpf) induced a dose-dependent reduction of the opercular bone size and a depletion of osteocalcin-positive cells, indicating an effect on osteoblast maturation. Chronic exposure of zebrafish larvae to BaP from 3 to 30 dpf affected the development of the axial skeleton and increased the incidence and severity of skeletal deformities. In young adults, BaP affected the mineralization of newly formed fin rays and scales, and impaired fin ray patterning and scale shape, through mechanisms that involve an imbalanced bone remodeling. Gene expression analyses indicated that BaP induced the activation of xenobiotic and metabolic pathways, while negatively impacting extracellular matrix formation and organization. Interestingly, BaP exposure positively regulated inflammation markers in larvae and increased the recruitment of neutrophils. A direct interaction between neutrophils and bone extracellular matrix or bone forming cells was observed in vivo, suggesting a role for neutrophils in the mechanisms underlying BaP osteotoxicity. Our work provides novel data on the cellular and molecular players involved in BaP osteotoxicity and brings new insights into a possible role for neutrophils in inflammatory bone reduction.

New Insights on Vitamin K Metabolism in Senegalese sole (Solea senegalensis) Based on Ontogenetic and Tissue-Specific Vitamin K Epoxide Reductase Molecular Data.

Vitamin K (VK) is a key nutrient for several biological processes (e.g., blood clotting and bone metabolism). To fulfill VK nutritional requirements, VK action as an activator of pregnane X receptor (Pxr) signaling pathway, and as a co-factor of γ-glutamyl carboxylase enzyme, should be considered. In this regard, VK recycling through vitamin K epoxide reductases (Vkors) is essential and should be better understood. Here, the expression patterns of vitamin K epoxide reductase complex subunit 1 (vkorc1) and vkorc1 like 1 (vkorc1l1) were determined during the larval ontogeny of Senegalese sole (Solea senegalensis), and in early juveniles cultured under different physiological conditions. Full-length transcripts for ssvkorc1 and ssvkorc1l1 were determined and peptide sequences were found to be evolutionarily conserved. During larval development, expression of ssvkorc1 showed a slight increase during absence or low feed intake. Expression of ssvkorc1l1 continuously decreased until 24 h post-fertilization, and remained constant afterwards. Both ssvkors were ubiquitously expressed in adult tissues, and highest expression was found in liver for ssvkorc1, and ovary and brain for ssvkorc1l1. Expression of ssvkorc1 and ssvkorc1l1 was differentially regulated under physiological conditions related to fasting and re-feeding, but also under VK dietary supplementation and induced deficiency. The present work provides new and basic molecular clues evidencing how VK metabolism in marine fish is sensitive to nutritional and environmental conditions.

Does a ghrelin stimulus during zebrafish embryonic stage modulate its performance on the long-term?

Metabolic programming refers to the induction, deletion, or impaired development of a somatic structure or "setting" of a physiological system by an early life stimulus operated at a critical period during development. Ghrelin is the only known orexigenic gut hormone, is an acylated peptide that acts as an endogenous ligand specific for growth-hormone secretagogue-receptor. The aim of the present work was to evaluate if an in ovo ghrelin administration could positively influence the zebrafish performance in the long-term and to gain insight on the mechanisms associated to ghrelin regulation of food intake during the larval phase. Food intake, growth potential, protein metabolism, expression of target genes involved in ghrelin, feeding behaviour regulation and locomotor activity were assessed in zebrafish (Danio rerio) larvae at 25 days post-fertilization. Elevated levels of acylated ghrelin in zebrafish eggs did not result in increased growth or food intake. Differences in mRNA expression between larvae fasted for 16 h before and 1 h after feeding were found for igf1ra, gh1 and pomca. Moreover, ghrelin treated larvae showed higher swimming activity, indicating that the peptide may have an important role on foraging activity. The present study addressed for the first time the effects of an early stimulus of ghrelin during the embryonic stage of zebrafish, however, further studies are needed to clarify the metabolic pathways affected by the early stimulus as well as focus on the effects on metabolic regulation of energy balance through lipid and carbohydrate metabolism.

Warfarin-exposed zebrafish embryos resembles human warfarin embryopathy in a dose and developmental-time dependent manner - From molecular mechanisms to environmental concerns.

Warfarin is the most worldwide used anticoagulant drug and rodenticide. Since it crosses placental barrier it can induce warfarin embryopathy (WE), a fetal mortality in neonates characterized by skeletal deformities in addition to brain hemorrhages. Although the effects of warfarin exposure in aquatic off target species were already described, the particular molecular toxicological mechanisms during early development are still unclear. Here, we used zebrafish (Danio rerio) to describe and compare the developmental effects of warfarin exposure (0, 15.13, 75.68 and 378.43 mM) on two distinct early developmental phases (embryos and eleuthero-embryos). Although exposure to both developmental phases induced fish mortality, only embryos exposed to the highest warfarin level exhibited features mimicking mammalian WE, e.g. high mortality, higher incidence of hemorrhages and altered skeletal development, among other effects. To gain insights into the toxic mechanisms underlying warfarin exposure, the transcriptome of embryos exposed to warfarin was explored through RNA-Seq and compared to that of control embryos. 766 differentially expressed (564 up- and 202 down-regulated) genes were identified. Gene Ontology analysis revealed particular cellular components (cytoplasm, extracellular matrix, lysosome and vacuole), biological processes (mainly amino acid and lipid metabolism and response to stimulus) and pathways (oxidative stress response and apoptosis signaling pathways) being significantly overrepresented in zebrafish embryos upon warfarin exposure. Protein-protein interaction further evidenced an altered redox system, blood coagulation and vasculogenesis, visual phototransduction and collagen formation upon warfarin exposure. The present study not only describes for the first time the WE in zebrafish, it provides new insights for a better risk assessment, and highlights the need for programming the rat eradication actions outside the fish spawning season to avoid an impact on off target fish community. The urge for the development of more species-specific anticoagulants for rodent pest control is also highlighted.

Osteotoxicity of 3-methylcholanthrene in fish.

Many chemicals produced by human activities end up in the aquatic ecosystem causing adverse developmental and reproductive effects in aquatic organisms. There is evidence that some anthropogenic chemicals disturb bone formation and skeletal development but the lack of suitable in vitro and in vivo systems for testing has hindered the identification of underlying mechanisms of osteotoxicity. Several fish systems - an in vitro cell system to study extracellular matrix mineralization and in vivo systems to evaluate bone formation and skeletogenesis - were combined to collect data on the osteotoxic activity of 3-methylcholanthrene (3-MC), a polycyclic aromatic hydrocarbon. Anti-mineralogenic effects, increased incidence of skeletal deformities and reduced bone formation and regeneration were observed in zebrafish upon exposure to 3-MC. Pathway reporter array revealed the role of the aryl hydrocarbon receptor 2 (Ahr2) in the mechanisms underlying 3-MC osteotoxicity in mineralogenic cell lines. Analysis of gene expression in zebrafish larvae confirmed the role of Ahr2 in the signaling of 3-MC toxicity. It also indicated a possible complementary action of the pregnane X receptor (Pxr) in the regulation of genes involved in bone cell activity and differentiation but also in xenobiotic metabolism. Data reported here demonstrated the osteotoxicity of 3-MC but also confirmed the suitability of fish systems to gain insights into the toxic mechanisms of compounds affecting skeletal and bone formation.

Central role of betaine-homocysteine S-methyltransferase 3 in chondral ossification and evidence for sub-functionalization in neoteleost fish.

BACKGROUND: To better understand the complex mechanisms of bone formation it is fundamental that genes central to signaling/regulatory pathways and matrix formation are identified. Cell systems were used to analyze genes differentially expressed during extracellular matrix mineralization and bhmt3, coding for a betaine-homocysteine S-methyltransferase, was shown to be down-regulated in mineralizing gilthead seabream cells. METHODS: Levels and sites of bhmt3 expression were determined by qPCR and in situ hybridization throughout seabream development and in adult tissues. Transcriptional regulation of bhmt3 was assessed from the activity of promoter constructs controlling luciferase gene expression. Molecular phylogeny of vertebrate BHMT was determined from maximum likelihood analysis of available sequences. RESULTS: bhmt3 transcript is abundant in calcified tissues and localized in cartilaginous structures undergoing endo/perichondral ossification. Promoter activity is regulated by transcription factors involved in bone and cartilage development, further demonstrating the central role of Bhmt3 in chondrogenesis and/or osteogenesis. Molecular phylogeny revealed the explosive diversity of bhmt genes in neoteleost fish, while tissue distribution of bhmt genes in seabream suggested that neoteleostean Bhmt may have undergone several steps of sub-functionalization. CONCLUSIONS: Data on bhmt3 gene expression and promoter activity evidences a novel function for betaine-homocysteine S-methyltransferase in bone and cartilage development, while phylogenetic analysis provides new insights into the evolution of vertebrate BHMTs and suggests that multiple gene duplication events occurred in neoteleost fish lineage. GENERAL SIGNIFICANCE: High and specific expression of Bhmt3 in gilthead seabream calcified tissues suggests that bone-specific betaine-homocysteine S-methyltransferases could represent a suitable marker of chondral ossification.

Comparative analysis of zebrafish bone morphogenetic proteins 2, 4 and 16: molecular and evolutionary perspectives.

BMP2, 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 down-regulate 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.

Zebrafish vitamin K epoxide reductases: expression in vivo, along extracellular matrix mineralization and under phylloquinone and warfarin in vitro exposure.

Vitamin K (VK) acts as a cofactor driving the biological activation of VK-dependent proteins and conferring calcium-binding properties to them. As a result, VK is converted into VK epoxide, which must be recycled by VK epoxide reductases (Vkors) before it can be reused. Although VK has been shown to play a central role in fish development, particularly during skeletogenesis, pathways underlying VK actions are poorly understood, while good and reliable molecular markers for VK cycle/homeostasis are still lacking in fish. In the present work, expression of 2 zebrafish vkor genes was characterized along larval development and in adult tissues through qPCR analysis. Zebrafish cell line ZFB1 was used to evaluate in vitro regulation of vkors and other VK cycle-related genes during mineralization and upon 24 h exposure to 0.16 and 0.8 µM phylloquinone (VK1), 0.032 µM warfarin, or a combination of both molecules. Results showed that zebrafish vkors are differentially expressed during larval development, in adult tissues, and during cell differentiation/mineralization processes. Further, several VK cycle intermediates were differentially expressed in ZFB1 cells exposed to VK1 and/or warfarin. Present work provides data identifying different developmental stages and adult tissues where VK recycling is probably highly required, and shows how genes involved in VK cycle respond to VK nutritional status in skeletal cells. Expression of vkor genes can represent a reliable indicator to infer VK nutritional status in fish, while ZFB1 cells could represent a suitable in vitro tool to get insights into the mechanisms underlying VK action on fish bone.

Sturgeon osteocalcin shares structural features with matrix Gla protein: evolutionary relationship and functional implications.

Osteocalcin (OC) and matrix Gla protein (MGP) are considered evolutionarily related because they share key structural features, although they have been described to exert different functions. In this work, we report the identification and characterization of both OC and MGP from the Adriatic sturgeon, a ray-finned fish characterized by a slow evolution and the retention of many ancestral features. Sturgeon MGP shows a primary structure, post-translation modifications, and patterns of mRNA/protein distribution and accumulation typical of known MGPs, and it contains seven possible Gla residues that would make the sturgeon protein the most γ-carboxylated among known MGPs. In contrast, sturgeon OC was found to present a hybrid structure. Indeed, although exhibiting protein domains typical of known OCs, it also contains structural features usually found in MGPs (e.g. a putative phosphorylated propeptide). Moreover, patterns of OC gene expression and protein accumulation overlap with those reported for MGP; OC was detected in bone cells and mineralized structures but also in soft and cartilaginous tissues. We propose that, in a context of a reduced rate of evolution, sturgeon OC has retained structural features of the ancestral protein that emerged millions of years ago from the duplication of an ancient MGP gene and may exhibit intermediate functional features.

Matrix Gla protein and osteocalcin: from gene duplication to neofunctionalization.

Osteocalcin (OC or bone Gla protein, BGP) and matrix Gla protein (MGP) are two members of the growing family of vitamin K-dependent (VKD) proteins. They were the first VKD proteins found not to be involved in coagulation and synthesized outside the liver. Both proteins were isolated from bone although it is now known that only OC is synthesized by bone cells under normal physiological conditions, but since both proteins can bind calcium and hydroxyapatite, they can also accumulate in bone. Both OC and MGP share similar structural features, both in terms of protein domains and gene organization. OC gene is likely to have appeared from MGP through a tandem gene duplication that occurred concomitantly with the appearance of the bony vertebrates. Despite their relatively close relationship and the fact that both can bind calcium and affect mineralization, their functions are not redundant and they also have other unrelated functions. Interestingly, these two proteins appear to have followed quite different evolutionary strategies in order to acquire novel functionalities, with OC following a gene duplication strategy while MGP variability was obtained mostly by the use of multiple promoters and alternative splicing, leading to proteins with additional functional characteristics and alternative gene regulatory pathways.

Mir-20a regulates in vitro mineralization and BMP signaling pathway by targeting BMP-2 transcript in fish.

MicroRNAs (miRNAs) are important regulators of vertebrate development but their role during skeletogenesis remains unknown. In this regard, we investigated the mineralogenic activity of miR-20a, a miRNA associated with osteogenesis, in fish bone-derived cells. Expression of miR-20a was up-regulated during differentiation and its overexpression inhibited mineralization, suggesting a role in fish tissue calcification. In this regard, a conserved miR-20a binding site was identified in bone morphogenetic protein 2 (BMP-2) 3'UTR and its functionality was evidenced through luciferase assays, and further confirmed by western-blot and qPCR. Type II BMP receptor (BMPR2) is also targeted by miR-20a in mammalian systems and evidence was collected for the presence of a binding site in fish sequences. We propose that miR-20a is a regulator of BMP pathway through specific action on BMP-2 and possibly BMPR2. Overexpression of miR-20a was also shown to up-regulate matrix Gla protein (MGP) transcript, a physiological inhibitor of calcification previously found to form a complex with BMP-2. We propose that MGP may play a role in the anti-mineralogenic effect promoted by miR-20a by decreasing availability of BMP-2. This study gives new insights into miRNA-mediated regulation of BMP-2, and sheds light into the potential role of miR-20a as a regulator of skeletogenesis.

Teleost fish osteocalcin 1 and 2 share the ability to bind the calcium mineral phase.

The occurrence of a second osteocalcin (OC2) has been reported in teleost fish, where it coexists with OC1 in some species. While it has been proposed that OC2 gene originated from OC1 through the fish whole-genome duplication event, little information is available on its molecular function and physiological role. The present study brings biological data supporting the presence of OC2 in the mineral phase of teleost fish bone and its association with the mineral phase together with OC1. The occurrence of OC2 forms with different levels of phosphorylation or γ-carboxylation, and with amino acid substitutions was observed. Comparative analysis of mature peptide sequences revealed the high conservation existing between OC1 and OC2, in particular within the core γ-carboxyglutamic acid domain, and suggests that both protein forms may have the same function, i.e., binding of calcium ions or hydroxyapatite crystals.

Four-and-a-half LIM domains protein 2 (FHL2) is associated with the development of craniofacial musculature in the teleost fish Sparus aurata.

Four-and-a-half LIM domains protein 2 (FHL2) is involved in major cellular mechanisms such as regulation of gene transcription and cytoskeleton modulation, participating in physiological control of cardiogenesis and osteogenesis. Knowledge on underlying mechanisms is, however, limited. We present here new data on FHL2 protein and its role during vertebrate development using a marine teleost fish, the gilthead seabream (Sparus aurata L.). In silico comparison of vertebrate protein sequences and prediction of LIM domain three-dimensional structure revealed a high degree of conservation, suggesting a conserved function throughout evolution. Determination of sites and levels of FHL2 gene expression in seabream indicated a central role for FHL2 in the development of heart and craniofacial musculature, and a potential role in tissue calcification. Our data confirmed the key role of FHL2 protein during vertebrate development and gave new insights into its particular involvement in craniofacial muscle development and specificity for slow fibers.

ESSA1 embryonic stem like cells from gilthead seabream: a new tool to study mesenchymal cell lineage differentiation in fish.

Embryonic stem (ES) cells are a promising tool for generation of transgenic animals and an ideal experimental model for in vitro studies of embryonic cell development, differentiation and gene manipulation. Here we report the development and initial characterization of a pluripotent embryonic stem like cell line, designated as ESSA1, derived from blastula stage embryos of the gilthead seabream (Sparus aurata, L). ESSA1 cells are cultured in Leibovitz's L-15 medium supplemented with 5% fetal bovine serum and, unlike other ES cells, without a feeder layer. They have a round or polygonal morphology, grow exponentially in culture and form dense colonies. ESSA1 cells also exhibit intense alkaline phosphatase activity, normal karyotype and are positive for stage-specific embryonic antigen-1 (SSEA1) and octamer-binding transcription factor 4 (Oct4) markers for up to 30 passages. Upon treatment with all-trans retinoic acid, ESSA1 cells differentiate into neuron-like, oligodendritic, myocyte and melanocyte cells; they can also form embryoid bodies when seeded in bacteriological plates, a characteristic usually associated with pluripotency. The capacity of ESSA1 cells to differentiate into osteoblastic, chondroblastic or osteoclastic cell lineages and to produce a mineralized extracellular matrix in vitro was demonstrated through histochemical techniques and further confirmed by immunocytochemistry using lineage-specific markers. Furthermore, ESSA1 cells can be used to produce chimera, where they contribute to the development of a variety of tissues including the trunk and gut of zebrafish embryos and fry. Thus, ESSA1 cells represent a promising model for investigating bone-lineage cell differentiation in fish and also highlight the potential of piscine stem cell research.

Short-term exposure to pharmaceuticals negatively impacts marine flatfish species: Histological, biochemical and molecular clues for an integrated ecosystem risk assessment.

The marine habitat and its biodiversity can be impacted by released pharmaceuticals. The short-term (7 days) effect of 3 commonly used drugs - warfarin, dexamethasone and imidazole - on Senegalese sole (Solea senegalensis) juveniles was investigated. Occurrence of hemorrhages, histopathological alterations, antioxidant status, activity of antioxidant enzymes and expression of genes involved in the xenobiotic response (pxr, abcb1 and cyp1a), were evaluated. The results showed a time and drug-dependent effect. Warfarin exposure induced hemorrhages, hepatocyte vacuolar degeneration, and altered the activity of glutathione peroxidase (GPx) and the expression of all the studied genes. Dexamethasone exposure increased liver glycogen content, altered antioxidant status, GPx and superoxide dismutase activities, as well as abcb1 and cyp1a expression. Imidazole induced hepatocyte vacuolar degeneration and ballooning, and altered the antioxidant status and expression of the tested genes. The present work anticipates a deeper impact of pharmaceuticals on the aquatic environment than previously reported, thus underlining the urgent need for an integrated risk assessment.