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.

Type I Diabetes in Zebrafish Reduces Sperm Quality and Increases Insulin and Glucose Transporter Transcripts.

Type I diabetes is a prominent human pathology with increasing incidence in the population; however, its cause is still unknown. This disease promotes detrimental effects on reproduction, such as lower sperm motility and DNA integrity. Hence, the investigation of the underlying mechanisms of this metabolic disturbance in reproduction and its transgenerational consequences is of the utmost importance. The zebrafish is a useful model for this research considering its high homology with human genes as well as its fast generation and regeneration abilities. Therefore, we aimed to investigate sperm quality and genes relevant to diabetes in the spermatozoa of Tg(ins:nfsb-mCherry) zebrafish, a model for type I diabetes. Diabetic Tg(ins:nfsb-mCherry) males showed significantly higher expression of transcripts for insulin a (insa) and glucose transporter (slc2a2) compared to controls. Sperm obtained from the same treatment group showed significantly lower sperm motility, plasma membrane viability, and DNA integrity compared to that from the control group. Upon sperm cryopreservation, sperm freezability was reduced, which could be a consequence of poor initial sperm quality. Altogether, the data showed similar detrimental effects related to type I diabetes in zebrafish spermatozoa at the cellular and molecular levels. Therefore, our study validates the zebrafish model for type I diabetes research in germ cells.

Resveratrol-Mediated Reversal of Doxorubicin-Induced Osteoclast Differentiation.

Secondary osteoporosis has been associated with cancer patients undertaking Doxorubicin (DOX) chemotherapy. However, the molecular mechanisms behind DOX-induced bone loss have not been elucidated. Molecules that can protect against the adverse effects of DOX are still a challenge in chemotherapeutic treatments. We investigated the effect and mechanism of DOX in osteoclast differentiation and used the Sirt 1 activator resveratrol (RES) to counteract DOX-induced effects. RAW 264.7 cells were differentiated into osteoclasts under cotreatment with DOX and RES, alone or combined. RES treatment inhibited DOX-induced osteoclast differentiation, reduced the expression of osteoclast fusion marker Oc-stamp and osteoclast differentiation markers Rank, Trap, Ctsk and Nfatc1. Conversely, RES induced the upregulation of antioxidant genes Sod 1 and Nrf 2 while DOX significantly reduced the FoxM1 expression, resulting in oxidative stress. Treatment with the antioxidant MitoTEMPO did not influence DOX-induced osteoclast differentiation. DOX-induced osteoclastogenesis was studied using the cathepsin-K zebrafish reporter line (Tg[ctsk:DsRed]). DOX significantly increased ctsk signal, while RES cotreatment resulted in a significant reduction in ctsk positive cells. RES significantly rescued DOX-induced mucositis in this model. Additionally, DOX-exposed zebrafish displayed altered locomotor behavior and locomotory patterns, while RES significantly reversed these effects. Our research shows that RES prevents DOX-induced osteoclast fusion and activation in vitro and in vivo and reduces DOX-induced mucositis, while improving locomotion parameters.

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.

Altered bone microarchitecture in a type 1 diabetes mouse model Ins2Akita.

Type 1 diabetes mellitus (T1DM) has been associated to several cartilage and bone alterations including growth retardation, increased fracture risk, and bone loss. To determine the effect of long term diabetes on bone we used adult and aging Ins2 Akita mice that developed T1DM around 3-4 weeks after birth. Both Ins2 Akita and wild-type (WT) mice were analyzed at 4, 6, and 12 months to assess bone parameters such as femur length, growth plate thickness and number of mature and preapoptotic chondrocytes. In addition, bone microarchitecture of the cortical and trabecular regions was measured by microcomputed tomography and gene expression of Adamst-5, Col2, Igf1, Runx2, Acp5, and Oc was quantified by quantitative real-time polymerase chain reaction. Ins2 Akita mice showed a decreased longitudinal growth of the femur that was related to decreased growth plate thickness, lower number of chondrocytes and to a higher number of preapoptotic cells. These changes were associated with higher expression of Adamst-5, suggesting higher cartilage degradation, and with low expression levels of Igf1 and Col2 that reflect the decreased growth ability of diabetic mice. Ins2 Akita bone morphology was characterized by low cortical bone area (Ct.Ar) but higher trabecular bone volume (BV/TV) and expression analysis showed a downregulation of bone markers Acp5, Oc, and Runx2. Serum levels of insulin and leptin were found to be reduced at all-time points Ins2 Akita . We suggest that Ins2 Akita mice bone phenotype is caused by lower bone formation and even lower bone resorption due to insulin deficiency and to a possible relation with low leptin signaling.

Cryoprotectants synergy improve zebrafish sperm cryopreservation and offspring skeletogenesis.

The synergy obtained by the combination of cryoprotectants is a successful strategy that can be beneficial on the optimization of zebrafish sperm cryopreservation. Recently, a protocol was established for this species using an electric ultrafreezer (-150 °C) performing cooling rate (-66 °C/min) and storage within one step. The ultimate objective of sperm cryopreservation is to generate healthy offspring. Therefore, the objective of this study was to select the most adequate cryoprotectant combination, for the previously established protocol, that generate high quality offspring with normal skeletogenesis. Among the permeating cryoprotectant concentrations studied 12.5% and 15% of N,N-dimethylformamide (DMF) yielded high post-thaw sperm quality and hatching rates. For these two concentrations, the presence of bovine serum albumin (10 mg/mL), egg yolk (10%), glycine (30 mM) and bicine (50 mM) was evaluated for post-thaw sperm motility, viability, in vitro fertilization success and offspring skeletal development (30 days post fertilization). Higher concentration of permeating cryoprotectant (15%) decreased the incidence of deformed arches and severe skeletal malformations, which suggests higher capacity to protect the cell against cold stress and DNA damage. Extender containing 15% DMF with Ctrl, Bicine and egg yolk were the non-permeating cryoprotectants with higher post-thaw quality. The use of these compounds results in a reduction in vertebral fusions, compressions and severity of skeletal malformations in the offspring. Therefore, these extender compositions are beneficial for the quality of zebrafish offspring sired by cryopreserved sperm with -66 °C/min freezing rate. To the best of our knowledge, this is the first report on skeletal development of the offspring sired by cryopreserved sperm performed with different freezing media compositions in zebrafish.

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.

Electric ultrafreezer (- 150 °C) as an alternative for zebrafish sperm cryopreservation and storage.

Zebrafish sperm cryopreservation is a fundamental methodology to manage and back-up valuable genetic resources like transgenic and mutant strains. Cryopreservation usually requires liquid nitrogen for storage, which is expensive and hazardous. Our objective was to evaluate if electric ultrafreezers (- 150 °C) are a viable alternative for zebrafish sperm storage. Zebrafish sperm was cryopreserved in the same conditions (- 20 °C/min), stored either in liquid nitrogen or in an ultrafreezer, and thawed after 1 week, 1 month, and 3 months. Sperm motility, membrane integrity, and fertilization ability were assessed. There were no significant differences in motility and hatching rate throughout storage time. Additionally, we aimed at understanding if cryopreservation directly in an ultrafreezer (- 66 °C/min) could improve post-thaw sperm quality. Freezing at - 20 °C/min was performed as before, and compared to samples cryopreserved with a fast cooling rate by placing directly in an ultrafreezer (- 66 °C/min). Sperm quality was assessed according to motility, viability, DNA fragmentation, and apoptosis (annexin V). The - 66 °C/min cooling rate showed significantly higher membrane and DNA integrity, and lower number of cells in late apoptosis in comparison to the other treatments. This study showed that zebrafish sperm cryopreservation and storage in an ultrafreezer system is possible and a fast cooling rate directly in ultrafreezer improves post-thaw sperm quality.

Improved regeneration and de novo bone formation in a diabetic zebrafish model treated with paricalcitol and cinacalcet.

Bone changes related to diabetes have been well stablished, but few strategies have been developed to prevent this growing health problem. In our work, we propose to investigate the effects of calcitriol as well as of a vitamin D analog (paricalcitol) and a calcimimetic (cinacalcet), in fin regeneration and de novo mineralization in a zebrafish model of diabetes. Following exposure of diabetic transgenic Tg(ins:nfsb-mCherry) zebrafish to calcitriol, paricalcitol and cinacalcet, caudal fins were amputated to assess their effects on tissue regeneration. Caudal fin mineralized and regenerated areas were quantified by in vivo alizarin red staining. Quantitative real-time PCR was performed using RNA from the vertebral column. Diabetic fish treated with cinacalcet and paricalcitol presented increased regenerated and mineralized areas when compared with non-treated diabetic group, while no significant increase was observed in non-diabetic fish treated with both drugs. Gene expression analysis showed an up-regulation for runt-related transcription factor 2b (runx2b), bone gamma-carboxyglutamic acid-containing protein (bglap), insulin a (insa) and insulin b (insb) and a trend of increase for sp7 transcription factor (sp7) in diabetic groups treated with cinacalcet and paricalcitol. Expression of insra and vdra was up-regulated in both diabetic and nondiabetic fish treated with cinacalcet. In nondiabetic fish treated with paricalcitol and cinacalcet a similar increase in gene expression could be observed but not so pronounced. The increased mineralization and regeneration in diabetic zebrafish treated with cinacalcet and paricalcitol can be explained by increased osteoblastic differentiation and increased insulin expression indicating pro-osteogenic potential of both drugs.

Glucose metabolism and gene expression in juvenile zebrafish (Danio rerio) challenged with a high carbohydrate diet: effects of an acute glucose stimulus during late embryonic life.

Knowledge on the role of early nutritional stimuli as triggers of metabolic pathways in fish is extremely scarce. The objective of the present study was to assess the long-term effects of glucose injection in the yolk (early stimulus) on carbohydrate metabolism and gene regulation in zebrafish juveniles challenged with a high-carbohydrate low-protein (HC) diet. Eggs were microinjected at 1 d post-fertilisation (dpf) with either glucose (2 M) or saline solutions. Up to 25 dpf, fish were fed a low-carbohydrate high-protein (LC) control diet, which was followed by a challenge with the HC diet. Survival and growth of 35 dpf juveniles were not affected by injection or the HC diet. Glucose stimulus induced some long-term metabolic changes in the juveniles, as shown by the altered expression of genes involved in glucose metabolism. On glycolysis, the expression levels of hexokinase 1 (HK1) and phosphofructokinase-6 (6PFK) were up-regulated in the visceral and muscle tissues, respectively, of juveniles exposed to the glucose stimulus, indicating a possible improvement in glucose oxidation. On gluconeogenesis, the inhibition of the expression levels of PEPCK in fish injected with glucose suggested lower production of hepatic glucose. Unexpectedly, fructose-1,6-bisphosphatase (FBP) expression was induced and 6PFK expression reduced by glucose stimulus, leaving the possibility of a specific regulation of the FBP-6PFK metabolic cycle. Glucose metabolism in juveniles was estimated using a [¹4C]glucose tracer; fish previously exposed to the stimulus showed lower retention of [¹4C]glucose in visceral tissue (but not in muscle tissue) and, accordingly, higher glucose catabolism, in comparison with the saline group. Globally, our data suggest that glucose stimulus at embryo stage has the potential to alter particular steps of glucose metabolism in zebrafish juveniles.

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.

Glucose overload in yolk has little effect on the long-term modulation of carbohydrate metabolic genes in zebrafish (Danio rerio).

Some fish show a low metabolic ability to use dietary carbohydrates. The use of early nutritional stimuli to program metabolic pathways in fish is ill defined. Therefore, studies were undertaken with zebrafish to assess the effect of high glucose levels during the embryonic stage as a lifelong modulator of genes involved in carbohydrate metabolism. Genes related to carbohydrate metabolism were expressed at low levels at 0.2 and 1 day post-fertilization (dpf). However, from 4 dpf onwards there was a significant increase on expression of all genes, suggesting that all analysed pathways were active. By microinjection, we successfully enriched zebrafish egg yolk with glucose (a 43-fold increase of basal levels). Acute effects of glucose injection on gene expression were assessed in larvae up to 10 dpf, and the programming concept was evaluated in juveniles (41 dpf) challenged with a hyperglucidic diet. At 4 dpf, larvae from glucose-enriched eggs showed a downregulation of several genes related to glycolysis, glycogenolysis, lipogenesis and carbohydrate digestion in comparison with control (saline-injected) embryos. This inhibitory regulation was suppressed after 10 dpf. At the juvenile stage, and upon switching from a low to a high digestible carbohydrate diet, early glucose enrichment had no significant effect on most analysed genes. However, these same fish showed altered expression of the genes for cytosolic phosphoenolpyruvate carboxykinase, sodium-dependent glucose cotransporter 1 and glycogen synthase, suggesting changes to the glucose storage capacity in muscle and glucose production and transport in viscera. Overall, supplementation of egg yolk with high glucose levels had little effect on the long-term modulation of carbohydrate metabolic genes in zebrafish.

Solea senegalensis vasa transcripts: molecular characterisation, tissue distribution and developmental expression profiles.

The Vasa protein is an RNA helicase belonging the DEAD (Asp-Glu-Ala-Asp)-box family. The crucial role played by the vasa gene in the germ-cell lineage of both vertebrates and invertebrates has made this gene a useful molecular marker for germinal cells and a useful tool in surrogate broodstock production using primordial germ cell transplantation. With the aim of establishing a novel approach to improving Solea senegalensis broodstock management, the vasa gene in this species was characterised. Four S. senegalensis vasa transcripts were isolated: Ssvasa1, Ssvasa2, Ssvasa3 and Ssvasa4. Their phylogenetic relationship with other vasa homologues was determined confirming the high degree of conservation of this helicase throughout evolution. Our qPCR results showed that S. senegalensis vasa transcripts are prevalently expressed in gonads, with ovary-specific expression for Ssvasa3 and Ssvasa4. During embryonic and larval development, a switch between the longest and the shortest transcripts was observed. While Ssvasa1 and Ssvasa2 were maternally supplied, Ssvasa3 and Ssvasa4 depended on the de novo expression program of the growing juveniles, suggesting that vasa mRNA could be involved in Senegalese sole gonad differentiation. In situ hybridisation and immunohistochemical analysis performed in 150-days after hatching (DAH) larvae showed vasa product expression in the germinal region of early gonads. In our work we demonstrated the usefulness of Ssvasa mRNAs as molecular markers for primordial germ cells and germinal cells during embryonic development, larval ontogenesis and gonad differentiation. Furthermore, our results confirmed the potential of vasa to help investigate germinal cell biotechnology for Senegalese sole reproduction.

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.