Zinc transporter expression in zebrafish (Danio rerio) during development

Zinc is a micronutrient important in several biological processes including growth and development. We have limited knowledge on the impact of maternal zinc deficiency on zinc and zinc regulatory mechanisms in the developing embryo due to a lack of in vivo experimental models that allow us to directly study the effects of maternal zinc on embryonic development following implantation. To overcome this barrier, we have proposed to use zebrafish as a model organism to study the impact of zinc during development. The goal of the current study was to profile the mRNA expression of all the known zinc transporter genes in the zebrafish across embryonic and larval development and to quantify the embryonic zinc concentrations at these corresponding developmental time points. The SLC30A zinc transporter family (ZnT) and SLC39A family, Zir-,Irt-like protein (ZIP) zinc transporter proteins were profiled in zebrafish embryos at 0, 2, 6, 12, 24, 48 and 120 h post fertilization to capture expression patterns from a single cell through full development. We observed consistent embryonic zinc levels, but differential expression of several zinc transporters across development. These results suggest that zebrafish is an effective model organism to study the effects of zinc deficiency and further investigation is underway to identify possible molecular pathways that are dysregulated with maternal zinc deficiency.     

Liver development in zebrafish （Danio rerio）

Liver is one of the largest internal organs in the body and its importance for metabolism, detoxification and homeostasis has been well established. In this review, we summarized recent progresses in studying liver initiation and development during embryogenesis using zebrafish as a model system. We mainly focused on topics related to the specification of hepatoblasts from endoderm, the formation and growth of liver bud, the differentiation of hepatocytes and bile duct cells from hepatoblasts, and finally the role of mesodermal signals in controlling liver development in zebrafish.     

Phaeodactylum tricornutum biomass in microdiets enhances Senegalese sole (Solea senegalensis) larval growth performance during weaning

Journal of Applied Phycology - Microalgae are one of the most promising groups of functional ingredients for inclusion in microdiets for Senegalese sole (Solea senegalensis) larvae, supplying...     

c-Lysozyme from Senegalese sole (Solea senegalensis): cDNA cloning and expression pattern

Lysozymes are key molecules of innate immunity and proved high bactericidal activity in fish, thus becoming attractive as tools for enhancing fish defences. In this study, a full-length c-type lysozyme cDNA from Senegalese sole (Solea senegalensis) has been cloned and characterized. The cDNA sequence was inferred from two overlapping fragments obtained by RACE-PCR and consisting on 631bp coding for 143 aminoacids. Catalytic and other conserved residues required for lysozyme activity were identified. Pair wise alignments showed the higher identities with c-type lysozyme from other flatfish. Expression patterns under various conditions showed a basal level and a clear upregulation mostly in hematopoietic organs after stimulation with LPS or infection with Photobacterium damselae. This study represents a first step on the genetics and function of the c-lysozyme of Senegalese sole, though disclosing g-DNA structure, allelic variability and antibacterial activity must be requirements prior its immunological properties might have biotechnological applications.     

Muscle regulatory factor gene: zebrafish (Danio rerio) myogenin cDNA

Myogenin is one of the basic helix-loop-helix proteins that regulate muscle-specific gene expression. Using reverse transciption-polymerase chain reaction (RT-PCR), 5'- and 3'-rapid amplification of cDNA ends (RACE), zebrafish myogenin cDNA was cloned from mRNA of embryos at 10-96 h post-fertilization. The cDNA, at 1384 base pairs (bp), contained a 771-bp open reading frame with 113- and 500-bp flanking regions at the 5'- and 3'-ends, respectively. The deduced amino acid sequences of zebrafish myogenin encoded a 256-amino-acid polypeptide. In a comparison with myogenin of carp, trout, Xenopus, chicken and human, zebrafish myogenin shared 90.9, 77.6, 70.3, 62.9 and 51.5% amino acid identity, respectively. The basic helix-loop-helix domains in myogenin are all conserved. The molecular phylogenic tree demonstrated that myogenin of zebrafish is more closely related to that of fish than to the myogenin of other vertebrates.     

Cloning and expression analysis of Mx cDNA from Senegalese sole (Solea senegalensis)

Senegalese sole (Solea senegalensis) is a promising fish species of growing interest in European aquaculture. In fish farming, viral infections are a constant threat therefore, understanding fish defence mechanisms is a main priority to avoid economic losses. Mx proteins are involved in the innate antiviral response of fish. They are induced by type I interferons (alpha and beta) and are essential to investigate viral defence mechanisms in fish, due to the difficulty in tracking interferon activity in these species. In this study a full-length Senegalese sole Mx cDNA has been RT-PCR cloned, resulting in 2322bp coding for 623 amino acids. The sequence accounts for the main characteristics of Mx proteins but lacking nuclear localisation signal (NLS), which suggests cytoplasmic localisation. The alignments of Senegalese sole Mx sequence showed the highest identity with the flatfish species, 80.1% identity with flounder and 78.9% with halibut. The spatial and temporal expression pattern has been analysed in control and challenged fish by RT-PCR. In control fish a constitutive level of sole Mx expression has been obtained and a clear induction was observed after treatment with Poly[I:C], which supports a putative role for the Mx in Senegalese sole viral defence. These findings contribute to increasing the knowledge of the role of interferon pathway in fish innate immunity and to develop new tools to fight virus infections in the culture of this species.     

Sex- and tissue-specific expression of aspartic proteinases in Danio rerio (zebrafish)

Full-length zebrafish cDNAs encoding two aspartic proteinases were cloned and sequenced. One of the two cDNAs was a 1708 bp product with an open reading frame of 398 amino acid residues corresponding to a cathepsin D. The other was a 1383 bp product encoding a polypeptide chain of 416 amino acids homologous to nothepsin, an aspartic proteinase first identified by us in the liver of Antarctic Notothenioidei. Gene expression assessed by RT–PCR and northern blot hybridization of RNA from different tissues showed that the expression was tissue- and sex-specific. Whereas the cathepsin D gene was expressed in all the tissues examined independently of the sex, the nothepsin gene was expressed exclusively in female livers.     

Calbindin immunoreactivity in the enteric nervous system of larval and adult zebrafish (Danio rerio)

Calbindin is a calcium-binding protein, commonly found in certain subpopulations of the enteric nervous system in mammals. Recently, calbindin-immunoreactive enteric neurons have also been demonstrated in shorthorn sculpin (Myoxocephalus scorpius). In the present study, calbindin immunoreactivity has been investigated in the gut of adult and larval zebrafish (Danio rerio) and differences and similarities between the two species are discussed. Calbindin immunoreactivity is present in 40%?C50% of all enteric neurons in adult zebrafish. It first appears at 3?days post-fertilisation (dpf) and is present in all regions of the gut by 13 dpf. Calbindin-immunoreactive nerve cell bodies do not differ in size from calbindin-negative cells. Zebrafish calbindin-immunoreactive neurons are serotonin-negative, with at least some being choline acetyltransferase (ChAT)-positive, in contrast to the sculpin in which cells are generally smaller than the average enteric neuron and are serotonin-positive and ChAT-negative. These findings further emphasise the importance of comparative studies for understanding the diversity of chemical coding in the enteric nervous system of fish and other vertebrates. Improved knowledge of the role of the enteric nervous system is also essential for future studies of gut activity with regard to zebrafish being used as a model organism.     

Type I and type II cytokeratin cDNAs from the zebrafish (Danio rerio) and expression patterns during early development

Full-length cDNAs of a type I (zfCKI), and a type II (zfCKII) cytokeratin from the adult zebrafish, Danio rerio, were characterized and their expressions studied during early development and in the adult. The 1,426 bp long zfCKI cDNA encodes a 46.7 kD protein, whereas the 2,398 bp zfCKII cDNA encodes a protein of 58.6 kD. zfCKI and zfCKII each have a central rod domain that is characteristic of intermediate filaments and which share 73%-91% and 87%-93% similarity, respectively, with those of type I and type II cytokeratins from zebrafish, goldfish, and the rainbow trout. The central rod domains of zfCKI and zfCKII also contain the IF signature motif, IA[T/E]YR[K/R]LL[D/E]. zfCKI has, in addition, a leucine-zipper motif at a.a. residues 184-205 and 191-212. Both zfCKI and zfCKII mRNAs are expressed in the epidermis of the zebrafish. zfCKII mRNA was both maternally inherited and zygotically transcribed and was detected from the one-cell embryo to adult stages. zfCKII was also strongly expressed specifically during the 20-somites, protruding-mouth, and adult stages. In the adult, it was uniformly expressed in the skin, fins and scale epidermis. In contrast, zfCKI mRNA was undetectable in the oocyte but was zygotically transcribed from the epiboly stage onwards. Its expression in the skin was strong only up to the swimming larva stage and was weak and patchy in the adult. Both zfCKI and zfCKII were expressed in the neurons and glial cells of the brain and spinal cord. In the adult eye, zfCKI and zfCKII were expressed in the ganglion cell layer and the retina, but zfCKII was also strongly expressed in the cornea as well as in chondrocytes in the skull.     

Effects of simulated microgravity on the development of the swimbladder and buoyancy control in larval zebrafish (Danio rerio)

The gas-filled swimbladder of teleost fishes provides hydrodynamic lift which counteracts the high density of other body tissues, and thereby allows the fish to achieve neutral buoyancy with minimal energy expenditure. In this study, we examined whether the absence of a constant direction gravitational vector affects the ontogeny of the swimbladder and buoyancy control in zebrafish (Danio rerio). We exposed fertilized eggs to simulated microgravity (SMG) in a closed rotating wall vessel with control eggs placed in a similar but nonrotating container. All eggs hatched in both groups. At 96 hr of postfertilization (hpf), all larvae were removed from the experimental and control vessels. At this point, 62% of the control larvae, but only 14% of SMG-exposed larvae, were observed to have inflated their swimbladder. In addition, the mean volume of the inflated swimbladders was significantly greater in the control larvae compared with larvae raised in SMG. After transfer to open stationary observation tanks, larvae with uninflated swimbladders in both groups swam to the surface to complete inflation, but this process was significantly delayed in larvae exposed to SMG. Initial differences in swimbladder inflation and volume between groups disappeared by 144 hpf. Furthermore, there were no apparent changes in patterns of development and maturation of swimbladder musculature, vasculature, or innervation resulting from SMG exposure at later stages of ontogeny. These data indicate that, despite a transient delay in swimbladder inflation in zebrafish larvae exposed to SMG, subsequent swimbladder development in these animals proceeded similarly to that in normal larvae.     

Developmental expression of alcohol dehydrogenase (ADH3) in zebrafish (Danio rerio)

Alcohol dehydrogenase (ADH) is the primary enzyme responsible for metabolism of ethanol to acetaldehyde. One class of ADH has been described in fish, and has been found to be structurally similar to mammalian class III ADH (glutathione-dependent formaldehyde dehydrogenase) but functionally similar to class I ADH (primarily responsible for ethanol metabolism). We have cloned a cDNA by RT-PCR from zebrafish (Danio rerio) liver representing the zebrafish ADH3 gene product, with a coding region of 1131 nucleotides. The deduced amino acid sequences share 90% identity to ADH3 from the marine fish Sparus aurata, and 82 and 81% identity to the mouse and human sequences, respectively. Using a quantitative competitive RT-PCR assay, ADH3 mRNA was detected at all timepoints analyzed and was lowest between 8 and 24 h postfertilization. Thus, differential ADH3 expression may be at least partly responsible for temporal variations in the sensitivity of zebrafish embryos to developmental alcohol exposure.     

In vivo studies of liver-type fatty acid binding protein (L-FABP) gene expression in liver of transgenic zebrafish (Danio rerio)

Mammalian liver fatty acid binding protein (L-FABP) is a small cytosolic protein in various tissues including liver, small intestine and kidney and is thought to play a crucial role in intracellular fatty acid trafficking and metabolism. To better understand its tissue-specific regulation during zebrafish hepatogenesis, we isolated 5'-flanking sequences of the zebrafish L-FABP gene and used a green fluorescent protein (GFP) transgenic strategy to generate liver-specific transgenic zebrafish. The 2.8-kb 5'-flanking sequence of zebrafish L-FABP gene was sufficient to direct GFP expression in liver primordia, first observed in 2 dpf embryos and then continuously to the adult stage. This pattern of transgenic expression is consistent with the expression pattern of the endogenous gene. F2 inheritance rates of 42-51% in all the seven transgenic lines were consistent with the ratio of Mendelian segregation. Further, hhex and zXbp-1 morphants displayed a visible liver defect, which suggests that it is possible to establish an in vivo system for screening genes required for liver development.     

Oestrogen-induced expression of a novel liver-specific aspartic proteinase in Danio rerio (zebrafish)

Aspartic proteinases are a group of endoproteolytic proteinases active at acidic pH and characterized by the presence of two aspartyl residues in the active site. They include related paralogous proteins such as cathepsin D, cathepsin E and pepsin. Although extensively investigated in mammals, aspartic proteinases have been less studied in other vertebrates. In a previous work, we cloned and sequenced a DNA complementary to RNA encoding an enzyme present in zebrafish liver. The sequence resulted to be homologous to a novel form of aspartic proteinase firstly described by us in Antarctic fish. In zebrafish, the gene encoding this enzyme is expressed only in the female liver, in contrast with cathepsin D that is expressed in all the tissues examined independently of the sex. For this reason we have termed the new enzyme liver-specific aspartic proteinase (LAP).Northern blot analyses indicate that LAP gene expression is under hormonal control. Indeed, in oestrogen-treated male fish, cathepsin D expression was not enhanced in the various tissues examined, but the LAP gene product appeared exclusively in the liver. Our results provide evidence for an oestrogen-induced expression of LAP gene in liver. We postulate that the sexual dimorphic expression of the LAP gene may be related to the reproductive process.