Effect of nanoparticle stabilization and physicochemical properties on exposure outcome: acute toxicity of silver nanoparticle preparations in zebrafish (Danio rerio).

Nanotechnology has vast potential for expanded development and novel application in numerous sectors of society. With growing use and applications, substantial production volumes and associated environmental release can be anticipated. Exposure effect of nanoparticles (NP) on biological systems may be intrinsic to their physicochemical properties introducing unknown associated risk. Herein, we expand the knowledge of health and environmental impact of silver nanoparticles (AgNPs), testing the acute toxicity of 14 AgNP preparations on developing zebrafish embryos (Danio rerio). Toxicological end points, including mortality, hatching rate, and heart rate were recorded. Concentration, stabilization agent and physicochemical properties were monitored as contributing outcome factors. Our findings indicate wide ranging LC50 24 h postfertilization values (0.487 ppm (0.315, 0.744 95% CI) to 47.89 ppm (18.45, 203.49 95% CI)), and indicate surface charge and ionic dissolution as key contributory factors in AgNP exposure outcome.

Loss of plakophilin 2 disrupts heart development in zebrafish.

The desmosomal armadillo protein plakophilin 2 is the only plakophilin expressed in the heart, and mutations in the human plakophilin 2 gene result in arrhythmogenic right ventricular cardiomyopathy. To investigate loss of function, we knocked down plakophilin 2 by morpholino microinjection in zebrafish. This resulted in decreased heart rate, cardiac oedema, blood pooling, a failure of the heart to pattern correctly and a twisted tail. Co-injection of plakophilin 2 mRNA rescued the morphant phenotype, indicating the specificity of the knockdown. Desmosome numbers were decreased in morphant hearts and the plaque and midline structures of the desmosomes in the intercalated discs were disrupted when examined by electron microscopy. cmlc2 and vmhc expression at 48 hours post-fertilization (hpf) showed incomplete looping of the heart in morphant embryos by whole mount in situ hybridization, and bmp4 expression was expanded into the ventricle. The domain of expression of the heart marker nkx2.5 at 24 hpf was expanded. At the 18 somite stage, expression of the cardiogenic gene lefty2 was abolished in the left cardiac field, with concomitant increases in bmp4, spaw and lefty1 expression, likely resulting in the looping defects. These results indicate that plakophilin 2 has both structural and signalling roles in zebrafish heart development.

Insulin-like growth factor-2 regulates early neural and cardiovascular system development in zebrafish embryos.

The insulin-like growth factor (IGF) family is essential for normal embryonic growth and development and it is highly conserved through vertebrate evolution. However, the roles that the individual members of the IGF family play in embryonic development have not been fully elucidated. This study focuses on the role of IGF-2 in zebrafish embryonic development. Two igf-2 genes, igf-2a and igf-2b, are present in the zebrafish genome. Antisense morpholinos were designed to knock down both igf-2 genes. The neural and cardiovascular defects in IGF-2 morphant embryos were then examined further using wholemount in situ hybridisation, TUNEL analysis and O-dianisidine staining. Knockdown of igf-2a or igf-2b resulted in ventralised embryos with reduced growth, reduced eyes, disrupted brain structures and a disrupted cardiovascular system, with igf-2b playing a more significant role in development. During gastrulation, igf-2a and igf-2b are required for development of anterior neural structures and for regulation of genes critical to dorsal-ventral patterning. As development proceeds, igf-2a and igf-2b play anti-apoptotic roles. Gene expression analysis demonstrates that igf-2a and igf-2b play overlapping roles in angiogenesis and cardiac outflow tract development. Igf-2b is specifically required for cardiac valve development and cardiac looping. Injection of a dominant negative IGF-1 receptor led to similar defects in angiogenesis and cardiac valve development, indicating IGF-2 signals through this receptor to regulate cardiovascular development. This is the first study describing two functional igf-2 genes in zebrafish. This work demonstrates that igf-2a and igf-2b are critical to neural and cardiovascular development in zebrafish embryos. The finding that igf-2a and igf-2b do not act exclusively in a redundant manner may explain why both genes have been stably maintained in the genome.

Regulation of expression of zebrafish (Danio rerio) insulin-like growth factor 2 receptor: implications for evolution at the IGF2R locus.

The insulin-like growth factor 2 receptor (IGF2R) is an unusual multifunctional receptor that interacts with a diverse variety of ligands. While the receptor has been well-characterized in mammals, little is known of its biology in other vertebrates. In this report, we characterize the expression of the zebrafish (Danio rerio) ortholog of the IGF2R gene. We show that two distinct, cell-type-specific promoters drive transcription of zebrafish igf2r and that these encode receptor isoforms that differ in their amino termini. Both promoters are active in adult fish and during embryonic development, but the proximal promoter generates more abundant transcripts. The 5'-UTR of the more abundantly expressed transcript contains several AUGs upstream of the main start codon, and these negatively regulate translation of a downstream reporter gene. Comparative sequence analysis shows that upstream AUGs (uAUGs) are a feature of IGF2R genes in several other vertebrates, including Xiphophorus, Xenopus, chicken, platypus, and opossum, but not in eutherian mammals. The IGF2R is imprinted in marsupial and placental mammals, and this transcriptional control of receptor abundance was proposed to have evolved following acquisition of an insulin-like growth factor 2 (IGF2) binding site by the ancestral receptor. Our observations suggest that receptor abundance was regulated at translation in ancestral vertebrates, before acquisition of an IGF2 binding site. We propose that evolution of imprinting at the mammalian IGF2R may have facilitated the loss of negative regulation of translation conferred by uAUGs.

Plakoglobin has both structural and signalling roles in zebrafish development.

Plakoglobin, or gamma-catenin, is found in both desmosomes and adherens junctions and participates in Wnt signalling. Mutations in the human gene are implicated in the congenital heart disorder, arrhythmogenic right ventricular cardiomyopathy (ARVC), but the signalling effects of plakoglobin loss in ARVC have not been established. Here we report that knockdown of plakoglobin in zebrafish results in decreased heart size, reduced heartbeat, cardiac oedema, reflux of blood between heart chambers and a twisted tail. Wholemount in situ hybridisation shows reduced expression of the heart markers nkx2.5 at 24 hours post fertilisation (hpf), and cmlc2 and vmhc at 48 hpf, while there is lack of restriction of the valve markers notch1b and bmp4 at 48 hpf. Wnt target gene expression was examined by semi-quantitative RT-PCR and found to be increased in morphant embryos indicating that plakoglobin is antagonistic to Wnt signalling. Co-expression of the Wnt inhibitor, Dkk1, rescues the cardiac phenotype of the plakoglobin morphant. beta-catenin protein expression is increased in morphant embryos as is its colocalisation with E-cadherin in adherens junctions. Endothelial cells at the atrioventricular boundary of morphant hearts have an aberrant morphology, indicating problems with valvulogenesis. Morphants also have decreased numbers of desmosomes and adherens junctions in the intercalated discs. These results establish the zebrafish as a model for ARVC caused by loss of plakoglobin function and indicate that there are signalling as well as structural consequences of this loss.

Mouse induced pluripotent stem cells.

The recent discovery that it is possible to directly reprogramme somatic cells to an embryonic stem (ES) cell-like pluripotent state, by retroviral transduction of just four genes (Oct3/4, Sox2, c-Myc and Klf4), represents a major breakthrough in stem cell research. The reprogrammed cells, known as induced pluripotent stem (iPS) cells, possess many of the properties of ES cells, and represent one of the most promising sources of patient-specific cells for use in regenerative medicine. While the ultimate goal is the use of iPS cells in the treatment of human disease, much of the research to date has been carried out with murine cells, and improved mouse iPS cells have been shown to contribute to live chimeric mice that are germ-line competent. Very recently, it has been reported that iPS cells can be generated by three factors without c-Myc, and these cells give rise to chimeric mice with a reduced risk of tumour development.

Molecular cloning and developmental expression of plakophilin 2 in zebrafish.

Armadillo proteins are involved in providing strength and support to cells and tissues, nuclear transport, and transcriptional activation. In this report, we describe the identification and characterisation of the cDNA of the desmosomal armadillo protein plakophilin 2 in zebrafish. The 2448bp coding sequence encodes a predicted 815 amino acid protein, with nine armadillo repeats characteristic of the p120-catenin subfamily. It shares conserved N-glycosylation, myristoylation, and glycogen synthase kinase 3, casein kinase 2, and protein kinase C phosphorylation sites with mammalian armadillo proteins including plakoglobin and beta-catenin. Semi-quantitative reverse transcription polymerase chain reaction and whole mount in situ hybridisation show that it is expressed both maternally and zygotically. It is ubiquitously expressed during blastula stages but becomes restricted to epidermal and cardiac tissue during gastrulation. These results provide evidence that zebrafish plakophilin 2 is developmentally regulated with potential roles in cell adhesion, signalling, and cardiac and skin development.

Molecular cloning and expression analysis of two distinct beta-glucosidase genes, bg1 and aven1, with very different biological roles from the thermophilic, saprophytic fungus Talaromyces emersonii.

Recent sequencing of a number of fungal genomes has revealed the presence of multiple putative beta-glucosidases. Here, we report the cloning of two beta-glucosidase genes (bg1 and aven1), which have very different biological functions and represent two of a number of beta-glucosidases from Talaromyces emersonii. The bg1 gene, encoding a putative intracellular beta-glucosidase, shows significant similarity to other fungal glucosidases from glycosyl hydrolase family 1, known to be involved in cellulose degradation. Solka floc, methyl-xylose, gentiobiose, beech wood xylan, and lactose induced expression of bg1, whereas glucose repressed expression. A second beta-glucosidase gene isolated from T. emersonii, aven1, encodes a putative avenacinase, an enzyme that deglucosylates the anti-fungal saponin, avenacin, rendering it less toxic to the fungus. This gene displays high homology with other fungal saponin-hydrolysing enzymes and beta-glucosidases within GH3. A putative secretory signal peptide of 21 amino acids was identified at the N-terminus of the predicted aven1 protein sequence suggesting that this enzyme is extracellular. Furthermore, T. emersonii cultivated on oat plant biomass was shown to deglucosylate avenacin. The presence of the avenacinase transcript was confirmed by RT-PCR on RNA extracted from mycelia grown in the presence of avenacin. The expression pattern of aven1 on various carbon sources was distinctly different from that of bg1. Only methyl-xylose and gentiobiose induced transcription of aven1. Gentiobiose induces synthesis of a number of cellulase genes by T. emersonii and it may be a possible candidate for the natural cellulase inducer observed in Penicillium purpurogenum. This work represents the first report of an avenacinase gene from a thermophilic, saprophytic fungal source, and suggests that this gene is not exclusive to plant pathogens.

Identification of zygotic genes expressed at the midblastula transition in zebrafish.

Early development of the embryo is directed by maternal gene products and characterised by limited zygotic gene activity, cell division synchrony and no cell motility in several vertebrates including fish and frogs. At the midblastula transition (MBT), zygotic transcription is grossly activated, cells become motile and cell divisions become asynchronous. The aim of this study was to identify genes whose expression is up-regulated at the MBT in zebrafish. Suppression subtractive hybridisation (SSH) was employed to isolate 48 unique cDNAs, 28 of which show significant similarity to known genes and 20 represent novel cDNAs. Twenty one of these genes, with potential roles in transcriptional regulation, cell cycle control, and embryonic patterning showed increased expression at the MBT. Our results demonstrate the value of SSH as a tool to clone novel, zygotic, developmentally regulated genes that may be important in the progression of the MBT and embryonic patterning.

Mannose 6-phosphate receptors in an ancient vertebrate, zebrafish.

The endosome/lysosome system plays key roles in embryonic development, but difficulties posed by inaccessible mammalian embryos have hampered detailed studies. The accessible, transparent embryos of Danio rerio, together with the genetic and experimental approaches possible with this organism, provide many advantages over rodents. In mammals, mannose 6-phosphate receptors (MPRs) target acid hydrolases to endosomes and lysosomes, but nothing is known of acid hydrolase targeting in zebrafish. Here, we describe the sequence of the zebrafish cation-dependent MPR (CD-MPR) and cation-independent MPR (CI-MPR), and compare them with their mammalian orthologs. We show that all residues critical for mannose 6-phosphate (M6P) recognition are present in the extracellular domains of the zebrafish receptors, and that trafficking signals in the cytoplasmic tails are also conserved. This suggests that the teleost receptors possess M6P binding sites with properties similar to those of mammalian MPRs, and that targeting of lysosomal enzymes by MPRs represents an ancient pathway in vertebrate cell biology. We also determined the expression patterns of the CD-MPR and CI-MPR during embryonic development in zebrafish. Both genes are expressed from the one-cell stage through to the hatching period. In early embryos, expression is ubiquitous, but in later stages, expression of both receptors is restricted to the anterior region of the embryo, covering the forebrain, midbrain and hindbrain. The expression patterns suggest time- and tissue-specific functions for the receptors, with particular evidence for roles in neural development. Our study establishes zebrafish as a novel, genetically tractable model for in vivo studies of MPR function and lysosome biogenesis.

nanor, a novel zygotic gene, is expressed initially at the midblastula transition in zebrafish.

A novel, developmentally regulated gene, nanor, was identified by suppression subtractive hybridization. It is first expressed following the midblastula transition (MBT), a critical developmental stage in the early vertebrate embryo when the zygotic genome is activated. The nanor cDNA (626bp) includes a complete open reading frame but neither the gene nor the deduced amino acid sequence shows significant similarity to any known gene or protein. Nanor encodes a 175 amino acid putative protein with a protein kinase C and three casein kinase II phosphorylation sites, an N-myristoylation site and an NFX-type zinc-finger domain, indicating a potential role in transcriptional regulation. Semi-quantitative RT-PCR, Northern blot, and in situ hybridization analysis revealed that nanor expression is developmentally regulated. It is initially expressed after the MBT at the sphere stage and during epiboly it is expressed in the forerunner cells. At 24 h post-fertilization, expression is solely anterior.

Insulin-like growth factor (IGF) signalling is required for early dorso-anterior development of the zebrafish embryo.

The insulin-like growth factor (IGF) signalling pathway has been highly conserved in animal evolution and, in mammals and Xenopus, plays a key role in embryonic growth and development, with the IGF-1 receptor (IGF-1R) being a crucial regulator of the signalling cascade. Here we report the first functional role for the IGF pathway in zebrafish. Expression of mRNA coding for a dominant negative IGF-1R resulted in embryos that were small in size compared to controls and had disrupted head and CNS development. At its most extreme, this phenotype was characterized by a complete loss of head and eye structures, an absence of notochord and the presence of abnormal somites. In contrast, up-regulation of IGF signalling following injection of IGF-1 mRNA, resulted in a greatly expanded development of anterior structures at the expense of trunk and tail. IGF-1R knockdown caused a significant decrease in the expression of Otx2, Rx3, FGF8, Pax6.2 and Ntl, while excess IGF signalling expanded Otx2 expression in presumptive forebrain tissue and widened the Ntl expression domain in the developing notochord. The observation that IGF-1R knockdown reduced expression of two key organizer genes (chordin and goosecoid) suggests that IGF signalling plays a role in regulating zebrafish organizer activity. This is supported by the expression of IGF-1, IGF-2 and IGF-1R in shield-stage zebrafish embryos and the demonstration that IGF signalling influences expression of BMP2b, a gene that plays an important role in zebrafish pattern formation. Our data is consistent with a common pathway for integration of IGF, FGF8 and anti-BMPs in early vertebrate development.

Three-dimensional structure of a thermostable native cellobiohydrolase, CBH IB, and molecular characterization of the cel7 gene from the filamentous fungus, Talaromyces emersonii.

The X-ray structure of native cellobiohydrolase IB (CBH IB) from the filamentous fungus Talaromyces emersonii, PDB 1Q9H, was solved to 2.4 A by molecular replacement. 1Q9H is a glycoprotein that consists of a large, single domain with dimensions of approximately 60 A x 40 A x 50 A and an overall beta-sandwich structure, the characteristic fold of Family 7 glycosyl hydrolases (GH7). It is the first structure of a native glycoprotein and cellulase from this thermophilic eukaryote. The long cellulose-binding tunnel seen in GH7 Cel7A from Trichoderma reesei is conserved in 1Q9H, as are the catalytic residues. As a result of deletions and other changes in loop regions, the binding and catalytic properties of T. emersonii 1Q9H are different. The gene (cel7) encoding CBH IB was isolated from T. emersonii and expressed heterologously with an N-terminal polyHis-tag, in Escherichia coli. The deduced amino acid sequence of cel7 is homologous to fungal cellobiohydrolases in GH7. The recombinant cellobiohydrolase was virtually inactive against methylumberiferyl-cellobioside and chloronitrophenyl-lactoside, but partial activity could be restored after refolding of the urea-denatured enzyme. Profiles of cel7 expression in T. emersonii, investigated by Northern blot analysis, revealed that expression is regulated at the transcriptional level. Putative regulatory element consensus sequences for cellulase transcription factors have been identified in the upstream region of the cel7 genomic sequence.

Cellular longevity: role of apoptosis and replicative senescence.

Cellular longevity refers to the lifespan of an individual cell. Normal cells have a finite lifespan and typically die by undergoing apoptosis, or enter into a state of irreversible growth arrest, termed replicative senescence, at the end of that lifespan. The lifespan of a cell is a balance between pro-survival/anti-apoptotic and pro-apoptotic death-promoting factors. The role of heat shock proteins, Bcl-2 family members, antioxidant molecules, and telomere length and telomerase activity in the regulation of apoptosis and replicative senescence, will be discussed.

Cyclin-dependent kinase 8 is expressed both maternally and zygotically during zebrafish embryo development.

Cyclin-dependent kinase 8 (cdk8) regulates transcription by phosphorylating RNA polymerase II and TFIIH. The mechanism of zygotic transcription activation during vertebrate embryonic development is poorly understood. Here we describe the cloning and developmental expression pattern of zebrafish cdk8 mRNA. It is highly conserved, sharing 79% DNA and 95% amino acid sequence identity with human cdk8, thereby indicating an important role for the protein. Northern blotting and whole mount in situ hybridisation revealed expression of zebrafish cdk8 maternally, following the onset of zygotic transcription at the mid-blastula transition (MBT) and throughout embryonic development.