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PubMed2001

The use of microangiography in detecting aberrant vasculature in zebrafish embryos exposed to cadmium.

Embryonic vascular patterns in zebrafish (Danio rerio) could be visualised by confocal microscopy coupled with microinjected fluorescent microbeads. This microangiographic technique was adopted here, for the first time, to study the effects of cadmium on cardiovascular development in zebrafish embryos. Zebrafish embryos were incubated in culture medium containing 100 microM cadmium from 5 h post fertilisation (hpf) to 48 hpf. At 48 hpf, embryos were examined for viability and occurrence of malformations. The 100 microM cadmium caused 32.21 +/- 3.65% mortality and 20.33 +/- 4.04% visible malformations in surviving embryos. In the remaining embryos with no visible signs of malformations, further assessments for less obvious abnormalities were performed. Assessments on craniofacial development were made by digital measurements on areas of brains and eyes. Cardiac development was assessed by immunostaining the heart with the antibody MF20 specific for myosin heavy chain. Body lengths of the embryos were also measured. Embryonic development of brains, eyes, hearts and body lengths of visibly healthy embryos in the cadmium treatment group showed no significant difference from the controls. Embryonic vasculature of these visibly healthy embryos was then studied by microinjecting fluorescent microbeads of diameter 0.02 microm into the circulation. All the cadmium treated embryos showed localised vascular defects in the dorsal aortae, segmental and cranial vessels while none of the control embryos showed any aberrant patterns in the networking of the vasculature. Improved image analyses on the anterior regions revealed that cadmium treated embryos had markedly less complex networks of cranial vessels with fewer vessels perfusing the craniofacial regions. The number of branch points in the vascular network was counted. In untreated embryos, there were 135.6 +/- 51 branches in the vasculature in entire body. In the cadmium treated embryos, there were 64.5+/-31 branches. The difference was significant when assessed with Student's t-test. It appeared that although cadmium did not cause any signs of external malformations in these visibly healthy embryos, nonetheless induced impaired branching and anastomsis of the cranial vessels. This study revealed, for the first time, that vital vascular structures in fish embryos could be affected by exposure to cadmium. This technique allowed visualisation of vascular anomalies in embryos showing no external signs of malformations. The impairment of anatomical features during embryonic development might serve as meaningful health endpoints in ecotoxicological studies and in risk assessment.

Angiography↗

PubMed2001

Interaction of cadmium toxicity in embryos and larvae of zebrafish (Danio rerio) with calcium and humic substances.

The influence of humic substances (HS) and calcium (Ca) on cadmium (Cd) toxicity was determined using zebrafish (Danio rerio). Embryo and larvae of the zebrafish were exposed to various Cd concentrations (1.8; 2.8; 4.2; 6.2; 9.3 mg/l Cd) for 144 h. Combinations of low (0.2 mmol/l) and high (2 mmol/l) Ca, +HS (5 mg/l C) or -HS were used during Cd exposure. The toxicity of Cd was affected by (1) exposure concentration; (2) exposure time; (3) presence of HS; and (4) the Ca concentration. The results show that Ca and HS protect against Cd toxicity in zebrafish embryos. The best protection was in the high Ca-HS group, followed by high Ca+HS group and low Ca+HS group. The survival in the low Ca-HS group was the worse. Survival in the high Ca-HS group and the high Ca+HS group was similar with the exception of the highest Cd concentration (9.3 mg/l) where the survival of the high Ca+HS group was less than in the high Ca-HS group. The exposure system was modelled using a chemical equilibrium program (MINEQL+) to determine if the likely mechanism causing the anomalous result in the highest Cd concentration. The equilibrium model cannot explain these results, which suggests that this effect has a kinetic basis, such as time needed for Cd to displace Ca already bound by HS.

Animals↗

PubMed2003

Developmental estrogenic exposure in zebrafish (Danio rerio): I. Effects on sex ratio and breeding success.

In vivo studies of fishes exposed to xenoestrogens have reported vitellogenin (Vtg) induction, ovatestes, altered sex ratios, and impaired reproductive capacity. The objective of this study was to determine concentration dependent effects of a weak estrogen receptor agonist, 4-nonylphenol (NP) and a potent estrogen receptor agonist, 17alpha-ethinylestradiol (EE) on sex ratios, gonad morphology, Vtg induction and breeding success in zebrafish (Danio rerio). Fish were exposed from 2 to 60 days post-hatch (dph) to NP (10, 30, or 100 microg/l nominal), EE (1, 10, or 100 ng/l nominal), or solvent control (acetone; 0.2% v/v) in a static-renewal system with replacement every 48 h. At 60 dph, 20 fish from each treatment were euthanized for histological examination of gonads and Western blotting for Vtg using pooled heart homogenates. Remaining fish were reared in clean water until adulthood (120 dph) for breeding studies. Due to high mortality in the 100 ng/l EE group, insufficient fish were available for analyses. The percentage of males at 60 dph changed from 45% (9/20) in solvent controls to 0% at 10 ng/l EE and 10% at 100 microg/l NP. A concentration dependent increase in the number of fish with undeveloped gonads at 60 dph was observed in the EE exposure group. Two fish with ovatestes were observed at 100 microg/l NP, while one was observed at 30 microg/l NP. Western blotting showed induction of Vtg at 30 and 100 microg/l NP and 10 ng/l EE. The sex ratios of adults determined at 160 dph revealed no significant departure from 1 male:1 female, suggesting that exposure of zebrafish to estrogenic chemicals during sexual differentiation and early gametogenesis did not irreversibly alter phenotypic sex. The condition factor of adult fish and ovo-somatic index of adult female fish were also unaffected by developmental exposure to NP or EE. Despite this, breeding trials conducted in adult fish from 120 to 160 dph revealed significant reductions in the percent of viable eggs, hatchability, and swim-up success at 10 ng/l EE and 100 microg/l NP. Our results suggest that functional reproductive capacity (breeding success) may be more sensitive than gross morphological endpoints (condition, ovo-somatic index, sex ratio) in adult zebrafish exposed to xenoestrogens during sexual differentiation and early gametogenesis.

Animals↗

PubMed1999

Cloning, sequencing and differential expression of alphaB-crystallin in the zebrafish, Danio rerio.

Here we report the cloning and expression of alphaB-crystallin from the zebrafish. 5'- and 3'-RACE was used to isolate a 900-bp transcript that contained insertions and deletions that differentiate it from both alphaA-crystallin and HSP-27. The deduced amino acid sequence of zebrafish alphaB-crystallin revealed that it lacked four residues in the C-terminus implicated in protein-protein interactions in other vertebrate species. In addition, the sequence contained two substitutions at sites implicated in phosphorylation in other vertebrate species. Northern analysis and semi-quantitative RT-PCR indicate that zebrafish alphaB-crystallin is expressed at extremely low levels outside of the lens.

Amino Acid Sequence↗

PubMed1999

Molecular cloning of a cold-shock domain protein, zfY1, in zebrafish embryo(1).

Cold-shock domain proteins in vertebrates contain a highly conserved domain which is related to the Escherichia coli cold-shock proteins. Here we report the cloning of a cold-shock domain protein from zebrafish embryo. Using the combination of PCR techniques with degenerate primers, 5'RACE and 3'RACE, the full length cDNA of a cold-shock domain protein in the zebrafish embryo was successfully cloned without constructing and screening a library. Determined from the deduced amino acid sequence, this protein is most similar to Xenopus, FRGY1, and this newly cloned zebrafish gene was therefore designated as zfY1.

Amino Acid Sequence↗

PubMed2003

Teleost multiple tissue (tmt) opsin: a candidate photopigment regulating the peripheral clocks of zebrafish?

Isolated organs and cell lines from zebrafish exhibit circadian oscillations in clock gene expression that can be entrained to a 24-h light/dark cycle. The mechanism underlying this cellular photosensitivity is unknown. We report the identification of a novel opsin family, tmt-opsin, that has a genomic structure characteristic of vertebrate photopigments, an amino acid identity equivalent to the known photopigment opsins, and the essential residues required for photopigment function. Significantly, tmt-opsin is expressed in a wide variety of neural and non-neural tissues, including a zebrafish embryonic cell line that exhibits a light entrainable clock. Collectively the data suggest that tmt-opsin is a strong candidate for the photic regulation of zebrafish peripheral clocks.

Amino Acid Sequence↗

PubMed2003

From cells to circuits: development of the zebrafish spinal cord.

The ability of an animal to carry out its normal behavioral repertoire requires generation of an enormous diversity of neurons and glia. The relative simplicity of the spinal cord makes this an especially attractive part of the nervous system for addressing questions about the development of vertebrate neural specification and function. The last decade has witnessed an explosion in our understanding of spinal cord development and the functional interactions among spinal cord neurons and glia. Cellular, genetic, molecular, physiological and behavioral studies in zebrafish have all been important in providing insights into questions that remained unanswered by studies from other vertebrate model organisms. This is the case because many zebrafish spinal neurons can be individually identified and followed over time in living embryos and larvae. In this review, we discuss what is currently known about the cellular, genetic and molecular mechanisms involved in specifying distinct cell types in the zebrafish spinal cord and how these cells establish the functional circuitry that mediates particular behaviors. We start by describing the early signals and morphogenetic movements that form the nervous system, and in particular, the spinal cord. We then provide an overview of the cell types within the spinal cord and describe how they are specified and patterned. We begin ventrally with floor plate and proceed dorsally, through motoneurons and oligodendrocytes, interneurons, astrocytes and radial glia, spinal sensory neurons and neural crest. We next describe axon pathfinding of spinal neurons. Finally, we discuss the roles of particular spinal cord neurons in specific behaviors.

Animals↗

PubMed2001

Analysis of pancreatic development in living transgenic zebrafish embryos.

Using DNA constructs containing regulatory sequences of the zebrafish Pdx-1 and insulin genes, germline transgenic zebrafish expressing the green fluorescent protein (GFP) reporter gene in the pancreas were generated. For both constructs, the GFP expression patterns in transgenic embryos were consistent with the mRNA expression patterns detected by RNA in situ hybridization. A deletion promoter analysis revealed that positive and negative cis-acting elements were involved in regulation of insulin gene expression. Three-dimensional reconstructions imaged from living embryos using two-photon laser-scanning microscopy (TPLSM) demonstrated that the zebrafish pancreas is formed from a single dorsal pancreatic cell mass. This is in contrast to mammals where the pancreas derives from both dorsal and ventral anlage. These transgenic fish should be useful for in vivo studies of factors involved in specifying and regulating pancreatic development and function.

5' Untranslated Regions↗

PubMed2002

Zebrafish insulin-like growth factor-I receptor: molecular cloning and developmental expression.

The biological actions of the insulin-like growth factors (IGFs) are mediated primarily by the IGF-I receptor (IGF-IR), and the IGF family has been highly conserved throughout vertebrate evolution. In this study we report the isolation of a 3 kb cDNA clone for the zebrafish IGF-IR that includes the complete 3' untranslated region and polyA tail and mapping of the receptor gene to zebrafish linkage group 7. The open reading frame deduced from the cDNA sequence encompasses the juxtamembrane and protein tyrosine kinase portions of the receptor, and is 70 and 67% identical to the corresponding regions of the IGF-IRs of the turbot and Xenopus, respectively. By RT-PCR, zebrafish IGF-IR expression was detected from early blastula to early larval stages of development. Using whole mount in situ hybridization, IGF-IR expression was detected after gastrulation. Expression was evident in most tissues but was particularly evident in the tail, in eye and ear primordia and in the brain.

3' Untranslated Regions↗

PubMed1999

Cloning of zebrafish activin type IIB receptor (ActRIIB) cDNA and mRNA expression of ActRIIB in embryos and adult tissues.

A full-length cDNA encoding for activin type IIB receptor (ActRIIB) was cloned from zebrafish embryos. It encodes a protein with 509 amino acids consisting of a signal peptide, an extracellular ligand binding domain, a single transmembrane region, and an intracellular kinase domain with predicted serine/threonine specificity. The extracellular domain shows 74-91% sequence identity to human, bovine, mouse, rat, chicken, Xenopus and goldfish activin type IIB receptors, while the transmembrane region and the kinase domain show 67-78% and 82-88% identity to these known activin IIB receptors, respectively. In adult zebrafish, ActRIIB mRNA was detected by RT-PCR in the gonads, as well as in non-reproductive tissues, including the brain, heart and muscle. In situ hybridization on ovarian sections further localized ActRIIB mRNA to cytoplasm of oocytes at different stages of development. Using whole-mount in situ hybridization, ActRIIB mRNA was found to be expressed at all stages of embryogenesis examined, including the sphere, shield, tail bud, and 6-7 somite. These results provide the first evidence that ActRIIB mRNA is widely distributed in fish embryonic and adult tissues. Cloning of zebrafish ActRIIB demonstrates that this receptor is highly conserved during vertebrate evolution and provides a basis for further studies on the role of activin in reproduction and development in lower vertebrates.

Activin Receptors, Type II↗

PubMed2000

Temporal expression and T3 induction of thyroid hormone receptors alpha1 and beta1 during early embryonic and larval development in zebrafish, Danio rerio.

The effects of thyroid hormones on metabolism and development are mediated by thyroid hormone receptors (TRs). We report the cloning and characterization of a TR beta1 cDNA from zebrafish. Southern blot analysis revealed that there is a single genomic locus for the TR beta gene, while the TR alpha gene potentially has two loci. Multiple TR alpha and TR beta transcripts were detected in adult tissues. Using a semiquantitative RT-PCR assay, zygotic expression of TR alpha1 and TR beta1 were shown to occur before the midblastula transition stage. In transiently transfected HeLa cells, TR alpha1 displayed constitutive transactivation in the absence of ligands, which was slightly enhanced by triiodothyronine (T3). The transactivating activity of TR beta1 was strictly ligand-dependent and repressed in the absence of T3. Finally, the T3 induction of TR alpha1 and TR beta1 mRNAs was demonstrated in zebrafish embryos and larvae. The auto-induction of TR alpha1 and TR beta1 may serve a regulatory role during the embryonic and larval development of zebrafish.

Amino Acid Sequence↗

PubMed2003

The crypt neurons in the olfactory epithelium of the adult zebrafish express TrkA-like immunoreactivity.

The olfactory neurons of vertebrates, including teleosts, regenerate through adult life. This ability seems to depend partially on neurotrophic factors, which include some neurotrophins, mainly nerve growth factor (NGF), and their cognate Trk receptors. Zebrafish is an ideal model for studying neurogenesis in the olfactory system, but what kinds of olfactory sensory neurons (ciliated, microvillous or crypt neurons) are under the control of neurotrophins during adult life is unknown. Here we examined the expression of Trk proteins in the olfactory epithelium of adult zebrafish using immunohistochemistry. TrkA-like immunoreactivity was the only Trk detected, and it was restricted to the somata of crypt sensory neurons, their central processes being apparently unreactive. These results provide indirect evidence for a possible role of NGF-like molecules (i.e. neurotrophin-6 and neurotrophin-7) in the peripheral olfactory system of adult zebrafish, acting on TrkA-like receptor expressing crypt neurons. It remains to be established whether or not TrkA-like receptors and their ligands play a function in the neurogenesis or differentiation of these cells.

Animals↗

PubMed2001

Isolation and characterization of an alpha 2-type zebrafish glycine receptor subunit.

The complementary DNA for a novel alpha subunit of the glycine receptor, alphaZ2, was isolated from a zebrafish adult brain library. The molecular characteristics, phylogenetic relationships and messenger RNA length of this alphaZ2 subunit show it to be an alpha2-type glycine receptor subunit isoform. The leader peptide however, diverges from those of known glycine receptor alpha isoforms. Recombinantly expressed in Xenopus oocytes, alphaZ2 formed functional glycine receptor channels. These homomeric channels were activated by glycine and taurine, with apparent affinities similar to those reported for zebrafish alphaZ1 glycine receptor, and were also effectively antagonized by nanomolar concentrations of strychnine. However, during prolonged applications of agonists, ionic currents of alphaZ2 receptor channels declined to a much lower steady-state level than those of alphaZ1, indicating different desensitization properties. Analysis of messenger RNA revealed that alphaZ2 is specifically expressed in adult brain tissue and present in both adult and embryonic zebrafish. This report contributes to the characterization of the diversity of glycine receptor isoforms in vertebrates.

Aging↗

PubMed2003

A novel glycine receptor alpha Z1 subunit variant in the zebrafish brain.

Alpha subunits of the inhibitory glycine receptor (GlyR) display genetic heterogeneity in mammals and zebrafish. This diversity is increased in mammals by the alternative splicing mechanism. We report here in zebrafish, the characterization of a new alphaZ1 subunit likely arising from alphaZ1 gene by an alternative splice process (alphaZ1L). This novel cDNA possesses 45 supplementary nucleotides at the putative exon2/exon3 boundary. The corresponding protein contains 15 additional amino acids in the NH2-terminal domain. Heterologous expression of homomeric GlyRalphaZ1L in human embryonic kidney-293 cells generates glycine-gated strychnine-sensitive chloride channels with no obvious discrepancy with pharmacological properties of GlyRalphaZ1. Moreover, zinc modulation of glycine-induced currents is identical in alphaZ1 and alphaZ1L glycine receptors. During ontogenesis, simultaneous alphaZ1 and alphaZ1L mRNA synthesis have been observed. Embryonic and adult alphaZ1 and alphaZ1L mRNA expressions are restricted to the CNS. Embryonic alphaZ1L mRNA anatomical pattern of expression is, however, highly restrained and strictly limited to the rostral part of the brain revealing a highly regionalized function of alphaZ1L in the CNS. This report contributes to the characterization of the diversity of glycine receptor isoforms in zebrafish and emphasizes the common mechanism used among vertebrates for creating GlyR variety and specificity.

Alternative Splicing↗

PubMed2000

Control of motor axon guidance in the zebrafish embryo.

The zebrafish neuromuscular system has been an exemplary model for studying motor axon guidance since its detailed characterization almost two decades ago. In particular, characterization and detailed analysis has focused on the development and axogenesis of early developing primary motoneurons. During the first day of development, neuromuscular connections are limited to three primary motoneurons per spinal cord hemisegment innervating three discreet myotome territories. Observations of dye labeled primary motor axons in living embryos revealed that axogenesis is highly stereotyped with each primary motor axon extending along specific pathways and displaying particular characteristics. Exploiting the unique attributes of zebrafish, notably the ability to analyze motoneurons in living embryos and the capability to induce mutations, has allowed a comprehensive cellular, molecular and genetic approach to discerning the mechanisms that control the formation of neuromuscular connectivity. Knowledge gained from this body of work not only relates to zebrafish, but to vertebrate axon guidance in general.

Animals↗

PubMed2001

Characterization of mouse epithelial protein lost in neoplasm (EPLIN) and comparison of mammalian and zebrafish EPLIN.

EPLIN is a cytoskeleton-associated protein that was initially identified as the product of a gene that is transcriptionally down-regulated in cancer cells. In human, there are two known isoforms, EPLIN-alpha and -beta, generated by alternative promoter usage from a single gene. With the exception of a single LIM (lin-11, isl-1, and mec-3) domain, the sequence of EPLIN is unique and does not provide any clues to its function. To identify conserved regions of EPLIN that may be important for its function, we have characterized mouse (m) and zebrafish (zf) EPLIN. As in human, two isoforms, the 593 aa mEPLIN-alpha (77% identity; 83% similarity) and 753 aa mEPLIN-beta (75% identity; 83% similarity), were present in mouse. mEPLIN-alpha is highly expressed in embryonic tissue and adult lung and spleen, whereas mEPLIN-beta is preferentially expressed in kidney, testis, lung and liver. The analysis of mEPLIN gene revealed that the overall organization of the exons in mouse and human are conserved. In zebrafish, there was only one form, the 629 aa zfEPLIN, corresponding to the mammalian EPLIN-beta. Like its mammalian counterparts, ectopically expressed zfEPLIN is co-localized to the actin cytoskeleton. While the overall homology between mammalian and zebrafish EPLIN was not striking (37% identity; 50% similarity), there were seven highly conserved regions, which should be useful in structure-function studies of this novel protein.

Amino Acid Sequence↗

PubMed1997

Cloning and expression of three members of the zebrafish Bmp family: Bmp2a, Bmp2b and Bmp4.

In vertebrates, Bmps (bone morphogenetic proteins) play critical roles in establishing the basic embryonic body plan and are involved in the development of a large variety of organs and tissues. To study the evolution of Bmps, we isolated cDNAs for three members of the zebrafish Bmp gene family: Bmp2a, Bmp2b and Bmp4. The deduced amino acid sequences of Bmp2a and Bmp4 consist of 386 and 400 aa, respectively and show high homologies to their counterparts in mouse, chick and Xenopus. The deduced Bmp2b aa sequence consists of 411 aa and the mature protein shows 88% and 86% identities to zebrafish Bmp2a and Bmp4, respectively. The expression of the mRNA of these three genes has been analyzed by whole mount in situ hybridization and RT-PCR. Areas of zebrafish Bmp2 and Bmp4 expression suggest evolutionary conserved mechanisms of Bmp2/4 dependent differentiation between lower and higher vertebrates.

Amino Acid Sequence↗

PubMed1997

Brain-derived neurotrophic factor gene expression in the developing zebrafish.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family of polypeptides that includes NGF, NT-3, NT-4/5 and NT-6. Although neurotrophins are known to be expressed in teleost fishes little is known about their functions in the development of these vertebrates. We are therefore studying BDNF in the zebrafish, Danio rerio. The structure of zebrafish BDNF mRNA was established using PCR and cDNA cloning. The encoded BDNF was 91% identical to mammalian BDNF. Southern blot analysis revealed a unique BDNF gene. Northern blot analysis detected two heterogeneous populations of BDNF transcripts centered at 1.6 and 2 kb. BDNF transcripts were first measurable 24 h post-fertilization (pf). Their abundance relative to total transcripts increased 6-fold between 1 day and 3 days pf and again 2-fold by 7 days pf. In situ hybridization analyses of 4-day-old larvae revealed BDNF transcripts in the retina, brain, otic vesicle, pectoral fin and the hair cells of the neuromast. The early onset and cellular sites of expression suggest that BDNF functions in nervous system and fin development in the zebrafish.

Aging↗