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PubMed2004

Global changes in genomic methylation levels during early development of the zebrafish embryo.

We have examined the methylation status of the zebrafish genome during early embryogenesis and we find evidence that methylation fluxes do occur in that organism. The parental genetic contributions to the zygote are, initially, differently methylated with the genome of the sperm being hypermethylated relative to the genome of the oocyte. Post-fertilization there is an immediate decrease in methylation of the embryonic genome but the methylation begins to increase rapidly and is re-established by the gastrulation stage. These results are consistent with the results of Santos et al. (Dev Biol 241:172-182, 2002), who examined the methylation of early mouse embryos, and this conservation argues that demethylation/re-methylation is an important part of vertebrate development.

Animals↗
PubMed2002

Alternative transcripts of a polyhomeotic gene homolog are expressed in distinct regions of somites during segmentation of zebrafish embryos.

Here we describe isolation and characterization of two zebrafish cDNAs, designated ph2alpha and ph2beta, which were identified as structural homologs of the Drosophila polyhomeotic, mouse Mph2, and human HPH2 genes, collectively termed the Polycomb group. The alpha and beta transcripts shared a 1.9-kb sequence at their 3'-termini. Alpha had an additional 1.6-kb sequence extending toward its 5'-terminus. Only a short 0.1-kb segment was unique to beta. Sequencing of a genomic clone corresponding to the two cDNAs indicated that the mRNAs were transcribed from a single gene locus by alternative promoters. Northern blots revealed expression of alpha transcripts during the segmentation period, while beta expression occurred at all developmental stages examined. Whole-mount in situ hybridizations with an alpha-specific probe and a probe recognizing both transcripts revealed distinct spatio-temporal expression patterns along developing somites. Alpha transcripts were detected initially at the 7-8 somite stage; beta transcripts appeared in the first somites. As segmentation proceeded, alpha and beta expression shifted position toward the tailbud in parallel with the formation of each somite. Within individual somites, the signal corresponding to alpha was strongest at the posterior border and weakest in the anterior region. Conversely, that corresponding to beta was strongest at the anterior border and weakest in the posterior region. The data support the idea that Ph2alpha and Ph2beta are involved in spatio-temporal generation of somites as well as in specification of antero-posterior regional differences within individual somites.

Amino Acid Sequence↗
PubMed2004

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.

Animals↗
PubMed2004

Phenylthiourea as a weak activator of aryl hydrocarbon receptor inhibiting 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 transcription in zebrafish embryo.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that can be activated by a diverse synthetic and naturally-occurring chemicals, such as the halogenated aromatic hydrocarbons (HAHs) and the non-halogenated polycyclic aromatic hydrocarbons (PAHs). The liganded AHR modulates the genetic activity of a variety of xenobiotic-responsive genes, including cytochrome P4501A1 (CYP1A1). The tyrosinase inhibitor 1-phenyl-2-thiourea (PTU) is widely used in zebrafish research to suppress pigmentation in developing embryos/fry. Here we showed that 0.2 mM PTU induced a basal level of CYP1A1 transcription in zebrafish embryonic integument as early as 24 h postfertilization (hpf) stage. Subsequently, PTU induced CYP1A1 transcription in blood vessels at 36 hpf. During larval stage, the liver and all pharyngeal arch vessels of PTU-treated embryos exhibited CYP1A1 transcription as well. Comparing to TCDD, PTU induces CYP1A1 transcription with much lower efficacy in zebrafish embryos. Coincubating the embryos with PTU and TCDD led to repressing TCDD-induced CYP1A1 transcription. Mechanistic studies indicated that both of PTU- and TCDD-mediated CYP1A1 transcriptions are modulated by the same AHR-ARNT signaling pathway.

Animals↗
PubMed2003

The expression of gelatinase A (MMP-2) is required for normal development of zebrafish embryos.

Gelatinase A, also called matrix metalloproteinase 2 (MMP-2), belongs to the matrix metalloproteinase (MMP) family. MMP-2 cleaves type IV collagen, denatured collagen (gelatin), and other extracellular matrix (ECM) components. MMP-2 has been reported to be involved in a number of biological and pathological processes, but previous studies have not indicated that its expression is essential for early embryogenesis. In the current study, we have utilized zebrafish as a developmental model to study the role of MMP-2 during embryogenesis. We have successfully isolated a zebrafish MMP-2 (zMMP-2) homologue showing over 80% identity and over 90% similarity to its human counterpart. In situ analysis showed that zMMP-2 was expressed as early as the one-cell stage implying a maternal origin during oogenesis, and embryos continued to express zMMP-2 through at least the 72-h stage of development. RT-PCR analysis confirmed the in situ expression pattern and gelatin zymography indicated that a metalloproteinase with the same gel mobility as vertebrate MMP-2 was present in zebrafish embryos. Injection of zMMP-2 antisense morpholino oligonucleotides into 1- to 4-cell embryos resulted in a truncated axis, monitored through 72 h of development indicating that this metalloproteinase plays an important role in zebrafish embryogenesis. Monpholino-induced alterations in development began to be observed at 12 h of embryogenesis based on morphological and axis marker studies. The results obtained in zebrafish are in contrast to murine knockout studies that indicate that MMP-2 does not have a major role in mouse embryogenesis.

Amino Acid Sequence↗
PubMed2004

Novel vector systems optimized for injecting in vitro-synthesized mRNA into zebrafish embryos.

Microinjection of nucleic acids or proteins is a useful way of studying embryonic development. In particular, injection of in vitro-transcribed capped RNA is commonly employed to achieve ectopic or increased expression of genes. Two vector systems, pCS2+ and pT7Ts, have been used for this purpose in zebrafish. However, they were initially optimized for Xenopus embryos not for zebrafish. Here we describe a vector, pcGlobin2, optimized for zebrafish, and its derivative, pcGlobin2-GST. This new vector system offers several advantages. First, pcGlobin 2 contains three critical elements 15' and 3' zebrafish beta-globin UTRs, and a poly(A) tail] for generating stable mRNAs and greatly improving translation efficiency. Second, subcloning and preparation of template DNA is easier because of the larger number of restriction sites. Third, protein-binding assays can be performed directly on the injected embryos with pcGlobin2-GST. Lastly, this vector system can be transfected into animal cells without additional subcloning.

Animals↗
PubMed1997

Developmental expression of thrombin in zebrafish embryos: a novel model to study hemostasis.

A partial cDNA encoding zebrafish prothrombin has been cloned and used as a probe to study the temporal expression of prothrombin mRNA during early embryonic development. The results revealed accumulation of prothrombin mRNA in diverse tissues such as the eyes and myotomes in early embryogenesis. We have also examined the enzymatic activity of thrombin in converting fibrinogen to fibrin in individual embryos at different stages of development. We found that the fibrin-forming activity does not temporally correlate with the first presence of thrombin mRNA in the early stages of embryogenesis, but does correlate with the initiation of blood formation. Our ability to observe the fibrin-forming activity in single individual embryo will facilitate studies on identifying recessive mutations affecting blood coagulation, such as the regulatory gene mutations controlling the clotting factor genes. Furthermore, the observation of thrombin activity will also facilitate studies on the blood coagulation pathways in the early embryogenesis in this zebrafish model.

Animals↗
PubMed2003

[Impact of aldicarb and its complex pollution on DNA of zebrafish embryo].

This paper deals with the impact of a complex pollution system composed of a very toxic carbamate pesticide, aldicarb and the widely used anionic surfactant, sodium dodecylbenzenesulfonate(SDBS), on DNA of zebrafish (Brachydanio rerio) embryo. The results indicated that DNA damage caused by aldicarb became more serious with its increasing concentration, but single-strand break caused by aldicarb in low concentration and short time could be repaired, and high concentration led to double-strand break which was difficult to repaire. SDBS in certain concentration (20 mg.L-1) could reduce the toxicity of aldicarb in the complex pollution system.

Aldicarb↗
PubMed2006

Electroporation of DNA, RNA, and morpholinos into zebrafish embryos.

The combination of accessible embryology and forward genetic techniques has made zebrafish a powerful model system for the study of vertebrate development. One limitation of genetic analysis is that the study of gene function is usually limited to the first developmental event affected by a gene. In vivo electroporation has recently matured as a method for studying gene function at different developmental time points and in specific regions of the organism. The focal application of current allows macromolecules to be efficiently introduced into a targeted region at any time in the life cycle. Here we describe a rapid protocol by which DNA, RNA and morpholinos can all be precisely electroporated into zebrafish in a temporally and spatially controlled manner. This versatile technique allows gene function to be determined by both gain and loss of function analyses in specific regions at specific times. This is the first report that describes the electroporation of three different molecules into embryonic and larval zebrafish cells.

Animals↗
PubMed2003

Role of aryl hydrocarbon receptor in mesencephalic circulation failure and apoptosis in zebrafish embryos exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent and potent developmental toxicant in various animals, with developing fish being the most sensitive organisms. Although the expression of aryl hydrocarbon receptor (AHR) as well as the partner molecule, AHR nuclear translocator (ARNT) in the brain has been reported, the effect of TCDD on the brain remains to be clarified in detail. Previously, we reported local circulation failure and apoptosis in dorsal midbrain caused by TCDD in developing zebrafish. In the present experiments, we investigated the effects of morpholino antisense oligos against aryl hydrocarbon receptor 2 (zfAHR2) (AHR2-MO) on toxicological endpoints caused by TCDD in developing zebrafish. AHR2-MO but not its negative homologue (4mis-AHR2-MO) improved TCDD-evoked circulation failure in mesencephalic vein and reduced the occurrence of apoptosis in dorsal midbrain, with concomitant inhibition of CYP1A induction in vascular endothelium. Injection of bovine serum albumin (BSA) into the general circulation, followed by immunohistochemistry with anti-BSA, showed that TCDD raised vascular permeability to albumin in dorsal midbrain, which was blocked by AHR2-MO and N-acetlycystein. In the absence of TCDD, development of embryos injected with AHR2-MO appeared normal at least until 60 h after fertilization. It is concluded that AHR2 activation in the vascular endothelium of the zebrafish embryo midbrain is involved in the mesencephalic circulation failure and apoptosis elicited by TCDD. This is the further evidence that vascular endothelium is the target of TCDD in relation to local circulation failure and apoptosis in dorsal midbrain.

Abnormalities, Drug-Induced↗
PubMed2000

Genetic analysis of melanophore development in zebrafish embryos.

Vertebrate pigment cells are derived from neural crest, a tissue that also forms most of the peripheral nervous system and a variety of ectomesenchymal cell types. Formation of pigment cells from multipotential neural crest cells involves a number of common developmental processes. Pigment cells must be specified; their migration, proliferation, and survival must be controlled and they must differentiate to the final pigment cell type. We previously reported a large set of embryonic mutations that affect pigment cell development from neural crest (R. N. Kelsh et al., 1996, Development 123, 369-389). Based on distinctions in pigment cell appearance between mutants, we proposed hypotheses as to the process of pigment cell development affected by each mutation. Here we describe the cloning and expression of an early zebrafish melanoblast marker, dopachrome tautomerase. We used this marker to test predictions about melanoblast number and pattern in mutant embryos, including embryos homozygous for mutations in the colourless, sparse, touchdown, sunbleached, punkt, blurred, fade out, weiss, sandy, and albino genes. We showed that in homozygous mutants for all loci except colourless and sparse, melanoblast number and pattern are normal. colourless mutants have a pronounced decrease in melanoblast cell number from the earliest stages and also show poor melanoblast differentiation and migration. Although sparse mutants show normal numbers of melanoblasts initially, their number is reduced later. Furthermore, their distribution indicates a defect in melanoblast dispersal. These observations permit us to refine our model of the genetic control of melanophore development in zebrafish embryos.

Amino Acid Sequence↗
PubMed2003

Interactions between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and hypoxia signaling pathways in zebrafish: hypoxia decreases responses to TCDD in zebrafish embryos.

The aryl hydrocarbon receptor (AHR) interacts with the aryl hydrocarbon receptor nuclear translocator (ARNT) to form a heterodimer that binds to promoters in target genes to alter transcription in response to xenobiotics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The ARNT protein also forms heterodimers with other proteins such as HIF-1alpha and HIF-2alpha to alter gene expression in response to low oxygen conditions. Because ARNT is shared between multiple signaling pathways it is possible that activation of one ARNT-requiring pathway could inhibit the activation of other pathways that depend on ARNT. One hypothesis to explain TCDD toxicity in early life stage fish is that TCDD activation of zfAHR2 sequesters zfARNT2 away from the hypoxia signaling pathway. To test this hypothesis we measured the ability of TCDD to prevent induction of heme oxygenase by hypoxia (40% saturation), as well as the ability of hypoxia to increase the sensitivity of zebrafish to the effects of TCDD during the first week of life. As a further test of the model we examined mutant zebrafish that lack zfARNT2 for phenotypes that resemble the effects of TCDD exposure. Our results demonstrate that sequestration of zfARNT2 is not causing TCDD toxicity. TCDD did not inhibit hypoxia induction of heme oxygenase, hypoxia and TCDD exposures were not additive in causing developmental toxicity, and mutant embryos that lack zfARNT2 do not develop defects mimicking TCDD toxicity. However, our results demonstrate some level of cross talk between the two pathways in the zebrafish embryo. Hypoxia decreased TCDD induction of zfCYP1A mRNA, and decreased the potency of TCDD in causing edema. It is not clear whether this is mediated through competition for zfARNT2, or through other mechanisms.

Animals↗
PubMed2000

An inherited functional circadian clock in zebrafish embryos.

Circadian clocks are time-keeping systems found in most organisms. In zebrafish, expression of the clock gene Period3 (Per3) oscillates throughout embryogenesis in the central nervous system and the retina. Per3 rhythmic expression was free-running and was reset by light but not by the developmental delays caused by low temperature. The time of fertilization had no effect on Per3 expression. Per3 messenger RNA accumulates rhythmically in oocytes and persists in embryos. Our results establish that the circadian clock functions during early embryogenesis in zebrafish. Inheritance of maternal clock gene products suggests a mechanism of phase inheritance through ovogenesis.

Amino Acid Sequence↗
PubMed1996

dino and mercedes, two genes regulating dorsal development in the zebrafish embryo.

We describe two genes, dino and mercedes, which are required for the organization of the zebrafish body plan. In dino mutant embryos, the tail is enlarged at the expense of the head and the anterior region of the trunk. The altered expression patterns of various marker genes reveal that, with the exception of the dorsal most marginal zone, all regions of the early dino mutant embryo acquire more ventral fates. These alterations are already apparent before the onset of gastrulation. mercedes mutant embryos show a similar but weaker phenotype, suggesting a role in the same patterning processes. The phenotypes suggests that dino and mercedes are required for the establishment of dorsal fates in both the marginal and the animal zone of the early gastrula embryo. Their function in the patterning of the ventrolateral mesoderm and the induction of the neuroectoderm is similar to the function of the Spemann organizer in the amphibian embryo.

Animals↗
PubMed2001

Effects of endosulfan and nonylphenol on the primordial germ cell population in pre-larval zebrafish embryos.

A variety of chemicals released into the aquatic environment are capable of targeting the reproductive system in fish and other vertebrates. Some of the effects observed in exposed adults may arise by permanent organizational changes that occur during embryogenesis, including changes in gonad structure and function. Little work has addressed the effects of pesticides and industrial chemicals, many of which are recognized as endocrine disrupting chemicals, on early embryos. The recent cloning of the vasa gene in zebrafish, the mRNA of which is found in fertilized eggs and is later segregated into the primordial germ cells (PGCs), has provided a unique opportunity to examine PGC migration and positioning in early embryos. We utilized antisense RNA probes to vasa mRNA in whole mount in situ hybridization analysis in order to examine the early migration and distribution of PGCs in embryos exposed to endosulfan and nonylphenol. The data reveal that these chemicals cause alterations in the distribution of PGCs along the anterior-posterior axis in 24-h-old embryos. This suggests that the previously reported alterations in juvenile and adult gonad structure of various aquatic vertebrates following exposure to pesticides and industrial chemicals could be related in part to alterations in early PGC distribution.

Animals↗
PubMed1995

ES-like cell cultures derived from early zebrafish embryos.

Pluripotent embryonic stem (ES) cell cultures provide an efficient method for genome manipulation with many applications in marine biotechnology. To develop this technology we have been working to derive fish ES cell lines for in vitro studies of embryo cell growth and differentiation and for the generation of transgenic fish. Zebrafish embryonal cell cultures were derived from blastula-stage embryos in LDF medium supplemented with fetal bovine serum, trout serum, trout embryo extract, selenium, insulin, and leukemia inhibitory factor. Cultures derived under these conditions on feeder layers of zebrafish embryonic fibroblasts possessed a diploid karyotype and exhibited an ES-like morphology with elevated levels of alkaline phosphatase enzyme activity. Injection of primary cell cultures derived from embryos of transgenic fish carrying neo produced chimeric fish detected by polymerase chain reaction analysis. Embryo cells cultured on poly-D-lysine substrate in the presence of retinoic acid or Buffalo rat liver cell-conditioned medium (BRL-CM) and a reduced serum concentration differentiated into neuronal cell types exhibiting elevated levels of acetylcholinesterase enzyme activity and expression of neurofilament and glial fibrillary acidic protein.

Acetylcholinesterase↗
PubMed2005

Cell proliferation in the developing lateral line system of zebrafish embryos.

The sensory organs of the embryonic lateral line system are deposited by migrating primordia that originate in the otic region. Here, we examine the pattern of cell proliferation in the posterior lateral line system. We conclude that three phases of cell proliferation are involved in the generation of this system, separated by two phases of mitotic quiescence. The first phase corresponds to generalized proliferation during gastrulation, followed by a first period of quiescence that may be related to the determination of the lateral line precursor cells. A second phase of proliferation takes place in the placode and migrating primordium. This region is organized in annuli that correspond to the expression of proneural/neurogenic genes. A second period of quiescence follows, corresponding to deposition and differentiation of the sensory organs. The third period of proliferation corresponds to continued renewal of hair cells by division of support cells within each sensory organ.

Animals↗