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At least 1,243 records · Page 69Linked to original sources
PubMed1999

Transgene expression in zebrafish: A comparison of retroviral-vector and DNA-injection approaches.

To assess alternative methods for introducing expressing transgenes into the germ line of zebrafish, transgenic fish that express a nuclear-targeted, enhanced, green fluorescent protein (eGFP) gene were produced using both pseudotyped retroviral vector infection and DNA microinjection of embryos. Germ-line transgenic founders were identified and the embryonic progeny of these founders were evaluated for the extent and pattern of eGFP expression. To compare the two modes of transgenesis, both vectors used the Xenopus translational elongation factor 1-alpha enhancer/promoter regulatory cassette. Several transgenic founder fish which transferred eGFP expression to their progeny were identified. The gene expression patterns are described and compared for the two modes of gene transfer. Transient expression of eGFP was detected 1 day after introducing the transgenes via either DNA microinjection or retroviral vector infection. In both cases of gene transfer, transgenic females produced eGFP-positive progeny even before the zygotic genome was turned on. Therefore, GFP was being provided by the oocyte before fertilization. A transgenic female revealed eGFP expression in her ovarian follicles. The qualitative patterns of gene expression in the transgenic progeny embryos after zygotic induction of gene expression were similar and independent of the mode of transgenesis. The appearance of newly synthesized GFP is detectable within 5-7 h after fertilization. The variability of the extent of eGFP expression from transgenic founder to transgenic founder was wider for the DNA-injection transgenics than for the retroviral vector-produced transgenics. The ability to provide expressing germ-line transgenic progeny via retroviral vector infection provides both an alternative mode of transgenesis for zebrafish work and a possible means of easily assessing the insertional mutagenesis frequency of retroviral vector infection of zebrafish embryos. However, because of the transfer of GFP from oocyte to embryo, the stability of GFP may create problems of analysis in embryos which develop as quickly as those of zebrafish.

Animals↗
PubMed1999

Molecular cloning, expression, and activity of zebrafish semaphorin Z1a.

Semaphorins/collapsins are a large family of secreted and cell surface molecules that are thought to guide growth cones to their targets. Although some members are clearly repulsive to specific growth cones in vitro, the in vivo role of many of these molecules in vertebrate embryos is still unclear. As a first step towards clarifying the in vivo role of semaphorins/collapsins, we analyzed semaZ1a in the simple and well-characterized zebrafish embryo. SemaZ1a is a secreted molecule that is highly homologous to Sema III/D/collapsin-1, and it can collapse chick dorsal root ganglion growth cones in vitro. It is expressed in highly specific patterns within the developing embryo, which suggests that it influences outgrowth by a variety of growth cones including those of the posterior lateral line ganglion. Consistent with this hypothesis, the peripherally extending growth cones of posterior lateral line neurons retract and partially collapse during normal outgrowth.

Amino Acid Sequence↗
PubMed2004

Phosphatidylserine receptor is required for the engulfment of dead apoptotic cells and for normal embryonic development in zebrafish.

During development, the role of the phosphatidylserine receptor (PSR) in the removal of apoptotic cells that have died is poorly understood. We have investigated this role of PSR in developing zebrafish. Programmed cell death began during the shield stage, with dead cells being engulfed by a neighboring cell that showed a normal-looking nucleus and the nuclear condensation multi-micronuclei of an apoptotic cell. The zebrafish PSR engulfing receptor was cloned (zfpsr), and its nucleotide sequence was compared with corresponding sequences in Drosophila melanogaster (76% identity), human (74%), mouse (72%) and Caenorhabditis elegans (60%). The PSR receptor contained a jmjC domain (residues 143-206) that is a member of the cupin metalloenzyme superfamily, but in this case serves an as yet unknown function(s). psr knockdown by a PSR morpholino oligonucleotide led to accumulation of a large number of dead apoptotic cells in whole early embryo. These cells interfered with embryonic cell migration. In addition, normal development of the somite, brain, heart and notochord was sequentially disrupted up to 24 hours post-fertilization. Development could be rescued in defective embryos by injecting psr mRNA. These results are consistent with a PSR-dependent system in zebrafish embryos that engulfs apoptotic cells mediated by PSR-phagocytes during development, with the system assuming an important role in the normal development of tissues such as the brain, heart, notochord and somite.

Amino Acid Sequence↗
PubMed2003

Ogon/Secreted Frizzled functions as a negative feedback regulator of Bmp signaling.

The zebrafish mutant ogon (also called mercedes and short tail) displays ventralized phenotypes similar to the chordino (dino) mutant, in which the gene for the Bmp antagonist Chordin is mutated. We isolated the gene responsible for ogon by a positional cloning strategy and found that the ogon locus encodes a zebrafish homolog of Secreted Frizzled (Sizzled), which has sequence similarity to a Wnt receptor, Frizzled. Unlike other secreted Frizzled-related proteins (sFrps) and the Wnt inhibitor Dickkopf1, the misexpression of Ogon/Sizzled dorsalized, but did not anteriorize, the embryos, suggesting a role for Ogon/Sizzled in Bmp inhibition. Ogon/Sizzled did not inhibit a Wnt8-dependent transcription in the zebrafish embryo. ogon/sizzled was expressed on the ventral side from the late blastula through the gastrula stages. The ventral ogon/sizzled expression in the gastrula stage was reduced or absent in the swirl/bmp2b mutants but expanded in the chordino mutants. Misexpression of ogon/sizzled did not dorsalize the chordino mutants, suggesting that Ogon/Sizzled required Chordin protein for dorsalization and Bmp inhibition. These data indicate that Ogon/Sizzled functions as a negative regulator of Bmp signaling and reveal a novel role for a sFrp in dorsoventral patterning.

Amino Acid Sequence↗
PubMed2004

Mosaic eyes, genomic instability mutants, and cancer susceptibility.

We now know that genomic instability contributes to cancer. The zebrafish mosaic eye assay developed by George Streisinger takes advantage of the organism's transparency to provide an excellent assay for detecting somatic mutation. This assay allowed us to identify zebrafish mutants with increased frequencies of somatic mutation and spontaneous cancer. Here, we have described details of mutagenesis, the basis and practical use of the mosaic eye assay, and the histological methods used to study genomic instability mutants and cancer susceptibility. These techniques should prove useful to other zebrafish researchers, as they are broadly applicable to many other biological investigations of embryos, larvae, and adult zebrafish.

Animals↗
PubMed2005

Developmental regulation and expression of the zebrafish connexin43 gene.

We cloned and sequenced the zebrafish (Danio rerio) connexin43 (Cx43alpha1) gene. The predicted protein sequence shows a high degree of sequence conservation. Transcript analyses revealed multiple transcription start sites and a potential alternative transcript encoding a N-terminally truncated Cx43alpha1 protein. Maternal Cx43alpha1 transcripts were detected, with zygotic expression initiated before gastrulation. In situ hybridization revealed many Cx43alpha1 expression domains, including the notochord and brain, heart and vasculature, many resembling patterns seen in mammalian embryos. Of interest, a reporter construct under control of the mouse Cx43alpha1 promoter was observed to drive green fluorescent protein expression in zebrafish embryos in domains mimicking the native Cx43alpha1 expression pattern in fish and mice. Sequence comparison between the mouse and zebrafish Cx43alpha1 promoter sequences showed the conservation of several transcription factor motifs, which otherwise shared little overall sequence homology. The conservation of protein sequence and developmental gene regulation would suggest that Cx43alpha1 gap junctions are likely to have conserved roles in vertebrate embryonic development.

Amino Acid Sequence↗
PubMed1995

Pou-2--a zebrafish gene active during cleavage stages and in the early hindbrain.

We have cloned the zebrafish pou-2 gene which encodes a novel type (class VII) of POU domain. Maternal pou-2 transcripts are initially found in all blastomeres. However, during later cleavage stages pou-2 expression disappears in the marginal cells. Some of their progeny will form the first lineage restricted compartment during zebrafish development. Blastula pou-2 expression in confined exclusively to the deep embryonic layer (DEL) forming the embryo proper. No expression is found in extraembryonic tissues, i.e. the yolk syncytial layer (YSL) and the enveloping layer (EVL). Thus pou-2 expression during early embryogenesis correlates with the continuing absence of cell lineage restriction. Towards the end of gastrulation, pou-2 expression becomes confined to the neural plate, predominantly to the prospective hindbrain and to the spinal cord. pou-2 expression in the forming hindbrain is restricted to future rhombomeres r2 and r4. Retinoic acid treatment during epiboly alters the hindbrain domains of pou-2, suggesting that the entire anterior hindbrain acquires r4-like properties. This finding is supported by analysis of early pax-2 and krx-20 expression patterns in RA-treated zebrafish embryos. The changes resemble similar hindbrain transformations observed in other vertebrates, supporting an evolutionary conservation of the mechanisms segmenting the hindbrain of vertebrates. pou-2 appears to respond to the same signals as other presumed patterning genes. This observation, together with pou-2 expression in the hindbrain prior to morphological segmentation, suggests an important role for this putative transcription factor in establishing and specifying rhombomeric segments.

Animals↗
PubMed2000

Laser-induced gene expression in specific cells of transgenic zebrafish.

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.

Animals↗
PubMed2004

In vivo biofluid dynamic imaging in the developing zebrafish.

Flow-structure interactions are ubiquitous in nature, and are important factors in the proper development of form and function in living organisms. In order to uncover the mechanisms by which flow-structure interactions affect vertebrate development, we first need to establish the techniques necessary to quantitatively describe the fluid flow environment within the embryo. To do this, we must bring dynamic, in vivo imaging methods to bear on living systems. Traditional avian and mammalian model systems can be problematic in this regard. The zebrafish (Danio rerio) is widely accepted as an excellent model organism for the study of vertebrate biology, as it shows substantial anatomical and genetic conservation with higher vertebrates, including humans. Their small size, optical transparency, and external development make zebrafish the ideal model system for dynamic imaging. This article reviews the current state of research in imaging biofluid flow within and around developing zebrafish embryos, with an emphasis on dynamic imaging modalities.

Animals↗
PubMed1998

The effect of pertussis toxin on zebrafish development: a possible role for inhibitory G-proteins in hedgehog signaling.

Recent results have indicated that cAMP-dependent protein kinase (PKA) acts as a negative regulator of Hedgehog signaling in target cells of the vertebrate embryo. Consequently, suppression of PKA activity is sufficient to mimic the effect of receiving a Hedgehog signal. We have explored whether PKA-inhibiting Gi-proteins (GiPs) may also be involved in the regulation of Hedgehog signaling. Zebrafish embryos were injected with RNA encoding pertussis toxin (Ptx), a specific inhibitor of GiPs. These embryos developed phenotypic traits opposite to embryos expressing a dominant negative form of the PKA regulatory subunit (dnPKA), including a fusion of the eyes, a lack of ventral specification in the forebrain, and an expansion of the sclerotome at the expense of adaxial fates in the posterior somites. These effects can be partially rescued by coexpression of dnPKA, but not by coexpression of Indian Hedgehog, suggesting that GiPs act upstream of PKA and downstream of Hedgehogs. Other Hedgehog- and PKA-dependent processes, sclerotomal specification and adaxial specification in the first five somites, are not negatively affected by Ptx. Thus, GiPs may be involved in Hedgehog signaling in some, but not all target cells.

Animals↗
PubMed2006

Cooperation of sonic hedgehog enhancers in midline expression.

In zebrafish, as in other vertebrates, the secreted signalling molecule Sonic hedgehog (Shh) is expressed in organiser regions such as the embryonic midline and the zona limitans intrathalamica (zli). To investigate the regulatory mechanisms underlying the pattern of shh expression, we carried out a systematic analysis of the intronic regulatory sequences of zebrafish shh using stable transgenesis. Deletion analysis identified the modules responsible for expression in the embryonic shield, the hypothalamus and the zli and confirmed the activities of previously identified notochord and floor plate enhancers. We detected a strong synergism between regulatory regions. The degree of synergy varied over time in the hypothalamus suggesting different mechanisms for initiation and maintenance of expression. Our data show that the pattern of shh expression in the embryonic central nervous system involves an intricate crosstalk of at least 4 different regulatory regions. When compared to the enhancer activities of the mouse Shh gene, we observed a remarkable divergence of function of structurally conserved enhancer sequences. The activating region ar-C (61% identical to SFPE2 in mouse Shh), for example, mediates floor plate expression in the mouse embryo while it directs expression in the forebrain and the notochord and only weakly in the floor plate in the zebrafish embryo. This raises doubts on the predictive power of phylogenetic footprinting and indicates a stunning divergence of function of structurally conserved regulatory modules during vertebrate evolution.

Animals↗
PubMed1996

Expression of zebrafish connexin43.4 in the notochord and tail bud of wild-type and mutant no tail embryos.

Communication via gap junctions provides a mechanism for the cell-cell transfer and coordination of developmental signals. The spatial restriction of gap junctions may also serve to organize cells into domains of coordinated behavior. To investigate the role of gap junctions during embryogenesis, we have characterized the expression of a member of the gap junction gene family, zebrafish connexin43.4, a homolog of connexin45 in chicken and mammals. Expression of connexin43.4 was induced in the early gastrula, coincident with the first definitive assignments of axial cell fate and the onset of the cell movements comprising convergence and extension in zebrafish. In situ hybridization and immunohistochemistry revealed that during gastrulation connexin43.4 mRNA and protein were progressively enriched in the germ ring and in the notochord primordia on the dorsal side of the embryo. Later in development connexin43.4 expression was detected in the notochord, the paraxial mesoderm, and the tail bud but was not observed after the differentiation of these tissues. In no tail mutant embryos which are defective in tail formation and proper morphogenesis of the notochord, connexin43.4 expression was absent during gastrulation from the caudal embryonic shield and notochord primordia. During somite stages in no tail embryos, connexin43.4 expression remained absent in the notochordal precursor cells and was lost in the tail bud. Thus, the no tail gene product, a transcription factor, was required for the expression of connexin43.4 in both the notochord and tail bud during morphogenesis. By microinjection of mRNA coding for a connexin43.4/green fluorescent protein fusion in the 1-cell zebrafish embryo, we showed that connexin43.4 is capable of assembling into structures reminiscent of gap junctions. The progressively restricted, developmental expression of the zebrafish connexin43.4 gene suggests that this gap junctional protein participates in the coordination of gastrulation and the formation of the notochord and tail.

Animals↗
PubMed2006

Characterization of the heme synthesis enzyme coproporphyrinogen oxidase (CPO) in zebrafish erythrogenesis.

Hemoglobin consists of heme and globin proteins and is essential for oxygen transport in all vertebrates. Although biochemical features of heme synthesis enzymes have been well characterized, the function of these enzymes in early embryogenesis is not fully understood. We found that the sixth heme synthesis enzyme, coproporphyrinogen oxidase (CPO), is predominantly expressed in the intermediate cell mass (ICM) that is a major site of zebrafish primitive hematopoiesis. Knockdown of zebrafish CPO using anti-sense morpholinos (CPO-MO) leads to a significant suppression of hemoglobin production without apparent reduction of blood cells. Injection of human CPO RNA, but not a mutant CPO RNA that is similar to a mutant responsible for a hereditary coproporphyria (HCP), restores hemoglobin production in the CPO-MO-injected embryos. Furthermore, expression of CPO in the ICM is severely suppressed in both vlad tepes/gata1 mutants and in biklf-MO-injected embryos. In contrast, over-expression of biklf and gata1 significantly induces ectopic CPO expression. The function of CPO in heme biosynthesis is apparently conserved between zebrafish and human, suggesting that CPO-MO-injected zebrafish embryos might be a useful in vivo assay system to measure the biological activity of human CPO mutations.

Amino Acid Sequence↗
PubMed1998

Activin-induced factors maintain goosecoid transcription through a paired homeodomain binding site.

Previous studies in both Xenopus and zebrafish have shown that goosecoid is one of the first genes to be transcribed at the onset of gastrulation. Goosecoid transcription still initiates when embryos are treated with protein synthesis inhibitors, indicating that it is mediated by preexisting factors and suggesting that goosecoid transcription is immediately downstream of the maternal mesoderm-inducing signal. However, goosecoid transcription continues long after this maternal signal has ceased to be active, indicating that there are mechanisms to maintain activin-induced transcription. Our study has focused on understanding the factors required to maintain this transcription. We have defined an element within the zebrafish goosecoid promoter that is sufficient for activin inducibility in both Xenopus and zebrafish embryos. This element, the goosecoid activin element, interacts with two developmentally regulated proteins from Xenopus embryos. A maternal protein interacts through cleavage stages until the midblastula transition, and a second protein binds from the onset of gastrulation. The second protein is zygotically expressed, and its binding is required for activin inducibility in our assay system. We suggest that the zygotic protein we have identified is a good candidate to be involved in the maintenance of goosecoid transcription. Furthermore, this zygotic protein is likely to contain a paired class homeodomain since a consensus binding site for such proteins is present within the goosecoid activin element and is essential for its function.

Activins↗
PubMed1992

Segregation and early dispersal of neural crest cells in the embryonic zebrafish.

We have exploited our ability to visualize and follow individual cells in situ, in the living embryo, to study the development of trunk neural crest in the embryonic zebrafish. In most respects, the development of zebrafish trunk neural crest is similar to the development of trunk neural crest in other species: zebrafish trunk neural crest cells segregate from the dorsal neural keel in a rostrocaudal sequence, migrate ventrally along two pathways, and give rise to neurons of the peripheral nervous system, Schwann cells, and pigment cells. However, some aspects of the development of zebrafish trunk neural crest differ from those of other vertebrates: zebrafish trunk neural crest cells are significantly larger and fewer in number than those in avian embryos and the locations of their migratory pathways are slightly different. This initial description of neural crest development in the zebrafish embryo provides the foundation for future experimental studies.

Animals↗
PubMed2006

Phylogenetic relationships and gene expression pattern of three different cathepsin L (Ctsl) isoforms in zebrafish: Ctsla is the putative yolk processing enzyme.

Certain cysteine proteases, such as cathepsin L (Ctsl), have been involved in yolk processing mechanisms in oocytes and embryos of lower vertebrates. In zebrafish (Danio rerio), three different ctsl genes, ctsla, ctslb and ctslc, have been found in the genome, but their pattern of expression, as well as information on which the encoded enzymes are potentially involved in yolk absorption during embryogenesis, is unknown. Here, phylogenetic and gene structure analysis revealed that zebrafish ctsla and ctslb genes are similar, showing a highly conserved structure in comparison with human ctsl, while ctslc presents different exon organization together with an earlier evolution. Thus, ctslc appears to be evolved from a common ancestral ctsl-like gene, possibly through an early duplication event, whereas ctsla and ctslb may be originated from a second duplication mechanism. Zebrafish ctsla, ctslb and ctslc also showed different patterns of mRNA expression during embryogenesis and in adult tissues. While Ctsla transcripts were accumulated in embryos throughout development and in the adult ovary, those encoding Ctslb were detected only in embryos around the time of hatching as previously reported, and those for Ctslc appeared only in larvae and in some adult tissues, but not in the ovary. In zebrafish and killifish (Fundulus heteroclitus) embryos, Ctsla mRNA was first detected in blastomers, and later in development it was localized in cells of the yolk syncytial layer, an embryonic structure involved in yolk absorption. These data therefore suggested that Ctsla is most likely the putative protease involved in yolk processing in fish embryos, while Ctslc seems not to be required during early embryogenesis in zebrafish.

Amino Acid Sequence↗
PubMed1997

Conservation of BMP signaling in zebrafish mesoderm patterning.

To investigate the conservation of mechanisms for mesodermal patterning between zebrafish and Xenopus, we isolated two cDNA clones encoding bone morphogenetic protein (BMP)-related proteins from a zebrafish cDNA library. Based on their predicted amino acid sequences, these two clones were designated as zbmp-2 and zbmp-4. Whole-mount in situ hybridization analysis revealed that in gastrula embryo, both genes were localized in the ventral part of the embryo, consistent with the proposed function of Xenopus BMP-4 in ventral mesoderm specification. zbmp-4 expression, however, was also seen in the embryonic shield, the most dorsal mesodermal structure. To examine the ability of zbmp-2 to ventralize mesoderm, we injected synthetic mRNA into zebrafish embryos and found that overexpression of this gene eliminated dorsal structures including notochord at both morphological and molecular level. In contrast, expression of ventral marker gene eve1 was expanded to the dorsal side. These effects are analogous to the ventralization of embryos caused by ectopic xBMP-4 expression. Taken together, one may conclude that the developmental mechanisms for mesodermal patterning regulated by BMPs are evolutionarily conserved between amphibians and teleosts.

Amino Acid Sequence↗