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PubMed1998

Excision of the tol2 transposable element of the medaka fish, Oryzias latipes, in zebrafish, Danio rerio.

The Tol2 element is a transposable element in Oryzias latipes (the medaka fish) found in the tyrosinase gene locus of the tyrosinase-deficient mutant medaka fish and has been shown to be excised from the genome during medaka embryogenesis (Koga, A., Suzuki, M., Inagaki, H., Bessho, Y., Hori, H., 1996. Transposon element in fish. Nature 383, 30). It is, however, not known whether the Tol2 element is an autonomous element. To determine whether the cloned Tol2 element is an autonomous element and whether excision can occur also in the other fish species, the plasmid DNA harboring the Tol2 element was injected to fertilized eggs of zebrafish, Danio rerio, and the total DNA extracted from the embryos 9-10h after the injection was analyzed by PCR. When a plasmid with the full-length Tol2 element was used for the microinjection, in 39 out of 43 injected embryos, we found generation of short PCR products indicative of the loss of the Tol2 element from the injected plasmid. Ten of these cases were analyzed at the DNA sequence level, and nine of them showed either precise excision of the Tol2 element (three cases) or nearly precise excision of the element with the addition of a few nucleotides of the target duplication (six cases). When a deletion version of the Tol2 element that retained the terminal inverted repeats but lacked about one-fourth of the open reading frame-coding region was used for the microinjection, such short PCR products could not be amplified from any of the injected embryos (0 out of 30). Thus, the Tol2 element is capable of excision in zebrafish embryos, presumably dependent on a putative transposase encoded by the Tol2 element itself. This transient embryonic excision assay using zebrafish should be useful to analyze the structure and the function of the transposase and cis-elements necessary for excision. Also, this study implies the potential use of the Tol2 element in transgenesis and insertional mutagenesis in both zebrafish and the medaka fish.

Animals↗

PubMed1995

The LIM class homeobox gene lim5: implied role in CNS patterning in Xenopus and zebrafish.

LIM homeobox genes are characterized by encoding proteins in which two cysteine-rich LIM domains are associated with a homeodomain. We report the isolation of a gene, named Xlim-5 in Xenopus and lim5 in the zebrafish, that is highly similar in sequence but quite distinct in expression pattern from the previously described Xlim-1/lim1 gene. In both species studied the lim5 gene is expressed in the entire ectoderm in the early gastrula embryo. The Xlim-5 gene is activated in a cell autonomous manner in ectodermal cells, and this activation is suppressed by the mesoderm inducer activin. During neurulation, expression of the lim5 gene in both the frog and fish embryo is rapidly restricted to an anterior region in the developing neural plate/keel. In the 2-day Xenopus and 24-hr zebrafish embryo, this region becomes more sharply defined, forming a strongly lim5-expressing domain in the diencephalon anterior to the midbrain-forebrain boundary. In addition, regions of less intense lim5 expression are seen in the zebrafish embryo in parts of the telencephalon, in the anterior diencephalon coincident with the postoptic commissure, and in restricted regions of the midbrain, hindbrain, and spinal cord. Expression in ventral forebrain is abolished from the 5-somite stage onward in cyclops mutant fish. These results imply a role for lim5 in the patterning of the nervous system, in particular in the early specification of the diencephalon.

Amino Acid Sequence↗

PubMed2000

Patterning the early zebrafish by the opposing actions of bozozok and vox/vent.

Fish and frog embryos are patterned along the dorsal-ventral axis during the gastrula stage by opposing gradients of Bmps and Bmp inhibitory proteins. Three transcriptional repressors with partially overlapping expression domains have been proposed to be important mediators of Bmp function in Xenopus. We find that two related factors are expressed in the early zebrafish embryo. Although these factors are considerably divergent from the related Xenopus genes, they are expressed in domains similar to those of their Xenopus relatives throughout embryogenesis. Both of the zebrafish genes, which we have named vox and vent, are potent ventralizing factors in both zebrafish and Xenopus embryos. Using mutants in the Bmp pathway, we find that there are Bmp-dependent and Bmp-independent domains of vox expression, whereas vent is mostly dependent upon Bmp signaling. We show that ectopic vox or vent negatively regulates expression of the early dorsal gene bozozok (boz) and that ectopic boz eliminates vox and vent expression. Moreover, the normal exclusion of vox and vent from the organizer region is lost in boz mutant embryos. Our results show that boz and vox/vent are mutually antagonistic and indicate that the early establishment of the size of the organizer domain is dependent on an interplay between these early expressed transcriptional repressors.

Amino Acid Sequence↗

PubMed2003

Zebrafish sperm cryopreservation with N,N-dimethylacetamide.

High fecundity, rapid generation time, and external development of optically clear embryos make the zebrafish (Danio rerio) a convenient vertebrate model for genetic, developmental, and disease studies. Efficient sperm cryopreservation enhances the zebrafish model system by optimizing productive use of facility space, extending the reproductive lifetime of males, providing an alternative to live stocks for strain recovery, and ensuring the survival of valuable mutant lines. Here we identify a cryoprotective medium, 10% N,N-dimethylacetamide (DMA) (v/v) diluted in buffered sperm motility-inhibiting solution (BSMIS), as well as parameters for zebrafish sperm cryopreservation that enhance cryopreservation efficiency and significantly increase the yield of live embryos from archived stocks. Our experiments emphasize the effect of the ratio of sperm and medium volume and the use of large egg clutches to maximize the recovery of viable embryos.

Acetamides↗

PubMed1998

Follistatin and noggin are excluded from the zebrafish organizer.

The patterning activity of the Spemann organizer in early amphibian embryos has been characterized by a number of organizer-specific secreted proteins including Chordin, Noggin, and Follistatin, which all share the same inductive properties. They can neuralize ectoderm and dorsalize ventral mesoderm by blocking the ventralizing signals Bmp2 and Bmp4. In the zebrafish, null mutations in the chordin gene, named chordino, lead to a severe reduction of organizer activity, indicating that Chordino is an essential, but not the only, inductive signal generated by the zebrafish organizer. A second gene required for zebrafish organizer function is mercedes, but the molecular nature of its product is not known as yet. To investigate whether and how Follistatin and Noggin are involved in dorsoventral (D-V) patterning of the zebrafish embryo, we have now isolated and characterized their zebrafish homologues. Overexpression studies demonstrate that both proteins have the same dorsalizing properties as their Xenopus homologues. However, unlike the Xenopus genes, zebrafish follistatin and noggin are not expressed in the organizer region, nor are they linked to the mercedes mutation. Expression of both genes starts at midgastrula stages. While no patterned noggin expression was detectable by in situ hybridization during gastrulation stages, later expression is confined to presumptive cartilage cells in the branchial arches and the neurocranium and to proximal regions of the pectoral fin buds. follistatin transcripts in gastrulating embryos are confined to anterior paraxial regions, which give rise to head mesoderm and the first five somites. The dorsolateral extent of this expression domain is regulated by Bmp2b, Chordino, and Follistatin itself. In addition, transient expression was observed in a subset of cells in the posterior notochord anlage. Later, follistatin is expressed in brain, eyes, and somites. Comparison of the spatiotemporal expression pattern of follistatin and noggin with those of bmp2b and bmp4 and overexpression studies suggest that Noggin and Follistatin may function as Bmp antagonists in later processes of zebrafish development, including late phases of D-V patterning, to refine the early pattern set up by the interaction of Chordino and Bmp2/4. It thus appears that many, but not all, aspects of early dorsoventral patterning are shared among different vertebrate species.

Amino Acid Sequence↗

PubMed2006

Distinct genetic interactions between multiple Vegf receptors are required for development of different blood vessel types in zebrafish.

Recent evidence indicates a specific role for vascular endothelial growth factor a (Vegfa) during artery development in both zebrafish and mouse embryos, whereas less is known about signals that govern vein formation. In zebrafish, loss of vegfa blocks segmental artery formation and reduces artery-specific gene expression, whereas veins are largely unaffected. Here, we describe a mutation in the zebrafish vegf receptor-2 homolog, kdra, which eliminates its kinase activity and leads to specific defects in artery development. We further find that Flt4, a receptor for Vegfc, cooperates with Kdr during artery morphogenesis, but not differentiation. We also identify an additional zebrafish vegfr-2 ortholog, referred to as kdrb, which can partially compensate for loss of kdra but is dispensable for vascular development in wild-type embryos. Interestingly, we find that these Vegf receptors are also required for formation of veins but in distinct genetic interactions that differ from those required for artery development. Taken together, our results indicate that formation of arteries and veins in the embryo is governed in part by different Vegf receptor combinations and suggest a genetic mechanism for generating blood vessel diversity during vertebrate development.

Animals↗

PubMed1997

Defective "pacemaker" current (Ih) in a zebrafish mutant with a slow heart rate.

At a cellular level, cardiac pacemaking, which sets the rate and rhythm of the heartbeat, is produced by the slow membrane depolarization that occurs between action potentials. Several ionic currents could account for this pacemaker potential, but their relative prominence is controversial, and it is not known which ones actually play a pacemaking role in vivo. To correlate currents in individual heart cells with the rhythmic properties of the intact heart, we have examined slow mo (smo), a recessive mutation we discovered in the zebrafish Danio rerio. This mutation causes a reduced heart rate in the embryo, a property we can quantitate because the embryo is transparent. We developed methods for culture of cardiocytes from zebrafish embryos and found that, even in culture, cells from smo continue to beat relatively slowly. By patch-clamp analysis, we discovered that a large repertoire of cardiac currents noted in other species are present in these cultured cells, including sodium, T-type, and L-type calcium and several potassium currents, all of which appear normal in the mutant. The only abnormality appears to be in a hyperpolarization-activated inward current with the properties of Ih, a current described previously in the nervous system, pacemaker, and other cardiac tissue. smo cardiomyocytes have a reduction in Ih that appears to result from severe diminution of one kinetic component of the Ih current. This provides strong evidence that Ih is an important contributor to the pacemaking behavior of the intact heart.

Animals↗

PubMed2005

Study on fish embryo responses to the treatment of cryoprotective chemicals using impedance spectroscopy.

Investigations using electrical impedance spectroscopy to measure the responses of fish embryos to the cryoprotective chemicals, methanol and dimethyl sulphoxide (DMSO), were carried out. Zebrafish (Danio rerio) embryos were used as a model to study the newly proposed technique. The normalised permittivity and conductivity changes of the embryos were measured continuously over a 20-min period in a customised embryo-holding chamber. The normalised permittivity and conductivity spectra were obtained during embryo exposure to different concentrations of methanol (1.0, 2.0 and 3.0 M) and DMSO (0.5, 1.0 and 2.0 M) solutions. The results showed significant permittivity and conductivity changes after embryo exposure to methanol and DMSO at the optimum embryo loading level (six embryos). Embryos in different concentrations of methanol and DMSO also resulted in quantitative responses shown in the normalised permittivity and conductivity spectra. The results demonstrated that fish embryo membrane permeability to cryoprotective chemicals could be monitored in real-time. The measurement of permittivity at a lower frequency range (10-10(3) Hz) and conductivity at a higher frequency range (10(4)-10(6) Hz) during fish embryo exposure to cryoprotective chemicals using impedance spectroscopy can be used as a new tool for the fast screening of most effective cryoprotective chemicals. The results from the present study also demonstrated the possibility of quantifying the level of cryoprotective chemicals penetrating the fish embryos.

Animals↗

PubMed1996

Contact inhibition/collapse and pathfinding of neural crest cells in the zebrafish trunk.

Neural crest cells in the trunk of vertebrate embryos have a choice of pathways after emigrating from the neural tube: they can migrate in either the medial pathway between somites and neural tube, or the lateral pathway between somites and epidermis. In zebrafish embryos, the first cells to migrate all choose the medial pathway. High resolution imaging of cells in living embryos suggests that neural crest cells do so because of repulsion by somites: cells take the medial pathway because the lateral somite surface triggers a paralysis and retraction of protrusions (contact inhibition or collapse) when the medial surface does not. Partial deletion of somites, using the spadetail mutation allows precocious entry into the lateral pathway, but only where somites are absent, supporting the notion that an inhibitory cue on somites delays entry. Growth cones of Rohon-Beard cells enter the lateral pathway before neural crest cells, demonstrating that there is no absolute barrier to migration. These data, in addition to providing a detailed picture of neural crest cells migrating in vivo, suggest that neural crest cells, like neuronal growth cones, are guided by a specific cue that triggers 'collapse' of active protrusions.

Animals↗

PubMed2025

Toxicological Assessment of Melamine-Functionalized Graphene Oxide and Carbon Nanotubes Using Zebrafish Models.

Graphene oxide (GO) and carbon nanotube (CNT)-based nanomaterials have attracted significant interest in various industrial and biomedical applications due to their unique physicochemical properties; however, concerns about their potential toxicity, especially when modified with additives like melamine (M), remain largely unresolved. This study investigates the toxicological effects and underlying mechanisms of graphene oxide-melamine (GO-M) and carbon nanotube-melamine (CNT-M) nanoparticles in zebrafish (Danio rerio) embryos and larvae. To this end, developmental toxicity, phenotypic and behavioral changes, as well as histopathological and immunofluorescence alterations, were evaluated following acute exposure to GO-M and CNT-M nanoparticles at concentrations of 5, 10, and 20 mg/L. Results showed that both nanoparticles delayed larval hatching, particularly at higher concentrations (10 and 20 mg/L). Malformations were observed at 20 mg/L in the GO-M group and at 10 and 20 mg/L in the CNT-M group. Additionally, significant changes in larval length and eye area were observed at all concentrations for both nanoparticles. Behavioral assessments revealed that CNT-M exposure at 10 and 20 mg/L significantly impaired head sensorimotor reflexes, while all concentrations affected tail reflexes. In contrast, GO-M exposure did not significantly alter sensorimotor responses. These findings suggest differential toxic mechanisms and neurobehavioral effects of GO-M and CNT-M nanoparticles during early zebrafish development.

Animals↗

PubMed2005

Determination of pH by microfluorometry: intracellular and interstitial pH regulation in developing early-stage fish embryos (Danio rerio).

Microfluorometric techniques were applied in vivo for continuous monitoring of specific acid-base parameters in zebrafish (Danio rerio) embryos during early stages of ontogeny. Dextran-coupled pH-sensitive single-excitation/dual-emission dye SNARF-1 was pressure-injected into individual cells or the interstitial space of 16- to 256-cell embryos, and pH was continuously recorded during subsequent development for time periods of up to 8 h. A novel calibration technique was developed, essentially characterized by in vitro inorganic buffer calibration of the optical system and mathematical post-processing according to the effects of in vivo dye modifiers through a correlation established by direct comparison of optical techniques with pH microelectrodes. This approach results in high accuracy of microfluorometry, comparable with that of pH electrodes, and a recovery only limited by the physical stability of the utilized optical system. Intracellular pH (pHi) in Danio rerio embryos between 1k-cells stage and the end of epiboly was found to be well regulated to a mean value of 7.55+/-0.13 (+/-s.d.), a range distinctly more alkaline than typical values for adult fish but in accordance with embryonic pHi of a few non-fish species shortly after fertilization. Also, interstitial pH (pHint) was significantly higher (8.08+/-0.25) than values for extracellular pH in adult fish. Distributions of HCO3- across membranes and between interstitium and ambient fluid compared with respective potentials strongly suggest that pH in these early stages of ontogeny is already adjusted by active transfer processes. Non-respiratory changes in ambient pH between 7.7 and 8.5 did not significantly affect pHi, a result potentially attributable to low membrane leakage rate or to the potency of active transfer mechanisms. In order to assess the pH regulatory systems more quantitatively, embryos were exposed to ambient changes of carbon dioxide partial pressure (PCO2). The direct impact of PCO2 changes on cell pH was alleviated by cell non-bicarbonate buffering and subsequent rapid, almost complete, compensation by changes in cell [HCO3-] as an expression of transmembrane transfer of acid-base relevant ions. On the basis of these results, we conclude that the regulatory potency of embryonic cells is well developed, is active to resist extensive homoiostatic stress and is efficient to maintain critical metabolism in adverse conditions, even at early stages of ontogeny.

Animals↗

PubMed1985

Cell lineage of zebrafish blastomeres. I. Cleavage pattern and cytoplasmic bridges between cells.

Patterns of cleavage and cytoplasmic connections between blastomeres in the embryo of the zebrafish, Brachydanio rerio have been described. The cell division pattern is often very regular; in many embryos a blastomere's lineage may be ascertained from its position in the cluster through the 64-cell stage. At the 5th cleavage, however, significant variability in pattern is observed, and alternative patterns of the 5th cleavage are described. The early cleavages are partial, incompletely separating blastomeres from the giant yolk cell. The tracer fluorescein-dextran (FD) was injected into blastomeres to learn the extent of the cytoplasmic bridging. It was observed that until the 10th cleavage, blastomeres located along the blastoderm margin maintain cytoplasmic bridges to the yolk cell. Beginning with the 5th cleavage, FD injected into a nonmarginal blastomere either remains confined to the injected cell, or if the injection was early in the cell cycle, the tracer spreads to the cell's sibling, through a bridge persisting from the previous cleavage. On the other hand, injected Lucifer yellow spreads, presumably via gap junctions, widely among blastomeres in a pattern unrelated to lineage.

Animals↗

PubMed2000

The zebrafish young mutation acts non-cell-autonomously to uncouple differentiation from specification for all retinal cells.

Embryos from mutagenized zebrafish were screened for disruptions in retinal lamination to identify factors involved in vertebrate retinal cell specification and differentiation. Two alleles of a recessive mutation, young, were isolated in which final differentiation and normal lamination of retinal cells were blocked. Early aspects of retinogenesis including the specification of cells along the inner optic cup as retinal tissue, polarity of the retinal neuroepithelium, and confinement of cell divisions to the apical pigmented epithelial boarder were normal in young mutants. BrdU incorporation experiments showed that the initiation and pattern of cell cycle withdrawal across the retina was comparable to wild-type siblings; however, this process took longer in the mutant. Analysis of early markers for cell type differentiation revealed that each of the major classes of retinal neurons, as well as non-neural Müller glial cells, are specified in young embryos. However, the retinal cells fail to elaborate morphological specializations, and analysis of late cell-type-specific markers suggests that the retinal cells were inhibited from fully differentiating. Other regions of the nervous system showed no obvious defects in young mutants. Mosaic analysis demonstrated that the young mutation acts non-cell-autonomously within the retina, as final morphological and molecular differentiation was rescued when genetically mutant cells were transplanted into wild-type hosts. Conversely, differentiation was prevented in wild-type cells when placed in young mutant retinas. Mosaic experiments also suggest that young functions at or near the cell surface and is not freely diffusible. We conclude that the young mutation disrupts the post-specification development of all retinal neurons and glia cells.

Animals↗

PubMed1995

Nodal induces ectopic goosecoid and lim1 expression and axis duplication in zebrafish.

One of the first intercellular signalling events in the vertebrate embryo leads to mesoderm formation and axis determination. In the mouse, a gene encoding a new member of the TGF-beta superfamily, nodal, is disrupted in a mutant deficient in mesoderm formation (Zhou et al., 1993, Nature 361, 543). nodal mRNA is found in prestreak mouse embryos, consistent with a role in the development of the dorsal axis. To examine the biological activities of nodal, we have studied the action of this factor in eliciting axis determination in the zebrafish, Danio rerio. Injection of nodal mRNA into zebrafish embryos caused the formation of ectopic axes that included notochord and somites. Axis duplication was preceded by the generation of an apparent ectopic shield (organizer equivalent) in nodal-injected embryos, as indicated by the appearance of a region over-expressing gsc and lim1; isolation and expression in the shield of the lim1 gene is reported here. These results suggest a role for a nodal-like factor in pattern formation in zebrafish.

Amino Acid Sequence↗

PubMed1997

Differential regulation of chordin expression domains in mutant zebrafish.

Patterning along the dorsal-ventral (D-V) axis of Xenopus and Drosophila embryos is believed to occur through a conserved molecular mechanism, with homologous proteins Chordin and Short gastrulation (Sog) antagonizing signaling by bone morphogenetic protein 4 (BMP-4) and Decapentaplegic (Dpp), respectively. We have isolated a zebrafish gene that is highly homologous to chordin and sog within cysteine-rich domains and exhibits conserved aspects of expression and function. As in Xenopus embryos, zebrafish chordin is expressed in the organizer region and transiently in axial mesoderm. Injection of zebrafish chordin mRNA to the ventral side of Xenopus embryos induced secondary axes. Ectopic overexpression in zebrafish resulted in an expansion of paraxial mesoderm and neurectoderm at the expense of more lateral and ventral derivatives, producing a range of defects similar to those of dorsalized zebrafish mutants (Mullins et al., 1996). In accordance with the proposed function of chordin in D-V patterning, dorsalized zebrafish mutants showed expanded domains of chordin expression by midgastrulation, while some ventralized mutants had reduced expression; however, in all mutants examined, early organizer expression was unaltered. In contrast to Xenopus, zebrafish chordin is also expressed in paraxial mesoderm and ectoderm and in localized regions of the developing brain, suggesting that there are additional roles for chordin in zebrafish embryonic development. Surprisingly, paraxial mesodermal expression of chordin appeared unaltered in spadetail mutants that later lack trunk muscle (Kimmel et al., 1989), while axial mesodermal expression was affected. This finding reveals an unexpected function for spadetail in midline mesoderm and in differential regulation of chordin expression during gastrulation.

Amino Acid Sequence↗

PubMed2001

The homeobox genes vox and vent are redundant repressors of dorsal fates in zebrafish.

Ventralizing transcriptional repressors in the Vox/Vent family have been proposed to be important regulators of dorsoventral patterning in the early embryo. While the zebrafish genes vox (vega1) and vent (vega2) both have ventralizing activity in overexpression assays, loss-of-function studies are needed to determine whether these genes have distinct or redundant functions in dorsoventral patterning and to provide critical tests of the proposed regulatory interactions among vox, vent and other genes that act to establish the dorsoventral axis. We show that vox and vent are redundant repressors of dorsal fates in zebrafish. Mutants that lack vox function have little or no dorsoventral patterning defect, and inactivation of either vox or vent by injection of antisense morpholino oligonucleotides has little or no effect on the embryo. In contrast, embryos that lack both vox and vent function have a dorsalized phenotype. Expression of dorsal mesodermal genes, including chordin, goosecoid and bozozok, is strongly expanded in embryos that lack vox and vent function, indicating that the redundant action of vox and vent is required to restrict dorsal genes to their appropriate territories. Our genetic analysis indicates that the dorsalizing transcription factor Bozozok promotes dorsal fates indirectly, by antagonizing the expression of vox and vent. In turn, vox and vent repress chordin expression, restricting its function as an antagonist of ventral fates to the dorsal side of the embryo. Our results support a model in which BMP signaling induces the expression of ventral genes, while vox and vent act redundantly to prevent the expression of chordin, goosecoid and other dorsal genes in the lateral and ventral mesendoderm.

Animals↗

PubMed1992

The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo.

Embryos mutant for the T gene, in mice, make insufficient mesoderm and fail to develop a notochord. We report the cloning and sequencing of the T gene in the zebrafish (Brachydanio rerio) and show the nuclear localization of the protein product. Both RNA and protein are found in cells of the germ ring, including enveloping layer cells, prior to and during gastrulation of zebrafish embryos. Nuclei of the yolk syncytial layer do not express Zf-T. High levels of expression are maintained throughout early development in the notochord, while in paraxial mesoderm cells the gene is turned off during gastrulation. Exposure of animal cap cells to activinA induces Zf-T expression, as does transplantation into the germ ring.

Amino Acid Sequence↗

PubMed1998

Isolation and characterization of tilapia (Oreochromis mossambicus) insulin-like growth factors gene and proximal promoter region.

To understand the molecular mechanism which controls the transcription of the insulin-like growth factors (IGFs) gene, we have cloned and sequenced the cDNA for the proximal promoter region of the tilapia IGFs gene and have characterized its activity by chloramphenicol acetyltransferase (CAT) transient transfected expression assays. Tilapia (Oreochromis mossambicus) IGF-I cDNA (549 bp) was amplified by PCR from single-stranded cDNA of growth hormone (GH)-induced liver RNA using a pair of oligonucleotides specific for fish IGF-I as amplification primers. Tilapia IGF-I and IGF-II 5' termini were analyzed by rapid amplification of cDNA 5' ends (5'RACE). Analysis of the 5'RACE results revealed two transcription start sites in IGF-I and one transcription start site in IGF-II. Different fragments of the 5' flanking region were transfected into human lung adenocarcinoma cells. In the cell line, maximum promoter activity was located in the distal 657 basepairs of the IGF-I 5' flanking region and in the distal 450 basepairs of the IGF-II 5' flanking region. The in vivo actions of the IGFs promoter on developmental stage expression were investigated further in transgenic zebrafish in which an IGFs promoter-driven green fluorescent protein (GFP) encoding the cDNA transgene was microinjected into embryos. Morphologic and RT-PCR studies of the transgenic zebrafish indicated that IGF-I promoter-driven GFP transcripts appeared for the first time in the 1-K-cell stage and the IGF-II promoter-driven GFP transcripts appeared for the first time in the 32-cell stage. Fluorescent (GFP) distribution was apparent within 48 h in IGF-II-transgenic zebrafish embryos, especially in eye, muscle, corpuscle, floor plate, horizontal myoseptum, yolk sac extension, and yolk sac. These results indicate that the IGF-I and IGF-II promoters are active in tissue and in a development-specific manner. Our findings also indicate that the IGF-II promoter influences the growth of fish embryos earlier than does IGF-I, and IGF-II has higher levels of expression than does IGF-I. These results suggest that the IGF-II promoter plays a growth factor role in teleost embryo development.

Amino Acid Sequence↗