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At least 343 records · Page 19Linked to original sources
PubMed1998

Patterns of free calcium in zebrafish embryos.

Direct knowledge of Ca2+ patterns in vertebrate development is largely restricted to early stages, in which they control fertilization, ooplasmic segregation and cleavage. To explore new roles of Ca2+ in vertebrate development, we injected the Ca2+ indicator aequorin into zebrafish eggs and imaged Ca2+ throughout the first day of development. During early cleavages, a high Ca2+ zone is seen in the cleavage furrows. The high Ca2+ zone during first cleavage spreads as a slow wave (0.5 microm/second) and is preceded by three Ca2+ pulses within the animal pole region of the egg. When Ca2+ concentrations are clamped at the resting level by BAPTA buffer injection into the zygote, all signs of development are blocked. In later development, Ca2+ patterns are associated with cell movements during gastrulation, with neural induction, with brain regionalization, with formation of the somites and neural keel, with otic placode formation, with muscle movements and with formation of the heart. Particularly remarkable is a sharp boundary between high Ca2+ in the presumptive forebrain and midbrain versus low Ca2+ in the presumptive hindbrain starting at 10 hours of development. When Ca2+ changes are damped by injection of low concentrations of BAPTA, fish form with greatly reduced eyes and hearts. The present study provides a first overview of Ca2+ patterns during prolonged periods of vertebrate development and points to new roles of Ca2+ in cellular differentiation and pattern formation.

Animals↗
PubMed1997

Cloning and expression of the quaking gene in the zebrafish embryo.

The responsible gene for hypomyelinating quaking deficiency, qkI, encoding a KH RNA binding protein, is expressed abundantly in the developing mouse nervous system, whereas who/how/struthio, a homologue of the qkI in Drosophila, is expressed predominantly in the mesoderm. Here we describe the isolation and early developmental expression of a zebrafish homologue of qkI. The zebrafish quaking cDNA, zqk, exhibits striking conservation with qkI across the coding region, accompanied by a unique 123 nucleotide insertion sequence. Maternal and zygotic zqk transcripts are ubiquitously distributed during cleavage and blastula periods, and then accumulate in the dorsal midline of the body trunk during gastrulation. During segmentation and pharyngula periods zqk transcripts are expressed in the neural tissue of the head region, and in the paraxial mesoderm of the body trunk. Subsequently they diminish until the hatching period, when they are expressed only in the cardiac sac and pectoral finbuds. We also found that the zqk transcript is alternatively spliced with the transcript containing a 123 nucleotide additional segment localized in neural tissue in the head region, but not in the paraxial mesoderm in the body trunk. The data suggest that the quaking gene family originated in the mesoderm and evolved to become expressed in the nervous system in lower vertebrates. The insertion of the 123 nucleotide sequence could be related to the acquisition of a neural function for the gene.

Alternative Splicing↗
PubMed1995

Analysis of FGF function in normal and no tail zebrafish embryos reveals separate mechanisms for formation of the trunk and the tail.

To analyse the roles of FGF activity and brachyury during gastrulation we have directly compared the consequences of inhibition of FGF-receptor signalling with the phenotype of the zebrafish brachyury mutant, no tail (ntl). We show that expression of ntl is regulated by FGF and that inhibition of FGF receptor-signalling leads to complete loss of the trunk and tail. Since the ntl mutant lacks the tail and notochord but has an otherwise normal trunk, this demonstrates that trunk development is dependent upon an unidentified gene, or set of genes, referred to as no trunk (ntk) which is regulated by FGF. We propose a model to explain the FGF-dependent regulation of ntl and ntk that accounts for the above phenotypes. Consistent with this model, over-expression of eFGF led to suppression of anterior fates and development of trunk and tail derivatives only. In addition, widespread activation of convergence and extension movements resulted in the formation of multiple axis-like structures. Expression of eve1 and cad1 was also regulated by FGF activity, suggesting that during gastrulation FGF activity is normally restricted to the germ ring where these genes, and ntl, are expressed. Taken together these data suggest that the germ ring acts as a posteriorising centre during AP patterning, mediated by FGF activity in this tissue.

Animals↗
PubMed2005

Combinatorial activity of Flamingo proteins directs convergence and extension within the early zebrafish embryo via the planar cell polarity pathway.

The seven-transmembrane protocadherin, Flamingo, functions in a number of processes during Drosophila development, including planar cell polarity (PCP). To assess the role(s) of Flamingo1/Celsr1 (Fmi1) during vertebrate embryogenesis we have exploited the zebrafish system, identifying two Fmi1 orthologues (zFmi1a and zFmi1b) and employing morpholinos to induce mis-splicing of zebrafish fmi1 mRNAs, to both imitate mutations identified in Drosophila flamingo and generate novel aberrant Flamingo proteins. We demonstrate that in the zebrafish gastrula, Fmi1 proteins function in concert with each other and with the vertebrate PCP proteins, Wnt11 and Strabismus, to mediate convergence and extension during gastrulation, without altering early dorso-ventral patterning. We show that zebrafish Fmi1a promotes extension of the entire antero-posterior axis of the zebrafish gastrula including prechordal plate and ventral diencephalic precursors. However, while we show that control over axial extension is autonomous, we find that Fmi1a is not required within lateral cells undergoing dorsal convergence.

Animals↗
PubMed2005

Sinup, a novel Siaz-interacting nuclear protein, modulates neural plate formation in the zebrafish embryos.

Siah, the vertebrate homologue of the Drosophila seven in absentia (sina) gene, is well conserved from Drosophila to mammal and involved in ubiquitination and proteasome-dependent degradation of various target proteins. To identify cellular proteins interacting with Siah, we screened a zebrafish cDNA library with zebrafish Siah (Siaz) as bait in a yeast two-hybrid assay. We identified a cDNA encoding a novel protein composed of 145 amino acids and termed it as Sinup (Siaz-interacting-nuclear-protein). Sinup is a novel nuclear protein that binds to the highly conserved C-terminal protein-interacting domain of Siaz both in vivo and in vitro. During development, sinup transcripts are abundant from the one-cell stage to the early blastula and then markedly diminished, suggesting sinup largely exists as maternal transcripts. sinup overexpression induced lateral expansion of the neural plate and in consequence caused ectopic expression of otx-2 and hoxb1b during the late gastrula stage. In addition, the lateral/paraxial expression of wnt8 at the onset of gastrulation is suppressed by the forced expression of sinup while the expression levels of various dorso-ventral markers are unaffected. In contrast, interfering with sinup functions using sinup morpholino oligonucleotides gradually diminished the anterior neuroectoderm from the posterior region, and resulted in compete loss of hindbrain at the 3-somites stage. Our report suggests that sinup expression should be tightly regulated during early embryonic development for the proper neural plate formation.

Amino Acid Sequence↗
PubMed2005

Dual roles of zygotic and maternal Scribble1 in neural migration and convergent extension movements in zebrafish embryos.

In the developing vertebrate hindbrain, the characteristic trajectory of the facial (nVII) motor nerve is generated by caudal migration of the nVII motor neurons. The nVII motor neurons originate in rhombomere (r) 4, and migrate caudally into r6 to form the facial motor nucleus. In this study, using a transgenic zebrafish line that expresses green fluorescent protein (GFP) in the cranial motor neurons, we isolated two novel mutants, designated landlocked (llk) and off-road (ord), which both show highly specific defects in the caudal migration of the nVII motor neurons. We show that the landlocked locus contains the gene scribble1 (scrb1), and that its zygotic expression is required for migration of the nVII motor neurons mainly in a non cell-autonomous manner. Taking advantage of the viability of the llk mutant embryos, we found that maternal expression of scrb1 is required for convergent extension (CE) movements during gastrulation. Furthermore, we show a genetic interaction between scrb1 and trilobite(tri)/strabismus(stbm) in CE. The dual roles of the scrb1 gene in both neuronal migration and CE provide a novel insight into the underlying mechanisms of cell movement in vertebrate development.

Animals↗
PubMed2001

The type I serine/threonine kinase receptor Alk8/Lost-a-fin is required for Bmp2b/7 signal transduction during dorsoventral patterning of the zebrafish embryo.

Ventral specification of mesoderm and ectoderm depends on signaling by members of the bone morphogenetic protein (Bmp) family. Bmp signals are transmitted by a complex of type I and type II serine/threonine kinase transmembrane receptors. Here, we show that Alk8, a novel member of the Alk1 subgroup of type I receptors, is disrupted in zebrafish lost-a-fin (laf) mutants. Two alk8/laf null alleles are described. In laf(tm110), a conserved extracellular cysteine residue is replaced by an arginine, while in laf(m100), Alk8 is prematurely terminated directly after the transmembrane domain. The zygotic effect of both mutations leads to dorsalization of intermediate strength. A much stronger dorsalization, similar to that of bmp2b/swirl and bmp7/snailhouse mutants, however, is obtained by inhibiting both maternally and zygotically supplied alk8 gene products with morpholino antisense oligonucleotides. The phenotype of laf mutants and alk8 morphants can be rescued by injected mRNA encoding Alk8 or the Bmp-regulated transcription factor Smad5, but not by mRNA encoding Bmp2b or Bmp7. Conversely, injected mRNA encoding a constitutively active version of Alk8 can rescue the strong dorsalization of bmp2b/swirl and bmp7/snailhouse mutants, whereas smad5/somitabun mutant embryos do not respond. Altogether, the data suggest that Alk8 acts as a Bmp2b/7 receptor upstream of Smad5.

Activin Receptors↗
PubMed2001

The u-boot mutation identifies a Hedgehog-regulated myogenic switch for fiber-type diversification in the zebrafish embryo.

Developmental programs that govern the embryonic diversification of distinct kinds of muscles in vertebrates remain obscure. For instance, the most widely recognized attribute of early diversity among skeletal myoblasts is their ability to differentiate exclusively into fibers with slow or fast contractile properties. However, we know little about the developmental basis and genetic regulation of this seminal event in vertebrate myogenesis. Here we show that in the zebrafish, the u-boot gene acts as a myogenic switch that regulates the choice of myoblasts to adopt slow versus fast fiber developmental pathways. In u-boot mutant embryos, slow muscle precursors abort their developmental program, failing to activate expression of the homeobox gene prox1 and transfating into muscle cells with fast fiber properties. Using oligonucleotide-mediated translational inhibition, we have investigated the role of prox1 in this program. We find that it functions in the terminal step of the u-boot controlled slow fiber developmental pathway in the regulation of slow myofibril assembly. Our findings provide new insight into the genetic control of slow versus fast fiber specification and differentiation and indicate that dedicated developmental pathways exist in vertebrates for the elaboration of distinct elements of embryonic muscle pattern.

Animals↗
PubMed2005

Cardiovascular gene expression profiles of dioxin exposure in zebrafish embryos.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contaminant that causes altered heart morphology, circulatory impairment, edema, hemorrhage, and early life stage mortality in fish. TCDD toxicity is dependent, in large part, on the aryl hydrocarbon receptor (AHR), but understanding of the molecular mechanism of cardiovascular embryotoxicity remains incomplete. To identify genes potentially involved in cardiovascular effects, we constructed custom cDNA microarrays consisting of 4896 zebrafish adult heart cDNA clones and over 200 genes with known developmental, toxicological and housekeeping roles. Gene expression profiles were obtained for 3-day-old zebrafish after early embryonic exposure to either 0.5 or 5.0 nM TCDD. In all, 516 clones were significantly differentially expressed (p < 0.005) under at least one treatment condition; 123 high-priority clones were selected for further investigation. Cytochromes P450 1A and 1B1, and other members of the AHR gene battery, were strongly and dose-dependently induced by TCDD. Importantly, altered expression of cardiac sarcomere components, including cardiac troponin T2 and multiple myosin isoforms, was consistent with the hypothesis that TCDD causes dilated cardiomyopathy. Observed increases in expression levels of mitochondrial energy transfer genes also may be related to cardiomyopathy. Other TCDD-responsive genes included fatty acid and steroid metabolism enzymes, ribosomal and signal-transduction proteins, and 18 expressed sequence tags (ESTs) with no known protein homologs. As the first broad-scale study of TCDD-modulated gene expression in a non-mammalian system, this work provides an important perspective on mechanisms of TCDD toxicity.

Animals↗
PubMed2003

The expression of tissue inhibitor of metalloproteinase 2 (TIMP-2) is required for normal development of zebrafish embryos.

MMP activities are controlled by a combination of proteolytic pro-enzyme activation steps and inhibition by endogenous inhibitors like alpha2-macroglobulin and the tissue inhibitors of metalloproteinases (TIMPs). TIMPs are the key inhibitors in tissue. The expression of both MMPs and TIMPs is controlled during tissue remodeling to maintain a balance in the turnover of extracellular matrix. Disruption of this balance may result in a broad spectrum of diseases. Additionally, TIMP-2 has been reported to have growth factor activities. To further study the function of TIMP-2 in development, we utilized zebrafish as an experimental model system. We have successfully isolated a TIMP-2 homologue from zebrafish (zTIMP-2). This zebrafish TIMP-2 showed high similarity to human TIMP-2 with all critical features conserved. Whole-mount in situ analysis showed that zTIMP-2 was expressed as early as the one-cell stage indicating a maternal origin. This expression continued through later stages of development. RT-PCR analysis confirmed the early expression pattern from the 16-cell stage through blastula, gastrula and 24-h stages. In addition, at the protein level, immunoreactive zTIMP-2 was detected using antibody against recombinant human TIMP-2. RFP-reporter analysis indicated that TIMP-2 can be secreted into the extracellular space where ECM is forming. Functional studies showed that the balance of TIMP-2 expression is important to normal development as reflected by the fact that both blockage of TIMP-2 translation using antisense morpholino oligonculeotides or increased translation of TIMP-2 using a mRNA microinjection approach resulted in abnormal zebrafish development. This is in contrast to murine knockout studies that indicate that TIMP-2 does not have a major role in mouse embryogenesis.

Amino Acid Sequence↗
PubMed2002

The elongation factors Pandora/Spt6 and Foggy/Spt5 promote transcription in the zebrafish embryo.

Precise temporal and spatial control of transcription is a fundamental component of embryonic development. Regulation of transcription elongation can act as a rate-limiting step during mRNA synthesis. The mechanisms of stimulation and repression of transcription elongation during development are not yet understood. We have identified a class of zebrafish mutations (pandora, sk8 and s30) that cause multiple developmental defects, including discrete problems with pigmentation, tail outgrowth, ear formation and cardiac differentiation. We demonstrate that the pandora gene encodes a protein similar to Spt6, a proposed transcription elongation factor. Additionally, the sk8 and s30 mutations are null alleles of the foggy/spt5 locus, which encodes another transcription elongation factor. Through real-time RT-PCR analysis, we demonstrate that Spt6 and Spt5 are both required for efficient kinetics of hsp70 transcription in vivo. Altogether, our results suggest that Spt6 and Spt5 play essential roles of comparable importance for promoting transcription during embryogenesis. This study provides the first genetic evidence for parallel functions of Spt6 and Spt5 in metazoans and establishes a system for the future analysis of transcription elongation during development.

Animals↗
PubMed2005

Developmentally regulated gene expression of the small heat shock protein Hsp27 in zebrafish embryos.

Mammalian small heat shock proteins including Hsp27 and alpha-B crystallin are constitutively expressed in various adult and embryonic tissues including skeletal and cardiac muscle. However, the function of these proteins during embryonic development is not understood, and information on their expression during the earliest stages of development is limited. We have recently demonstrated constitutive and stress inducible expression of a homologue of human Hsp27 in adult zebrafish, an important experimental model of vertebrate developmental processes. Here, we assessed the temporospatial dynamics of zebrafish Hsp27 (hsp27) and alpha-B crystallin (cryab) gene expression using reverse-transcriptase PCR (RT-PCR) and hsp27 expression by in situ hybridization. Our results reveal that initial upregulation of hsp27 expression occurs during early gastrulation. Expression of hsp27 is detected transiently in developing myotomes, lens and brain, and more persistently in developing heart. The constitutive expression level of hsp27 in embryos at some stages of development is considerably greater than that observed in unstressed adult tissues. Expression of hsp27 was also observed in all tissues examined in embryos recovering from heat stress. The pattern of expression observed for hsp27 overlaps partially, but not completely, with that reported for other heat shock proteins.

Animals↗
PubMed1999

Delta-mediated specification of midline cell fates in zebrafish embryos.

BACKGROUND: Fate mapping studies have shown that progenitor cells of three vertebrate embryonic midline structures - the floorplate in the ventral neural tube, the notochord and the dorsal endoderm - occupy a common region prior to gastrulation. This common region of origin raises the possibility that interactions between midline progenitor cells are important for their specification prior to germ layer formation. RESULTS: One of four known zebrafish homologues of the Drosophila melanogaster cell-cell signaling gene Delta, deltaA (dlA), is expressed in the developing midline, where progenitor cells of the ectodermal floorplate, mesodermal notochord and dorsal endoderm lie close together before they occupy different germ layers. We used a reverse genetic strategy to isolate a missense mutation of dlA, dlAdx2, which coordinately disrupts the development of floorplate, notochord and dorsal endoderm. The dlAdx2 mutant embryos had reduced numbers of floorplate and hypochord cells; these cells lie above and beneath the notochord, respectively. In addition, mutant embryos had excess notochord cells. Expression of a dominant-negative form of Delta protein driven by mRNA microinjection produced a similar effect. In contrast, overexpression of dlA had the opposite effect: fewer trunk notochord cells and excess floorplate and hypochord cells. CONCLUSION: Our results indicate that Delta signaling is important for the specification of midline cells. The results are most consistent with the hypothesis that developmentally equivalent midline progenitor cells require Delta-mediated signaling prior to germ layer formation in order to be specified as floorplate, notochord or hypochord.

Animals↗
PubMed2006

Sp5l is a mediator of Fgf signals in anteroposterior patterning of the neuroectoderm in zebrafish embryo.

The neuroectoderm is patterned along the anterior-posterior axis in vertebrate embryos. Fgf signals are required to induce the posterior neuroectodermal fates, but they repress the anterior fate. Sp5l/Spr2, an Sp1-like transcription factor family member, has been shown to be required for development of mesoderm and posterior neuroectoderm. We demonstrate here that repression of the anterior neuroectodermal markers fez and otx1 by fgf17b or fgf3 coincides with induction of sp5l in the anterior neuroectoderm, and that this repression is efficiently rescued by simultaneous sp5l knockdown. On the other hand, sp5l knockdown is able to inhibit inductive activity of ectopic Fgf signals on the expression of the posterior neuroectodermal markers gbx2, hoxb1b, and krox20. Furthermore, effect of overexpression of a dominant negative Fgf receptor on anteroposterior patterning of the neuroectoderm is rescued by sp5l overexpression. Taken together, these data suggest that sp5l mediates the functions of Fgf signals in anteroposterior patterning of the neuroectoderm during zebrafish embryogenesis.

Animals↗