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At least 433 records · Page 24Linked to original sources

PubMed2002

A quantitative analysis of the kinetics of Gal4 activator and effector gene expression in the zebrafish.

Using a temperature-inducible hsp70:Gal4 activator and UAS:myc-notch1a-intra as effector, we determined quantitatively the kinetics of expression of both transgenes and analysed the effects of varying their expressivity on several phenotypic traits in the developing zebrafish. hsp70:Gal4 is transcribed within 15 min after temperature-mediated induction, but Gal4 RNA decays rapidly. The Gal4 protein was found to be quite stable, as functional Gal4, which was detectable 1.5 h after heat shock (HS), persisted for at least 13 h. myc-notch1a-intra RNA is expressed approximately 1.5 h after HS, but unlike the Gal4 RNA, it was found to be very stable; it continues to accumulate during the succeeding 17 h after HS. Fully penetrant phenotypic effects are obtained after a relatively long activator induction with a 30-min HS.

Animals↗

PubMed2002

Developmental biology of zebrafish myeloid cells.

The zebrafish (Danio rerio) has emerged as an informative vertebrate model for developmental studies, particularly those employing genetic approaches such as mutagenesis and screening. Zebrafish myelopoiesis has recently been characterized, paving the way for the experimental strengths of this model organism to contribute to an improved understanding of the genetic regulation of myeloid development. Zebrafish have a multi-lineage myeloid compartment with two types of granulocyte (heterophil/neutrophil and eosinophil granulocytes), and monocyte/macrophages, each with characteristic morphological features and histochemical staining properties. Molecular markers have been characterised for various myeloid cell types and their precursor cells, for example: stem cells (scl, hhex, lmo2), myeloid lineage precursors (spi1/pu.1, c/ebp1), heterophil granulocytes (mpx/mpo), macrophages (L-plastin, fms). In zebrafish, the sites of early myeloid and erythroid commitment are anatomically separated, being located in the rostral and caudal lateral plate mesoderm respectively. Functional macrophages appear before cells displaying granulocytic markers. By the second day of life, cells expressing granulocyte- and macrophage-specific genes are scattered throughout the embryo, but tend to aggregate in the ventral venous plexus, which may be a site of their production or a preferred site for their residence. Even in early embryos, macrophages are phagocytically active, and granulocytes participate in acute inflammation. Equipped with an understanding of the developmental biology of these various myeloid cells and a set of tools for their identification and functional study, we will now be able to exploit the experimental strengths of this model organism to better understand the genetic regulation of myelopoiesis.

Animals↗

PubMed2006

Zebrafish bashful/laminin-alpha 1 mutants exhibit multiple axon guidance defects.

Laminin is known to provide a highly permissive substratum and in some cases directional information for axon outgrowth in vitro. However, there is still little known about laminin function in guiding axons in vivo. We investigated the axon guidance role of laminin-alpha1 in the developing zebrafish nervous system. Analysis of zebrafish bashful (bal)/laminin-a1 mutants revealed multiple functions for laminin-alpha1 in the outgrowth and guidance of central nervous system (CNS) axons. Most CNS axon pathways are defective in bal embryos. Some axon types, including retinal ganglion cell axons, early forebrain axons, and hindbrain reticulospinal axons, make specific pathfinding errors, suggesting laminin-alpha1 is required for directional decisions. Other axon tracts are defasciculated or not fully extended in bal embryos, suggesting a function for laminin-alpha1 in regulating adhesion or providing a permissive substratum for growth. In addition, some neurons have excessively branched axons in bal, indicating a potential role for laminin-alpha1 in branching. In contrast to CNS axons, most peripheral axons appear normal in bal mutants. Our results, thus, reveal important and diverse functions for laminin-alpha1 in guiding developing axons in vivo.

Alleles↗

PubMed2001

Selective rescue of early haematopoietic progenitors in Scl(-/-) mice by expressing Scl under the control of a stem cell enhancer.

The stem cell leukaemia gene (Scl) encodes a basic helix-loop-helix transcription factor with a pivotal role in both haematopoiesis and endothelial development. During mouse development, Scl is first expressed in extra-embryonic mesoderm, and is required for the generation of all haematopoietic lineages and normal yolk sac angiogenesis. Ectopic expression of Scl during zebrafish development specifies haemangioblast formation from early mesoderm. These results suggest that SCL is essential for establishing the transcriptional programme responsible for the formation of haematopoietic stem cells and have focused attention on the transcriptional regulation of Scl itself. Previous studies have identified a panel of Scl enhancers each of which directed expression to a subdomain of the normal Scl expression pattern. Among them, a 3' enhancer directed expression during development to vascular endothelium and haematopoietic progenitors but not to Ter119(+) erythroid cells. The expression in haematopoietic stem cells, however, remained undetermined. We demonstrate that this 3' enhancer directs lacZ expression in transgenic mice to most foetal and adult long-term repopulating haematopoietic stem cells, and therefore functions as a stem cell enhancer. Consistent with these results, expression in Scl(-/-) embryos of exogenous Scl driven by the stem cell enhancer rescued the formation of early haematopoietic progenitors and also resulted in normal yolk sac angiogenesis. By contrast, erythropoiesis remained markedly deficient in rescued embryos. This observation is consistent with the inactivity of the stem cell enhancer in erythroid cells and reveals an essential role for SCL during erythroid differentiation in vivo.

Animals↗

PubMed1997

A vasa-like gene in zebrafish identifies putative primordial germ cells.

The vasa gene is essential for germline formation in Drosophila. Vasa-related genes have been isolated from several organisms including nematode, frog and mammals. In order to gain insight into the early events in vertebrate germline development, zebrafish was chosen as a model. Two zebrafish vasa-related genes were isolated, pl10a and vlg. The pl10a gene was shown to be widely expressed during embryogenesis. The vlg gene and vasa belong to the same subfamily of RNA helicase encoding genes. Putative maternal vlg transcripts were detected shortly after fertilization and from the blastula stage onwards, expression was restricted to migratory cells most likely to be primordial germ cells.

Amino Acid Sequence↗

PubMed2005

Bacillus anthracis oedema toxin as a cause of tissue necrosis and cell type-specific cytotoxicity.

Oedema factor (OF) and protective antigen (PA) are secreted by Bacillus anthracis, and their binary combination yields oedema toxin (OT). Following PA-mediated delivery to the cytosol, OF functions as an adenylate cyclase generating high levels of cAMP. To assess OT as a possible cause of tissue damage and cell death, a novel approach was developed, which utilized a developing zebrafish embryo model to study toxin activity. Zebrafish embryos incubated with OT exhibited marked necrosis of the liver, cranium and gastrointestinal tract, as well as reduced swim bladder inflation. The OT-treated embryos survived after all stages of development but succumbed to the toxin within 7 days. Additional analysis of specific cell lines, including macrophage and non-macrophage, showed OT-induced cell death is cell type-specific. There was no discernible correlation between levels of OF-generated cAMP and cell death. Depending on the type of cell analysed, cell death could be detected in low levels of cAMP, and, conversely, cell survival was observed in one cell line in which high levels of cAMP were found following treatment with OT. Collectively, these data suggest OT is cytotoxic in a cell-dependent manner and may contribute to disease through direct cell killing leading to tissue necrosis.

Adenylyl Cyclases↗

PubMed2001

Bmp2b and Oep promote early myocardial differentiation through their regulation of gata5.

Members of both the bone morphogenetic protein (Bmp) and EGF-CFC families have been implicated in vertebrate myocardial development. Zebrafish swirl (swr) encodes Bmp2b, a member of the Bmp family required for patterning the dorsoventral axis. Zebrafish one-eyed pinhead (oep) encodes a maternally and zygotically expressed member of the EGF-CFC family essential for Nodal signaling. Both swr/bmp2b and oep mutants exhibit severe defects in myocardial development. swr/bmp2b mutants exhibit reduced or absent expression of nkx2.5, an early marker of the myocardial precursors. Embryos lacking zygotic oep (Zoep mutants) display cardia bifida and, as we show here, also display reduced or absent nkx2.5 expression. Recently, we have demonstrated that the zinc finger transcription factor Gata5 is an essential regulator of nkx2.5 expression. In this paper, we investigate the relationships between bmp2b, oep, gata5, and nkx2.5. We show that both swr/bmp2b and Zoep mutants exhibit defects in gata5 expression in the myocardial precursors. Forced expression of gata5 in swr/bmp2b and Zoep mutants restores robust nkx2.5 expression. Moreover, overexpression of gata5 in Zoep mutants restores expression of cmlc1, a myocardial sarcomeric gene. These results indicate that both Bmp2b and Oep regulate gata5 expression in the myocardial precursors, and that Gata5 does not require Bmp2b or Oep to promote early myocardial differentiation. We conclude that Bmp2b and Oep function at least partly through Gata5 to regulate nkx2.5 expression and promote myocardial differentiation. We integrate these and other data to propose a pathway of the molecular events regulating early myocardial differentiation in zebrafish.

Animals↗

PubMed2001

The pitx2 homeobox protein is required early for endoderm formation and nodal signaling. .

Nodal and Nodal-related factors play fundamental roles in a number of developmental processes, including mesoderm and endoderm formation, patterning of the anterior neural plate, and determination of bilateral asymmetry in vertebrates. pitx2, a paired-like homeobox gene, has been proposed to act downstream of Nodal in the gene cascade providing left-right cues to the developing organs. Here, we report that pitx2 is required early in the Nodal signaling pathway for specification of the endodermal and mesodermal germ layers. We found that pitx2 is expressed very early during Xenopus and zebrafish development and in many regions where Nodal signaling is required, including the presumptive mesoderm and endoderm at the blastula and gastrula stages and the prechordal mesoderm at later stages. In Xenopus embryos, overexpression of pitx2 caused ectopic expression of goosecoid and sox-17 and interfered with mesoderm formation. Overexpression of pitx2 in Xenopus animal cap explants partially mimics the effects of Nodal overexpression, suggesting that pitx2 is a mediator of Nodal signaling during specification of the endoderm and prechordal plate, but not during mesoderm induction. We further demonstrate that pitx2 is induced by Nodal signaling in Xenopus animal caps and that the early expression of zebrafish pitx2 is absent when the Nodal signaling pathway is inactive. Inhibition of pitx2 function using a chimeric EnR-pitx2 blocked specification of the mesoderm and endoderm and caused severe embryonic defects resembling those seen when Nodal signaling is inhibited. Following inhibition of pitx2 function, the fate of ventral vegetal blastomeres was shifted from an endodermal to a more mesodermal fate, an effect that was reversed by wild-type pitx2. Finally, we show that inhibition of pitx2 function interferes with the response of cells to Nodal signaling. Our results provide direct evidence that pitx2 function is required for normal specification of the endodermal and mesodermal germ layers.

Amino Acid Sequence↗

PubMed1999

Confocal microscopic analysis of morphogenetic movements.

Confocal microscopy is an excellent means of imaging cellular dynamics within living zebrafish embryos because it provides a means of optically sectioning tissues that have been labeled with specific fluorescent probe molecules. In order to study genetically encoded patterns of cell behavior that are involved in the formation of germ layers and various organ primordia, it is possible to vitally stain an entire zebrafish embryo with one or more fluorescent probe molecules and then examine morphogenetic behaviors within specific cell populations of interest using time-lapse confocal microscopy. There are two major advantages to this "bulk-labeling" approach: (1) the applied fluorescent probe (a contrast-enhancing agent) allows all of the cells within an intact zebrafish embryo to be rapidly stained; (2) the morphogenetic movements and shape changes of hundreds of cells can then be examined simultaneously in vivo using time-lapse confocal microscopy. The neutral fluorophore Bodipy 505/515 and its sphingolipid-derivative Bodipy-C5-ceramide are particularly useful, nonteratogenic vital stains for imaging cellular dynamics in living zebrafish embryos. These photostable fluorescent probes (when applied with 2% DMSO) percolate through the enveloping layer epithelium of the embryo, and localize in yolk-containing cytoplasm and interstitial space, respectively, owing to their different physiochemical characteristics. Bodipy-ceramide, for instance, remains highly localized to interstitial fluid once it accumulates within a zebrafish embryo, allowing the boundaries of deep cells to be clearly discerned throughout the entire embryo. Through the use of either of these fluorescent vital stains, it is possible to rapidly convert a developing zebrafish embryo into a strongly fluorescent specimen that is ideally suited for time-lapse confocal imaging. For zebrafish embryos whose deep cells have been intentionally "scatter-labeled" with fluorescent lineage tracers (e.g., fluorescent dextrans), sequential confocal z-series (i.e., focus-throughs) of the embryo can be rendered into uniquely informative 3D time-lapse movies using readily available image-processing programs. Similar time-lapse imaging, combined with rapidly advancing computer-assisted visualization techniques, may soon be applied to study the dynamics of GFP-fusion proteins in vivo, as well as other types of synthetic probe molecules designed to reveal the cytological processes associated with the patterning and morphological transformations of the zebrafish's embryonic tissues.

Animals↗

PubMed2001

Differential gene expression of Xenopus Pitx1, Pitx2b and Pitx2c during cement gland, stomodeum and pituitary development.

The members of the Pitx family of homeobox transcription factors have been involved in many aspects of vertebrate embryogenesis, like for example, development of teeth, eyes and limbs. We previously reported expression patterns and function of Pitx2c in the generation of laterality and asymmetric morphogenesis of heart and gastro-intestinal tract in mouse, frog and zebrafish (Development 126 (1999) 1225; Mech. Dev. 90 (2000) 41). Here we describe the differential expression of Pitx1, Pitx2b and Pitx2c during anterior ectodermal pattern formation and differentiation of cement gland, stomodeum and pituitary in the frog Xenopus laevis.

Amino Acid Sequence↗

PubMed2006

Development of the zebrafish lymphatic system requires VEGFC signaling.

Lymphangiogenesis results in the formation of a vascular network distinct from arteries and veins that serves to drain interstitial fluid from surrounding tissues and plays a pivotal role in the immune defense of vertebrates as well as in the progression of cancer and other diseases . In mammals, lymph vessels are lined by endothelial cells possibly sprouting from embryonic veins, and their development appears to be critically dependent on the function of PROX1 and VEGFC signaling . The existence of a lymphatic system in teleosts has been a matter of debate for decades. Here we show on the morphological, molecular, and functional levels that zebrafish embryos develop a lymphatic vasculature that serves to retrieve components of the interstitium to the lymph system. We demonstrate the existence of vessels that are molecularly and functionally distinct from blood vessels and show that the development of these vessels depends on Vegfc and VEGFR-3/Flt4 signaling. These findings imply that the molecular components controlling lymphangiogenesis in zebrafish and mammals are conserved and that the zebrafish lymphatic system develops early enough to allow in vivo observations, lineage tracing, and genetic as well as pharmacological screens.

Angiopoietin-2↗

PubMed2001

Developmental expression of cytochrome P450 aromatase genes (CYP19a and CYP19b) in zebrafish fry (Danio rerio).

Cytochrome P450 aromatase (CYP19) is the terminal enzyme in the steroidogenic pathway that converts androgens (e.g., testosterone) into estrogens (e.g., estradiol). Regulation of this gene dictates the ratio of androgens to estrogens; therefore, appropriate expression of this enzyme is critical for reproduction as well as being pivotal in sex differentiation for most vertebrates. It is assumed that most vertebrates have a single CYP19 gene that is regulated by multiple tissue-specific promoter regions. However, the zebrafish (Danio rerio) has two genes (CYP19a and CYP19b), each encoding a significantly different protein and possessing its own regulatory mechanism. The primary purpose of this study was to determine the pattern of expression of each of the CYP19 genes in the developing zebrafish. A fluorescent-based method of real-time, quantitative RT-PCR provided the sensitivity and specificity to determine transcript abundance in single embryos/juveniles harvested at days 0 through 41 days post-fertilization (dpf), which encompasses the developmental events of sex determination and gonadal differentiation. CYP19 transcripts could be detected as early as 3 or 4 dpf, (CYP19a and CYP19b, respectively) and peak abundance was detected on day five. In general, the CYP19 genes differed significantly in the ontogeny of their expression. In most cases, the gonadal form of CYP19 (CYP19a) was more abundant than the brain form (CYP19b); however, unlike CYP19a, the pattern of CYP19b expression could be clearly segregated into two populations, suggesting an association with sex differentiation. Pharmacological steroids (ethinylestradiol and 17 alpha-methyltestosterone) enhanced the expression of the CYP19b gene at all three days examined (4, 6, and 10 dpf). These data suggest that the timely and appropriate expression of CYP19 is important in development and that the expression of CYP19b (the "extra-gonadal" form) may be associated with sexual differentiation if not sexual determination. J. Exp. Zool. 290:475-483, 2001.

Animals↗

PubMed2004

Water permeability and TCDD-induced edema in zebrafish early-life stages.

A common response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in teleost embryos is blue-sac disease, characterized by pericardial and yolk-sac edema. The cellular and extracellular fluids of freshwater fish are hyperosmotic compared to the surrounding water. In order to be in osmotic balance, freshwater fish must maintain a barrier to minimize water entry and excrete excess water that passes the barrier. We hypothesized that edema observed in TCDD-exposed zebrafish was caused by a failure of a barrier to incoming water. As a test of this hypothesis, we removed the osmotic gradient that drives water entry by increasing the osmolarity of the surrounding water with mannitol. Abolishing the osmotic gradient between the interior body fluids and the water environment of the developing zebrafish significantly reduced both pericardial and yolk-sac edema. When added after edema formation had already started, mannitol only partially reversed pre-existing edema. An alternate hypothesis is that TCDD impairs water excretion, allowing water to accumulate as edema fluid. However, we were unable to demonstrate an alteration in kidney function: expression of early markers for kidney development appeared normal, and we did not observe TCDD-induced changes in kidney filtration. An alteration in the overall shape of the kidney was observed, but this may be a consequence of compression by edema. In conclusion, TCDD exposure may inhibit the function of a permeability barrier to water, which is critical for maintaining osmotic balance in early development.

Aging↗

PubMed2006

An essential role for zebrafish Fgfrl1 during gill cartilage development.

The vertebrate craniofacial skeleton develops via a complex process involving signaling cascades in all three germ layers. Fibroblast growth factor (FGF) signaling is essential for several steps in pharyngeal arch development. In zebrafish, Fgf3 and Fgf8 in the mesoderm and hindbrain have an early role to pattern the pouch endoderm, influencing craniofacial integrity. Endodermal FGF signaling is required for the differentiation and survival of postmigratory neural crest cells that form the pharyngeal skeleton. We identify a novel role for zebrafish Fgf receptor-like 1a (Fgfrl1a) that is indispensable during gill cartilage development. We show that depletion of Fgfrl1a is sufficient to abolish cartilage derivatives of the ceratobranchials. Using an Fgfrl1a-deficient model, we analyzed expression of genes critical for chondrogenesis in the different compartments of the developing pharyngeal arch. Fgfrl1a-depleted animals demonstrate typical neural crest specification and migration to populate the arch primordia as well as normal pouch segmentation. However, in the absence of Fgfrl1a, larvae fail to express the transcription factor glial cells missing 2 (gcm2), a gene necessary for cartilage and gill filament formation, in the ectodermal lining of the branchial arches. In addition, two transcription factors essential for chondrogenesis, sox9a and runx2b, fail to express within the mesenchymal condensations of the branchial arches. A duplicate zebrafish gene, fgfrl1b, has now been identified. We show that Fgfrl1b is also required for proper formation of all ventral cartilage elements and acts cooperatively with Fgfrl1a during gill cartilage formation.

Amino Acid Sequence↗

PubMed2005

Hematopoietic stem cell fate is established by the Notch-Runx pathway.

Identifying the molecular pathways regulating hematopoietic stem cell (HSC) specification, self-renewal, and expansion remains a fundamental goal of both basic and clinical biology. Here, we analyzed the effects of Notch signaling on HSC number during zebrafish development and adulthood, defining a critical pathway for stem cell specification. The Notch signaling mutant mind bomb displays normal embryonic hematopoiesis but fails to specify adult HSCs. Surprisingly, transient Notch activation during embryogenesis via an inducible transgenic system led to a Runx1-dependent expansion of HSCs in the aorta-gonad-mesonephros (AGM) region. In irradiated adults, Notch activity induced runx1 gene expression and increased multilineage hematopoietic precursor cells approximately threefold in the marrow. This increase was followed by the accelerated recovery of all the mature blood cell lineages. These data define the Notch-Runx pathway as critical for the developmental specification of HSC fate and the subsequent homeostasis of HSC number, thus providing a mechanism for amplifying stem cells in vivo.

Animals↗

PubMed2006

BMP signaling restricts hemato-vascular development from lateral mesoderm during somitogenesis.

The bone morphogenetic protein (BMP) signaling pathway is essential during gastrulation for the generation of ventral mesoderm, which makes it a challenge to define functions for this pathway at later stages of development. We have established an approach to disrupt BMP signaling specifically in lateral mesoderm during somitogenesis, by targeting a dominant-negative BMP receptor to Lmo2+ cells in developing zebrafish embryos. This results in expansion of hematopoietic and endothelial cells, while restricting the expression domain of the pronephric marker pax2.1. Expression of a constitutively active receptor and transplantation experiments were used to confirm that BMP signaling in lateral mesoderm restricts subsequent hemato-vascular development. The results show that the BMP signaling pathway continues to function after cells are committed to a lateral mesoderm fate, and influences subsequent lineage decisions by restricting hemato-vascular fate in favor of pronephric development.

Animals↗

PubMed2006

Dose-dependent effects of chemical immobilization on the heart rate of embryonic zebrafish.

The small size and optical transparency of zebrafish embryos and larvae greatly facilitate modern intravital microscopic phenotyping of these experimentally tractable laboratory animals. Neither the experimentally derived dose-response relationships for chemicals commonly used in the mounting of live fish larvae, nor their effect on the stress of the animal, are currently available in the research literature. This is particularly problematic for IACUCs attempting to maintain the highest ethical standards of animal care in the face of a recent spate in investigator-initiated requests to use embryonic zebrafish as experimental models. The authors address this issue by describing the dose-dependent efficacy of several commonly used chemical mounting treatments and their effect on one stress parameter, embryo heart rate. The results of this study empirically define, for the first time, effective, minimally stressful treatments for immobilization and in vivo visualization during early zebrafish development.

Aminobenzoates↗

PubMed1994

Efficient recovery of ENU-induced mutations from the zebrafish germline.

We studied the efficiency with which two chemical mutagens, ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) can induce mutations at different stages of spermatogenesis in zebrafish (Brachydanio rerio). Both EMS and ENU induced mutations at high rates in post-meiotic germ cells, as indicated by the incidence of F1 progeny mosaic for the albino mutation. For pre-meiotic germ cells, however, only ENU was found to be an effective mutagen, as indicated by the frequencies of non-mosaic mutant progeny at four different pigmentation loci. Several mutagenic regimens that varied in either the number of treatments or the concentration of ENU were studied to achieve an optimal ratio between the mutagenicity and toxicity. For the two most mutagenic regimens: 4 x 1 hr in 3 mM ENU and 6 x 1 hr in 3 mM ENU, the minimum estimate of frequencies of independent mutations per locus per gamete was 0.9-1.3 X 10(-3). We demonstrate that embryonic lethal mutations induced with ENU were transmitted to offspring and that they could be recovered in an F2 screen. An average frequency of specific-locus mutations of 1.1 X 10(-3) corresponded to approximately 1.7 embryonic lethal mutations per single mutagenized genome. The high rates of mutations achievable with ENU allow for rapid identification of large numbers of genes involved in a variety of aspects of zebrafish development.

Alleles↗