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D Y Stainier

Publications and source records attributed to D Y Stainier.

At least 19 recordsLinked to original sources
PubMed2001

Zebrafish genetics and vertebrate heart formation.

Forward-genetic analyses in Drosophila and Caenorhabditis elegans have given us unprecedented insights into many developmental mechanisms. To study the formation of organs that contain cell types and structures not present in invertebrates, a vertebrate model system amenable to forward genetics would be very useful. Recent work shows that a newly initiated genetic approach in zebrafish is already making significant contributions to understanding the development of the vertebrate heart, an organ that contains several vertebrate-specific features. These and other studies point to the utility of the zebrafish system for studying a wide range of vertebrate-specific processes.

Animals↗
PubMed2001

Multiple roles for Gata5 in zebrafish endoderm formation.

Previous studies have indicated that gata5, a zinc-finger transcription factor gene, is required for the development of the zebrafish gut tube. Here, we show that gata5 mutants also display defects in the development of other endodermal organs such as the liver, pancreas, thyroid and thymus. gata5 is expressed in the endodermal progenitors from late blastula stages, suggesting that it functions early during endoderm development. We indeed find that during gastrulation stages, gata5 mutants form fewer endodermal cells than their wild-type siblings. In addition, the endodermal cells that form in gata5 mutants appear to express lower than wild-type levels of endodermal genes such as sox17 and axial/foxA2. Conversely, overexpression of gata5 leads to expanded endodermal gene expression. These data indicate that Gata5 is involved both in the generation of endodermal cells at late blastula stages and in the maintenance of endodermal sox17 expression during gastrulation. We have also analyzed the relationship of Gata5 to other factors involved in endoderm formation. Using complementary mutant and overexpression analyses, we show that Gata5 regulates endoderm formation in cooperation with the Mix-type transcription factor Bon, that Gata5 and Bon function downstream of Nodal signaling, and that cas function is usually required for the activity of Gata5 in endoderm formation. Finally, we show that fau/gata5, bon and cas exhibit dominant genetic interactions providing additional support that they function in the same pathway. Together, these data demonstrate that Gata5 plays multiple roles in endoderm development in zebrafish, and position Gata5 relative to other regulators of endoderm formation.

Animals↗
PubMed2000

A sphingosine-1-phosphate receptor regulates cell migration during vertebrate heart development.

Coordinated cell migration is essential in many fundamental biological processes including embryonic development, organogenesis, wound healing and the immune response. During organogenesis, groups of cells are directed to specific locations within the embryo. Here we show that the zebrafish miles apart (mil) mutation specifically affects the migration of the heart precursors to the midline. We found that mutant cells transplanted into a wild-type embryo migrate normally and that wild-type cells in a mutant embryo fail to migrate, suggesting that mil may be involved in generating an environment permissive for migration. We isolated mil by positional cloning and show that it encodes a member of the lysosphingolipid G-protein-coupled receptor family. We also show that sphingosine-1-phosphate is a ligand for Mil, and that it activates several downstream signalling events that are not activated by the mutant alleles. These data reveal a new role for lysosphingolipids in regulating cell migration during vertebrate development and provide the first molecular clues into the fusion of the bilateral heart primordia during organogenesis of the heart.

Alleles↗
PubMed2000

The zebrafish bonnie and clyde gene encodes a Mix family homeodomain protein that regulates the generation of endodermal precursors.

Vertebrate endoderm development has recently become the focus of intense investigation. In this report, we first show that the zebrafish bonnie and clyde (bon) gene plays a critical early role in endoderm formation. bon mutants exhibit a profound reduction in the number of sox17-expressing endodermal precursors formed during gastrulation, and, consequently, a profound reduction in gut tissue at later stages. The endodermal precursors that do form in bon mutants, however, appear to differentiate normally indicating that bon is not required at later steps of endoderm development. We further demonstrate that bon encodes a paired-class homeodomain protein of the Mix family that is expressed transiently before and during early gastrulation in both mesodermal and endodermal progenitors. Overexpression of bon can rescue endodermal gene expression and the formation of a gut tube in bon mutants. Analysis of a newly identified mutant allele reveals that a single amino acid substitution in the DNA recognition helix of the homeodomain creates a dominant interfering form of Bon when overexpressed. We also show through loss- and gain-of-function analyses that Bon functions exclusively downstream of cyclops and squint signaling. Together, our data demonstrate that Bon is a critical transcriptional regulator of early endoderm formation.

Amino Acid Sequence↗
PubMed2000

A conserved role for H15-related T-box transcription factors in zebrafish and Drosophila heart formation.

T-box transcription factors are critical regulators of early embryonic development. We have characterized a novel zebrafish T-box transcription factor, hrT (H15-related T box) that is a close relative of Drosophila H15 and a recently identified human gene. We show that Drosophila H15 and zebrafish hrT are both expressed early during heart formation, in strong support of previous work postulating that vertebrate and arthropod hearts are homologous structures with conserved regulatory mechanisms. The timing and regulation of zebrafish hrT expression in anterior lateral plate mesoderm suggest a very early role for hrT in the differentiation of the cardiac precursors. hrT is coexpressed with gata4 and nkx2.5 not only in anterior lateral plate mesoderm but also in noncardiac mesoderm adjacent to the tail bud, suggesting that a conserved regulatory pathway links expression of these three genes in cardiac and noncardiac tissues. Finally, we analyzed hrT expression in pandora mutant embryos, since these have defects in many of the tissues that express hrT, including the heart. hrT expression is much reduced in the early heart fields of pandora mutants, whereas it is ectopically expressed subsequently. Using hrT expression as a marker, we describe a midline patterning defect in pandora affecting the anterior hindbrain and associated midline mesendodermal derivatives. We discuss the possibility that the cardiac ventricular defect previously described in pandora and the midline defects described here are related.

Amino Acid Sequence↗
PubMed2000

Universal GFP reporter for the study of vascular development.

We report the generation and characterization of transgenic mouse and zebrafish expressing green fluorescent protein (GFP) specifically in vascular endothelial cells in a relatively uniform fashion. These reporter lines exhibit fluorescent vessels in developing embryos and throughout adulthood, allowing visualization of the general vascular patterns with single cell resolution. Furthermore, we show the ability to purify endothelial cells from whole embryos and adult organs by a single step fluorescence activated cell sorting. We expect that these transgenic reporters will be useful tools for imaging vascular morphogenesis, global gene expression profile analysis of endothelial cells, and high throughput screening for vascular mutations.

Animals↗
PubMed2000

The bHLH transcription factor hand2 plays parallel roles in zebrafish heart and pectoral fin development.

The precursors of several organs reside within the lateral plate mesoderm of vertebrate embryos. Here, we demonstrate that the zebrafish hands off locus is essential for the development of two structures derived from the lateral plate mesoderm - the heart and the pectoral fin. hands off mutant embryos have defects in myocardial development from an early stage: they produce a reduced number of myocardial precursors, and the myocardial tissue that does form is improperly patterned and fails to maintain tbx5 expression. A similar array of defects is observed in the differentiation of the pectoral fin mesenchyme: small fin buds form in a delayed fashion, anteroposterior patterning of the fin mesenchyme is absent and tbx5 expression is poorly maintained. Defects in these mesodermal structures are preceded by the aberrant morphogenesis of both the cardiogenic and forelimb-forming regions of the lateral plate mesoderm. Molecular analysis of two hands off alleles indicates that the hands off locus encodes the bHLH transcription factor Hand2, which is expressed in the lateral plate mesoderm starting at the completion of gastrulation. Thus, these studies reveal early functions for Hand2 in several cellular processes and highlight a genetic parallel between heart and forelimb development.

Animals↗
PubMed2000

Induction and differentiation of the zebrafish heart requires fibroblast growth factor 8 (fgf8/acerebellar).

Vertebrate heart development is initiated from bilateral lateral plate mesoderm that expresses the Nkx2.5 and GATA4 transcription factors, but the extracellular signals specifying heart precursor gene expression are not known. We describe here that the secreted signaling factor Fgf8 is expressed in and required for development of the zebrafish heart precursors, particularly during initiation of cardiac gene expression. fgf8 is mutated in acerebellar (ace) mutants, and homozygous mutant embryos do not establish normal circulation, although vessel formation is only mildly affected. In contrast, heart development, in particular of the ventricle, is severely abnormal in acerebellar mutants. Several findings argue that Fgf8 has a direct function in development of cardiac precursor cells: fgf8 is expressed in cardiac precursors and later in the heart ventricle. Fgf8 is required for the earliest stages of nkx2.5 and gata4, but not gata6, expression in cardiac precursors. Cardiac gene expression is restored in acerebellar mutant embryos by injecting fgf8 RNA, or by implanting a Fgf8-coated bead into the heart primordium. Pharmacological inhibition of Fgf signalling during formation of the heart primordium phenocopies the acerebellar heart phenotype, confirming that Fgf signaling is required independently of earlier functions during gastrulation. These findings show that fgf8/acerebellar is required for induction and patterning of myocardial precursors.

Animals↗
PubMed2000

Hhex and scl function in parallel to regulate early endothelial and blood differentiation in zebrafish.

During embryogenesis, endothelial and blood precursors are hypothesized to arise from a common progenitor, the hemangioblast. Several genes that affect the differentiation of, or are expressed early in, both the endothelial and blood lineages may in fact function at the level of the hemangioblast. For example, the zebrafish cloche mutation disrupts the differentiation of both endothelial and blood cells. The transcription factor gene scl is expressed in both endothelial and blood lineages from an early stage and can regulate their differentiation. Here we report that in zebrafish the homeobox gene hhex (previously called hex) is also expressed in endothelial and blood lineages from an early stage. We find that hhex expression in these lineages is significantly reduced in cloche mutant embryos, indicating that hhex functions downstream of cloche to regulate endothelial and blood differentiation. Ectopic expression of hhex through injection of a DNA construct leads to the premature and ectopic expression of early endothelial and blood differentiation genes such as fli1, flk1 and gata1, indicating that Hhex can positively regulate endothelial and blood differentiation. However, analysis of a hhex deficiency allele shows that hhex is not essential for early endothelial and blood differentiation, suggesting that another gene, perhaps scl, compensates for the absence of Hhex function. Furthermore, we find that hhex and scl can induce each other's expression, suggesting that these two genes cross-regulate each other during early endothelial and blood differentiation. Together, these data provide the initial framework of a pathway that can be used to further integrate the molecular events regulating hemangioblast differentiation.

Animals↗
PubMed1999

casanova plays an early and essential role in endoderm formation in zebrafish.

The cellular and molecular mechanisms that regulate endoderm development in vertebrates have only recently begun to be explored. Here we show that the zebrafish locus casanova plays an early and essential role in this process. casanova mutants lack a gut tube and do not express any molecular markers of endoderm differentiation. The early endodermal expression of genes such as axial, gata5, and fkd2 does not initiate in casanova mutants, indicating that the endoderm is defective from the onset of gastrulation. Mosaic analysis demonstrates that casanova functions cell autonomously within the endodermal progenitors. We also report the isolation of a zebrafish homologue of Mixer, a gene important for early endoderm formation in Xenopus. casanova does not encode zebrafish Mixer, and mixer expression is normal in casanova mutants, indicating that casanova acts downstream of, or parallel to, mixer to promote endoderm formation. We further find that the forerunner cells, a specialized group of noninvoluting dorsal mesendodermal cells, do not form in casanova mutants. Studies of casanova mutants do not support an important role for the forerunner cells in either dorsal axis or tail development, as has been previously proposed. In addition, although different populations of mesodermal precursors are generated normally in casanova mutants, morphogenetic defects in the heart, vasculature, blood, and kidney are apparent, suggesting a possible role for the endoderm in morphogenesis of these organs.

Amino Acid Sequence↗
PubMed1999

Gata5 is required for the development of the heart and endoderm in zebrafish.

The mechanisms regulating vertebrate heart and endoderm development have recently become the focus of intense study. Here we present evidence from both loss- and gain-of-function experiments that the zinc finger transcription factor Gata5 is an essential regulator of multiple aspects of heart and endoderm development. We demonstrate that zebrafish Gata5 is encoded by the faust locus. Analysis of faust mutants indicates that early in embryogenesis Gata5 is required for the production of normal numbers of developing myocardial precursors and the expression of normal levels of several myocardial genes including nkx2.5. Later, Gata5 is necessary for the elaboration of ventricular tissue. We further demonstrate that Gata5 is required for the migration of the cardiac primordia to the embryonic midline and for endodermal morphogenesis. Significantly, overexpression of gata5 induces the ectopic expression of several myocardial genes including nkx2.5 and can produce ectopic foci of beating myocardial tissue. Together, these results implicate zebrafish Gata5 in controlling the growth, morphogenesis, and differentiation of the heart and endoderm and indicate that Gata5 regulates the expression of the early myocardial gene nkx2.5.

Amino Acid Sequence↗
PubMed1999

A molecular pathway leading to endoderm formation in zebrafish.

BACKGROUND: Several potentially important regulators of vertebrate endoderm development have been identified, including Activin-related growth factors and their receptors; transcriptional regulators encoded by the genes Mixer, Xsox17, and HNF3beta; zebrafish One-eyed pinhead (Oep), a member of the Cripto/FRL-1/Cryptic family of epidermal growth factor related proteins (EGF-CFC); and the product of the zebrafish locus casanova, which plays an essential cell-autonomous role in endoderm formation. RESULTS: Using overexpression studies and the analysis of different zebrafish mutants, we have assembled a molecular pathway that leads to endoderm formation. We report that a zebrafish Sox17 homologue is expressed during gastrulation exclusively in the endoderm and that casanova mutants lack all sox17 expression. Overexpression of mixer induces ectopic sox17-expressing cells in wild-type embryos and promotes endoderm formation in oep mutants, but does not rescue sox17 expression or endoderm formation in casanova mutants. Overexpression of a constitutively active form of the type I transforming growth factor beta (TGF-beta) receptor TARAM-A also promotes sox17 expression in wild-type and oep mutant embryos, but not in casanova mutants. We also show that the Nodal-related molecules Cyclops and Squint and the transmembrane protein Oep are essential for normal mixer expression. CONCLUSIONS: The data indicate that the following pathway leads to zebrafish endoderm formation: Cyclops and Squint activate receptors such as TARAM-A; Oep also appears to act upstream of such receptors; signals transduced by these receptors lead to the expression of mixer, Mixer then acts through casanova to promote the expression of sox17 and differentiation of the endoderm.

Amino Acid Sequence↗
PubMed1999

A role for the extraembryonic yolk syncytial layer in patterning the zebrafish embryo suggested by properties of the hex gene.

Recent studies in mouse suggest that the extraembryonic endoderm has an important role in early embryonic patterning [1]. To analyze whether similar mechanisms operate in other vertebrates, we cloned the zebrafish homologue of Hex, a homeobox gene that is expressed asymmetrically in the mouse visceral endoderm [2]. Early expression of zebrafish hex is restricted to the dorsal portion of the yolk syncytial layer (YSL), an extraembryonic tissue. By the onset of gastrulation, hex is expressed in the entire dorsal half of the YSL, which directly underlies the cells fated to form the neural plate. We show that hex expression is initially regulated by the maternal Wnt pathway and later by a Bmp-mediated pathway. Overexpression experiments of wild-type and chimeric Hex constructs indicate that Hex functions as a transcriptional repressor and its overexpression led to the downregulation of bmp2b and wnt8 expression and the expansion of chordin expression. These findings provide further evidence that the zebrafish YSL is the functional equivalent of the mouse visceral endoderm and that extraembryonic structures may regulate early embryonic patterning in many vertebrates.

Animals↗
PubMed1999

Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish.

The embryonic vertebrate heart is divided into two major chambers, an anterior ventricle and a posterior atrium. Although the fundamental differences between ventricular and atrial tissues are well documented, it is not known when and how cardiac anterior-posterior (A-P) patterning occurs. The expression patterns of two zebrafish cardiac myosin genes, cardiac myosin light chain 2 (cmlc2) and ventricular myosin heavy chain (vmhc), allow us to distinguish two populations of myocardial precursors at an early stage, well before the heart tube forms. These myocardial subpopulations, which may represent the ventricular and atrial precursors, are organized in a medial-lateral pattern within the precardiac mesoderm. Our examinations of cmlc2 and vmhc expression throughout the process of heart tube assembly indicate the important role of an intermediate structure, the cardiac cone, in the conversion of this early medial-lateral pattern into the A-P pattern of the heart tube. To gain insight into the genetic regulation of heart tube assembly and patterning, we examine cmlc2 and vmhc expression in several zebrafish mutants. Analyses of mutations that cause cardia bifida demonstrate that the achievement of a proper cardiac A-P pattern does not depend upon cardiac fusion. On the other hand, cardiac fusion does not ensure the proper A-P orientation of the ventricle and atrium, as demonstrated by the heart and soul mutation, which blocks cardiac cone morphogenesis. Finally, the pandora mutation interferes with the establishment of the early medial-lateral myocardial pattern. Altogether, these data suggest new models for the mechanisms that regulate the formation of a patterned heart tube and provide an important framework for future analyses of zebrafish mutants with defects in this process.

Animals↗
PubMed1999

Patterning during organogenesis: genetic analysis of cardiac chamber formation.

A classical genetic approach, in which mutagenized organisms are screened for phenotypes of interest, is appealing for the analysis of developmental processes. Here, we describe the advantages of zebrafish genetics for the study of heart development. As an example of the utility of this strategy, we discuss its potential to illuminate the molecular mechanisms of cardiac chamber formation. The signals that specify ventricular and atrial lineages and the differentiation pathways that produce distinct chambers are poorly understood. Recently identified zebrafish mutations that disrupt ventricular or atrial development promise to reveal genes essential for these processes.

Animals↗
PubMed1999

Cell-autonomous and non-autonomous requirements for the zebrafish gene cloche in hematopoiesis.

Vertebrate embryonic hematopoiesis is a complex process that involves a number of cellular interactions, notably those occurring between endothelial and blood cells. The zebrafish cloche mutation affects both the hematopoietic and endothelial lineages from an early stage (Stainier, D. Y. R., Weinstein, B. M., Detrich, H. W. R., Zon, L. I. and Fishman, M. C. (1995) Development 121, 3141-3150). cloche mutants lack endocardium, as well as head and trunk endothelium, and nearly all blood cells. Cell transplantation studies have revealed that the endocardial defect in cloche is cell-autonomous: wild-type cells can form endocardium in mutant hosts, but mutant cells never contribute to the endocardium in wild-type or mutant hosts. In this paper, we analyze the cell-autonomy of the blood defect in cloche. The blood cell deficiency in cloche mutants could be an indirect effect of the endothelial defects. Alternatively, cloche could be required cell-autonomously in the blood cells themselves. To distinguish between these possibilities, we cotransplanted wild-type and mutant cells into a single wild-type host in order to compare their respective hematopoietic capacity. We found that transplanted wild-type cells were much more likely than mutant cells to contribute to circulating blood in a wild-type host. Furthermore, in the few cases where both wild-type and mutant donors contributed to blood in a wild-type host, the number of blood cells derived from the wild-type donor was always much greater than the number of blood cells derived from the mutant donor. These data indicate that cloche is required cell-autonomously in blood cells for their differentiation and/or proliferation. When we assessed early expression of the erythropoietic gene gata-1 in transplant recipients, we found that mutant blastomeres were as likely as wild-type blastomeres to give rise to gata-1-expressing cells in a wild-type host. Together, these two sets of data argue that cloche is not required cell-autonomously for the differentiation of red blood cells, as assayed by gata-1 expression, but rather for their proliferation and/or survival, as assayed by their contribution to circulating blood. In addition, we found that transplanted wild-type cells were less likely to express gata-1 in a mutant environment than in a wild-type one, suggesting that cloche also acts non-autonomously in red blood cell differentiation. This non-autonomous function of cloche in red blood cell differentiation may reflect its cell-autonomous requirement in the endothelial lineage. Thus, cloche appears to be required in erythropoiesis cell non-autonomously at a step prior to gata-1 expression, and cell-autonomously subsequently.

Animals↗
PubMed1998

Characterization of the Huntington's disease (HD) gene homologue in the zebrafish Danio rerio.

The Huntington's disease (HD) gene contains a trinucleotide repeat that is expanded and unstable in patients with the disease (HDCRG, 1993). As the first step toward investigating a potential role for this gene in early vertebrate development, we isolated the homolog of the Huntington's disease (ZHD) cDNA in zebrafish. This cDNA encodes a predicted protein product of 3121 amino acids with 70% identity to human huntingtin. The first exon is predicted to encode four glutamines, followed by only one proline, demonstrating that the polymorphic polyproline stretch found in mammalian HD sequences is absent in the fish. We sequenced approximately 900bp upstream from the predicted start codon and found that it lacks a TATA box, CCAAT box, or Sp1 binding sites. Western blot analysis revealed that the protein is expressed at a high level in late embryonic development and at moderate levels in the adult head.

Amino Acid Sequence↗