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Didier Y R Stainier

Publications and source records attributed to Didier Y R Stainier.

At least 19 recordsLinked to original sources
PubMed2006

Microsomal triglyceride transfer protein is required for yolk lipid utilization and absorption of dietary lipids in zebrafish larvae.

Although the absorption, transport, and catabolism of dietary lipids have been studied extensively in great detail in mammals and other vertebrates, a tractable genetic system for identifying novel genes involved in these physiologic processes is not available. To establish such a model, we monitored neutral lipid by staining fixed zebrafish larvae with oil red o (ORO). The head structures, heart, vasculature, and swim bladder stained with ORO until the yolk was consumed 6 days after fertilization (6 dpf). Thereafter, the heart and vasculature no longer had stainable neutral lipids. Following a high-fat meal, ORO stained the intestine and vasculature of 6 dpf larvae, and whole-larval triacylglycerol (TAG) and apolipoprotein B levels increased. Levels of microsomal triglyceride transfer protein (Mtp), the protein responsible for packaging TAG and betalipoproteins into lipoprotein particles, were unchanged by feeding. Since the developing zebrafish embryo expresses mtp in the yolk cell layer, liver, and intestine, we determined the effect of targeted knockdown of Mtp expression using an antisense morpholino oligonucleotide approach (Mtp MO) on the transport of yolk and dietary lipids. Mtp MO injection led to loss of Mtp expression and of lipid staining in the vasculature, heart, and head structures. Mtp MO-injected larvae were smaller than age-matched, uninjected larvae, consumed very little yolk, and did not absorb dietary neutral lipids; however, they absorbed a short chain fatty acid that does not require Mtp for transport. Importantly, the vasculature appeared unaffected in Mtp MO-injected larvae. These studies indicate that zebrafish larvae are suitable for genetic studies of lipid transport and metabolism.

Animals↗
PubMed2006

The HeArt of regeneration.

Fish and amphibian hearts are known to regenerate after partial resection, but the molecular mechanisms underlying this process remain unclear. In this issue of Cell, Lipilina et al. analyze regeneration in the zebrafish heart. Their work indicates that new cardiomyocytes originate from undifferentiated progenitor cells and reveals a critical role for the epicardium, the cellular layer that covers the heart.

Animals↗
PubMed2006

A common progenitor for haematopoietic and endothelial lineages in the zebrafish gastrula.

It has been proposed that haematopoietic and endothelial cells share a common progenitor, termed the haemangioblast. This idea was initially conceived as a result of the observation that these two cell types develop in close proximity to each other within the embryo. Support for this hypothesis was provided by studies on single-cell-derived colonies that can produce both haematopoietic and endothelial cells in vitro. Although these data point towards the existence of a common progenitor for these two lineages, the presence of a bipotential progenitor cell has yet to be demonstrated in vivo. Through the construction of single-cell-resolution fate maps of the zebrafish late blastula and gastrula, we demonstrate that individual cells can give rise to both haematopoietic and endothelial cells. These bipotential progenitors arise along the entire extent of the ventral mesoderm and contribute solely to haematopoietic and endothelial cells. We also find that only a subset of haematopoietic and endothelial cells arise from haemangioblasts. The endothelial descendants of the haemangioblasts all clustered in a specific region of the axial vessels regardless of the location of their progenitors. Our results provide in vivo evidence supporting the existence of the haemangioblast and reveal distinct features of this cell population.

Animals↗
PubMed2006

Mesodermal Wnt2b signalling positively regulates liver specification.

Endodermal organs such as the lung, liver and pancreas emerge at precise locations along the primitive gut tube. Although several signalling pathways have been implicated in liver formation, so far no single gene has been identified that exclusively regulates liver specification. In zebrafish, the onset of liver specification is marked by the localized endodermal expression of hhex and prox1 at 22 hours post fertilization. Here we used a screen for mutations affecting endodermal organ morphogenesis to identify a unique phenotype: prometheus (prt) mutants exhibit profound, though transient, defects in liver specification. Positional cloning reveals that prt encodes a previously unidentified Wnt2b homologue. prt/wnt2bb is expressed in restricted bilateral domains in the lateral plate mesoderm directly adjacent to the liver-forming endoderm. Mosaic analyses show the requirement for Prt/Wnt2bb in the lateral plate mesoderm, in agreement with the inductive properties of Wnt signalling. Taken together, these data reveal an unexpected positive role for Wnt signalling in liver specification, and indicate a possible common theme for the localized formation of endodermal organs along the gut tube.

Animals↗
PubMed2006

In vivo cell biology: following the zebrafish trend.

A deeper understanding of the mechanisms of cell behavior is essential if we want to comprehend how an organism develops and functions. Changes in cellular processes, including the orientation of cell divisions, cell shape, polarity, differentiation and migration, account for tissue rearrangements during development and homeostasis. The in vivo relevance of in vitro findings is being constantly debated and the need for in vivo systems becoming more pressing. The zebrafish (Danio rerio) might become the vertebrate system of choice for a wide spectrum of biological questions that need to be investigated in vivo at cellular and subcellular resolutions. Here, we discuss some recent studies in which the zebrafish was used to gain insight into cell-biological mechanisms. Although this model system has been predominantly appreciated for its amenability to forward genetics, current advances in imaging technology and an increasing number of transgenic lines are bringing it closer to its full potential.

Animals↗
PubMed2006

The yolk syncytial layer regulates myocardial migration by influencing extracellular matrix assembly in zebrafish.

The roles of extra-embryonic tissues in early vertebrate body patterning have been extensively studied, yet we know little about their function during later developmental events. Here, we analyze the function of the zebrafish extra-embryonic yolk syncytial layer (YSL) specific transcription factor, Mtx1, and find that it plays an essential role in myocardial migration. Downregulating the function of Mtx1 in the YSL leads to cardia bifida, a phenotype in which the myocardial cells fail to migrate to the midline. Mtx1 in the extra-embryonic YSL appears to regulate the embryonic expression of fibronectin, a gene previously implicated in myocardial migration. We further show dosage-sensitive genetic interactions between mtx1 and fibronectin. Based on these data, we propose that the extra-embryonic YSL regulates myocardial migration, at least in part by influencing fibronectin expression and subsequent assembly of the extracellular matrix in embryonic tissues.

Animals↗
PubMed2005

Cellular and molecular analyses of vascular tube and lumen formation in zebrafish.

Tube and lumen formation are essential steps in forming a functional vasculature. Despite their significance, our understanding of these processes remains limited, especially at the cellular and molecular levels. In this study, we analyze mechanisms of angioblast coalescence in the zebrafish embryonic midline and subsequent vascular tube formation. To facilitate these studies, we generated a transgenic line where EGFP expression is controlled by the zebrafish flk1 promoter. We find that angioblasts migrate as individual cells to form a vascular cord at the midline. This transient structure is stabilized by endothelial cell-cell junctions, and subsequently undergoes lumen formation to form a fully patent vessel. Downregulating the VEGF signaling pathway, while affecting the number of angioblasts, does not appear to affect their migratory behavior. Our studies also indicate that the endoderm, a tissue previously implicated in vascular development, provides a substratum for endothelial cell migration and is involved in regulating the timing of this process, but that it is not essential for the direction of migration. In addition, the endothelial cells in endodermless embryos form properly lumenized vessels, contrary to what has been previously reported in Xenopus and avian embryos. These studies provide the tools and a cellular framework for the investigation of mutations affecting vasculogenesis in zebrafish.

Animals↗
PubMed2005

Formation of the digestive system in zebrafish: III. Intestinal epithelium morphogenesis.

Recent analysis of a novel strain of transgenic zebrafish (gutGFP) has provided a detailed description of the early morphological events that occur during the development of the liver and pancreas. In this paper, we aim to complement these studies by providing an analysis of the morphological events that shape the zebrafish intestinal epithelium. One of our goals is to provide a framework for the future characterization of zebrafish mutant phenotypes in which intestinal epithelial morphogenesis has been disrupted. Our analysis encompasses the period between 26 and 126 h post-fertilization (hpf) and follows the growth, lumen formation and differentiation of a continuous layer of endoderm into a functional intestinal epithelium with three morphologically distinct segments: the intestinal bulb, mid-intestine and posterior intestine. Between 26 hpf and 76 hpf, the entire intestinal endoderm is a highly proliferative organ. To make a lumen, the zebrafish endoderm cells undergo apical membrane biogenesis, adopt a bilayer configuration and form small cavities that coalesce without cell death. Thereafter, the endoderm cells polarize and differentiate into distinct cell lineages. Enteroendocrine cells are distinguished first at 52 hpf in the caudal region of the intestine in a new stable transgenic line, Tg[nkx2.2a:mEGFP]. The differentiation of mucin-containing goblet cells is first evident at 100 hpf and is tightly restricted to a middle segment of the intestine, designated the mid-intestine, that is also demarcated by the presence of enterocytes with large supranuclear vacuoles. Meanwhile, striking expansion of the lumen in the rostral intestine forms the intestinal bulb. Here the epithelium elaborates folds and proliferating cells become progressively restricted to a basal compartment analogous to the crypts of Lieberkühn in mammals. At 126 hpf, the posterior intestine remains an unfolded monolayer of simple columnar epithelium.

Animals↗
PubMed2005

Vertebrate Smoothened functions at the primary cilium.

The unanticipated involvement of several intraflagellar transport proteins in the mammalian Hedgehog (Hh) pathway has hinted at a functional connection between cilia and Hh signal transduction. Here we show that mammalian Smoothened (Smo), a seven-transmembrane protein essential for Hh signalling, is expressed on the primary cilium. This ciliary expression is regulated by Hh pathway activity; Sonic hedgehog or activating mutations in Smo promote ciliary localization, whereas the Smo antagonist cyclopamine inhibits ciliary localization. The translocation of Smo to primary cilia depends upon a conserved hydrophobic and basic residue sequence homologous to a domain previously shown to be required for the ciliary localization of seven-transmembrane proteins in Caenorhabditis elegans. Mutation of this domain not only prevents ciliary localization but also eliminates Smo activity both in cultured cells and in zebrafish embryos. Thus, Hh-dependent translocation to cilia is essential for Smo activity, suggesting that Smo acts at the primary cilium.

Amino Acid Motifs↗
PubMed2005

Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development.

Defects in cardiac valve morphogenesis and septation of the heart chambers constitute some of the most common human congenital abnormalities. Some of these defects originate from errors in atrioventricular (AV) endocardial cushion development. Although this process is being extensively studied in mouse and chick, the zebrafish system presents several advantages over these models, including the ability to carry out forward genetic screens and study vertebrate gene function at the single cell level. In this paper, we analyze the cellular and subcellular architecture of the zebrafish heart during stages of AV cushion and valve development and gain an unprecedented level of resolution into this process. We find that endocardial cells in the AV canal differentiate morphologically before the onset of epithelial to mesenchymal transformation, thereby defining a previously unappreciated step during AV valve formation. We use a combination of novel transgenic lines and fluorescent immunohistochemistry to analyze further the role of various genetic (Notch and Calcineurin signaling) and epigenetic (heart function) pathways in this process. In addition, from a large-scale forward genetic screen we identified 55 mutants, defining 48 different genes, that exhibit defects in discrete stages of AV cushion development. This collection of mutants provides a unique set of tools to further our understanding of the genetic basis of cell behavior and differentiation during AV valve development.

Animals↗
PubMed2005

nanor, a novel zygotic gene, is expressed initially at the midblastula transition in zebrafish.

A novel, developmentally regulated gene, nanor, was identified by suppression subtractive hybridization. It is first expressed following the midblastula transition (MBT), a critical developmental stage in the early vertebrate embryo when the zygotic genome is activated. The nanor cDNA (626bp) includes a complete open reading frame but neither the gene nor the deduced amino acid sequence shows significant similarity to any known gene or protein. Nanor encodes a 175 amino acid putative protein with a protein kinase C and three casein kinase II phosphorylation sites, an N-myristoylation site and an NFX-type zinc-finger domain, indicating a potential role in transcriptional regulation. Semi-quantitative RT-PCR, Northern blot, and in situ hybridization analysis revealed that nanor expression is developmentally regulated. It is initially expressed after the MBT at the sphere stage and during epiboly it is expressed in the forerunner cells. At 24 h post-fertilization, expression is solely anterior.

Animals↗
PubMed2005

No organ left behind: tales of gut development and evolution.

The function of an organ is dependent on its cellular constituents as well as on their assembly into a cohesive unit. The developing gut faces unique challenges as one of the longest and largest organs in the body and also because it is constantly interfacing with external factors through the diet. Its location deep within the body has until recently hampered investigation into its formation. The patterning of the gut along its longitudinal, dorsoventral, left-right, and radial axes is one of the fascinating issues that pertain to the development, function, and homeostasis of this understudied organ.

Animals↗
PubMed2005

Hand2 regulates epithelial formation during myocardial diferentiation.

Myocardial differentiation is initiated by the activation of terminal-differentiation gene expression within a subset of cells in the anterior lateral plate mesoderm. We have previously shown that shortly after this activation, myocardial cells undergo epithelial maturation [1], suggesting that myocardial differentiation encompasses both molecular and cellular changes. To address the question of how the molecular programs driving myocardial gene expression and the formation of the myocardial epithelium are integrated, we analyzed the role of two essential myocardial terminal-differentiation factors, Hand2 and Gata5, in myocardial epithelia formation. hand2 and gata5 mutants exhibit a much-reduced number of myocardial cells and defects in myocardial gene expression [2,3]. We find that the few myocardial precursors that are present in hand2 mutants do not polarize. In contrast, embryos with reduced Gata5 function exhibit polarized myocardial epithelia despite a similar reduction in myocardial precursor number, indicating that proper cell number is not required for epithelial formation. Taken thogether, these results indicate that Hand2 is uniquely required for myocardial polarization, a previously unappreciated role for this critical transcription factor. Furthermore, these results demonstrate that two independent processes, the polarizaton of myocardial precursors and the allocation of proper cell number, contribute to myocardial development.

Animals↗
PubMed2005

Microarray analysis of zebrafish cloche mutant using amplified cDNA and identification of potential downstream target genes.

Zebrafish is an excellent model organism for studying vertebrate development and human disease. With the availability of increased numbers of zebrafish mutants and microarray chips, gene expression profiling has become a powerful tool for identification of downstream target genes perturbed by a specific mutation. One of the obstacles often encountered, however, is to isolate large numbers of zebrafish mutant embryos that are indistinguishable in morphology from the wild-type siblings for microarray analysis. Here, we report a method using amplified cDNA derived from five embryos for gene expression profiling of the 18-somite zebrafish cloche (clo) mutant, in which development of hematopoietic and endothelial lineages is severely impaired. In total, 31 differentially expressed target genes are identified, of which 13 have not been reported previously. We further determine that of these 13 new targets, 8 genes, including coproporphyrinogen oxidase (cpo), carbonic anhydrase (cahz), claudin g (cldn g), zinc-finger-like gene 2 (znfl2), neutrophil cytosol factor 1 (ncf1), matrix metalloproteinase 13 (mmp13), dual specificity phosphatase 5 (dusp5), and a novel gene referred as zebrafish vessel-specific gene 1 (zvsg1) are predominantly expressed in hematopoietic and endothelial cells. Comparative analysis demonstrates that this method is comparable and complementary to that of the conventional approach using unamplified sample. Our study provides valuable information for studying hematopoiesis and vessel formation. The method described here offers a powerful tool for gene expression profiling of zebrafish mutants in general.

Animals↗
PubMed2004

Early myocardial function affects endocardial cushion development in zebrafish.

Function of the heart begins long before its formation is complete. Analyses in mouse and zebrafish have shown that myocardial function is not required for early steps of organogenesis, such as formation of the heart tube or chamber specification. However, whether myocardial function is required for later steps of cardiac development, such as endocardial cushion (EC) formation, has not been established. Recent technical advances and approaches have provided novel inroads toward the study of organogenesis, allowing us to examine the effects of both genetic and pharmacological perturbations of myocardial function on EC formation in zebrafish. To address whether myocardial function is required for EC formation, we examined silent heart (sih(-/-)) embryos, which lack a heartbeat due to mutation of cardiac troponin T (tnnt2), and observed that atrioventricular (AV) ECs do not form. Likewise, we determined that cushion formation is blocked in cardiofunk (cfk(-/-)) embryos, which exhibit cardiac dilation and no early blood flow. In order to further analyze the heart defects in cfk(-/-) embryos, we positionally cloned cfk and show that it encodes a novel sarcomeric actin expressed in the embryonic myocardium. The Cfk(s11) variant exhibits a change in a universally conserved residue (R177H). We show that in yeast this mutation negatively affects actin polymerization. Because the lack of cushion formation in sih- and cfk-mutant embryos could be due to reduced myocardial function and/or lack of blood flow, we approached this question pharmacologically and provide evidence that reduction in myocardial function is primarily responsible for the defect in cushion development. Our data demonstrate that early myocardial function is required for later steps of organogenesis and suggest that myocardial function, not endothelial shear stress, is the major epigenetic factor controlling late heart development. Based on these observations, we postulate that defects in cardiac morphogenesis may be secondary to mutations affecting early myocardial function, and that, in humans, mutations affecting embryonic myocardial function may be responsible for structural congenital heart disease.

Actins↗
PubMed2004

The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation.

Vascular development is a complex but orderly process that is tightly regulated. A number of secreted factors produced by surrounding cells regulate endothelial cell (EC) differentiation, proliferation, migration and coalescence into cord-like structures. Vascular cords then undergo tubulogenesis to form vessels with a central lumen. But little is known about how tubulogenesis is regulated in vivo. Here we report the identification and characterization of a new EC-derived secreted factor, EGF-like domain 7 (Egfl7). Egfl7 is expressed at high levels in the vasculature associated with tissue proliferation, and is downregulated in most of the mature vessels in normal adult tissues. Loss of Egfl7 function in zebrafish embryos specifically blocks vascular tubulogenesis. We uncover a dynamic process during which gradual separation and proper spatial arrangement of the angioblasts allow subsequent assembly of vascular tubes. This process fails to take place in Egfl7 knockdown embryos, leading to the failure of vascular tube formation. Our study defines a regulator that controls a specific and important step in vasculogenesis.

Animals↗
PubMed2004

Notch signaling can regulate endoderm formation in zebrafish.

Early in vertebrate development, the processes of gastrulation lead to the formation of the three germ layers: ectoderm, mesoderm, and endoderm. The mechanisms leading to the segregation of the endoderm and mesoderm are not well understood. In mid-blastula stage zebrafish embryos, single marginal cells can give rise to both endoderm and mesoderm (reviewed by Warga and Stainier [2002] The guts of endoderm formation. In: Solnica-Krezel L, editor. Pattern formation in zebrafish. Berlin: Springer-Verlag. p 28-47). By the late blastula stage, however, single marginal cells generally give rise to either endoderm or mesoderm. To investigate this segregation of the blastoderm into cells with either endodermal or mesodermal fates, we analyzed the role of Notch signaling in this process. We show that deltaC, deltaD, and notch1 are expressed in the marginal domain of blastula stage embryos and that this expression is dependent on Nodal signaling. Activation of Notch signaling from an early stage leads to a reduction of endodermal cells, as assessed by sox17 and foxA2 expression. We further find that this reduction in endoderm formation by the activation of Notch signaling is preceded by a reduction in the expression of bonnie and clyde (bon) and faust/gata5, two genes necessary for endoderm formation (Reiter et al. [1999] Genes Dev 13:2983-2995; Reiter et al. [2001] Development 128:125-135; Kikuchi et al. [2001] Genes Dev 14:1279-1289). However, activation of Notch signaling in bon mutant embryos leads to a further reduction in endodermal cells, also arguing for a bon-independent role for Notch signaling in endoderm formation. Altogether, these results suggest that Notch signaling plays a role in the formation of the endoderm, possibly in its segregation from the mesoderm.

Animals↗