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At least 325 records · Page 18Linked to original sources
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↗
PubMed2000

Cloning and characterization of a novel nuclear Bcl-2 family protein, zfMcl-1a, in zebrafish embryo.

The importance of the Bcl-2 family proteins in normal vertebrate embryogenesis is being recognized; however, their regulatory mechanism is poorly understood. To elucidate the embryonic roles of Bcl-2 family proteins, we cloned and characterized the first zebrafish Bcl-2 family protein, zfMcl-1a. Zebrafish Mcl-1a shows the highest homology to rat Mcl-1 and contains several conserved BH domains of the Bcl-2 family proteins. It also contains a nuclear localization signal (NLS). Using EGFP reporter analysis, we verified the nuclear localization of zfMcl-1a. Deletion of the NLS resulted in distribution of the fusion protein in the cytoplasm. Northern blot analysis indicated that zfMcl-1a mRNA is 1.5 kb and was expressed in oocytes and throughout embryonic development. Notably, the expression of zfMcl-1a transcript was significantly downregulated during gastrulation. These results suggest that zfMcl-1a is a novel nuclear Bcl-2 family protein and is likely to play an important role in zebrafish oogenesis and embryogenesis.

3T3 Cells↗
PubMed2006

Sequestration of retinyl esters is essential for retinoid signaling in the zebrafish embryo.

For vertebrate development, vitamin A (all-trans retinol) is required in quantitative different amounts and spatiotemporal distribution for the production of retinoic acid, a nuclear hormone receptor ligand, and 11-cis retinal, the chromophore of visual pigments. We show here for zebrafish that embryonic retinoid homeostasis essentially depends on the activity of a leci-thin:retinol acyltransferase (Lratb). During embryogenesis, lratb is expressed in mostly non-overlapping domains opposite to retinal dehydrogenase 2 (raldh2), the key enzyme for retinoic acid synthesis. Blocking retinyl ester formation by a targeted knock down of Lratb results in significantly increased retinoic acid levels, which lead to severe embryonic patterning defects. Thus, we provide evidence that a balanced competition between Lratb and Raldh2 for yolk vitamin A defines embryonic compartments either for retinyl ester or retinoic acid synthesis. This homeostatic mechanism dynamically adjusts embryonic retinoic acid levels for gene regulation, concomitantly sequestering excess yolk vitamin A in the form of retinyl esters for the establishment of larval vision later during development.

Acyltransferases↗
PubMed2002

Tissue-specific expression of AHR2, ARNT2, and CYP1A in zebrafish embryos and larvae: effects of developmental stage and 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure.

To better understand the role of the aryl hydrocarbon receptor (AHR) signaling pathway in causing tissue-specific signs of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity in zebrafish, the temporal and spatial expression of the zebrafish aryl hydrocarbon receptor 2 (zfAHR2), aryl hydrocarbon receptor nuclear translocator 2 (zfARNT2), and an AHR regulated gene, cytochrome P4501A (zfCYP1A), were assessed in larvae exposed to vehicle or TCDD (1.55 nM) from 3-4 h postfertilization (hpf). Coexpression of a transcriptionally active AHR pathway was apparent by the expression of zfCYP1A mRNA and protein in certain larval tissues. zfCYP1A protein was first detected in the skin and vasculature of TCDD-exposed larvae at 36 hpf. Vascular-specific zfCYP1A protein expression continued from 36 to120 hpf at which time it was also detected in the heart, kidney, and liver. zfCYP1A mRNA was observed in TCDD treated larvae as early as 24 hpf in the developing vascular system. Vascular specific zfCYP1A mRNA expression in the head, trunk, and tail by 36 hpf in TCDD-exposed larvae, confirmed immunohistochemical localization. The expression of zfAHR2 and zfARNT2 mRNAs was generally similar in control and TCDD-exposed larvae. Coexpression of zfAHR2, zfARNT2, and zfCYP1A mRNAs was evident in TCDD-exposed larvae by 36 hpf and in the vasculature, heart, and trunk kidney by 48 hpf, well before the first signs of overt developmental toxicity are observed. In addition to their function in response to AHR agonists, zfAHR2 and zfARNT2 may be involved in development and function of the nervous system. zfAHR2 and zfARNT2 were detected in the brain, spinal cord, and sensory organs. However, TCDD-induced zfCYP1A expression was not detected in these tissues. Taken together, these results are consistent with the notion that the cardiovascular system is a primary target of TCDD developmental toxicity in zebrafish.

Animals↗
PubMed2004

Hedgehog and Fgf signaling pathways regulate the development of tphR-expressing serotonergic raphe neurons in zebrafish embryos.

Serotonin (5HT) plays major roles in the physiological regulation of many behavioral processes, including sleep, feeding, and mood, but the genetic mechanisms by which serotonergic neurons arise during development are poorly understood. In the present study, we have investigated the development of serotonergic neurons in the zebrafish. Neurons exhibiting 5HT-immunoreactivity (5HT-IR) are detected from 45 h postfertilization (hpf) in the ventral hindbrain raphe, the hypothalamus, pineal organ, and pretectal area. Tryptophan hydroxylases encode rate-limiting enzymes that function in the synthesis of 5HT. As part of this study, we cloned and analyzed a novel zebrafish tph gene named tphR. Unlike two other zebrafish tph genes (tphD1 and tphD2), tphR is expressed in serotonergic raphe neurons, similar to tph genes in mammalian species. tphR is also expressed in the pineal organ where it is likely to be involved in the pathway leading to synthesis of melatonin. To better understand the signaling pathways involved in the induction of the serotonergic phenotype, we analyzed tphR expression and 5HT-IR in embryos in which either Hh or Fgf signals are abrogated. Hindbrain 5HT neurons are severely reduced in mutants lacking activity of either Ace/Fgf8 or the transcription factor Noi/Pax2.1, which regulates expression of ace/fgf8, and probably other genes encoding signaling proteins. Similarly, serotonergic raphe neurons are absent in embryos lacking Hh activity confirming a conserved role for Hh signals in the induction of these cells. Conversely, over-activation of the Hh pathway increases the number of serotonergic neurons. As in mammals, our results are consistent with the transcription factors Nk2.2 and Gata3 acting downstream of Hh activity in the development of serotonergic raphe neurons. Our results show that the pathways involved in induction of hindbrain serotonergic neurons are likely to be conserved in all vertebrates and help establish the zebrafish as a model system to study this important neuronal class.

Animals↗
PubMed1997

Pattern formation in janus-mutant zebrafish embryos.

Mechanisms that underlie the formation of the vertebrate body appear to be highly conserved between amphibia and teleosts. For teleosts, however, mesoderm induction and the establishment of dorsoventral polarity are poorly understood. In this study, we present an analysis of early pattern formation in the zebrafish maternal-effect mutation janus. This mutation frequently results in a separation of the cleavage stage blastoderm into two halves that undergo separate development until fusion occurs at the end of gastrulation. Here, we employ janus-mutant embryos to analyze the mechanisms of mesoderm formation and ventral specification in a teleost. Analysis of the expression of the panmesodermal marker no tail in janus-mutant embryos indicates that mesoderm induction depends on a marginal position. In an analysis of ventral specification, we show that the early expression of the ventral marker GATA-2 is confined to the area on both blastodermal halves opposite the dorsal shield region. Since, in janus-mutant embryos, the dorsal position is random with respect to the division plane bisecting the two blastodermal halves, a variety of dorsoventral asymmetries arise within individual embryos. In one constellation, the dorsal position is localized to the plane of bisection and two ventral positions develop at opposite ends of the blastodermal halves. Hence, ventral fates can be specified at any position around the blastodermal margins and are excluded from the dorsal position. The diblastodermic system of the janus-mutant embryo allows for the study of the interactions of dorsal and ventral determinants in varying spatial arrangements. We have studied pattern formation in dorsal half-blastoderms that contain the entire shield region but only a reduced ventrolateral marginal zone. As assessed by the presence of the most ventral cell type, blood, ventral specification within a dorsal half-blastoderm is not suppressed.

Animals↗
PubMed2006

Loss of selenoprotein N function causes disruption of muscle architecture in the zebrafish embryo.

Mutations in the gene coding for selenoprotein N (SelN), a selenium containing protein of unknown function, cause different forms of congenital muscular dystrophy in humans. These muscular diseases are characterized by early onset of hypotonia which predominantly affect in axial muscles. We used zebrafish as a model system to understand the function of SelN in muscle formation during embryogenesis. Zebrafish SelN is highly homologous to its human counterpart and amino acids corresponding to the mutated positions in human muscle diseases are conserved in the zebrafish protein. The sepn1 gene is highly expressed in the somites and notochord during early development. Inhibition of the sepn1 gene by injection of antisense morpholinos does not alter the fate of the muscular tissue, but causes muscle architecture disorganization and greatly reduced motility. Ultrastructural analysis of the myotomes reveals defects in muscle sarcomeric organization and in myofibers attachment, as well as altered myoseptum integrity. These studies demonstrate the important role of SelN for muscle organization during early development. Moreover, alteration of myofibrils architecture and tendon-like structure in embryo deficient for SelN function provide new insights into the pathological mechanism of SelN-related myopathy.

Amino Acid Sequence↗
PubMed2001

Direct DNA delivery into zebrafish embryos employing tissue culture techniques.

The production of transfected fish embryos requires expertise in injecting the fertilized eggs and/or expensive equipment for electroporation or microprojectiles. This article demonstrates that by exposure to DNA constructs conjugated with transfecting reagents dechorionated Danio rerio embryos are capable of acquiring extracellular DNA and expressing reporter genes. Embryos incubated with pCMVluc complexed with GeneJammer or GenePORTER expressed luciferase 24-48 h after exposure. pCMVGFP DNA mixed with the same agents generated embryos that exhibited differential patterns of expression of green fluorescent protein (GFP). Embryonic development varied depending on the procedure employed and the reporter gene utilized. Expression of the luciferase gene did not interfere with the subsequent development of the embryos. In contrast, the embryos expressing a high level of GFP were affected, probably due to a very active promoter. These results demonstrate the ease of obtaining transfected fish embryos, which facilitate the mass production of new genotypes and extend the procedure to laboratories with limited resources.

Animals↗
PubMed2006

The cytosolic loop of the gamma-secretase component presenilin enhancer 2 protects zebrafish embryos from apoptosis.

The gamma-secretase complex, composed of presenilin, presenilin enhancer 2 (Pen-2), nicastrin, and Aph-1, catalyzes the final cleavage of amyloid precursor protein to generate the toxic amyloid beta protein, the major component of plaques in the brains of Alzheimer disease patients. To understand the in vivo function of Pen-2, we used morphant technology available in zebrafish and transiently knocked down the expression of endogenous Pen-2 by injecting the morpholino (MO) against Pen-2. Two truncated Pen-2 proteins lacking either the cytosolic or the C-terminal domain were expressed in MO-injected embryos. This deletion analysis demonstrated that the Pen-2 cytosolic loop is essential for protecting developing embryos from caspase-dependent apoptosis caused by the reduction of Pen-2. Twelve amino acids in the C terminus of Pen-2 were dispensable and could not rescue the Pen-2 knockdown-induced apoptotic phenotype. Surprisingly, double knockdown of Pen-2 and nuclear factor kappaB component p65 abrogated the single Pen-2 MO-induced caspase activation, indicating that a previously reported pro-apoptotic role of NF-kappaB in some cell types could be manifested in a whole animal and that knockdown of Pen-2 may trigger pro-apoptotic activation of NF-kappaB.

Amyloid Precursor Protein Secretases↗
PubMed2006

Germ cell-less expression in zebrafish embryos.

The gene of germ cell-less (gcl) has been shown to be important in early differentiation of germ cells in Drosophila. Although the gcl homologue genes have been identified in some organisms, there is little data on the expression pattern and functional analysis of the gcl gene in zebrafish. In this research, real-time quantitative reverse transcription-polymerase chain reaction showed that the level of gcl mRNA expression rapidly decreases from the 4-cell stage to the sphere stage at which it reaches a minimum, gradually increases from the 50%-epiboly stage, and then remains stable during the posterior stages. Results of in situ hybridization indicated that the transcripts of zebrafish gcl are evenly distributed in all blastomeres from the 2-cell stage to the blastula period, different from that of vasa, nonas1 and dead end mRNA, and condense into some clusters of cells located along the blastoderm margin from the gastrulation period. During subsequent development, the transcripts are segregated as subcellular clumps to a small number of cells that would migrate to the position of the gonad in the dorsal side. In the adult, gcl mRNA was widely expressed in developing germ cells of both ovary and testis. These data suggest that zebrafish gcl have potentially important roles in the formation of primordial germ cells.

Animals↗
PubMed1997

Expression of genes encoding the collagen-binding heat shock protein (Hsp47) and type II collagen in developing zebrafish embryos.

Hsp47 is a heat-shock protein which interacts with newly synthesize procollagen chains in the endoplasmic reticulum (ER) of collagen-secreting cells and is thought to assist in procollagen triple helix assembly and subsequent transport to the cis-Golgi. This is supported by studies which have reported that genes encoding collagen and Hsp47 are subject to co-ordinate increases and decreases in expression in cultured cells. However, limited information is available regarding hsp47 expression in vivo, particularly during early embryonic development when a variety of collagen genes are expressed. Here we show that the zebrafish hsp47 gene is expressed in a dynamic spatiotemporal pattern in developing embryos. Strong expression of hsp47 mRNA is co-incident predominantly with expression of the type II collagen gene (col2a1) in a number of chondrogenic and non-chondrogenic tissues including the notochord, otic vesicle and developing fins. Notochordal expression of both genes is disrupted in floating head (flh) and no tail (ntl) embryos, which lack properly differentiated notochords. Surprisingly, no hsp47 mRNA is detectable in the strongly col2a1-expressing cells of the floor plate and hypochord, indicating that the two genes are not strictly co-regulated. Finally, Northern blot analysis revealed two alternative transcripts of col2a1 which are expressed in distinct temporal patterns. Appearance of the larger transcript occurs following somitogenesis, a time which coincides with the co-activation of hsp47 and col2a1 gene expression in tissues outside of the notochord.

Age Factors↗
PubMed2006

Tob1 controls dorsal development of zebrafish embryos by antagonizing maternal beta-catenin transcriptional activity.

Maternal beta-catenin and Nodal signals are essential for the formation of the dorsal organizer, which, in turn, induces neural and other dorsal tissue development in vertebrate embryos. Tob (Transducer of ErbB2) proteins possess antiproliferative properties and are known to influence BMP signaling, but their relationship to other signaling pathways and to embryonic patterning in general was unclear. In this study, we demonstrate that zebrafish tob1a is required for correct dorsoventral patterning. Mechanistically, Tob1a inhibits beta-catenin transcriptional activity by physically associating with beta-catenin and preventing the formation of beta-catenin/LEF1 complexes. Although Tob1a can also inhibit the transcriptional activity of the Nodal effector Smad3, its role in limiting dorsal development is executed primarily by antagonizing the beta-catenin signal. We further demonstrate that Tob family members across species share similar biochemical properties and biological activities.

Animals↗
PubMed2002

Cyclin-dependent kinase 8 is expressed both maternally and zygotically during zebrafish embryo development.

Cyclin-dependent kinase 8 (cdk8) regulates transcription by phosphorylating RNA polymerase II and TFIIH. The mechanism of zygotic transcription activation during vertebrate embryonic development is poorly understood. Here we describe the cloning and developmental expression pattern of zebrafish cdk8 mRNA. It is highly conserved, sharing 79% DNA and 95% amino acid sequence identity with human cdk8, thereby indicating an important role for the protein. Northern blotting and whole mount in situ hybridisation revealed expression of zebrafish cdk8 maternally, following the onset of zygotic transcription at the mid-blastula transition (MBT) and throughout embryonic development.

Amino Acid Sequence↗