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PubMed2006

Chemical modulation of receptor signaling inhibits regenerative angiogenesis in adult zebrafish.

We examined the role of angiogenesis and the need for receptor signaling using chemical inhibition of the vascular endothelial growth factor receptor in the adult zebrafish tail fin. Using a small-molecule inhibitor, we were able to exert precise control over blood vessel regeneration. An angiogenic limit to tissue regeneration was determined, as avascular tissue containing skin, pigment, neuronal axons and bone precursors could regenerate up to about 1 mm. This indicates that tissues can regenerate without direct interaction with endothelial cells and at a distance from blood supply. We also investigated whether the effects of chemical inhibition could be enhanced in zebrafish vascular mutants. We found that adult zebrafish, heterozygous for a mutation in the critical receptor effector phospholipase Cgamma1, show a greater sensitivity to chemical inhibition. This study illustrates the utility of the adult zebrafish as a new model system for receptor signaling and chemical biology.

Angiogenesis Inhibitors↗
PubMed2003

Recent advances in the study of fatty acid desaturases from animals and lower eukaryotes.

The biosynthesis of polyunsaturated fatty acids (PUFAs) in different organisms can involve a variety of pathways, catalyzed by a complex series of desaturation and elongation steps. A range of different desaturases have been identified to date, capable of introducing double bonds at various locations on the fatty acyl chain. Some recently identified novel desaturases include a delta4 desaturase from marine fungi, and a bi-functional delta5/delta6 desaturase from zebrafish. Using molecular genetics approaches, these desaturase genes have been isolated, identified, and expressed in variety of heterologous hosts. Results from these studies will help increase our understanding of the biochemistry of desaturases and the regulation of PUFA biosynthesis. This is of significance because PUFAs play critical roles in multiple aspects of membrane physiology and signaling mechanisms which impact human health and development.

Animals↗
PubMed2026

Discovery of NAT-6-321056 as a novel modulator of VEGFR2 signaling to suppress tumor angiogenesis.

Vascular endothelial growth factor receptor 2 (VEGFR2) is a master regulator of angiogenesis and cancer progression. However, current VEGFR2 modulators face significant challenges, including off-target toxicity and acquired resistance, underscoring the urgent need for novel therapeutic agents with improved efficacy and safety profiles. Here, we reported that virtual screening of 39,442 natural products from the ZINC natural products-derived library, coupled with molecular docking and molecular dynamics (MD) simulations to evaluate the binding stability of candidate compounds, identified NAT-6-321056 as a highly promising modulator of VEGFR2 signaling. Biological evaluations demonstrated that NAT-6-321056 exerted potent inhibition on the growth of a broad spectrum of cancer cells, including both solid tumors and hematological malignancies. In EA.hy 926 endothelial cells and SK-N-DZ neuroblast cells, the compound significantly suppressed proliferation, migration, and invasion. Microscale thermophoresis (MST) confirmed direct binding of NAT-6-321056 to VEGFR2 with favorable affinity. Kinase profiling against a panel of 33 kinases indicated that NAT-6-321056 exhibited a multi-kinase modulation profile. Mechanistic studies revealed that NAT-6-321056 suppressed the expression of hypoxia-inducible factor 1-alpha (HIF-1α) and was associated with reduced VEGFR2 phosphorylation and attenuation of the downstream ERK/JNK/AKT signaling pathways. Moreover, NAT-6-321056 exhibited robust in vivo anti-angiogenic effects in both the chick chorioallantoic membrane (CAM) assay and transgenic zebrafish vascular fluorescence imaging models. Computational absorption, distribution, metabolism, excretion, and toxicity (ADMET) prediction suggested acceptable drug-like properties. Collectively, these findings demonstrated that NAT-6-321056 is a promising modulator of VEGFR2 signaling with potent anti-angiogenic activity and represents a viable candidate for cancer therapy.

Vascular Endothelial Growth Factor Receptor-2↗
PubMed2001

Zebrafish myelopoiesis and blood cell development.

The zebrafish (Danio rerio) animal model offers a unique opportunity to discover novel genes required for the control of normal vertebrate myeloid cell development. It is well suited for both developmental and genetic analyses: eg, genome-wide chemical mutagenesis screens have led to the identification of specific new genes affecting vertebrate erythropoiesis. Mutants defective in one or more hematopoietic functions will be useful as models of human disease and will assist in the elucidation of lineage-specific developmental programs. By using a combination of forward genetic mutagenesis screens and emerging strategies based on transgenic and antisense knockdown approaches, it should be possible to dissect the genetic programs that lead to myeloproliferative/myelodysplastic syndromes and to acute myeloid leukemia.

Animals↗
PubMed2000

Zebrafish comparative genomics and the origins of vertebrate chromosomes.

To help understand mechanisms of vertebrate genome evolution, we have compared zebrafish and tetrapod gene maps. It has been suggested that translocations are fixed more frequently than inversions in mammals. Gene maps showed that blocks of conserved syntenies between zebrafish and humans were large, but gene orders were frequently inverted and transposed. This shows that intrachromosomal rearrangements have been fixed more frequently than translocations. Duplicated chromosome segments suggest that a genome duplication occurred in ray-fin phylogeny, and comparative studies suggest that this event happened deep in the ancestry of teleost fish. Consideration of duplicate chromosome segments shows that at least 20% of duplicated gene pairs may be retained from this event. Despite genome duplication, zebrafish and humans have about the same number of chromosomes, and zebrafish chromosomes are mosaically orthologous to several human chromosomes. Is this because of an excess of chromosome fissions in the human lineage or an excess of chromosome fusions in the zebrafish lineage? Comparative analysis suggests that an excess of chromosome fissions in the tetrapod lineage may account for chromosome numbers and provides histories for several human chromosomes.

Animals↗
PubMed2003

Steps during the development of the zebrafish locomotor network.

This review summarizes recent data from our lab concerning the development of motor activities in the developing zebrafish. The zebrafish is a leading model for studies of vertebrate development because one can obtain a large number of transparent, externally and rapidly developing embryos with motor behaviors that are easy to assess (e.g. for mutagenic screens). The emergence of embryonic motility was studied behaviorally and at the cellular level. The embryonic behaviors appear sequentially and include an early, transient period of spontaneous, alternating tail coilings, followed by responses to touch, and swimming. Patch clamp recording in vivo revealed that an electrically coupled network of a subset of spinal neurons generates spontaneous tail coiling, whereas a chemical (glutamatergic and glycinergic) synaptic drive underlies touch responses and swimming and requires input from the hindbrain. Swimming becomes sustained in larvae once serotonergic neuromodulatory effects are integrated. We end with a brief overview of the genetic tools available for the study of the molecular determinants implicated in locomotor network development in the zebrafish. Combining genetic, behavioral and cellular experimental approaches will advance our understanding of the general principles of locomotor network assembly and function.

Animals↗
PubMed2003

Loss of maternal Smad5 in zebrafish embryos affects patterning and morphogenesis of optic primordia.

The mechanisms of patterning and morphogenesis of vertebrate eye primordia are heavily debated. Taking advantage of the maternal effect of a zebrafish smad5 null mutation (Mm169), we investigate the effect of early signaling by members of the bone morphogenetic proteins (Bmps) on eye field patterning and optic vesicle morphogenesis. In contrast to previous Xenopus and chick studies demonstrating a late dorsalizing effect of Bmp4 expressed in the dorsal neural retina itself, we show that patterning of the eye primordia already starts during blastula and early gastrula stages. At these stages, bmps expressed on the ventrolateral side of the embryo promote dorso-distal fates in the entire neuroectoderm, including the eye primordia. Despite a normal split of the eye field in the midline, the eye primordia of Mm169 embryos fail to evaginate laterally. They display a concentric pattern with retinal cells in the center and optic stalk cells in the periphery, representing a flattened version of the topologic relationships present in the mature wild-type eye. Different interpretations of these latter findings are presented. They can be best explained with a model according to which zebrafish eye morphogenesis occurs as a telescopic extension of disc-like, concentric primordia, similar to the development of appendages from imaginal discs in Drosophila.

Animals↗
PubMed2002

cDNA cloning and expressions of cytochrome P450 1A in zebrafish embryos.

Cytochrome P450 1A (CYP1A) is well known for being induced by aromatic hydrocarbons, including 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD). We determined the complete cDNA sequence of a CYP1A open reading frame with both 5'- and 3'-ends in zebrafish (zfCYP1A), a useful model for environmental toxicology. zfCYP1A shows high percentage identity with CYP1As of mammals, domestic fowl and xenopus (51.9-60.4%), as well as the other fish species (63.8-89.2%). As revealed by in situ hybridization and immunohistochemistry, zfCYP1A was scarcely detected in control embryos but was markedly induced by TCDD especially in heart, vascular endothelial cells, intestinal epithelium, pronephros and outer integument in both prehatched and hatched embryos. These expression patterns are consistent with possible involvement of zfCYP1A in TCDD-induced toxicities.

Amino Acid Sequence↗
PubMed2003

Comparing protein stabilities during zebrafish embryogenesis.

The stabilities of many key proteins are regulated, e.g. via ubiquitination and proteasomal degradation, with important biological consequences. We present a convenient method that allows the analysis and comparison of protein stabilities during embryogenesis using early zebrafish development as a model system. Basically, this method involves ectopic overexpression of epitope-tagged proteins via mRNA injections in one-to-four-cell stage embryos and subsequent protein detection after various time points. Indeed, the protein stability of the ubiquitin ligase RLIM, which is able to autoubiquitinate and target itself for proteasomal degradation, was much shorter when compared to a protein consisting of a Myc epitope-tag and a nuclear localization domain. Thus, this method may be used more widely for the study of developmental protein stability.

Animals↗
PubMed2004

Zebrafish as a pharmacological tool: the how, why and when.

Zebrafish combine the relevance of a vertebrate with the scalability of an invertebrate. They can live in 96-well plate format and readily absorb chemicals from the water. These features have stimulated the use of zebrafish by medical researchers to model human disease and then assess the action of compounds in a whole organism. Examples of the power of this system have been illustrated with the cloning of zebrafish human ether-a-go-go-related gene (HERG), which shows near 100% homology in key domains, and the associated ability to identify drugs that prolong the QT interval both rapidly and with tiny amounts (micrograms) of compound.

Animals↗
PubMed2000

Identification and expression of alternatively spliced aryl hydrocarbon nuclear translocator 2 (ARNT2) cDNAs from zebrafish with distinct functions.

In order to further establish zebrafish as a vertebrate model for studying the mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity it is necessary to characterize the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator (AhR/ARNT) signaling pathways in this species. In this study, three zfARNT2 cDNAs were isolated, expressed, and characterized and named zfARNT2b, zfARNT2c, and zfARNT2a. zfARNT2b, zfARNT2c, and zfARNT2a encode proteins with theoretical molecular weights of 81, 79, and 45 kDa, respectively. zfARNT2b and zfARNT2a proteins are identical over the first 403 amino acids but differ in their C-terminal domains as a result of alternative mRNA splicing. zfARNT2c is nearly identical to zfARNT2b, with the exception of an in frame 15 amino acid deletion adjacent to the basic region of zfARNT2c. Using quantitative RT-PCR methods the tissue distribution of each zfARNT2 isoform was determined. In COS-7 cells expressing zfARNT2b and zfAhR2, 10 nM TCDD causes a nine-fold induction of a dioxin responsive reporter gene. In COS-7 cells expressing zfARNT2a or zfARNT2c, TCDD does not induce reporter gene expression. In contrast, all three zfARNT2 proteins induce reporter gene activity under control of hypoxia responsive elements when cotransfected with the zebrafish endothelial specific PAS protein 1. DNA gel shift analysis suggests that the decreased function of zfARNT2a is due to inefficient binding of zfARNT2a/zfAhR2 complexes to dioxin responsive elements. These results also indicate that alternative mRNA splicing results in formation of ARNT proteins with distinct functional properties.

Alternative Splicing↗
PubMed2006

Light reaches the very heart of the zebrafish clock.

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early-stage zebrafish embryos contain clocks that are directly light-responsive. We describe recent experiments using single-cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light-responsiveness of early-stage zebrafish embryos.

Animals↗
PubMed2002

Molecular dissection of craniofacial development using zebrafish.

The zebrafish, Danio rerio, is a small, freshwater teleost that only began to be used as a vertebrate genetic model by the late George Streisinger in the early 1980s. The strengths of the zebrafish complement genetic studies in mice and embryological studies in avians. Its advantages include high fecundity, externally fertilized eggs and transparent embryos that can be easily manipulated, inexpensive maintenance, and the fact that large-scale mutagenesis screens can be performed. Here we review studies that have used the zebrafish as a model for craniofacial development. Lineage studies in zebrafish have defined the origins of the cranial skeleton at the single-cell level and followed the morphogenetic behaviors of these cells in skeletal condensations. Furthermore, genes identified by random mutational screening have now revealed genetic pathways controlling patterning of the jaw and other pharyngeal arches, as well as the midline of the skull, that are conserved between fish and humans. We discuss the potential impact of specialized mutagenesis screens and the future applications of this versatile, vertebrate developmental model system in the molecular dissection of craniofacial development.

Animals↗
PubMed1996

Early ear development in the embryo of the zebrafish, Danio rerio.

The zebrafish provides an important model for vertebrate inner ear development. The otic placode becomes visible at approximately 16 hours (at 28.5 degrees C) and forms a vesicle with a lumen by cavitation at approximately 18 hours. Two otoliths appear in the lumen by 19.5 hours, and at about 24 hours the first sensory hair cells are seen, grouped in two small patches, one beneath each otolith, corresponding to future maculae. Staining with fluorescent phalloidin reveals 10-20 hair cells in each macula by 42 hours; between 3 days and 7 days the numbers grow to approximately 80 per macula. Neurons of the statoacoustic ganglion are first visible by staining with HNK-1 antibody at about 24 hours. Serial sections and time-lapse films show that the neuronal precursors originate by delamination from the ventral face of the otocyst; the peak period of delamination is from 22 hours to 30 hours. The system of semicircular canals forms between 42 hours and 72 hours by outgrowth of protrusions from the walls of the otocyst to form pillars of tissue spanning the lumen. Three further clusters of hair cells also become visible in this period, forming the three cristae. Thus, by the end of the first week, all key components of the ear are present. Subsequent growth produces thousands more hair cells; additional neurons probably derive from proliferation of neuronal precursors within the ganglion. Although the timetable is species-specific, the principles of inner ear development in the zebrafish seem to be the same as in other vertebrates.

Animals↗
PubMed1996

Magnetic resonance microscopy and spectroscopy reveal kinetics of cryoprotectant permeation in a multicompartmental biological system.

Successful cryopreservation of most multicompartmental biological systems has not been achieved. One prerequisite for success is quantitative information on cryoprotectant permeation into and amongst the compartments. This report describes direct measurements of cryoprotectant permeation into a multicompartmental system using chemical shift selective magnetic resonance (MR) microscopy and MR spectroscopy. We used the developing zebrafish embryo as a model for studying these complex systems because these embryos are composed of two membrane-limited compartments: (i) a large yolk (surrounded by the yolk syncytial layer) and (ii) differentiating blastoderm cells (each surrounded by a plasma membrane). MR images of the spatial distribution of three cryoprotectants (dimethyl sulfoxide, propylene glycol, and methanol) demonstrated that methanol permeated the entire embryo within 15 min. In contrast, the other cryoprotectants exhibited little or no permeation over 2.5 h. MR spectroscopy and microinjections of cryoprotectants into the yolk inferred that the yolk syncytial layer plays a critical role in limiting the permeation of some cryoprotectants throughout the embryo. This study demonstrates the power of MR technology combined with micromanipulation for elucidating key physiological factors in cryobiology.

Animals↗
PubMed1999

Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters.

Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole-mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker-mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2-kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5-kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8-kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters.

Animals↗
PubMed2002

High-efficiency gene transfer into adult fish: a new tool to study fin regeneration.

Zebrafish represents an excellent model to study the function of vertebrate genes (e.g., well-developed genetics, large number of mutants, and genomic sequencing in progress), inasmuch as we have tools to manipulate gene expression. Recent use of injected morpholinos in eggs provides a good method to " knockdown " gene expression in early development (Nasevicius and Ekker, 2000), and the "caged" RNA injected in eggs allows to overexpress a gene in a specific set of cells (Ando et al., 2001). However, a method to specifically modify gene expression in the juvenile or in the adult is still missing. Such a method would be a very powerful tool to understand gene function in differentiated tissues. We describe here an electroporation-based approach, which allows gene transfer in adult tissues. Its efficiency was assessed using a GFP (green fluorescent protein) dependent assay. We then used this method to disrupt the Fgf signalling pathway during the process of regeneration.

Animals↗
PubMed2006

Profiling of prostanoids in zebrafish embryonic development.

Prostanoids (PG) play important roles in vascular, pulmonary, reproductive and renal physiology. Little is known about their roles in the embryonic development. Using the oviparous zebrafish embryo as a model, we determined the temporal expression of PGs synthesized from exogenous prostaglandin H(2). Prostaglandin E(2) is the major PG throughout first 120 h post-fertilization (hpf), whereas prostaglandin F(2)(alpha) is at a lower but also a constant level. Reverse transcription-polymerase chain reaction (RT-PCR) showed that transcripts of cytosolic and membrane-bound PGE synthases were evident during the 120 hpf period. Compared with thromboxane A(2), the level of prostacyclin (PGI(2))is higher at first 24 hpf, the stage before the formation of blood vessel. RT-PCR showed that transcript of prostacyclin synthase appeared at 7 hpf whereas thromboxane synthase appeared at 48 hpf, suggesting that PGI(2) has additional functions besides hemostasis. Interestingly, level of prostaglandin D(2) (PGD(2)) followed an exponential decay over 120 hpf with a rate constant of 0.048 h(-1) and transcript of lipocalin-type PGD synthase was expressed at a higher level at early stage of development, suggesting that PGD(2) is highly regulated during embryogenesis.

Animals↗