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PubMed2004

Organization and expression analysis of the zebrafish hepcidin gene, an antimicrobial peptide gene conserved among vertebrates.

Hepcidin is an antimicrobial peptide and iron-regulatory molecule that is conserved among vertebrates. Mutations or over-expression of the human hepcidin gene have been found in patients with hemochromatosis and refractory anemia. To further understand the function and regulation of hepcidin, animal models are needed. We sequenced cDNA, genes and upstream regions of zebrafish hepcidin and analyzed gene expression by kinetic PCR. Zebrafish hepcidin genes consist of two introns and three exons that encode a prepropeptide (91 amino acids). The amino acid sequences and gene organization were remarkably conserved between zebrafish and other species. Elevated gene expression was observed in abdominal organs, skin, and heart in fish that developed signs of infection following bacterial injection. Zebrafish may be a suitable model organism for further study of hepcidin gene regulation.

Amino Acid Sequence↗
PubMed2005

Young thrombocytes initiate the formation of arterial thrombi in zebrafish.

The zebrafish system is an excellent vertebrate genetic model to study hemostasis and thrombosis because saturation mutagenesis screens can identify novel genes that play a role in this vital physiologic pathway. To study hemostatic mutations, it is important to understand the physiology of zebrafish hemostasis and thrombosis. Previously, we identified zebrafish thrombocytes and have shown that they participate in arterial thrombus formation. Here, we recognized 2 populations of thrombocytes distinguishable by DiI-C18 (DiI) staining. DiI+ thrombocytes have a high density of adhesive receptors and are functionally more active than DiI- thrombocytes. We classified DiI+ thrombocytes as young and DiI- thrombocytes as mature thrombocytes. We found young and mature thrombocytes each formed independent clusters and that young thrombocytes clustered first. We have also shown that young thrombocytes initiate arterial thrombus formation. We propose that due to the increased adhesive receptor density on young thrombocytes, they adhere first to the subendothelial matrix, get activated rapidly, release agonists, and recruit more young thrombocytes, which further release more agonists. This increase in agonists activates the less active mature thrombocytes, drawing them to the growing thrombus. Since arterial thrombus formation is a fundamental hemostatic event, this mechanism may be conserved in mammals and may open new avenues for prevention of arterial thrombosis.

Animals↗
PubMed2005

Assembly of trigeminal sensory ganglia by chemokine signaling.

Sensory neurons with related functions form ganglia, but how these precisely positioned clusters are assembled has been unclear. Here, we use the zebrafish trigeminal sensory ganglion as a model to address this question. We find that some trigeminal sensory neurons are born at the position where the ganglion is assembled, whereas others are born at a distance and have to migrate against opposing morphogenetic movements to reach the site of ganglion assembly. Loss of Cxcr4b-mediated chemokine signaling results in the formation of mispositioned ganglia. Conversely, ectopic sources of the chemokine SDF1a can attract sensory neurons. Transplantation experiments reveal that neuron-neuron interaction and the adhesion molecules E- and N-Cadherin also contribute to ganglion assembly. These results indicate that ganglion formation depends on the interplay of birthplace, chemokine attraction, cell-cell interaction, and cadherin-mediated adhesion.

Animals↗
PubMed2003

Cloning and characterization of zebrafish tbx1.

Tbx1 is one of the genes within the DiGeorge Critical Region (DGCR) and has been recently identified as the critical gene for the cardiovascular anomalies in the DiGeorge mouse models. We have cloned, sequenced and analyzed the zebrafish (Danio rerio) tbx1 cDNA. It encodes a protein of 460 amino acids that shares 64% identity and 67% similarity with the human TBX1 orthologue at the amino acid level. Although maternal expression was detected by RT-PCR, only zygotic expression could be detected by whole-mount in situ hybridization. Expression of zebrafish tbx1 by whole-mount in situ hybridization was first detected at 40% epiboly, 5.0 hours post fertilization (hpf) in the dorsal blastoderm margin. Through the stage of embryonic shield formation, tbx1 expression is restricted to the hypoblast, in the region of cells fated to become head and lateral plate mesoderm and pharyngeal endoderm. At 18 hpf, when the heart tube is beginning to assemble, three domains of tbx1 expression can be seen: cardiac precursors, pharyngeal arch precursors and otic vesicle. These three domains will remain the sites of tbx1 expression to varying degrees through at least 72 hpf. By 51 hpf, tbx1 expression can be seen in the cardiac outflow tract, the ventricle and the atrium, although by 72 hpf cardiac expression is strongest in the cardiac outflow tract. This newly identified tbx1 expression pattern in cardiac regions other than the cardiac outflow tract offers a new insight into the role of the tbx1 transcription factor in cardiac development.

Amino Acid Sequence↗
PubMed2004

New models for the study of Mycobacterium-host interactions.

The outcome of Mycobacterium infection is determined by a series of complex interactions between the bacteria and host immunity. Traditionally, mammalian models and cultured cells have been used to study these interactions. Recently, ameba (Dictyostelium), fruit flies (Drosophila) and zebrafish, amenable to forward genetic screens, have been developed as models for mycobacterial pathogenesis. Infection of these hosts with mycobacteria has allowed the dissection of intracellular trafficking pathways (Dictyostelium) and the roles of phagocytic versus antimicrobial peptide responses (Drosophila). Real-time visualization of the optically transparent zebrafish embryo/larva has elucidated mechanisms by which Mycobacterium-infected leukocytes migrate and subsequently aggregate into granulomas, the hallmark pathological structures of tuberculosis.

Animals↗
PubMed2005

Pigment cell distributions in different tissues of the zebrafish, with special reference to the striped pigment pattern.

The orderly pigment pattern of zebrafish (Danio rerio) is a good model system for studying how spatial patterns form in animals. Recent molecular genetic studies have shown that interactions between the pigment cells play major roles in pattern formation. In the present study, we performed comparative transmission electron microscopy of pigment cells, in order to clarify the structural interactions of pigment cells in tissues with and without a striped pattern. In patterned tissues, pigment cells were distributed as a one-cell-thick sheet. The layer order of the sheets is always kept strictly. In tissues without a striped pattern, the layer order was often disturbed or the cells were distributed in a scattered, double-sheeted, or an accumulated pile. Our observations suggest that the underlying mechanism that controls the vertical order of the pigment cells is related to that controlling the stripe pattern.

Animals↗
PubMed2006

The role of heparan sulphate proteoglycans in angiogenesis.

The presence of HS (heparan sulphate) proteoglycans on the cell surface and in the extracellular environment is critical to many physiological processes including the growth of new blood vessels from pre-existing vasculature (angiogenesis). A plethora of growth factors and their receptors, extracellular matrix molecules and enzymes bind to specific sites on the HS sugar chain. For example, HS proteoglycans have profound effects on the bioactivity of the key angiogenic factor VEGF (vascular endothelial growth factor) (VEGF(165)), affecting its diffusion, half-life and interaction with its tyrosine kinase receptors. A number of HS structural features that mediate the specific binding of VEGF(165), including sulphation requirements, have been determined. In parallel, zebrafish embryos were used as a vertebrate model system to study the role in vascular development of the biosynthetic enzymes that create these specific binding sites on HS. It was discovered that knockdown of one of the HS 6-O-sulphotransferases in zebrafish with morpholino antisense oligonucleotides reduced vascular branching and corresponded to changes in the HS structure. The roles of the extracellular 6-O-sulphatase enzymes, the sulfs, in vascular development are now being investigated. Both oligosaccharides and small molecule biosynthetic enzyme inhibitors could be valuable HS-based strategies for controlling aberrant angiogenesis in diseases as diverse as cancer and heart disease.

Animals↗
PubMed2006

Caudal neurosecretory system of the zebrafish: ultrastructural organization and immunocytochemical detection of urotensins.

The caudal neurosecretory system is described here for the first time in the zebrafish, one of the most important models used to study biological processes. Light- and electron-microscopical approaches have been employed to describe the structural organization of Dahlgren cells and the urophysis, together with the immunohistochemical localization of urotensin I and II (UI and UII) peptides. Two latero-ventral bands of neuronal perikarya in the caudal spinal cord project axons to the urophysis. The largest secretory neurons (approximately 20 microm) are located rostrally. UII-immunoreactive perikarya are much more numerous than those immunoreactive for UI. A few neurons are immunopositive for both peptides. Axons contain 75-nm to 180-nm dense-core vesicles comprising two populations distributed in two axonal types (A and B). Large dense vesicles predominate in type A axons and smaller ones in type B. Immunogold double-labelling has revealed that some fibres contain both UI and UII, sometimes even within the same neurosecretory granule. UII is apparently the major peptide present and predominates in type A axons, with UI predominating in type B. A surprising finding, not previously reported in other fish, is the presence of dense-core vesicles, similar to those in neurons, in astrocytes including their end-feet around capillaries. Secretory type vesicles are also evident in ependymocytes and cerebrospinal-fluid-contacting neurons in the terminal spinal cord. Thus, in addition to the urophysis, this region may possess further secretory systems whose products and associated targets remain to be established. These results provide the basis for further experimental, genetic and developmental studies of the urophysial system in the zebrafish.

Animals↗
PubMed2005

Transcriptome analysis of zebrafish embryogenesis using microarrays.

Zebrafish (Danio rerio) is a well-recognized model for the study of vertebrate developmental genetics, yet at the same time little is known about the transcriptional events that underlie zebrafish embryogenesis. Here we have employed microarray analysis to study the temporal activity of developmentally regulated genes during zebrafish embryogenesis. Transcriptome analysis at 12 different embryonic time points covering five different developmental stages (maternal, blastula, gastrula, segmentation, and pharyngula) revealed a highly dynamic transcriptional profile. Hierarchical clustering, stage-specific clustering, and algorithms to detect onset and peak of gene expression revealed clearly demarcated transcript clusters with maximum gene activity at distinct developmental stages as well as co-regulated expression of gene groups involved in dedicated functions such as organogenesis. Our study also revealed a previously unidentified cohort of genes that are transcribed prior to the mid-blastula transition, a time point earlier than when the zygotic genome was traditionally thought to become active. Here we provide, for the first time to our knowledge, a comprehensive list of developmentally regulated zebrafish genes and their expression profiles during embryogenesis, including novel information on the temporal expression of several thousand previously uncharacterized genes. The expression data generated from this study are accessible to all interested scientists from our institute resource database (http://giscompute.gis.a-star.edu.sg/~govind/zebrafish/data_download.html).

Journal Article↗
PubMed2026

A conserved Notch-Meis1-Pbx cascade specifies secretory progenitors into spatially diverse intestinal best4 + cells.

best4 + cells are a recently described vertebrate intestinal epithelial cell type. best4 + cells are altered in inflammatory bowel disease and colorectal cancer, suggesting that stimulation of their homeostatic replenishment may have therapeutic potential. However, the development and function of best4 + cells remain unclear. Since mice lack best4 + cells, we established zebrafish as a tractable in vivo model to observe, manipulate, and remove best4 + cells in an organismal context. We dissected best4 + cell developmental regulation in vivo from birth to differentiation and specialization, focusing on factors conserved in best4 + cells across vertebrates. Lineage tracing demonstrated that best4 + cells arise from secretory progenitors, where Notch/Dll4 signaling mediates a decision between best4 + and enterochromaffin cells by triggering meis1b expression. Following specification by meis1b, pbx3a spatially diversifies best4 + cells, which develop regional heterogeneity in gene expression, intracellular pH, and function. In vivo live imaging and removal of best4 + cells showed that best4+ cells sense luminal pH changes and extend dynamic luminal and stromal projections, but are not required to restore global luminal pH after challenge. Altogether, this study experimentally delineates best4 + cell developmental regulation and develops a genetic toolkit to examine their function in vivo, both of which will aid investigating how best4 + cells are altered or can be restored during disease.

Animals↗
PubMed2004

Activation of apoptosis and caspase-3 in zebrafish early gastrulae.

Nonmammalian vertebrate embryos do not manifest apoptosis before gastrulation, and it has been suggested that their cells are inhibited from undergoing apoptosis. To study this interesting possibility, the zebrafish (Danio rerio) embryo is an excellent model. However, the appearance of apoptosis varies among species, and many components of cell death are not highly conserved. To undertake the larger investigation, we first need to document by several criteria that cell death in the zebrafish embryo is apoptotic. Exposure of gastrulating germ-ring stage embryos to cycloheximide or staurosporine elicits an arrest in development and cell death within 8 hr. Caspase-3 activity increases, followed by translocation of phosphatidylserine, loss of cell-cell adhesion, cleavage of poly (ADP-ribose) polymerase, terminal deoxynucleotidyl transferase-mediated dNTP-fluorescein nick end labeling (TUNEL)-positive nuclei, internucleosomal DNA fragmentation, chromatin condensation and margination, and blebbing of the nuclear membrane. Thus, by many criteria, cell death in zebrafish is apoptotic; many of the markers of apoptosis found in mammals are conserved in zebrafish; and post-midblastula transition embryos have the capacity to activate a caspase-dependent apoptotic response well before naturally occurring programmed cell death is seen.

Adenosine Diphosphate Ribose↗
PubMed1994

cDNA sequence of zebrafish (Brachydanio rerio) translation elongation factor-1 alpha: molecular phylogeny of eukaryotes based on elongation factor-1 alpha protein sequences.

We have isolated and determined the nucleotide sequence of a cDNA clone containing the complete coding region for elongation factor-1 alpha (EF-1 alpha) from an embryonic zebrafish cDNA library. A secondary structure model based on all known EF-1 alpha and EF-Tu protein sequences is presented and the presence of conserved putative protein kinase C phosphorylation sites in loop regions of eukaryotic EF-1 alpha is demonstrated. Using distance matrix and maximum parsimony methods we constructed multi-kingdom phylogenetic trees containing 22 different eukaryotic sequences. Strikingly, both tree constructions show Fungi to be the closest relative of Animalia among eukaryotic kingdoms. A 12 amino acid stretch present in all animal and fungal sequences known to date was found to be absent from all plant, protist an archaebacterial EF-1 alpha sequences suggesting that this sequence was inserted following the separation of plants from the lineage leading to fungi and animals. In contrast to our results, molecular phylogenies based on small subunit ribosomal RNA sequences as well as other protein sequences have failed to yield consistent results regarding the branching order among the kingdoms Plantae, Fungi and Animalia. The slow evolutionary rate and universal occurrence of EF-1 alpha (EF-Tu in eubacteria) makes this protein a particularly interesting tool for probing distant evolutionary relationships.

Animals↗
PubMed2026

The maternal-to-zygotic transition is a critical window for PFOA-induced disruption of developmental programming.

Early embryogenesis is governed by precisely timed gene regulatory programs that coordinate cell fate specification, tissue patterning, and morphogenesis. The maternal-to-zygotic transition (MZT) represents a pivotal developmental milestone during which regulatory control shifts from maternally deposited transcripts to activation of the zygotic genome. Disruption of this transition has the potential to alter developmental trajectories with lasting consequences. Per- and polyfluoroalkyl substances (PFAS), environmentally persistent contaminants, have been linked to developmental abnormalities, yet their impact on core embryonic gene regulatory networks especially with exposure during MZT is not well understood. Using zebrafish (Danio rerio), a tractable vertebrate model and New Approach Methodology (NAM), we investigated how PFAS exposure during the MZT alters early developmental programming. Embryos were exposed starting at different times before and within the MZT time window and collected at 24 h post-fertilization (hpf) for transcriptomic analysis. Targeted qRT-PCR revealed dysregulation of genes controlling transcriptional activation, lineage specification, proliferation, and differentiation. Whole-transcriptome RNA sequencing (RNA-seq) further identified widespread perturbations in gene networks governing transcriptional regulation, cell signaling, and embryonic morphogenesis. Temporal analysis revealed that exposure beginning at 3.5 hpf, followed by 8 hpf, corresponding to early zygotic genome activation and near completion of zygotic activation, respectively, resulted in the greatest differential gene expression changes at 24 hpf. Consistent with these early gene regulatory perturbations, larvae exposed starting at 8 hpf also exhibited altered behavior at 5 days post-fertilization. Together, these findings demonstrate that PFAS exposure during MZT disrupts the establishment of embryonic gene regulatory networks, linking environmental toxicant exposure to altered developmental patterning and organismal outcomes. This work underscores the vulnerability of early developmental transitions to environmental perturbation and positions MZT as a critical window of susceptibility during development.

NAMs (new approach methodologies)↗
PubMed2002

Dealing with saturation at the amino acid level: a case study based on anciently duplicated zebrafish genes.

The ray-finned fishes (Actinopterygii) seem to have two copies of many tetrapod (Sarcopterygii) genes. The origin of these duplicate fish genes is the subject of some controversy. One explanation for the existence of these extra fish genes could be an increase in the rate of independent gene duplications in fishes. Alternatively, gene duplicates in fish may have been formed in the ancestor of all or most Actinopterygii during a complete genome duplication event. A third possibility is that tetrapods have lost more genes than fish after gene or genome duplication events in the common ancestor of both lineages. These three hypotheses can be tested by phylogenetic reconstruction. Previously, we found that a large number of anciently duplicated genes of zebrafish are sister sequences in evolutionary trees suggesting that they were produced in Actinopterygii after the divergence of Sarcopterygii [Phil. Trans. R. Soc. Lond. B 356 (2001) 119]. On the other hand, several well-supported trees showed one of the two fish genes as the sister sequence to a monophyletic clade that included the second fish gene and genes from frog, chicken, mouse and human. These so-called outgroup topologies suggest that the origin of many fish duplicates predates the divergence of the Sarcopterygii and Actinopterygii and support the hypothesis that tetrapods have lost duplicates that have been retained in fish. Here we show that many of these 'outgroup' tree topologies are erroneous and can be corrected when mutational saturation is taken into account. To this end, a Java-based application has been developed to visualize the amount of saturation in amino acid sequences. The program graphically displays the number of observed frequent and rare amino acid replacements between pairs of sequences against their overall evolutionary distance. Discrimination between frequent and rare amino acid replacements is based on substitution probability matrices (e.g. PAM and BLOSUM). Evolutionary distances between sequences can be computed from the fraction of unsaturated sites only and evolutionary trees inferred by pairwise distance methods. When trees are computed by omitting the saturated fraction of sites, most fish duplicates are sister sequences.

Amino Acids↗
PubMed2006

Inhibition of BMP signaling during zebrafish fin regeneration disrupts fin growth and scleroblasts differentiation and function.

The zebrafish caudal fin provides a simple model to study molecular mechanisms of dermal bone regeneration. We previously showed that misexpression of Bone morphogenetic protein 2b (Bmp2b) induces ectopic bone formation within the regenerate. Here we show that in addition to bmp2b and bmp4 another family member, bmp6, is involved in fin regeneration. We further investigated the function of BMP signaling by ectopically expressing the BMP signaling inhibitor Chordin which caused: (1) inhibition of regenerate outgrowth due to a decrease of blastema cell proliferation and downregulation of msxb and msxC expression and (2) reduced bone matrix deposition resulting from a defect in the maturation and function of bone-secreting cells. We then identified targets of BMP signaling involved in regeneration of the bone of the fin rays. runx2a/b and their target col10a1 were downregulated following BMP signaling inhibition. Unexpectedly, the sox9a/b transcription factors responsible for chondrocyte differentiation were detected in the non-cartilaginous fin rays, sox9a and sox9b were not only differentially expressed but also differentially regulated since sox9a, but not sox9b, was downregulated in the absence of BMP signaling. Finally, this analysis revealed the surprising finding of the expression, in the fin regenerate, of several factors which are normally the signatures of chondrogenic elements during endochondral bone formation although fin rays form through dermal ossification, without a cartilage intermediate.

Animals↗
PubMed2002

The Hox Paradox: More complex(es) than imagined.

An understanding of the origin of different body plans requires knowledge of how the genes and genetic pathways that control embryonic development have evolved. The Hox genes provide an appealing starting point for such studies because they play a well-understood causal role in the regionalization of the body plan of all bilaterally symmetric animals. Vertebrate evolution has been characterized by gene, and possibly genome, duplication events, which are believed to have provided raw genetic material for selection to act upon. It has recently been established that the Hox gene organization of ray-finned fishes, such as the zebrafish, differs dramatically from that of their lobe-finned relatives, a group that includes humans and all the other widely used vertebrate model systems. This unusual Hox gene organization of zebrafish is the result of a duplication event within the ray-finned fish lineage. Thus, teleosts, such as zebrafish, have more Hox genes arrayed over more clusters (or "complexes") than do tetrapod vertebrates. Here, I review our understanding of Hox cluster architecture in different vertebrates and consider the implications of gene duplication for Hox gene regulation and function and the evolution of different body plans.

Animals↗
PubMed2004

Ecotoxicological evaluation of 4-aminobiphenyl using a test battery.

Experimental bioassays are currently used in ecotoxicology and environmental toxicology to provide information for risk assessment evaluation of new chemicals and to investigate their effects and mechanisms of action; in addition, ecotoxicological models are used for the detection, control, and monitoring of the presence of pollutants in the environment. As a single bioassay will never provide a full picture of the quality of the environment, a representative, cost-effective, and quantitative test battery should be developed. In this study, the effects of 4-aminobiphenyl (4-ABP) were investigated using a battery of ecotoxicological model systems, including immobilization of Daphnia magna, development of zebrafish embryos, and inhibition of mammalian cell proliferation. The growth inhibition effects of 4-ABP were assessed on mouse connective tissue fibroblast cells (L929 cells) and human hepatocelluar carcinoma cells (HepG2 cells) by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-trazolium bromide reduction assay. The results reveal that 4-aminobiphenyl is toxic for aquatic organisms and mammalian cells. The system most sensitive to 4-aminobiphenyl is D. magna immobilization, followed by development of zebrafish embryos, and inhibition of cell proliferation. L929 and HepG2 cell growth inhibition bioassays show low sensitivity. These findings indicate that a single model for the possible harmful effect of 4-ABP has its limitations; only a test battery, composed of bioassays on different species, can provide an accurate assessment of the action of 4-ABP in the whole environment.

Aminobiphenyl Compounds↗
PubMed2006

Antipsychotics produce locomotor impairment in larval zebrafish.

Zebrafish has been a favored vertebrate genetic model organism for studying developmental processes. It also holds a great potential for understanding the genetic basis of behavior and associated behavioral disorders. Despite such potential, their use in the study of behavior is greatly under-explored. It is well known that multiple classes of drugs used to treat psychiatric diseases produce extrapyramidal side (EPS) effects and consequent movement disorders in humans. The underlying molecular causes of these drug-induced movement disorders are poorly understood. Here we report that zebrafish treated with the antipsychotics fluphenazine and haloperidol (both of which can induce severe EPS in humans) develop movement defects. In contrast, another antipsychotic olanzapine, which produces mild to little EPS in humans, leads to minimal movement defects in zebrafish. These results establish a rapid assay system in which the effects of EPS-inducing agents can be assessed. Thus, future genetic screening in zebrafish shall identify genes and pathways that elucidate drug-induced movement disorder in human as well as provide insights into the brain control of locomotor activity. Future chemical screening in zebrafish may act as a preclinical test for the EPS effect of certain drugs, as well as a test used to researching drugs made to counteract the effects of EPS.

Animals↗