Translate this page to:
In JoVE (1)
Other Publications (74)
- Molecular Plant-microbe Interactions : MPMI
- Carbohydrate Research
- Molecular Plant-microbe Interactions : MPMI
- Journal of Experimental Botany
- Nucleic Acids Research
- Plant Molecular Biology
- Molecular Immunology
- Biophysical Journal
- Development (Cambridge, England)
- Journal of Cell Science
- Trends in Microbiology
- Nucleic Acids Research
- Plant Molecular Biology
- Molecular Immunology
- Journal of Cell Science
- American Journal of Physiology. Regulatory, Integrative and Comparative Physiology
- Infection and Immunity
- Molecular Plant-microbe Interactions : MPMI
- Molecular Microbiology
- Gene Expression Patterns : GEP
- Biophysical Journal
- FEBS Letters
- Molecular Plant-microbe Interactions : MPMI
- Bio Systems
- BMC Genomics
- Journal of Cell Science
- Developmental Biology
- Comparative Biochemistry and Physiology. Toxicology & Pharmacology : CBP
- BMC Physiology
- Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry
- Journal of Biomolecular Screening
- Developmental and Comparative Immunology
- Journal of Immunology (Baltimore, Md. : 1950)
- Molecular Immunology
- The International Journal of Developmental Biology
- Molecular Immunology
- Genome Biology
- Biophysical Journal
- General and Comparative Endocrinology
- The International Journal of Developmental Biology
- BMC Research Notes
- Reproductive Biology and Endocrinology : RB&E
- BMC Research Notes
- Molecular Immunology
- PloS One
- BMC Genomics
- Current Drug Targets
- PloS One
- BMC Developmental Biology
- Integrative Biology : Quantitative Biosciences from Nano to Macro
- Birth Defects Research. Part C, Embryo Today : Reviews
- BMC Research Notes
- The Journal of Pathology
- Journal of Integrative Bioinformatics
- Methods in Cell Biology
- BMC Immunology
- Fish & Shellfish Immunology
- The Journal of Pathology
- PloS One
- The Journal of Pathology
- PloS One
This translation into Hindi was automatically generated.
English Version | Other Languages
Articles by Herman P. Spaink in JoVE
Zebrafish भ्रूण की Intracellular बैक्टीरियल रोगज़नक़ों के साथ संक्रमण
Erica L. Benard1, Astrid M. van der Sar2, Felix Ellett3, Graham J. Lieschke3, Herman P. Spaink1, Annemarie H. Meijer1
1Department of Molecular Cell Biology, Institute of Biology, Leiden University, 2Department of Medical Microbiology and Infection Control, VU University Medical Center, 3Australian Regenerative Medicine Institute, Monash University
पारदर्शी zebrafish भ्रूण उपयोगी मॉडल कल्पना करने के लिए मेजबान और इस तरह के रूप में सहज प्रतिरक्षा कोशिकाओं और intracellular बैक्टीरियल रोगज़नक़ों के बीच में कार्यात्मक अध्ययन बातचीत, साबित कर दिया है
Other articles by Herman P. Spaink on PubMed
Lotus Japonicus Gene Ljsbp is Highly Conserved Among Plants and Animals and Encodes a Homologue to the Mammalian Selenium-binding Proteins
Molecular Plant-microbe Interactions : MPMI. Apr, 2002 | Pubmed ID: 12026169
We have isolated and characterized a Lotus japonicus gene (Ljsbp) encoding a putative polypeptide with striking homology to the mammalian 56-kDa selenium-binding protein (SBP). cDNA clones homologous to LjSBP were also isolated from soybean, Medicago sativa, and Arabidopsis thaliana. Comparative expression studies in L japonicus and A. thaliana showed that sbp transcripts are present in various tissues and at different levels. Especially in L japonicus nodules and seedpods and A. thaliana siliques, sbp expression appears to be developmentally up-regulated. sbp Gene transcripts were localized by in situ hybridization in the infected cells and vascular bundles of young nodules, while in mature nodules, low levels of expression were only detected in the parenchymatous cells. Expression of sbp transcripts in young seedpods and siliques was clearly visible in vascular tissues and embryos, while in embryos, low levels of expression were detected in the root epidermis and the vascular bundles. Polyclonal antibodies raised against a truncated LjSBP recombinant protein recognized a polypeptide of about 60 kDa in nodule extracts. Immunohistochemical experiments showed that accumulation of LjSBP occurred in root hairs, in the root epidermis above the nodule primordium, in the phloem of the vasculature, and abundantly in the infected cells of young nodules. Irrespective of the presence of rhizobia, expression of SBP was also observed in root tips, where it was confined in the root epidermis and protophloem cells. We hypothesize that LjSBP may have more than one physiological role and can be implicated in controlling the oxidation/reduction status of target proteins, in vesicular Golgi transport, or both.
Nature. Jun, 2002 | Pubmed ID: 12087390
Carbohydrate Research. Jul, 2002 | Pubmed ID: 12110194
The novel lipochitin oligosaccharide (LCOs) structures produced by Rhizobium etli KIM5s were characterized using a nanoHPLC reverse-phase system coupled to an ion-trap mass spectrometer. This technique was shown to be more sensitive for structural elucidation of LCOs than previously used mass spectrometric methods. The structures of the LCOs of R. etli KIM5s, the majority containing six monosaccharide residues, differed from those synthesized by all other rhizobia analyzed to date. In addition, novel structures in which the chitin backbone was deacetylated at one or more GlcNAc moieties were found as minor compounds. The difference in host range of this strain compared to that of other known bean microsymbionts is discussed.
Genetic Analysis of a PH-regulated Operon from Rhizobium Tropici CIAT899 Involved in Acid Tolerance and Nodulation Competitiveness
Molecular Plant-microbe Interactions : MPMI. Feb, 2003 | Pubmed ID: 12575750
Rhizobium tropici CIAT899 is highly acid tolerant and a good competitor for Phaseolus vulgaris nodule occupancy at low pH values. Using Tn5 mutagenesis, we identified an operon required for acid tolerance and nodulation competitiveness. The insertion was mapped to the 5' end of atvA, encoding a product with high sequence identity to the agro-bacterial AcvB virulence protein. Complementation analyses indicated that atvA is an ortholog of acvB, both genes being required for acid tolerance. A Ser/Ala substitution in the LIPASE_SER motif of AtvA resulted in an acid sensitive Fix+ but very poorly competing strain, demonstrating that Ser-313 is essential for AtvA function. atvA is the second gene in an operon that is transcriptionally upregulated by acid shock. The acid-responsive promoter was mapped to a 469-bp intergenic region located upstream of lpiA, the first gene in the operon. lpiA-like genes are found in several alpha, beta, and gamma Proteobacteria that interact with eukaryotic host cells, and they are predicted to encode membrane proteins related to the FmtC/MprF family from low G+C Firmicutes. The latter proteins are involved in resistance to cationic antimicrobial peptides. A nonpolar deletion in lpiA caused a sevenfold decrease in relative nodulation competitiveness.
Journal of Experimental Botany. Mar, 2003 | Pubmed ID: 12598573
The members of the 14-3-3 isoform family have been shown to be developmentally regulated during animal embryogenesis, where they take part in cell differentiation processes. 14-3-3 isoform-specific expression patterns were studied in plant embryogenic processes, using barley (Hordeum vulgare L.) microspore embryogenesis as a model system. After embryogenesis induction by stress, microspores with enlarged morphology showed higher viability than non-enlarged ones. Following microspore culture, cell division was only observed among the enlarged microspores. Western blot and immunolocalization of three barley 14-3-3 isoforms, 14-3-3A, 14-3-3B and 14-3-3C were carried out using isoform-specific antibodies. The level of 14-3-3C protein was higher in enlarged microspores than in non-enlarged ones. A processed form of 14-3-3A was associated with the death pathway of the non-enlarged microspores. In the early embryogenesis stage, 14-3-3 subcellular localization differed among dividing and non-dividing microspores and the microspore-derived multicellular structures showed a polarized expression pattern of 14-3-3C and a higher 14-3-3A signal in epidermis primordia. In the late embryogenesis stage, 14-3-3C was specifically expressed underneath the L(1) layer of the shoot apical meristem and in the scutellum of embryo-like structures (ELSs). 14-3-3C was also expressed in the scutellum and underneath the L(1) layer of the shoot apical meristem of 21 d after pollination (DAP) zygotic embryos. These results reveal that 14-3-3A processing and 14-3-3C isoform tissue-specific expression are closely related to cell fate and initiation of specific cell type differentiation, providing a new insight into the study of 14-3-3 proteins in plant embryogenesis.
Glycobiology. Oct, 2003 | Pubmed ID: 12881410
Chitin oligosaccharides (COs) play a role in plant development and are presumed to affect body plan formation during vertebrate embryogenesis. The mechanisms of COs recognition and cellular processes underlying embryonic development are still not understood. We analyze the possible link with the mitogen-activated protein kinase pathway that is conserved in evolution through the plant and animal kingdom and has been implicated in diverse cellular processes, including cell growth, proliferation, differentiation, survival, and vertebrate development. We show that in vivo stimulation of embryonic zebrafish cells ZF13 and ZF29 with chitin tetrasaccharides at 10-9 M concentration transiently induced activation/phosphorylation of extracellular regulated kinases (ERKs), with a maximum after 15 min. Furthermore the biological specificity of chitin tetrasaccharides and various derivatives was examined. The replacement of one or two GlcNAc residues of the chitin backbone by glucose and fucosylation of chitin tetrasaccharides at the reducing terminus caused a complete loss of their activity. We also tested a chitin tetrasaccharide analogue in which the oxygen atoms in glycosidic linkages were replaced by sulfur atoms. This analog, which could not be enzymatically hydrolyzed, was as potent an inducer as chitin tetrasaccharide. These results suggest that the observed activation of ERKs is chitin tetrasaccharide-specific and does not require further enzymatic processing. We examined possible signaling pathways leading to ERK activation by COs by use of phosphospecific antibodies and inhibitors. We conclude that a high-affinity CO receptor system exists that links to the Raf, MEK, and ERK pathway in zebrafish cells.
Nucleic Acids Research. Sep, 2003 | Pubmed ID: 12930950
The plant gene enod40 is highly conserved among legumes and also present in various non-legume species. It is presumed to play a central regulatory role in the Rhizobium-legume interaction, being expressed well before the initiation of cortical cell divisions resulting in nodule formation. Two small peptides encoded by enod40 mRNA as well as its secondary structure have been shown to be key elements in the signalling processes underlying nodule organogenesis. Here results concerning the secondary structure of mRNA of enod40 in soybean are presented. This study combined a theoretical approach, involving structure prediction and comparison, as well as structure probing. Our study indicates five conserved domains in enod40 mRNA among numerous leguminous species. Structure comparison suggests that some domains are also conserved in non-leguminous species and that an additional domain exists that was found only in leguminous species developing indeterminate nodules. Enzymatic and chemical probing data support the structure for three of the domains, and partially for the remaining two. The rest of the molecule appears to be less structured. Some of the domains include motifs, such as U-containing internal loops and bulges, which seem to be conserved. Therefore, they might be involved in the regulatory role of enod40 RNA.
Plant Molecular Biology. Aug, 2003 | Pubmed ID: 14682616
For this work, Lotus japonicus transgenic plants were constructed expressing a fusion reporter gene consisting of the genes beta-glucuronidase (gus) and green fluorescent protein (gfp) under control of the soybean auxin-responsive promoter GH3. These plants expressed GUS and GFP in the vascular bundle of shoots, roots and leafs. Root sections showed that in mature parts of the roots GUS is mainly expressed in phloem and vascular parenchyma of the vascular cylinder. By detecting GUS activity, we describe the auxin distribution pattern in the root of the determinate nodulating legume L. japonicus during the development of nodulation and also after inoculation with purified Nod factors, N-naphthylphthalamic acid (NPA) and indoleacetic acid (IAA). Differently than white clover, which forms indeterminate nodules, L. japonicus presented a strong GUS activity at the dividing outer cortical cells during the first nodule cell divisions. This suggests different auxin distribution pattern between the determinate and indeterminate nodulating legumes that may be responsible of the differences in nodule development between these groups. By measuring of the GFP fluorescence expressed 21 days after treatment with Nod factors or bacteria we were able to quantify the differences in GH3 expression levels in single living roots. In order to correlate these data with auxin transport capacity we measured the auxin transport levels by a previously described radioactive method. At 48 h after inoculation with Nod factors, auxin transport showed to be increased in the middle root segment. The results obtained indicate that L. japonicus transformed lines expressing the GFP and GUS reporters under the control of the GH3 promoter are suitable for the study of auxin distribution in this legume.
Molecular Immunology. Jan, 2004 | Pubmed ID: 14687934
The zebrafish genomic sequence database was analysed for the presence of genes encoding members of the Toll-like receptors (TLR) and interleukin receptors (IL-R) and associated adaptor proteins containing a TIR domain. The resulting predictions show the presence of one or more counterparts for the human TLR1, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, IL-1R and IL-18R genes and one copy of the adaptor genes MyD88, MAL, TRIF and SARM. In contrast to data for the pufferfish Fugu rubripes, zebrafish has two genes that are highly similar to human TLR4. In addition, one fish-specific TLR group can be distinguished that is closely related to the Drosophila melanogaster Toll-9 gene. The sequence of cloned cDNAs for TLR4, TLR2 and MyD88 show the same intron-exon organisation as in the human counterparts. Expression analysis using reverse transcriptase-PCR (RT-PCR) shows that 17 of the predicted zebrafish TLR genes and all the genes encoding adaptor proteins are expressed in the adult stage. A subset of the TLR genes are expressed at higher levels in fish infected with the pathogen Mycobacterium marinum. The induced genes include the homologues of the human TLR1 and TLR2 genes, whose functions are associated with mycobacterial infections, underscoring the suitability of zebrafish as a model for analysis of the vertebrate innate immune system.
Single-molecule Imaging of the H-ras Membrane-anchor Reveals Domains in the Cytoplasmic Leaflet of the Cell Membrane
Biophysical Journal. Jan, 2004 | Pubmed ID: 14695305
In the last decade evidence has accumulated that small domains of 50-700 nm in diameter are located in the exoplasmic leaflet of the plasma membrane. Most of these domains supposedly consist of specific sets of lipids and proteins, and are believed to coordinate signal transduction cascades. Whether similar domains are also present in the cytoplasmic leaflet of the plasma membrane is unclear so far. To investigate the presence of cytoplasmic leaflet domains, the H-Ras membrane-targeting sequence was fused to the C-terminus of the enhanced yellow fluorescent protein. Using single-molecule fluorescence microscopy, trajectories of individual molecules diffusing in the cytoplasmic leaflet of the plasma membrane were recorded. From these trajectories, the diffusion of individual membrane-anchored enhanced yellow fluorescent protein molecules was studied in live cells on timescales from 5 to 200 ms. The results show that the diffusion of 30-40% of the molecules is constrained in domains with a typical size of 200 nm. Neither breakdown of actin nor cholesterol extraction changed the domain characteristics significantly, indicating that the observed domains may not be related to the membrane domains identified so far.
Has2 is Required Upstream of Rac1 to Govern Dorsal Migration of Lateral Cells During Zebrafish Gastrulation
Development (Cambridge, England). Feb, 2004 | Pubmed ID: 14729574
The large extracellular polysaccharide Hyaluronan (HA) and its synthesizing enzymes (Has) have been implicated in regulating the migratory potential of metastatic cancer cells. Here, we analyze the roles of zebrafish Has2 in normal development. Antisense morpholino oligonucleotide (MO)-mediated knockdown of zebrafish Has2 leads to the loss of HA, and severe migratory defects during gastrulation, somite morphogenesis and primordial germ cell migration. During gastrulation, ventrolateral cells of has2 morphant embryos fail to develop lamellipodia and to migrate dorsally, resulting in a blockage of dorsal convergence, whereas extension of the dorsal axis is normal. The effect is cell autonomous, suggesting that HA acts as an autocrine signal to stimulate the migration of HA-generating cells. Upon ectopic expression in axial cells, has2 causes the formation of supernumerary lamellipodia and a blockage of axis extension. Epistasis analyses with constitutively active and dominant-negative versions of the small GTPase Rac1 suggest that HA acts by Rac1 activation, rather than as an essential structural component of the extracellular matrix. Together, our data provide evidence that convergence and extension are separate morphogenetic movements of gastrulation. In addition, they suggest that the same HA pathways are active to auto-stimulate cell migration during tumor invasion and vertebrate embryogenesis.
Isoform-specific Differences in Rapid Nucleocytoplasmic Shuttling Cause Distinct Subcellular Distributions of 14-3-3 Sigma and 14-3-3 Zeta
Journal of Cell Science. Mar, 2004 | Pubmed ID: 14996909
Nucleocytoplasmic transport of proteins plays an important role in the regulation of many cellular processes. Differences in nucleocytoplasmic shuttling can provide a basis for isoform-specific biological functions for members of multigene families, like the 14-3-3 protein family. Many organisms contain multiple 14-3-3 isoforms, which play a role in numerous processes, including signalling, cell cycle control and apoptosis. It is still unclear whether these isoforms have specialised biological functions and whether this specialisation is based on isoform-specific ligand binding, expression regulation or specific localisation. Therefore, we studied the subcellular distribution of 14-3-3 sigma and 14-3-3 zeta in vivo in various mammalian cell types using yellow fluorescent protein fusions and isoform-specific antibodies. 14-3-3 sigma was mainly localised in the cytoplasm and only low levels were present in the nucleus, whereas 14-3-3 zeta was found at relatively higher levels in the nucleus. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the 14-3-3 proteins rapidly shuttle in and out of the nucleus through active transport and that the distinct subcellular distributions of 14-3-3 sigma and 14-3-3 zeta are caused by differences in nuclear export. 14-3-3 sigma had a 1.7x higher nuclear export rate constant than 14-3-3 zeta, while import rate constants were equal. The 14-3-3 proteins are exported from the nucleus at least in part by a Crm1-dependent, leptomycin B-sensitive mechanism. The differences in subcellular distribution of 14-3-3 that we found in this study are likely to reflect a molecular basis for isoform-specific biological specialisation.
Trends in Microbiology. May, 2004 | Pubmed ID: 15120137
Nucleic Acids Research. 2004 | Pubmed ID: 15388798
DNA computing aims at using nucleic acids for computing. Since micromolar DNA solutions can act as billions of parallel nanoprocessors, DNA computers can in theory solve optimization problems that require vast search spaces. However, the actual parallelism currently being achieved is at least a hundred million-fold lower than the number of DNA molecules used. This is due to the quantity of DNA molecules of one species that is required to produce a detectable output to the computations. In order to miniaturize the computation and considerably reduce the amount of DNA needed, we have combined DNA computing with single-molecule detection. Reliable hybridization detection was achieved at the level of single DNA molecules with fluorescence cross-correlation spectroscopy. To illustrate the use of this approach, we implemented a DNA-based computation and solved a 4-variable 4-clause instance of the computationally hard Satisfiability (SAT) problem.
Different Subcellular Localization and Trafficking Properties of KNOX Class 1 Homeodomain Proteins from Rice
Plant Molecular Biology. Aug, 2004 | Pubmed ID: 15604716
Genes of the KN1-like homeobox (KNOX) class 1 encode transcription factors involved in shoot apical meristem development and maintenance. We studied the subcellular localization of Green Fluorescent Protein-tagged rice KNOX proteins (Oskn1-3) after particle bombardment of onion and rice cells and after transformation of Arabidopsis and rice with constitutive and inducible expression constructs. In all test systems, the three rice KNOX proteins showed nuclear and cytoplasmic localization patterns. However, Oskn1 additionally showed in some cells a distribution over punctae moving randomly in the cytosol. Use of an inducible expression system indicated a nuclear presence of Oskn1 in cells of the shoot apical meristem and post-transcriptional down-regulation in early leaf primordia. Arabidopsis and rice test systems were used to study effects of plant hormones and auxin transport inhibition on KNOX protein localization. Application of GA3 or 1-NAA shifted protein localization completely to the cytoplasm and resulted in loss of the punctae formed by Oskn1. Conversely, NPA application induced a complete nuclear localization of the KNOX proteins. To study intercellular movement of the KNOX proteins we set up a novel co-bombardment assay in which trafficking of untagged KNOX proteins was visualized through the co-trafficking of green fluorescent or blue fluorescent marker proteins. In multiple independent experiments Oskn1 trafficked more extensively to neighboring cells than Oskn2 and Oskn3. Differences in the localization and trafficking properties of Oskn1, Oskn2 and Oskn3 correlate with differences in mRNA localization patterns and functional differences between the rice KNOX genes and their putative orthologues from other species.
Programmed Cell Death During the Transition from Multicellular Structures to Globular Embryos in Barley Androgenesis
Planta. Jun, 2005 | Pubmed ID: 15645302
Androgenesis represents one of the most fascinating examples of cell differentiation in plants. In barley, the conversion of stressed uninucleate microspores into embryo-like structures is highly efficient. One of the bottlenecks in this process is the successful release of embryo-like structures out of the exine wall of microspores. In the present work, morphological and biochemical studies were performed during the transition from multicellular structures to globular embryos. Exine wall rupture and subsequent globular embryo formation were observed only in microspores that divided asymmetrically. Independent divisions of the generative and the vegetative nuclei gave rise to heterogeneous multicellular structures, which were composed of two different cellular domains: small cells with condensed chromatin structure and large cells with normal chromatin structure. During exine wall rupture, the small cells died and their death marked the site of exine wall rupture. Cell death in the small cell domain showed typical features of plant programmed cell death. Chromatin condensation and DNA degradation preceded cell detachment and cytoplasm dismantling, a process that was characterized by the formation of vesicles and vacuoles that contained cytoplasmic material. This morphotype of programmed cell death was accompanied by an increase in the activity of caspase-3-like proteases. The orchestration of such a death program culminated in the elimination of the small generative domain, and further embryogenesis was carried out by the large vegetative domain. To date, this is the first report to show evidence that programmed cell death takes part in the development of microspore-derived embryos.
Transcriptome Profiling of Adult Zebrafish at the Late Stage of Chronic Tuberculosis Due to Mycobacterium Marinum Infection
Molecular Immunology. Jun, 2005 | Pubmed ID: 15829308
The Mycobacterium marinum-zebrafish infection model was used in this study for analysis of a host transcriptome response to mycobacterium infection at the organismal level. RNA isolated from adult zebrafish that showed typical signs of fish tuberculosis due to a chronic progressive infection with M. marinum was compared with RNA from healthy fish in microarray analyses. Spotted oligonucleotide sets (designed by Sigma-Compugen and MWG) and Affymetrix GeneChips were used, in total comprising 45,465 zebrafish transcript annotations. Based on a detailed comparative analysis and quantitative reverse transcriptase-PCR analysis, we present a validated reference set of 159 genes whose regulation is strongly affected by mycobacterial infection in the three types of microarrays analyzed. Furthermore, we analyzed the separate datasets of the microarrays with special emphasis on the expression profiles of immune-related genes. Upregulated genes include many known components of the inflammatory response and several genes that have previously been implicated in the response to mycobacterial infections in cell cultures of other organisms. Different marker genes of the myeloid lineage that have been characterized in zebrafish also showed increased expression. Furthermore, the zebrafish homologs of many signal transduction genes with relationship to the immune response were induced by M. marinum infection. Future functional analysis of these genes may contribute to understanding the mechanisms of mycobacterial pathogenesis. Since a large group of genes linked to immune responses did not show altered expression in the infected animals, these results suggest specific responses in mycobacterium-induced disease.
Single-molecule Diffusion Measurements of H-Ras at the Plasma Membrane of Live Cells Reveal Microdomain Localization Upon Activation
Journal of Cell Science. May, 2005 | Pubmed ID: 15860728
Recent studies show that the partitioning of the small GTPase H-Ras in different types of membrane microdomains is dependent on guanosine 5'-triphosphate (GTP)-loading of H-Ras. Detailed knowledge about the in vivo dynamics of this phenomenon is limited. In this report, the effect of the activation of H-Ras on its microdomain localization was studied by single-molecule fluorescence microscopy. Individual human H-Ras molecules fused to the enhanced yellow fluorescent protein (eYFP) were imaged in the dorsal plasma membrane of live mouse cells and their diffusion behavior was analyzed. The diffusion of a constitutively inactive (S17N) and constitutively active (G12V) mutant of H-Ras was compared. Detailed analysis revealed that for both mutants a major, fast-diffusing population and a minor, slow-diffusing population were present. The slow-diffusing fraction of the active mutant was confined to 200 nm domains, which were not observed for the inactive mutant. In line with these results we observed that the slow-diffusing fraction of wild-type H-Ras became confined to 200 nm domains upon insulin-induced activation of wild-type H-Ras. This activation-dependent localization of H-Ras to 200 nm domains, for the first time directly detected in live cells, supports the proposed relationship between H-Ras microdomain localization and activation.
Genomic Annotation and Expression Analysis of the Zebrafish Rho Small GTPase Family During Development and Bacterial Infection
Genomics. Jul, 2005 | Pubmed ID: 15894457
The zebrafish genomic sequence database was analyzed for the presence of genes encoding members of the Rho small GTPases. The analysis shows the presence of 32 zebrafish Rho genes representing one or more homologs of the human RHOA, RND3, RHOF, RHOG, RHOH, RHOJ, RHOU, RHOV, CDC42, RAC1, RAC2, RAC3, RND1, RHOBTB1, RHOBTB2, RHOBTB3, and RHOT1 genes. By expression analysis using reverse transcriptase-PCR we show that at least 20 of the predicted zebrafish small GTPase genes are expressed in the adult stage. Interestingly, only 5 of these were found to be expressed at early embryonic stages, including rhoab, rhoad, cdc42a, cdc42c, and rac1a. We observed a strong upregulation of zebrafish rhogb expression after Mycobacterium marinum infection of adult fish. This complete annotation study provides a firm basis for the use of zebrafish as a model for analysis of Rho GTPase function in vertebrate development and the innate immune system.
Gene Expression Profiling of the Long-term Adaptive Response to Hypoxia in the Gills of Adult Zebrafish
American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. Nov, 2005 | Pubmed ID: 15994372
Low oxygen levels (hypoxia) play a role in clinical conditions such as stroke, chronic ischemia, and cancer. To better understand these diseases, it is crucial to study the responses of vertebrates to hypoxia. Among vertebrates, some teleosts have developed the ability to adapt to extremely low oxygen levels. We have studied long-term adaptive responses to hypoxia in adult zebrafish. We used zebrafish that survived severe hypoxic conditions for 3 wk and showed adaptive behavioral and phenotypic changes. We used cDNA microarrays to investigate hypoxia-induced changes in expression of 15,532 genes in the respiratory organs (the gills). We have identified 367 differentially expressed genes of which 117 showed hypoxia-induced and 250 hypoxia-reduced expressions. Metabolic depression was indicated by repression of genes in the TCA cycle in the electron transport chain and of genes involved in protein biosynthesis. We observed enhanced expression of the monocarboxylate transporter and of the oxygen transporter myoglobin. The hypoxia-induced group further included the genes for Niemann-Pick C disease and for Wolman disease [lysosomal acid lipase (LAL)]. Both diseases lead to a similar intra- and extracellular accumulation of cholesterol and glycolipids. The Niemann-Pick C protein binds to cholesterol from internal lysosomal membranes and is involved in cholesterol trafficking. LAL is responsible for lysosomal cholesterol degradation. Our data suggest a novel adaptive mechanism to hypoxia, the induction of genes for lysosomal lipid trafficking and degradation. Studying physiological responses to hypoxia in species tolerant for extremely low oxygen levels can help identify novel regulatory genes, which may have important clinical implications.
Time-lapse Tracking of Barley Androgenesis Reveals Position-determined Cell Death Within Pro-embryos
Planta. Feb, 2005 | Pubmed ID: 15449059
Following abiotic stress to induce barley (Hordeum vulgare L.) androgenesis, the development of 794 enlarged microspores in culture was monitored by time-lapse tracking. In total, 11% of the microspores tracked developed into embryo-like structures (type-I pathway), 36% formed multicellular structures (type-II pathway) and 53% of the microspores followed gametophytic divisions, accumulated starch and died in the first days of tracking (type-III pathway). Despite the microspore fate, enlarged microspores showed similar morphologies directly after stress treatment. Ultrastructural analysis, however, revealed two morphologically distinct cell types. Cells with a thin intine layer and an undifferentiated cytoplasm after stress treatment were associated with type-I and type-II pathways, whereas the presence of differentiated amyloplasts and a thick intine layer were associated with the type-III pathway. Tracking revealed that the first morphological change associated with embryogenic potential was a star-like morphology, which was a transitory stage between uninucleate vacuolated microspores after stress and the initiation of cell division. The difference between type-I and type-II pathways was observed during the time they displayed the star-like morphology. During the transition phase, embryo-like structures in the type-I pathway were always released out of the exine wall at the opposite side of the pollen germ pore, whereas in the type-II pathway multicellular structures were unable to break the exine and to release embryo-like structures. Moreover, by combining viability studies with cell tracking, we show that release of embryo-like structures was preceded by a decrease in viability of the cells positioned at the site of exine wall rupture. These cells were also positively stained by Sytox orange, a cell death indicator. Thereby, we demonstrate, for the first time, that a position-determined cell death process marks the transition from a multicellular structure into an embryo-like structure during barley androgenesis.
Infection and Immunity. Apr, 2006 | Pubmed ID: 16552074
Innate immunity signaling mechanisms during vertebrate embryogenesis are largely unknown. To study Toll-like receptor (TLR) signaling function in the zebrafish embryo model, we designed an experimental setup for antisense morpholino knockdown under conditions of bacterial infection. Clearance of Salmonella enterica serovar Typhimurium Ra bacteria was significantly impaired after knockdown of myeloid differentiation factor 88 (MyD88), a common adaptor protein in TLR and interleukin-1 receptor signaling. Thereby, we demonstrate for the first time that the innate immune response of the developing embryo involves MyD88-dependent signaling, which further establishes the zebrafish embryo as a model for the study of vertebrate innate immunity.
The Production of Species-specific Highly Unsaturated Fatty Acyl-containing LCOs from Rhizobium Leguminosarum Bv. Trifolii is Stringently Regulated by NodD and Involves the NodRL Genes
Molecular Plant-microbe Interactions : MPMI. Mar, 2006 | Pubmed ID: 16570652
A proportion of the Nod factors of some Rhizobium leguminosarum bv. trifolii strains is characterized by the presence of highly unsaturated fatty acyl chains containing trans double bonds in conjugation with the carbonyl group of the glycan oligosaccharide backbone. These fatty acyl chains are C18:3, C20:3, C18:4, or C20:4 and have UV-absorption maxima at 303 and 330 nm. These Nod factors are presumed to be important for host-specific nodulation on clover species. However, in wild-type R. leguminosarum bv. trifolii ANU843, Nod factors with these characteristic acyl chains were not observed using standard growth conditions. They were observed only when nod genes were present in multiple copies or when transcription was artificially increased to higher levels by introduction of extra copies of the transcriptional regulator gene nodD. In a screen for the genetic requirements for production of the Nod factors with these characteristic structures, it was found that the region downstream of nodF and nodE is essential for the presence of highly unsaturated fatty acyl moieties. Mu-lacZ insertion in this region produced a mutant that did not produce detectable levels of the highly unsaturated fatty acyl-bearing Nod factors. The Mu-lacZ insertion was translationally fused to a putative new gene, designated nodR, in the nodE-nodL intergenic region; however, no predicted function for the putative NodR protein has been obtained from database homology searches. In a set of 12 wild-type strains of R. leguminosarum by. trifolii originating from various geographical regions that were analyzed for the presence of a nodR-like gene, it was found that seven strains carry a homologous NodR open reading frame. Taken together, our results suggest a tightly controlled regulation of nod genes, in which we propose that it is the balance of transcriptional levels of nodFE and the nodRL genes that is critical for determining the presence of highly unsaturated fatty acyl moieties in the Nod factors produced by R. leguminosarum bv. trifolii.
Cloning, Functional Expression and Characterization of Mesorhizobium Loti Arylamine N-acetyltransferases: Rhizobial Symbiosis Supplies Leguminous Plants with the Xenobiotic N-acetylation Pathway
Molecular Microbiology. Apr, 2006 | Pubmed ID: 16573698
Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes involved in the detoxification of numerous aromatic chemicals. The NAT-dependent N-acetylation pathway has not previously been detected in plants. We demonstrate here the occurrence of the NAT-dependent pathway in leguminous plants, due to symbiosis with Mesorhizobium loti. We cloned two NAT enzymes from M. loti and showed that these two recombinant enzymes catalysed the N-acetylation of several known NAT substrates, including aniline-derived pesticide residues. We also demonstrate the existence of a functional NAT-dependent acetylation pathway in the root nodules of Lotus japonicus inoculated with M. loti. M. loti is the first non-eukaryotic organism shown to express two catalytically active NAT isoforms. This work also provides the first evidence for acquisition of a xenobiotic detoxification pathway by a plant through symbiosis with a soil microbe.
Gene Expression Patterns : GEP. Oct, 2006 | Pubmed ID: 16774848
The mitogen activated protein kinases (MAPK) family pathway is conserved in evolution through the plant and animal kingdoms. These proteins have been implicated in diverse cellular processes including cell growth, proliferation, differentiation, survival and development. In this study we annotated and cloned members of the zebrafish MAPK gene-family, containing the ERK, JNK and p38 subfamilies. Their sequences were compared to orthologs of other vertebrates (human, mouse and rat) and the temporal and spatial expression levels of the zebrafish mapk genes were determined during early zebrafish development. Semi-quantitative reverse transcriptase-PCR analysis revealed that most mapk genes are expressed throughout zebrafish development. Erk2,3 and p38a were expressed at a constant level throughout zebrafish embryogenesis, whereas erk1,4,5,6,7 and p38b showed specific temporal expression patterns. The spatial expression patterns were obtained by whole mount in situ hybridization at 24 h post fertilization (hpf) and 48 hpf embryos. The expression patterns were localized in specific regions at both stages and were tightly regulated during embryogenesis. For p38b, no staining was detected at 24 and 48 hpf. However, its expression was demonstrated at blastula-stage. Together, we identified the zebrafish orthologs of the zebrafish MAPK gene family and determined their specific spatial and temporal expression and distribution patterns during zebrafish embryogenesis.
Zebrafish. 2006 | Pubmed ID: 18377223
Magnetic resonance microscopy (MRM) is an imaging modality that allows for noninvasive acquisition of high-resolution images in intact opaque animals. The zebrafish (Danio rerio) is an important model organism for the study of vertebrate biology. However, optical in vivo studies in zebrafish are restricted to very early developmental stages due to the opacity of the juvenile and adult stages. Application of high resolution MRM has not yet been explored in adult zebrafish. In this study we applied and optimized high resolution MRM methods to examine anatomical structures noninvasively in adult zebrafish. Clear morphological proton images were obtained by T(2)-weighted spin echo and rapid acquisition with rapid acquisition with relaxation enhancement (RARE) sequences which revealed many anatomical details in the entire intact zebrafish at a magnetic field strength of 9.4 T. In addition, in vivo imaging of adult zebrafish revealed sufficient anatomical details. To our knowledge this is the first report of the application of high resolution MRM to study detailed anatomical structures in adult zebrafish.
Zebrafish. 2006 | Pubmed ID: 18377224
Compared with the increasing use of zebrafish as a model organism in many laboratories, zebrafish cell lines are still unexploited and limited in application, partly due to their unknown genetic and physiological properties. We characterize two zebrafish embryonic fibroblast cell lines, ZF4 and PAC2. We demonstrate the genetic stability of these two zebrafish cell lines and achieved genetic manipulation by either lipid-mediated transfection or an electroporation- based nucleofection method. Data from zebrafish chip analysis (Affymetrix) demonstrate unique characteristics of these two cell lines in gene expression levels, showing that different zebrafish cell lines can be classified by their transcriptome profile. Their transcriptional responses to serum growth factor exposure suggest that zebrafish fibroblast cell lines may be used to study processes related to wound-healing or cancer.
ZebraFISH: Fluorescent in Situ Hybridization Protocol and Three-dimensional Imaging of Gene Expression Patterns
Zebrafish. 2006 | Pubmed ID: 18377226
We present a method and protocol for fluorescent in situ hybridization (FISH) in zebrafish embryos to enable three-dimensional imaging of patterns of gene expression using confocal laser scanning microscopy. We describe the development of our protocol and the processing workflow of the three-dimensional images from the confocal microscope. We refer to this protocol as zebraFISH. FISH is based on the use of tyramide signal amplification (TSA), which results in highly sensitive and very localized fluorescent staining. The zebraFISH protocol was extensively tested and here we present a panel of five probes for genes expressed in different tissues or single cells. FISH in combination with confocal laser scanning microscopy provides an excellent tool to generate three-dimensional images of patterns of gene expression. We propose that such three-dimensional images are suitable for building a repository of gene expression patterns, complementary to our previously published three-dimensional anatomical atlas of zebrafish development (bio-imaging.liacs.nl/). Our methodology for image processing of three-dimensional confocal images allows an analytical approach to the definition of gene expression domains based on the three-dimensional anatomical atlas.
Biophysical Journal. Aug, 2006 | Pubmed ID: 16920696
Recent evidence on the occurrence of small (5-700 nm diameter) lipid microdomains in the exoplasmic leaflet of the plasma membrane has evoked interest in the possibility that similar domains may also be present in the cytoplasmic leaflet of the plasma membrane. However, current knowledge about these "lipid rafts", in live cells is limited. One way to obtain insight into the occurrence and the size of lipid rafts is the use of single-molecule microscopy, which allows one to study the diffusive motion of individual molecules with high positional and temporal accuracy. Using this technique, we compared the diffusion behavior of the Lck membrane anchor, which has a high affinity for lipid rafts, to the diffusion behavior of the K-Ras membrane anchor, which has negligible affinity for rafts and compared the results with those of the H-Ras membrane anchor. Surprisingly, we found only minor differences in the diffusion behavior of the various lipid anchors, indicating that putative cytoplasmic leaflet lipid rafts would have to be small (<137 nm diameter) and do not affect the mobility of membrane-anchored molecules much on timescales up to 60 ms.
FEBS Letters. Sep, 2006 | Pubmed ID: 16949582
The mitogen activated protein kinase (MAPK) family, consisting of the extracellular signal regulated protein kinase, c-Jun amino terminal MAPK and p38 subfamilies, is conserved in evolution throughout the plant and animal kingdoms. These proteins have been implicated in diverse cellular processes including cell growth, migration, proliferation, differentiation, survival and development. Gene-targeting approaches in mice, chickens, frogs and zebrafish revealed crucial roles of MAPK in vertebrate development. Gene-disruption or -silencing often lead to lethal effects, therefore the zebrafish ex utero development provides an excellent in vivo model to study the function of MAPK in early embryogenesis. In this review, we summarize the current understanding of the MAPK family function in vertebrate-development and place this into the perspective of possibilities for future research.
Characterization of Genomic Clones and Expression Analysis of the Three Types of Superoxide Dismutases During Nodule Development in Lotus Japonicus
Molecular Plant-microbe Interactions : MPMI. Mar, 2007 | Pubmed ID: 17378429
Superoxide dismutases (SODs) are metalloenzymes that play a primary role in the protection against oxidative stress in plants and other organisms. We have characterized four SOD genes in Lotus japonicus and have analyzed their expression in roots and four developmental stages of nodules. The expression of cytosolic CuZnSOD, at the mRNA, protein, and enzyme activity levels, decreases with nodule age, and the protein is localized in the dividing cells and infection threads of emergent nodules and in the infected cells of young nodules. The mitochondrial MnSOD was downregulated, whereas the bacteroidal MnSOD displayed maximal protein and enzyme activity levels in older nodules. Two additional genes, encoding plastidic (FeSOD1) and cytosolic (FeSOD2) FeSOD isoforms, were identified and mapped. The genes are located in different chromosomes and show differential expression. The FeSOD1 mRNA level did not change during nodule development, whereas FeSOD2 was upregulated. The distinct expression patterns of the SOD genes may reflect different regulatory mechanisms of the enzyme activities during nodule ontogeny. In particular, at the mRNA and activity levels, the virtual loss of cytosolic CuZnSOD in mature and old nodules, concomitant with the induction of FeSOD2, suggests that the two enzymes may functionally compensate each other in the cytosol at the late stages of nodule development.
Model Systems for Infectious Disease and Cancer in Zebrafish: a Report on an EMBO Workshop Held at the Lorentz Center, Leiden, The Netherlands, July 16-18, 2007
Zebrafish. 2007 | Pubmed ID: 18284335
Bio Systems. Mar, 2007 | Pubmed ID: 16860927
One line of DNA computing research focuses on parallel search algorithms, which can be used to solve many optimization problems. DNA in solution can provide an enormous molecular library, which can be searched by molecular biological techniques. We have implemented such a parallel search for solutions to knapsack problems, which ask for the best way to pack a knapsack of limited volume. Several instances of knapsack problems were solved using DNA. We demonstrate how the computations can be extended by in vivo translation of the DNA library into protein. This combination of DNA and protein allows for multi-criterion optimization. The knapsack computations performed can then be seen as protein optimizations, one of the most complex computations performed by natural systems.
BMC Genomics. 2008 | Pubmed ID: 18442396
The MAPK signaling proteins are involved in many eukaryotic cellular processes and signaling networks. However, specific functions of most of these proteins in vertebrate development remain elusive because of potential redundancies. For instance, the upstream activation pathways for ERK1 and ERK2 are highly similar, and also many of their known downstream targets are common. In contrast, mice and zebrafish studies indicate distinct roles for both ERKs in cellular proliferation, oncogenic transformation and development. A major bottleneck for further studies is that relatively little is known of in vivo downstream signaling specific for these kinases.
A Spatially Restricted Increase in Receptor Mobility is Involved in Directional Sensing During Dictyostelium Discoideum Chemotaxis
Journal of Cell Science. May, 2008 | Pubmed ID: 18469015
The directed cell migration towards a chemotactic source, chemotaxis, involves three complex and interrelated processes: directional sensing, cell polarization and motility. Directional sensing allows migrating eukaryotic cells to chemotax in extremely shallow gradients (<2% across the cell body) of the chemoattractant. Although directional sensing has been observed as spatially restricted responses along the plasma membrane, our understanding of the ;compass' of the cell that controls the gradient-induced translocation of proteins during chemotactic movements is still largely lacking. Until now, the dynamical behaviour and mobility of the chemoattractant-receptor molecule has been neglected in models describing the directional sensing mechanisms. Here, we show by single-molecule microscopy an agonist-induced increase in the mobile fraction of cAMP-receptor at the leading edge of chemotacting Dictyostelium discoideum cells. The onset of receptor mobility was correlated to the uncoupling and activation of the Galpha2-protein. A finite-element simulation showed that the increase in mobile fraction of the activated receptor enabled the amplified generation of activated Gbetagamma-dimers at the leading edge of the cell, faithfully representing a primary linear amplification step in directional sensing. We propose here that modulation of the receptor mobility is directly involved in directional sensing and provides a new mechanistic basis for the primary amplification step in current theoretical models that describe directional sensing.
Developmental Biology. Jul, 2008 | Pubmed ID: 18514184
The MAPKs are key regulatory signaling molecules in many cellular processes. Here we define differential functions for ERK1 and ERK2 MAPKs in zebrafish embryogenesis. Morpholino knockdown of ERK1 and ERK2 resulted in cell migration defects during gastrulation, which could be rescued by co-injection of the corresponding mRNA. Strikingly, Erk2 mRNA cross-rescued ERK1 knockdown, but erk1 mRNA was unable to compensate for ERK2 knockdown. Cell-tracing experiments revealed a convergence defect for ERK1 morphants without a severe posterior-extension defect, whereas ERK2 morphants showed a more severe reduction in anterior-posterior extension. These defects were primary changes in gastrulation cell movements and not caused by altered cell fate specification. Saturating knockdown conditions showed that the absence of FGF-mediated dual-phosphorylated ERK2 from the blastula margin blocked initiation of epiboly, actin and tubulin cytoskeleton reorganization processes and further arrested embryogenesis, whereas ERK1 knockdown had only a mild effect on epiboly progression. Together, our data define distinct roles for ERK1 and ERK2 in developmental cell migration processes during zebrafish embryogenesis.
Comparative Biochemistry and Physiology. Toxicology & Pharmacology : CBP. Nov, 2008 | Pubmed ID: 18602498
Progestins have a broad range of functions in reproductive biology. Many rapid nongenomic actions of progestins have been identified, including induction of oocyte maturation, modulation of reproductive signaling in the brain, rapid activation of breast cancer cell signaling, induction of the acrosomal reaction and hypermotility in mammalian sperm. Currently, there are three receptor candidates for mediating rapid progestin actions: (1) membrane progestin receptors (mPRs); (2) progestin receptor membrane components (PGRMCs); and (3) nuclear progestin receptors (nPRs). The recently-described mPR family of proteins has seven integral transmembrane domains and mediates signaling via G-protein coupled pathways. The PGRMCs have a single transmembrane with putative Src homology domains for potential activation of second messengers. The classical nPRs, in addition to having well defined transcriptional activity, can also mediate rapid activation of intracellular signaling pathways. However, details of the mechanisms by which these three classes of progestin receptors mediate rapid intracellular signaling and their subcellular localization remain unclear. In addition, mPRs, nPRs and PGRMCs exhibit overlapping expression and functions in multiple tissues, implying potential interactions during oocyte maturation, parturition, and breast cancer signaling in individual cells. However, the overwhelming majority of studies to date have focused on the functions of one of these groups of receptors in isolation. This review will summarize recent findings on the three major progestin receptor candidates, emphasizing the different approaches used, some experimental pitfalls, and current controversies. We will also review evidence for the involvement of mPRs and nPRs in one of the most well-characterized nongenomic steroid actions in basal vertebrates, oocyte maturation, and conclude by suggesting some future areas of research. Clarification of the controversies surrounding the identities and localization of membrane progestin receptors may help direct future research that could advance our understanding of rapid actions of steroids.
BMC Physiology. 2008 | Pubmed ID: 18616800
If European silver eels are prevented from reproductive migration, they remain in a prepubertal stage by dopaminergic inhibition of pituitary activity. Because this inhibition is likely a requirement for an extended female growth stage, we tested if it is sex-specific by subjecting both sexes to stimulation by GnRHa (Gonadotropin-Releasing Hormone agonist) - injection or 3-months swimming in seawater.
Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry. Aug, 2008 | Pubmed ID: 18666264
We use photothermal microscopy to detect and image individual gold nanoparticles that are either embedded in a polymer film or immobilized in an aqueous environment. Reducing the numerical aperture of the detection optics allows us to achieve a 200-fold-enlarged detection volume while still retaining sufficient detectivity. We characterize the capabilities of this approach for the detection of gold colloids with a diameter of 20 nm, with emphasis on practical aspects that are important for high-throughput-screening applications. The extended detection volume in combination with the stability of the photothermal signal are major advantages compared to fluorescence-based approaches, which are limited by photoblinking and photobleaching. Careful consideration is given to the trade-off between the maximum increase in local temperature that can be tolerated by a biological specimen and the minimum integration time needed to reliably determine whether a given volume contains a target species. We find that our approach has the potential to increase the detection-limited flow rate (i.e. the limit given by the detection volume divided by the minimum detection time) by two to three orders of magnitude.
Analysis of Interactions of Signaling Proteins with Phage-displayed Ligands by Fluorescence Correlation Spectroscopy
Journal of Biomolecular Screening. Sep, 2008 | Pubmed ID: 18753688
Fluorescent correlation spectroscopy (FCS) was used to measure binding affinities of ligands to ligates that are expressed by phage-display technology. Using this method we have quantified the binding of the 14-3-3 signaling protein to artificial peptide ligand. As a ligand we used the R18 artificial peptide expressed as a fusion in the cpIII coat protein that is present in 3 to 5 copies in an M13 phage. Comparisons of binding affinities were made with free R18 ligands using FCS. The result showed a relatively high binding affinity for the phage-displayed R18 peptide compared with binding to free fluorescently labeled R18. Quantification was supported by titration of the phage numbers using atomic force microscopy (AFM). AFM was shown to accurately determine phage numbers in solution as a good alternative for electron microscopy. It was shown to give reliable data that correlated perfectly with those of the viable phage numbers determined by classical bacterial infection studies. In conclusion, a very fast and sensitive method for the selection of new peptide ligands or ligates based on a quantitative assay in solution has been developed.
Identification and Real-time Imaging of a Myc-expressing Neutrophil Population Involved in Inflammation and Mycobacterial Granuloma Formation in Zebrafish
Developmental and Comparative Immunology. 2008 | Pubmed ID: 17553562
By enhancer trap screening we identified a transgenic zebrafish line showing leukocyte-specific YFP expression during late embryo and early larval development. Its enhancer detection insertion was mapped near a novel member of the myc proto-oncogene family, encoding transcription factors known to be important for regulating human myelopoiesis. Characterization of the zebrafish myc family showed that only this particular myc gene is strongly expressed in leukocytes. To identify the myc/YFP-expressing cell type, we re-examined specificity of described myeloid markers by multiplex fluorescent in situ hybridization, showing that lcp1 can be considered as a general leukocyte marker, csf1r as a macrophage-specific marker, and mpx and lyz as neutrophil-specific markers. Subsequent colocalization analysis defined the YFP-positive cells as a subset of the neutrophil population. Using real-time confocal imaging we demonstrate that these cells migrate to sites of inflammation and are involved in innate immune responses towards infections, including Mycobacterium marinum-induced granuloma formation.
Endocrinology. Apr, 2008 | Pubmed ID: 18096659
In humans, two glucocorticoid receptor (GR) splice variants exist: GRalpha and GRbeta, which are identical between amino acids 1-727 and then diverge. Whereas GRalpha (the canonical GR) acts as a ligand-activated transcription factor, GRbeta does not bind traditional glucocorticoid agonists, lacks GRalpha's transactivational activity, and acts as a dominant-negative inhibitor of GRalpha. It has been suggested that this receptor isoform is involved in the induction of glucocorticoid resistance in asthma patients. Unfortunately, a GR beta-isoform has been detected in only humans, and therefore, an animal model for studies on this isoform is lacking. In the present study, we demonstrate that in zebrafish a GR isoform exists that diverges from the canonical zebrafish GR at the same position as human GRbeta from human GRalpha. The zebrafish GR beta-isoform acts as a dominant-negative inhibitor in reporter assays, and the extent of inhibition and the effective GRalpha/GRbeta ratio is similar to studies performed with the human GR isoforms. In addition, the subcellular localization of zebrafish GRbeta is similar to its human equivalent. Finally, expression levels of GRalpha and GRbeta were determined in adult zebrafish tissues and at several developmental stages. Both receptor isoforms were detected throughout the body, and GRbeta mRNA levels were relatively low compared with GRalpha mRNA levels, as in humans. Thus, for the first time, a GR beta-isoform has been identified in a nonhuman animal species, shedding new light on the relevance of this GR splice variant and providing a versatile animal model for studies on the GR system.
Disease Modeling in Zebrafish: Cancer and Immune Responses--a Report on a Workshop Held in Spoleto, Italy, July 20-22, 2009
Zebrafish. Dec, 2009 | Pubmed ID: 20047471
The growing interest in using zebrafish for genetic and functional dissection of malignancy and infection was highlighted by the second international workshop on Zebrafish Models of Cancer and the Immune Response in Spoleto, Italy (July 20-22, 2009). The overarching theme of the state-of-the-art reports featured the unique suitability of zebrafish for in vivo monitoring of fundamental biologic and pathologic processes. For example, in vivo imaging was employed for the first demonstration of direct development of hematopoietic stem cells from hemogenic epithelium and for visualization of T-cell homing and interaction with thymic epithelial cells. In addition, in vivo monitoring was instrumental for developing disease models of solid tumors, leukemia, and of inflammatory conditions, and for assessing the efficacy of small molecule drugs under physiologic and pathologic conditions. The success of zebrafish small molecule screens was underscored by the identification of prostaglandin E2 (PGE2) as an efficient inducer of stem cell expansion that led to the initiation of the first human trial on the efficacy of PGE2 in bone marrow transplantation. Further, zebrafish models of infectious diseases such as tuberculosis have been established that are now amenable to high-throughput in vivo drug screens, a much-needed development in the fight against drug-resistant microorganisms. The success of this workshop and the rapidly growing field of cancer and the immune response in zebrafish have spawned follow-up meetings in Boston (June 2010) and Edinburgh (2011).
Transcriptome Profiling and Functional Analyses of the Zebrafish Embryonic Innate Immune Response to Salmonella Infection
Journal of Immunology (Baltimore, Md. : 1950). May, 2009 | Pubmed ID: 19380811
Due to the clear separation of innate immunity from adaptive responses, the externally developing zebrafish embryo represents a useful in vivo model for identification of innate host determinants of the response to bacterial infection. Here we performed a time-course transcriptome profiling study and gene ontology analysis of the embryonic innate immune response to infection with two model Salmonella strains that elicit either a lethal infection or an attenuated response. The transcriptional response to infection with both the lethal strain and the avirulent LPS O-Ag mutant strain showed clear conservation with host responses detected in other vertebrate models and human cells, including induction of genes encoding cell surface receptors, signaling intermediates, transcription factors, and inflammatory mediators. Furthermore, our study led to the identification of a large set of novel immune response genes and infection markers, the future functional characterization of which will support vertebrate genome annotation. From the time series and bacterial strain comparisons, matrix metalloproteinase genes, including mmp9, were among the most consistent infection-responsive genes. Purified Salmonella flagellin also strongly induced mmp9 expression. Using knockdown analysis, we showed that this gene was downstream of the zebrafish homologs of the flagellin receptor TLR5 and the adaptor MyD88. Additionally, flagellin-mediated induction of other inflammation markers, including il1b, il8, and cxcl-C1c, was reduced upon Tlr5 knockdown as well as expression of irak3, a putative negative TLR pathway regulator. Finally, we showed that induction of il1b, mmp9, and irak3 requires Myd88-dependent signaling, while ifn1 and il8 were induced Myd88 independently during Salmonella infection.
Specificity of the Zebrafish Host Transcriptome Response to Acute and Chronic Mycobacterial Infection and the Role of Innate and Adaptive Immune Components
Molecular Immunology. Jul, 2009 | Pubmed ID: 19409617
Pathogenic mycobacteria have the ability to survive within macrophages and persist inside granulomas. The complex host-pathogen interactions that determine the outcome of a mycobacterial infection process result in marked alterations of the host gene expression profile. Here we used the zebrafish model to investigate the specificity of the host response to infections with two mycobacterium strains that give distinct disease outcomes: an acute disease with early lethality or a chronic disease with granuloma formation, caused by Mycobacterium marinum strains Mma20 and E11, respectively. We performed a microarray study of different stages of disease progression in adult zebrafish and found that the acute and the chronic strains evoked partially overlapping host transcriptome signatures, despite that they induce profoundly different disease phenotypes. Both strains affected many signaling cascades, including WNT and TLR pathways. Interestingly, the strongest differences were observed at the initial stage of the disease. The immediate response to the acute strain was characterized by higher expression of genes encoding MHC class I proteins, matrix metalloproteinases, transcription factors, cytokines and other common immune response proteins. In contrast, small GTPase and histone gene groups showed higher expression in response to the chronic strain. We also found that nearly 1000 mycobacterium-responsive genes overlapped between the expression signatures of infected zebrafish adults and embryos at different stages of granuloma formation. Since adult zebrafish possess an adaptive immune system similar to mammals and zebrafish embryos rely solely on innate immunity, this overlap indicates a major contribution of the innate component of the immune system in the response to mycobacterial infection. Taken together, our comparison of the transcriptome responses involved in acute versus chronic infections and in the embryonic versus adult situation provides important new leads for investigating the mechanism of mycobacterial pathogenesis.
The International Journal of Developmental Biology. 2009 | Pubmed ID: 19557689
Basic research in pattern formation is concerned with the generation of phenotypes and tissues. It can therefore lead to new tools for medical research. These include phenotypic screening assays, applications in tissue engineering, as well as general advances in biomedical knowledge. Our aim here is to discuss this emerging field with special reference to tools based on zebrafish developmental biology. We describe phenotypic screening assays being developed in our own and other labs. Our assays involve: (i) systemic or local administration of a test compound or drug to zebrafish in vivo; (ii) the subsequent detection or "readout" of a defined phenotypic change. A positive readout may result from binding of the test compound to a molecular target involved in a developmental pathway. We present preliminary data on assays for compounds that modulate skeletal patterning, bone turnover, immune responses, inflammation and early-life stress. The assays use live zebrafish embryos and larvae as well as adult fish undergoing caudal fin regeneration. We describe proof-of-concept studies on the localised targeting of compounds into regeneration blastemas using microcarriers. Zebrafish are cheaper to maintain than rodents, produce large numbers of transparent eggs, and some zebrafish assays could be scaled-up into medium and high throughput screens. However, advances in automation and imaging are required. Zebrafish cannot replace mammalian models in the drug development pipeline. Nevertheless, they can provide a cost-effective bridge between cell-based assays and mammalian whole-organism models.
Molecular Immunology. Sep, 2009 | Pubmed ID: 19631987
Novel high-throughput deep sequencing technology has dramatically changed the way that the functional complexity of transcriptomes can be studied. Here we report on the first use of this technology to gain insight into the wide range of transcriptional responses that are associated with an infectious disease process. Using Solexa/Illumina's digital gene expression (DGE) system, a tag-based transcriptome sequencing method, we investigated mycobacterium-induced transcriptome changes in a model vertebrate species, the zebrafish. We obtained a sequencing depth of over 5 million tags per sample with strong correlation between replicates. Tag mapping indicated that healthy and infected adult zebrafish express over 70% of all genes represented in transcript databases. Comparison of the data with a previous multi-platform microarray analysis showed that both types of technologies identified regulation of similar functional groups of genes. However, the unbiased nature of DGE analysis provided insights that microarray analysis could not have achieved. In particular, we show that DGE data sets are instrumental for verification of predicted gene models and allowed us to detect mycobacterium-regulated switching between different transcript isoforms. Moreover, genomic mapping of infection-induced DGE tags revealed novel transcript forms for which any previous EST-based evidence of expression was lacking. In conclusion, our deep sequencing analysis revealed in depth the high degree of transcriptional complexity of the host response to mycobacterial infection and resulted in the discovery and validation of new gene products with induced expression in infected individuals.
Genome Biology. 2009 | Pubmed ID: 19646228
Addiction is a pathological dysregulation of the brain's reward systems, determined by several complex genetic pathways. The conditioned place preference test provides an evaluation of the effects of drugs in animal models, allowing the investigation of substances at a biologically relevant level with respect to reward. Our lab has previously reported the development of a reliable conditioned place preference paradigm for zebrafish. Here, this test was used to isolate a dominant N-ethyl-N-nitrosourea (ENU)-induced mutant, no addiction (nad(dne3256)), which fails to respond to amphetamine, and which we used as an entry point towards identifying the behaviorally relevant transcriptional response to amphetamine.
Single-molecule Microscopy Reveals Membrane Microdomain Organization of Cells in a Living Vertebrate
Biophysical Journal. Aug, 2009 | Pubmed ID: 19686669
It has been possible for several years to study the dynamics of fluorescently labeled proteins by single-molecule microscopy, but until now this technology has been applied only to individual cells in culture. In this study, it was extended to stem cells and living vertebrate organisms. As a molecule of interest we used yellow fluorescent protein fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. We used a wide-field fluorescence microscopy setup to visualize individual molecules in a zebrafish cell line (ZF4) and in primary embryonic stem cells. A total-internal-reflection microscopy setup was used for imaging in living organisms, in particular in epidermal cells in the skin of 2-day-old zebrafish embryos. Our results demonstrate the occurrence of membrane microdomains in which the diffusion of membrane proteins in a living organism is confined. This membrane organization differed significantly from that observed in cultured cells, illustrating the relevance of performing single-molecule microscopy in living organisms.
Temporal Expression of Hepatic Estrogen Receptor 1, Vitellogenin1 and Vitellogenin2 in European Silver Eels
General and Comparative Endocrinology. Mar, 2010 | Pubmed ID: 19766647
Because European silver eels have never been caught during or after their 6000-km reproductive migration to the Sargasso Sea, all existing knowledge on their sexual maturation comes from hormonal stimulation. Silver eels that start their oceanic migration are still immature with pre-vitellogenic oocytes. Hence we assumed that vitellogenesis should start with the expression of the estrogen receptor in the liver before the circulating 17beta-estradiol (E2) can have any effect. In this study we followed the hepatic vitellogenesis upon 4 weekly injections with carp pituitary extracts (CPE). New molecular primers for the expression of the estrogen receptor 1 (esr1), vitellogenin1 (vtg1) and vitellogenin2 (vtg2) in the liver were developed. Sequences of vtg2 and esr1 were not previously described in Anguilla anguilla. All eels showed weekly increase of the eye size and pectoral fin length, which are signs of early maturation. The same occurred with the gonadosomatic index, the oocyte stage and diameter, and number of deposited fat droplets. Early vitellogenesis appeared as a 3-step process (1) E2-levels and esr1 expression were significantly increased already after one injection, (2) vtg1 and vtg2 expression were significantly increased after one and two injections, respectively, and (3) vtg1 and vtg2 expression increased further after three and four injections. Then also plasma calcium (corresponds with plasma vitellogenin) increased and yolk globuli appeared in the oocytes. These results show that esr1 is the first of the three genes examined that is expressed during the onset of hepatic vitellogenesis. Furthermore, ovarian vitellogenesis (appearance of yolk globuli in oocytes) occurs 1-2 weeks later than the onset of hepatic vitellogenesis.
Identification of Hoxb1b Downstream Genes: Hoxb1b As a Regulatory Factor Controlling Transcriptional Networks and Cell Movement During Zebrafish Gastrulation
The International Journal of Developmental Biology. 2010 | Pubmed ID: 19876844
Hox proteins are homeobox containing transcription factors that play important roles in patterning the presumptive central nervous system and the axial mesoderm in the early vertebrate embryo. Hox genes are first expressed during gastrula stages and recent studies suggest that their function goes beyond their role as patterning determinants. To improve our understanding of the role of Hox proteins during early vertebrate development, we designed a strategy to identify target genes of the zebrafish hoxb1b using overexpression and whole-genome microarray analysis. We directly compared the hoxb1b microarray data with those resulting from heterologous over-expression of the Xenopus XhoxD1 gene in zebrafish embryos. Both genes are the first expressed hox genes in their respective native embryos and display similar spatial expression patterns. The zebrafish transcriptome was analysed prior to the start of the expression of the endogenous hoxb1b gene and we observed extensive overlap between the hoxb1b and XhoxD1 putative downstream genes suggesting evolutionary functional conservation between these hox genes. Furthermore, genes encoding transcription factors and proteins that are known to be involved in cell adhesion and movement were over-represented among the candidate downstream genes, indicating the involvement of the developmentally earliest expressed hox genes in transcriptional networks and cell movement processes.
BMC Research Notes. 2010 | Pubmed ID: 20233395
Transcriptome analysis during embryogenesis usually requires pooling of embryos to obtain sufficient RNA. Hence, the measured levels of gene-expression represent the average mRNA levels of pooled samples and the biological variation among individuals is confounded. This can irreversibly reduce the robustness, resolution, or expressiveness of the experiment. Therefore, we developed a robust method to isolate abundant high-quality RNA from individual embryos to perform single embryo transcriptome analyses using zebrafish as a model organism. Available methods for embryonic zebrafish RNA isolation minimally utilize ten embryos. Further downscaling of these methods to one embryo is practically not feasible.
Swimming Suppresses Hepatic Vitellogenesis in European Female Silver Eels As Shown by Expression of the Estrogen Receptor 1, Vitellogenin1 and Vitellogenin2 in the Liver
Reproductive Biology and Endocrinology : RB&E. 2010 | Pubmed ID: 20302623
When European silver eels (Anguilla anguilla) venture into the Atlantic Ocean for their 6,000 km semelparous spawning run to the Sargasso Sea, they are still in a prepubertal stage. Further sexual development appears to be blocked by dopaminergic inhibition of hypothalamus and pituitary activity. Recently, we found that swimming for several weeks in freshwater stimulated the incorporation of fat droplets in the oocytes. So, it was hypothesized that long term swimming in seawater would release the inhibition further and would also stimulate the production of vitellogenin by the liver.
Blood. Jul, 2010 | Pubmed ID: 20424185
The Spi1/Pu.1 transcription factor plays a crucial role in myeloid cell development in vertebrates. Despite extensive studies of Spi1, the controlled gene group remains largely unknown. To identify genes dependent on Spi1, we used a microarray strategy using a knockdown approach in zebrafish embryos combined with fluorescence-activated cell sorting of myeloid cells from transgenic embryos. This approach of using knockdowns with specific green fluorescent protein-marked cell types was highly successful in identifying macrophagespecific genes in Spi1-directed innate immunity. We found a gene group downregulated on spi1 knockdown, which is also enriched in fluorescence-activated cell-sorted embryonic myeloid cells of a spi1:GFP transgenic line. This gene group, representing putative myeloidspecific Spi1 target genes, contained all 5 previously identified Spi1-dependent zebrafish genes as well as a large set of novel immune-related genes. Colocalization studies with neutrophil and macrophage markers revealed that genes cxcr3.2, mpeg1, ptpn6, and mfap4 were expressed specifically in early embryonic macrophages. In a functional approach, we demonstrated that gene cxcr3.2, coding for chemokine receptor 3.2, is involved in macrophage migration to the site of bacterial infection. Therefore, based on our combined transcriptome analyses, we discovered novel early macrophage-specific marker genes, including a signal transducer pivotal for macrophage migration in the innate immune response.
Zebrafish. Jun, 2010 | Pubmed ID: 20515295
Zebrafish cancer models are fast gaining ground in cancer research. Most tumors in zebrafish develop late in life, when fish are no longer transparent, limiting in vivo optical imaging methods. Thus, noninvasive imaging to track tumor in adult zebrafish remains challenging. In this study, we applied magnetic resonance microimaging (microMRI) to track spontaneous melanomas in stable transgenic zebrafish models expressing an RAS oncoprotein and lacking P53 (mitf:Ras::mitf:GFP X p53(-/-)). Tumors in live adult zebrafish were observed at various locations using a T(2)-weighted fast spin echo sequence at 9.4 T. Further, live imaging of tumors at ultrahigh field (17.6 T) revealed significant tumor heterogeneity. This heterogeneity was also confirmed by the significant differences in transverse relaxation time, T(2) measured in various regions of tumor. To our knowledge, this is the first report demonstrating the application of microMRI to detect the locations, invasion status, and characteristics of internal melanomas in zebrafish and suggesting that noninvasive microMRI can be applied for longitudinal studies to track tumor development and real-time assessment of therapeutic effects in zebrafish tumor models.
Integrating Heterogeneous Sequence Information for Transcriptome-wide Microarray Design; a Zebrafish Example
BMC Research Notes. 2010 | Pubmed ID: 20626891
A complete gene-expression microarray should preferably detect all genomic sequences that can be expressed as RNA in an organism, i.e. the transcriptome. However, our knowledge of a transcriptome of any organism still is incomplete and transcriptome information is continuously being updated. Here, we present a strategy to integrate heterogeneous sequence information that can be used as input for an up-to-date microarray design.
Molecular Immunology. Nov-Dec, 2010 | Pubmed ID: 20851470
TRAF6 is a key player at the cross-roads of development and immunity. The analysis of its in vivo molecular function is a great challenge since severe developmental defects and early lethality caused by Traf6 deficiency in knock-out mice interfere with analyses of the immune response. In this study we have used a new strategy to analyze the function of Traf6 in a zebrafish-Salmonella infectious disease model. In our approach the effect of a Traf6 translation-blocking morpholino was titrated down to avoid developmental defects and the response to infection under these conditions was studied using the combination of microarray analysis and whole transcriptome deep sequencing. Transcriptome profiling of the traf6 knock-down allowed the identification of a gene set whose responsiveness during infection is highly dependent on Traf6. Expression trend analysis based on the resulting datasets identified nine clusters of genes with characteristic transcription response profiles, demonstrating Traf6 has a dynamic role as a positive and negative regulator. Among the Traf6-dependent genes was a large set of well known anti-microbial and inflammatory genes. Additionally, we identified several genes which were not previously linked to a response to microbial infection, such as the fertility hormone gene gnrh2 and the DNA-damage regulated autophagy modulator 1 gene dram1. With the use of the zebrafish embryo model we have now analyzed the in vivo function of Traf6 in the innate immune response without interference of adaptive immunity.
Establishing Zebrafish As a Novel Exercise Model: Swimming Economy, Swimming-enhanced Growth and Muscle Growth Marker Gene Expression
PloS One. 2010 | Pubmed ID: 21217817
Zebrafish has been largely accepted as a vertebrate multidisciplinary model but its usefulness as a model for exercise physiology has been hampered by the scarce knowledge on its swimming economy, optimal swimming speeds and cost of transport. Therefore, we have performed individual and group-wise swimming experiments to quantify swimming economy and to demonstrate the exercise effects on growth in adult zebrafish.
The Epigenetic Regulator Histone Deacetylase 1 Promotes Transcription of a Core Neurogenic Programme in Zebrafish Embryos
BMC Genomics. 2011 | Pubmed ID: 21226904
The epigenetic regulator Histone Deacetylase 1 (Hdac1) is required for specification and patterning of neurones and myelinating glia during development of the vertebrate central nervous system (CNS). This co-ordinating function for Hdac1 is evolutionarily conserved in zebrafish and mouse, but the mechanism of action of Hdac1 in the developing CNS is not well-understood.
Current Drug Targets. Jun, 2011 | Pubmed ID: 21366518
The zebrafish holds much promise as a high-throughput drug screening model for immune-related diseases, including inflammatory and infectious diseases and cancer. This is due to the excellent possibilities for in vivo imaging in combination with advanced tools for genomic and large scale mutant analysis. The context of the embryo's developing immune system makes it possible to study the contribution of different immune cell types to disease progression. Furthermore, due to the temporal separation of innate immunity from adaptive responses, zebrafish embryos and larvae are particularly useful for dissecting the innate host factors involved in pathology. Recent studies have underscored the remarkable similarity of the zebrafish and human immune systems, which is important for biomedical applications. This review is focused on the use of zebrafish as a model for infectious diseases, with emphasis on bacterial pathogens. Following a brief overview of the zebrafish immune system and the tools and methods used to study host-pathogen interactions in zebrafish, we discuss the current knowledge on receptors and downstream signaling components that are involved in the zebrafish embryo's innate immune response. We summarize recent insights gained from the use of bacterial infection models, particularly the Mycobacterium marinum model that illustrate the potential of the zebrafish model for high-throughput antimicrobial drug screening.
PloS One. 2011 | Pubmed ID: 21390204
One-third of the world population is infected with Mycobacterium tuberculosis and multi-drug resistant strains are rapidly evolving. The noticeable absence of a whole organism high-throughput screening system for studying the progression of tuberculosis is fast becoming the bottleneck in tuberculosis research. We successfully developed such a system using the zebrafish Mycobacterium marinum infection model, which is a well-characterized model for tuberculosis progression with biomedical significance, mimicking hallmarks of human tuberculosis pathology. Importantly, we demonstrate the suitability of our system to directly study M. tuberculosis, showing for the first time that the human pathogen can propagate in this vertebrate model, resulting in similar early disease symptoms to those observed upon M. marinum infection. Our system is capable of screening for disease progression via robotic yolk injection of early embryos and visual flow screening of late-stage larvae. We also show that this system can reliably recapitulate the standard caudal vein injection method with a throughput level of 2,000 embryos per hour. We additionally demonstrate the possibility of studying signal transduction leading to disease progression using reverse genetics at high-throughput levels. Importantly, we use reference compounds to validate our system in the testing of molecules that prevent tuberculosis progression, making it highly suited for investigating novel anti-tuberculosis compounds in vivo.
BMC Developmental Biology. 2011 | Pubmed ID: 21396126
Studies on artificial hybridization of different Anguilla species were conducted recently, i.e. female A. australis with male A. dieffenbachii, and female A. japonica with male A. anguilla. The existence of these artificial hybrids was however not demonstrated by independent genetic methods. Two species - A. anguilla and A. australis - that are phylogenetically close but have different sexual maturation times (12-25 weeks and 6-8 weeks, respectively), were expected to produce favourable hybrids for reproduction studies.
Integrative Biology : Quantitative Biosciences from Nano to Macro. Jun, 2011 | Pubmed ID: 21541374
Receptor internalization upon ligand stimulation is a key component of a cell's response and allows a cell to correctly sense its environment. Novel fluorescent methods have enabled the direct visualization of the agonist-stimulated G-protein-coupled receptors (GPCR) trafficking in living cells. However, it is difficult to observe internalization of GPCRs in vivo due to intrinsic autofluorescence and cytosolic signals of fluorescently labeled GPCRs. This study uses the superior positional accuracy of single-molecule fluorescence microscopy to visualize in real time the internalization of Dictyostelium discoideum cAMP receptors, cAR1, genetically encoded with eYFP. This technique made it possible to follow the number of receptors in time revealing that the fraction of cytosolic receptors increases after persistent agonist stimulation and that the majority of the receptors were degraded after internalization. The observed internalization process was phosphorylation dependent, as shown with the use of a phosphorylation deficient cAR1 mutant, cm1234-eYFP, or stimulation with an antagonist, Rp-cAMPS that does not induce receptor phosphorylation. Furthermore, experiments done in mound-stage cells suggest that intrinsic, phosphorylation-induced internalization of cAR1 is necessary for Dictyostelium wild type cells to progress properly through multicellular development. To our knowledge, this observation illustrates for the first time phosphorylation-dependent internalization of single cAR1 molecules in living cells and its involvement in multicellular development. This very sensitive imaging of receptor internalization can be a useful and universal approach for pharmacological characterization of GPCRs in other cell types.
Birth Defects Research. Part C, Embryo Today : Reviews. Jun, 2011 | Pubmed ID: 21671352
Technological innovation has helped the zebrafish embryo gain ground as a disease model and an assay system for drug screening. Here, we review the use of zebrafish embryos and early larvae in applied biomedical research, using selected cases. We look at the use of zebrafish embryos as disease models, taking fetal alcohol syndrome and tuberculosis as examples. We discuss advances in imaging, in culture techniques (including microfluidics), and in drug delivery (including new techniques for the robotic injection of compounds into the egg). The use of zebrafish embryos in early stages of drug safety-screening is discussed. So too are the new behavioral assays that are being adapted from rodent research for use in zebrafish embryos, and which may become relevant in validating the effects of neuroactive compounds such as anxiolytics and antidepressants. Readouts, such as morphological screening and cardiac function, are examined. There are several drawbacks in the zebrafish model. One is its very rapid development, which means that screening with zebrafish is analogous to "screening on a run-away train." Therefore, we argue that zebrafish embryos need to be precisely staged when used in acute assays, so as to ensure a consistent window of developmental exposure. We believe that zebrafish embryo screens can be used in the pre-regulatory phases of drug development, although more validation studies are needed to overcome industry scepticism. Finally, the zebrafish poses no challenge to the position of rodent models: it is complementary to them, especially in early stages of drug research.
Rapid Screening of Innate Immune Gene Expression in Zebrafish Using Reverse Transcription - Multiplex Ligation-dependent Probe Amplification
BMC Research Notes. 2011 | Pubmed ID: 21676242
With the zebrafish increasingly being used in immunology and infectious disease research, there is a need for efficient molecular tools to evaluate immune gene expression in this model species. RT-MLPA (reverse transcription - multiplex ligation-dependent probe amplification) provides a sensitive and reproducible method, in which fluorescently labelled amplification products of unique lengths are produced for a defined set of target transcripts. The method employs oligonucleotide probes that anneal to adjacent sites on a target sequence and are then joined by a heat-stable ligase. Subsequently, multiplex PCR with universal primers gives rise to amplicons that can be analyzed with standard sequencing equipment and relative quantification software. Allowing the simultaneous quantification of around 40 selected markers in a one-tube assay, RT-MLPA is highly useful for high-throughput screening applications.
A ΔRaf1-ER-inducible Oncogenic Zebrafish Liver Cell Model Identifies Hepatocellular Carcinoma Signatures
The Journal of Pathology. Sep, 2011 | Pubmed ID: 21744342
Although the underlying molecular mechanism of hepatocellular carcinoma remains unclear, signalling pathways essential in cell survival and growth are altered, including the Raf-MEK-MAPK pathway. This pathway can be activated by hepatitis B or C virus infections and the ectopic expression of the Raf-1 oncogene is frequently seen in hepatocellular carcinomas. In addition, the Raf-MEK-MAPK pathway was also shown to be deregulated in zebrafish liver tumours. Based on the genetic conservation between zebrafish and human liver tumours, the zebrafish was used as an animal model to better understand the molecular basis of hepatocellular carcinoma. Here we establish an inducible oncogenic zebrafish cell model, in which oncogenic human Raf-1(ΔRaf1) can be post-transcriptionally activated in zebrafish liver cells by administration of 4-hydroxytamoxifen (4HT). The ΔRaf1 activation resulted in the hyperactivation of the zebrafish MEK-ERK cascade, promoted cell growth and proliferation, and inhibited apoptosis. The mitogenic transformation of the ZFL-ΔRaf1-ER cells was confirmed by in vivo allo-transplantation and in silico microarray analyses. Gene expression profiling of cells treated with 4HT and a MEK-inhibitor identified a Raf-MEK-dependent signature set. This transcriptome response was compared to zebrafish and human liver cancer transcriptomes. We identified, and validated by quantitative PCR, a set of genes transcriptionally regulated by hyperactive MAPK signalling in ZFL-ΔRaf1-ER cells, zebrafish liver tumours and human liver tumours, suggesting that the in vitro zebrafish liver cell model can be used for further study of the molecular basis of human hepatocellular carcinoma. The molecular targeting of the commonly regulated hepatocellular carcinoma genes using the ZFL-ΔRaf1-ER cell model can be applied for high-throughput preclinical target discovery.
Journal of Integrative Bioinformatics. 2011 | Pubmed ID: 21908900
The common carp is a candidate model system for immunology research. Using next-generation sequencing technology, we have generated a huge amount of sequence reads from the carp genome and transcriptome. Currently, our aim is to identify carp genes involved in the development of the innate immune response, particularly TIR domain-containing genes, from a preliminary genome assembly. To achieve this, we developed a comprehensive gene identification pipeline. This analysis allowed us to estimate that the carp has 39 TIR domain-containing transcript isoforms and genes.
Methods in Cell Biology. 2011 | Pubmed ID: 21951535
The major cell types of the innate immune system, macrophages and neutrophils, develop during the first two days of zebrafish embryogenesis. The interaction of these immune cells with pathogenic microbes can excellently be traced in the optically transparent zebrafish embryos. Various tools and methods have recently been developed for visualizing and isolating the zebrafish embryonic innate immune cells, for establishing infections by different micro-injection techniques, and for analyzing the host innate immune response following microbial recognition. Here we provide practical guidelines for the application of these methodologies and review the current state of the art in zebrafish infectious disease research.
Comparison of Static Immersion and Intravenous Injection Systems for Exposure of Zebrafish Embryos to the Natural Pathogen Edwardsiella Tarda
BMC Immunology. 2011 | Pubmed ID: 22003892
The zebrafish embryo is an important in vivo model to study the host innate immune response towards microbial infection. In most zebrafish infectious disease models, infection is achieved by micro-injection of bacteria into the embryo. Alternatively, Edwardsiella tarda, a natural fish pathogen, has been used to treat embryos by static immersion. In this study we used transcriptome profiling and quantitative RT-PCR to analyze the immune response induced by E. tarda immersion and injection.
Deep Sequencing of the Innate Immune Transcriptomic Response of Zebrafish Embryos to Salmonella Infection
Fish & Shellfish Immunology. Nov, 2011 | Pubmed ID: 20816807
Salmonella enterica serovar Typhimurium (S. typhimurium) bacteria cause an inflammatory and lethal infection in zebrafish embryos. To characterize the embryonic innate host response at the transcriptome level, we have extended and validated previous microarray data by Illumina next-generation sequencing analysis. We obtained 10 million sequence reads from control and Salmonella-infected zebrafish embryos using a tag-based sequencing method (DGE or Tag-Seq) and 15 million reads using whole transcript sequencing (RNA-Seq), which respectively mapped to circa 65% and 85% of 28,716 known Ensembl transcripts. Both sequencing methods showed a strong correlation of sequence read counts per transcript and an overlap of 241 transcripts differentially expressed in response to infection. A lower overlap of 165 transcripts was observed with previous microarray data. Based on the combined sequencing-based and microarray-based transcriptome data we compiled an annotated reference set of infection-responsive genes in zebrafish embryos, encoding transcription factors, signal transduction proteins, cytokines and chemokines, complement factors, proteins involved in apoptosis and proteolysis, proteins with anti-microbial activities, as well as many known or novel proteins not previously linked to the immune response. Furthermore, by comparison of the deep sequencing data of S. typhimurium infection in zebrafish embryos with previous deep sequencing data of Mycobacterium marinum infection in adult zebrafish we derived a common set of infection-responsive genes. This gene set consists of known and putative innate host defense genes that are expressed both in the absence and presence of a fully developed adaptive immune system and that provide a valuable reference for future studies of host-pathogen interactions using zebrafish infection models.
An Osteosarcoma Zebrafish Model Implicates Mmp-19 and Ets-1 As Well As Reduced Host Immune Response in Angiogenesis and Migration
The Journal of Pathology. Feb, 2012 | Pubmed ID: 22297719
About 40% of osteosarcoma patients die of metastases. Novel strategies to improve treatment of metastatic patients require a better understanding of the processes involved, like angiogenesis, migration and the immune response. However the rarity of osteosarcoma and its heterogeneity make this neoplasm difficult to study. Recently we reported malignant transformation of mouse mesenchymal stem cells (MSCs) which formed osteosarcoma upon transplantation into mice. Here we studied these cells in zebrafish embryos and found that transformed MSCs induced angiogenesis and migrated through the bodies of the embryos, but this was never observed with non-transformed normal MSCs (progenitors of the transformed MSCs). Whole genomic expression analysis of both the cells and the host showed that angiogenesis and migration related genes matrix metalloproteinase 19 (Mmp-19) and erythroblastosis virus E26 oncogene homologue 1 (Ets-1) were over-expressed in transformed MSCs compared to normal MSCs. Investigating the host response, embryos injected with transformed MSCs showed decreased expression of immune related genes, especially major histocompatibility complex class 1 (mhc1ze), as compared to embryos injected with normal MSCs. These findings contribute to the identification of genetic events involved in angiogenesis, migration and host response providing targets as well as an appropriate model for high-throughput drug screens. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
PloS One. 2012 | Pubmed ID: 22347456
A quantitative bio-imaging platform is developed for analysis of human cancer dissemination in a short-term vertebrate xenotransplantation assay. Six days after implantation of cancer cells in zebrafish embryos, automated imaging in 96 well plates coupled to image analysis algorithms quantifies spreading throughout the host. Findings in this model correlate with behavior in long-term rodent xenograft models for panels of poorly- versus highly malignant cell lines derived from breast, colorectal, and prostate cancer. In addition, cancer cells with scattered mesenchymal characteristics show higher dissemination capacity than cell types with epithelial appearance. Moreover, RNA interference establishes the metastasis-suppressor role for E-cadherin in this model. This automated quantitative whole animal bio-imaging assay can serve as a first-line in vivo screening step in the anti-cancer drug target discovery pipeline.
Neutrophil-mediated Experimental Metastasis is Enhanced by VEGFR Inhibition in a Zebrafish Xenograft Model
The Journal of Pathology. Feb, 2012 | Pubmed ID: 22374800
Inhibition of VEGF signaling effectively suppresses localized tumor growth but accelerates tumor invasiveness and micrometastasis by unknown mechanisms. To study the dynamic and reciprocal interactions between tumor cells and their microenvironment during these processes, we established a xenograft model by injecting tumor cells into the blood circulation of transparent zebrafish embryos. This reproducibly results in rapid simultaneous formation of a localized tumor and experimental micrometastasis, allowing time-resolved imaging of both processes at single-cell resolution within one week. The tumor vasculature was initiated de novo by remodeling of primitive endothelial cells into a functional network. Roles of myeloid cells in critical tumorigenesis steps such as vascularization and invasion were revealed by genetic and pharmaceutical approaches. We discovered that the physiological migration of neutrophils controlled tumor invasion by conditioning the collagen matrix and forming the metastatic niche, as detected by two-photon confocal microscopy and second harmonic generation. Administration of VEGFR inhibitors blocked tumor vascularization and a localized tumor growth but enhanced migration of neutrophils, which in turn promoted tumor invasion and formation of micrometastasis. This demonstrates the in vivo cooperation between VEGF signaling and myeloid cells in metastasis and provides a new mechanism underlying the recent findings that VEGFR targeting can promote tumor invasiveness. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
PloS One. 2012 | Pubmed ID: 22384188
The enigmatic life cycle and elongated body of the European eel (Anguilla anguilla L., 1758) have long motivated scientific enquiry. Recently, eel research has gained in urgency, as the population has dwindled to the point of critical endangerment. We have assembled a draft genome in order to facilitate advances in all provinces of eel biology. Here, we use the genome to investigate the eel's complement of the Hox developmental transcription factors. We show that unlike any other teleost fish, the eel retains fully populated, duplicate Hox clusters, which originated at the teleost-specific genome duplication. Using mRNA-sequencing and in situ hybridizations, we demonstrate that all copies are expressed in early embryos. Theories of vertebrate evolution predict that the retention of functional, duplicate Hox genes can give rise to additional developmental complexity, which is not immediately apparent in the adult. However, the key morphological innovation elsewhere in the eel's life history coincides with the evolutionary origin of its Hox repertoire.