Developmental Regulation and Downstream Effects of the Knox Class Homeobox Genes Oskn2 and Oskn3 from Rice

Plant Molecular Biology. Mar, 2002  |  Pubmed ID: 11908517

Plant homeobox genes of the class 1 knox (knotted1-like) type are involved in the regulation of shoot apical meristem formation and function. Their expression generally occurs either throughout the meristem or specifically at the lateral organ boundaries. Down-regulation in the organ primordia is tightly controlled and misexpression in leaves leads to a perturbed development. Here, the transcriptional control of two rice knox genes, Oskn2 and Oskn3, was addressed, showing that the promoter sequences of both genes mediate the initial down-regulation during lateral organ formation, but are insufficient to keep expression in lateral organs stably off. Therefore, maintenance of the repressed state requires control elements outside the promoter regions. Ectopic expression of Oskn2 or Oskn3 induced similar defects in panicle branching. internode elongation and leaf patterning. However, small differences suggested that their target gene specificities are not completely overlapping. This was supported by the observation that Oskn3 protein but not Oskn2 could interact with two reported recognition sequences of a KNOX protein from barley. Finally, protein-protein interactions may contribute to the functioning of KNOX proteins, as the ability of Oskn3 and Oskn2 to form heterodimers could be demonstrated.

Characterization of Five Novel Dehydration-responsive Homeodomain Leucine Zipper Genes from the Resurrection Plant Craterostigma Plantagineum

Plant Molecular Biology. Aug, 2002  |  Pubmed ID: 12081368

Homeodomain leucine zipper (HDZip) genes encode putative transcription factors that are unique to plants. A function in regulating processes that are specific for plants is postulated, such as responses to environmental cues and developmental signals. This is supported by a growing body of evidence resulting from studies of HDZip genes in a variety of species. In addition to the previously isolated CPHB-1 and -2 genes, this paper reports the isolation of members of five families of Craterostigma plantagineum homeobox leucine zipper genes (CPHB) via a yeast one-hybrid screening approach. Based on the sequence homology and protein interactions the encoded proteins (CPHB-3/4/5/6/7) were classified into HDZip class II and I genes. Homo- and heterodimerization of CPHB proteins within the same structurally related class has been demonstrated and the DNA-binding activity of CPHB proteins to two homeodomain recognition elements (HDE1 and HDE2) has been compared in yeast. All families of CPHB genes were modulated in their expression in response to dehydration in leaves and roots. CPHB-6 and CPHB-7 transcripts accumulated in leaves during early stages of dehydration and decreased after prolonged dehydration. Both transcripts were also induced in ABA-treated callus. CPHB-3/4/5 were down-regulated by dehydration in both leaves and roots. The results support the role of HDZips in regulating programs of gene expression in C. plantagineum that lead to desiccation tolerance.

The Procambium Specification Gene Oshox1 Promotes Polar Auxin Transport Capacity and Reduces Its Sensitivity Toward Inhibition

Plant Physiology. Nov, 2002  |  Pubmed ID: 12428000

The auxin-inducible homeobox gene Oshox1 of rice (Oryza sativa) is a positive regulator of procambial cell fate commitment, and its overexpression reduces the sensitivity of polar auxin transport (PAT) to the PAT inhibitor 1-N-naphthylphthalamic acid (NPA). Here, we show that wild-type rice leaves formed under conditions of PAT inhibition display vein hypertrophy, reduced distance between longitudinal veins, and increased distance between transverse veins, providing experimental evidence for a role of PAT in vascular patterning in a monocot species. Furthermore, we show that Oshox1 overexpression confers insensitivity to these PAT inhibitor-induced vascular-patterning defects. Finally, we show that in the absence of any overt phenotypical change, Oshox1 overexpression specifically reduces the affinity of the NPA-binding protein toward NPA and enhances PAT and its sensitivity toward auxin. These results are consistent with the hypothesis that Oshox1 promotes fate commitment of procambial cells by increasing their auxin conductivity properties and stabilizing this state against modulations of PAT by an endogenous NPA-like molecule.

The RADICLELESS1 Gene is Required for Vascular Pattern Formation in Rice

Development (Cambridge, England). Feb, 2003  |  Pubmed ID: 12505996

The molecular mechanisms through which the complex patterns of plant vascular tissues are established are largely unknown. The highly ordered, yet simple, striate array of veins of rice leaves represents an attractive system to study the dynamics underlying pattern formation. Here we show that mutation in the RADICLELESS1 (RAL1) gene results in distinctive vascular pattern defects. In ral1 embryonic scutella, secondary veins are absent and in the prematurely aborted and discontinuous primary veins, cells are misaligned to each other. In ral1 leaves, longitudinal and commissural (transverse) veins display altered spacing and the commissural veins additionally show atypical branching and interruptions in their continuity. The vascular pattern alterations of ral1 occur in the context of normally shaped leaf primordia. Anatomical inspection and analysis of the expression of the procambium specification marker Oshox1-GUS and of the auxin-inducible reporter DR5-GUS demonstrates that all the vascular patterning aberrations of ral1 originate from defects in the procambium, which represents the earliest identifiable stage of vascular development. Furthermore, the ral1 mutant is unique in that procambium formation in leaf primordium development is delayed. Finally, the ral1 vascular patterning distortions are associated with a defective response to auxin and with an enhanced sensitivity to cytokinin. ral1 is the first mutant impaired in both procambium development and vascular patterning to be isolated in a monocot species.

Expression Analysis of the Toll-like Receptor and TIR Domain Adaptor Families of Zebrafish

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.

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.

Gene Expression and Digit Homology in the Chicken Embryo Wing

Evolution & Development. Jan-Feb, 2005  |  Pubmed ID: 15642086

The bird wing is of special interest to students of homology and avian evolution. Fossil and developmental data give conflicting indications of digit homology if a pentadactyl "archetype" is assumed. Morphological signs of a vestigial digit I are seen in bird embryos, but no digit-like structure develops in wild-type embryos. To examine the developmental mechanisms of digit loss, we studied the expression of the high-mobility group box containing Sox9 gene, and bone morphogenetic protein receptor 1b (bmpR-1b)-markers for precondensation and prechondrogenic cells, respectively. We find an elongated domain of Sox9 expression, but no bmpR-1b expression, anterior to digit II. We interpret this as a digit I domain that reaches precondensation, but not condensation or precartilage stages. It develops late, when the tissue in which it is lodged is being remodeled. We consider these findings in the light of previous Hoxd-11 misexpression studies. Together, they suggest that there is a digit I vestige in the wing that can be rescued and undergo development if posterior patterning cues are enhanced. We observed Sox9 expression in the elusive "element X" that is sometimes stated to represent a sixth digit. Indeed, incongruity between digit domains and identities in theropods disappears if birds and other archosaurs are considered primitively polydactyl. Our study provides the first gene expression evidence for at least five digital domains in the chick wing. The failure of the first to develop may be plausibly linked to attenuation of posterior signals.

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.

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.

Characterization of a Polyclonal Antiserum Against the Monoterpene Monooxygenase, Geraniol 10-hydroxylase from Catharanthus Roseus

Journal of Plant Physiology. Apr, 2005  |  Pubmed ID: 15900881

Geraniol 10-hydroxylase (G10H) is a P450 containing enzyme which is the first committed step in the biosynthesis of monoterpene indole alkaloids (MIAs), including the Catharanthus roseus-anticancer drugs vinblastine and vincristine. It is thought that G10H has a regulatory role in MIA production. In the present paper, we report the characterization of a polyclonal serum raised against the purified G10H polypeptide. Anti-G10H IgG was able to inhibit the G10H activity and also recognized the G10H polypeptide from C. roseus and other plants producing MIAs. These results establish the usefulness of this antiserum as a biochemical tool for the study of G10H regulation.

Multiple Regulatory Elements Contribute to the Vascular-specific Expression of the Rice HD-Zip Gene Oshox1 in Arabidopsis

Plant & Cell Physiology. Aug, 2005  |  Pubmed ID: 15964905

The primary vascular tissues of plants differentiate from a single precursor tissue, the procambium. The role of upstream regulatory sequences in the transcriptional control of early vascular-specific gene expression is largely unknown. The onset of expression of the rice homeodomain-leucine zipper (HD-Zip) gene Oshox1 marks procambial cells that have acquired their distinctive anatomical features but do not yet display any overt signs of terminal vascular differentiation. The expression pattern of Oshox1 in rice appears to be mainly controlled by the activity of the 1.6 kb upstream promoter region. Here, we show that the Oshox1 promoter directs vascular, auxin- and sucrose-responsive reporter gene expression in Arabidopsis plants in a fashion comparable with that in rice. This is the case not only during normal development but also upon experimental manipulation, suggesting that the cis-acting regulatory elements that are instrumental in Oshox1 expression pattern are conserved between rice and Arabidopsis. Finally, through analysis of reporter gene expression profiles conferred by progressive 5' deletions of the Oshox1 promoter in transgenic Arabidopsis, we have identified upstream regulatory regions required for auxin and sucrose inducibility, and for cell type-, tissue- and organ-specific aspects of Oshox1 expression. Our study suggests that Oshox1 embryonic vascular expression is mainly achieved through suppression of expression in non-vascular tissues.

EU-OSTID: a Collection of Transposon Insertional Mutants for Functional Genomics in Rice

Plant Molecular Biology. Sep, 2005  |  Pubmed ID: 16217605

A collection of 1373 unique flanking sequence tags (FSTs), generated from Ac/Ds and Ac transposon lines for reverse genetics studies, were produced in japonica and indica rice, respectively. The Ds and Ac FSTs together with the original T-DNAs were assigned a position in the rice genome sequence represented as assembled pseudomolecules, and found to be distributed evenly over the entire rice genome with a distinct bias for predicted gene-rich regions. The bias of the Ds and Ac transposon inserts for genes was exemplified by the presence of 59% of the inserts in genes annotated on the rice chromosomes and 41% present in genes transcribed as disclosed by their homology to cDNA clones. In a screen for inserts in a set of 75 well annotated transcription factors, including homeobox-containing genes, we found six Ac/Ds inserts. This high frequency of Ds and Ac inserts in genes suggests that saturated knockout mutagenesis in rice using this strategy will be efficient and possible with a lower number of inserts than expected. These FSTs and the corresponding plant lines are publicly available through OrygenesDB database and from the EU consortium members.

MyD88 Innate Immune Function in a Zebrafish Embryo Infection Model

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.

A Homeodomain Leucine Zipper Gene from Craterostigma Plantagineum Regulates Abscisic Acid Responsive Gene Expression and Physiological Responses

Plant Molecular Biology. Jun, 2006  |  Pubmed ID: 16830180

A subset of homeodomain leucine zipper proteins (HDZip) play a role in regulating adaptation responses including developmental adjustment to environmental cues in plants. Here we report the structural and functional characterisation of a dehydration responsive nuclear-targeted HDZip transcriptional regulator, CpHB-7. DNA-protein interaction studies suggest that CDeT6-19, a known ABA and dehydration responsive dehydrin gene, is a potential target gene of CpHB-7 in the desiccation-tolerant plant Craterostigma plantagineum. Transgenic plants that ectopically express CpHB-7 display reduced sensitivity towards ABA during seed germination and stomatal closure. Expression analysis reveals that genes with induced or repressed expression in CpHB-7 ectopic expression lines are either mostly repressed or induced by ABA, drought or salt treatment respectively, thus demonstrating that CpHB-7 modifies ABA-responsive gene expression as a negative regulator. CpHB-7 gene expression is also linked to early organ development, leading to the suggestion that CpHB-7 is functionally similar to the Arabidopsis transcription factor, ATHB-6.

Genetic and Transcriptome Characterization of Model Zebrafish Cell Lines

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.

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

Distinct Functions for ERK1 and ERK2 in Cell Migration Processes During Zebrafish Gastrulation

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.

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.

A Genome-wide Survey of HD-Zip Genes in Rice and Analysis of Drought-responsive Family Members

Plant Molecular Biology. Jan, 2008  |  Pubmed ID: 17999151

The homeodomain leucine zipper (HD-Zip) genes encode transcription factors that have diverse functions in plant development and have often been implicated in stress adaptation. The HD-Zip genes are the most abundant group of homeobox (HB) genes in plants and do not occur in other eukaryotes. This paper describes the complete annotation of the HD-Zip families I, II and III from rice and compares these gene families with Arabidopsis in a phylogeny reconstruction. Orthologous pairs of rice and Arabidopsis HD-Zip genes were predicted based on neighbour joining and maximum parsimony (MP) trees with support of conserved intron-exon organization. Additionally, a number of HD-Zip genes appeared to be unique to rice. Searching of EST and cDNA databases and expression analysis using RT-PCR showed that 30 out of 31 predicted rice HD-Zip genes are expressed. Most HD-Zip genes were broadly expressed in mature plants and seedlings, but others showed more organ specific patterns. Like in Arabidopsis and other dicots, a subset of the rice HD-Zip I and II genes was found to be regulated by drought stress. We identified both drought-induced and drought-repressed HD-Zip genes and demonstrate that these genes are differentially regulated in drought-sensitive versus drought-tolerant rice cultivars. The drought-repressed HD-Zip family I gene, Oshox4, was selected for promoter-GUS analysis, showing that drought-responsiveness of Oshox4 is controlled by the promoter and that Oshox4 expression is predominantly vascular-specific. Loss-of-function analysis of Oshox4 revealed no specific phenotype, but overexpression analysis suggested a role for Oshox4 in elongation and maturation processes.

Discovery of a Functional Glucocorticoid Receptor Beta-isoform in Zebrafish

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.

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.

Zebrafish Development and Regeneration: New Tools for Biomedical Research

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.

Deep Sequencing of the Zebrafish Transcriptome Response to Mycobacterium Infection

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.

Zebrafish Reward Mutants Reveal Novel Transcripts Mediating the Behavioral Effects of Amphetamine

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.

Bell-like Homeodomain Selectively Regulates the High-irradiance Response of Phytochrome A

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2009  |  Pubmed ID: 19666535

Plant responses mediated by phytochrome A display a first phase saturated by transient light signals and a second phase requiring sustained excitation with far-red light (FR). These discrete outcomes, respectively so-called very-low-fluence response (VLFR) and high-irradiance response (HIR), are appropriate in different environmental and developmental contexts but the mechanisms that regulate the switch remain unexplored. Promoter analysis of a light-responsive target gene revealed a motif necessary for HIR but not for VLFR. This motif is required for binding of the Bell-like homeodomain 1 (BLH1) to the promoter in in vitro and in yeast 1-hybrid experiments. Promoter substitutions that increased BLH1 binding also enhanced HIR. blh1 mutants showed reduced responses to continuous FR and to deep canopy shadelight, but they retained normal responses to pulsed FR or red light and unfiltered sunlight. BLH1 enhanced BLH1 expression and its promotion by FR. We conclude that BLH1 specifically regulates HIR and not VLFR of phytochrome A.

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).

Burkholderia Cenocepacia Creates an Intramacrophage Replication Niche in Zebrafish Embryos, Followed by Bacterial Dissemination and Establishment of Systemic Infection

Infection and Immunity. Apr, 2010  |  Pubmed ID: 20086083

Bacteria belonging to the "Burkholderia cepacia complex" (Bcc) often cause fatal pulmonary infections in cystic fibrosis patients, yet little is know about the underlying molecular mechanisms. These Gram-negative bacteria can adopt an intracellular lifestyle, although their ability to replicate intracellularly has been difficult to demonstrate. Here we show that Bcc bacteria survive and multiply in macrophages of zebrafish embryos. Local dissemination by nonlytic release from infected cells was followed by bacteremia and extracellular replication. Burkholderia cenocepacia isolates belonging to the epidemic electrophoretic type 12 (ET12) lineage were highly virulent for the embryos; intravenous injection of <10 bacteria of strain K56-2 killed embryos within 3 days. However, small but significant differences between the clonal ET12 isolates K56-2, J2315, and BC7 were evident. In addition, the innate immune response in young embryos was sufficiently developed to control infection with other less virulent Bcc strains, such as Burkholderia vietnamiensis FC441 and Burkholderia stabilis LMG14294. A K56-2 cepR quorum-sensing regulator mutant was highly attenuated, and its ability to replicate and spread to neighboring cells was greatly reduced. Our data indicate that the zebrafish embryo is an excellent vertebrate model to dissect the molecular basis of intracellular replication and the early innate immune responses in this intricate host-pathogen interaction.

Macrophage-specific Gene Functions in Spi1-directed Innate Immunity

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.

Transcriptome Analysis of Traf6 Function in the Innate Immune Response of Zebrafish Embryos

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.

Yeast One-hybrid Screens for Detection of Transcription Factor DNA Interactions

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 20931383

The yeast one-hybrid system is widely recognized as a valuable and straightforward technique to study interactions between transcription factors and DNA. By means of one-hybrid screens, transcription factors or other DNA-binding proteins, expressed from cDNA expression libraries, can be identified due to the interactions with a DNA sequence-of-interest that is linked to a reporter gene, such as the yeast HIS3 gene. Usually, the library is constructed in an E. coli-yeast shuttle vector designed for production of hybrid proteins consisting of a library protein and the trans-activating domain (AD) from the yeast GAL4 transcription factor. Here, we describe an optimized system of vectors for one-hybrid screenings together with detailed step-wise protocols, an elaborate trouble-shooting guide and many technical tips to conduct successful screenings. This system and other yeast genetic selection procedures derived from one-hybrid methodology proved highly useful to help understanding the regulatory networks controlling expression of the genome.

Host-pathogen Interactions Made Transparent with the Zebrafish Model

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.

A High-throughput Screen for Tuberculosis Progression

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.

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.

Infectious Disease Modeling and Innate Immune Function in Zebrafish Embryos

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.

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.