Abstract: Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed protein kinase C substrate that has emerged as a potential therapeutic target for amelioration of mucin secretion and inflammation in patients with chronic obstructive pulmonary disease. MARCKS also plays a key role in regulation of neutrophil adhesion, migration and degranulation. Given its biological role in epithelial and immune cells, we hypothesized that MARCKS may play an integral role in cytokine secretion by neutrophils. As the amino terminus of MARCKS is highly conserved across vertebrate species, we successfully applied the well-characterized human MARCKS inhibitory peptide, MANS, to attenuate MARCKS function in isolated canine neutrophils. Pretreatment of canine neutrophils with MANS peptide significantly reduced both mRNA and protein expression of a broad range of LPS-induced cytokines, including IL-8, a CXCL1 orthologue and TNF-α, in comparison to untreated cells or those treated with a control peptide. This reduction in cytokine expression was observed even when neutrophils were treated with MANS 2h after LPS exposure. The observed reduction in cytokine secretion was not due to protein retention nor cell death, but was associated with reduced cytokine transcript synthesis. These observations identify MARCKS protein as a promising therapeutic target in treatment of inflammatory diseases or syndromes attributed to neutrophil influx and inflammatory cytokine production such as sepsis, acute lung injury (ALI) and acute respiratory distress syndrome (ARDS).
Abstract: The novel immune-type receptors (NITRs), which have been described in numerous bony fish species, are encoded by multigene families of inhibitory and activating receptors and are predicted to be functional orthologs to the mammalian natural killer cell receptors (NKRs). Within the zebrafish NITR family, nitr9 is the only gene predicted to encode an activating receptor. However, alternative RNA splicing generates three distinct nitr9 transcripts, each of which encodes a different isoform. Although nitr9 transcripts have been detected in zebrafish lymphocytes, the specific hematopoietic lineage(s) that expresses Nitr9 remains to be determined. In an effort to better understand the role of NITRs in zebrafish immunity, anti-Nitr9 monoclonal antibodies were generated and evaluated for the ability to recognize the three Nitr9 isoforms. The application of these antibodies to flow cytometry should prove to be useful for identifying the specific lymphocyte lineages that express Nitr9 and may permit the isolation of Nitr9-expressing cells that can be directly assessed for cytotoxic (e.g., NK) function.
Abstract: To uncover the molecular mechanisms of embryonic development, the ideal loss-of-function strategy would be capable of targeting specific regions of the living embryo with both temporal and spatial precision. To this end, we have developed a novel pharmacological agent that can be light activated to achieve spatiotemporally limited inhibition of Rho kinase activity in vivo. A new photolabile caging group, 6-nitropiperonyloxymethyl (NPOM), was installed on a small-molecule inhibitor of Rho kinase, Rockout, to generate a 'caged Rockout' derivative. Complementary biochemical, cellular, molecular and morphogenetic assays in both mammalian cell culture and Xenopus laevis embryos validate that the inhibitory activity of the caged compound is dependent on exposure to light. Conveniently, this unique reagent retains many of the practical advantages of conventional small-molecule inhibitors, including delivery by simple diffusion in the growth medium and concentration-dependent tuneability, but can be locally activated by decaging with standard instrumentation. Application of this novel tool to the spatially heterogeneous problem of embryonic left-right asymmetry revealed a differential requirement for Rho signaling on the left and right sides of the primitive gut tube, yielding new insight into the molecular mechanisms that generate asymmetric organ morphology. As many aromatic/heterocyclic small-molecule inhibitors are amenable to installation of this caging group, our results indicate that photocaging pharmacological inhibitors might be a generalizable technique for engendering convenient loss-of-function reagents with great potential for wide application in developmental biology.
Abstract: A heretofore-unrecognized multigene family encoding diverse immunoglobulin (Ig) domain-containing proteins (DICPs) was identified in the zebrafish genome. Twenty-nine distinct loci mapping to three chromosomal regions encode receptor-type structures possessing two classes of Ig ectodomains (D1 and D2). The sequence and number of Ig domains, transmembrane regions and signaling motifs vary between DICPs. Interindividual polymorphism and alternative RNA processing contribute to DICP diversity. Molecular models indicate that most D1 domains are of the variable (V) type; D2 domains are Ig-like. Sequence differences between D1 domains are concentrated in hypervariable regions on the front sheet strands of the Ig fold. Recombinant DICP Ig domains bind lipids, a property shared by mammalian CD300 and TREM family members. These findings suggest that novel multigene families encoding diversified immune receptors have arisen in different vertebrate lineages and affect parallel patterns of ligand recognition that potentially impact species-specific advantages.
Abstract: Devices containing III–V semiconductors such as InAs are increasingly being used in the electronic industry for a variety of optoelectronic applications. Furthermore, the attractive chemical, material, electronic properties make such materials appealing for use in devices designed for biological applications, such as biosensors. However, in biological applications the leaching of toxic materials from these devices could cause harm to cells or tissue. Additionally, after disposal, toxic inorganic materials can leach from devices and buildup in the environment, causing long-term ecological harm. Therefore, the toxicity of these materials along with their stability in physiological conditions are important factors to consider. Surface modifications are one common method of stabilizing semiconductor materials in order to chemically and electronically passivate them. Such surface modifications could also prevent the leaching of toxic materials by preventing the regrowth of the unstable surface oxide layer and by creating an effective barrier between the semiconductor surface and the surrounding environment. In this study, various surface modifications on InAs are developed with the goal of decreasing the leaching of indium and arsenic. The leaching of indium and arsenic from modified substrates was assessed in physiological conditions using inductively coupled plasma mass spectrometry (ICP-MS). Substrates modified with 11-mercapto-1-undecanol (MU) and graft polymerized with poly(ethylene) glycol (PEG) were most effective at preventing indium and arsenic leaching. These surfaces were characterized using contact angle analysis, ellipsometry, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). Substrates modified with collagen and synthetic polyelectrolytes were least effective, due to the destructive nature of acidic environments on InAs. The toxicity of modified and unmodified InAs, along with raw indium, arsenic, and PEG components was assessed using zebrafish embryos.
Abstract: Natural killer (NK) cells affect a form of innate immunity that recognizes and eliminates cells that are infected with certain viruses or have undergone malignant transformation. In mammals, this recognition can be mediated through immunoglobulin- (Ig) and/or lectin-type NK receptors (NKRs). NKR genes in mammals range from minimally polymorphic single-copy genes to complex multigene families that exhibit high levels of haplotypic complexity and exhibit significant interspecific variation. Certain single-copy NKR genes that are present in one mammal are present as expanded multigene families in other mammals. These observations highlight NKRs as one of the most rapidly evolving eukaryotic gene families and likely reflect the influence of pathogens, especially viruses, on their evolution. Although well characterized in human and mice, cytotoxic cells that are functionally similar to NK cells have been identified in species ranging from birds to reptiles, amphibians and fish. Although numerous receptors have been identified in non-mammalian vertebrates that share structural relationships with mammalian NKRs, functionally defining these lower vertebrate molecules as NKRs is confounded by methodological and interpretive complexities. Nevertheless, several lines of evidence suggest that NK-type function or its equivalent has sustained a long evolutionary history throughout vertebrate species.
Abstract: The dog is both a valued veterinary species and a widely used translational model for sepsis research. However, relatively little work has been performed evaluating potential biomarkers present during canine infection. Triggering receptor expressed on myeloid cells-1 (TREM-1) has shown promise as a biomarker for infection and pneumonia in humans. Here we describe, for the first time, the expression and function of the canine orthologue of TREM-1. Expression of TREM-1 on canine neutrophils is significantly up-regulated by stimulation with microbial agonists of TLR2/6, TLR1/2, and TLR4/MD2. Kinetics of TREM-1 protein up-regulation are rapid, with significant increases observed within 2 hr of neutrophil activation. Functionally, canine TREM-1 synergistically enhances LPS-induced production of IL-8, TNF-α and a canine orthologue of CXCL1. Collectively, these data suggest that TREM-1 expression in dogs, as it is in humans, is an amplifier of pro-inflammatory responses to microbial products. These results have direct application to veterinary diagnostics as well as the potential to enhance the utility of canine disease models in the assessment of potential therapeutics in the treatment of human sepsis.
Abstract: The Sp family of transcription factors is required for the expression of cell cycle- and developmentally regulated genes, and the deregulated expression of a handful of family members is associated with human tumorigenesis. Sp2 is a relatively poorly characterized member of the Sp family that, although widely expressed, exhibits little or no DNA binding or transcriptional activity in human and mouse cell lines. To begin to address the role(s) played by Sp2 in early metazoan development we have cloned and characterized Sp2 from zebrafish (Danio rerio). We report that 1) the intron/exon organization and amino acid sequence of zebrafish Sp2 is closely conserved with its mammalian orthologues, 2) zebrafish Sp2 weakly stimulates an Sp-dependent promoter in vitro and associates with the nuclear matrix in a DNA-independent fashion, 3) zebrafish Sp2 is inherited as a maternal transcript, is transcribed in zebrafish embryos and adult tissues, and is required for completion of gastrulation, and 4) zebrafish lines carrying transgenes regulated by the Sp2 promoter recapitulate patterns of endogenous Sp2 expression.
Abstract: Novel immune-type receptors (NITRs) are encoded by large multi-gene families and share structural and signaling similarities to mammalian natural killer receptors (NKRs). NITRs have been identified in multiple bony fish species, including zebrafish, and may be restricted to this large taxonomic group. Thirty-nine NITR genes that can be classified into 14 families are encoded on zebrafish chromosomes 7 and 14. Herein, we demonstrate the expression of multiple NITR genes in the zebrafish ovary and during embryogenesis. All 14 families of zebrafish NITRs are expressed in hematopoietic kidney, spleen and intestine as are immunoglobulin and T cell antigen receptors. Furthermore, all 14 families of NITRs are shown to be expressed in the lymphocyte lineage, but not in the myeloid lineage, consistent with the hypothesis that NITRs function as NKRs. Sequence analyses of NITR amplicons identify known alleles and reveal additional alleles within the nitr1, nitr2, nitr3, and nitr5 families, reflecting the recent evolution of this gene family.
Abstract: Morpholino oligonucleotides, or morpholinos, have emerged as powerful antisense reagents for evaluating gene function in both in vitro and in vivo contexts. However, the constitutive activity of these reagents limits their utility for applications that require spatiotemporal control, such as tissue-specific gene disruptions in embryos. Here we report a novel and efficient synthetic route for incorporating photocaged monomeric building blocks directly into morpholino oligomers and demonstrate the utility of these caged morpholinos in the light-activated control of gene function in both cell culture and living embryos. We demonstrate that a caged morpholino that targets enhanced green fluorescent protein (EGFP) disrupts EGFP production only after exposure to UV light in both transfected cells and living zebrafish (Danio rerio) and Xenopus frog embryos. Finally, we show that a caged morpholino targeting chordin, a zebrafish gene that yields a distinct phenotype when functionally disrupted by conventional morpholinos, elicits a chordin phenotype in a UV-dependent manner. Our results suggest that photocaged morpholinos are readily synthesized and highly efficacious tools for light-activated spatiotemporal control of gene expression in multiple contexts.
Abstract: Cystic fibrosis (CF) is a genetic disease caused by recessive mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is associated with prevalent and chronic Pseudomonas aeruginosa lung infections. Despite numerous studies that have sought to elucidate the role of CFTR in the innate immune response, the links between CFTR, innate immunity, and P. aeruginosa infection remain unclear. The present work highlights the zebrafish as a powerful model organism for human infectious disease, particularly infection by P. aeruginosa. Zebrafish embryos with reduced expression of the cftr gene (Cftr morphants) exhibited reduced respiratory burst response and directed neutrophil migration, supporting a connection between cftr and the innate immune response. Cftr morphants were infected with P. aeruginosa or other bacterial species that are commonly associated with infections in CF patients, including Burkholderia cenocepacia, Haemophilus influenzae, and Staphylococcus aureus. Intriguingly, the bacterial burden of P. aeruginosa was found to be significantly higher in zebrafish Cftr morphants than in controls, but this phenomenon was not observed with the other bacterial species. Bacterial burden in Cftr morphants infected with a P. aeruginosa ΔLasR mutant, a quorum sensing-deficient strain, was comparable to that in control fish, indicating that the regulation of virulence factors through LasR is required for enhancement of infection in the absence of Cftr. The zebrafish system provides a multitude of advantages for studying the pathogenesis of P. aeruginosa and for understanding the role that innate immune cells, such as neutrophils, play in the host response to acute bacterial infections commonly associated with cystic fibrosis.
Abstract: Novel immune-type receptors (NITRs) are encoded by clusters of multigene families and have been identified in multiple bony fish species. All NITRs possess one extracellular immunoglobulin (Ig) domain of the variable (V) type and recent crystal structures of NITR V domains demonstrate their high degree of similarity to V domains of antigen receptors. Many NITRs possess a second extracellular Ig domain of the intermediate (I) type which helps differentiate NITRs from other V domain receptors. The majority of NITRs are type I transmembrane receptors; however, a small number are predicted to encode secreted proteins. Based on their sequence and structure, NITRs have been proposed to be "functional orthologs" of mammalian natural killer receptors (NKRs). Like NKRs, most NITRs possess short functional motifs permitting their classification as inhibitory or activating. NITRs lacking these motifs are functionally ambiguous. Inhibitory and activating NKRs utilize opposing signaling mechanisms to influence the response of NK cells to target cells; studies employing recombinant NITRs suggest that these signaling pathways are conserved between NKRs and NITRs. An analysis of all published NITR sequences demonstrates the conserved nature of multiple residues within the NITR Ig domains permitting the identification of NITR ESTs from salmon, cod, halibut, lake whitefish and stickleback species. Complete data sets of NITRs from the sequencing of the zebrafish and medaka genomes provide insight into the evolution of the NITRs within bony fish species. It is likely that all teleost species encode NITRs which function within innate immunity to regulate cell mediated cytotoxicity.
Abstract: A recessive nonsense mutation in the zebrafish recombination activating gene 1 (rag1) gene results in defective V(D)J recombination; however, animals homozygous for this mutation (rag1(-/-)) are reportedly viable and fertile in standard, nonsterile aquarium conditions but display increased mortality after intraperitoneal injection with mycobacteria. Based on their survival in nonsterile environments, we hypothesized that the rag1(-/-) zebrafish may possess an "enhanced" innate immune response to compensate for the lack of an adaptive immune system. To test this hypothesis, microarray analyses were used to compare the expression profiles of the intestines and hematopoietic kidneys of rag1 deficient zebrafish to the expression profiles of control (heterozygous) siblings. The expression levels of 12 genes were significantly altered in the rag1(-/-) kidney including the up regulation of a putative interferon stimulated gene, and the down regulation of genes encoding fatty acid binding protein 10, keratin 5 and multiple heat shock proteins. The expression levels of 87 genes were shown to be significantly altered in the rag1(-/-) intestine; the majority of these differences reflect increased expression of innate immune genes, including those of the coagulation and complement pathways. Subsequent analyses of orthologous coagulation and complement genes in Rag1(-/-) mice indicate increased transcription of the complement C4 gene in the Rag1(-/-) intestine.
Abstract: Large granular lymphocyte (LGL) leukemia, or LGLL, is characterized by increased numbers of circulating clonal LGL cells in association with neutropenia, anemia, rheumatoid arthritis, and pulmonary artery hypertension (PAH). Emerging evidence suggests that LGLL cells with a CD8(+)CD28(null) phenotype induce these clinical manifestations through direct destruction of normal tissue. Compared with CD8(+)CD28(null) T cells from healthy controls, CD8(+)CD28(null) T cells from LGLL patients have acquired the ability to directly lyse pulmonary artery endothelial cells and human synovial cells. Here, we show that LGLL cells from patients possess enhanced cytotoxic characteristics and express elevated levels of activating natural killer receptors as well as their signaling partners, DAP10 and DAP12. Moreover, downstream targets of DAP10 and DAP12 are constitutively activated in LGLL cells, and expression of dominant-negative DAP10 and DAP12 dramatically reduces their lytic capacity. These are the first results to show that activating NKR-ligand interactions play a critical role in initiating the DAP10 and DAP12 signaling events that lead to enhanced lytic potential of LGLL cells. Results shown suggest that inhibitors of DAP10 and DAP12 or other proteins involved in this signaling pathway will be attractive therapeutic targets for the treatment of LGLL and other autoimmune diseases and syndromes.
Abstract: Novel immune-type receptors (NITRs) are immunoglobulin-variable (V) domain-containing cell surface proteins that possess characteristic activating/inhibitory signaling motifs and are expressed in hematopoietic cells. NITRs are encoded by multigene families and have been identified in bony fish species. A single gene cluster, which encodes 36 NITRs that can be classified into 12 families, has been mapped to zebrafish chromosome 7. We report herein the presence of a second NITR gene cluster on zebrafish chromosome 14, which is comprised of three genes (nitr13, nitr14a, and nitr14b) representing two additional NITR gene families. Phylogenetic analyses indicate that the V domains encoded by the nitr13 and nitr14 genes are more similar to each other than any other zebrafish NITR suggesting that these genes arose from a tandem gene duplication event. Similar analyses comparing zebrafish Nitr13 and Nitr14 to NITRs from other fish species indicate that the nitr13 and nitr14 genes are phylogenetically related to the catfish IpNITR13 and IpNITR15 genes. Sequence features of the chromosomal region encoding nitr13 suggest that this gene arose via retrotransposition.
Abstract: Large granular lymphocyte (LGL) leukemia is commonly associated with poor hematopoiesis. The first case of pulmonary artery hypertension (PAH) was observed in a 57-year-old woman with natural killer (NK)-LGL leukemia and transfusion-dependent anemia. Using a genetic approach, we demonstrated that killing of pulmonary endothelial cells by patient NK cells was mediated by dysregulated balance in activating and inhibitory NK-receptor signaling. Elevated pulmonary artery pressure and erythroid differentiation improved after disrupting the NK-receptor signaling pathway with 4 courses of a farnesyltransferase inhibitor, tipifarnib. Coincidental association between PAH and LGL leukemia suggest a causal relationship between the expanded lymphocyte population and these clinical manifestations. This trial is registered at www.ClinicalTrials.gov as NCI 6823.
Abstract: BACKGROUND: Novel immune-type receptor (NITR) genes are members of diversified multigene families that are found in bony fish and encode type I transmembrane proteins containing one or two extracellular immunoglobulin (Ig) domains. The majority of NITRs can be classified as inhibitory receptors that possess cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). A much smaller number of NITRs can be classified as activating receptors by the lack of cytoplasmic ITIMs and presence of a positively charged residue within their transmembrane domain, which permits partnering with an activating adaptor protein. RESULTS: Forty-four NITR genes in medaka (Oryzias latipes) are located in three gene clusters on chromosomes 10, 18 and 21 and can be organized into 24 families including inhibitory and activating forms. The particularly large dataset acquired in medaka makes direct comparison possible to another complete dataset acquired in zebrafish in which NITRs are localized in two clusters on different chromosomes. The two largest medaka NITR gene clusters share conserved synteny with the two zebrafish NITR gene clusters. Shared synteny between NITRs and CD8A/CD8B is limited but consistent with a potential common ancestry. CONCLUSION: Comprehensive phylogenetic analyses between the complete datasets of NITRs from medaka and zebrafish indicate multiple species-specific expansions of different families of NITRs. The patterns of sequence variation among gene family members are consistent with recent birth-and-death events. Similar effects have been observed with mammalian immunoglobulin (Ig), T cell antigen receptor (TCR) and killer cell immunoglobulin-like receptor (KIR) genes. NITRs likely diverged along an independent pathway from that of the somatically rearranging antigen binding receptors but have undergone parallel evolution of V family diversity.
Abstract: The antigen combining sites of immunoglobulin (Ig) and T cell antigen receptors (TCRs), which are present in all jawed vertebrates, consist of a paired variable (V) domain heterodimer that exhibits varying degrees of germline- and extraordinarily high levels of somatically-derived variation. The near limitless variation in receptor specificity on the surface of individual lymphocytes is the basis for clonal selection in the adaptive immune response. A basic question arises as to whether or not there are other forms of immune-type receptors in vertebrates as well as in invertebrates that derive immune specificity through sequence differences in V domains. Our laboratory has discovered two such families of molecules, the novel immune-type receptors and the variable region-containing chitin-binding proteins. Both families of molecules encode V domains that share some characteristics of adaptive immune receptors but likely mediate innate functions.
Abstract: Both inhibitory and activating forms of natural killer (NK) cell receptors are found in mammals. The activating receptors play a direct role in the recognition of virally infected or transformed cells and transduce activating signals into the cell by partnering with an adaptor protein, which contains a cytoplasmic activation motif. Activating NK receptors encoded by the mammalian leukocyte receptor complex (e.g., killer cell immunoglobulin-like receptors) and the natural killer complex (e.g., Ly49s) partner with the adaptor protein DAP12, whereas NK receptors encoded in the CD94/NKG2 complex partner with the adaptor protein DAP10. Novel immune-type receptors (NITRs) found in bony fish share several common features with immunoglobulin-type NK receptors. Nitr9 is a putative activating receptor in zebrafish that induces cytotoxicity within the context of human NK cells. One isoform of Nitr9, Nitr9L, is shown here to preferentially partner with a zebrafish ortholog of Dap12. Cross-linking the Nitr9L-Dap12 complex results in activation of the phosphytidylinositol 3-kinase-->AKT-->extracellular signal-regulated kinase pathway suggesting that the DAP12-based activating pathway is conserved between bony fish and mammals.
Abstract: Transmembrane bound receptors comprised of extracellular immunoglobulin (Ig) or lectin domains play integral roles in a large number of immune functions including inhibitory and activating responses. The function of many of the activating receptors requires a physical interaction with an adaptor protein possessing a cytoplasmic regulatory motif. The partnering of an activating receptor with an adaptor protein relies on complementary charged residues in the two transmembrane domains. The mammalian natural killer (NK) and Fc receptors (FcR) represent two of many receptor families, which possess activating receptors that partner with adaptor proteins for signaling. Zebrafish represent a powerful experimental model for understanding developmental regulation at early stages of embryogenesis and for efficiently generating transgenic animals. In an effort to understand developmental aspects of immune receptor function, we have accessed the partially annotated zebrafish genome to identify six different adaptor molecules: Dap10, Dap12, Cd3zeta, Cd3zeta-like, FcRgamma and FcRgamma-like that are homologous to those effecting immune function in mammals. Their genomic organizations have been characterized, cDNA transcripts have been recovered, phylogenetic relationships have been defined and their cell lineage-specific expression patterns have been established.
Abstract: The sequence and the structure of DNA methyltransferase-2 (Dnmt2) bear close affinities to authentic DNA cytosine methyltransferases. A combined genetic and biochemical approach revealed that human DNMT2 did not methylate DNA but instead methylated a small RNA; mass spectrometry showed that this RNA is aspartic acid transfer RNA (tRNA(Asp)) and that DNMT2 specifically methylated cytosine 38 in the anticodon loop. The function of DNMT2 is highly conserved, and human DNMT2 protein restored methylation in vitro to tRNA(Asp) from Dnmt2-deficient strains of mouse, Arabidopsis thaliana, and Drosophila melanogaster in a manner that was dependent on preexisting patterns of modified nucleosides. Indirect sequence recognition is also a feature of eukaryotic DNA methyltransferases, which may have arisen from a Dnmt2-like RNA methyltransferase.
Abstract: The identification of C-type lectin (Group V) natural killer (NK) cell receptors in bony fish has remained elusive. Analyses of the Fugu rubripes genome database failed to identify Group V C-type lectin domains (Zelensky and Gready, BMC Genomics 5:51, 2004) suggesting that bony fish, in general, may lack such receptors. Numerous Group II C-type lectin receptors, which are structurally similar to Group V (NK) receptors, have been characterized in bony fish. By searching the zebrafish genome database we have identified a multi-gene family of Group II immune-related, lectin-like receptors (illrs) whose members possess inhibiting and/or activating signaling motifs typical of Group V NK receptors. Illr genes are differentially expressed in the myeloid and lymphoid lineages, suggesting that they may play important roles in the immune functions of multiple hematopoietic cell lineages.
Abstract: The looping events that establish left-right asymmetries in the vertebrate gut tube are poorly understood. Retinoic acid signaling is known to impact left-right development in multiple embryonic contexts, although its role in asymmetric digestive organ morphogenesis is unknown. Here, we show that the genes for retinaldehyde dehydrogenase (RALDH2) and a retinoic acid hydroxylase (CYP26A1) are expressed in complementary patterns in the Xenopus gut during looping. A late-stage chemical genetic assessment reveals that agonists and antagonists of retinoid signaling generate abnormal gut looping topologies, digestive organ heterotaxias, and intestinal malrotations. Accessory organ deformities commonly associated with intestinal malrotation in humans, such as annular pancreas, pancreas divisum, and extrahepatic biliary tree malformations, are also induced by distinct retinoid receptor agonists. Thus, late-stage retinoic acid signaling is likely to play a critical role in asymmetric gut tube morphogenesis and may underlie the etiology of several clinically relevant defects in the digestive system.
Abstract: The zebrafish has become a powerful tool for dissecting vertebrate gene function during embryogenesis. Numerous molecular systems have been developed to examine gene function in zebrafish, including transgenics for creating lineage-tracer lines of zebrafish that express a fluorescent protein as a marker for specific populations of cells, and antisense strategies, primarily morpholinos, for knocking down gene function. The focus of this review is to summarize the pros and cons of the currently available systems for functional genomics in zebrafish, and to discuss the need for future methodologies.
Abstract: The novel immune-type receptor (NITR) genes encode a unique multigene family of leukocyte regulatory receptors, which possess an extracellular Ig variable (V) domain and may function in innate immunity. Artificial chromosomes that encode zebrafish NITRs have been assembled into a contig spanning approximately 350 kb. Resolution of the complete NITR gene cluster has led to the identification of eight previously undescribed families of NITRs and has revealed the presence of C-type lectins within the locus. A maximum haplotype of 36 NITR genes (138 gene sequences in total) can be grouped into 12 distinct families, including inhibitory and activating receptors. An extreme level of interindividual heterozygosity is reflected in allelic polymorphisms, haplotype variation, and family-specific isoform complexity. In addition, the exceptional diversity of NITR sequences among species suggests divergent evolution of this multigene family with a birth-and-death process of member genes. High-confidence modeling of Nitr V-domain structures reveals a significant shift in the spatial orientation of the Ig fold, in the region of highest interfamily variation, compared with Ig V domains. These studies resolve a complete immune gene cluster in zebrafish and indicate that the NITRs represent the most complex family of activating/inhibitory surface receptors thus far described.
Abstract: Among members of the Ig superfamily (IgSF), antigen receptors have the unique capacity to rearrange their variable domains, thereby creating an extensive repertoire for antigen recognition. It is assumed that antigen receptors evolved from a non-rearranging IgSF member by insertion of a transposable element. Although the nature of this predecessor is unknown, two multigene families of innate immune receptors that bear a close structural resemblance to antigen receptor chains have been identified in mammals and bony fish, respectively: signal-regulatory proteins (SIRPs) and novel immune-type receptors (NITRs). Members of both families encode V-set Ig domains with a typical antigen receptor-like joining (J) motif and possess the potential to signal through immunoreceptor tyrosine-based inhibition motifs (ITIMs) or immunoreceptor tyrosine-based activation motifs (ITAMs). By analogy to the T-cell receptor (TCR) and certain innate receptors [e.g. killer cell inhibitory receptors (KIRs)] that recognize MHC molecules, SIRP members regulate immune function by interaction with broadly expressed 'self' ligands. We propose the existence of an evolutionary and functional link between innate and adaptive immune receptors that sheds light on the nature of the antigen receptor predecessor(s).
Abstract: Bony fish (teleosts) possess multiple cytotoxic cell lineages that recognize and destroy virally infected and transformed cells. In general, these lineages parallel their functional equivalents in mammals and include neutrophilic granulocytes, macrophages, cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. These four cell types have been morphologically identified in multiple fish species but only limited information is available about their function. In contrast, much work has gone into examining the function of a fifth cytotoxic cell lineage, termed nonspecific cytotoxic cells (NCC), that has been referred to as the bony fish equivalent of NK cells. However, evidence suggesting that NCC do not represent the NK lineage has come through the development of multiple cytotoxic catfish cell lines that are morphologically and functionally similar to human NK cells and are distinct from NCC. In addition to characterizing cytotoxic cells from fish, recent work has identified the novel immune-type receptors (NITR) and cichlid killer leukocyte receptors (cKLR) that are structurally related to mammalian NK receptors and likely play a role in cytotoxic function in fish. This review summarizes the morphological and functional evidence for cytotoxic cells within bony fish and discusses future directions for examining cytotoxicity through genomics and transgenics.
Abstract: Novel immune-type receptor ( NITR) genes that encode two extracellular immunoglobulin domains and cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been described previously in three lineages of bony fish. In the current study, four ITIM-containing NITR cDNAs are identified in the rainbow trout ( Oncorhynchus mykiss), and their expression patterns and genomic complexity are characterized. The ITIM-containing NITR2 gene maps 1.3 cM from an ITIM-containing C-type lectin receptor ( TCL-2) on linkage group XXI. A comprehensive, phylogenetic analysis of NITRs from rainbow trout and three other major lineages of bony fish defines conserved families of NITRs and suggests an ancient lineage of distinct groups of genes. Several probable scenarios that explain the origins of variant forms of NITRs are described.
Abstract: Two decades of research have established the zebrafish (Danio rerio) as a significant model system for studying vertebrate development and gene structure-function relationships. Recent advances in mutation screening, the creation of genomic resources, including the Zebrafish Genome Project and the development of efficient transgenesis procedures, make this model increasingly attractive for immunological study.
Abstract: A novel gene, BIVM (for basic, immunoglobulin-like variable motif-containing), has been identified using an electronic search based on the conservation of short sequence motifs within the variable region of immunoglobulin (Ig) genes. BIVM maps to human chromosome 13q32-q33 and is predicted to encode a 503-amino-acid protein with a pI of 9.1. The 5' untranslated region of BIVM is encoded in two exons; the coding portion is encoded in nine exons. BIVM is tightly linked (41 bp) and in the opposite transcriptional orientation to MGC5302 (also known as KDEL1 and EP58) in human. The ubiquitous expression of BIVM in normal tissues and the presence of a 5' CpG island suggest that BIVM is a housekeeping gene. Characterization of BIVM in representative species demonstrates significant conservation throughout deuterostomes; no sequence with significant identity to BIVM has been detected in proteostomes. However, an unusual gene has been identified in the protozoan pathogen Giardia lamblia that is similar to the core sequence of BIVM, suggesting the possibility of a horizontal gene transfer.
Abstract: Immune inhibitory receptor genes that encode a variable (V) region, a unique V-like C2 (V/C2) domain, a transmembrane region, and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been described previously in two lineages of bony fish. In the present study, eleven related genes encoding distinct structural forms have been identified in Ictalurus punctatus (channel catfish), a well characterized immunological model system that represents a third independent bony fish lineage. Each of the different genes encodes an N-terminal V region but differs in the number of extracellular Ig domains, number and location of joining (J) region-like motifs, presence of transmembrane regions, presence of charged residues in transmembrane regions, presence of cytoplasmic tails, and/or distribution of ITIM(s) within the cytoplasmic tails. Variation in the numbers of genomic copies of the different gene types, their patterns of expression, and relative levels of expression in mixed leukocyte cultures (MLC) is reported. V region-containing immune-type genes constitute a far more complex family than recognized originally and include individual members that might function in inhibitory or, potentially activatory manners.
Abstract: DNMT2 is a human protein that displays strong sequence similarities to DNA (cytosine-5)-methyltransferases (m(5)C MTases) of both prokaryotes and eukaryotes. DNMT2 contains all 10 sequence motifs that are conserved among m(5)C MTases, including the consensus S:-adenosyl-L-methionine-binding motifs and the active site ProCys dipeptide. DNMT2 has close homologs in plants, insects and Schizosaccharomyces pombe, but no related sequence can be found in the genomes of Saccharomyces cerevisiae or Caenorhabditis elegans. The crystal structure of a deletion mutant of DNMT2 complexed with S-adenosyl-L-homocysteine (AdoHcy) has been determined at 1.8 A resolution. The structure of the large domain that contains the sequence motifs involved in catalysis is remarkably similar to that of M.HHAI, a confirmed bacterial m(5)C MTase, and the smaller target recognition domains of DNMT2 and M.HHAI are also closely related in overall structure. The small domain of DNMT2 contains three short helices that are not present in M.HHAI. DNMT2 binds AdoHcy in the same conformation as confirmed m(5)C MTases and, while DNMT2 shares all sequence and structural features with m(5)C MTases, it has failed to demonstrate detectable transmethylase activity. We show here that homologs of DNMT2, which are present in some organisms that are not known to methylate their genomes, contain a specific target-recognizing sequence motif including an invariant CysPheThr tripeptide. DNMT2 binds DNA to form a denaturant-resistant complex in vitro. While the biological function of DNMT2 is not yet known, the strong binding to DNA suggests that DNMT2 may mark specific sequences in the genome by binding to DNA through the specific target-recognizing motif.
Abstract: Novel immune-type receptor (NITR) genes, which initially were identified in the Southern pufferfish (Spheroides nephelus), encode products which consist of an extracellular variable (V) and V-like C2 (V/C2) domain, a transmembrane region, and a cytoplasmic tail, which typically possesses an immunoreceptor tyrosine-based inhibition motif (ITIM). Multiple NITR genes have been identified in close, contiguous chromosomal linkage. The V regions of NITRs resemble prototypic forms defined for immunoglobulin (Ig) and T-cell antigen receptor (TCR), are present in multiple families and exhibit regionalized variation in sequence, which also occurs in Ig and TCR. Comparisons of exons encoding transmembrane and cytoplasmic regions of multiple NITRs suggest that exon shuffling has factored in the diversification of the NITR gene complex. Zebrafish (Danio rerio) NITRs exhibit many of these characteristics. NITRs that have been identified in additional species of bony fish demonstrate additional variation in the number of extracellular domains as well as in the presence of intramembranous charged residues, cytoplasmic tails and ITIMs. The presence in NITRs of V regions that are related closely to those found in Ig and TCR, as well as regulatory motifs and other structural features that are characteristic of immune inhibitory receptors encoded at the leukocyte receptor cluster, suggests that the NITRs are representative of an integral stage in the evolution of innate and adaptive immune function.
Abstract: An extensive, highly diversified multigene family of novel immune-type receptor (nitr) genes has been defined in Danio rerio (zebrafish). The genes are predicted to encode type I transmembrane glycoproteins consisting of extracellular variable (V) and V-like C2 (V/C2) domains, a transmembrane region and a cytoplasmic tail. All of the genes examined encode immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic tail. Radiation hybrid panel mapping and analysis of a deletion mutant line (b240) indicate that a minimum of approximately 40 nitr genes are contiguous in the genome and span approximately 0.6 Mb near the top of zebrafish linkage group 7. One flanking region of the nitr gene complex shares conserved synteny with a region of mouse chromosome 7, which shares conserved synteny with human 19q13.3-q13.4 that encodes the leukocyte receptor cluster. Antibody-induced crosslinking of Nitrs that have been introduced into a human natural killer cell line inhibits the phosphorylation of mitogen-activated protein kinase that is triggered by natural killer-sensitive tumor target cells. Nitrs likely represent intermediates in the evolution of the leukocyte receptor cluster.
Abstract: The zebrafish has become a well-established animal model for the analysis of development and of several disease phenotypes. Several of the favorable traits that make it a popular model organism would also be beneficial for the study of normal and abnormal vertebrate development in which DNA methylation may play a role. We report the determination of the full-length cDNA sequence corresponding to the zebrafish DNA (cytosine-5-) methyltransferase gene, Dnmt1. It is 4,907 bases long and has an open reading frame predicted to encode a 1,499 amino acid protein that is similar in size and sequence to a number of other methyltransferases identified in other organisms.
Abstract: Immunoglobulin gene diversity has been characterized to varying degrees in modern representatives of all of the major radiations of cartilaginous fish. A pattern of overall chromosomal relationships of the various types of joined and unjoined Ig gene clusters is suggested in which the essential features are: (a) both Ig heavy and light-chain gene clusters occur on multiple chromosomes, (b) various classes of Ig are interspersed, (c) not all individual gene loci appear to be closely linked (Fig. 2). The cluster-type Ig gene system appears to be a series of (potentially) individually regulated loci analogous in part to the olfactory receptor gene system (BUCK and AXEL 1991) and markedly distinct from Ig loci in other vertebrate groups and TCR genes. Such a system would be ideal for the creation of variation in both form and function in a large number of clusters while preserving or partially preserving specificity in a number of other gene clusters. The full range of joined genes and the relative number of joined genes (as relates to unjoined genes), have yet to be determined. Nevertheless, a number of conclusions can be drawn: (a) four distinct forms of heavy-chain joining have been identified (VDD-J, VD-DJ, V-D-DJ, and VDJ; Fig. 1); (b) light-chain genes, which possess only two recombining elements, can be found in either unjoined (V-J) or joined (VJ) forms (Fig. 1); (c) physical linkage between individual joined and unjoined genes has not been established, although such investigations have not been pursued in a significantly rigorous manner as to rule out this possibility; (d) joined light-chain genes are expressed and can be somatically mutated. Can germline joining be viewed as an ancestral character? The answer to this needs to be considered in the context of an overall system in which the level of structural and functional redundancy is extremely high. Joining is an adaptation that is unique to multicluster gene families. The phenomenon overcomes the possibility of not generating a specific form of a receptor, a major shortcoming of conventional rearranging Ig and TCR gene systems. The limitation of encoding specific receptors is compensated through large numbers of additional gene clusters that retain the capacity to rearrange and generate new specificities. Commitment of a V region to diverse, fixed specificity also is a property of the NITR genes, which although not related closely to Ig in a structural sense, may reflect an analogous phenomena. The possibility that immune-type diversity is achieved in the absence of somatic rearrangement and that remnants of such systems could be operative in immune recognition in contemporary vertebrates is of extraordinary significance in terms of our overall understanding of the relationships between adaptive and innate immune recognition.
Abstract: In addition to being an excellent model system for studying vertebrate development, the zebrafish has become a great tool for gene discovery by mutational analysis. The recent availability of the zebrafish EST database and radiation hybrid mapping panels has dramatically expanded the framework for genomic research in this species. Developing comparative maps of the zebrafish and human genomes is of particular importance for zebrafish mutagenesis studies in which human orthologs are sought for zebrafish genes. However, only partial cDNA sequences are determined routinely for mapped ESTs, leaving the identity of the EST in question. It previously had been reported that zebrafish linkage group 7 shares conserved synteny with human chromosome 11q13. In an effort to further define this relationship, five full-length zebrafish cDNAs, fth1, slc3a2, prkri, cd81, and pc, as well as one putative human gene, DBX were identified and their map positions ascertained. These six genes, along with men1, fgf3 and cycd1 define two regions of conserved synteny between linkage group 7 and 11q13.
Abstract: The immunoglobulin superfamily (IgSF) is an extensively diversified multigene family whose members share a common structural feature, the Ig fold. Members of the Ig/T-cell antigen receptor (TCR) subset of the IgSF mediate antigen-specific recognition in adaptive immune responses. Antigen-binding receptors belonging to this subset are present in all species of jawed vertebrates. To explore whether there are additional structurally related but otherwise distinct members of this subset, we have developed a technique termed the short-primer polymerase chain reaction (PCR) that targets structurally conserved short motifs in the Ig fold. Large-scale sequencing efforts and recent advances in information biotechnology, including "electronic PCR," provide additional computational means to implement similarly directed searches within databases. The use of these approaches has led to the discoveries of Ig/TCR homologues in a variety of phylogenetically diverse organisms, a diversified family of novel immune-type receptor genes, as well as a novel human IgSF member. The potential of random sequencing efforts and virtual screening of databases is described in the context of two novel genes in bony fish. The various methodologies that are discussed and the examples shown provide means for further investigating, and/or elucidating novel, IgSF receptors as well as components of pathways that are involved in immune responses in both traditional and nontraditional model systems.
Abstract: The spermatozoon and oocyte genomes bear sex-specific methylation patterns that are established during gametogenesis and are required for the allele-specific expression of imprinted genes in somatic tissues. The mRNA for Dnmt1, the predominant maintenance and de novo DNA (cytosine-5)-methyl transferase in mammals, is present at high levels in postmitotic murine germ cells but undergoes alternative splicing of sex-specific 5' exons, which controls the production and localization of enzyme during specific stages of gametogenesis. An oocyte-specific 5' exon is associated with the production of very large amounts of active Dnmt1 protein, which is truncated at the N terminus and sequestered in the cytoplasm during the later stages of oocyte growth, while a spermatocyte-specific 5' exon interferes with translation and prevents production of Dnmt1 during the prolonged crossing-over stage of male meiosis. During the course of postnatal oogenesis, Dnmt1 is present at high levels in nuclei only in growing dictyate oocytes, a stage during which gynogenetic developmental potential is lost and biparental developmental potential is gained.
Abstract: Trace levels of 5-methylcytosine persist in the DNA of mouse embryonic stem cells that are homozygous for null mutations in Dnmt1 , the gene for the one previously recognized metazoan DNA methyltransferase. This residual 5-methylcytosine may be the product of a candidate second DNA methyltransferase, Dnmt2, that has now been identified in human and mouse. Dnmt2 contains all the sequence motifs diagnostic of DNA (cytosine-5)-methyltransferases but appears to lack the large N-terminal regulatory domain common to other eukaryotic methyltransferases. Dnmt2 is more similar to a putative DNA methyltransferase of the fission yeast Schizosaccharomyces pombe than to Dnmt1. Dnmt2 produces multiple mRNA species that are present at low levels in all tissues of human and mouse and is not restricted to those cell types known to be active in de novo methylation. The human DNMT2 gene was mapped to chromosome 10p12-10p14 in a panel of radiation hybrids. Dnmt2 is a candidate for the activity that methylates newly integrated retroviral DNA and maintains trace levels of 5-methylcytosine in the DNA of embryonic stem cells homozygous for null mutations in Dnmt1.
Abstract: The mechanisms that establish and maintain methylation patterns in the mammalian genome are very poorly understood, even though perturbations of methylation patterns lead to a loss of genomic imprinting, ectopic X chromosome inactivation, and death of mammalian embryos. A family of sequence-specific DNA methyltransferases has been proposed to be responsible for the wave of de novo methylation that occurs in the early embryo, although no such enzyme has been identified. A universal mechanism-based probe for DNA (cytosine-5)-methyltransferases was used to screen tissues and cell types known to be active in de novo methylation for new species of DNA methyltransferase. All identifiable de novo methyltransferase activity was found to reside in Dnmt1. As this enzyme is the predominant de novo methyltransferase at all developmental stages inspected, it does not fit the definition of maintenance methyltransferase or hemimethylase. Recent genetic data indicate that de novo methylation of retroviral DNA in embryonic stem cells is likely to involve one or more additional DNA methyltransferases. Such enzymes were not detected and are either present in very small amounts or are very different from Dnmt1. A new method was developed and used to determine the sequence specificity of intact Dnmt1 in whole-cell lysates. Specificity was found to be confined to the sequence 5'-CpG-3'; there was little dependence on sequence context or density of CpG dinucleotides. These data suggest that any sequence-specific de novo methylation mediated by Dnmt1 is either under the control of regulatory factors that interact with Dnmt1, or is cued by alternative secondary structures in DNA.
Abstract: Most of the 5-methylcytosine in mammalian DNA resides in transposons, which are specialized intragenomic parasites that represent at least 35% of the genome. Transposon promoters are inactive when methylated and, over time, C-->T transition mutations at methylated sites destroy many transposons. Apart from that subset of genes subject to X inactivation and genomic imprinting, no cellular gene in a non-expressing tissue has been proven to be methylated in a pattern that prevents transcription. It has become increasingly difficult to hold that reversible promoter methylation is commonly involved in developmental gene control; instead, suppression of parasitic sequence elements appears to be the primary function of cytosine methylation, with crucial secondary roles in allele-specific gene expression as seen in X inactivation and genomic imprinting.
Abstract: DNA (cytosine-5)-methyltransferases (EC 2.1.1.37) maintain patterns of methylated cytosine residues in the mammalian genome; faithful maintenance of methylation patterns is required for normal development of mice, and aberrant methylation patterns are associated with certain human tumors and developmental abnormalities. The organization of coding sequences at the 5'-end of the murine and human DNA methyltransferase genes was investigated, and the DNA methyltransferase open reading frame was found to be longer than previously suspected. Expression of the complete open reading frame by in vitro transcription-translation and by transfection of expression constructs into COS7 cells resulted in the production of an active DNA methyltransferase of the same apparent mass as the endogenous protein, while translation from the second in-frame ATG codon produced a slightly smaller but fully active protein. Characterization of mRNA 5' sequences and the intron-exon structure of the 5' region of the murine and human genes indicated that a previously described promoter element (Rouleau, J., Tanigawa, G., and Szyf, M. (1992) J. Biol. Chem. 267, 7368-7377) actually lies in an intron that is more than 5 kilobases downstream of the transcription start sites.
Abstract: While it is now accepted that methylation of cytosine residues plays a role in various epigenetic phenomena in mammals and flowering plants, the involvement of methylation patterns in the regulation of normal development has remained a controversial and essentially untested issue in the 20 years since such a role was first proposed. Antisense suppression of a DNA methyltransferase in Arabidopsis and characterization of methylation-defective mutants of Arabidopsis have shown that perturbations of methylation patterns disrupt the development of plants, and targeted mutation of the murine gene that encodes the one known from of DNA methyltransferase has shown that methylation is required for cellular differentiation, genomic imprinting, and X chromosome inactivation in mammals. Ectopic expression of homeotic genes and homeotic transformations of floral organs in methylation-defective plants suggest that (in plants and perhaps mammals) heritable methylation patterns reinforce and may have supplanted heritable gene control mediated by chromosomal proteins of the Polycomb and trithorax groups. It is also possible that the developmental abnormalities are the result of ectopic gene expression caused by activation of transcription from nearby parasitic sequence elements that are normally repressed by methylation. Application of modern methods of genetic analysis promises to give definite answers to long-standing questions as to the roles and significance of genomic methylation patterns in normal development and genome defense.
Abstract: ESA152 is a highly hydrophobic 18 kDa sialoglycoprotein, which becomes expressed on ram sperm in the proximal cauda epididymis. ESA 152 is expressed on all regions of the sperm surface, most strongly on the posterior region of the head, most weakly on the anterior region of the head. In this paper, we show that induction of the acrosome reaction with Ca2+ ionophore causes ESA152 to be redistributed from the posterior to the anterior region of the head plasma membrane. Cross-linking ESA152 with bivalent antibody causes similar redistribution and induces the acrosome reaction. Induction of the acrosome reaction with ESA152 antibody requires Ca2+ but is insensitive to (10 ng/ml) pertussis toxin.
