Innate immune cells and bacterial infection in zebrafish.

The physical attributes of the zebrafish, including optical transparency during embryogenesis, large clutch sizes, external development, and rapid organogenesis were features that initially attracted developmental biologists to use this vertebrate as an experimental model system. With the progressive development of an extensive genetic "tool kit" and an ever-growing number of transgenic reporter lines, the zebrafish model has evolved into an informative system in which to mimic and study aspects of human disease, including those associated with bacterial infections. This chapter provides detailed protocols for microinjection of bacterial strains into zebrafish larvae and subsequent experiments to investigate single-larva bacterial burdens, live imaging of specific neutrophil and macrophage bactericidal functions, and how these protocols may be applied to drug discovery approaches to uncover novel immunomodulatory drugs.

In situ hybridization screen in zebrafish for the selection of genes encoding secreted proteins.

An in situ hybridization expression screen using a signal sequence trap system has been conducted in zebrafish to isolate cDNAs that encode secreted proteins. Random clones (secreted expressed sequence tags; sESTs) were sequenced from zebrafish embryonic (18-24 hr postfertilization) and adult kidney libraries. From the two RNA sources, 627 random sEST cDNAs were identified as being homologous or identical to known genes and 166 clones encode currently unidentified genes. The sESTs represent a broad range of enzymes and other regulatory molecules. Whole-mount in situ hybridization analysis was carried out by using antisense probes generated from 244 selected sESTs, and a range of expression patterns was obtained. Genetic mapping undertaken with sEST sequences demonstrated that assignment of map position was attainable by using 5' primers. The signal sequence trap system used in this work has yielded a range of cDNAs that encode secreted proteins and, together with analysis of patterns of expression and genetic mapping, has the potential to facilitate analysis of signaling pathways central to development and physiology.

The smad proteins and TGFbeta signalling: uncovering a pathway critical in cancer.

The critical role of TGFbeta in development and growth control is well established but the signalling pathway has only recently been elucidated. Identification of the Smads as TGFbeta's intracellular signalling mediators has led to an explosion of information on a novel signalling network that links the cell surface to the nucleus. Many cancers develop resistance to the growth-inhibitory effects of TGFbeta and mutations in signalling pathway components have been discovered that may underly tumour progression.

The TGF-beta type II receptor in chronic myeloid leukemia: analysis of microsatellite regions and gene expression.

Genomic instability is one mechanism proposed to play a role in the disease progression of chronic myeloid leukemia (CML). Microsatellite regions in the type II transforming growth factor-beta receptor (TGF-beta RII) gene appear to be targets for mutation in some cancers displaying microsatellite instability (replication error phenotype, RER+). Furthermore, TGF-beta RII mutations in RER+ tumors have been associated with decreased TGF-beta RII mRNA levels. As TGF-beta is a potent negative growth regulator of hematopoietic cells, investigations were undertaken to determine whether inactivation of the receptor by microsatellite alteration might be involved in the progression of CML. Analysis of TGF-beta RII mRNA expression by RNase protection, with comparison of cells from the chronic, accelerated and blast phases of CML, showed no change in TGF-beta RII transcript levels during disease progression. However, during each phase of the disease, low levels of TGF-beta RII were detected when compared with the hematopoietic cells of normal donors. Furthermore, this decreased expression was also observed in the other myeloproliferative disorders, polycythemia rubra vera (PRV) and essential thrombocythemia (ET). The leukemia cell lines K562 and HL-60 had no detectable TGF-beta RII mRNA. Two microsatellite regions found altered in RER+ colon cancers were analyzed to establish if these sequences were aberrant in CML. No alteration was detected in either of these regions in any phase of the disease. These results suggest that alterations of the microsatellite regions in the TGF-beta RII gene are not involved in the progression of CML. Decreased expression of TGF-beta RII in CML cells and leukemia cell lines raises the possibility that altered expression of the receptor may play a role in the initiation and/or maintenance of the disease state.

Isolation of zebrafish gdf7 and comparative genetic mapping of genes belonging to the growth/differentiation factor 5, 6, 7 subgroup of the TGF-beta superfamily.

The Growth/differentiation factor (Gdf) 5, 6, 7 genes form a closely related subgroup belonging to the TGF-beta superfamily. In zebrafish, there are three genes that belong to the Gdf5, 6, 7 subgroup that have been named radar, dynamo, and contact. The genes radar and dynamo both encode proteins most similar to mouse GDF6. The orthologous identity of these genes on the basis of amino acid similarities has not been clear. We have identified gdf7, a fourth zebrafish gene belonging to the Gdf5, 6, 7 subgroup. To assign correct orthologies and to investigate the evolutionary relationships of the human, mouse, and zebrafish Gdf5, 6, 7 subgroup, we have compared genetic map positions of the zebrafish and mammalian genes. We have mapped zebrafish gdf7 to linkage group (LG) 17, contact to LG9, GDF6 to human chromosome (Hsa) 8 and GDF7 to Hsa2p. The radar and dynamo genes have been localized previously to LG16 and LG19, respectively. A comparison of syntenies shared among human, mouse, and zebrafish genomes indicates that gdf7 is the ortholog of mammalian GDF7/Gdf7. LG16 shares syntenic relationships with mouse chromosome (Mmu) 4, including Gdf6. Portions of LG16 and LG19 appear to be duplicate chromosomes, thus suggesting that radar and dynamo are both orthologs of Gdf6. Finally, the mapping data is consistent with contact being the zebrafish ortholog of mammalian GDF5/Gdf5.

Expression and functional analysis of two isoforms of the human GM-CSF receptor alpha chain in myeloid development and leukaemia.

The human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) alpha chain RNA is alternatively spliced to yield receptor isoforms. Two of these, alpha 1 and alpha 2, differ in their cytoplasmic domains. Because the GM-CSFR beta chain (beta c) is shared with the receptors for interleukins 3 and 5 it is possible that the alpha chain confers specificity on the GM-CSF response and that the different isoforms might refine this response further. Studies have been directed at determination of the respective biological roles of the alpha 1 and alpha 2 isoforms. Expression of the isoforms was examined by RNase protection analysis in normal granulocytes and a variety of cell lines of haemopoietic origin, at different stages of differentiation and activation. Expression was also analysed in cells from patients with a variety of leukaemic subtypes. Results demonstrated that the relative abundance of the isoforms was similar in all cell populations examined. The human GM-CSFR alpha 1 or alpha 2 receptors were independently expressed in the murine factor-dependent cell line FDC-P1, so that the properties of the receptors could be compared. Cell lines that expressed either receptor could be converted to growth in response to human GM-CSF and assumed a more differentiated phenotype when compared with the parental cell line. However, the morphology, expression of cell surface antigens and dose-growth response characteristics did not differ significantly between cells that expressed either the alpha 1 or alpha 2 receptor. These studies demonstrate that the alpha 1 and alpha 2 subunits of the GM-CSF receptor are co-ordinately regulated in both normal and malignant haemopoiesis. Furthermore, each receptor is able to deliver both proliferative and differentiative signals to myeloid cells.

New insights into the control of cell growth; the role of the AxI family.

AxI, Dtk and Mer are recently described receptors that constitute a new receptor tyrosine kinase subfamily. They bind the vitamin K-dependent protein growth-arrest-specific gene 6 (Gas6) that is structurally related to the anticoagulation factor protein S. Studies suggest a role for these receptors in developmental processes. In the function of the hematopoietic and nervous systems and in tumorigenesis.

Expression of murine interleukin 11 and its receptor alpha-chain in adult and embryonic tissues.

Interleukin 11 (IL-11) is a multifunctional cytokine that has diverse effects on blood cells and their precursors and on a number of cell types outside of the hematopoietic system. The cDNAs encoding murine IL-11 and its receptor alpha-chain (IL-11R alpha) have recently been isolated. We have used the RNase protection assay to examine the expression of murine IL-11 and IL-11R alpha in a range of adult mouse tissues, in embryos, and during development of embryonic stem (ES) cells into cystic embryoid bodies in vitro. The testis showed a high level of IL-11 gene expression while a much lower level of expression was detected in the lung, stomach, small intestine, and large intestine. Expression of IL-11 was not detected between day 10.5 and day 18.5 post coitum of embryonic development or in differentiating ES cells in vitro. In contrast, the IL-11R alpha was found to be expressed in all adult tissues examined, during embryonic development, and in totipotent and differentiating ES cells.

Production of tumour necrosis factor-alpha by cultured human peripheral blood leucocytes in response to the anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (NSC 640488).

The investigative anti-tumour agent 5,6-dimethylxanthenonone-4-acetic acid (DMXAA, NSC 640488), developed in this laboratory as an improved analogue of flavone acetic acid (FAA, NSC 347512), is currently in clinical trial. The ability of DMXAA to up-regulate tumour necrosis factor (TNF) mRNA and protein synthesis in cultured human peripheral blood leucocytes (HPBLs) has been investigated and compared with that of flavone acetic acid (FAA) and of bacterial lipopolysaccharide (LPS). Human peripheral blood leucocytes were isolated from buffy coats obtained from a blood transfusion centre and also from blood samples from laboratory volunteers. At a concentration of 400 microg ml(-1) and an incubation time of 2 h, DMXAA up-regulated mRNA synthesis in six of eight individuals tested, as measured by Northern blotting. The degree of up-regulation varied in different individuals from one to nine times that of control levels. In contrast, FAA caused no induction above that of control levels and in some cases suppressed expression relative to controls, extending previous data that DMXAA but not FAA up-regulates TNF mRNA in the human HL-60 tumour cell line. At the same concentration but with longer incubation times (6-12 h), DMXAA induced increases in TNF protein in 11 of 15 samples of HPBLs from buffy coats and also in 11 of 15 samples of HPBLs from volunteers, as measured by cytotoxicity assays with L929 cells. FAA caused no increase in TNF protein, while LPS induced TNF to approximately 20-fold higher levels than did DMXAA. Considerable heterogeneity of response was observed with both sources of HPBLs, and there was little or no correlation between the extent of TNF induction by DMXAA and LPS in individual samples. In vitro analysis of the response of human peripheral blood leucocytes to DMXAA may be a useful test in clinical trials of agents such as DMXAA.

The Dtk receptor tyrosine kinase, which binds protein S, is expressed during hematopoiesis.

Dtk (Tyro 3/Sky/Rse/Brt/Tif) belongs to a recently recognized subfamily of receptor tyrosine kinases that also includes Ufo (Axl/Ark) and Mer (Eyk). Ligands for Dtk and Ufo have been identified as protein S and the related molecule Gas6, respectively. This study examined expression of Dtk during ontogeny of the hematopoietic system and compared the pattern of expression with that of Ufo. Both receptors were abundantly expressed in differentiating embryonic stem cells, yolk sac blood islands, para-aortic splanchnopleural mesoderm, fractionated AA4+ fetal liver cells, and fetal thymus from day 14 until birth. Although Ufo was expressed at moderate levels in adult bone marrow, expression of Dtk in this tissue was barely detectable. In adult bone marrow subpopulations fractionated using counterflow centrifugal elutriation, immunomagnetic bead selection for lineage-depletion and FACS sorting for c-kit expression, very low levels of Dtk and/or Ufo were detected in some cell fractions. These results suggest that Dtk and Ufo are likely to be involved in the regulation of hematopoiesis, particularly during the embryonic stages of blood cell development.

Analysis of the murine Dtk gene identifies conservation of genomic structure within a new receptor tyrosine kinase subfamily.

The receptor tyrosine kinase Dtk/Tyro 3/Sky/rse/brt/tif is a member of a new subfamily of receptors that also includes Axl/Ufo/Ark and Eyk/Mer. These receptors are characterized by the presence of two immunoglobulin-like loops and two fibronectin type III repeats in their extracellular domains. The structure of the murine Dtk gene has been determined. The gene consists of 21 exons that are distributed over 21 kb of genomic DNA. An isoform of Dtk is generated by differential splicing of exons from the 5' region of the gene. The overall genomic structure of Dtk is virtually identical to that determined for the human UFO gene. This particular genomic organization is likely to have been duplicated and closely maintained throughout evolution.

Identification of a novel receptor tyrosine kinase expressed in acute myeloid leukemic blasts.

Using the polymerase chain reaction with degenerate oligonucleotides derived from conserved motifs within the catalytic kinase domain of protein tyrosine kinases, and RNA extracted from embryonic stem cells, sequences that encode a segment of the kinase domain of several potentially novel receptor tyrosine kinases (RTKs) have been identified. One of these was selected for further study because in Northern analysis it hybridized to RNA from multipotential hematopoietic cell lines, but not from lines representative of lineage-committed cells. A cDNA for this receptor, designated developmental tyrosine kinase (DTK), was isolated and encodes a protein with structural similarities to AXL. Together these receptors form a new class of RTK. DTK is expressed in a number of human leukemic cell lines, and in the blasts of 6 of 11 patients with acute myeloid leukemia (AML) analyzed. The structure of DTK suggests that it may function as a cell adhesion molecule, and mediate cell-to-cell or cell-matrix interactions between hematopoietic cells and their respective microenvironments.

Induction of tumor necrosis factor-alpha messenger RNA in human and murine cells by the flavone acetic acid analogue 5,6-dimethylxanthenone-4-acetic acid (NSC 640488).

The investigational antitumor agent, 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA; NSC 640488) induced greater expression of tumor necrosis factor-alpha (TNF-alpha) mRNA in murine spleen cells in vivo at its optimal dose of 27.5 mg/kg than flavone acetic acid (FAA; NSC 347512) at its optimal dose of 220 mg/kg. Up-regulation of TNF-alpha mRNA was obtained using 5,6-MeXAA in vitro in cultures of murine splenocytes, the murine J774 macrophage cell line, and the human HL-60 myelomonocytic leukemia cell line. Maximal induction occurred at a 5,6-MeXAA concentration of 200 micrograms/ml for both murine J774 and human HL-60 cells. A direct comparison of FAA and 5,6-MeXAA (100-600 micrograms/ml) to stimulate TNF-alpha mRNA in HL-60 cells showed activity by 5,6-MeXAA at all doses but minimal activity with FAA. The results demonstrate that 5,6-MeXAA is equally potent in up-regulating TNF-alpha mRNA in human and murine cells of the monocyte/macrophage lineage, whereas FAA has demonstrable activity in murine cells only. The results suggest that 5,6-MeXAA would be a more active clinical agent than FAA because TNF-alpha induction appears to be a critical factor in the antitumor effects of this class of compounds.

Isolation of a receptor tyrosine kinase (DTK) from embryonic stem cells: structure, genetic mapping and analysis of expression.

Analysis of receptor tyrosine kinases expressed during mouse embryonic stem cell differentiation resulted in the cloning of a receptor designated developmental tyrosine kinase (DTK). The 850 amino acid mature receptor protein comprises an extracellular domain with two immunoglobulin-like motifs and two fibronectin type III modules, a 25 amino acid transmembrane domain and a cytoplasmic region with a catalytic kinase domain. In embryonic stem cells growing in the presence of leukemia inhibitory factor DTK is abundantly expressed and this level of expression is maintained in differentiating embryonic stem cells and cystic embryoid bodies. In mid-gestational embryos (E14.5), DTK RNA is expressed in many tissues including brain, eye, thymus, lung, heart, gut, liver, testis and limbs. In contrast, expression of DTK in adult mice becomes restricted to brain, portions of the gastrointestinal tract, bladder, testis and ovary. There is enrichment of transcripts encoding DTK in purified fetal liver hematopoietic stem cells, when compared with unfractionated fetal liver. The DTK gene maps to mouse chromosome 2, band F.

Isolation and characterization of the human DTK receptor tyrosine kinase.

A cDNA encoding the human homologue of the murine DTK receptor tyrosine kinase has been isolated from a human brain library. The DTK cDNA encodes a mature protein of 850 amino acids with similar structural features to those of the murine receptor. The extracellular domain contains two immunoglobulin-like motifs and two fibronectin type III modules; features which define a new class of receptor tyrosine kinase. The human DTK gene has been mapped by fluorescent in situ hybridization to chromosome 15q15, and a DTK-related gene identified at chromosome 15q24. In fetal tissues, transcripts for DTK were detected in brain, kidney, lung and heart. Prominent expression was observed in the embryonal carcinoma cell line NT2/D1. Expression of the gene is up-regulated in adult tissues with high levels of expression in many regions of the adult brain. DTK is also abundantly expressed in adult kidney, testis, and ovary.

Expression of isoforms of the human receptor tyrosine kinase c-kit in leukemic cell lines and acute myeloid leukemia.

Because mutations in receptor tyrosine kinases may contribute to cellular transformation, studies were undertaken to examine c-kit in human leukemia. Isoforms of c-kit have been characterized in the human megakaryoblastic leukemia cell line M-07. Deletion of the four amino acids Gly-Asn-Asn-Lys in the extracellular domain represents an alternatively spliced isoform that has been shown by others, in mice, to be associated with constitutive receptor autophosphorylation (Reith et al, EMBO J 10:2451, 1991). Additional isoforms differ in the inclusion or exclusion of a serine residue in the interkinase domain, a region that contains the binding site for phosphatidylinositol 3-kinase. By RNase protection analysis, we have shown coexpression of the Gly-Asn-Asn-Lys+ and Gly-Asn-Asn-Lys- isoforms, with dominance of the Gly-Asn-Asn-Lys- transcript, in normal human bone marrow, normal melanocytes, a range of tumor cell lines, and the blasts of 23 patients with acute myeloid leukemia. Analysis of transcripts for the Ser+ and Ser- isoforms also showed coexpression in all normal and leukemic cells examined. The ratios of isoform expression for both the Gly-Asn-Asn-Lys and Ser variants were relatively constant, providing no evidence in the tumors examined that upregulation of one isoform contributes to the neoplastic process.

A functional isoform of the human granulocyte/macrophage colony-stimulating factor receptor has an unusual cytoplasmic domain.

The granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (GMR) transduces a signal that results in the proliferation, differentiation, and functional activation of hematopoietic cells. This study sought to determine whether functional isoforms of the receptor exist that may be important in generating this diversity of cellular response. We have isolated a cDNA encoding an isoform of the low-affinity human GMR that is a product of alternative splicing of the GMR gene and results in a predicted 410-amino acid protein with a cytoplasmic domain that is rich in serine residues, a feature of regions critical in signal transduction for other receptors of the hematopoietin receptor superfamily. This receptor bound ligand and was functionally active when introduced into a murine factor-dependent cell line; mRNA transcripts representative of this isoform were coexpressed with those for a previously isolated 400-amino acid isoform of the GMR in normal hematopoietic and leukemic cells. In view of the recent isolation of a cDNA, designated GM-CSF R beta, that confers high-affinity binding of GM-CSF in cotransfection experiments with the low-affinity receptor, we suggest that the previously isolated low-affinity receptor be designated GM-CSF R alpha 1 and the one described in this report be designated GM-CSF R alpha 2.

Evidence that anti-asialo GM1 in vivo improves engraftment of T cell-depleted bone marrow in hybrid recipients.

Engraftment of T cell-depleted bone marrow was studied in a P----F1 murine bone marrow transplant model which features long-term stability of mixed chimerism of donor (B6) and host (B6AF1) cells after BMT. We report that a polyclonal antibody to asialo GM1 (anti-ASGM1) given in vivo after transplant was able to increase long-term donor bone marrow engraftment. In vivo anti-ASGM1 eliminated NK activity but did not affect the generation of cytotoxic T cells nor did it stimulate hematopoiesis in vitro. Anti-Thy 1.2, a pan-T cell monoclonal antibody, had no effect on donor engraftment. We conclude that ASGM1+ cells with NK activity inhibit the long-term engraftment of bone marrow stem cells in this model and that antibodies to NK cells can be used in vivo as an effective component of the transplant conditioning regimen.