Elisabeth Gateff

Nimrod, a Putative Phagocytosis Receptor with EGF Repeats in Drosophila Plasmatocytes

The hemocytes, the blood cells of Drosophila, participate in the humoral and cellular immune defense reactions against microbes and parasites [1-8]. The plasmatocytes, one class of hemocytes, are phagocytically active and play an important role in immunity and development by removing microorganisms as well as apoptotic cells. On the surface of circulating and sessile plasmatocytes, we have now identified a protein, Nimrod C1 (NimC1), which is involved in the phagocytosis of bacteria. Suppression of NimC1 expression in plasmatocytes inhibited the phagocytosis of Staphylococcus aureus. Conversely, overexpression of NimC1 in S2 cells stimulated the phagocytosis of both S. aureus and Escherichia coli. NimC1 is a 90-100 kDa single-pass transmembrane protein with ten characteristic EGF-like repeats (NIM repeats). The nimC1 gene is part of a cluster of ten related nimrod genes at 34E on chromosome 2, and similar clusters of nimrod-like genes are conserved in other insects such as Anopheles and Apis. The Nimrod proteins are related to other putative phagocytosis receptors such as Eater and Draper from D. melanogaster and CED-1 from C. elegans. Together, they form a superfamily that also includes proteins that are encoded in the human genome.

In vitro induction of cecropin genes--an immune response in a Drosophila blood cell line.

The Drosophila melanogaster cell line mbn-2 was explored as a model system to study insect immune responses in vitro. This cell line is of blood cell origin, derived from larval hemocytes of the mutant lethal (2) malignant blood neoplasm (1(2)mbn). The mbn-2 cells respond to microbial substances by the activation of cecropin genes, coding for bactericidal peptides. The response is stronger than that previously described for SL2 cells, and four other tested Drosophila cell lines were totally unresponsive. Bacterial lipopolysaccharide, algal laminarin (a beta-1,3-glucan), and bacterial flagellin were strong inducers, bacterial peptidoglycan fragments gave a weaker response, whereas a formyl-methionine-containing peptide had no effect. Experiments with different drugs indicate that the response may be mediated by a G protein, but not by protein kinase C or eicosanoids, and that it requires a protein factor with a high rate of turnover.

Ultrastructure and Cytochemistry of the Cell Types in the Larval Hematopoietic Organs and Hemolymph of Drosophila Melanogaster

The ultrastructure of the primordial blood cells in the first and second hematopoietic lobes of the late second and third instar larva and prepupa of Drosophila melanogaster was compared with the ultrastructure of the blood cells found freely in the larval hemolymph. Within the hematopoietic lobes two principal cell‐types were detected: (i) the prohemocytes and proplasmatocytes, and (ii) different developmental stages of crystal cells., Prohemocytes are characterized by a ribsome‐rich cytoplasm, showing small amounts of mitochondria, rough ER and Golgi complexes and few primary lyosomes. Prohemocytes differentiate into proplasmatocytes. When released into the hemolymph they transform further into plasmato‐, podo‐, and lamellocytes. This differentiation pathway is characterized by a gradual, numerical increase of cytoplasmic organelles, the development of the lysosomal system and the aquisition of the capacity for phagocytosis and melanin formation. The differentiation of a procrystal cell into a crystal cell involves a number of intermediate stages, during which the crystalline material is produced, accumulated, and crystallized. Primary and secondary lysosomes in the primordial blood cells of the hematopoietic organs as well as the free blood cells in the hemolymph were identified cytochemically with the help of the acid phosphatase test. The capacity for melanin synthesis was studied with the phenol‐ and polyphenol oxidase test.

The Drosophila melanogaster tumor suppressor gene lethal(3)malignant brain tumor encodes a proline-rich protein with a novel zinc finger

The lethal(3)malignant brain tumor [t(3)mbt] gene causes, when mutated, malignant growth of the adult optic neuroblasts and ganglion mother cells in the larval brain and imaginal disc overgrowth. Via overlapping deficiencies a genomic region of approximately 6.0 kb was identified, containing l(3)mbt+ gene sequences. The l(3)mbt+ gene encodes seven transcripts of 5.8 kb, 5.65 kb, 5.35 kb, 5.25 kb, 5.0 kb, 4.4 kb and 1.8 kb. The putative MBT163 protein, encompassing 1477 amino acids, is proline-rich and contains a novel zinc finger. In situ hybridizations of whole mount embryos and larval tissues revealed l(3)mbt+ RNA ubiquitously present in stage 1 embryos and throughout embryonic development in most tissues. In third instar larvae l(3)mbt+ RNA is detected in the adult optic anlagen and the imaginal discs, the tissues directly affected by l(3)mbt mutations, but also in tissues, showing normal development in the mutant, such as the gut, the goblet cells and the hematopoietic organs.

Definition of Drosophila hemocyte subsets by cell-type specific antigens.

We analyzed the heterogeneity of Drosophila hemocytes on the basis of the expression of cell-type specific antigens. The antigens characterize distinct subsets which partially overlap with those defined by morphological criteria. On the basis of the expression or the lack of expression of blood cell antigens the following hemocyte populations have been defined: crystal cells, plasmatocytes, lamellocytes and precursor cells. The expression of the antigens and thus the different cell types are developmentally regulated. The hemocytes are arranged in four main compartments: the circulating blood cells, the sessile tissue, the lymph glands and the posterior hematopoietic tissue. Each hemocyte compartment has a specific and characteristic composition of the various cell types. The described markers represent the first successful attempt to define hemocyte lineages by immunological markers in Drosophila and help to define morphologically, functionally, spatially and developmentally distinct subsets of hemocytes.

A temperature-sensitive brain tumor suppressor mutation of Drosophila melanogaster: Developmental studies and molecular localization of the gene

The recessive-lethal, temperature-sensitive (ts) mutation of the tumor suppressor gene lethal(3)malignant brain tumor (l(3)mbt) causes in a single step the malignant transformation of the adult optic neuroblasts and ganglion mother cells in the larval brain at the restrictive temperature of 29 degrees C. The transformed cells are differentiation-incompetent and grow autonomously in a lethal and invasive fashion in situ in the brain as well as after transplantation in vivo into wild-type adult hosts. The imaginal discs show epithelial overgrowth. At the permissive temperature of 22 degrees C development is completely normal. The ts-period of gene activity responsible for 100% brain tumor suppression and normal imaginal disc development encompasses the first six hours of embryonic development. The l(3)mbt gene function is, however, also required thereafter for the proper differentiation of the brain and the imaginal discs. The l(3)mbt gene is located cytologically in the salivary gland chromosome bands 97E8-F11, and in molecular terms in 29 kb of DNA detected via a P-element insertional deletion.

Tumor-suppressor genes, hematopoietic malignancies and other hematopoietic disorders of Drosophila melanogaster.

The main components of the Drosophila circulatory system are the dorsal vessel with the attached hematopoietic organs (lymph glands), the hemolymph, and the hemocytes.’ Two blood cell types originate in the hematopoietic organs, the plasmatocytes and the crystal At the end of larval life and in particular during the larval-pupal transition, plasmatocytes differentiate into podoand lamellocytes. This differentiation is characterized by a steady increase of mitochondria, rER, and primary and secondary lyso~omes.~ The maturation of the plasmatocytes is further characterized by increased phagocytosis. Thus, plasmatocytes and their descendants are involved in phagocytosis, encapsulation, and melanization of foreign bodies. Drosophila plasmatocytes can, therefore, be compared with vertebrate macrophages in their capacity to recognize “self” from “non-self,” to phagocytose, and to encapsulate. The nonphagocytic crystal cells seem to be able to coagulate and melanize hem~lyrnph.~ Blood cells also play an important role during metamorphosis during which most cells are of the lamellocyte type and are involved in sequestering disintegrating larval tissues. Hereditary hematopoietic disorders were reported in the earliest days of Drosophilu genetics. The first lethal mutation, affecting hematopoiesis and designated as 1(1)7was discovered in 1916 by C. B. Bridges. The studies by Stark”-’of this mutant revealed a plasmatocyte tumor that she classified as a lymphorsarcoma. The loss of the mutant strain posed the questions whether the mutant plasmatocyte growth was truly malignant and whether Drosophilu can develop true malignancies in general. identified four genes, which in the homozygously mutated state cause the malignant transformation of the larval plasmatocytes (TABLE 1). Two further blood tumor mutants are the Turn’.” and the air16 or hen” mutations. With the exception of the Turn’ mutation, which displays a dominant conditional lethality, the remaining five mutations show a recessive mode of inheritance and, thus, by definition can be classified as tumor-suppressor genes. On the other extreme is a recently found gene mutation that causes blood cell deficiency (E. Gateff, unpublished). In addition, about 40 gene mutations are known to induce In mutagenesis experiments Gateff

Genetic and Molecular Analysis of Six Tumor Suppressor Genes in Drosophila melanogaster

Six Drosophila melanogaster tumor suppressor genes causing malignant or benign tumors in specific cell types are described. The wild-type alleles of these genes are instrumental in the differentiation of particular cell types. In the homozygous state, recessive mutations in the genes interrupt the differentiation of the cells and thus cause their uncontrolled, autonomous, lethal proliferation. The tumors show all major characteristics of malignant and benign neoplastic growth. Genomic sequences of four of the genes have been identified and are currently being characterized. ImagesFIGURE 1.FIGURE 2.FIGURE 2.

Comparative Ultrastructure of Wild-Type and Tumorous Cells of Drosophila

In Drosophila genetic factors cause malignant and benign neoplasms (Gateff, 1978a,b,c). In addition, compactly growing lethal tumors have been obtained from eye-antennal imaginal discs during serial subculture in the abdomens of female flies (Gateff, 1978a,b). The following tumor types have been found: (1) lethal-benign imaginal disc neoplasms, (2) malignant neuroblastomas, (3) malignant blood cell neoplasms, and (4) a benign gonial cell neoplasm. The fine structure of some of the above tumors has been studied and compared to the fine structure of corresponding wild-type cells. These will be discussed below.