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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.

SummaryThe 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.

Larvae homozygous for the recessive lethal allele without children(rgl) (woc(rgl)) fail to pupariate. Application of exogenous 20-hydroxyecdysone elicits puparium formation and pupation. Ecdysteroid titer measurements on mutant larvae show an endocrine deficiency in the brain-ring gland complex, which normally synthesizes ecdysone, resulting in a failure of the larvae to achieve a threshold whole body hormone titer necessary for molting. Ultrastructural investigation revealed extensive degeneration of the prothoracic cells of the ring gland in older larvae. The woc gene, located in polytene chromosomal region 97F, consists of 11 exons. A 6.8-kb transcript is expressed throughout development but is absent in the mutant woc(rgl) larvae. The woc gene encodes a protein of 187 kDa. Eight zinc fingers of the C2-C2 type point to a possible function as a transcription factor. The woc protein shows considerable homology to human proteins which have been implicated in both mental retardation and a leukemia/lymphoma syndrome.

The Drosophila melanogaster tumor suppressor gene lethal(2)tumorous imaginal discs (l(2)tid) causes in homozygotes malignant growth of cells of the imaginal discs and the death of the mutant larvae at the time of puparium formation. We describe the molecular cloning of the l(2)tid+ gene and its temporal expression pattern in the wild-type and mutant alleles. Germ line rescue of the tumor phenotype was achieved with a 7.0 kb Hindlll-fragment derived from the polytene chromosome band 59F5. The l(2)tid+ gene spans approximately 2.5 kb of genomic DNA. The protein coding region, 1,696 bps long, is divided by an intron into two exons. The predicted Tid56 protein contains 518 amino acids and possesses a theoretical molecular weight of 56 kDa. It shows significant homology to all known DnaJ related proteins from bacteria, yeast, and man. The possible function of the Tid56 protein in tumor suppression is delineated.

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.

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.

Three of the twenty recessive-lethal tumor suppressor genes of Drosophila cause imaginal disc tumors in the homozygously mutated state. One of these is the lethal(2)tumorous imaginal discs (l(2)tid) gene. Histo-logical preparations show the tumorous imaginal disc epithelium to consist of a mosaic of cells in monolayer and cells in clumped arrangement. In contrast, the wild-type imaginal disc epithelium is comprised exclusively of cells in monolayer arrangement. Mutant imaginal disc tissue pieces implanted into ready-to-pupariate wild-type larvae fail to differentiate. Implantation of l(2)tid imaginal disc tissue pieces in vivo into wild-type adult flies revealed a lethal, tumorous growth comparable to that in situ, thus characterizing the l(2)tid imaginal discs as truly malignant. The phenotypes of double mutants between two l(2)tid alleles and tumor suppressor genes, such as lethal(2)giant larvae and lethal(2)brain tumor , and the epithelial overgrowth mutant lethal(2)fat are described and discussed. Finally, we present the genetic, cytogenetic and molecular localization of the l(2)tid gene to the giant chromosome bands 59F4–6.

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.

Hemocytes, cellular elements of the innate immune system in insects, play a crucial role in the cellular and humoral immune response. Although a significant amount of information has been collected on their differentiation and function, our understanding of hemocyte development is far from complete. Their characterisation is mostly based on morphological criteria. However, molecular markers were recently identified, defining functional subsets by the aid of monoclonal antibodies. Isolated subsets of hemocytes, in sufficient quantity and purity could help to analyse their development in vitro and also to further define their molecular characteristics. Here we describe an antibody-based rosetting technique for the physical separation of Drosophila hemocyte sub-populations. We have applied anti-hemocyte antibodies coupled to sheep red blood cells for separation. The method relies on the formation of rosettes between hemocytes and sheep erythrocytes, sensitised with discriminative anti-hemocyte monoclonal antibodies. Using this method the rosetting and non-rosetting hemocytes can be separated from each other by gradient centrifugation. Rosette-forming cells from the pellet and non-rosetting cells from the interface can be isolated in high recovery. The method can be used for functional and molecular characterisation of hemocyte sub-populations. The procedure is sensitive, reproducible and easy to perform.

The lethal(3)malignant brain tumor [l(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 zine 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.

The tumor suppressor gene, lethal(3)malignant blood neoplasm-1+, of Drosophila melanogaster is required for the differentiation of the phagocytic blood-cell type, the plasmatocyte. In the homozygously mutated state it causes the malignant transformation of these blood cells. We present here the cloning, sequencing, structure, and expression of the l(3)mbn-1+ gene during development. The cloned gene was identified by germ-line transformation, generation of revertants, and the detection of the corresponding mRNA in blood cells and other tissues. Homologies of the G-S-rich C-terminus of the putative MBN83 protein to human cytokeratins K1, K10, and mouse loricrin were found. The structure and possible function of the wild-type l(3)mbn-1+ gene are discussed.

Krebs ist ein entwicklungsgenetisches und somit ein Zelldifferenzierungsproblem. Der Differenzierungsvorgang wird durch viele zell- und entwicklungsspezifische Gene gesteuert. Die kausale Beziehung zwischen einer rezessiven Genmutation und der malignen Entartung wurde erstmals bei der Fruchtfliege Drosophila melanogaster an einer Reihe von Genen nachgewiesen [21, 22, 36]. Diese mutierten, also funktionslosen Entwicklungsgene zeigen einen rezessiv-letalen Vererbungsmodus. Ihre Wildtyp-Allele sind dominant und unterbinden die maligne Entartung und werden folglich als Tumorsuppressorgene bezeichnet.

The development of the rhabdomeric pattern in the compound eye ofDrosophila has been studied using combined transplantation and electron microscope techniques. In a first series of experiments eye imaginal discs of increasing age were implanted into larvae ready to pupate, thus losing variable amounts of the normal time for development. A sequence of differentiative abilities was found in the metamorphosed test pieces. As far as the photoreceptor cells are concerned, the most prominent steps of this sequence are: ability to form groups with other similar elements, anatomical polarization of microvilli, establishment of the rhabdomeric pattern and formation of an equator line. The stability of determination of the equator line was tested in a second experimental series. Fragment of different topographical origin within the mature eye anlage were brought to metamorphosis by implantation into larvae ready to pupate. It was found that an equator line differentiates only in those pieces which according to the published anlage maps contain the prospective equator region prior to metamorphosis. The mitotic abilities of implanted eye imaginal discs were investigated by means of “in vitro”3H-thymidine pulse-labelling and light microscope autoradiography of the differentiated test pieces. During the third larval stage the eye anlage is traversed by two consecutive mitotic waves, each one of them producing different categories of receptor cells. The first, anterior wave predominantly produces cells oriented toward the poles of the eye within the ommatidia, while the second, posterior wave gives rise to elements exclusively in an equatorial position. The dynamics of this proliferation are discussed in relation to the findings in the implantation experiments. Silver-grain counts support the possibility that at least two successive cell divisions occur in the eye anlage between labeling with tritiated thymidine and beginning of morphological differentiation. The relevance of this finding for the understanding of the concept of acquisition of competence is discussed.

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.

Malignant neoplasms that develop in 12 recessive-lethal, larval mutants of Drosophila melanogaster are discussed. These mutations affect the adult optic neuroblasts and ganglion-mother cells in the larval brain, the imaginal discs, and the hematopoietic organs. The malignant neoplasms exhibit fast, autonomous growth, loss of the capacity for differentiation, increased mobility and invasiveness, lethality in situ and after transplantation, and histological, fine structural, and karyotypic abnormalities. Intermediate neoplasms are also found. These combine both benign and malignant qualities. They grow in a noninvasive, compact fashion, typical of benign tumors, yet they also exhibit malignant qualities such as fast, autonomous, and lethal growth, loss of differentiation capacity, changes in cellular morphology, and lethal growth after transplantation into wild-type hosts. Thus Drosophila and vertebrate neoplasms show striking similarities.

The fine structure and histochemistry of the neoplastic primordial blood cell-types in the larval hematopoietic organs and the mature cell-types in the hemolymph of the blood tumor mutant lethal (1) malignant blood neoplasm (l(1)mbn) of Drosophila melanogaster were investigated. In this mutant the cell-types of the plasmatocyte-line are neoplastic while the cell-types of the crystal-cell-line are not and are much reduced in numbers (1, 2). In contrast to the wild-type the mutant hematopoietic organs are enlarged and contain, in addition to primordial blood-cells, large numbers of mature plasmato-, podo-, and lamellocytes. All cell-types of the plasmatocyte-line differ in their fine structure and behavior from their wild-type counterparts. The mutant blood cells show generally a numerical increase of cell organelles and acid phosphatase positive primary and secondary lysosomes. In the phenol oxidase test they showed a vigorous melanization reaction. Plasmato- and podocytes invade into the tissues of the larva and show high phagocytic activity.

In the recessive mutant lethal(3)malignant blood neoplasm, (l(3)mbn), the precursors of the plasmatocytes, the proplasmatocytes, are malignantly transformed. The hypertrophied hematopoietic organs produce excessive amounts of plasmatocytes which are released into the hemolymph throughout the third larval instar. As a result, their number in the hemolymph increases up to 15-fold compared with that in the wild-type hemolymph. Most free blood cells in the mutant hemolymph are plasmatocytes. Podocytes and lamellocytes of normal size are rare. Approximately 10–12% of the blood cells are giant plasmato-, podo-, and lamellocytes which show numerous primary and secondary lysosomes in their cytoplasm. Furthermore, the malignant plasmatocytes invade all larval tissues which become histolyzed by a yet unknown process. The high level of polyphenol oxidase expression, coupled with the inability of the cells to distinguish between their own and foreign tissue, leads to the formation of melanotic masses and eventually to the melanization of the entire larva.

A new virus-like particle has been found in Drosophila. Thus far it has been detected only in electron micrographs of certain cells capable of division, such as those in larval imaginal disks, cell lines derived from imaginal disks, cells from a genetically controlled brain tumor, and adult gut cells. It appears to be slightly elliptical in shape, about 37 millimicrons by 45 millimicrons, and occurs in both the nucleus and cytoplasm. From the evidence it is suggested that the particle is a new virus.

A bewildering array of causative factors for cancer have been found. They range from purely environmental, such as diet or environmental substances, over radiations of all types, carcinogens and mutagens (Berenblum, 1974) up to viruses (Bishop, 1978) and transforming factors (Todaro et al., 1981).

This paper describes two ecdysone-deficient, recessive-lethal mutants,lethal(1)giant ring gland (grg) andlethal(1)suppressor of forkedmad-ts (mad-ts: Jurgens and Gateff 1979) and compares their ecdysteroid titers with that of the wild-type. Mutant larvae show a much reduced ecdysteroid content, amounting to 1/10 to 1/30 of the wild-type values, but never a true titer peak. They fail to pupate and die after 1–3 weeks. Ecdysteroid feeding elicits different responses in the larvae of the two mutants.mad-ts larvae pupate within 24 h, thus showing that their low ecdysteroid titer is directly connected to their inability to pupate.mad-ts resembles the mutantlethal (3)ecdysone-1ts (Garen et al. 1977). Thegrg mutant larvae, on the other hand, fail to pupate after 20-hydroxyecdysone feeding as well as injection. The primary defect of thegrg mutant is not entirely clear. Thegrg larval salivary gland cells appear to possess normal ecdysteroid receptors. Furthermore, the low ecdysteroid titer ingrg is not the result of an increased ecdysteroid catabolism. The primary defect in the mutant may lie in the malfunctioning neurosecretory cells which do not show neurosecretion in histological preparations. Further support for this notion comes from electronmicrographs of the enlargedgrg ring glands which, in contrast to the wild-type, do not possess nerve endings.

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.

Publisher Summary This chapter describes the different types of benign and malignant neoplasms found in 25 Drosophila mutants and discusses these mutants in the light of the recent findings. In Drosophila , 25 of a total of 5000 genes have been found to cause malignant or benign neoplasms in the mutated state. These genes play crucial roles in the differentiation of specific cell types and tissues. The wild-type alleles of these genes control particular pathways of development in specific tissues or cells at particular times. The cells and tissues involved include the adult optic neuroblasts, the imaginal discs, and the primordial blood cells in the larva, the male and female gonial cells in the adult, and neuroblasts, blood cells, and other not yet identified cell types in the embryo. Moreover, to understand the primary events leading to malignant neoplastic transformation, insight has to be gained into the genetics of normal development.