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.

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 rapid rosetting method for separation of hemocyte sub-populations of Drosophila melanogaster.

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.

Ultrastructure and Cytochemistry of the Cell-types in the Tumorous Hematopoietic Organs and the Hemolymph of the Mutant Lethal (1) Malignant Blood Neoplasm (l(1)mbn) of Drosophila Melanogaster: (drosophila/mutant blood cells/ultrastructure/cytochemistry).

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.

Ultrastructure and Cytochemistry of the Cell Types in the Larval Hematopoietic Organs and Hemolymph of Drosophila Melanogaster: (drosophila/hematopoiesis/blool cells/ultrastructure/cytochemistry).

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.

Pattern specification in imaginal discs ofDrosophila melanogaster : Developmental analysis of a temperature-sensitive mutant producing duplicated legs.

l(1)su(f)mad-ts (mad) is a new temperature-sensitive (ts) lethal mutant ofDrosophila melanogaster which produces duplicated legs after temperature pulse treatment during larval development. The ts-lethality was studied in temperature experiments and genetic mosaics. Temperature pulses given during two distinct TSPs of larval development result in two different types of leg pattern duplication. "Total" differ from "partial" duplications with respect to the affected leg compartments and the orientation of the planes of symmetry which are perpendicular to the dorso-ventral and the proximo-distal leg axes in total and partial duplications, respectively. Genetic mosaic studies indicate (i) disc autonomy of leg pattern duplication, (ii) clonal separation of the anlagen of the two pattern copies, and (iii) clonal restriction along the antero-posterior compartment border in the two pattern copies of totally duplicated legs.The results suggest thatmad leg pattern duplication is caused by a change in positional information rather than by cell death and subsequent regeneration. Our data are compatible with the assumption that during normal development the leg disc cells acquire information about their position within the disc with respect to the different leg axes independently and at different times.

Developmental capacities of immature eye-antennal imaginal discs ofDrosophila melanogaster.

The location of the immature eye-antennal discs ofDrosophila melanogaster in embryos and young larvae was established by means of transplantation experiments. The developmental capacities of these immature discs was then investigated by implanting them into larvae which were ready to metamorphose, thus bypassing large portions of embryonic and larval development. Imaginal eye-antennal discs of embryos and first instar larvae are unable to synthesize eye pigments or secrete cuticle. The discs acquire the first detectable competence in the middle of the second instar, 32-36 hours after hatching, when the eye region of the disc becomes competent to synthesize ommochrome pigments and the rest of the disc becomes competent to secrete a thin untanned, transparent cuticle. Competence to synthesize pteridine pigments becomes evident later, 36-42 hours after hatching. The competence to produce specific bristle and hair patterns is acquired still later, 42-56 hours after hatching. Different regions of the eye-antennal disc acquire competence at different times and the acquisition of competence seems to occur in a proximo-distal sequence within both eye and antennal regions of the disc. In the eye region of the disc, the competence to produce proximal structures such as facets appears before the competence to produce ocelli. Similarly, in the antennal region of the disc, the competence to produce the first antennal segment appears before the competence to produce the third antennal segment or arista. Also, the acquisition of competence to produce a specific cuticular pattern occurs four to six hours earlier in the eye region of the disc than in the antennal region. It was also found that the temporal sequence in which differentiation events actually occur during adult development is similar to the temporal sequence in which specific competences are acquired by the growing immature eye-antennal discs.

Developmental studies on two ecdysone deficient mutants ofDrosophila melanogaster.

This paper describes two ecdysone-deficient, recessive-lethal mutants,lethal(1)giant ring gland (grg) andlethal(1)suppressor of forked mad-ts (mad-ts: Jürgens 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-1 ts (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 the wild-type three ecdysteroid peaks were found: one shortly before puparium formation, the second at approximately 12 h and the third at about 30 h after pupation. The ecdysteroid titer peak in late third instar, wild-type larvae is mainly due to the presence of 20-dydroxyecdysone as shown by radioimmunoassays after thin layer chromatography and derivatization followed by gas liquid chromatography and mass spectroscopy. In addition, a number of unidentified polar and apolar metabolites were also present.

The development of ommatidial patterning in metamorphosed eye imaginal discs implants ofDrosophila melanogaster.

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.