Lethal(1) aberrant immune response mutations leading to melanotic tumor formation in Drosophila melanogaster

Using P element-mediated mutagenesis we have isolated 20 X-linked lethal mutations, representing at least 14 complementation groups, which exhibit melanotic tumor phenotypes. We present the systematic analysis of this interesting group of lethal mutations that were selected for their visible melanotic or immune response. The lethal and melanotic tumor phenotypes of each lethal(1) aberrant immune response (air) mutation are pleiotropic effects of single genetic lesions. Lethality occurs throughout the larval and early pupal periods of development and larval development is extended in some air mutants. The air mutant lethal syndromes include abnormalities associated with the brain, haematopoietic organs, gut, salivary glands, ring glands, and imaginal discs. Additional characterization of the melanotic tumor mutations Tuml and tu(1)Szts have indicated that the melanotic tumor phenotype is similar to that observed in the air mutants. These studies have led to the proposal that two distinct classes of melanotic tumor mutations exist. Class 1 includes mutants in which melanotic tumors result from "autoimmune responses" or the response of an apparently normal immune system to the presence of abnormal target tissues. The Class 2 mutants display obvious defects in the haematopoietic organs or haemocytes, manifested as overgrowth, and the resulting aberrant immune system behavior may contribute to melanotic tumor formation.     

Screening of larval/pupal P-element induced lethals on the second chromosome in Drosophila melanogaster : clonal analysis and morphology of imaginal discs

We have carried out screens for lethal mutations on the second chromosome of Drosophila melanogaster that are associated with abnormal imaginal disc morphologies, particularly in the wing disc. From a collection of 164 P element-induced mutations with a late larva/pupa lethal phase we have identified 56 new loci whose gene products are required for normal wing disc development and for normal morphology of other larval organs. Genetic mosaics of these 56 mutant lines show clonal mutant phenotypes for 23 cell-viable mutations. These phenotypes result from altered cell parameters. Causal relationships between disc and clonal phenotypes are discussed. Received: 19 June 1997 / Accepted: 4 August 1997     

P-Lacw Insertional Mutagenesis on the Second Chromosome of Drosophila Melanogaster: Isolation of Lethals with Different Overgrowth Phenotypes

A single P-element insertional mutagenesis experiment was carried out for the second chromosome of Drosophila melanogaster using the P-lacW transposon. Out of 15,475 insertions on the second chromosome, 2,308 lethal and 403 semilethal mutants (altogether 2,711) were recovered. After eliminating clusters, 72% of the mutants represent independent insertions. Some of the mutants with larval, prepupal or pupal lethal phases have a prolonged larval period and show gradual overgrowth of the imaginal discs, brain and/or the hematopoietic organs (lymph glands). In this paper, 16 overgrowth mutants are described. As revealed by in situ hybridization, none of the mutations corresponds to any of the previously known overgrowth mutations on the second chromosome.     

An <Emphasis Type="Italic">in vivo</Emphasis> RNA interference screen identifies gene networks controlling <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>blood cell homeostasis

Background  

In metazoans, the hematopoietic system plays a key role both in normal development and in defense of the organism. In Drosophila, the cellular immune response involves three types of blood cells: plasmatocytes, crystal cells and lamellocytes. This last cell type is barely present in healthy larvae, but its production is strongly induced upon wasp parasitization or in mutant contexts affecting larval blood cell homeostasis. Notably, several zygotic mutations leading to melanotic mass (or "tumor") formation in larvae have been associated to the deregulated differentiation of lamellocytes. To gain further insights into the gene regulatory network and the mechanisms controlling larval blood cell homeostasis, we conducted a tissue-specific loss of function screen using hemocyte-specific Gal4 drivers and UAS-dsRNA transgenic lines.     

The recovery, penetrance, and pleiotropy of X-linked, cold sensitive mutants in Drosophila

Summary The viability at 16°, 22°, and 30°C, loci, visible phenotypes if any, possible effective lethal phase, and female fertility of seven X-linked, recessive cold sensitive mutations are reported. Five of the seven are female sterile at the restrictive temperature of 16°C; two of these five are also female sterile at the permissive temperature of 25°C. For two of the five mutations which are female sterile, escapers at the restrictive temperature exhibit visible phenotypes characteristic of mutants which affect protein synthesis. The possibility that some of the mutants affect ribosomes is considered. One of the mutants, l(1)TW-6 ^cs , is probably a cold sensitive meiotic mutant as well as a cold sensitive zygotic lethal. One of the mutants is a non-conditional visible allele of lozenge.Supported by NSF Grants GB 7707 and GB 20910.     

Mutations affecting the pattern of the larval cuticle inDrosophila melanogaster

Summary The present report describes the recovery and genetic characterization of mutant alleles at zygotic loci on the third chromosome ofDrosophila melanogaster which alter the morphology of the larval cuticle. We derived 12600 single lines from ethyl methane sulfonate (EMS)-treatedst e orrucuca chromosomes and assayed them for embryonic lethal mutations by estimating hatch rates of egg collections. About 7100 of these lines yielded at least a quarter of unhatched eggs and were then scored for embryonic phenotypes. Through microscopic examination of unhatched eggs 1772 lines corresponding to 24% of all lethal hits were classified as embryonic lethal. In 198 lines (2.7% of all lethal hits), mutant embryos showed distinct abnormalities of the larval cuticle. These embryonic visible mutants define 45 loci by complementation analysis. For 32 loci, more than one mutant allele was recovered, with an average of 5.8 alleles per locus. Complementation of all other mutants was shown by 13 mutants. The genes were localized on the genetic map by recombination analysis, as well as cytologically by complementation analysis with deficiencies. They appear to be randomly distributed along the chromosome. Allele frequencies and comparisons with deficiency phenotypes indicate that the 45 loci represent most, if not all, zygotic loci on the third chromosome, where lack of function recognizably affects the morphology of the larval cuticle.     

Developmental analysis of a temperature-sensitive melanotic tumor mutant inDrosophila melanogaster

Summary A sex-linked, temperature-sensitive melanotic tumor mutation inDrosophila melanogaster, tu (1) Sz ^ts, was mapped at 34.3±and localized to bands 10A10-11 of the polytene chromosomes. At 26°Ctu-Sz ^ts larvae develop melanotic tumors whereas 18°C is non-permissive for tumor formation. Tumorigenesis at 26°C involves the encapsulation of abnormal caudal fat body regions by precociously differentiated hemocytes. Low temperature blocks the development of the abnormal adipose cells and the overlying aberrant tissue surfaces but does not inhibit precocious differentiation of the hemocytes to the lamellocytic form. This phenotypic difference at the two temperatures indicates that lamellocyte encapsulation to form melanotic tumors is directed against abnormal tissue surfaces. On the basis of these observations and an earlier study (Rizki and Rizki 1979) we propose that hereditary melanotic tumors inD. melanogaster are a calss of autoimmune disorders in which affected tissue surfaces arouse the body's cellmediated defense response.     

X-linked loci of Drosophila melanogaster causing defects in the morphology of the embryonic salivary glands

To identify X chromosomal genes required for salivary gland development in the Drosophila embryo, we screened embryos hemizygous for EMS-induced lethal mutations to find mutations causing gross morphological defects in salivary gland development. The parental strain carried a lac Z transgene on the second chromosome, which was specifically expressed in the salivary glands so the mutations could be unambiguously identified. Embryos from 3,383 lines were tested for salivary gland abnormalities following lacZ staining. From 63 lines exhibiting aberrant salivary gland phenotypes, 52 stable lines were established containing mutations affecting salivary gland development. From these, 39 lines could be assigned to nine complementation groups: armadillo, brinker, folded gastrulation, giant, hindsight, Notch, runt, stardust and twisted gastrulation. Received: 10 April 2000 / Accepted: 31 May 2000     

Screening of larval/pupal P-element induced lethals on the second chromosome in Drosophila melanogaster: clonal analysis and morphology of imaginal discs

We have carried out screens for lethal mutations on the second chromosome of Drosophila melanogaster that are associated with abnormal imaginal disc morphologies, particularly in the wing disc. From a collection of 164 P element-induced mutations with a late larva/pupa lethal phase we have identified 56 new loci whose gene products are required for normal wing disc development and for normal morphology of other larval organs. Genetic mosaics of these 56 mutant lines show clonal mutant phenotypes for 23 cell-viable mutations. These phenotypes result from altered cell parameters. Causal relationships between disc and clonal phenotypes are discussed.     

The black-pearl gene of Drosophila defines a novel conserved protein family and is required for larval growth and survival

Using a transposon insertion line of the Drosophila Genome Project we have cloned the black-pearl gene (blp), analyzed cDNA clones, generated various mutants, and characterized their phenotypes. The blp gene codes for a protein of 15.7 kDa calculated molecular weight that has been conserved from yeast to plants and mammals with high homology. A domain of these new proteins shows distant similarity to DnaJ domains indicating a functionally relevant interaction with other proteins. The P element insertion in line P1539 lies within the 5' untranslated leader of the black-pearl gene. Flies homozygous for this insertion are semi-lethal, escapers produce very few offspring and show melanotic inclusions in the hemocoel ('black pearls') similar to various melanotic 'tumor' mutants. Two small deletions confined to the blp gene and two EMS-induced mutations are homozygous lethal. These null mutants appear normal up to a prolonged first instar larval stage but fail to grow and die. Thus in Drosophila the blp gene is specifically required for larval growth. The evolutionary conservation in both unicellular and multicellular organisms suggests for the new protein family described here a fundamental role in cell growth.     

Genetic and molecular analysis of the dpy-14 region in Caenorhabditis elegans.

Summary Essential genes have been identified in the 1.5 map unit (m.u.)dpy-14-unc-29 region of chromosome I inCaenorhabditis elegans. Previous work defined nine genes with visible mutant phenotypes and nine genes with lethal mutant phenotypes. In this study, we have identified an additional 28 essential genes with 97 lethal mutations. The mutations were mapped using eleven duplication breakpoints, eight deficiencies and three-factor recombination experiments. Genes required for the early stages of development were common, with 24 of the 37 essential genes having mutant phenotypes arresting at an early larval stage. Most mutants of a gene have the same time of arrest; only four of the 20 essential genes with multiple alleles have alleles with different phenotypes. From the analysis of complementing alleles oflet-389, alleles with the same time-of-arrest phenotype were classified as either hypomorphic or amorphic. Mutants oflet-605, let-534 andunc-37 have both uncoordinated and lethal phenotypes, suggesting that these genes are required for the coordination of movement and for viability. The physical and genetic maps in thedpy-14 region were linked by positioning two N2/BO polymorphisms with respect to duplications in the region, and by localizing the right breakpoint of the deficiencyhDf8 on the physical map. Using cross-species hybridization toC. briggsae, ten regions of homology have been identified, eight of which are known to be coding regions, based on Northern analysis and/or the isolation of cDNA clones.     

Growth, metastasis, and invasiveness of Drosophila tumors caused by mutations in specific tumor suppressor genes

 More than 50 genes have been identified in Drosophila by loss-of-function mutations that lead to overgrowth of specific tissues. Loss-of-function mutations in the lethal giant larvae, discs large, or brain tumor genes cause neoplastic overgrowth of larval brains and imaginal discs. In the present study, the growth and metastatic potential of tumors resulting from mutations in these genes were quantified. Overgrown brains and imaginal discs were transplanted into adults and β-galactosidase accumulation was used as a marker to identify donor cells. Mutations in these three genes generated tumors with similar metastatic patterns. For brain tumors, the metastatic index (a measure we defined as the fraction of hosts that acquired secondary tumors normalized for the amount of primary tumor growth) of each of the three mutants was similar. Analysis of cell proliferation in mutant brains suggests that the tumors arise from a population of several hundred cells which represent only 1–2% of the cells in third instar larval brains. For imaginal disc tumors from lethal giant larvae and brain tumor mutants, it is shown for the first time that they can be metastatic and invasive. Primary imaginal disc tumors from lethal giant larvae and brain tumor mutants formed secondary tumors in 43 and 53% of the hosts, respectively, although the secondary tumors were, in general, smaller than the secondary tumors derived from primary brain tumors. Received: 18 August 1997 / Accepted: 16 October 1997     

Lethals, Steriles and Deficiencies in a Region of the X Chromosome of CAENORHABDITIS ELEGANS

Twenty-one X-linked recessive lethal and sterile mutations balanced by an unlinked X-chromosome duplication have been identified following EMS treatment of the small nematode, Caenorhabditis elegans. The mutations have been assigned by complementation analysis to 14 genes, four of which have more than one mutant allele. Four mutants, all alleles, are temperature-sensitive embryonic lethals. Twelve mutants, in ten genes, are early larval lethals. Two mutants are late larval lethals, and the expression of one of these is influenced by the number of X chromosomes in the genotype. Two mutants are maternal-effect lethals; for both, oocytes made by mutant hermaphrodites are rescuable by wild-type sperm. One of the maternal-effect lethals and two larval lethals are allelic. One mutant makes defective sperm. The lethals and steriles have been mapped by recombination and by complementation testing against 19 deficiencies identified after X-ray treatment. The deficiencies divide the region, about 15% of the X-chromosome linkage map, into at least nine segments. The deficiencies have also been used to check the phenotypes of hemizygous lethal and sterile hermaphrodites.     

Behavioral mutants of Drosophila melanogaster. IV. Analysis of developmentally temperature-sensitive mutations affecting flight

Mutations in 13 genes with temperature-sensitive (ts), flightless phenotypes have been examined. All hop and fly well when raised at the permissive temperature, but fly poorly, or not at all, when raised at the restrictive temperature. The mutations were divided into three groups on the basis of their temperature-sensitive periods (TSPs) for flightlessness. The TSPs for mutations at five loci, fli-C¹, D¹, E¹, I¹, and shak A¹, in the first group are confined to 24 to 48 hr interval during early pupal development. Mutations in the second group, including eag¹⁰¹, fli B¹, and fu^ts1 have continuous TSPs 3 to 4 days in length, extending from late larval through the early pupal stages. The flight TSPs for mutations in the third class, including fli J¹, fli K², flrd H³, and flrd N¹, are almost continuous, and span most of the larval and pupal periods. Many of the mutations have pleiotropic phenotypes, including semilethality and lethality, and wing posture and cuticle abnormalities, with discernible TSPs. One of the more intriguing pleiotropic phenotypes is the ts optomotor response exhibited by fli J², the TSP for which extends from late larval through late pupal stages.     

COP9 signalosome subunits 4 and 5 regulate multiple pleiotropic pathways in Drosophila melanogaster

The COP9 signalosome (CSN) is an essential eight-subunit repressor of light-regulated development in ARABIDOPSIS: This complex has also been identified in animals, though its developmental role remains obscure. CSN subunits have been implicated in various cellular processes, suggesting a possible role for the CSN as an integrator of multiple signaling pathways. In order to elucidate the function of the CSN in animals, a Drosophila model system has previously been established. Gel-filtration analysis with antibodies against CSN subunits 4, 5 and 7 revealed that these proteins act as a complex in Drosophila that is similar in size to the plant and mammalian complexes. Null mutations in either one of two subunits, CSN4 or CSN5, are larval lethal. Successful embryogenesis appears to be a consequence of maternal contribution of the complex. Biochemical analysis indicates that the different subunits are found in both CSN-dependent and CSN-independent forms, and that these forms are differentially affected by the mutations. Phenotypic characterization of these two mutants indicates that they show both shared and unique phenotypes, which suggest specific roles for each subunit. Both mutants have defective oocyte and embryo patterning, and defects in response to DNA damage, while csn5 mutants develop melanotic tumors and csn4 mutants have phenotypes reminiscent of defects in ecdysone signaling.     

Analysis of molting and metamorphosis in the ecdysteroid-deficient mutant L(3)3DTS of Drosophila melanogaster

The dominant temperature-sensitive mutation L(3)3^DTS (DTS-3) in Drosophila melanogaster causes lethality of heterozygotes during the third larval instar at the restrictive temperature (29°C). Temperature-shift experiments revealed two distinct temperature-sensitive periods, with lethal phases during the third larval instar (which may persist for 4 weeks) and during the late pupal stage. At 29°C mutant imaginal discs are unable to evert in situ, but did evert normally if cultured in the presence of exogenous ecdysterone or when implanted into wild-type larval hosts. The only morphologically abnormal tissue present in the lethal larvae is the ring gland, the prothoracic gland being greatly hypertrophied in third instar DTS-3 larvae. Injection of a single wild-type ring gland rescued these mutant larvae, indicating that the mutant gland is functionally, as well as morphologically, abnormal. Finally, the mutant larvae were shown to have less than 10% of the wild-type ecdysteroid levels. These results are all consistent with a proposed lesion in ecdysteroid hormone production in DTS-3 larvae. A comparison with the phenotypes of other “ecdysone-less” mutants is presented.     

Mechanism of conjugation and recombination in bacteria XVI

Summary Protein synthesis by ribosomes from several cryptopleurine-resistant yeast mutants is also resistant to emetine and tubulosine. These mutants can be classified into two different types: Class I mutants which display high levels of resistance to emetine and tubulosine and Class II mutants that are only weakly resistant to tubulosine and are slightly more sensitive to emetine than those of Class I. Apparently all mutants have similar levels of resistance to cryptopleurine. The distinct phenotypes of Class I and Class II strains are expressed through their 40S ribosomal subunit. Genetic analysis has shown that the mutations to cryptopleurine resistance are allelic and that in a particular case (strain CRY6) the pleiotropic phenotype is a result of the expression of the cryl locus. It is suggested that Class I and Class II mutants arise from two independent mutational events within the cryl allele. in heterozygous (+/cryl) diploids both the sensitive and the resistant genes are expressed as shown by studies of the action of cryptopleurine on polyphenylalanine-synthesizing system derived from each parental sensitive and resistant haploid strain and heterozygous diploid strains. The apparent dominance of sensitivity over resistance which may be observed in vivo in heterozygous (+/cryl) diploids has been explained in terms of the mode of action of the inhibitors.     

Hemocyte responses to implanted tissues inDrosophila melanogaster larvae

Summary At 26° C temperature-sensitivetu(1) Sz ^ts larvae ofDrosophila melanogaster develop melanotic tumors consisting of aberrant caudal adipose tissue encapsulated by precociously differentiated hemocytes (lamellocytes). Whentu-Sz ^ts larvae are grown at 18° C, lamellocytes are present but the caudal fat body surfaces remain normal and melanotic tumors do not develop (Rizki and Rizki, preceding paper). In this paper we demonstrate that the lamellocytes intu-Sz ^ts larvae at 18° C encapsulate implants of mechanically-damaged fat bodies and adipose cells devoid of basement membrane, while leaving host fat bodies or implanted fat bodies with intact basement membrane unencapsulated. Therefore, low temperature blocks melanotic tumor formation by normalizing the surfaces of the prospective tumor-forming sites intu-Sz ^ts.The discriminatory ability oftu-Sz ^ts lamellocytes was examined by challenging them with undamaged heterospecific tissues. Tissues from sibling species ofD. melanogaster were not encapsulated whereas tissues fromDrosophila species outside theD. melanogaster species subgroup were. Ultrastructural examination of encapsulated heterospecific tissues showed intact basement membrane, so we propose that distinction between self and not self by lamellocytes depends upon the molecular architecture of the basement membrane. In similar series of experiments usingD. virilis donor tissues inOre-R wild type larval hosts, fat bodies remained unencapsulated and imaginal disks metamorphosed. These studies suggest that continued presence of lamellocytes in the larval host is a prerequisite for encapsulation.