Genotoxicity of p-aminophenol in somatic and germ line cells of Drosophila melanogaster

p-Aminophenol (PAP; as a component of, e.g., hair dyes, photographic developers, as adsorbent in gas filters, as a metabolite of various fungicides, pesticides and drugs) has been tested for genotoxicity in Drosophila by means of the sex-linked recessive lethal test (SLRLT) and the somatic mutation and recombination test (SMART) of the wing. While the SLRLT was not significant, the SMART clearly indicated that the compound has genotoxic properties in this in vivo test in agreement with a majority of mammalian short-term tests in vitro and in vivo. The reducing agent dithiothreitol enhanced the genotoxic effects of PAP in the SMART; the reasons for this interaction remain to be elucidated.     

Rapid fluorescence-based screening for Wolbachia endosymbionts in Drosophila germ line and somatic tissues

Wolbachia is a globally distributed bacterial endosymbiont present in arthropods and nematodes. The advent of sensitive PCR-based approaches has greatly facilitated the identification of Wolbachia-infected individuals and analysis of population infection levels. Here, a complementary visual fluorescence-based Wolbachia screening approach is described. Through the use of the fluorescent dye Syto-11, Wolbachia can be efficiently detected in various Drosophila tissues, including ovaries. Syto-11 also stains Wolbachia in other insects. Because Wolbachia is inherited through the maternal germ line, bacteria reside in the ovaries of flies in infected populations. An advantage of this staining approach is that it informs about Wolbachia titer as well as its tissue and cellular distribution. Using this method, the infection status of insect populations in two central California locations was determined, and variants with unusually low or high Wolbachia titers were isolated. In addition, a variant with ovarioles containing both infected and uninfected egg chambers was identified. Syto-11 staining of Cardinium- and Spiroplasma-infected insects was also analyzed.     

Effects of DNA-repair-deficient mutants on somatic and germ line mutagenesis in the UZ system in Drosophila melanogaster

To analyze the underlying mechanisms of the UZ system in Drosophila melanogaster, i.e., the unstable white locus with an IS element included originally described by Rasmuson and Green (1974), the repair deficient mutants mei-9b and mei-4lD5 and the meiotic mutant mei-2l8 were introduced into X-chromosomes containing the UZ system. These strains were submitted to larval feeding of MMS to analyze differences occurring in mutation rate and survival. The mei-9b and mei-4l strains were markedly sensitive to MMS treatment, while mei-2l8 did not affect survival. Only the mutant mei-4l, which is lacking in post-replication repair, affected the somatic mutation rate of the UZ system, decreasing it by 50%. The spontaneous germ line mutation rate of UZ, on the other hand, was increased by introducing mei-4l or mei-9b, possibly as a result of the high frequency of unrepaired spontaneous chromosome breaks that occur in these mutants.     

A clonal analysis of the roles of somatic cells and germ line during oogenesis in Drosophila

In order to test whether particular female sterile mutations block functions which normally occur in somatic or germ line derivatives, clones homozygous for each mutation were X-ray induced in heterozygous females. Using the germ line-dependent egg marker, fs(1)K10, it was possible to identify the eggs derived from clones which had been induced in the germ line. Mutations were classified as germ line dependent when these eggs also showed the phenotype associated with the female sterile mutation. Two mutations which caused early abnormalities in oogenesis (fs(1)116, fs(1)1304) were shown to affect germ cells, whereas two mutations which caused egg retention (fs(1)462, fs(1)1001) were somatically dependent. A mutation altering egg dimensions without affecting egg volume (short egg) was also shown to depend on somatic cells in the ovary. With one exception (fs(1)K4), mutations which caused production of fragile, collapsed eggs (fs(1)180, fs(1)473, fs(1)384, and fs(1)1163) were somatically dependent. Patches of mutant fs(1)384 morphology were found in the chorions of the eggs not derived from germ line clones. These patches are interpreted as being caused by homozygous clones in the somatically derived follicle cell epithelium and suggest that fs(1)384 affects processes occurring in these cells during the synthesis of the egg coverings.     

Cell-autonomous and inductive signals can determine the sex of the germ line of drosophila by regulating the gene Sxl

To investigate the mechanism of sex determination in the germ line, we analyzed the fate of XY germ cells in ovaries, and the fate of XX germ cells in testes. In ovaries, germ cells developed according to their X:A ratio, i.e., XX cells underwent oogenesis, XY cells formed spermatocytes. In testes, however, XY and XX germ cells entered the spermatogenic pathway. Thus, to determine their sex, the germ cells of Drosophila have cell-autonomous genetic information, and XX cells respond to inductive signals of the soma. Results obtained with amorphic and constitutive mutations of Sxl show that both the genetic and the somatic signals act through Sxl to achieve sex determination in germ cells.     

Mutagenicity of sumithion tested in Drosophila somatic and germ cells

Sumithion, a broad-spectrum insecticide, was tested for its mutagenicity in the Drosophila wing-spot test and sex-linked recessive lethal test. Strains carrying the recessive mutant markers mwh and flr3 in their third chromosomes, expressed phenotypically as multiple trichomes or thickened and misshapen wing hairs in the adult wings, were used in the wing-spot test. Larvae transheterozygous for these markers were exposed to the insecticide in instant food and the sex-linked recessive lethal test was performed by the standard technique using the Basc strain. The compound is mutagenic in the wing primordial cells and induces recombination at high doses. Further, the frequency of induction of sex-linked recessive lethals is significant only at high treatment doses.     

Interactions between germ cells and somatic cells in Drosophila melanogaster

Interactions between germ cells and somatic cells are important at several stages of Drosophila development. The types of interactions that will be discussed include: (1) molecules physically transferred from one cell to another; (2) long range interactions by hormones; and (3) local interactions between germ cells and somatic cells when they are in close proximity. These interactions have been mostly characterized during oogenesis.     

The Drosophila sex determination gene daughterless has different functions in the germ line versus the soma

As a regulator of the female-specific gene Sxl, da+ provides an essential maternal component in the control of sex determination and dosage compensation; nevertheless, neither the maternal nor zygotic phenotypes of the original mutant da allele is sex-specific. Here we clarify the role of da+ in Drosophila development, finding: this sex determination gene is indeed pleiotropic; zygotic functioning of da+ is essential in both sexes for somatic cell development, but not for germ cell development; da female sterility results from a somatic, rather than germ-line, defect; and expression of da+ in the maternal germ line is required only for daughters in the subsequent generation, as expected for a specific regulator of Sxl+. These conclusions follow from the characterization of new da null alleles isolated by a selection for defects in maternally acting positive regulators of Sxl.     

Chromatin organization in the male germ line of Drosophila hydei

The chromatin organization in developing germ cells of Drosophila hydei males was studied with the highly sensitive DNA stain DAPI (4, 6-diamidino-2-phenylindole dichloride). The prophase of meiosis I is characterized by decondensed chromosomes and only late during this stage do they condense rapidly. The sex chromosomes show allocycly. During postmeiotic development the final condensation of chromatin is preceded by a cycle of condensation and subsequent decondensation. Meiotic chromosomes were studied in more detail after orcein staining. Pairing sites of the sex chromosomes could be localized in the distal end of the heterochromatic arm of the X chromosome and distally in both arms of the Y chromosome. The various heterochromatic parts of the genome condense differentially in meiosis. Chromatin reorganization was studied cytochemically with antibodies raised against histones H1 and H2A of D. melanogaster. The core histone H2A is present in spermatid nuclei until the late elongation stage. However, histone H1 is not found in the chromatin later than the early primary spermatocyte stage. Thus, chromatin reorganization during spermatogenesis in D. hydei is complex. The process is discussed with regard to possible functions.     

Acrylamide is genotoxic to the somatic and germ cells of Drosophila melanogaster

The genotoxic effects of acrylamide, a recently detected carcinogen, have been studied in the somatic (wing primordia) and germ cells of Drosophila melanogaster by the wing mosaic assay and the sex-linked recessive lethal test respectively. Larvae, 72 +/- 4 h old, were exposed to 6 different concentrations of acrylamide ranging between 0.25 mM and 5.0 mM in instant medium for 48 h. It is observed that acrylamide is both mutagenic and recombinogenic in the wing disc cells and induces sex-linked recessive lethals.     

Mammalian DNMTs in the male germ line DNA of Drosophila

It is controversial whether DNA methylation plays a functional role in Drosophila. We have studied testis DNA of Drosophila melanogaster Meigen, 1830 with antisera against 5-methylcytosine (5mC) and found no evidence for the presence of significant amounts of 5mC. Reactions occur only with 1 of 3 5mC antisera, but they are restricted to nuclear regions without detectable amounts of DNA. The antisera apparently cross-react with other nuclear components. If the murine de novo DNA methyltransferases, DNMT3A and DNMT3B, are expressed under the control of the spermatocyte-specific beta2-tubulin promoter in testes, DNA methylation is not increased and no effects on the fertility of the fly are seen. DNA methylation has, therefore, no functional relevance in the male germ line of Drosophila.     

Drosophila oogenesis: coordinating germ line and soma

A new function for Delta-Notch signaling has been discovered in Drosophila oogenesis: Delta expressed in the germ cells activates Notch in the surrounding somatic follicle cells to control their differentiation, proliferation and morphogenesis.     

Stage-specific inductive signals in the Drosophila neuroectoderm control the temporal sequence of neuroblast specification

One of the initial steps of neurogenesis in the Drosophila embryo is the delamination of a stereotype set of neural progenitor cells (neuroblasts) from the neuroectoderm. The time window of neuroblast segregation has been divided into five successive waves (S1-S5) in which subsets of neuroblasts with specific identities are formed. To test when identity specification of the various neuroblasts takes place and whether extrinsic signals are involved, we have performed heterochronic transplantation experiments. Single neuroectodermal cells from stage 10 donor embryos (after S2) were transplanted into the neuroectoderm of host embryos at stage 7 (before S1) and vice versa. The fate of these cells was uncovered by their lineages at stage 16/17. Transplanted cells adjusted their fate to the new temporal situation. Late neuroectodermal cells were able to take over the fate of early (S1/S2) neuroblasts. The early neuroectodermal cells preferentially generated late (S4/S5) neuroblasts, despite their reduced time of exposure to the neuroectoderm. Furthermore, neuroblast fates are independent from divisions of neuroectodermal progenitor cells. We conclude from these experiments that neuroblast specification occurs sequentially under the control of non-cell-autonomous and stage-specific inductive signals that act in the neuroectoderm.     

A Wolbachia-associated fitness benefit depends on genetic background in Drosophila simulans

The alpha-proteobacteria Wolbachia infect a number of insect species and influence host reproduction to favour the spread of infected females through a population. The fitness effect of this infection is important in understanding the spread and maintenance of Wolbachia within and among host populations. However, a full elucidation of fitness effect requires careful control of host genetic background. Here, I transferred a single clone of Wolbachia (the wHa strain) into three genetically distinct isofemale lines of the fly Drosophila simulans using microinjection methodology. These lines carried one of the three described mitochondrial haplogroups (siI, siII or siIII) and differ in nuclear genome as well. Population cage assays showed that wHa-infected siIII flies enjoyed a dramatic fitness benefit compared to uninfected siIII. In contrast, wHa did not affect the fitness of siI or siII flies. This study points to the importance of host-by-symbiont interaction terms that may play an important role in organismal-fitness.     

Differential requirements of a mitotic acetyltransferase in somatic and germ line cells

During mitosis different types of cells can have differential requirements for chromosome segregation. We isolated two new alleles of the separation anxiety gene (san). san was previously described in both Drosophila and in humans to be required for centromeric sister chromatid cohesion (Hou et al., 2007; Williams et al., 2003). Our work confirms and expands the observation that san is required in vivo for normal mitosis of different types of somatic cells. In addition, we suggest that san is also important for the correct resolution of chromosomes, implying a more general function of this acetyltransferase. Surprisingly, during oogenesis we cannot detect mitotic defects in germ line cells mutant for san. We hypothesize the female germ line stem cells have differential requirements for mitotic sister chromatid cohesion.