Isolation and Characterization of Dominant Female Sterile Mutations of Drosophila Melanogaster. II. Mutations on the Second Chromosome

Twenty-four, second chromosome, dominant female sterile (Fs) mutations in Drosophila are described. Fs(2) were isolated at a frequency of approximately 1 per 1000 EMS-treated chromosomes screened. In comparison the isolation of frequency for second chromosome zygotic recessive lethal mutations was approximately 550 per 1000. Complementation analysis of the Fs(2) revertants showed that the 24 Fs(2) mutations identify 13-15 loci, calculated to be about 65-75% of the second chromosome genes EMS mutable to dominant female sterility. Two of the Fs(2) mutations are useful tools for the dominant female sterile technique: Fs(2)1 for induction and detection of germ-line clones and Fs(2)Ugra for follicle cell clones. Several of the Fs(2) mutations bring about novel mutant phenotypes. Seven of them alter egg shape, whereas the others arrest development primarily at two stages: around fertilization by five Fs(2) and during cleavage divisions [by Fs(2) in three loci]. The remaining that allow development to the larval stage of differentiation include four new dorsal alleles and one dominant torso allele. Analysis of germ-line chimeras revealed that with two exceptions all the Fs(2) mutations are germ-line dependent. The Fs(2) mutations were mapped mainly on the basis of mitotic recombination induced in the female germ-line cells of adult females. That most of the Fs(2) may be gain-of-function mutations is indicated by the unusual behavior of the Fs+ germ-line clones and also by the fact that 90% of the could be induced to revert.     

Isolation and Characterization of Dominant Female Sterile Mutations of Drosophila Melanogaster. I. Mutations on the Third Chromosome

Fifty-one dominant female sterile (Fs) mutations linked to the third chromosome of Drosophila melanogaster are described. EMS induced Fs mutations arise with the frequency of one Fs per about 2500 recessive lethals. Complementation analysis of the revertants showed that these Fs mutations represent 27-34 loci, about 60% of the third chromosome units mutable to dominant female sterility by EMS. The Fs mutations were mapped on the basis of mitotic recombination induced in the female (in 16 cases also in the male) germ-line. Behavior of the revertants and the Fs+ germ-line clones demonstrate the gain-of-function nature of the Fs alleles. With two exceptions, the Fs(3) mutations are germ-line dependent. Novel phenotypes appeared in most of the Fs mutations. With eight exceptions, the Fs(3) mutations are fully penetrant, in some cases with variable expressivity. One of the Fs(3) mutations is a non-ovary-dependent egg retention mutation, two others alter egg shape, and 27 bring about arrest in development at about the time of fertilization. In 21 of the Fs(3) mutations embryos develop to the larval stage of differentiation; this group includes 5 new alleles of Toll and 4 of easter.     

Genetic Evidence That the Sans Fille Locus Is Involved in Drosophila Sex Determination

Females homozygous for sans fille1621 (= fs(1)1621) have an abnormal germ line. Instead of producing eggs, the germ-line cells proliferate forming ovarian tumors or excessive numbers of nurse cells. The Sex-lethal gene product(s) regulate the branch point of the dosage compensation and sex determination pathways in the soma. The role of Sex-lethal in the germ line is not clear but the germ line of females homozygous for female sterile Sex-lethal alleles or germ-line clones of loss-of-function alleles are characterized by ovarian tumors. Females heterozygous for sans fille1621 or Sex-lethal are phenotypically wild type with respect to viability and fertility but females trans-heterozygous for sans fille1621 and Sex-lethal show ovarian tumors, somatic sexual transformations, and greatly reduced viability.     

Requirement for Cell-Proliferation Control Genes in Drosophila Oogenesis

Genes that are required for cell proliferation control in Drosophila imaginal discs were tested for function in the female germ-line and follicle cells. Chimeras and mosaics were produced in which developing oocytes and nurse cells were mutant at one of five imaginal disc overgrowth loci (fat, lgd, lgl, c43 and dco) while the enveloping follicle cells were normal. The chimeras were produced by transplantation of pole cells and the mosaics were produced by X-ray-induced mitotic recombination using the dominant female-sterile technique. The results show that each of the genes tested plays an essential role in the development or function of the female germ line. The fat, lgl and c43 homozygous germ-line clones fail to produce eggs, indicating a germ-line requirement for the corresponding genes. Perdurance of the fat+ gene product in mitotic recombination clones allows the formation of a few infertile eggs from fat homozygous germ-line cells. The lgd homozygous germ-line clones give rise to a few eggs with abnormal chorionic appendages, a defect thought to result from defective cell communication between the mutant germ-line and the nonmutant follicle cells. One allele of dco (dcole88) prevents egg development when homozygous in the germ line, whereas the dco18 allele has no effect on germ-line development. Fs(2)Ugra, a recently described follicle cell-dependent dominant female-sterile mutation, allowed the analysis of egg primordia in which fat, lgd or lgl homozygous mutant follicle cells surrounded normal oocytes. The results show that the fat and lgd genes are not required for follicle cell functions, while absence of lgl function in follicles prevents egg development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Viability of Female Germ-Line Cells Homozygous for Zygotic Lethals in DROSOPHILA MELANOGASTER

We have analyzed the viability of different types of X chromosomes in homozygous clones of female germ cells. The chromosomes carried viable mutations, single-cistron zygotic-lethal and semi-lethal mutations, or small (about six chromosome band) deletions. Homozygous germ-line clones were produced by recombination in females heterozygous for an X-linked, dominant, agametic female sterile.

All the zygotic-viable mutants are also viable in germ cells. Of 16 deletions tested (uncovering a total of 93 bands) only 2 (of 4 and 5 bands) are germ-cell viable. Mutations in 15 lethal complementation groups in the zeste-white region were tested. When known, the most extreme alleles at each locus were tested. Only in five loci (33%) were the mutants viable in the germ line. Similar studies of the same deletions and point-mutant lethals in epidermal cells show that 42% of the bands and 77% of the lethal alleles are viable. Thus, germ-line cells have more stringent cell-autonomous genetic requirements than do epidermal cells.

The eggs recovered from clones of three of the germ-cell viable zw mutations gave embryos arrested early in embryogenesis, although genotypically identical embryos derived from heterozygous oogonia die as larvae or even hatch as adult escapers. For two genes, homozygosis of the mutations tested also caused embryonic arrest of heterozygous female embryos, and in one case, the eggs did not develop at all. Germ-line clones of one quite leaky mutation gave eggs that were indistinguishable from normal. The abundance of genes whose products are required for oogenesis, whose products are required in the oocyte, and whose activity is required during zygotic development is discussed.

     

High male:female ratio of germ-line mutations: an alternative explanation for postulated gestational lethality in males in X-linked dominant disorders.

In this paper I suggest that a vastly higher rate of de novo mutations in males than in females would explain some, if not most, X-linked dominant disorders associated with a low incidence of affected males. It is the inclusion of the impact of a high ratio of male:female de novo germ-line mutations that makes this model new and unique. Specifically, it is concluded that, if an X-linked disorder results in a dominant phenotype with a significant reproductive disadvantage (genetic lethality), affected females will, in virtually all cases, arise from de novo germ-line mutations inherited from their fathers rather than from their mothers. Under this hypothesis, the absence of affected males is explained by the simple fact that sons do not inherit their X chromosome (normal or abnormal) from their fathers. Because females who are heterozygous for a dominant disorder will be clinically affected and will, in most cases, either be infertile or lack reproductive opportunities, the mutant gene will not be transmitted by them to the next generation (i.e., it will be a genetic lethal). This, not gestational lethality in males, may explain the absence of affected males in most, if not all, of the 13 known X-linked dominant diseases characterized by high ratios of affected female to male individuals. Evidence suggesting that this mechanism could explain the findings in the Rett syndrome is reviewed in detail.     

The effects of two mutations connected with chromatin functions on female germ-line cells of Drosophila

Summary We have studied the developmental effects of two dominant suppressor mutations of position-effect variegation mutations on female germ-line cells. Su-var (2) 1⁰¹, which has been shown to affect chromatin structure though altering histone deacetylation, and Su-var (3) 3⁰³are recessive female steriles and zygotic lethals in the presence of butyrate or an additional Y chromosome. We have analysed mosaic females with mutant germ-line and normal soma and concluded that intact functions of the Su-var (2) 1 and the Su-var (3) 3 genes are required for development of both the soma and the germ-line and that as indirect evidence suggest, their maternally provided products are needed for normal embryonic development. It is suggested that there is possibly a common control of chromatin structure and gene expression in the soma, female germ-line and embryonic cells of Drosophila.     

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.     

Feeding, fecundity and lifespan in female Drosophila melanogaster

Male seminal fluid proteins induce a profound remodelling of behavioural, physiological and gene signalling pathways in females of many taxa, and typically cause elevated egg production and decreased sexual receptivity. In Drosophila melanogaster, these effects can be mediated by an ejaculate 'sex peptide' (SP), which, in addition, contributes significantly to the cost of mating in females. Recent research has revealed that SP can stimulate female post-copulatory feeding, raising the possibility that the widespread female cost of mating could be due to over-feeding. In this study, we used D. melanogaster as a model to test this hypothesis. We first show that elevated post-mating feeding is dependent upon egg production and does not occur in sterile ovoD1 mutant females. This conclusion was also supported by the increase in feeding of virgin females whose egg production was experimentally elevated. We then demonstrated that sterile ovoD1 and fertile females experienced identical survival costs of mating, related to their frequency of mating and not to female feeding rate or to egg production. We conclude that female mating costs are not the result of over-feeding, but may be due to other, potentially more direct, effects of ejaculate molecules.     

Analysis of follicle-cell functions in Drosophila: the Fs(3)Apc mutation and the development of chorionic appendages

Fs(3)Apc is a dominant female-sterile mutation of Drosophila melanogaster which causes an incomplete migration of follicle cells between the oocyte and the nurse cells. This leads to leakage of anterior egg cytoplasm followed by degeneration of the egg primordium or deposition of flaccid eggs with reduced anterior egg coverings including the dorsal appendages. Analysis of ovarian and germ-line chimeras revealed that the focus of the Apc phenotype is located in the ovarian soma. Apc+ clones, induced by mitotic recombination, lead to the formation of "exceptional" eggs with (often partial) rescue of the mutant phenotype. Analysis of Apc+ mosaics shows that the Apc mutant phenotype depends on the genotype of the anterior follicle cells. The patterns of apparent Apc+ clones suggest that there is no lineage restriction between the follicle cells that form the anterior egg coverings and those that form the dorsal appendages at the follicle envelope stage.     

Genotoxicity of zineb detected through the somatic and germ-line mosaic assays and the sex-linked recessive-lethal test in Drosophila melanogaster

The genotoxicity of zineb, a carbamate fungicide, has been tested through eye, wing and female germ line mosaic assays and the sex-linked recessive-lethal test in Drosophila melanogaster. Larvae of different instars, heterozygous for appropriate recessive genetic markers, were exposed to the fungicide in food for different durations of time. The adult eyes and wings were screened for induction of mosaic spots and the eggs laid by the females were checked for induction of female germ-line mosaicism. It is concluded that zineb is genotoxic to both somatic and germ-line cells of Drosophila.     

Genetic Analysis of Three Dominant Female-Sterile Mutations Located on the X Chromosome of DROSOPHILA MELANOGASTER

Three dominant female-sterile mutations were isolated following ethyl methanesulfonate (EMS) mutagenesis. Females heterozygous for two of these mutations show atrophy of the ovaries and produce no eggs (ovo ^D1) or few eggs (ovo^D2); females heterozygous for the third mutation, ovo^D3, lay flaccid eggs. All three mutations are germ line-dependent and map to the cytological region 4D-E on the X chromosome; they represent a single allelic series. Two doses of the wild-type allele restore fertility to females carrying ovo^D3 and ovo^D2, but females carrying ovo^D1 and three doses of the wild-type allele remain sterile. The three mutations are stable in males but are capable of reversion in females; reversion of the dominant mutations is accompanied by the appearance, in the same region, of a recessive mutation causing female sterility. We discuss the utility of these mutations as markers of clones induced in the female germ line by mitotic recombination as well as the nature of the mutations.     

Heterozygosity and fitness in a California population of the labyrinth spider Metepeira ventura (Araneae, Araneidae)

Abstract. The relationship between individual heterozygosity and characteristics likely to be associated with fitness was investigated in the labyrinth spider Metepeira ventura . Adult females and their egg sacs were collected at a coastal site in southern California, and three measures of bodily condition (carapace width, weight, residual index) and six measures of reproductive output (number of egg sacs, variation in egg number among sacs [coefficient of variation], total number of eggs, mean eggs/sac, mean eggs/sac divided by carapace width, mean eggs/sac divided by weight) were determined for each spider. The sample was polymorphic at three allozyme loci that were in Hardy–Weinberg equilibrium, and individual females were heterozygous at up to two of the three loci, forming three heterozygosity classes (0, 1, and 2). None of the bodily condition measures were significantly related to the number of heterozygous loci, while four of the reproductive output estimators (total number of eggs, mean eggs/sac, mean eggs/sac divided by carapace width, mean eggs/sac divided by weight) were significantly influenced by heterozygosity. In each significant case, values for class 2 females were less than those for class 0 and 1 females, whose values were usually more similar. Thus, while female bodily condition was comparable among classes, the most heterozygous females produced fewer total eggs and eggs per sac than their less heterozygous peers. The fact that females of M. ventura engage in a reproductive investment-number trade-off suggests that high-variability and low-variability females may be pursuing distinct reproductive strategies in the wild, with more heterozygous females being K -selected (smaller clutches, heavier eggs) and more homozygous females being r -selected (larger clutches, lighter eggs). Further investigation will be needed to assess more fully the fitness value of heterozygosity in M. ventura .     

Use of a Yeast Site-Specific Recombinase to Produce Female Germline Chimeras in Drosophila

We describe an efficient method for generating female germline mosaics by inducing site-specific homologous mitotic recombination with a yeast recombinase (FLP) which is driven by a heat shock promoter. These germline mosaics are produced in flies heterozygous for the agametic, germline-dependent, dominant female sterile (DFS) mutation ovoD1, where only flies possessing germline clones are able to lay eggs. This method, the "FLP-DFS" technique, is very efficient because more than 90% of females with germline clones can be recovered. We show that this heat-inducible, site-specific mitotic recombination system does not affect viability and that the germline clones recovered are physiologically the same as those created by X-ray induced mitotic recombination. We describe the parameters of FLP-recombinase induced germline mitotic recombination and the use of the "FLP-DFS" technique to analyze the maternal effect of X-linked zygotic lethal mutations.     

A dominant, recombination-defective allele of Dmc1 causing male-specific sterility

DMC1 is a meiosis-specific homolog of bacterial RecA and eukaryotic RAD51 that can catalyze homologous DNA strand invasion and D-loop formation in vitro. DMC1-deficient mice and yeast are sterile due to defective meiotic recombination and chromosome synapsis. The authors identified a male dominant sterile allele of Dmc1, Dmc1^Mei11, encoding a missense mutation in the L2 DNA binding domain that abolishes strand invasion activity. Meiosis in male heterozygotes arrests in pachynema, characterized by incomplete chromosome synapsis and no crossing-over. Young heterozygous females have normal litter sizes despite having a decreased oocyte pool, a high incidence of meiosis I abnormalities, and susceptibility to premature ovarian failure. Dmc1^Mei11 exposes a sex difference in recombination in that a significant portion of female oocytes can compensate for DMC1 deficiency to undergo crossing-over and complete gametogenesis. Importantly, these data demonstrate that dominant alleles of meiosis genes can arise and propagate in populations, causing infertility and other reproductive consequences due to meiotic prophase I defects.     

A Dominant, Recombination-Defective Allele of Dmc1 Causing Male-Specific Sterility

DMC1 is a meiosis-specific homolog of bacterial RecA and eukaryotic RAD51 that can catalyze homologous DNA strand invasion and D-loop formation in vitro. DMC1-deficient mice and yeast are sterile due to defective meiotic recombination and chromosome synapsis. The authors identified a male dominant sterile allele of Dmc1, Dmc1^Mei11, encoding a missense mutation in the L2 DNA binding domain that abolishes strand invasion activity. Meiosis in male heterozygotes arrests in pachynema, characterized by incomplete chromosome synapsis and no crossing-over. Young heterozygous females have normal litter sizes despite having a decreased oocyte pool, a high incidence of meiosis I abnormalities, and susceptibility to premature ovarian failure. Dmc1^Mei11 exposes a sex difference in recombination in that a significant portion of female oocytes can compensate for DMC1 deficiency to undergo crossing-over and complete gametogenesis. Importantly, these data demonstrate that dominant alleles of meiosis genes can arise and propagate in populations, causing infertility and other reproductive consequences due to meiotic prophase I defects.     

A cost of reproduction in Drosophila melanogaster: stress susceptibility

Little is known about physiological mechanisms that underlie the cost of reproduction. We tested the hypothesis that stress susceptibility is a cost of reproduction. In one test of our hypothesis, Drosophila melanogaster females were exposed to a juvenile hormone analog (methoprene) to stimulate egg production followed by stress assays. A sterile stock of D. melanogaster was employed as a control for reproduction. Exposure of fertile females to methoprene resulted in an increase in female reproduction and increased susceptibility to oxidative stress and starvation (compared to solvent controls). Sterile females did not exhibit a decrease in stress resistance. Mating also stimulated egg production. As a second test of our hypothesis, mated females were compared to virgin females. Mated fertile females were relatively susceptible to oxidative stress, but this relationship was not evident when mated and virgin sterile females were compared. The results of the present study support the hypothesis that stress susceptibility is a cost of reproduction.     

Identification of Grandchildless Loci Whose Products Are Required for Normal Germ-Line Development in the Nematode Caenorhabditis Elegans

To identify genes that encode maternal components required for development of the germ line in the nematode Caenorhabditis elegans, we have screened for mutations that confer a maternal-effect sterile or "grandchildless" phenotype: homozygous mutant hermaphrodites produced by heterozygous mothers are themselves fertile, but produce sterile progeny. Our screens have identified six loci, defined by 21 mutations. This paper presents genetic and phenotypic characterization of four of the loci. The majority of mutations, those in mes-2, mes-3 and mes-4, affect postembryonic germ-line development; the progeny of mutant mothers undergo apparently normal embryogenesis but develop into agametic adults with 10-1000-fold reductions in number of germ cells. In contrast, mutations in mes-1 cause defects in cytoplasmic partitioning during embryogenesis, and the resulting larvae lack germ-line progenitor cells. Mutations in all of the mes loci primarily affect the germ line, and none disrupt the structural integrity of germ granules. This is in contrast to grandchildless mutations in Drosophila melanogaster, all of which disrupt germ granules and affect abdominal as well as germ-line development.     

A male destroying an egg in a cooperative breeding attempt in Lesser Spotted Woodpecker Dendrocopos minor

A cooperative breeding case was found in Lesser Spotted Woodpecker (LSW) in which a male and three different females were involved. The contributions of each member of the cooperative breeding team were quantified during nest excavation and incubation. This is the first such report on LSW. During daytime, the nest was mainly occupied by the male during the final excavation phase, nest guarding, and egg laying. Aggressive interactions were recorded between two of the females during nest excavation, egg laying and incubation. The male was recorded destroying an egg presumably laid by the dominant female. The highest contribution during daytime incubation was made by the dominant female (39%), followed by the male (34%) and then by a second female (27%). The third female contributed very little at any stage. A picture of the male removing the egg was analyzed to estimate the egg maximum width, ruling out a possible runt egg. Motivations behind the behaviour of the male destroying the egg remain unclear.