Autophagy in Drosophila ovaries is induced by starvation and is required for oogenesis.

Autophagy, an evolutionarily conserved lysosome-mediated degradation, promotes cell survival under starvation and is controlled by insulin/target of rapamycin (TOR) signaling. In Drosophila, nutrient depletion induces autophagy in the fat body. Interestingly, nutrient availability and insulin/TOR signaling also influence the size and structure of Drosophila ovaries, however, the role of nutrient signaling and autophagy during this process remains to be elucidated. Here, we show that starvation induces autophagy in germline cells (GCs) and in follicle cells (FCs) in Drosophila ovaries. This process is mediated by the ATG machinery and involves the upregulation of Atg genes. We further demonstrate that insulin/TOR signaling controls autophagy in FCs and GCs. The analysis of chimeric females reveals that autophagy in FCs, but not in GCs, is required for egg development. Strikingly, when animals lack Atg gene function in both cell types, ovaries develop normally, suggesting that the incompatibility between autophagy-competent GCs and autophagy-deficient FCs leads to defective egg development. As egg morphogenesis depends on a tightly linked signaling between FCs and GCs, we propose a model in which autophagy is required for the communication between these two cell types. Our data establish an important function for autophagy during oogenesis and contributes to the understanding of the role of autophagy in animal development.

Polymorphic microsatellite DNA markers in the penduline tit, Remiz pendulinus.

To describe the exceptional mating system of the penduline tit, Remiz pendulinus, we aim to combine field observation records with DNA analysis based on polymorphic microsatellite DNA markers. Here we describe features of nine loci and their corresponding polymerase chain reaction primers. The observed number of alleles varied from four to seven and the observed heterozygosity ranged from 0.419 to 0.802. Neither of the loci is sex-linked and as linkage disequilibrium analysis showed they assort independently. Seven of the nine loci were polymorphic in the Cape penduline tit, Anthoscopus minutus.

TPPP/p25 promotes tubulin assemblies and blocks mitotic spindle formation.

Recently, we isolated from bovine brain a protein, TPPP/p25 and identified as p25, a brain-specific protein that induced aberrant tubulin assemblies. The primary sequence of this protein differs from that of other proteins identified so far; however, it shows high homology with p25-like hypothetical proteins sought via blast. Here, we characterized the binding of TPPP/p25 to tubulin by means of surface plasmon resonance; the kinetic parameters are as follows: kon, 2.4 x 10(4) M(-1) x s(-1); koff, 5.4 x 10(-3) s(-1); and Kd, 2.3 x 10(-7) M. This protein at substoichometric concentration promotes the polymerization of tubulin into double-walled tubules and polymorphic aggregates or bundles paclitaxel-stabilized microtubules as judged by quantitative data of electron and atomic force microscopies. Injection of bovine TPPP/p25 into cleavage Drosophila embryos expressing tubulin-GFP fusion protein reveals that TPPP/p25 inhibits mitotic spindle assembly and nuclear envelope breakdown without affecting other cellular events like centrosome replication and separation, microtubule nucleation by the centrosomes, and nuclear growth. GTP counteracts TPPP/p25 both in vitro and in vivo.

Dominant-negative mutant dynein allows spontaneous centrosome assembly, uncouples chromosome and centrosome cycles.

Cleavage cycles commence and chromosome and centrosome cycles proceed in harmony following fertilization of Drosophila eggs and completion of the meiotic divisions. The sperm-introduced centrioles replicate, separate, and while recruit pericentriolar material centrosomes (CS) form. The CS nucleate asters of microtubules (MT). Spindles form following interaction of some astral MT with kinetochores. In unfertilized eggs, chromosomes do not replicate, and CS and MT asters never form, although their components are present in the egg cytoplasm; unknown mechanisms prevent chromosome replication and CS and MT assembly. In unfertilized Laborc(D) eggs, rudimentary CS assemble spontaneously and instantaneously and nucleate small MT asters. In fertilized Laborc(D) eggs, normal CS form and organize normal asters. However, the CS replicate prior to accomplishment of the first mitosis, and spindles with multiple CS develop. In fertilized Laborc(D) eggs, while the chromosome cycles cease, CS cycles proceed as in wild type. Knowing that Laborc(D) is a dominant-negative mutation and encodes the formation of mutant cytoplasmic dynein heavy chain molecules, we show here that cytoplasmic dynein is involved in prevention of CS assembly in unfertilized eggs and establishing harmony between the chromosome and the CS cycles.

pitkin(D), a novel gain-of-function enhancer of position-effect variegation, affects chromatin regulation during oogenesis and early embryogenesis in Drosophila.

The vast majority of the >100 modifier genes of position-effect variegation (PEV) in Drosophila have been identified genetically as haplo-insufficient loci. Here, we describe pitkin(Dominant) (ptn(D)), a gain-of-function enhancer mutation of PEV. Its exceptionally strong enhancer effect is evident as elevated spreading of heterochromatin-induced gene silencing along euchromatic regions in variegating rearrangements. The ptn(D) mutation causes ectopic binding of the SU(VAR)3-9 heterochromatin protein at many euchromatic sites and, unlike other modifiers of PEV, it also affects stable position effects. Specifically, it induces silencing of white+ transgenes inserted at a wide variety of euchromatic sites. ptn(D) is associated with dominant female sterility. +/+ embryos produced by ptn(D)/+ females mated with wild-type males die at the end of embryogenesis, whereas the ptn(D)/+ sibling embryos arrest development at cleavage cycle 1-3, due to a combined effect of maternally provided mutant product and an early zygotic lethal effect of ptn(D). This is the earliest zygotic effect of a mutation so far reported in Drosophila. Germ-line mosaics show that ptn+ function is required for normal development in the female germ line. These results, together with effects on PEV and white+ transgenes, are consistent with the hypothesis that the ptn gene plays an important role in chromatin regulation during development of the female germ line and in early embryogenesis.

The Ketel gene encodes a Drosophila homologue of importin-beta.

The Drosophila melanogaster Ketel gene was identified via the Ketel(D) dominant female sterile mutations and their ketel(r) revertant alleles that are recessive zygotic lethals. The maternally acting Ketel(D) mutations inhibit cleavage nuclei formation. We cloned the Ketel gene on the basis of a common breakpoint in 38E1. 2-3 in four ketel(r) alleles. The Ketel(+) transgenes rescue ketel(r)-associated zygotic lethality and slightly reduce Ketel(D)-associated dominant female sterility. Ketel is a single copy gene. It is transcribed to a single 3.6-kb mRNA, predicted to encode the 97-kD Ketel protein. The 884-amino-acid sequence of Ketel is 60% identical and 78% similar to that of human importin-beta, the nuclear import receptor for proteins with a classical NLS. Indeed, Ketel supports import of appropriately designed substrates into nuclei of digitonin-permeabilized HeLa cells. As shown by a polyclonal anti-Ketel antibody, nurse cells synthesize and transfer Ketel protein into the oocyte cytoplasm from stage 11 of oogenesis. In cleavage embryos the Ketel protein is cytoplasmic. The Ketel gene appears to be ubiquitously expressed in embryonic cells. Western blot analysis revealed that the Ketel gene is not expressed in several larval cell types of late third instar larvae.

The Ketel(D) dominant-negative mutations identify maternal function of the Drosophila importin-beta gene required for cleavage nuclei formation.

The Ketel(D) dominant female-sterile mutations and their ketel(r) revertant alleles identify the Ketel gene, which encodes the importin-beta (karyopherin-beta) homologue of Drosophila melanogaster. Embryogenesis does not commence in the Ketel(D) eggs deposited by the Ketel(D)/+ females due to failure of cleavage nuclei formation. When injected into wild-type cleavage embryos, cytoplasm of the Ketel(D) eggs does not inhibit nuclear protein import but prevents cleavage nuclei formation following mitosis. The Ketel(+) transgenes slightly reduce effects of the Ketel(D) mutations. The paternally derived Ketel(D) alleles act as recessive zygotic lethal mutations: the Ketel(D)/- hemizygotes, like the ketel(r)/ketel(r) and the ketel(r)/- zygotes, perish during second larval instar. The Ketel maternal dowry supports their short life. The Ketel(D)-related defects originate most likely following association of the Ketel(D)-encoded mutant molecules with a maternally provided partner. As in the Ketel(D) eggs, embryogenesis does not commence in eggs of germline chimeras with ketel(r)/- germline cells and normal soma, underlining the dominant-negative nature of the Ketel(D) mutations. The ketel(r) homozygous clones are fully viable in the follicle epithelium in wings and tergites. The Ketel gene is not expressed in most larval tissues, as revealed by the expression pattern of a Ketel promoter-lacZ reporter gene.

Genetic requirement of epidermal and female germ line cells in Drosophila in the light of clonal analysis.

Whether the function of a gene is required in a given cell type is often determined through the analysis of clones homozygous for a mutant allele of the gene. The clones usually develop following X-ray induced mitotic recombination. The paper summarizes the conclusions of clonal analyses of different types of mutations in both the epidermis and the female germ line cells of Drosophila. Principles of the so called dominant female sterile technique -for the germ line and the follicle cells- and its use are summarized. Special attention is paid to the genetic requirement of the female germ line due to its fundamental function in the regulation of early embryogenesis.

The Tomaj mutant alleles of alpha Tubulin67C reveal a requirement for the encoded maternal specific tubulin isoform in the sperm aster, the cleavage spindle apparatus and neurogenesis during embryonic development in Drosophila.

The three dominant TomajD and their eleven revertant (TomajR) alleles have been localized to the alpha Tubulin67C gene of Drosophila melanogaster. Although the meiotic divisions are normally completed in eggs laid by TomajD/+, TomajD/-, TomajR/- females, embryogenesis arrests prior to the gonomeric division. The arrest is caused by: (1) the failure of prominent sperm aster formation; and (2) a consequent lack of female pronuclear migration towards the male pronucleus. Concomitant with the sperm aster defect, the four female meiotic products fuse (tetra-fusion), similar to what is seen in eggs of wild-type virgin females. In eggs of females heterozygous for weaker TomajR alleles, embryogenesis comes to a cessation before or shortly after cortical migration of cleavage nuclei. The apparent source of embryonic defect is the cleavage spindle apparatus. One of the three TomajD alleles is cold-sensitive and its cold-sensitive period coincides with the completion of female meiosis and pronuclear migration. Disorganized central and peripheral nervous systems are also characteristic of embryos derived from the temperature-sensitive TomajD/+ females. The Tomaj mutant phenotypes indicate an involvement of the normal alpha Tubulin67C gene product in: (1) the formation of the sperm aster; (2) cleavage spindle apparatus formation/function; and (3) the differentiation of the embryonic nervous system. The TomajD alleles encode a normal-sized alpha Tubulin67C isotype. Sequence analyses of the TomajD alleles revealed the replacement in different positions of a single negatively charged or neutral amino acid with a positively charged one. These residues presumably identify important functional sites.

Genetic and developmental analysis of mutant Ketel alleles that identify the Drosophila importin-beta homologue.

The Ketel gene of Drosophila melanogaster was identified by four KetelD dominant female-sterile mutations and their 27 revertants. The X-ray and the P-induced KetelR alleles delineated the Ketel locus to the 38E1.2-3 cytological position. Although oogenesis proceeds, normally in the KetelD/+ females, embryogenesis comes to a deadlock shortly after fertilization inside the normal-looking eggs of the KetelD/+ females. The KetelD alleles are dominant negative mutations of antimorph type. Cytoplasm of the KetelD/(+)-derived eggs induce lesions when injected into wild-type eggs and the KetelD alleles can be reverted. Zygotes homozygous for loss-of-function (revertant) KetelR alleles die in second larval instar. Analysis of the cold-sensitive Ketel alleles and the genetic interactions between importin-alpha and KetelR mutant alleles indicate an involvement of the Ketel gene product in oo-, embryogenesis and larval life and show interaction of the KETEL protein with different components of nuclear processes. Molecular analysis (to be published elsewhere) confirmed the genetic data and revealed that the Ketel gene encodes the Drosophila homologue of importin-beta, an essential component of nuclear protein import.

Horka, a dominant mutation of Drosophila, induces nondisjunction and, through paternal effect, chromosome loss and genetic mosaics.

Fs(3)Horka (Horka) was described as a dominant female-sterile mutation of Drosophila melanogaster. Genetic and cytological data show that Horka induces mostly equational nondisjunction during spermatogenesis but not chromosome loss and possesses a dominant paternal effect: the X, second, third and the fourth chromosomes, but not the Y, are rendered unstable while undergoing spermatogenesis and may be lost in the descending zygotes. The frequency of Horka-induced chromosome loss is usually 2-4% but varies with the genetic background and can be over 20%. The X chromosome loss occurs during the gonomeric and the initial cleavage divisions. Loss of the X and fourth chromosomes shows no correlation. We propose, based on similarities in the mutant phenotypes with the chromosome destabilizing mutations nonclaret disjunctional and paternal loss, that the normal Horka+ product is required for function of the centromeres and/or nearby regions. Horka is a convenient tool for the generation of gynandromorphs, autosome mosaics and for the study of gene expression in mosaics.

A gain-of-function mutation in Drosophila MAP kinase activates multiple receptor tyrosine kinase signaling pathways.

In the Drosophila eye, activation of the sevenless (sev) receptor tyrosine kinase is required for the specification of the R7 photoreceptor cell fate. In a genetic screen for mutations that result in the activation of the sev signaling pathway in the absence of the inducing signal, we identified a gain-of-function mutation in rolled (rlSevenmaker [rlSem]), which encodes a homolog of mitogen-activated protein (MAP) kinase. In addition to the sev pathway, this mutation activates the pathways controlled by torso and the epidermal growth factor receptor homology. The rlSem mutation results in the substitution of a single conserved amino acid in the kinase domain. Activation of MAP kinase by the rlSem mutation is both necessary and sufficient to activate multiple signaling pathways controlled by receptor tyrosine kinases.

Gynandromorphs of Drosophila suggest one common primordium for the somatic cells of the female and male gonads in the region of abdominal segments 4 and 5.

In mosaic gonads of gynandromorphs of Drosophila, the amount of female and the amount of male somatic tissues add up to roughly one unit. This suggests that the somatic component of the gonads in males and females derives from a single common primordium, i.e. testes and ovaries appear to be homologous. Fate-mapping places this primordium ventrally of the sternites into the mesodermal region of the fourth and fifth abdominal segment. This location is corroborated by the observation that defects in and around abdominal segment 4 and absence of the gonads are strongly correlated in animals damaged by the mutation osk301. Gonads were mosaic with a frequency of 10.5% which indicates that the gonadal primordium originates from about 10 progenitor cells, and together with other evidence, suggests that these progenitor cells are located within a single segment (or parasegment).

Gonad formation and development requires the abd-A domain of the bithorax complex in Drosophila melanogaster.

The abdominal-A (abd-A) gene, a member of the bithorax complex, is required for the correct identity of parasegments (PS) 7 through 13. Mutations in iab-4, one of the cis-regulatory regions of abd-A, transform epidermal structures of PS 9 and also cause loss of gonads in adult flies. Here, we describe a developmental and molecular analysis of the role of iab-4 functions in gonadal development. In flies homozygous for a strong iab-4 allele, gonadogenesis is not initiated in the embryo because the mesodermal cells fail to encapsulate the pole cells. Flies homozygous for weaker iab-4 mutations sometimes form ovaries. The ovary-oviduct junctions are abnormal, however, and egg transfer from the ovary to the uterus is blocked in the adult. To localize the sites that require iab-4 function, we have analyzed animals chimeric for the mutant and wild-type cells. These chimeras were generated by three kinds of transplantation experiments: pole cells, embryonic somatic nuclei or larval ovaries. Our results suggest that iab-4 is required in the somatic cells of the gonadal primordia, but not the germ line. In addition, the formation of functional ovary-oviduct junctions and egg transfer also requires iab-4 functions in the somatic cells of the ovary and in at least one additional somatic tissue.

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)

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.

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.

Genetic analysis of chorion formation in Drosophila melanogaster: I. The effects of one somatic-specific and seven germ-line-specific mutations.

Eight X-linked recessive female sterile mutations, derived from a hybrid dysgenic screen of Drosophila melanogaster and representing eight distinct loci, have been characterized by genetic and ultrastructural analysis. Four have abnormal respiratory appendages, three have essentially normal appendages but show moderate defects in the endochorion, and one mutant, fs(1)ne1a, exhibits major defects in both the endochorion and the respiratory appendages. Germ line clones of all eight mutants were generated using the dominant female sterile technique. Seven of the eight mutations are germ line specific, indicating that, although the eggshell is produced by the follicular cells, germ line functions play a significant role in its elaboration. The mutant that shows major defects, fs(1)ne1a, is somatic line specific, and exerts its effect in the ovary.

Function of torso in determining the terminal anlagen of the Drosophila embryo.

The formation of the unsegmented terminal regions of the Drosophila larva, acron and telson requires the function of at least five maternal genes (terminal genes class). In their absence, the telson and acron are not formed. One of them, torso (tor), has gain-of-function alleles which have an opposite phenotype to the lack-of-function (tor-) alleles: the segmented regions of the larval body, thorax and abdomen, are missing, whereas the acron is not affected and the telson is enlarged. In strong gain-of-function mutants, the pair-rule gene fushi tarazu (ftz) is not expressed, demonstrating the suppression of the segmentation process in an early stage of development. The tor gain-of-function effect is neutralized, and segmentation is restored in double mutants with the zygotic gene tailless (tll), which has a phenotype similar (but not identical) to that of tor-. This suggests that tor acts through tll, and that in the gain-of-function alleles of tor, the tll gene product is ectopically expressed at middle positions of the embryo, where it inhibits the expression of segmentation genes like ftz.