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Myosin VI is a motor protein which is necessary for the morphogenesis of epithelial tissues during Drosophila development. The spatial and temporal expression of Myosin VI was examined by expressing a GFP (Green Fluorescent Protein) tagged Myosin VI molecule (PGM), under the control of a Myosin VI-Gal4 line. PGM was present in tissues that were shown previously to express Myosin VI, such as the ovarian follicle epithelium, and the individualization complex; and in other tissues, including the trachea, the midgut, the salivary glands and the imaginal discs. The GFP-tagged Myosin V1 rescued the male sterile phenotype of Jaguar showing it is functional in vivo. Within individual cells, the role of the head and neck domain and the tail domain in targeting of the Myosin V1 molecule was examined by investigating the localisation of the separate domains tagged to GFP. In salivary glands and follicle cells the head and neck domains were concentrated in the cell nucleus, where the minus end of each actin filament is located. We found that the tail domain anchors the whole molecule outside of the nucleus. Similarly, in the individualization complex in the testes, the tail anchors the whole molecule to the base of the complex while the separated head with neck domain becomes scattered along the entire actin molecule suggesting the cellular location may be determined by cargo proteins that bind to the tail domain rather than by the movement of Myosin VI along the actin filaments.

There are 16 classes of unconventional myosins. Class V myosins have been shown to be involved in transporting cargo to and from the cell periphery. Class VI myosins have also been shown to transport cargo from the cell periphery, although it seems that these proteins have many roles which include the mediation of cell migration and stereocillia stabilisation. With the requirement of myosin VI for Drosophila oogenesis, the localised expression of Myosin V in the developing egg chamber and recent mounting evidence which links myosin VI to the migration of human ovarian cancer cell lines, we wanted to investigate the expression pattern of these two myosin classes in the normal mouse ovary. Here we show that these myosins are expressed, localised and regulated within the oocyte and granulosa cells of the developing mouse follicle.

extramacrochaetae (emc) functions during many developmental processes in Drosophila, such as sensory organ formation, sex determination, wing vein differentiation, regulation of eye photoreceptor differentiation, cell proliferation and development of the Malpighian tubules, trachea and muscles in the embryo. It encodes a Helix-Loop-Helix transcription factor that negatively regulates bHLH proteins. We show here that emc mRNA and protein are present throughout oogenesis in a dynamic expression pattern and that emc is involved in the regulation of chorionic appendage formation during late oogenesis. Expression of sense and antisense emc constructs as well as emc follicle cell clones leads to eggs with shorter, thicker dorsal appendages that are closer together at base than in the wild type. We demonstrate that emc lies downstream of fs(1)K10, gurken and EGFR in the Grk/EGFR signalling pathway and that it participates in controlling Broad-Complex expression at late stages of oogenesis.

The effects of dopamine (DA) on juvenile hormone (JH) metabolism and fitness (estimated as fecundity and viability levels under heat stress (38 degrees C)) in Drosophila virilis have been studied. An increase of DA level obtained by feeding with DA reduced fitness of wild-type (wt) flies under stress, and decreased JH degradation in young wt females while increasing it in sexually mature wt females. A decrease in DA levels resulted from 3-iodo-tyrosine treatment and caused a decrease in JH degradation in sexually mature wt and heat sensitive (hs) mutant females (DA level in hs females is twice as high in wt females). A dramatic decrease in viability under stress and fecundity under normal conditions in wt, but not hs, females was observed. 3-iodo-tyrosine treatment also reduced the number of oocytes at stages 8-14, delayed oocyte transition to stage 10 and resulted in the accumulation of mature eggs in wt females. It delayed maturation of wt, but not hs, males as well, but did not affect their fertility. This advances our understanding of the regulation of JH metabolism by DA in Drosophila and suggests a crucial role for the basal DA level in fitness.

The loco gene encodes members of a family of RGS proteins responsible for the negative regulation of G-protein signalling. At least two transcripts of loco are expressed in oogenesis, loco-c2 is observed in the anterior-dorsal follicle cells and is downstream of the epidermal growth factor receptor signalling pathway, initiated in the oocyte. loco-c3 is a new transcript of loco, which is expressed in the nurse cells from stage 6 onwards. Analysis of newly generated mutants and antisense technology enabled us to establish that disrupting loco in follicle cells results in ventralized eggs, while disrupting loco in nurse cells results in short eggs, due to defective dumping of the nurse cell cytoplasm into the oocyte.

In many sexually mature insects egg production and oviposition are tightly coupled to copulation. Sex-Peptide is a 36-amino-acid peptide synthesized in the accessory glands ofDrosophila melanogastermales and transferred to the female during copulation. Sex-Peptide stimulates vitellogenic oocyte progression through a putative control point at about stage 9 of oogenesis. Here we show that application of the juvenile hormone analogue methoprene mimics the Sex-Peptide-mediated stimulation of vitellogenic oocyte progression in sexually mature virgin females. Apoptosis is induced by 20-hydroxyecdysone in nurse cells of stage 9 egg chambers at physiological concentrations (10−7M). 20-Hydroxyecdysone thus acts as an antagonist of early vitellogenic oocyte development. Simultaneous application of juvenile hormone analogue, however, protects early vitellogenic oocytes from 20-hydroxyecdysone-induced resorption. These results suggest that the balance of these hormones in the hemolymph regulates whether oocytes will progress through the control point at stage 9 or undergo apoptosis. These data are further supported by a molecular analysis of the regulation of yolk protein synthesis and uptake into the ovary by the two hormones. We conclude that juvenile hormone is a downstream component in the Sex-Peptide response cascade and acts by stimulating vitellogenic oocyte progression and inhibiting apoptosis. Since juvenile hormone analogue does not elicit increased oviposition and reduced receptivity, Sex-Peptide must have an additional, separate effect on these two postmating responses.

Background: The activation of protein kinase B (PKB, also known as c-Akt) is stimulated by insulin or growth factors and results from its phosphorylation at Thr308 and Ser473. We recently identified a protein kinase, termed PDK1, that phosphorylates PKB at Thr308 only in the presence of lipid vesicles containing phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) or phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ). Results: We have cloned and sequenced human PDK1. The 556-residue monomeric enzyme comprises a catalytic domain that is most similar to the PKA, PKB and PKC subfamily of protein kinases and a carboxy-terminal pleckstrin homology (PH) domain. The PDK1 gene is located on human chromosome 16p 13.3 and is expressed ubiquitously in human tissues. Human PDK1 is homologous to the Drosophila protein kinase DSTPK61, which has been implicated in the regulation of sex differentiation, oogenesis and spermatogenesis. Expressed PDK1 and DSTPK61 phosphorylated Thr308 of PKB α only in the presence of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 . Overexpression of PDK1 in 293 cells activated PKB α and potentiated the IGF1-induced phosphorylation of PKB α at Thr308. Experiments in which the PH domains of either PDK1 or PKB α were deleted indicated that the binding of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 to PKB α is required for phosphorylation and activation by PDK1. IGF1 stimulation of 293 cells did not affect the activity or phosphorylation of PDK1. Conclusions: PDK1 is likely to mediate the activation of PKB by insulin or growth factors. DSTPK61 is a Drosophila homologue of PDK1. The effect of PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 in the activation of PKB α is at least partly substrate directed.

genes which are expressed in the adult female fat body are Many eukaryotic genes generate alternately spliced trantargets of the DSX protein (Burtis et al., 1991) and their yolk scripts which can produce different proteins or which have protein products are essential for oocyte development. altered translational controls. One of the most direct demHowever, the use of alternate splicing in sexual developonstrations that alternately spliced forms of transcripts lead ment in Drosophila is not limited to dsx. Alternate splicing to different developmental consequences lies within the sex of dsx transcripts is controlled by the products of the transdetermination pathway of Drosophila. The doublesex (dsx) former (tra) and transformer-2 (tra-2) genes (Nagoshi et al., gene at the end of this pathway in somatic cells encodes 1988). tra RNA is also alternately spliced, in this case with two differently spliced transcripts, one specific for males the dramatic consequence that in males no functional proand one specific for females (Burtis and Baker, 1989). These tein product is made (Butler et al., 1986; McKeown et al., encode proteins with a common DNA binding region and 1987), whereas in females an RNA binding protein is proa sexually unique carboxy terminus; DSX and DSX act as duced which interacts with the tra-2-encoded RNA binding transcription factors and have opposing activities. The main protein (Belote and Baker, 1982), directing the female-spedevelopmental consequences are the repression of a set of cific splicing of dsx. It seems likely that tra and tra-2 have downstream female-specific differentiation genes by DSX other targets in addition to dsx that are important for sexual in males and the repression of certain male characteristics development, since several aspects of sexual dimorphism by DSX in females (for reviews see Slee and Bownes, 1990; depend upon the tra/tra-2 genes but are independent of dsx Steinmann-Zwicky et al., 1990; Ryner and Swain, 1995). (Taylor et al., 1994). This includes courtship behaviour; the Many of the sexual differences between male and female development of abdominal cells, which produce a female Drosophila are controlled by the two alternate products of pheromone; and the correct innervation of nerves needed the dsx gene (Burtis and Baker, 1989). They direct both the for the development of a male-specific muscle (Lawrence determination of sex-specific characteristics in the imaginal and Johnston, 1986; Taylor, 1992). cells and the maintenance of determination throughout The female-specific splicing of tra RNA is itself directed subsequent cell divisions. This regulation leads to the final by an alternately spliced gene product. This is encoded by differentiation of male or female genitalia, the differences the Sex-lethal (Sxl) gene that is at the head of the sex-deterin pigmentation patterns in the abdomen of each sex and mination hierarchy (Cline, 1984, 1993). Sxl produces many in other sex-specific bristle patterns, such as the sex comb transcripts, including several specific to female somatic on the first leg of the male. Differences between the sexes cells, that generate a functional RNA binding protein. The in the pattern of nerve cell divisions are also directed by male mRNA from Sxl does not encode a functional protein the two related DSX proteins (Taylor and Truman, 1992), (Bell et al., 1988). The SXL protein is known to direct the as is the selection between the male or female development female-specific splicing of its own RNAs as well as that of

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.

During vitellogenesis the 3 major yolk proteins of Drosophila are synthesized and transported from the tissues which produce them to the oocyte. Here they are endocytosed by the oocyte and stored in yolk granules for utilization during embryogenesis. In this paper the regulation of vitellogenesis is discussed, including the tissue-specific and hormonal regulation of the yolk protein genes; transport of yolk proteins to the oocyte; storage of the proteins; and their possible functions during embryogenesis.

Male adults of Drosophila , which do not normally produce yolk polypeptides can be stimulated to do so by 20-hydroxy-ecdysone. The response to a single dose of hormone is a short burst of yolk-polypeptide synthesis by the fat body. The magnitude of the response is dose dependent. A second treatment with the hormone leads to a similar response. Long-term feeding of males does not lead to permanent activation of the yolk-polypeptide genes. It was not possible to stimulate male fat bodies to synthesise yolk polypeptides with 20-hydroxy-ecdysone in vitro . It is concluded that 20-hydroxy-ecdysone, possibly in the presence of another hormone or a carrier protein, induces transcription of the yolk-polypeptide genes of the male fat body and its presence is required to keep the genes active. In females, however, mature fat bodies can produce yolk polypeptides in vitro without the requirement for added hormone. It is argued that male and female fat bodies have different hormonal requirements for the maintenance of expression of the genes coding for the yolk polypeptides.

Methionine in the form of free amino acids, small peptides and several proteins is transferred from ejaculate to females in Drosophila melanogaster and D. pseudoobscura . The male contribution is found mostly in the ovary and the sizes of the polypeptides transferred have been established. Some components of the ejaculate are rapidly transferred to the somatic tissues outside the ovary and reproductive system where they co-migrate with the yolk polypeptides. The incorporation of free methionine into polypeptides in non-reproductive tissues is higher in females that have been deprived of a source of protein prior to mating. The molecules transferred from ejaculate may have roles in regulating reproductive behaviour and may also be used as a nutrient source. We suggest that free amino acids in the ejaculate are rapidly utilised in protein synthesis in the somatic tissues outside the ovary and could provide a boost to yolk protein and hence egg production in females in nature, where nutrients may be in limited supply.

The three yolk-protein genes of D. melanogaster (YP1, YP2 and YP3) are normally expressed in the female from the time of eclosion, but not in males. Expression can be induced in males of any age by treatment with a steroid hormone, 20-hydroxyecdysone. Yolk polypeptides can be identified in haemolymph from females of several Drosophila spp by their cross-reactivity to anti-yolk polypeptides antibodies prepared against D. melanogaster yolk proteins. Western blotting was used to show the accumulation of yolk polypeptides in males of several D. melanogaster sibling spp ( D. simulans, D. mauritiana, D. orena, D. erecta, D. yakuba and D. teissieri ) and one other spp ( D. virilis ), 16 hr after 20-hydroxyecdysone treatment. YP-homologous transcripts were identified in females of the Drosophila melanogaster sibling spp by RNA hybridization to cloned D. melanogaster YP genes (pYP1, pYP2 and pYP3). YP-homologous transcripts accumulated in males of D. melanogaster sibling spp 16 hr after treatment with 20-hydroxyecdysone; but comparisons between hybridizations to a mixed YP1/2/3 gene probe and YP1-specific probes suggested that the yolk polypeptide genes may not always be transcribed in a coordinated fashion in these males. This difference was no longer apparent after translation into yolk polypeptides had occurred. Steroid treatment of males appeared not to stimulate expression of all genes, since the levels of some haemolymph polypeptides did not appear to be affected by 20-hydroxyecdysone, while other such polypeptides were detected at reduced levels after the hormone treatment which led to expression of the yolk polypeptide-genes in this sex.

The vestigial (vg) mutant of Drosophila melanogaster shows reduced wing size and lacks margin structures from the wing blade. The expressivity is temperature-sensitive, more structures being formed at 29°C than at 25°C. There is cell death in the third instar wing disc which to some extent parallels the fate map locations of the structures absent in the adult. Vestigial wing discs are unable to regenerate margin structures even when given extra time for growth by culturing them in an adult abdomen before metamorphosis. If the region of cell death is excised from the disc before culture, there is still no regeneration of margin structures, indicating that the dead cells do not physically prevent regulation. Furthermore, by metamorphosing young vg wing discs, it was discovered that cells never acquire competence to make margin during wing disc development. Experiments mixing fragments of vg wing disc with non- vg wing disc fragments of ebony multiple wing hairs (e mwh) genotype showed that the vg cells interacted with the e mwh cells and wing blade was intercalated of both genotypes. However, structures such as wing margin, and alar lobe, usually affected in vg wings, were always made from e mwh cells and not from vg cells. Analysis of mutants which are unable to differentiate particular cell types may help us to understand the mechanism of pattern establishment in developing imaginal discs.

Yolk protein synthesis can be induced in male Drosophila melanogaster by the injection of high doses of 20-hydroxyecdysone, but the response is short-lived with the rate of yolk protein synthesis reaching a peak at 8–12 hr after injection, and declining to barely detectable levels after 24 hr. The injected 20-hydroxyecdysone was rapidly metabolised in vivo to compounds of higher polarity, with as little as 17% of the original steroid remaining within 10 min of a 480 ng injection, of which about 60% was in the form of more polar metabolites. These metabolites were inactive at inducing yolk protein synthesis when injected into previously untreated male flies, at a concentration where 20-hydroxyecdysone reproducibly induced yolk protein synthesis. In in vitro incubations of isolated fly regions the limited amount of metabolism observed was towards compounds of lower polarity than 20-hydroxyecdysone, tentatively identified as acetyl derivatives rather than the pattern of highly polar metabolites observed in vivo .

The ovaries and fat bodies of Drosophila melanogaster adult females both synthesize yolk polypeptides. In a series of experiments it has been shown that the ovaries become competent to mature in an adult male host, where only ovarian synthesis occurs, very early in metamorphosis and synthesis of yolk polypeptides begins in a time-dependent sequence related to the age of the ovary when transplanted. Maturation of ovaries occurs prior to eclosion when they are transplanted to an earlier developmental stage showing that neither the event of eclosion not the adult environment is essential in triggering yolk-polypeptide synthesis by the ovary. When metamorphosing ovaries are transplanted to a female host they take up host yolk polypeptides from the haemolymph, but this does not lead to the implanted ovary developing substantially better than in a male host where only synthesis by the ovary can occur. The regulation of ovarian yolk-polypeptide synthesis therefore appears to be autonomous to the ovary itself. There may be a trigger early in metamorphosis which induces competence in the ovary so that it subsequently initiates yolk-polypeptide gene expression at eclosion.

Ecdysteroid titres in whole flies and different tissues of adult male and female Drosophila were determined at various times after eclosion using a radioimmunoassay. The ecdysteroid titre decreased as the flies matured after eclosion. The differences in titre between males and females can be accounted for by their difference in body weight. The ecdysteroids were found to be distributed throughout several tissues. At eclosion not all of the ecdysteroid complement present could be accounted for by that found localised in tissues. After maturation of the flies the ecdysteroids in various tissues can account for the majority of that detected in whole-fly extracts. Ecdysteroids were produced during in vitro culture of various tissues, but the quantities detected were low by comparison with ring glands of wandering 3rd-instar larvae. Neither the ovaries nor the abdominal body walls (fat body) seem to be a major source of hormone, and they are only able to convert minute quantities of ecdysone to the biologically active form, 20-hydroxyecdysone, in vitro. The amounts of 20-hydroxyecdysone present were measured using high performance liquid chromatography and radioimmunoassay. We tentatively suggest that the differential experession of the yolk-protein-genes in the fat bodies of males and females does not result from differences in hormone titres between them.

The 3 yolk-protein genes of Drosophila are usually expressed in the adult female fat body and follicle cells of the ovary. Both a juvenile hormone analogue, ZR515, and 20-hydroxyecdysone affect the process of vitellogenesis, and 20-hydroxyecdysone can activate transcription of the genes in male fat bodies. We have investigated what might regulate the expression of the genes in relation to egg production during adult life. Feeding flies on a sugar diet reduces egg laying and the number of vitellogenic oocytes present in flies. Vitellogenesis is rapidly resumed if the flies are treated with ZR515. Restoring a normal diet or treatment with 20-hydroxyecdysone does not bring about rapid recovery of oogenesis and with 20-hydroxyecdysone vitellogenesis fails to recommence at all. Analysis of the RNA coding for the yolk proteins showed that sugar feeding does reduce the number of transcripts present in the fat body and both ZR515 hormone and 20-hydroxyecdysone can stimulate the amount of this RNA present in both sugar-fed and normally fed flies. Thus nutrition seems gradually to affect egg production and yolk-protein gene expression; yet ZR515 hormone and 20-hydroxyecdysone can overcome these effects, presumably by redirecting resources to give priority to the transcription and translation of these genes in the fat body.

A staging system devised for the identification of D. melanogaster individuals at equivalent points during metamorphosis was employed to generate a profile of whole-body free ecdysteroid titers throughout this period, in each sex, using a radioimmune assay (RIA). Few major differences between the sexes were detected, and no sex-specific peaks in titer were demonstrated. Males and females show elevated titers shortly before the onset of cuticle-deposition, both pupal (stage P1) and adult (stage P7), and also at melanization of the bristles in the pharate adult (stage P11). RIA was used in conjunction with high-performance liquid chromatography to measure and identify certain ecdysteroids present at these stages (P1, P7 and P10–13), at each of which ecdysone was found to be more abundant than 20-hydroxy-ecdysone. The measured free titers at stages P7 and P10–13 were, respectively, about 1.4 and 0.6 times that of stage P1. Makisterone A appeared to be present at stage P1, and a parallel analysis by gas-liquid chromatography/mass fragmentography suggested the presence of 20,26-dihydroxyecdysone at stage P7.

Vitellogenesis in Drosophila is regulated by the sex-determination gene pathway, the hormones ecdysone and juvenile hormone and the availability of nutrients. In this paper I show that the nutritional regulation is unlikely to be mediated via titres of ecdysone or juvenile hormone. Starved flies do not have reduced juvenile hormone or ecdysteroid titres. I show that flies without ovaries have normal levels of free ecysteroids after 2 days suggesting that the ovaries do not produce substantial quantities of free ecdysteroids continuously in the adult for use in regulating vitellogenesis, however, they probably do contribute to the whole body titre in mature flies since titres do become less in ovary-less flies at 3 days. This is consistent with data on the measurement of ecdysteroids produced by ovaries in vitro . Flies defective in vitellogenesis, namely apterous 4 , long thought to be a juvenile hormone mutant, are shown by gas chromatography/mass spectrometry to have a reduced juvenile hormone level. The interactions between these hormones, the genetic control of yolk-protein transcription, the ovary and the actual occurrence of vitellogenesis are discussed.