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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.

Heterotrimeric G proteins mediate asymmetric division of Drosophila neuroblasts. Free Gβγ appears to be crucial for the generation of an asymmetric mitotic spindle and consequently daughter cells of distinct size. However, how Gβγ is released from the inactive heterotrimer remains unclear. Here we show that Locomotion defects (Loco) interacts and colocalizes with Gαi and, through its GoLoco motif, acts as a guanine nucleotide dissociation inhibitor (GDI) for Gαi. Simultaneous removal of the two GoLoco motif proteins, Loco and Pins, results in defects that are essentially indistinguishable from those observed in Gβ13F or Gγ1 mutants, suggesting that Loco and Pins act synergistically to release free Gβγ in neuroblasts. Furthermore, the RGS domain of Loco can also accelerate the GTPase activity of Gαi to regulate the equilibrium between the GDP- and the GTP-bound forms of Gαi. Thus, Loco can potentially regulate heterotrimeric G-protein signaling via two distinct modes of action during Drosophila neuroblast asymmetric divisions.

The link between reproduction and environmental signals is poorly understood at the physiological, genetic and molecular levels. We describe a mutant strain of Drosophila virilis that has altered responses to heat stress. Heat stress in wild-type females results in oocyte maturation delays, degradation of early vitellogenic egg chambers, inhibition of yolk protein gene expression in follicle cells and accumulation of mature oocytes. The mutant females have increased levels of ecdysteroids and decreased juvenile hormone degradation, and show all of the heat-stress-induced reproductive effects observed in wild-type flies, without exposure to heat stress. During oogenesis in mutant females following heat stress there is an increase in early vitellogenic oocyte degradation and some degradation of late egg chambers. 20-Hydroxyecdysone levels, but not juvenile hormone degradation, change following heat stress in mutant females.

Despite similar functions, the yolk proteins of the higher dipteran flies and the vitellogenins found in other insects are unrelated at the sequence level and have evolved from different genes. Both are selectively endocytosed into the ovary via receptors belonging to the LDLR receptor subfamily. We cloned the Drosophila yp1 gene into an E. coli expression vector and showed that the yolk protein produced by E. coli is taken up into ovaries of both Drosophila melanogaster and the malaria mosquito Anopheles gambiae, which normally uses vitellogenin.

Myosins constitute a superfamily of motor proteins that convert energy from ATP hydrolysis into mechanical movement along the actin filaments. Phylogenetic analysis currently places myosins into 17 classes based on class-specific features of their conserved motor domain. Traditionally, the myosins have been divided into two classes depending on whether they form monomers or dimers. The conventional myosin of muscle and nonmuscle cells forms class II myosins. They are complex molecules of four light chains bound to two heavy chains that form bipolar filaments via interactions between their coiled-coil tails (type II). Class I myosins are smaller monomeric myosins referred to as unconventional myosins. Now, at least 15 other classes of unconventional myosins are known. How many myosins are needed to ensure the proper development and function of eukaryotic organisms? Thus far, three types of myosins were found in budding yeast, six in the nematode Caenorhabditis elegans, and at least 12 in human. Here, we report on the identification and classification of Drosophila melanogaster myosins. Analysis of the Drosophila genome sequence identified 13 myosin genes. Phylogenetic analysis based on the sequence comparison of the myosin motor domains, as well as the presence of the class-specific domains, suggests that Drosophila myosins can be divided into nine major classes. Myosins belonging to previously described classes I, II, III, V, VI, and VII are present. Molecular and phylogenetic analysis indicates that the fruitfly genome contains at least five new myosins. Three of them fall into previously described myosin classes I, VII, and XV. Another myosin is a homolog of the mouse and human PDZ-containing myosins, forming the recently defined class XVIII myosins. PDZ domains are named after the postsynaptic density, disc-large, ZO-1 proteins in which they were first described. The fifth myosin shows a unique domain composition and a low homology to any of the existing classes. We propose that this is classified when similar myosins are identified in other species.

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.

The Drosophila melanogaster staufen gene encodes an RNA binding protein (Dm Stau) required for the localization and translational repression of mRNAs within the Drosophila oocyte. In mammals translational repression is important for normal spermatogenesis in males and storage of mRNAs in the oocytes of females. In the present study we identified two mouse cDNA expressed sequence tags (ESTs), encoding proteins with significant homology to Dm Stau and used these firstly to screen a mouse kidney cDNA library and secondly to determine whether staufen mRNAs are expressed in the ovaries and testes of mice and rats. Sequence analysis of the cDNAs revealed that they originated from two different genes. Using Northern blots of RNAs from kidneys, ovaries and testes, both cDNAs hybridized to mRNA species of approximately 3 kb in all three tissues. On sections of mouse ovaries, staufen mRNA was localized specifically to oocytes. On sections of mouse testes, staufen mRNA was expressed in spermatocytes found in seminiferous tubules at stages VI-XII of the spermatogenic cycle. In conclusion, we have shown that the mammalian homologues of Dm stau are expressed in germ cells in both male and female mice, consistent with a role for these RNA binding proteins in mammalian gametogenesis.

Many genetic cascades are conserved in evolution, yet they trigger different responses and hence determine different cell fates at specific times and positions in development. At stage 10 of oogenesis, mirror is expressed in anterior-dorsal follicle cells, and we show that this is dependent upon the Gurken signal from the oocyte. The fringe gene is expressed in a complementary pattern in posterior-ventral follicle cells at the same stage. Ectopic expression of mirror represses fringe expression, thus linking the epidermal growth factor receptor (EGFR) signalling pathway to the Fringe signalling pathway via Mirror. The EGFR pathway also triggers the cascade that leads to dorsal-ventral axis determination in the embryo. We used twist as an embryonic marker for ventral cells. Ectopic expression of mirror in the follicle cells during oogenesis ultimately represses twist expression in the embryo, and leads to similar phenotypes to the ectopic expression of the activated form of EGFR. Thus, mirror also controls the Toll signalling pathway, leading to Dorsal nuclear transport. In summary, we show that the Mirror homeodomain protein provides a link that coordinates the Gurken/EGFR signalling pathway (initiated in the oocyte) with the Fringe/Notch/Delta pathway (in follicle cells). This coordination is required for epithelial morphogenesis, and for producing the signal in ventral follicle cells that determines the dorsal/ventral axis of the embryo.

We have isolated and sequenced a cDNA encoding a predicted 524 amino acid protein from a Drosophila melanogaster ovarian library. Sequence comparisons suggest that this protein encodes a sodium-dependent inorganic phosphate co-transporter similar to a sequence isolated from a rat brain library. In situ hybridisation to messenger RNA in ovaries shows strong expression in germarium at stage 2 of oogenesis. Expression is then weak in follicle cells until stage 10, when high transcript levels are seen in the nurse cells and transferred to the oocyte. This presumably reflects functions in oogenesis and the production of stored mRNAs for use in embryogenesis.

We report that Drosophila unconventional myosin VI, encoded by Myosin heavy chain at 95F (Mhc95F), is required for both imaginal disc and egg chamber morphogenesis. During oogenesis, Mhc95F is expressed in migrating follicle cells, including the border cells, which migrate between the nurse cells to lie at the anterior of the oocyte; the columnar cells that migrate over the oocyte; the centripetal cells that migrate between the oocyte and nurse cells; and the dorsal-anterior follicle cells, which migrate to secrete the chorionic appendages. Its function during development has been studied using a targeted gene silencing technique, combining the Gal4-UAS targeted expression system and the antisense RNA technique. Antibody staining shows that the expression of myosin 95F is greatly decreased in follicle cells when antisense Mhc95F RNA is expressed. Interfering with expression of Drosophila myosin VI at various developmental stages frequently results in lethality. During metamorphosis it results in adult flies with malformed legs and wings, indicating that myosin VI is essential for imaginal disc morphogenesis. During oogenesis, abnormal follicle cell shapes and aberrant follicle cell migrations are observed when antisense Mhc95F is expressed in follicle cells during stages 9 to 10, suggesting that the Drosophila myosin VI is required for follicle cell epithelial morphogenesis.

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.

Epidermal growth factor receptor (EGFR) signaling pathways are frequently involved in generating cell fate diversity in a number of organisms. During anterior–posterior and dorso–ventral polarity in the Drosophila egg chamber and eggshell, EGFR signaling leads to a number of determinative events in the follicle cell layer. A high level of Gurken signal leads to the expression of argos in dorsal midline cells. Lateral follicle cells, receiving a lower level of Gurken signal, can continue to express the Broad-Complex (BR-C) and differentiate into cells which produce chorionic appendages. Misexpression of argos in mid-oogenesis causes the midline cells to retain expression of BR-C, resulting in a single fused large appendage. Evidence that argos can directly repress Gurken-induced EGFR signaling is seen when premature expression of argos is induced earlier in oogenesis. It represses the Gurken signal at stage 5–6 of oogenesis which determines posterior follicle cells and occasionally leads to eggs with anteriors at both ends. We propose that the Gurken signal at stage 9 of oogenesis induces follicle cells to take on two fates, dorsal midline and lateral, each producing different parts of the eggshell and that argos is one of the key downstream genes required to select between these two fates. Dev. Genet. 25:375–386, 1999. © 1999 Wiley-Liss, Inc.

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.

An abundant ovarian cDNA from Drosophila melanogaster has been cloned and sequenced. The predicted protein sequence is similar to that of the ribosomal protein 1024 of Dictyostelium discoideum, the 40S ribosomal protein ys11 of Saccharomyces cerevisiae and a 22 kd protein from Trypanosoma brucei. It seems, therefore, that the Drosophila cDNA also encodes a ribosomal protein. Transcripts are found at all stages of the life cycle but are especially abundant in the ovary, suggesting that this mRNA is maternally stored for utilization in embryogenesis to enable the rapid production of ribosomal proteins and assembly of ribosomes.

The number of Drosophila egg chambers is controlled by the nutritional status of the female. There is a developmental checkpoint at stage 8, which is controlled by BR-C in the follicle cells along with ecdysteroid. During this period, developmental decision is made in each egg chamber to determine if it will develop or die. During nutritional shortage, inducing apoptosis in the nurse cells of stages 8 and 9 egg chambers reduces the number of egg chambers. We show that ecdysone response genes E75A and E75B are involved in inducing or suppressing apoptosis. It is thus possible that the E75 isoforms A and B are involved in the decision to develop or die in oogenesis. We have established part of the pathway by which ecdysone response genes control apoptosis of the nurse cells and hence select between degeneration or development of individual egg chambers at stages 8 and 9.

Egg chambers of Drosophila are reabsorbed under conditions of nutritional shortage by inducing apoptosis at stages 8 and 9, midway through oogenesis. Nutritional shortage leads to an increase in ecdysone concentration in flies. Apoptosis at stage 8/9 is also induced by 20-hydroxyecdysone injection into the females maintained with adequate nutrition. The expression pattern in the ovary of some ecdysone response genes, E75A, BR-C, is different according to the nutritional environment and the overexpression of these genes induces apoptosis. Apoptosis is suppressed by Juvenile hormone analog treatment of females under nutritional shortage. We predict nutritional and stress response genes control hormone levels and the increase in ecdysone concentration in the flies following starvation induces the ovarian apoptosis. We therefore used a microarray approach to identify the genes involved in receiving the nutritional signal from the environment and translating it in the ovary, thus initiating and executing apoptosis.

Myosin VI is an unconventional Myosin that has been implicated in vesicle transport and membrane trafficking. We isolated lethal mutants of Myosin VI, which lack protein once maternal supplies have been utilised during embryogenesis. Dorsal closure, where there is a ring of Myosin VI at the edge of the migrating epithelial sheet, is often abnormal. The sheet of migrating cells is irregular, rather than a smooth epithelium and cells begin to detach. Some embryos hatch into larvae, containing detached cells loose in the haemolymph. Myosin VI is crucial for correct cell morphology and maintenance of adhesive cellular contacts within epithelial cell layers.

We have identified partial loss of function mutations in class VI unconventional myosin, 95F myosin, which results in male sterility. During spermatogenesis the germ line precursor cells undergo mitosis and meiosis to form a bundle of 64 spermatids. The spermatids remain interconnected by cytoplasmic bridges until individualization. The process of individualization involves the formation of a complex of cytoskeletal proteins and membrane, the individualization complex (IC), around the spermatid nuclei. This complex traverses the length of each spermatid resolving the shared membrane into a single membrane enclosing each spermatid. We have determined that 95F myosin is a component of the IC whose function is essential for individualization. In wild-type testes, 95F myosin localizes to the leading edge of the IC. Two independent mutations in 95F myosin reduce the amount of 95F myosin in only a subset of tissues, including the testes. This reduction of 95F myosin causes male sterility as a result of defects in spermatid individualization. Germ line transformation with the 95F myosin heavy chain cDNA rescues the male sterility phenotype. IC movement is aberrant in these 95F myosin mutants, indicating a critical role for 95F myosin in IC movement. This report is the first identification of a component of the IC other than actin. We propose that 95F myosin is a motor that participates in membrane reorganization during individualization.

We report the analysis of a number of lines of Drosophila melanogaster containing insertions of the yeast gal4 gene. By crossing a UAS-lacZ fusion gene as a reporter into these lines, we analysed the expression patterns of beta-galactosidase during oogenesis. Since there is no expression of GAL4 in the germ-line in these experiments, this is an ideal system for the analysis of expression patterns in sub-sets of follicle cells. These lines provide ideal markers for sets of follicle cells, e.g. anterior or posterior polar cells for studying genetic interactions in oogenesis; however, they can also be used in the same way as conventional enhancer traps to clone nearby genes with similar expression patterns. The advantages of this dual gal4/UAS system over conventional enhancer trapping includes the possibility of GAL4-directed misexpression and antisense expression studies to establish the function of the genes we identified during follicle cell determination and differentiation. These studies could lead to the isolation of homologous genes crucial in mammalian oogenesis. Understanding how the somatic cells and germ cells interact to promote growth and maturation of the mammalian follicle and oocyte could well be crucial for improving the fertility of eggs used for in-vitro fertilization programmes, and could provide methods for assessing the quality of eggs.

Ubiquitin is expressed in eukaryotic cells as precursors, fused via its carboxyl terminus either to other ubiquitin sequences in linear polyubiquitin arrays or to specific ribosomal proteins. In some of the polyubiquitin fusions a single amino acid (e.g., valine in humans) is attached to the carboxyl terminus. These gene products are rapidly (probably cotranslationally) cleaved by ubiquitin carboxyl-terminal hydrolase (UCH) enzymes; therefore, although ubiquitin precursors are suitable substrates for assays of UCH activity, they are difficult to isolate from nucleated cells. While the recombinant approach allows the production of ubiquitin precursors in prokaryotic cells (which do not contain the ubiquitin system), proteins produced in this manner require purification and may also be susceptible to modification by bacterial enzymes, e.g., adventitious proteolysis. As an alternative we have chemically synthesized human ubiquitin–valine. In the assay described here the cleavage of ubiquitin–valine to ubiquitin (77 and 76 residue proteins, respectively) by a purified recombinant Drosophila UCH was monitored by capillary electrophoresis. Mass spectrometry verified the precise cleavage of ubiquitin–valine, confirming that this synthetic protein is a UCH substrate. Synthetic ubiquitin–valine may serve as a generic substrate for UCHs allowing the purification and identification of new members of this enzyme family.

The yolk protein genes (yps) are expressed in a temporal, tissue- and sex-specific fashion in Drosophila melanogaster. Here we report the sequence of two related genes in Calliphora erythrocephala. The predicted Calliphora yolk protein (YP) sequences are well conserved, especially at the C-terminal end when compared to those of D. melanogaster and Ceratitis capitata. Database searches with the Calliphora yolk protein B (CeYPB) sequence identify the vertebrate lipase similarity reported for the YPs of Drosophila and Ceratitis. Moreover, sequences with identity to divalent ion-binding sites were observed, which colocalized with putative tyrosine sulfation sites. Calliphora oogenesis differs from Drosophila in that it is cyclic in response to a meat feed. The Calliphora yp genes are expressed in the follicle cells of the egg chamber during vitellogenesis, as shown by in situ hybridization, and the yp message levels correlate with YP synthesis. The synthesis of the yp transcripts in ovaries of Calliphora occurs in the same pattern as that for ovarian transcripts in Drosophila. In the carcass, yp transcript levels are correlated with the production of a batch of eggs.

Using the enhancer trap approach we have searched for genes with important functions in oogenesis. We selected a line of flies with a P insertion, carrying the Escherichia coli lac Z gene, which showed β-galactosidase expression in the nurse cell nuclei during oogenesis. Surrounding the P insertion we discovered a cluster of transcription units with enriched expression in the ovary. One of these encodes a protein with extensive sequence similarity to the human and yeast ubiquitin carboxyl terminal hydrolase. Analysis of a fusion protein including the putative ubiquitin carboxyl terminal hydrolase indicated that this protein does have the appropriate enzyme activity, and the gene was assigned the name ubiquitin carboxyl terminal hydrolase uch-D . The expression of this gene is enriched in the nurse cells and transcripts are transported to the embryo. Transcripts are abundant for the first few hours of development. The transcripts are found to be enriched on the ventral side of the oocyte and nurse cells. Rather little is known about the ubiquitin pathway in Drosophila and the discovery of this gene enables us to make predictions as to the roles it may play daring early embryogenesis.

Yolk proteins are transported from the hemolymph into the oocytes of insects during vitellogenesis by receptor-mediated endocytosis. Since other hemolymph proteins, both native and foreign, are not accumulated in the oocyte, the process of uptake is selective for yolk proteins. Peptide domains within the yolk proteins must therefore be involved in receptor recognition. With the long-term aim of identifying these domains and to open the possibility of understanding the molecular basis of receptor-mediated endocytosis of yolk proteins, we began investigating how well this mechanism has been conserved in evolution. We studied the uptake of yolk proteins from 13 different Drosophila species and five other dipteran species, namely, Calliphora erythrocephala, Sarcophaga argyrostoma, Musca domestica, Lucilia servicata, and Protophormia terrae-novae, into the ovaries of Drosophila melanogaster and Drosophila funebris. The results from these experiments showed that in all cases the foreign yolk proteins were taken up by the host ovaries, indicating that the mechanism and peptide domains of the yolk proteins involved in recognition of the receptor have been well conserved in dipteran evolution.

Specific mutations in the yolk protein genes, yp1 and yp2, of Drosophila melanogaster cause the yolk proteins (YPs) they encode to precipitate, ultimately resulting in female sterility. YPs of the yp1 mutant fs(1)1163 are secreted normally but then precipitate as globules and occasionally as crystalline fibers in the subbasement membrane space of the fat body (Butterworth et al., 1991, J. Cell Biol. 112, 727-737). The present ultrastructural and immunological studies of the fat body of the yp2 mutant fs(1)K313 show that YP also precipitates as globules in the same tissue compartment. The globules are also incapable of passing into the hemolymph but they are morphologically distinct from those of fs(1)1163. Similar analyses were performed on developing oocytes in wild type and both mutant strains. YP-containing aggregates, ultrastructurally similar to those in the fat body of each respective mutant, were found in the space between the plasmalemma and the vitelline membrane and embedded within the membrane itself. The evidence suggests that the precipitates interfere with the correct assembly of the eggshell membranes, leading to the sterile phenotype. Immunogold studies demonstrate that newly synthesized YPs in the normal and mutant strains share secretory vesicles with putative, vitelline membrane proteins and that the translocation of follicle cell YP is not through the membrane along the interfollicular spaces but directly through the plasmalemma facing the oocyte. Further the YP precipitates in the mutants permit visualization of the polarity of exocytosis of YP from the follicle cells.

Summary The expression of the yolk protein genes has been investigated in male and female Drosophila melanogaster. The continued incorporation of radiolabeled ribonucleotides into yolk protein transcripts suggests that the genes are actively transcribed in the female fat body throughout the period of yolk protein production. In the male, the injection of high doses of 20-hydroxyecdysone transiently stimulates the production of yolk proteins. Investigation by HPLC and RIA of the fate of 20-hydroxyecdysone following its injection into male flies indicated a very rapid rate of metabolism to a group of compounds of higher polarity. Inhibition of yolk protein transcript accumulation by cycloheximide in both males and females suggested that the synthesis of other protein factors may be required for the initiation and maintenance of yolk protein gene transcription in Drosophila. A model suggesting how the yolk protein genes are regulated is presented.

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 extent and type of adult transformations in the tumorous head (tuh) mutation of Drosophila melanogaster were studied. The observations indicate homeotic transformations, duplications and deficiencies of eye antennal disc derivatives. Contrary to previous observations there is no transformation of eye to abdomen and the only homeotic transformations identifiable are antenna to leg and rostralhaut to genitalia. Embryonic and post-embryonic lethality was also examined. Specific anterior abnormalities were found in embryos leading to early death. The amount of embryonic lethality was not affected by increased temperature during oogenesis. When analysing the adult phenotype, however, the whole period of oogenesis was sensitive to pulses of increased temperature; the pulses however did not have to be restricted to any particular stage of oogenesis to be effective in increasing tuh penetrance. The increase in the penetrance of the adult transformations is also exhibited when eggs are moved from 25 °C to 29 °C from the 8th to the 12th hour of embryonic development. Experiments indicate that the tuh-3 gene is active before the 8th hour of embryogenesis.

The three yolk-protein genes of Drosophila are normally expressed only in adult female fat bodies and ovaries. 20-hydroxyecdysone can affect the transcription of these genes in males and females, as can mutations in the sex-determining genes tra, tra-2, ix and dsx. We have asked a number of basic questions about how these genes are regulated, using an inhibitor of protein synthesis (cycloheximide), labelling RNA in vivo, a temperature-sensitive sex-determination mutant (tra-2tsl), and 20-hydroxyecdysone. We have found that the yolk-protein genes are continuously transcribed in the fat bodies of adult females and that maintenance of this transcription requires protein synthesis. Hormone induction in males is also inhibited by cycloheximide, suggesting that the products of other genes are essential both for 20-hydroxyecdysone to be able to switch on the genes, and for their continuous transcription in the female fat body. The products of the tra-2 gene are also required for continuous transcription of the yolk-protein genes, suggesting that the pathway inhibited by the cycloheximide is that of the sex-determination hierarchy. 20-hydroxyecdysone can override the sex-determination system and induce yolk protein synthesis in normal males and tra-2ts reared and maintained at the restrictive temperature.

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.

Drosophila eggs at nuclear multiplication or blastoderm stages were regionally irradiated with monochromatic u.v. Twenty-four hours after irradiation, the results were classified as normal larvae, undifferentiated eggs, or defective embryos; the latter were subdivided into embryos with anterior or posterior defects, or embryos without anterior and posterior specificity. The irradiation of anterior (posterior) egg regions and the occurrence of anterior (posterior) defects were strongly correlated. These correlations were found using eggs of both stages. The size of the irradiated area did not obviously influence the types of defect. Dose-response curves were established irradiating anterior quarters of eggs at 245, 265, 285, or 305 nm wavelength. The frequency of embryonic defects increased with increasing u.v. doses, whereas the dosages applied did not increase the frequency of undifferentiated eggs over the control level. In eggs during nuclear multiplication 285 nm radiation was most effective in producing embryonic defects. After blastoderm formation, the efficiency of irradiation was generally increased but similar at all wavelengths employed. The induction of embryonic defects was photoreversible after u.v.-irradiation at the blastoderm stage. The data reported also include transmittance spectra of chorion and egg membrane preparations from Droso-phila eggs.

A number of female sterile mutations on the first and third chromosomes of Drosophila melanogaster have been screened for defects in the yolk proteins using polyacrylamide gel electrophoresis. Two new mutants were identified. 6m45 accumulates all three yolk proteins (YP1, YP2 and YP3) in the haemolymph but they are all absent from the ovaries suggesting it is a yolk-protein-uptake mutant. In contrast, 1163 is a temperature-sensitive mutation with a large reduction in the quantity of YP1 in the haemolymph and ovaries at 29 °C. Both mutants are autonomous in ovary transplant experiments.

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.

Nuclei isolated from a permanent cell line derived from Drosophila melanogaster embryos have been injected, along with a radioactive DNA precursor [3H]TTP, into Xenopus laevis eggs. In culture, less than 7 % of the cells were in S phase. After a 90 min incubation, following injection into eggs, 99% of the nuclei were shown by autoradiography to have synthesized DNA. In a similar experiment, a density label BrdUTP was injected into eggs along with the nuclei. Subsequent analysis on caesium chloride gradients showed that this DNA synthesis was semi-conservative replication. Therefore we conclude that signals present in Xenopus egg cytoplasm can initiate and sustain true semi-conservative DNA replication in nuclei from an invertebrate organism.

The yolk proteins stored in Drosophila, oocytes for utilisation during embryogenesis are an ideal system for studying the regulation of gene expression during development. The 3 major polypeptides found in yolk in D. melanogaster are synthesised in the fat body and ovarian follicle cells and selectively accumulated by the oocyte during vitellogenesis. In order to understand more about their regulation and the mechanism of uptake, studies on other species are necessary.

When complementary fragments of an imaginal disc ofDrosophila are cultured for several days prior to metamorphosis, usually one fragment will regenerate while the other will duplicate. It has been proposed that wound healing plays an important part in disc regulation (French et al. 1976; Reinhardt et al. 1977) by initiating cell proliferation and determining the mode of regulation. We tried to delay the wound healing process by leaving a region of dead cells between the wound edges. In "06" fragments (Bryant 1975a) wound healing has occurred after 1-2 days of culture and the regeneration of missing structures after 2-4 days of culture. We observed that leaving a region of dead cells between the wound edges delays both wound healing and the regeneration of missing structures by 2 days.When disc fragments are cultured in female abdomens and then exposed to

The three yolk protein genes (yps) of Drosophila melanogaster are expressed in the ovary and fat body of the adult female. Their levels of expression in the fat body depend upon both juvenile hormone (JH) and 20-hydroxyecdysone (20E). Using transformed lines of flies with various flanking sequences from the yp genes and lacZ, Adh, or native yp genes as reporters, the regulation of the three yp genes by 20E and the JH analogue ZR515 (methoprene) was investigated. For 20E, induction of reporter gene expression in males was assayed and, for JH, upregulation of the genes in nutritionally deprived females, which express yolk proteins (YPs) at very low levels, was followed. We were able to map 20E inducible sites upstream of yp3 and sites located 3' and within the coding sequence or introns of yp3 which can interact to respond to 20E. There are also sites in the intergenic spacer between yp1 and yp2. Evidence for repressors was also found upstream of the yp genes, suggesting downstream 20E inducible elements may be important in vivo. There appears to be a difference in the response to 20E in the fat body of the thorax and abdomen between different constructs in males. It is not clear whether those sequences which respond to 20E are genuine ecdysone response elements (i.e., binding sites for the ecdysone receptor) or if the effect is indirect. Methoprene upregulation of YPs, however, was only ever observed using native yp genes as reporters, suggesting that this hormone may act on intron sequences or yp coding sequences, or perhaps by influencing stability of the yp mRNA.

The non-vitellogenic ovaries from homozygous apterous4 mutants of Drosophila melanogaster synthesise yolk-proteins when cultured in vitro, which accounts for the ability of ap4/ap4 ovaries to become vitellogenic in a male-host environment. However, when ap4/ap4 ovaries are transplanted into D. arizonensis females, a large proportion of the ovarian yolk proteins are of the arizonensis type, indicating that ap4/ap4 ovaries are also able to take up yolk proteins in a suitable female-host environment.

Drosophila melanogaster flies were transformed with a yp1-Adh fusion gene with 890 bp of yp1 5' flanking sequence. In an Adh- background these flies show a stage, tissue and sex-specific pattern of alcohol dehydrogenase (ADH) activity characteristic of yolk protein genes. ADH activity is not present in dsxD/dsx pseudomales indicating that this fragment contains sites where the dsx gene product exerts its effect. Transformed male flies do not exhibit ADH activity when injected with 20-hydroxyecdysone while synthesis of native yolk proteins is induced. Thus the hormone inducibility and sex regulation have been separated in this construct.

Females of Drosophila melanogaster were centrifuged at 2400 g and 4200 g for 3 h in a number of different orientations. The oocytes in various stages of vitellogenesis become separated into three layers; a centrifugal yolk layer, a central cytoplasmic layer, and a centripetal lipid layer. The direction of layering is related to the orientation of the female. The process of recovery of the ovaries was followed and the development of the eggs laid analysed. Many of the eggs laid over the subsequent 3 days die very early in development and fail to produce any differentiated structures. Some hatch into normal larvae and others produce defective embryos. The most common defect being a misarrangement of the segmentation. The differences in the kinds of experimentally inducible pattern aberrations in Drosophila and other diptera is discussed.

The three yolk proteins in Drosophilamelanogaster [ 1 ] can be labelled with [35S]methionine during their synthesis either ‘in vitro’ or ‘in vivo’ [2,3]. It has been shown that mRNA isolated from the fat body and ovaries of DrosophiZa can be translated in the wheat germ cell free system into YPl and YP2, yet YP3 was not synthesised [3,4]. Consequently it was not known whether the wheat germ simply failed to translate the mRNA for YP3 or if perhaps YP3 was derived from YPl , YP2, or a larger precursor. We found no evidence of a precursor, and furthermore peptide analysis and the presence of YP3 in a mutant with reduced quantities of YPl in the haemolymph and ovary [5] indicated that YP3 is either a modification of YP2 or is translated from a separate mRNA. In this paper short labelling periods are used both ‘in vivo’ and ‘in vitro’ to indicate that is in unlikely that YP3 is a derivative of another protein.

It has been known for many years that when a wing disc ofDrosophila is bisected, and the fragments cultured in adult females, regulation occurs and either a complete disc is regenerated or the fragment is duplicated. We have investigated how this regeneration process occurs. To establish which cells contribute to the regenerate, and thus determine if regeneration is the result of epimorphic regulation, fragments of discs, after culture in an adult for one to five days, were exposed to3H-thymidine to label replicating cells. Imaginal discs, both whole and as regenerating fragments, undergo some DNA replication which is distributed throughout the disc, but cut discs frequently show clusters of labelled cells around the wound, indicating that regeneration is probably epimorphic.

In Drosophila three yolk polypeptides (YP1, YP2 and YP3) are synthesised at two sites in the adult female: in the fat body tissue, from which they are transported via the haemolymph to the ovary, and in the ovarian follicle cells which surround the developing oocytes. All three yolk polypeptides are synthesised at equal levels in the fat body. In this paper we show that the steady-state level of YP3 RNA is significantly reduced in the ovary in comparison with the fat body, and that none of the yolk protein genes is amplified either in the fat body or the follicle cells. In order to determine the basis of the reduced level of YP3 RNA in the ovary, which could result from a lower rate of transcription or through a decreased stability of the RNA, we have devised an in vivo method of determining relative rates of gene transcription. In both the fat body and the ovary all three yolk proteins are transcribed at similar rates. Thus we infer that YP3 RNA is destablished in the ovary, accounting for the reduction in its steadystate level.

An ovary transplanted from a Drosophila melanogaster female into a male will mature and form morphologically normal yolk-filled oocytes. Since it has been supposed that the yolk polypeptides come only from the female fat body, it was hypothesized that the implanted ovary induces the fat body of the male host to synthesize and secrete yolk polypeptides (YPs). To test this hypothesis, fat body preparations from females, untreated males, and males containing transplanted ovaries were cultured in vitro with 35S-methionine and the medium was examined for the presence of newly labeled YPs. Female fat body secreted newly labeled YPs, but no freshly synthesized YPs were secreted by fat bodies from untreated males or from males containing transplanted ovaries. In vitro cultured ovaries, however, both from females and from male hosts did secrete newly synthesized YPs. Therefore, the YPs in an ovary that matured in a male come mainly from endogenous synthesis by the implanted ovary. To find whether males were responsive to the hormones that stimulate YP production in isolated female abdomens, we treated males with the juvenile hormone analogue ZR-515 and with 20-hydroxyecdysone. The latter, but not the former, was able to cause synthesis and secretion of three bands migrating precisely as YPs in SDS gels. Partial peptide digests of the 20-hydroxyecdysone-stimulated polypeptides in males showed them to be identical with those stimulated by 20-hydroxyecdysone or ZR-515 in isolated female abdomens and with the three YPs found in normal female hemolymph. Finally, YP synthesis was assayed in mutants that affect the phenotypic sex of a fly. It was found that flies bearing two X chromosomes and the mutations dsx, dsxD, ix or three sets of autosomes continued to make YPs, but tra-3-pseudomales did not. These results suggest that the process of sex determination involves steps leading to synthesis of an ecdysteroid in females, which then activates synthesis of the YPs by the fat body. A hypothesis is suggested to explain the fact that two different hormones can stimulate YP synthesis and two different organs can synthesize YPs.

It has been established that the yolk proteins in Drosophila are synthesised and secreted by the fat body, transported in the haemolymph, and accumulated in the developing oocytes [l-5] . In this paper we describe experiments which show that YPl, and YP2 are translated in a cell-free system as proteins of the correct molecular weight rather than aslarger precisors, furthermore our data indicates that the smallest yolk protein, YP3, may be a post-translationally modified derivative of YP2.