Drosophila vitelline membrane assembly: a critical role for an evolutionarily conserved cysteine in the "VM domain" of sV23.

The vitelline membrane (VM), the oocyte proximal layer of the Drosophila eggshell, contains four major proteins (VMPs) that possess a highly conserved "VM domain" which includes three precisely spaced, evolutionarily conserved, cysteines (CX7CX8C). Focusing on sV23, this study showed that the three cysteines are not functionally equivalent. While substitution mutations at the first (C123S) or third (C140S) cysteines were tolerated, females with a substitution at the second position (C131S) were sterile. Fractionation studies showed that sV23 incorporates into a large disulfide linked network well after its secretion ceases, suggesting that post-depositional mechanisms are in place to restrict disulfide bond formation until late oogenesis, when the oocyte no longer experiences large volume increases. Affinity chromatography utilizing histidine tagged sV23 alleles revealed small sV23 disulfide linked complexes during the early stages of eggshell formation that included other VMPs, namely sV17 and Vml. The early presence but late loss of these associations in an sV23 double cysteine mutant suggests that reorganization of disulfide bonds may underlie the regulated growth of disulfide linked networks in the vitelline membrane. Found within the context of a putative thioredoxin active site (CXXS) C131, the critical cysteine in sV23, may play an important enzymatic role in isomerizing intermolecular disulfide bonds during eggshell assembly.

Morphogenesis of the eggshell in Drosophila.

The Drosophila eggshell is a specialized extracellular matrix that forms between the oocyte and overlaying somatic follicle cells during the latter stages of oogenesis. Largely proteinaceous, the eggshell is a highly organized multilayered structure with regional specializations designed to perform a variety of functions. Production of a functional eggshell features: (1) the differentiation of subsets of follicle cells in response to ovarian signals, (2) directed migrations of the follicle cells within the developing egg chamber, (3) expression of eggshell structural genes by the follicle cells in a defined temporal and spatial order, (4) postdepositional modifications of the eggshell proteins including several temporally regulated proteolytic cleavage events, and (5) regulated trafficking of several eggshell proteins in the assembling structure. By exploiting the genetic advantages of Drosophila and using evolution as a guide, the eggshell provides an excellent experimental system to study, in vivo, molecular mechanisms used to regulate protein-protein interactions throughout the assembly of a complex extracellular architecture in a developing organism.

The functions of the multiproduct and rapidly evolving dec-1 eggshell gene are conserved between evolutionarily distant species of Drosophila.

The Drosophila dec-1 gene encodes multiple proteins that are required for female fertility and proper eggshell morphogenesis. Genetic and immunolocalization data suggest that the different DEC-1 proteins are functionally distinct. To identify regions within the proteins with potential biological significance, we cloned and sequenced the D. yakuba and D. virilis dec-1 homologs. Interspecies comparisons of the predicted translation products revealed rapidly evolving sequences punctuated by blocks of conserved amino acids. Despite extensive amino acid variability, the proteins produced by the different dec-1 homologs were functionally interchangeable. The introduction of transgenes containing either the D. yakuba or the D. virilis dec-1 open reading frames into a D. melanogaster DEC-1 protein null mutant was sufficient to restore female fertility and wild-type eggshell morphology. Normal expression and extracellular processing of the DEC-1 proteins was correlated with the phenotypic rescue. The nature of the conserved features highlighted by the evolutionary comparison and the molecular resemblance of some of these features to those found in other extracellular proteins suggests functional correlates for some of the multiple DEC-1 derivatives.

Differential sorting of constitutively co-secreted proteins in the ovarian follicle cells of Drosophila.

Conventional and freeze-fracture electron microscopy, immuno-electron microscopy of ovarian cryosections and confocal immunofluorescence were used to analyze the ovarian distribution of the major protein classes being secreted by the follicle cells during the vitellogenic and choriogenic stages of Drosophila oogenesis. Our results clearly demonstrated that at vitellogenic stages the follicle cells co-secrete constitutively vitelline membrane and yolk proteins that are either sorted into distinct secretory vesicles or they are segregated in different parts of bipartite vesicles by differential condensation. Following their exocytosis only the vitelline membrane proteins are incorporated into the forming vitelline membrane. The yolk proteins (along with their hemolymph circulating counterparts) diffuse through gaps amongst the incomplete vitelline membrane and are internalized through endocytosis by the oocyte where they are finally stored into modified lysosomes referred to as alpha-yolk granules. The unexpected immunolocalization of vitelline membrane antigens in the associated body of the alpha-yolk granules may indicate that this structure is a transient repository for the proteins being internalized into the oocyte along with the yolk proteins. In the early choriogenic follicle cells the vitelline membrane and early chorion proteins were found to be co-secreted and to be evenly intermixed into the same secretory vesicles. These findings illuminate new details concerning the follicle cells secretory and oocyte endocytic pathways and provide for the first time evidence for condensation-mediated sorting of constitutively secreted proteins in Drosophila.

Nonsecreting myeloma variants with heavy-chain carbohydrate deficiencies.

Light- and heavy-chain synthesis was studied in six previously isolated S194-2 mouse myeloma variant lines and in the parent from which they were derived. Serological data and comparative analysis of the cyanogen bromide fragments obtained from variant and parent intracellular immunoglobulin showed that five variants which failed to secrete detectable amounts of IgA synthesized both heavy- and light-chain subunits. Whereas at least two heavy-chain populations were resolved in the parent line, one containing carbohydrate and one devoid of carbohydrate, only the heavy-chain fraction devoid of carbohydrate was detected in the variant lines. The correlation between carbohydrate deficiencies on the heavy chains and lack of immunoglobulin secretion in five independent subclones is discussed in terms of possible primary lesions and the role of carbohydrate in secretion.

Molecular analysis and rescue of a vitelline membrane mutant in Drosophila.

The eggshell in Drosophila is produced by ovarian follicle cells during the later stages of oogenesis. Eggshell formation involves the ordered synthesis and assembly of several protein components. Genes encoding the most abundant eggshell proteins have been identified by molecular cloning studies. Morphological examination of eggs produced by females carrying female sterile mutations on the X and third chromosomes have revealed additional loci involved in chorion formation. In this study we screened a collection of female sterile mutants carrying EMS-induced mutations on the second chromosome for eggshell mutants. A class of six mutants with potential vitelline membrane defects was identified on the basis of the response of mutant eggs to hypochlorite solutions. Biochemical analysis showed that one mutant, fs(2)QJ42, failed to produce a major vitelline membrane protein, sV23. The mutation was mapped cytogenetically to 26A, a region previously implicated in vitelline membrane formation by molecular cloning studies. Northern blot analysis using a cloned copy of the sV23 gene as probe showed a 10- to 15-fold reduction of sV23 RNA levels in the mutant. sV23 synthesis and fertility were restored when a normal copy of the sV23 gene was introduced into the mutant via germ line transformation. Transposons carrying the sV23 gene with as little as 147 bp of 5' flanking DNA were capable of restoring fertility and sV23 protein to wild type levels.

Cloning and analysis of the dec-1 female-sterile locus, a gene required for proper assembly of the Drosophila eggshell.

Female-sterile mutations at the dec-1 (defective chorion-1) locus of Drosophila severely disrupt the organization of the eggshell late in oogenesis. Previous characterization of dec-1 mutations has correlated the defects with failure to accumulate an early eggshell protein that undergoes proteolytic cleavage during choriogenesis. To enable further study of the regulation and processing of dec-1 products, we have molecularly cloned the locus and characterized its transcripts. Chromosome jumping was used to isolate a deficiency breakpoint within the locus. Overlapping genomic clones from a wild-type library were then obtained, and a region including the dec-1 locus was identified by hybridization to cDNA probes complementary to RNA from stage 9-10 egg chambers. Analysis of genomic rearrangements associated with the locus verified its identity. Two transcripts from the locus have been identified and characterized using cDNA clones, RNase protection, and primer extension analyses. A 4.0-kb transcript accumulates maximally in stages 9-10, when the primary follicle cell protein associated with dec-1 mutations is synthesized. A second transcript of 5.8 kb, generated by alternative splicing, accumulates during stages 11-12. These results are discussed in light of previous analysis of dec-1 mutations.

Parasitoid pressure and the radiation of a gallforming group (Cecidomyiidae: Asphondylia spp.) on creosote bush (Larrea tridentata).

We tested the Enemy Impact Hypothesis, which predicts that communities of one tropic level are organized by the tropic level above. In the case of gallforming insect communities, the hypothesis predicts that gall morphology will diverge, minimizing the number of parasitoids shared among species. We used the monophyletic group of gallforming cecidomyiids (Asphondylia spp.) on creosote bush (Larrea tridentata) to test this hypothesis, predicting that species with thicker gall walls should exclude species of parasitoids with shorter ovipositors and have lower levels of parasitism. Of 17 parasitoid species reared from Asphondylia galls on creosote bush, 9 accounted for over 98% of parasitism. Seven of these 9 species had ovipositors long enough to penetrate 10 of 13 gall morphs measured. There was no significant relationship between gall wall thickness and number of associated parasitoid species (r (2)=0.01, P>0.05, n=13). There was no relationship between gall wall thickness and types of parasitoid species colonizing galls: parasitoids with the shortest ovipositors colonized all types of gall morphs and were dominant members of the parasitoid assemblages in galls with the thickest walls. Ultimately, there were no significant differences in percent parasitism among Asphondylia species, regardless of gall wall thickness. We found no difference in numbers of associated parasitoids or percent parasitism in galls with different textures (e.g. hairy versus smooth), different locations on the plant or different phenologies. Our results suggest that enemy impact has not influenced the diversity of this gall community. Gall wall thickness, phenology, location on the plant and surface structure do not appear to influence the distribution of parasitoid species. Other explanations are offered to account for diversity in gall morphology among these species.

Cloning and characterization of a dispersed, multicopy, X chromosome sequence in Drosophila melanogaster.

We have isolated and characterized a dispersed middle repetitive DNA sequence from Drosophila melanogaster that is concentrated on the euchromatic portion of the X chromosome. In situ hybridization of the repeat unit to salivary gland chromosomes shows the sequence is distributed among approximately 10 major and 20 minor X chromosomal sites. Based on DNA sequence analysis of homologous sequences from three different cytogenetic regions, the 372-base-pair repeat unit appears to be (A + T)-rich and noncoding and shows strong sequence conservation among units from different chromosomal regions. The nature and distribution of this sequence are suggestive of the hypothetical X chromosome DNA sequences thought to be involved in the primary establishment of sex determination and dosage compensation in Drosophila.

7C female sterile mutants fail to accumulate early eggshell proteins necessary for later chorion morphogenesis in Drosophila.

Seven noncomplementing female sterile mutations that affect eggshell assembly in Drosophila have been mapped to the 7C1-3 region of the X-chromosome. TEM of the mature eggshell of one of the alleles, fs(1)410, shows a lack of organization within the endochorion and an accumulation of electron dense material in the vitelline membrane of stage 14 eggchambers. SDS-PAGE of radiolabeled eggshell proteins shows that two proteins, s67 and s85, fail to accumulate in the fs(1)410 eggshell. In wild-type flies s85 is produced during stage 10 of oogenesis and then processed to s67 in stages 13 and 14. Neither s85 nor an additional stage 10 specific follicle cell protein (s130) are detected in fs(1)410 or four of the mutant alleles. Short-term labeling studies, analyses of in vitro translation products, and the simultaneous occurrence of s85 and s130 as electrophoretic variants in geographic fly strains indicate s85 is derived from s130. Although major biochemical differences appear in stage 10, mutant and wild-type eggshells are morphologically indistinguishable until stages 13-14. These results suggest that follicle cell proteins synthesized during the time of vitelline membrane deposition (stage 10) are important for proper assembly of the chorion layers during stages 13 and 14.

Identification and time of synthesis of chorion proteins in Drosophila melanogaster.

The chorion of Drosophila melanogaster consists of proteins secreted by the follicular epithelium during late oogenesis. Petri, Wyman and Kafatos (1976) have described six major protein components of the Drosophila chorion and reported the synthesis of these proteins in vitro by mass-isolated egg chambers. We have used two-dimensional gel electrophoresis to identify approximately twenty components in highly purified chorion preparations. The synthesis patterns of these proteins in vivo were determined by isolating egg chambers of different developmental stages from flies injected with 14C amino acids. Chorion proteins constitute a large fraction of the protein synthesized by ovarian egg chambers in stages 12--14. The sizes and times of synthesis of the chorion proteins correlate closely with the production of poly(A)-containing RNAs by the follicle cells (Spradling and Mahowald, 1979).

Identification and cytogenetic localization of vitelline membrane messenger RNAs in Drosophila.

During stages 9 and 10 of oogenesis in Drosophila the major proteins involved in vitelline membrane (VM) formation are synthesized and secreted by the somatic follicle cells surrounding the oocyte. To identify potential mRNAs involved in VM protein synthesis, newly synthesized poly(A)-containing RNA from egg chambers of different developmental stages was studied. Urea-agarose gel electrophoresis revealed two RNA bands in stage 10 egg chambers in the size range expected for those which encode the smaller VM proteins. These RNA bands, T1 and T2, are specifically enriched in stage 10 follicle cell preparations. In vitro translations in reticulocyte lysates in the absence and presence of microsomal membranes showed both RNA bands code for products that are synthesized in precursor forms which are processed to species that comigrate with VM proteins. T2 directed the synthesis of processed species that comigrated with the 23- to 24-kDa and 17.5-kDa VM proteins (J. Fargnoli and G.L. Waring, 1982, Dev. Biol. 92, 306-314) while the T1 translation product comigrated with the 14-kDa protein. To determine the cytogenetic location of the genes encoding T1 and T2 RNAs, radiolabeled T1 and T2 RNAs were hybridized in situ to salivary gland chromosomes. The results suggest that the structural genes coding for the small vitelline membrane proteins are localized at two sites on the second chromosome: 39DE and 42A.

Regulated processing of dec-1 eggshell proteins in Drosophila.

The Drosophila dec-1 gene encodes multiple products that are necessary for proper eggshell assembly. During stages 9-12 of oogenesis the ovarian follicle cells synthesize three alternatively spliced dec-1 RNAs that encode proteins of 106, 125, and 177 kDa. All three of these primary translation products undergo developmentally regulated posttranslational cleavages. Antibodies to trpE fusion proteins containing different regions of the dec-1 proteins have been used to identify processing intermediates as well as stable derivatives of fc106, fc125, and fc177. fc106, the most abundant product of the locus, is cleaved at its N-terminus, producing an N-terminal 25-kDa derivative and s80, a stage 10 eggshell protein. During late oogenesis N-terminal cleavage of s80 yields a 20-kDa N-terminal derivative and a 60-kDa eggshell protein. fc125, which differs from fc106 by a C-terminal extension, is processed at its N-terminus through a series of transient intermediates to a stable 95-kDa C-terminal derivative in late stage 10 egg chambers. Unlike its s80 counterpart, the 95-kDa derivative is not subjected to later cleavage events. fc177, synthesized during stages 11-12, is cleaved to a stable C-terminal derivative of approximately 85 kDa. Although all of the cleavage sites fall within regions that are common to fc106, fc125, and fc177, each protein follows a different maturation pathway which results in a variety of proteins with distinctive N- and C-termini. Our data suggest that inter- or intramolecular interactions dictated by the C-terminal ends of the molecules determine the pathway followed by each dec-1 protein.

Organization and expression of a second chromosome follicle cell gene cluster in Drosophila.

Four genes expressed during the period of vitelline membrane formation are clustered within 8 kb of DNA in region 26A of the second chromosome. Temporal and quantitative difference in the profiles of accumulated RNA suggest that the genes are independently regulated although they are selectively expressed during the stages of vitelline membrane biosynthesis. In situ hybridization and S1 analyses of RNAs from fractionated eggchambers established that these genes are active only in the follicle cells. S1 mapping with in vitro synthesized RNA probes shows that three of the genes are tandemly oriented. All four appear to be intronless. In vitro translation products from hybrid-selected RNAs indicate that two of these genes code for major vitelline membrane proteins. Sequence analysis of these two genes support this conclusion. The cell- and stage-specific expression of the other two genes, encoding less abundant RNAs, suggests that they also play a role in early eggshell production.

Drosophila dec-1 eggshell proteins are differentially distributed via a multistep extracellular processing and localization pathway.

In Drosophila the multilayered eggshell forms during late oogenesis between the oocyte and the overlaying follicle cells. Proper eggshell assembly requires wild-type dec-1 gene function. Alternatively spliced dec-1 transcripts encode three proproteins that are cleaved extracellularly in a stage-specific manner to at least five distinct derivatives. Using polyclonal antibodies raised against fusion proteins containing different regions of the dec-1 proteins, we have localized several dec-1 derivatives in the assembling and completed eggshell. Although all of the dec-1 derivatives are generated in the oocyte proximal vitelline membrane layer, they are differentially distributed in the mature egg. Some derivatives are gradually released from the vitelline membrane and become localized within distinct regions of the chorion, while others are taken up by the oocyte or become concentrated in the endochorionic spaces or cavities. The diverse distributions of the dec-1 derivatives suggest that each derivative plays a distinct role in eggshell assembly. These results also suggest that the vitelline membrane layer, by acting as a transient storage site, may control the availability of molecules active in eggshell assembly and by extension perhaps other follicle cell products important in early embryonic pattern formation.

Characterization and sequence of follicle cell genes selectively expressed during vitelline membrane formation in Drosophila.

To isolate genes involved in vitelline membrane production, an ovarian cDNA library was screened with eggchamber RNAs labeled in vivo. Two cDNA clones encoding RNAs that are selectively expressed in follicle cells during the period of vitelline membrane formation were isolated. Following isolation of homologous genomic clones from a Drosophila library, one gene was localized by in situ hybridization to chromosomal region 26A, and the other to 3C. Developmental Northern blots demonstrated that both genes produce 700-800 nucleotide transcripts that accumulate during the stages of vitelline membrane synthesis. In vitro translation products from hybrid selected RNAs and DNA sequence analysis both indicate that the 26A region gene encodes a major protein component of the vitelline membrane. The structural properties of the 3C region follicle cell gene seem more compatible with an intracellular function.

Multiple proteins are produced from the dec-1 eggshell gene in Drosophila by alternative RNA splicing and proteolytic cleavage events.

The defective chorion-1 gene (dec-1) in Drosophila encodes follicle cell proteins necessary for proper eggshell assembly. A distinctive feature of the gene is the production of multiple products by both alternative RNA splicing and proteolytic processing events. DNA and protein sequencing studies have revealed several dec-1 protein products. The predominant translation product, fc106, has a vitelline membrane-like N-terminal domain followed by a glutamine, methionine-rich central region, largely in the form of 26 amino acid repeats. During late stage 10 the N-terminal portion of fc106 is cleaved, yielding s80, a major eggshell protein. Conceptual translation of the DNA sequence as well as molecular analyses of several dec-1 mutants suggest that the less abundant alternatively spliced RNAs encode primary translation products with different carboxy terminal ends. These results are discussed with respect to previous genetic analyses of dec-1 mutants as well as with respect to potential protein-protein interactions which may underlie stabilization of this complex extracellular structure.

Isolation of germ line-dependent female-sterile mutation that affects yolk specific sequestration and chorion formation in Drosophila.

Two loci on the X chromosome have been implicated in choriogenesis by in situ hybridization of poly A-containing RNA from choriogenic eggchambers to Drosophila polytene chromosomes (A.C. Spradling and A.P. Mahowald (1979). Cell 16, 589-598): 7E and 12E. At least two genes coding for major eggshell proteins map to region 7E (A.C. Spradling, M.E. Digan, A.P. Mahowald, M. Scott, and E.A. Craig (1980). Cell 19, 905-914). In an effort to elucidate the functional role of the 12E gene product, 3600 EMS-treated X chromosomes were screened for recessive female-sterile mutations that mapped within the region 11F10-12F1. Four independent female-sterile mutations were recovered, three of which fell into one complementation group (fs29, fs117, and fs445). Mapping by analysis of recombinant progeny as well as of trans heterozygotes utilizing other deficiency chromosomes showed that the three noncomplementing mutations all mapped to region 12E1-12F1. Studies comparing chorion morphology and protein synthesis indicate localized perturbations in the extracellular assembly of eggshell components in mutant eggchambers. The germ line dependence of the mutations was established using germ line mosaics constructed by pole cell transplantation. Analysis of eggchamber protein accumulation patterns showed reduced amounts of yolk polypeptides (YPs) in the mutants. The elevated concentrations of YPs found in mutant hemolymph coupled with the normal YP biosynthetic patterns and active uptake of trypan blue by mutant oocytes suggest that 12E sequences play a role in yolk-specific sequestration.

Eggshell assembly in Drosophila: processing and localization of vitelline membrane and chorion proteins.

The Drosophila eggshell consists of three major proteinaceous layers: the vitelline membrane, the inner chorionic layer, and the outer endochorion. During the latter stages of oogenesis, the proteins that comprise these layers are synthesized and secreted by epithelial follicle cells which surround the maturing oocyte. While there is considerable knowledge of the structural units which comprise the eggshell layers, there is little knowledge of how individual proteins function or interact with one another to form the structure. Immunoelectron microscopy was used to follow the distribution of four different eggshell proteins in the assembling and mature eggshell. sV23 and sV17, follicle cell proteins synthesized during the early stages of eggshell formation (stages 8-10), were distributed within the vitelline membrane layer at all stages. Despite marked temporal differences in their accumulation profiles, s36 and s18, putative chorion proteins, were similarly distributed throughout the floor, pillars, and roof of the endochorion. Although the vitelline membrane appears to be morphologically complete by stage 11, developmental Western blots and immunolocalization data indicate that molecular dynamism persists within the layer throughout the subsequent choriogenic stages. During early chorion formation the vitelline membrane appears to act as a reservoir for chorion proteins since s36 was found predominantly in the vitelline membrane layer of stage 12 egg chambers. During the late choriogenic stages (13-14), both sV17 and sV23 are processed to smaller derivatives. Interactions between the eggshell layers were suggested by ultrastructural analysis of a sV23 protein null mutant which showed that the structural integrity of the outer chorion is dependent upon the presence of a vitelline membrane component.

Carbon-nutrient balance hypothesis in within-species phytochemical variation ofSalix lasiolepis.

Predictions of the carbon-nutrient balance hypothesis were tested using a study of within-species phytochemical variation in the arroyo willow,Salix lasiolepis. The prediction that a balance between nutrients (total protein) and carbon-based secondary metabolites (total phenols) should exist was supported using water treatment and fertilizer experiments and wild willow clones. Leaf nitrogen content and net photosynthetic rates of plants potted in soil in which parental plants grew was low, indicating that wild plants exist under relatively low nutrient status-high carbon balance conditions. The hypothesis also correctly predicted positive relationships between shoot length and phenols in glasshouse plants, wild plants, and plants in the water treatment experiment and negative relationships between shoot length and phenols in the fertilizer treatment experiment. Total phenolic glycosides, fragilin, picein, salicortin, tremulacin, and tremuloidin all correlated positively with shoot length in glasshouse plants on a carbon-biased balance, and male willows had generally lower levels of phenolic glycosides than females. Salicortin and tremulacin showed the strongest positive relationships with shoot length.