Specific protein synthesis in cellular differentiation: II. The program of protein synthetic changes during chorion formation by silkmoth follicles,and its implementation in organ culture

During their differentiation, the follicular epithelial cells of the silkmoth, Antheraea polyphemus, produce the extracellular proteinaceous eggshell or chorion. Choriogenesis entails continuous changes in cell-specific protein synthesis; the various chorion proteins are synthesized with distinct kinetics. On the basis of protein synthetic profiles, 17 stages of choriogenesis are defined. The average duration of the stages is 3.0 hr, and thus choriogenesis lasts a total of approximately 51 hr. This program of protein synthetic changes is autonomous; i.e., it is implemented with normal kinetics by follicles cultured in isolation in a defined tissue culture medium.     

Early establishment and autonomous implementation of a developmental program controlling silkmoth chorion gene expression.

To address the question of whether prechoriogenic follicles of the silkmoth Bombyx mori have the capacity to enter choriogenesis in organ culture and define the stage at which choriogenesis becomes established as a follicle-autonomous program, we have cultured immature ovarioles dissected from developing pupae and examined the protein synthetic profiles of follicular cells of individual follicles at the end of the culture period. The protein synthetic profiles of the cultured follicles were also correlated with corresponding profiles of chorion mRNA accumulation. Our results demonstrate that the last 17 (+/- 2) vitellogenic follicles of Day 5 to 7 pupae are capable of initiating choriogenesis in organ culture. The earliest vitellogenic stage to enter choriogenesis in vitro does so after 34 (+/- 4) hr in culture and follicles entering choriogenesis in vitro are capable of proceeding through all choriogenic stages at a speed comparable to that occurring in vivo. Therefore, once the choriogenic program becomes established in follicular cells, it can be implemented autonomously in the absence of extrafollicular factors. Earlier vitellogenic stages lack this capacity, presumably because they require additional hemolymph factors to establish the choriogenic potential. Our results demonstrate that the choriogenic potential of cultured vitellogenic follicles cannot be influenced by addition of 20-hydroxyecdysone to the culture medium.     

Effect of the pleiotropic GrB mutation of Bombyx mori on chorion protein synthesis

The Gr^B mutation has a profound pleiotropic effect, leading in the homozygous state to the absence or extreme reduction of a substantial number of chorion proteins. The effect shows developmental specificity: most of the proteins normally synthesized beginning with stage III of choriogenesis or later, but possibly none of these normally beginning with stage II, are eliminated in the mutant. More subtle quantitative effects on certain other proteins are also observed, including prolongation of synthesis of some proteins which normally terminate at stage VIII. The proteins eliminated in the mutant are present in the heterozygote at intermediate levels, quantitatively close to those in the wild-type. The differences in chorion protein composition result from correspondingly altered protein synthesis rather than from post-translational degradation or modification. The missing proteins also fall to be synthesized in vitro when total RNA from mutant follicles is translated in the wheat germ system. It appears that as a consequence of the mutation, a set of mRNAs fails to be synthesized or accumulated. These results are consistent with the possibility that Gr^B is a regulatory mutation, or a deletion eliminating multiple chorion genes, clustered predominantly according to the developmental period of their expression.     

The silkmoth chorion: Morphogenesis of surface structures and its relation to synthesis of specific proteins

The morphogenesis of four spatially differentiated surface regions of the silkmoth eggshell (chorion) has been documented and correlated with differing patterns of chorion protein synthesis within the corresponding secretory cells. During the first half of choriogenesis the polygonal pattern of ridges which cover the entire chorion appears. Regional differences in the morphology of developing ridges are not accompanied by significant protein differences, and thus presumably reflect differences in secretory cell behavior and shape. During the second half of choriogenesis expanding domes of the chorion located immediately beneath three-cell junctions of the overlying secretory surface become prominent surface features exclusively in the aeropyle crown region. Domes are composed of a thin lamellar skin and an inner buttressing “filler.” Continued filler deposition appears to cause a ripping of the lamellar skin, transforming the dome into a multiple-pronged crown that overflows with filler. Continued synthesis of lamellar chorion components elongates and strengthens the crowns until they can stand alone without the support of filler. In the aeropyle crown region, synthesis of regionally specific proteins begins in the second half of choriogenesis and accelerates until the final stages, in parallel with dome/crown formation. The more numerous proteins which are common to all regions are synthesized at approximately equal rates within all regions, and their synthesis decelerates toward the end of choriogenesis. Fifteen of the proteins (excluding filler) which are found predominantly in the aeropyle crown region may be necessary but not sufficient for crown formation, since they also occur in the stripe region (1); presumably the secretory cell surfaces mold the same components differently in the two regions. Filler appears to play an important scaffolding role in crown formation. A group of eight aeropyle crown region-specific chorion proteins which compose filler have been identified on two-dimensional gels and shown to be restricted to one of five previously described classes of chorion proteins.     

Specific protein synthesis in cellular differentiation: IV. The chorion proteins of Bombyx mori and their program of synthesis

Detailed analysis of the chorion proteins of Bombyx mori reveals more than 70 components, each distinguished by electrophoretic mobility, relative abundance, and kinetics of synthesis. Many proteins are strain specific. A protein numbering system is established, based on isoelectric point and molecular weight. As in Antheraea polyphemus, chorion proteins are produced asynchronously, individual proteins showing characteristic developmental kinetics. The synthetic program is analyzed in detail. Stages of choriogenesis are defined according to the pattern of protein synthesis, and their relative and absolute durations are determined. In a few stages, synthesis of numerous protein cohorts is coordinately initiated.     

BmCAP,a silkmoth gene encoding multiple protein isoforms characterized by SoHo and SH3 domains: Expression analysis during ovarian follicular development

CAP/ArgBP2/vinexin family proteins, adaptor proteins characterized by three SH3 domains at their C-termini and a SoHo domain towards their N-termini, are known to regulate cell adhesion, cytoskeletal organization, and growth factor signaling. Here we present the isolation and ovarian expression of the BmCAP gene which encodes CAP/ArgBP2/vinexin family proteins in the silkmoth, Bombyx mori. Screening for full-length cDNA clones identified three mRNA isoforms, BmCAP-A1, BmCAP-A2 and BmCAP-B, which show expression throughout ovarian follicular development. Using an antibody raised against a unique region between the SoHo and SH3 domains, BmCAP-A protein isoforms were identified that show specific expression in different compartments of the ovarian follicles. Immunofluorescence staining of the cells of the follicular epithelium establishes a dynamic pattern of BmCAP-A protein localization during choriogenesis. During early choriogenesis, BmCAP-A has a diffuse localization in the cytoplasm but could also be found concentrated at the apical and basal sides at the cell–cell junctions. During late choriogenesis, the diffuse cytoplasmic staining of BmCAP-A disappears while the staining pattern at the apical side resembles a blueprint for the eggshell surface structure. We suggest that BmCAP-A isoforms have important functions during ovarian development, which involve not only the traditional roles in actin organization or cell–cell adhesion but also the regulation of secretion of chorion proteins and the sculpting of the chorion surface.     

Specific protein synthesis in cellular differentiation: VI. Temporal expression of chorion gene families in Bombyx mori strain C108

Detailed patterns of expression for putative members of 5 major chorion gene families have been obtained by separating labeled proteins using two-dimensional polyacrylamide gel electrophoresis. Proteins fall into 4 temporal cohorts called early, early middle, middle, and late on the basis of when they initiate and terminate synthesis. Proteins synthesized during the early, early middle, and late periods are highly synchronous, exhibiting abrupt onset times and relatively uniform termination times. Middle proteins begin synthesis in small groups at staggered times over a relatively long period, but most cease translation as the late proteins turn on. This data is correlated with a previous follicle staging system based on separation of newly synthesized chorion proteins by isoelectric focusing alone. The absolute timing of choriogenesis was determined in vivo, using trypan blue dye to mark vitellogenic follicles. The relative age difference between chorionating follicles was 2.2–2.6 hr; chorion biosynthesis took 4 days in all. These data are discussed in terms of patterns of activity of chorion gene families, the functions of the temporal cohorts, and regulation of the chorion multigene system.     

Accelerated poly(A) loss on alpha-tubulin mRNAs during protein synthesis inhibition in Chlamydomonas

Detachment of flagella in Chlamydomonas reinhardii stimulates a rapid accumulation of tubulin mRNAs. The induced tubulin mRNAs are normally rapidly degraded following flagellar regeneration, but inhibition of protein synthesis with cycloheximide prevents their degradation. alpha-Tubulin poly(A) tail lengths were measured during normal accumulation and degradation, and in cycloheximide-treated cells. To measure alpha-tubulin mRNA poly(A) chain lengths with high resolution, specific 3' fragments of alpha 1- and alpha 2-tubulin mRNAs, generated by RNase H digestion of mRNA-oligonucleotide hybrids, were sized by Northern analysis. Both alpha-tubulin mRNAs have a newly synthesized poly(A) chain of about 110 adenylate residues. The poly(A) tails shorten with time, and show an average length of 40 to 60 adenylate residues by 90 minutes after deflagellation, at which time induced alpha-tubulin mRNA is being rapidly degraded. Poly(A) loss is significantly accelerated in cycloheximide-treated cells, and this loss is not attributible simply to the longer time the stabilized molecules spend in the cytoplasm. A large fraction of alpha-tubulin mRNA accumulates as mRNA with very short poly(A) tails (less than 10 residues) in the presence of cycloheximide, indicating that deadenylated alpha-tubulin mRNAs can be stable in vivo, at least in the absence of protein synthesis. The rate and extent of poly(A) loss in cycloheximide are greater for alpha 2-tubulin mRNA than for alpha 1-tubulin mRNA. This difference cannot be attributed to differential ribosome loading. This finding is interesting in that the two mRNAs are very similar in sequence with the exception of their 3' untranslated regions.     

Cell-free translation of silkmoth chorion mRNAs: Identification of protein precursors and characterization of cloned DNAs by hybrid-selected translation

The secretory silkmoth chorion proteins are synthesized as precursors bearing signal peptides. Precursors are detected upon cell-free translation of chorion mRNAs in the wheat germ system; they are processed into products identical in size to authentic chorion proteins when translation is performed in the presence of microsomal membranes from dog pancreas. Precursors corresponding to specific protein size classes and subclasses are identified by three approaches: comparison of precursors and products encoded by stage-specific mRNAs, comparison of precursors and products encoded by mRNAs specifically hybridizing to individual chorion cDNA clones, and comparison of relative amino acid compositions of precursors and authentic chorion proteins. Translation of stage-specific mRNA preparations indicates that, in general, the developmental changes of in vivo chorion protein synthesis are based on changes in concentrations of the corresponding mRNAs. Characterization of the precursors makes it possible to identify, for any chorion DNA clone, the protein subclass, a member of which is encoded by the clone sequence.     

Role of protein synthesis in decay and accumulation of mRNA during spore germination in the cellular slime mold Dictyostelium discoideum.

Spore germination in Dictyostelium discoideum is a particularly suitable model for studying the regulation of gene expression, since developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. The previous isolation of three cDNA clones specific for mRNA developmentally regulated during spore germination allowed for the quantitation of the specific mRNAs during this process. The three mRNAs specific to clones pLK109, pLK229, and pRK270 have half-lives much shorter (minutes) than those of constitutive mRNAs (hours). Using spore germination as a model, we studied the roles of ribosome-mRNA interactions and protein synthesis in mRNA degradation by using antibiotics that inhibit specific reactions in protein biosynthesis. Cycloheximide inhibits the elongation step of protein synthesis. Polysomes accumulate in inhibited cells because ribosomes do not terminate normally and new ribosomes enter the polysome, eventually saturating the mRNA. Pactamycin inhibits initiation, and consequently polysomes break down in the presence of this drug. Under this condition, the mRNA is essentially free of ribosomes. pLK109, pLK229, and pRK270 mRNAs were stabilized in the presence of cycloheximide, but pactamycin had no effect on their normal decay. Since it seems likely that stability of mRNA reflects the availability of sites for inactivation by nucleases, it follows that in the presence of cycloheximide, these sites are protected, presumably by occupancy by ribosomes. No ribosomes are bound to mRNA in the presence of pactamycin, and therefore mRNA degrades at about the normal rate. The data further indicate that a labile protein is probably not involved in mRNA decay or stabilization, since protein synthesis is inhibited equally by both antibiotics. We conclude that it may be important to use more than one type of protein synthesis inhibitor to evaluate whether protein synthesis is required for mRNA decay. The effect of protein synthesis inhibition on mRNA synthesis and accumulation was also studied. mRNA synthesis continues in the presence of inhibitors, albeit at a diminished rate relative to that of the uninhibited control.     

BmVMP90, a large vitelline membrane protein of the domesticated silkmoth Bombyx mori, is an essential component of the developing ovarian follicle

We present the characterization of BmVMP90, a vitelline membrane protein (VMP) of the silkmoth Bombyx mori bearing similarities with dipteran VMPs whose existence had recently been suggested by an in silico analysis of the silkmoth genome and follicular cell RNA expression analyses. Using a specific antibody, we determine the presence of BmVMP90 protein in ovarian follicular cell extracts at the end of vitellogenesis and in vitelline membrane extracts but not in the chorion of fractionated eggshells isolated from ovulated follicles. Whole mount follicle immunofluorescence studies reveal a pattern of BmVMP90 deposition matching the ?imprinted? pattern of follicular cells on the vitelline membrane surface. Antisense DNA-directed inhibition BmVMP90 expression in ex?vivo cultures of early vitellogenic follicles produced a phenotype of kidney- or bean-shaped follicles with detached follicular epithelia, suggestive of the importance of BmVMP90 for the integrity of developing follicles and normal deposition of the chorion structure that follows vitelline membrane formation but no adverse effects on the execution of the follicular cell-imprinted program of choriogenesis per se.     

SPECIFIC PROTEIN SYNTHESIS IN CELLULAR DIFFERENTIATION : Production of Eggshell Proteins by Silkmoth Follicular Cells

Silkmoth follicles, arranged in a precise developmental sequence within the ovariole, yield pure and uniform populations of follicular epithelial cells highly differentiated for synthesis of the proteinaceous eggshell (chorion). These cells can be maintained and labeled efficiently in organ culture; their in vitro (and cell free) protein synthetic activity reflects their activity in vivo. During differentiation the cells undergo dramatic changes in protein synthesis. For 2 days the cells are devoted almost exclusively to production of distinctive chorion proteins of low molecular weight and of unusual amino acid composition. Each protein has its own characteristic developmental kinetics of synthesis. Each is synthesized as a separate polypeptide, apparently on monocistronic messenger RNA (mRNA), and thus reflects the expression of a distinct gene. The rapid changes in this tissue do not result from corresponding changes in translational efficiency. Thus, the peptide chain elongation rate is comparable for chorion and for proteins synthesized at earlier developmental stages (1.3–1.9 amino acids/sec); moreover, the spacing of ribosomes on chorion mRNA (30–37 codons per ribosome) is similar to that encountered in other eukaryotic systems.     

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).