Eggshell Assembly Research Articles

Comparative Transcriptomic Analysis Revealing the Potential Mechanisms of Erythritol-Caused Mortality and Oviposition Inhibition in Drosophila melanogaster.

Erythritol has shown excellent insecticidal performance against a wide range of insect species, but the molecular mechanism by which it causes insect mortality and sterility is not fully understood. The mortality and sterility of Drosophila melanogaster were assessed after feeding with 1M erythritol for 72 h and 96 h, and gene expression profiles were further compared through RNA sequencing. Enrichment analysis of GO and KEGG revealed that expressions of the adipokinetic hormone gene (Akh), amylase gene (Amyrel), α-glucosidase gene (Mal-B1/2, Mal-A1-4, Mal-A7/8), and triglyceride lipase gene (Bmm) were significantly up-regulated, while insulin-like peptide genes (Dilp2, Dilp3 and Dilp5) were dramatically down-regulated. Seventeen genes associated with eggshell assembly, including Dec-1 (down 315-fold), Vm26Ab (down 2014-fold) and Vm34Ca (down 6034-fold), were significantly down-regulated or even showed no expression. However, there were no significant differences in the expression of three diuretic hormone genes (DH44, DH31, CAPA) and eight aquaporin genes (Drip, Big brain, AQP, Eglp1, Eglp2, Eglp3, Eglp4 and Prip) involved in osmolality regulation (all p value > 0.05). We concluded that erythritol, a competitive inhibitor of α-glucosidase, severely reduced substrates and enzyme binding, inhibiting effective carbohydrate hydrolysis in the midgut and eventually causing death due to energy deprivation. It was clear that Drosophila melanogaster did not die from the osmolality of the hemolymph. Our findings elucidate the molecular mechanism underlying the mortality and sterility in Drosophila melanogaster induced by erythritol feeding. It also provides an important theoretical basis for the application of erythritol as an environmentally friendly pesticide.

Proteomic analysis of quail calcified eggshell matrix: a comparison to chicken and turkey eggshell proteomes.

BackgroundEggshell mineralization in commercially important species such as chicken, turkey or quail is of interest as a general model of calcium carbonate biomineralization. Knowledge of proteins and molecular mechanisms in eggshell assembly may also pave the way to manipulation of thickness of the calcified layer or other features. Comparison of eggshell matrix proteomes of different species may contribute to a better understanding of the mineralization process. The recent publication of the quail genome sequence now enables the proteomic analysis of the quail shell matrix and this comparison with those of chicken and turkey.ResultsThe quail eggshell proteome comprised 622 identified proteins, 311 of which were shared with chicken and turkey eggshell proteomes. Forty-eight major proteins (iBAQ-derived abundance higher than 0.1 % of total identified proteome) together covered 94 % of total proteome mass. Fifteen of these are also among the most abundant proteins in chicken and turkey eggshell matrix. Only three proteins with a percentage higher than 1.0 % of the total had not previously been identified as eggshell matrix proteins. These were an uncharacterized member of the latexin family, an uncharacterized protease inhibitor containing a Kunitz domain, and gastric intrinsic factor. The most abundant proteins were ovocleidin-116, ovalbumin and ovocalyxin-36 representing approximately 31, 13 and 8 % of the total identified proteome, respectively. The major phosphoproteins were ovocleidin-116 and osteopontin. While osteopontin phosphorylation sites were predominantly conserved between chicken and quail sequences, conservation was less in ovocleidin-116.ConclusionsOvocleidin-116 and ovocalyxin-36 are among the most abundant eggshell matrix proteins in all three species of the family Phasianidae analyzed so far, indicating that their presently unknown function is essential for eggshell mineralization. Evidence for other chicken eggshell-specific proteins in quail was inconclusive. Therefore measurement of additional eggshell proteomes, especially from species of different families and preferentially from outside the order Galliformes, will be necessary.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-015-0078-1) contains supplementary material, which is available to authorized users.

Hierarchical assembly of the eggshell and permeability barrier in C. elegans

In metazoans, fertilization triggers the assembly of an extracellular coat that constitutes the interface between the embryo and its environment. In nematodes, this coat is the eggshell, which provides mechanical rigidity, prevents polyspermy, and is impermeable to small molecules. Using immunoelectron microscopy, we found that the Caenorhabditis elegans eggshell was composed of an outer vitelline layer, a middle chitin layer, and an inner layer containing chondroitin proteoglycans. The switch between the chitin and proteoglycan layers was achieved by internalization of chitin synthase coincident with exocytosis of proteoglycan-containing cortical granules. Inner layer assembly did not make the zygote impermeable as previously proposed. Instead, correlative light and electron microscopy demonstrated that the permeability barrier was a distinct envelope that formed in a separate step that required fatty acid synthesis, the sugar-modifying enzyme PERM-1, and the acyl chain transfer enzyme DGTR-1. These findings delineate the hierarchy of eggshell assembly and define key molecular mechanisms at each step.

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 (CX 7CX 8C). 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.

Building up the Drosophila eggshell: First of all the eggshell genes must be transcribed

The Drosophila eggshell provides a model system for studying the assembly of extracellular matrix. Eggshell formation is a complex process that requires time-coordinated synthesis, cleavage, and transport of various proteins and finally cross-linking mediated by particular functional domains. It has been suggested that the eggshell can act as a storage site for spatial cues involved in embryonic pattern formation. Its structural components are synthesized in the somatic follicle cells in a precise temporally and spatially regulated manner. This review will summarize our knowledge of eggshell gene expression. We will discuss the amplification of the chorion gene clusters and the data acquired on the expression patterns and the regulatory elements controlling transcription of eggshell genes. We will then focus on the findings that correlate follicular epithelium patterning and eggshell gene expression, and discuss the interesting perspectives of an involvement in eggshell assembly of embryonic patterning cues.

A Mutant dec-1 Transgene Induces Dominant Female Sterility in Drosophila melanogaster

The Drosophila dec-1 gene produces three proproteins required for female fertility and eggshell assembly. The three proproteins are distinguished by their C termini. Fc106, the most abundant proprotein, is cleaved within the vitelline membrane to three mature derivatives in a developmentally regulated manner. To define sequences within fc106 that are critical for its function, we created wild-type and mutant versions of an fc106 cDNA transgene. The functional consequences of the mutations were assessed in dec-14, a female-sterile splicing mutant that does not produce the fc106 isoform. The fertility of dec-14 females was restored by the introduction of either a wild-type transgene or a transgene bearing a C-terminal deletion that included fc106-specific sequences. Surprisingly, the removal of internal coding sequences created an aberrant DEC-1 proprotein that induced female sterility when introduced into wild-type flies. Dominant female sterility was not associated with larger deletions that included the fc106 N terminus, suggesting that abnormal juxtaposition of N- and C-terminal sequences in the aberrant proprotein interfered with endogenous DEC-1 proteins. Changes in the fractionation behavior of the endogenous fc106 C-terminal derivative, s60, and morphological changes in the endochorion in response to expression of the aberrant proprotein support this interpretation.

Egfr signaling modulates VM32E gene expression during Drosophila oogenesis

Drosophila vitelline membrane gene VM32E is expressed in the follicle cells of the stage 10 egg chamber and shows a peculiar temporal and spatial expression pattern compared to the other members of the same gene family. Previous work has led us to demonstrate that Decapentaplegic (Dpp) signaling represses the expression of the VM32E gene in the centripetal follicle cells. In this paper, we describe another level of complexity of the VM32E gene expression regulation. Through clonal analyses, we show that the expression of the VM32E gene in the main body follicle cells is modulated by the epidermal growth factor receptor (Egfr) activity. In follicle cell clones expressing a constitutively active form of the Egfr, the VM32E gene is downregulated, while the loss of the Egfr activity upregulates VM32E expression. In addition, we show that the ectopic expression of the Egfr-induced ETS transcription factor PointedP2 (PntP2) affects the expression of the VM32E gene. From these results and our previously published data, it appears that the proper patterning of follicle cells, defined by Dpp and Egfr signaling pathways, controls the VM32E gene expression pattern. This may suggest that a fine tuning of the expression of specific eggshell structural genes could be part of the complex process that leads to a proper eggshell assembly.

Minor proteins and enzymes of the Drosophila eggshell matrix

The Drosophila eggshell provides an in vivo model system for extracellular matrix assembly, in which programmed gene expression, cell migrations, extracellular protein trafficking, proteolytic processing, and cross-linking are all required to generate a multi-layered and regionally complex architecture. While abundant structural components of the eggshell are known and are being characterized, less is known about non-abundant structural, regulatory, and enzymatic components that are likely to play critical roles in eggshell assembly. We have used sensitive mass spectrometry-based analyses of fractionated eggshell matrices to validate six previously predicted eggshell proteins and to identify eleven novel components, and have characterized the expression patterns of many of their mRNAs. Among these are several putative structural or regulatory (non-enzymatic) proteins, most larger in mass than the major eggshell proteins and often showing preferential expression in follicle cells overlying specific structural features of the eggshell. Of particular note are the putative enzymes, some likely to be involved in matrix cross-linking (two yellow family members previously implicated in eggshell integrity, a heme peroxidase, and a small-molecule oxidoreductase) and others possibly involved in matrix proteolysis or adhesion (proteins related to cathepsins B and D). This work provides a framework for future molecular studies of eggshell assembly.

Fc177, a minor dec-1 proprotein, is necessary to prevent ectopic aggregation of the endochorion during eggshell assembly in Drosophila

The Drosophila eggshell is a highly specialized extracellular matrix that forms between the oocyte and the surrounding epithelial follicle cells during late oogenesis. The dec-1 gene, which is required for proper eggshell assembly, produces three proproteins that are cleaved within the vitelline membrane layer to multiple derivatives. The different spatial distributions of the cleaved derivatives suggest that they play distinct roles in eggshell assembly. Using extant dec-1 mutations in conjunction with genetically engineered dec-1 transgenes, we show that, although all three dec-1 proproteins, fc106, fc125, and fc177, are required for female fertility, gross morphological abnormalities in the eggshell are observed only in the absence of fc177. The coalescence of the roof, pillar, and floor substructures of the tripartite endochorion suggested that quantitatively minor fc177 derivatives are necessary to prevent ectopic aggregation of endochorion proteins during the assembly process. Expression of a fc177 cDNA in dec-1 null mutants was sufficient to restore spaces within the endochorion layer. Fc177 may function as a scaffolding protein akin to those utilized in viral morphogenesis.

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.

Regulated Processing ofdec-1Eggshell Proteins inDrosophila

TheDrosophila dec-1gene encodes multiple products that are necessary for proper eggshell assembly. During stages 9–12 of oogenesis the ovarian follicle cells synthesize three alternatively spliceddec-1RNAs 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 thedec-1proteins 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 eachdec-1protein.

Evolution of thedec-1 eggshell locus inDrosophila. II. Intraspecific DNA sequence analysis reveals length mutations in a repetitive region inD. melanogaster

The dec-1 eggshell gene in Drosophila melanogaster encodes follicle cell proteins required for proper eggshell assembly. As shown by Southern and Northern analyses the dec-1 gene occurs in four alleles (Fc1-4) among wild-type strains. Its second exon has a distinct feature in the form of 12 repeats with 78-91 nucleotides; the first five show nearly 100% homology. DNA sequence comparison of the repeated region of the alleles revealed that the length polymorphisms are caused by changes in the numbers of the first five repeats. The results suggest that the alleles have been generated by unequal intragenic crossing-over and/or slippage during DNA replication and that the allelic length variants have arisen independently. The possibility that the most common allele, FC1, has a selective advantage over the other alleles is discussed.

Biomineralization and Eggshells: Cell-Mediated Acellular Compartments of Mineralized Extracellular Matrix

Publisher Summary This chapter summarizes cell biology, morphological organization, crystallography, chemical composition, process of mineralization, and biological function of avian eggshells. It also discusses emerging concepts of biomineralization and speculates about the mechanism of eggshell assembly and its implications for fabrication of polymer-ceramic composites. The eggshell is a microenvironmental compartment for housing developing embryos of a number of species. This unique microenvironment provides physical protection to the embryo and regulates gas, water, and ionic exchange. The avian eggshell can be characterized as a multilayered, polymer–ceramic composite. The three main layers include an outer mucous layer, an intermediate calcified zone, and an inner fibrous membrane layer. The shell membranes are the most internal layer of the eggshell and are formed by two nonmineralized fibrillar sublayers, the outer membrana testae externa, and the inner membrana testae interna or putaminis. The most external layer of the eggshell is referred to as the cuticle. This proteinaceous layer covers the entire calcified portion of the shell to a depth of about 10 μ m. The complementary use of biological, chemical, and crystallographic approaches demonstrates that the avian eggshell is a very promising model for the study of biomineralization. Avian eggshell is one of the most rapidly mineralizing biological systems known.

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.

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.