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Keywords:

  • Drosophila;
  • oogenesis;
  • eggshell;
  • gene expression;
  • gene amplification;
  • transcriptional control

Abstract

  1. Top of page
  2. Abstract
  3. OVERVIEW OF DROSOPHILA OOGENESIS
  4. EGGSHELL MORPHOLOGY
  5. EXPRESSION IN TIME AND SPACE OF EGGSHELL GENES
  6. ELEMENTS CONTROLLING THE EXPRESSION OF EGGSHELL GENES
  7. PERSPECTIVES
  8. Acknowledgements
  9. REFERENCES

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. Developmental Dynamics 237:2061–2072, 2008. © 2008 Wiley-Liss, Inc.


OVERVIEW OF DROSOPHILA OOGENESIS

  1. Top of page
  2. Abstract
  3. OVERVIEW OF DROSOPHILA OOGENESIS
  4. EGGSHELL MORPHOLOGY
  5. EXPRESSION IN TIME AND SPACE OF EGGSHELL GENES
  6. ELEMENTS CONTROLLING THE EXPRESSION OF EGGSHELL GENES
  7. PERSPECTIVES
  8. Acknowledgements
  9. REFERENCES

In Drosophila melanogaster, the events of oogenesis take place in egg chambers, which consist of the oocyte and 15 nurse cells, surrounded by a monolayer of approximately 1,000 follicle cells. Based on the morphology of the maturing egg chamber, oogenesis has been divided into 14 stages (Fig. 1) (King,1970; Spradling,1993). Each ovary of an adult female is composed of 14–16 ovarioles, each containing a string of developing egg chambers of progressive age. The ovariole is considered the morphological and functional unit of the ovary and is structurally divided into two different regions: a proximal one, called vitellarium, where egg chambers develop into mature eggs, and a distal one, called germarium, characterized by the presence of germline and somatic stem cell populations. In the germarium, each germline stem cell divides asymmetrically to renew itself and to produce a cystoblast that undergoes four mitotic divisions, each with incomplete cytokinesis, to produce a germline cyst of 16 cystocytes interconnected by ring canals. One of the two cells originating from the first mitotic division will develop as the oocyte, while the other 15 will develop as accessory nurse cells (Lin and Spradling,1993). Nearby, somatic stem cells give rise to precursor follicle cells and about 16 of them invade between adjoining cysts, cease division, and become pre-polar cells, which ultimately become polar cells and stalk cells. Inward migration of polar, stalk, and epithelial cells separate individual germline cysts into discrete egg chambers (Horne-Badovinac and Bilder,2005). As the cyst exits the germarium, the other somatic cells covering each chamber, the epithelial follicle cells, remain undifferentiated. Follicle cells that surround the egg chamber undergo mitotic divisions to follow the increase in size of the germline cells. By stage 7, the epithelial follicle cells cease proliferation and enter endocycles, a change in cell cycle triggered by Notch signalling (Shcherbata et al.,2004; Horne-Badovinac and Bilder,2005). Afterwards, these cells begin to show morphological and molecular signs of differentiation into the five main epithelial fates: border, stretched, centripetal, posterior, and main body follicle cells (Fig. 2).

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Figure 1. Stages of egg chamber development. Confocal cross-sections of wild type egg chambers. Staging of Drosophila egg chambers is facilitated by labelling with FITC-phalloidin (green) that reveals F-actin cytoskeleton and with nuclear dye Propidium Iodide (red). A high magnification of the germarium and of a stage-1 egg chamber is shown (top left). During mid-oogenesis (stages 9–11), the rearrangement of follicle cells and the increasing size of the oocyte become evident. In the late stages (stages 12–14), oocyte growth continues and the nurse cells, after carrying out their function, undergo death. germ., germarium; st., stage.

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Figure 2. Schematic view of different cell populations in the stage-10B egg chamber.

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Each of these follicle cell subpopulations has a specific function with respect to the production of a mature egg, such that the correct number and position of each type is critical to ultimate egg morphogenesis. These functions influence the production of structures that are essential to the egg, such as the dorsal respiratory appendages and the micropyle. These functions are also critical for proper anterior-posterior organization of the oocyte and, therefore, also for the resulting embryo (Xi et al.,2003). Follicle cells form a cuboidal epithelium until stage 8, but at the beginning of stage 9 they reorganize in a series of migrations. The 6–10 anterior-most follicle cells, the border cells, migrate through the nurse cells to the oocyte anterior end. Another 50 anterior cells, the stretched cells, form a flattened epithelium overlying the nurse cells. Polar cells reside at the anterior and posterior termini of egg chamber and organize follicle cell fates throughout oogenesis. They provide cues for axial patterning of the embryo, create a porthole for sperm entry, and organize the specialized eggshell domains thought to enhance embryonic survival (Beccari et al.,2002; Xi et al.,2003; Grammont and Irvine,2002). Coincidently with these events in the soma, the oocyte itself is busy re-arranging its polarity. At stage 6, the posterior terminal follicle cells differentiate upon Epidermal Growth Factor Receptor (Egfr) activation, and then produce an as-yet unidentified feedback signal to the germline. A consequence of this feedback is that the posterior oocyte microtubule organizing centre (MTOC) breaks down and a new one forms at the anterior pole, thus reversing the polarity of the oocyte's microtubule network.

Accompanying this process is a movement of the germinal vesicle away from the posterior pole to localize at the anterior corner of the oocyte, where it activates the Egfr once more to establish the polarity of the dorsal ventral axis (Lopez-Schier,2003). During oogenesis, nurse cells also undergo nuclear changes, which involve a series of endoreplication cycles that increase their polyploid values (from 512 to 2,048). Nurse cells develop a complex microtubular apparatus organized in ring canals through which they transfer to the oocyte mRNA, proteins, organelles, and lipidic droplets, contents that are important for the correct development of the future embryo. During early stages of oogenesis, this transfer is slow but continuous and increases strongly at stage 10B. From stage 10B to stage 12, nurse cells transfer all their cytoplasmic contents to the oocyte and finally undergo apoptosis (Cavaliere et al.,1998; Foley and Cooley,1998). At stage 14 of oogenesis, after the construction of eggshell, the follicle cells die by apoptosis (Nezis et al.2002).

EGGSHELL MORPHOLOGY

  1. Top of page
  2. Abstract
  3. OVERVIEW OF DROSOPHILA OOGENESIS
  4. EGGSHELL MORPHOLOGY
  5. EXPRESSION IN TIME AND SPACE OF EGGSHELL GENES
  6. ELEMENTS CONTROLLING THE EXPRESSION OF EGGSHELL GENES
  7. PERSPECTIVES
  8. Acknowledgements
  9. REFERENCES

The Drosophila eggshell is an extracellular structure functional to the different roles it absolves, from egg fertilization to the hatching of the larva at the end of embryogenesis (Margaritis,1985; Margaritis and Mazzini,1998; Waring,2000). This framework is laid down during late oogenesis between the oocyte and overlying follicle cells. The eggshell proteins, secreted by the follicle cells, assemble into a highly organized structure featuring both radial and regional complexity. This eggshell specialization depends on sequential and in some cases combined activities of the known major signaling pathways during the middle and late stages of oogenesis. Five morphologically distinct layers have been identified in the Drosophila eggshell: an oocyte proximal vitelline membrane (approximately 300 nm), a lipid wax layer, an inner chorion layer (40–50 nm), an endochorion (500–700 nm), and an exochorion (300–500 nm) (Fig. 3). The vitelline membrane is the first layer to be made up by follicular epithelium and appears as a continuous granular layer without prominent substructures. Its deposition begins during stage 9, when the vitelline membrane proteins appear accumulated on the surface of the oocyte in small vesicles called vitelline bodies. At stage 10B, these vesicles fuse to form a continuous thick layer, which gradually thins down as oogenesis proceeds (Margaritis et al.,1980; Margaritis,1985). Deposition of the wax layer begins in late stage 10 with the accumulation of lipid-filled vesicles between follicle cells and vitelline membrane. Synthesis and secretion of lipid vesicles proceed through stage 12. As the lipid vesicles accumulate on the vitelline membrane surface, they take on a flat appearance. During stage 14, the vesicles are compressed into three to four planes of overlapping plaques, which create a water-impermeable layer between the vitelline membrane and chorion (reviewed in Waring,2000). The inner chorion layer is also continuous and characterized by periodic structure. At the end of choriogenesis the inner chorion layer acquires its final thickness and unique crystalline substructure. Three-dimensional reconstructions reveal that the crystalline inner chorion layer consists of two types of subunits, which appear to be grouped into an octamer of four dimer pairs (Margaritis et al.,1984). Ultrastructural analyses of stage 14 egg chambers and isolated endochorions reveal the tripartite nature of this layer (Margaritis et al.,1980). A thin fenestrated floor is separated from a thick outer roof layer by vertical pillars creating cavities that facilitate gas exchange (Fig. 3). The continuous outer roof network displays ridges and it defines the borders of the follicle cell imprints. The imprints in the main body of the eggshell are fairly uniform, with those on the dorsal side being more elongate than those found ventrally. Freeze-fractured views of endochorion reveal globular structures interconnected via fine fibrils (Margaritis and Mazzini,1998). The exochorion consists of loose fibers that tend to be oriented parallel to the oocyte surface. It usually appears to consist of two layers, the innermost being less electron dense.

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Figure 3. Eggshell morphology. A: Schematic view of a multilayered eggshell as seen in the central main body region. The inner layers include the vitelline membrane (VM), the wax layer (WL), and the inner chorion layer (ICL). The outer layers include the endochorion (En), where a thin fenestrated floor (F) is separated from an outer roof (R) by vertical pillar (P) creating cavities as indicated, and the exochorion (Ex). B: Schematic structure of Drosophila egg illustrating the specialized regions of the shell. The prominent structures indicated are the micropylus (Mi), the operculum (Op), and the dorsal appendages (DA). Aeropyle (Ae) and collar (Co) are also indicated.

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Like most insect eggs, several regional specializations are apparent on the surface of the Drosophila eggshell. Its anterior end is characterized by a nearly flat plate called operculum, a specialized region that has evolved to facilitate hatching of the larva at the end of embryogenesis. Its anterior and lateral margins are called the collar region and mark an abrupt transition to the follicle cell imprints found on the ventral side of the main shell (Margaritis et al.,1984; reviewed in Waring,2000). The operculum is flanked by the micropyle at its anterior end and two long appendages at its posterior end (Fig. 3). The micropylar apparatus is a protuberance through which sperm gains access to the oocyte membrane. Its physical dimensions are believed to provide a block to polyspermy by restricting passage to a single sperm (Perotti,1974). Formation of micropyle begins in stage-10 egg chambers with the secretion of a paracrystalline region of the vitelline membrane by the border cells and is completed at stage 14 with the degeneration of border cells giving rise to the micropylar canal. The final result is an open-ended and hollow cone into which a protuberance of the vitelline membrane fits (Margaritis,1984; Margaritis et al.,1980). By serving as gills, the respiratory appendages on either side of the dorsal midline allow eggs to carry out gas exchange when they are submerged under water. These appendages are long and largely cylindrical, but slightly paddle-shaped at the tip. These structures have two distinct surfaces: a ventral one that consists of a continuous fine network and a dorsal one with isolated and porous plaques that cover the main area. The two sides of each appendage are interconnected via an extensive network of modified pillars with air spaces. Each appendage starts to be formed at stage 11 by a population of 65–70 columnar follicle cells, which migrate out over the stretched cells on either side of the dorsal midline. The follicle cells then spread out to form the flat paddle structure (Rittenhouse and Berg,1995). In the posterior pole, a group of approximately 20–30 follicle cells leave imprints distinctly smaller than those in the main body. Here, two nested rings of distinct cell imprints form the aeropyle (Fig. 3) (Margaritis et al.,1980; Dobens and Raftery,2000) a specialized region thought to be also involved in gas exchange.

EXPRESSION IN TIME AND SPACE OF EGGSHELL GENES

  1. Top of page
  2. Abstract
  3. OVERVIEW OF DROSOPHILA OOGENESIS
  4. EGGSHELL MORPHOLOGY
  5. EXPRESSION IN TIME AND SPACE OF EGGSHELL GENES
  6. ELEMENTS CONTROLLING THE EXPRESSION OF EGGSHELL GENES
  7. PERSPECTIVES
  8. Acknowledgements
  9. REFERENCES

Timing of Gene Expression

Precise timing of synthesis of different eggshell components is relevant for the ordered assembly of the five layers and it relies on a fine regulation of gene expression (Waring,2000). The genes encoding major eggshell structural proteins are transcribed in follicle cells during stages 8–14 of oogenesis in a well-defined temporal order (Table 1) (King,1970; Spradling,1993; Waring,2000). The vitelline membrane genes are mainly expressed during mid-oogenesis stages 8–10, while the chorion genes are transcribed from stage 11 onward. The chorion synthesis, which occurs in the last 5–6 hours of oogenesis, requires both rapid production of large amounts of protein as well as fine control over the timing of gene expression. These requirements are met in two ways: (1) by amplification of the two chorion gene clusters and (2) by precise transcriptional control of the individual chorion genes.

Table 1. Timing of Major Eggshell Gene Expression During Oogenesisa
GeneStage 8Stage 9Stage 10Stage 11Stage 12Stage 13Stage 14
  • a

    The different gene transcripts were described in: Mindrinos et al. (1985), Burke et al. (1987), Popodi et al. (1988), Jin and Petri (1993) (VM26A.1, VM26A.2, and VM34C), Gigliotti et al. (1989) (VM32E), Parks et al. (1986), Parks and Spradling (1987) (s36 and s38), Griffin-Shea et al. (1982), Parks and Spradling (1987) (s16, s19, s15, and s18). +++, maximum transcript level.

VM26A.1+++++++   
VM26A.2++++++   
VM34C++++++    
VM32E  +++    
s36  +++++++++
s38  +++++++++
s16   ++++++++
s19  +++++++
s15     ++++++
s18   ++++++++

Developmental control of eggshell gene expression relies in some instances on transcription of closely linked genes. Four vitelline membrane structural components are located on the left arm of the second chromosome. Their names refer to the map location on the polytene chromosome: VM32E, VM34C, VM26A.1, and VM26A.2 (Higgins et al.,1984; Mindrinos et al.,1985; Burke et al.,1987; Popodi et al.,1988; Gigliotti et al.,1989). While VM32E and VM34C are isolated, VM26A.1 and VM26A.2 appear to be clustered with other putative vitelline membrane genes (Popodi et al.,1988). Major chorion genes are set in two clusters, respectively, located at region 7F on the X chromosome (s36 and s38; Parks et al.,1986) and at region 66D on the third chromosome (s15, s16, s18, and s19; Spradling,1981; Griffin-Shea et al.,1982). Transcripts from genes s36 and s38 of the X-linked chorion cluster appear during early stages of chorion formation at stage 11, reach a peak at stage 12 and 13, and decline at stage 14 (Parks et al.,1986). Transcripts encoded by genes s15, s16, s18, and s19 of the third chromosome cluster accumulate at various overlapping late periods, mostly at stages 13 and 14 (Griffin-Shea et al.,1982). According to the choriogenic stages at which their corresponding genes are mainly expressed in the follicle cells, the major chorion proteins are designated as developmentally early (s38, s36), middle (s19, s16), and late (s18, s15). However, each gene has a unique mRNA accumulation profile indicating that temporal control is gene specific (Griffin-Shea et al.,1982; Parks et al.,1986). For example, already at stage 10 very low levels of s36 and s38 chorion genes can be detected by northern blot analysis (Parks and Spradling,1987). An earlier expression at stage 10 was also found for the s19 chorion gene but not for s16 (Griffin-Shea et al.,1982). In addition to these chorion genes, other genes have been identified that encode minor chorion components (Waring,2000). Among these, the Femcoat protein is specifically expressed in the follicle cells at late stages and it is required for endochorion formation (Kim et al.,2002). In a recent effort to find unknown proteins playing relevant structural or regulatory roles in eggshell biogenesis, 11 new distinct proteins have been identified as structural components of the eggshell (Fakhouri et al.,2006). Among these three are putative vitelline membrane proteins, seven are putative chorion components and one is a novel protein. The genes encoding the putative vitelline membrane proteins (CG9050, CG13992, and CG13997) map to the 26A region and are immediately adiacent to the VM26A.1 and VM26A.2 coding regions. Three of the putative chorion components (CG11381, CG14796, and CG15570) are expressed in a temporal pattern overlapping that of the early chorion genes. The proteins encoded by the CG14796 and CG15570 genes do not show any structural relationship with known proteins while the CG11381 product, a glutamine-rich protein, may correspond to the minor chorion protein s70. Two other putative chorion components (CG13083 and CG13084) are expressed in the same pattern of intermediate and late chorion genes. The two last predicted chorion proteins idientified (CG15350 and CG33962) are encoded by predicted genes mapping near the s36 and s38 early chorion genes. Finally, a small proline-rich protein was identified as putative component of the eggshell (CG13114).

The timing of eggshell protein synthesis is not always related to the final position of the proteins in the eggshell. Indeed, after their secretion the eggshell components could undergo trafficking between layers. Therefore, classifying them either as vitelline membrane or chorion proteins is not always clear. The highly dynamic state shown by some eggshell components, reflected by their trafficking between layers, appears evident by analyzing the VM32E protein (Andrenacci et al.,2001). At the time of its synthesis (stage 10), VM32E protein is not detectable in anterior and posterior follicle cells. However, it is able to diffuse in the extracellular space around the oocyte, and by stage 11 it is evenly distributed in the vitelline membrane. Moreover, as assessed by immunoelectron microscopy, at late stages of oogenesis VM32E protein is partially released from the vitelline membrane and becomes included in the endochorion layer too. A detailed functional analysis of the different VM32E domains showed that the C-terminal domain is required for this partial relocalization of the VM32E protein. Another example is provided by the early chorion protein s36, which initially localizes mainly in the vitelline membrane layer and, at late stages, it becomes distributed throughout the endochorion (Pascucci et al.,1996). Although s36 is described as a chorion protein and its mutant as producing egg chambers that lack endochorion organization (Digan et al.,1979), the mutant eggs show defects also in the vitelline membrane assembly (Cernilogar et al.,2001). This could imply that s36 protein might represent a structural component of both chorion and vitelline membrane layers. Cleavage and relocalization of eggshell components have been documented also for some products of the dec-1 locus (Hawley and Waring,1988). Stage-specific alternative RNA processing gives rise to three dec-1 transcripts, fc125, fc177, and fc106, encoding proteins with different C-terminal ends (Waring et al.,1990). The fc106 isoform is processed in the vitelline membrane giving rise to some products that remain in the vitelline membrane and other products that relocalize to the chorion (Nogueron et al.,2000). Similarly, the processing of the late isoform fc177 in the vitelline membrane results in products that move into the chorion or to the oocyte cytoplasm.

Chorion Gene Amplification

To compensate for the limited time for maximal expression, chorion genes undergo selective amplification in order to meet production of massive amounts of chorion proteins (Parks and Spradling,1987). The degree of amplification differs between the two chorion gene clusters. The X chromosome cluster is amplified ∼16-fold, while the third chromosome cluster undergoes ∼60 rounds of amplification (Delidakis et al.,1989; Orr-Weaver,1991). Besides these clusters, two other developmental amplicons have been isolated. These are localized, respectively, at 30B and 62D and are ampliflied to a less extent. Although they do not contain chorion genes, these sites include genes that are expressed in the follicle cells during late oogenesis and could probably function in eggshell formation (Claycomb et al.,2004).

Chorion gene amplification is a complex process that relies on both cis- and trans-acting elements (Fig. 4). Information on cis-acting elements come from studies on the third chromosome cluster. 2D gel analysis revealed that amplification of the third chromosome cluster uses three DNA replication origins, called ori-α, ori-β, and ori-γ, with a preference for ori-β (Delidakis and Kafatos,1989; Heck and Spradling,1990). Besides replication origins, other regions are required for attainment of high levels of amplification, as assessed by various analyses. Indeed, using different approaches, a 320-bp region required for high levels of amplification, called Amplification Control Element third chromosome (ACE3), and four distinct stimulatory regions, called Amplification Enhancing Regions (AER) (de Cicco and Spradling,1984; Delidakis and Kafatos,1989; Orr-Weaver et al.,1989), were identified. Testing amplification efficiency of constructs carrying different regions of the third chromosome cluster flanked by insulator elements proved that both ori-β and ACE3 are necessary and sufficient for amplification (Lu et al.,2001).

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Figure 4. Schematic organization of the third chromosome chorion gene cluster. Major (ori-β) and minor (ori-α and ori-γ) DNA replication origins (brackets) and cis-regulatory elements (box and solid bars) required for amplification are indicated in relation to the chorion genes (arrows). Bottom: An enlargement of the ACE3 region with the binding sites for Myb and Mip120 proteins.

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Genetic analysis led to the identification of various trans-acting factors including key S-phase regulators, initiation factors, and proteins used for normal genomic replication (for a comprehensive review see Tower,2004). Mutations in these genes impair chorion gene amplification resulting in thin and fragile eggshells and thus nonviable eggs. A crucial step in chorion gene amplification is the localization of pre-replicative complex at amplification loci (pre-RC; Orc2, Cdt1, Mcm6; Landis et al.,1997; Whittaker et al.,2000; Schwed et al.,2002). Both in vitro and in vivo experiments demonstrated that the origin recognition complex (ORC) binds to ACE3 and ori-β sequences (Austin et al.,1999), suggesting that replication origins are characterized by certain sequence requirements. However, quantitative analysis of ORC binding suggested that DNA topology might also be an important factor. Indeed, purified ORC has a 30-fold higher affinity for negative supercoiled DNA, which can be generated in the cell by removal of histones with chromatin remodeling complexes (Remus et al.,2004). Supporting data to this hypothesis came from chromatin immunoprecipitation experiments showing that Myb and Mip120 (Beall et al.,2002), components of the Myb complex, and E2f1 (Bosco et al.,2001), one of the two E2F subunits, bind to the chorion replication origin. As demonstrated by Fung et al. (2003), Myb interacts with chromatin-modifying complexes. Also E2f1 alters chromatin structure interacting with the tumor-suppressor and cell cycle regulator Rb, which recruits histone deacetylase complexes. In agreement with these results, nucleosomes are hyperacetylated at ACE3 and ori-β sequences (Aggarwal and Calvi,2004). More information on the dynamics of DNA replication at the chorion loci came from a study of Claycomb et al. (2002). Combining high-resolution confocal and deconvolution microscopy and quantitative realtime PCR, these authors revealed that ORC localizes to amplified regions only during amplification initiation, which extends from stage 10A to stage 11. During this process, ORC staining is coincident with BrdU incorporation. At stage 11, when initiation is completed and the elongation phase begins, ORC is no longer localized at origins and BrdU incorporation resolves in a double bar structure representing the bi-directional replication fork movement. The same pattern of BrdU incorporation is shown by the elongation factors PCNA and MCM2-7, which are loaded onto pre-RC by the double-parked protein DUP/Cdt1 and cell division cycle Cdc6/Cdc18. While replication forks move away from amplification origin, they create an onionskin-like DNA structure (Claycomb et al.,2002; Osheim et al.,1988; Spradling and Leys,1988). Since there are no obvious termination sites, amplified copy number falls off as a relatively smooth 40–100-kb bell curve centered over each locus (Claycomb et al.,2004; Orr-Weaver,1991; Spradling,1981). Recently Calvi et al. (2007), by visualizing BrdU incorporation during oogenesis, demonstrated that the amplification process begins at stage 10B in many Drosophila species, included Drosophila virilis, which diverged from Drosophila melanogaster at least 40 million years ago. They also showed that histone hyper acetylation and Orc2 localization at the amplifying loci are conserved, both being detectable in different species using specific antibodies. These results highlight the evolutionary conservation of at least some aspects of amplification and raise various questions that probably could be answered in coming years, thanks also to the genomic information now available for 11 sibling species.

Spatial Expression of Eggshell Genes

The Drosophila egg has a characteristic asymmetric shape and contains functionally important regional features that are constructed by subgroups of follicle cells. The existence of regional specializations suggests that eggshell gene expression is regulated spatially as well as temporally during oogenesis. Indeed, the spatial expression pattern of the different chorion genes has shown that while the genes of the third chromosome cluster (s15, s16, s18, and s19) are expressed throughout the follicular epithelium, the expression of X-linked chorion genes (s36 and s38) is initially detected in a small dorsal sub-population of stage 10B follicle cells. However, in later stages these genes are expressed in all follicle cells. In addition, some minor chorion genes are expressed in a more localized manner (Parks and Spradling,1987). Interestingly, among the putative chorion genes recently identified, several show preferential expression in follicle cells covering specific regions of the eggshell suggesting they encode components that could contribute to specific structural features of the eggshell (Fakhouri et al.,2006).

Since the vitelline membrane is made up of only a single eggshell layer distributed all around the egg membrane, the different vitelline membrane genes should have a similar expression pattern. The transcript of the VM26A.2 gene, encoding the most abundant component of the vitelline membrane, is detected in all follicle cells surrounding the oocyte from stage 8. The same expression pattern was observed for the VM26A.1 gene and for the CG9050 gene (Burke et al.,1987; Popodi et al.,1988, Fakhouri et al.,2006). Conversely, the VM32E gene shows a peculiar spatial transcriptional profile and its expression is restricted at stage 10 of oogenesis (Gargiulo et al.,1991). Its transcript is detected in the main body follicle cells and is absent in the anterior and posterior follicle cells. Within the main body follicle cells, the expression of VM32E is temporally regulated. The expression starts at stage 10A and it appears to spread from the ventral to the dorsal follicle cell domain. This unique expression pattern may be linked to some special functions that the VM32E protein would carry out during vitelline membrane assembly and/or in endochorion formation.

ELEMENTS CONTROLLING THE EXPRESSION OF EGGSHELL GENES

  1. Top of page
  2. Abstract
  3. OVERVIEW OF DROSOPHILA OOGENESIS
  4. EGGSHELL MORPHOLOGY
  5. EXPRESSION IN TIME AND SPACE OF EGGSHELL GENES
  6. ELEMENTS CONTROLLING THE EXPRESSION OF EGGSHELL GENES
  7. PERSPECTIVES
  8. Acknowledgements
  9. REFERENCES

cis-Acting Elements

Transcriptional regulation of vitelline membrane genes has been studied using different approaches.

Using transposon rescue experiments, Savant and Waring (1989) have reported that as little as 147 bp of the 5′ promoter region flanking VM26A.2 gene was capable of restoring fertility and wild type VM26A.2 protein levels in the fs(2)QJ42 mutant allele of VM26A.2. This result indicates that all the regulatory elements required to direct the proper expression of VM26A.2 gene are contained in a very short proximal region flanking the transcription initiation site. A fine promoter dissection analysis was carried out for VM26A.1 and VM32E genes indicating that the expression of these two genes in the follicular epithelium is composite and it results from a combination of DNA regulatory elements. The VM26A.1 promoter region has been dissected by Jin and Petri (1993). A DNA segment of 597 bp (−617/−20) of the proximal 5′ flanking DNA is sufficient to promote the specific expression of VM26A.1 gene. A developmental control element of 176 bp, capable of promoting the basic expression of the gene in the follicular epithelium, was found within this region. Moreover, additional control elements were found to be necessary for the high level and specific spatial pattern expression of VM26A.1 gene. A detailed analysis of the VM32E promoter region has been carried out (Gargiulo et al.,1991; Cavaliere et al.,1997; Andrenacci et al.,2000). The minimal regulatory regions that confer the proper temporal and spatial pattern of expression of this gene are contained within the −348/−39 region. A 74-bp proximal region (−112/−39) contains regulatory element(s) to lead the activation of the gene in the follicular epithelium when joined with two other cis-regulatory elements and is absolutely required for their output. The first element (−253/−113), individually unable to raise reporter expression, elicits gene activity in the ventral domain when joined to the proximal fragment; a second element (−348/−254) joined to the same proximal fragment sustains the full dorsal and ventral activity.

The bases of the developmental program regulating temporal chorion gene expression have been studied in detail for the s15 and s36 genes. Although chorion genes are part of clusters that are amplified, transformation experiments established that the s15 and s36 chorion genes can be expressed autonomously by the activity of their promoters when transferred to multiple chromosomal sites. This has allowed for dissection of the regulatory elements ruling temporal and spatial expression of these genes. The relevant regulatory elements controlling s15 chorion gene expression have been identified in a 73-bp segment (−118/−46) of the proximal 5′-flanking DNA. Within this region, at least three regulatory elements were found: an essential positive element (TCACGT) shared by s36 chorion gene and other chorion genes, irrespective of temporal specificity; a second positive element required for the normal late expression of s15 chorion gene; a negative element repressing precocious expression during early choriogenic stages (Mariani et al.,1988). Furthermore, a more detailed mutational analysis of the −189/−39 s15 promoter region has shown that it encompasses many positive and negative, at least partially degenerate, cis-regulatory elements, which are involved in specifying the highly precise expression pattern of the s15 gene during development (Mariani et al.,1996). The analysis of s36 chorion gene promoter has identified a 84-bp-long proximal regulatory region, PRR (−132/−49), sufficient to direct follicular expression of a reporter gene in a temporally and spatially correct pattern, mimicking that of the endogenous s36 gene (Tolias and Kafatos,1990). When the 84-bp segment is subdivided, the two halves lead to different, somewhat complementary staining patterns at the two poles of the follicle. Furthermore, a distal regulatory region, DRR (−1,213/−427), contains apparently redundant element(s) resulting in weak but exclusive expression at the anterior dorsal end of choriogenic follicles. Thus, s36 gene expression in the follicle cells is composite, resulting from a combination of cis-regulatory elements.

trans-Acting Elements

The existence of putative regulatory trans-acting factors responsible for coordinated chorion gene expression in follicle cells was identified by Shea et al. (1990). Two transcription factors, USP (originally named CF1) and CF2 that bind to the promoter of the s15 chorion gene, have been isolated. CF2 is a member of the C2H2 zinc finger protein family and its involvement in dorsoventral polarity of the egg chamber has been demonstrated (Hsu et al.,1996; Mantrova and Hsu1998), but its putative role on chorion gene regulation is still unknown. The USP factor, encoded by the ultraspiracle (usp) gene belongs to the nuclear receptor family of transcription factors and is homologous to the vertebrate RXR receptor (Henrich et al.1990; Oro et al.1990; Shea et al.1990). USP forms heterodimers with the Ecdysone Receptor (EcR). The ecdysone-EcR/USP ligand/receptor complex binds at Ecdysone Response Elements (EcREs) near responsive promoters to coordinate gene expression in diverse tissues (Riddiford et al.,2000). The USP binding site in the s15 promoter region overlaps with the conserved chorion hexamer TGACGT sequence. Although the in vivo functional significance of USP binding on chorion gene promoters has yet to be investigated, this may be related to a control of the ecdysone signaling on chorion gene expression. Supporting this hypothesis, Hackney et al. (2007) have recently shown that the EcR activity regulates chorion gene expression and amplification during eggshell formation.

More work must be carried out to identify the transcription factors controlling expression of different eggshell genes. The tissue specificity transcription of these genes may be under the control of factor(s) controlling the activity of more than one gene. In addition to the control of the ecdysone signaling on chorion gene expression, it has been reported also that the activity of the transcription factor Tramtrack69 (Ttk69) is required for chorion gene expression. Indeed, in homozygous females for the female sterile allele of tramtrack called twin peaks (ttktwk), the level of s36 and s15 and/or s18 chorion gene transcripts was reduced, but the amplification of chorion genes was not affected (French et al.,2003).

Furthermore, the complex expression profile displayed by some genes is achieved by the interplay of specific positive and negative transcription factors. Although these factors have yet to be identified, it has been suggested that they are controlled by the Egfr and Decapentaplegic (Dpp) signaling pathways. These two signaling pathways have been shown to function in follicle cell epithelium to pattern the eggshell (Deng and Bownes,1997; Dobens and Raftery,1998; Peri and Roth,2000; Price et al.,1989; Twombly et al.,1996). During oogenesis, the Egfr pathway establishes both the anterior-posterior and dorsoventral axes of the egg chamber. Axis specification requires spatially restricted activation of the Egfr in the somatically-derived follicular epithelium of the egg chamber by its germline specific ligand Gurken (Grk), a transforming growth factor-alpha (TGF-α) signaling molecule (Neuman-Silberberg and Schüpbach,1993,1996; Schüpbach,1987). During oogenesis, the egg chamber acquires a dorsoventral polarity when the asymmetrically localized Grk protein activates a gradient of Egfr activity resulting in the induction of distinct follicle cell fates along the dorsoventral axis. High levels of Grk induce dorsal follicle cell fates (Ray and Schüpbach,1996; Perrimon and Perkins,1997; Dobens and Raftery,2000), and activate both a positive and a negative feedback signaling leading to the definition of two separate populations of dorsal follicle cells that will guide the production of the two dorsal appendages (Wasserman and Freeman,1998; Peri et al.,1999). The most ventral follicle cells, which have the lowest level of Egfr activity, express the pipe gene, which determines the ventral follicle cell fate of the future embryo (Sen et al.,1998). Therefore, a gradient of Egfr activity along the dorsoventral axis determines the full range of follicle cell fates. The patterning of the eggshell along the anterior-posterior axis also requires Dpp signaling. At stage 10 of egg chamber development, Dpp is expressed in both the nurse cell–associated follicle cells and in centripetal cells, and is required for the formation of the specialized eggshell anterior regions. Indeed, the size and placement of the operculum and dorsal appendages are quite sensitive to altered levels of Dpp signal (Twombly et al.,1996). The complex spatial and temporal regulation adopted by the s36 and VM32E genes has suggested an involvement of the Egfr and/or Dpp signaling pathways in shaping their expression pattern. Tolias et al. (1993) have shown that spatial expression of the s36 chorion gene is influenced by the Egfr signalling. In wild type egg chambers, transcription of the s36 gene begins anteriorly at late stage 10B and becomes very strong in the anterior dorsal region at stage 11. This is followed by moderately strong but highly localized expression at the posterior pole, and then the expression spreads to the main body of the follicle at stages 12 and 13. By analyzing the lacZ reporter gene expression driven by the minimal s36 promoter region, Tolias and coauthors have shown that in Egfr mutant background the s36 promoter activity is affected. The activation of the reporter gene is delayed to stage 11, when the anterior end of the epithelium expresses the reporter gene as a uniform ring rather than predominantly on its dorsal side. Subsequently, the ring increases in intensity but without evident dorsal polarity.

The VM32E expression pattern is dependent on the Dpp and Egfr signaling pathways. By analyzing the native VM32E gene and the activity of specific VM32E regulatory regions in genetic backgrounds altering the Dpp pathway, it has been shown that the Dpp signaling pathway negatively controls VM32E gene expression in the centripetal follicle cells (Bernardi et al.,2006). The VM32E gene was ectopically expressed in centripetal follicle cell clones where the Dpp signaling was repressed. Within the main body follicle cells the temporal regulation of VM32E, gene expression, in which the gene is first active in the ventral follicle cells and later in the dorsal ones, is modulated by the Egfr signaling pathway (Bernardi et al.,2007). It has been shown that the VM32E gene is down-regulated in follicle cell clones expressing a constitutively active form of the Egfr while the loss of Egfr activity up-regulates VM32E expression. Therefore, it appears that the degree of VM32E gene expression depends on the level of Egfr activity.

PERSPECTIVES

  1. Top of page
  2. Abstract
  3. OVERVIEW OF DROSOPHILA OOGENESIS
  4. EGGSHELL MORPHOLOGY
  5. EXPRESSION IN TIME AND SPACE OF EGGSHELL GENES
  6. ELEMENTS CONTROLLING THE EXPRESSION OF EGGSHELL GENES
  7. PERSPECTIVES
  8. Acknowledgements
  9. REFERENCES

Genetic and molecular studies on eggshell genes have documented that their expression is based on a complex regulatory strategy, involving specific transcription factors and signaling pathways. This fine regulation is required for the developmental program leading to the proper eggshell assembly (Fig. 5). Moreover, much of the work carried out thus far has focused on identifying and characterizing the various eggshell proteins. Less is known about the function of specific eggshell components. Furthermore, the exquisite temporal and spatial regulation of some eggshell structural genes suggests a crucial role of their encoded proteins in the complex process of eggshell formation. Many aspects of eggshell biogenesis are still to be elucidated. Besides its structural contribution to eggshell formation, it has been suggested that the vitelline membrane may perform an important function for the localization of maternal signals. During oogenesis, the framework for early embryonic development is laid down. Follicle cells overlying the oocyte and germline cells interact critically to establish the polarity both of the egg chamber and the developing embryo (St Johnston and Nusslein-Volhard,1992; Gonzalez-Reyes et al.,1995; Ray and Schüpbach,1996). Spatial cues are crucial to patterning embryos during development. Some of these cues occur in the extracellular environment and may function in the activation or presentation of secreted signaling molecules. All the information for embryonic patterning that is derived from the follicular epithelium is stored in the perivitelline space, an extracellular compartment between the embryo plasma membrane and the vitelline membrane layer. The dorsoventral axis of the embryo is defined by a ventral signal that arises within the perivitelline space. It has been shown that nudel, one of the genes essential for dorsoventral development and expressed in follicle cells surrounding the oocyte, is required for eggshell formation (LeMosy and Hashimoto,2000), raising the possibility that spatial information for dorsoventral patterning may be stored in the vitelline membrane layer. In addition, it has been shown that fs(1)Nasrat and fs(1)polehole genes are required for vitelline membrane assembly (Cernilogar et al.,2001; Jimenez et al.,2002). These genes are needed for local activation of the Torso receptor tyrosine kinase that specifies head and tail structures in the early Drosophila embryo. Interestingly, it has been found that the Torsolike protein, which is synthesized in the follicle cells and is involved in Torso activation, is specifically localized to the polar regions of the vitelline membrane in laid eggs (Stevens et al.,2003). The incorporation of Torsolike into the vitelline membrane provides a mechanism for the transfer of spatial information from the follicle cells to the developing embryo. These findings open up very interesting issues of a linkage between eggshell assembly and embryonic patterning. The eggshell is stabilized by a progressive cross-linking process that renders its components largely insoluble. The chorion becomes insoluble during stage 14 as a result of a peroxidase-type enzyme activity that cross-links two or three tyrosine residues of the chorion proteins (Petri et al.,1976). At the time of its synthesis, the vitelline membrane is under a highly dynamic state (Andrenacci et al.,2001) and its proteins remain soluble till stage 14 and become insoluble only in laid eggs (reviewed by Waring,2000). The programmed late vitelline membrane hardening may allow the proper embedding into the vitelline membrane or the deposition of positional cues elaborated by the follicle cells into the perivitellinic space.

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Figure 5. Framework leading to the proper eggshell morphogenesis.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. OVERVIEW OF DROSOPHILA OOGENESIS
  4. EGGSHELL MORPHOLOGY
  5. EXPRESSION IN TIME AND SPACE OF EGGSHELL GENES
  6. ELEMENTS CONTROLLING THE EXPRESSION OF EGGSHELL GENES
  7. PERSPECTIVES
  8. Acknowledgements
  9. REFERENCES

We thank Tien Hsu for critical reading of the manuscript and helpful suggestions. We are also very grateful to Marco Privitera for the graphic work and to Angela Algeri for proofreading the manuscript. Our work is supported in part by 2008 MAE grant (Con il contributo del Ministero degli Affari Esteri, Direzione Generale per la Promozione e la Cooperazione Culturale). F.B., P.R., and S.D. have a fellowship from the University of Bologna.

REFERENCES

  1. Top of page
  2. Abstract
  3. OVERVIEW OF DROSOPHILA OOGENESIS
  4. EGGSHELL MORPHOLOGY
  5. EXPRESSION IN TIME AND SPACE OF EGGSHELL GENES
  6. ELEMENTS CONTROLLING THE EXPRESSION OF EGGSHELL GENES
  7. PERSPECTIVES
  8. Acknowledgements
  9. REFERENCES