Egg chambers emerge from the germarium with a uniform monolayer of cuboidal FCs. During vitellogenesis, when the oocyte swells due to yolk uptake, these cells will undergo a dramatic series of cell shape changes and migrations that result in the majority of the FCs moving to surround the oocyte. However, there is a period of several days between encapsulation and the FC migrations (stages 1–8) in which the FCE is morphogenetically quiescent. During this time, the FCs proliferate, the epithelial structure of the tissue continues to mature, and the FCE is patterned along its A-P axis. In this section, we discuss three topics that relate to general properties of the FCE and events that largely take place between stages 1 and 8. A-P patterning will be covered in the next section in conjunction with the discussion on FC migrations.
Proliferation, Endoreplication, and Growth
Approximately 80 FCs surround the germline cyst at the time that an egg chamber buds from the germarium (King and Vanoucek, 1960). The germ cells cease dividing before encapsulation but then enter a phase of endoreplication and growth, in which DNA synthesis occurs in the absence of cytokinesis and the germ cells dramatically increase in size. To accommodate the rapidly growing germ cells, the FCs continue to proliferate through the early stages of oogenesis. In well-fed females, the FCs have a doubling time of approximately 10 hr, but similar to SSC divisions, the proliferation rate can drop fourfold in response to poor nutrition (Margolis and Spradling, 1995; Drummond-Barbosa and Spradling, 2001). FC divisions cease at the end of stage 6, at which time the FCs undergo three rounds of endoreplication and growth and selectively amplify four chromosomal regions rich in genes required for eggshell production and patterning (reviewed in Calvi and Spradling, 1999; Botchan and Levine, 2004). King and Margaritis originally estimated that there are approximately 1,000 FCs in the egg chamber when divisions cease at the end of stage 6 (King and Vanoucek, 1960; Margaritis et al., 1980); however, direct counts of FC nuclei place the number closer to 650 (Margolis and Spradling, 1995).
The transition from proliferation to endoreplication occurs when a Delta signal from the germline activates Notch in the FCs. In egg chambers that contain either Delta germline clones or Notch FC clones, the FCs continue to proliferate beyond stage 6 (Deng et al., 2001; Grammont and Irvine, 2001; Lopez-Schier and St. Johnston, 2001). How is it that Notch signaling regulates this transition? In addition to continuing to divide, FCs that fail to receive the Delta signal also express FasIII, a protein whose expression is normally restricted to early stages of oogenesis (Grammont and Irvine, 2001; Lopez-Schier and St. Johnston, 2001). This ectopic FasIII expression has led to the suggestion that Notch mutant FCs continue to divide because they have failed to differentiate. However, FasIII is normally down-regulated long before the pulse of Delta signaling at stage 6, and Notch mutant FCs do express some late differentiation markers (Deng et al., 2001). A second, but not mutually exclusive, possibility is that the Notch pathway acts more directly on cell cycle components independent of FC differentiation. In fact, during the switch to endocycles, the transcription of key cell cycle components required for the G1/S, G2/M, and M/G1 transitions appear to be downstream of Notch activation (Deng et al., 2001; Schaeffer et al., 2004; Shcherbata et al., 2004). One area for further investigation is to determine what factors trigger the up-regulation of Delta in the germline to stop FC divisions.
FC proliferation occurs during the early stages of oogenesis, and there is, therefore, little overlap between cell division and major cell rearrangements within the FCE. Although the process of encapsulation occurs while the somatic cells are actively dividing, all subsequent movements of the FCE occur in the absence of cell division. These morphogenetic processes, thus, can be attributed entirely to changes in cell position and/or cell shape within a stable population of FCs.
Apicobasal Polarity and Junctions
The FCE, like other Drosophila epithelia, is architecturally similar to vertebrate epithelia and, thus, provides an outstanding system in which to study features such as apicobasal polarity. There are many excellent reviews on epithelial polarity (Tepass et al., 2001; Knust and Bossinger, 2002; Johnson and Wodarz, 2003), and we particularly refer the reader to a discussion of FCE polarity by Muller (2000). In this section, we will limit our discussion to some unique aspects of FC architecture, such as the interaction between the FCs and germ cells during polarization and the specialized array of cell junctions that FCs contain.
One of the most intriguing properties of the FCE is that its apical surface contacts the germ cells throughout much of oogenesis (Fig. 3). For most epithelia, the apical domain constitutes a free surface facing the exterior of the body or the lumen of a tube. Although unusual, this close proximity between the FCE and germline is essential for many aspects of FC biology. During later stages of oogenesis, FCs function as protein factories, synthesizing yolk proteins and eggshell components, which are secreted from their apical surfaces toward the oocyte. The interaction between the apical surface and germ cells also facilitates numerous signaling events between the germline and soma. We have discussed previously two signaling events during oogenesis where the Delta ligand in the germline activates the Notch receptor on the apical surface of the FCs. One interesting question that arises is whether the close apposition between the FCE and germline also provides a positional cue for the establishment and maintenance of apicobasal polarity in this tissue.
Figure 3. Apicobasal polarity in the follicle cell epithelium. The drawing shows a stage 8 egg chamber with a magnified view of two follicle cells. This magnification reveals the proximity of the apical surface to the germline and basal surface to a basement membrane as well as the relative positions of certain junctional complexes.
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Typically, the early cues that establish apicobasal polarity come in the form of basal cues, through cell–substrate adhesion, and lateral cues in the form of cadherin-based cell–cell adhesion. These adhesive events then trigger the formation of protein complexes at the cell surface, which further refine the apical and basolateral membrane domains and lead to the localization of a cadherin-based junctional complex, the zonula adherens (ZA), at their interface. These protein complexes have been best studied in the embryonic ectoderm where three groups, known as the Baz (Bazooka/Par-6/aPKC), Scrib (Scribble/Discs Large/Letha Giant Larvae), and Crumbs (Crumbs/Stardust/PATJ) complexes, have been shown to act in a functional hierarchy to delineate the apical and basolateral membrane domains (Bilder et al., 2003; Tanentzapf and Tepass, 2003). The Baz complex localizes to the marginal zone just apical to and overlapping with the ZA and acts first in the hierarchy to specify the apical domain. The Scrib complex is found just basolateral to the ZA and functions as a basolateral determinant by repressing the apicalizing activity of the Baz complex. Finally, the Baz complex recruits the Crumbs group to the apical domain, to antagonize the activity of the Scrib complex.
The FC monolayer is unusual among epithelia in that the cells have the potential to receive adhesion-based polarizing signals at their basal, lateral and apical surfaces. Before encapsulation, the FC precursors adhere to a basement membrane that surrounds the germarium and contact each other laterally through adherens junctions. These contacts appear to be sufficient to establish a basal membrane domain; however, apical and lateral markers are intermixed, suggesting that contact with the germ cells is necessary to resolve the apical and lateral domains (Tanentzapf et al., 2000). In support of this assertion, the apical determinant Crumbs is not expressed in the FCs of agametic ovaries (Tanentzapf et al., 2000). One interesting question is whether the interactions between the Baz, Scrib, and Crumbs complexes mirror those seen in the ectoderm or have been modified to exploit the unique cellular environment of this tissue. It is known that all three complexes are required for cell polarity in the FCE, as loss of function of any one component leads to rounded cells and multilayering at the follicle poles (Manfruelli et al., 1996; Goode and Perrimon, 1997; De Lorenzo et al., 1999; Bilder et al., 2000; Genova et al., 2000; Tanentzapf et al., 2000; Abdelilah-Seyfried et al., 2003; Benton and St. Johnston, 2003; Hutterer et al., 2004). More work will be required, however, to explore the interactions between these various players. Finally, contact between the apical surface and the germline also appears to be required to maintain polarity in the FCE, as loss of function of egghead or brainiac, which are both required in the germline, can lead to multilayering and loss of polarity during mid-oogenesis (Goode et al., 1996).
A second interesting property of the FCE is the array of cellular junctions that connect the FCs to one another and to the germline. The FCs assemble a functional ZA at the apical side of their lateral surfaces from the time that the egg chamber first buds from the germarium (Muller, 2000). Of interest, the adherens junctions of the ZA contain both E and N cadherin up to stage 10 of oogenesis, at which time N-cadherin disappears (Tanentzapf et al., 2000). Overlap of two classic cadherins in the same tissue is unusual in Drosophila and could exist because of the dual role E-cadherin plays in adhesion between the FCs and adhesion between the FCs germline. In fact, the large Maf transcription factor Traffic Jam, which is required for proper interactions between the germline and soma, regulates the expression of E-cadherin but not N-cadherin in the FCs (Li et al., 2003). The septate junction (SJ), which acts as a transepithelial diffusion barrier, forms along the lateral membrane, just basal to the ZA. SJ formation begins when proliferation is complete at stage 6, but these junctions do not fully mature until stage 10 after the posterior migration of the FCs is finished (see below; Mahowald, 1972; Muller, 2000). Functional gap junctions have been observed between cells within the FCE and between FCs and the germline (Mahowald, 1972; Giorgi and Postlethwait, 1985; Bohrmann and Haas-Assenbaum, 1993; Tazuke et al., 2002). What information passes through these gap junctions is currently unknown, but there is evidence that gap junctions between the FCs and oocyte are required for the oocyte to take up yolk (Waksmonski and Woodruff, 2002). Cytoplasmic bridges, presumably resulting from incomplete cytokinesis, have also been observed to connect groups of up to eight FCs within the FCE (Giorgi, 1978; Woodruff and Tilney, 1998). The cytoplasmic bridges in the FCs are much smaller than those found within the germline cyst, and their function remains to be determined.
Planar Polarity and Egg Elongation
In addition to apicobasal polarity, many epithelia also display polarity within the plane of the epithelium. Planar polarity can govern tissue morphogenesis in several ways (for review, see Adler, 2002). In the case of the FCE, it is involved in creating the elongated shape of the egg. Each FC contains a dense array of polarized actin filaments at its basal cortex that is arranged such that the fibers run perpendicular to the A-P axis of the egg chamber (Gutzeit, 1990). This pattern is first established during stages 5–6 in FCs that lie near the egg chamber poles. The effect of this bilateral initiation is that actin filaments appear to swirl around each pair of polar cells, which has led to the suggestion that these cells may function as organizers in this process (Frydman and Spradling, 2001). The circumferential orientation of the actin bundles then spreads medially until all FCs display this pattern by stage 7; this organization in maintained in cuboidal and columnar FCs through stage 14 (Gutzeit, 1990; Frydman and Spradling, 2001). Intriguingly, laminin A is organized into complementary circumferential fibers within the basal extracellular matrix (ECM) that surrounds each follicle (Gutzeit et al., 1991).
This striking circumferential organization of actin and laminin within the FCE has led to a model proposing that the planar array of filaments acts as a molecular corset that forces the follicle to grow preferentially along its A-P axis. Egg chambers are roughly spherical until stage 6, but lengthen along their A-P axis roughly concurrent with the polarization of the actin bundles. This A-P elongation is particularly apparent after nurse cell dumping at stage 11 and leads to the production of a lozenge-shaped egg. Although it is not yet known how these actin fibers exert morphogenetic forces to shape the egg, their proper orientation within the plane of the epithelium is absolutely required for them to perform this function. Several genes have been identified that, when mutated in the FCs, produce spherical eggs (Gutzeit et al., 1991; Bateman et al., 2001; Frydman and Spradling, 2001; Deng et al., 2003). Examining clones of these mutations within the FCE reveals that actin bundles form normally at the basal side of each cell, but their orientation is random with respect to the A-P axis. Surprisingly, this phenotype is not restricted to the mutant clone, but spreads to adjacent wild-type tissue, suggesting a need for cell communication within the epithelium to establish the planar pattern of actin. Evidence for communication between the cells is also seen within mutant clones where small groups of cells often coordinate their actin bundles with respect to each other, whether or not they match the global orientation of actin bundles in the tissue.
Several mutations that cause a spherical egg phenotype have been shown to disrupt proteins that mediate interactions between the actin cytoskeleton and the ECM. These disruptions include the Dystrophin-associated glycoprotein complex component Dystroglycan (Deng and Ruohola-Baker, 2000), the receptor-like tyrosine phosphatase Dlar (Bateman et al., 2001; Frydman and Spradling, 2001), and the β-integrin subunit encoded by the myospheroid (mys) gene (Duffy et al., 1998; Bateman et al., 2001). It is not yet known how these proteins work to align the actin filaments along the A-P axis, but studies of DLar indicate that it may play an early role in the establishment of the planar pattern. DLar and myospheroid genetically interact and their proteins colocalize at the actin filament termini. Whereas β-integrin is maintained at the termini throughout oogenesis, the localization of DLar is transient, occurring only during the stages when the planar pattern is established. Furthermore, genetic rescue experiments have shown that expression of DLar before stages 7–8 is sufficient to rescue the mutant phenotype, whereas expression at stage 10 is not. These data have led to a model postulating that DLar is required to modulate the early interaction between integrins, the basal actin filaments and the ECM (Bateman et al., 2001). An opposing model stipulates that DLar is actually required for polar cell specification and that the misorientation of actin in DLar mutants is secondary to this defect (Frydman and Spradling, 2001). This model is based on the observation that DLar mutant follicles often have additional polar cells and that the circumferential actin pattern is first seen in the polar region. Mosaic analysis has shown, however, that DLar is not required in polar cells to correctly orient the actin bundles (Frydman and Spradling, 2001). This observation, in conjunction with the protein localization pattern makes it more likely that DLar functions independently in these two processes.
An interesting and open question is to what extent the planar organization of actin filaments within the FCE is governed by conserved mechanisms that control other planar polarity systems in the fly such as the proximal–distal positioning of wing hairs (for reviews, see Adler, 2002; Tree et al., 2002). One striking feature shared by both systems is that mutant clones that disrupt the planar organization of the cytoskeleton have nonautonomous effects on neighboring wild-type cells. To date, however, no overlap has been found between the genes that appear to control planar polarity in the FCE and the genes that have been indicated in other planar polarity systems. This finding includes the serpentine receptor Frizzled and its downstream effector Disheveled, which are required for most examples of planar polarity in Drosophila as well as in vertebrates. Future experiments will be required to determine whether an entirely unique system is used to create planar polarity in the FCE or if conserved mechanisms are operating that have not yet been recognized.