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Correspondence to: Graham H. Thomas, Departments of Biology and of Biochemistry and Molecular Biology, The Pennsylvania State University, 208 Erwin W. Mueller Laboratory, University Park, PA 16802. Tel:(814) 863-0716 Fax:(814) 865-9131 E-mail:gxt5{at}psu.edu.
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Abstract |
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Changes in cell shape and position drive morphogenesis in epithelia and depend on the polarized nature of its constituent cells. The spectrin-based membrane skeleton is thought to be a key player in the establishment and/or maintenance of cell shape and polarity. We report that apical ßHeavy-spectrin (ßH), a terminal web protein that is also associated with the zonula adherens, is essential for normal epithelial morphogenesis of the Drosophila follicle cell epithelium during oogenesis. Elimination of ßH by the karst mutation prevents apical constriction of the follicle cells during mid-oogenesis, and is accompanied by a gross breakup of the zonula adherens. We also report that the integrity of the migratory border cell cluster, a group of anterior follicle cells that delaminates from the follicle epithelium, is disrupted.
Elimination of ßH prevents the stable recruitment of 
-spectrin to the apical domain, but does not result in a loss of apicobasal polarity, as would be predicted from current models describing the role of spectrin in the establishment of cell polarity. These results demonstrate a direct role for apical (ßH)2-spectrin in epithelial morphogenesis driven by apical contraction, and suggest that apical and basolateral spectrin do not play identical roles in the generation of apicobasal polarity.
Key Words: spectrin, oogenesis, cell polarity, zonula adherens, morphogenesis
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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EPITHELIAL cell sheets perform a number of coordinated morphogenetic movements in response to a variety of signals during the development of multicellular organisms. These include infoldings generated by localized apical constriction and concerted migrations driven either by convergent extension or radial intercalation (
The spectrin-based membrane skeleton (SBMS) is a ubiquitous cytoskeletal structure that has been postulated to play a role in the establishment and/or maintenance of apicobasal polarity and has a close relationship with cadherin-based adhesive complexes (
Several properties of the SBMS have led to the proposal that it plays a key role in the generation and/or maintenance of apicobasal cell polarity in epithelia (
Each spectrin molecule is comprised of two and two ß chains that are largely made up of spectrin repeats that give the molecules their extended rope-like morphology (ß)2 tetramers to cross-link F-actin. The presence of specialized spectrin repeats and of various nonrepetitive domains that provide binding sites for other proteins suggests that these molecules act as molecular scaffolds capable of nucleating specific complexes of proteins at the membrane (
In Drosophila melanogaster, the SBMS is built around two distinct spectrin isoforms; a single -spectrin chain assembles with one of two ß-spectrin chains, ß- or ßHeavy-spectrin (ßH), to form (ß)2 and (ßH)2 tetramers, respectively. Fly ß-spectrin has a typical ß-spectrin–like organization with an NH2-terminal actin binding domain followed by 17 spectrin repeats and a nonrepetitive COOH-terminal region containing a pleckstrin homology (PH) domain (ß)2 and (ßH)2 tetramers form actin cross-linking units of 180 and 250 nm, respectively, reflecting the difference in the number of spectrin repeats within each ß chain (ß)2 is always basolateral while (ßH)2 is always apical (ßH)2 is associated with the ZA where its abundance closely follows that of DE-cadherin (
The SBMS is essential for Drosophila development. Mutations have been recovered in all three spectrin genes and all result in extensive or complete lethality. Mutations affecting -spectrin are first instar larval lethals that disrupt both the apical and basolateral SBMS, resulting in the disruption of cell–cell contact (
Here, we examine the specific contribution of the apical SBMS to epithelial cell polarity, adhesion, and morphogenesis by analyzing the effects of the karst mutation on follicle cell morphogenesis in Drosophila oogenesis. During mid-oogenesis, the follicle cell epithelium undergoes a well-defined migration to envelop the oocyte. This involves apical constriction and results in a change in cell shape from cuboidal to columnar. We show that the karst mutation completely eliminates the apical spectrin membrane skeleton and prevents normal apical contraction of the follicle cells. However, lack of apical spectrin does not eliminate the ability of these cells to establish and maintain apicobasal polarity. Furthermore, we observe gross disruptions of the ZA in karst mutants. These results are consistent with the hypothesis that a primary role of the apical SBMS lies in facilitating changes in cell shape, perhaps by contributing to the integrity of the ZA. Our results also imply that it is the disruption of the basolateral SBMS specifically, or the basolateral plus the apical SBMS, that results in the loss of apicobasal polarity seen in -spectrin mutants (
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Materials and Methods |
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Fly Stocks
The karst stocks mwh ve kst1 e/TM6B, mwh ve kst2 e/TM6B, mwh kst14.1 red e/TM6B, mwh Df(3L)1226 red e/TM6B, and kst01318/TM6B have been previously described (
Antibodies
To detect ßH, we used serum #243 (-spectrin, we used ascites fluid #N3 at a dilution of 1/5,000 (
FITC, Cy3 or Cy5 conjugated, and affinity-purified secondary antibodies used were all made in goat and were obtained from Jackson ImmunoResearch Laboratories, Inc. These antibodies were rehydrated according to the manufacturer's instructions and used at dilutions of 1/100. Alexa 488 conjugated secondary antibody was obtained from Molecular Probes and used at a dilution of 1/200.
Immunofluorescent Staining of Ovaries
2–4-d old females fed with yeast paste at 25°C were dissected and their ovaries were teased apart into individual ovarioles in PBS (130 mM NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4, pH 7) using a tungsten needle. The tissue was fixed in buffer B (16.7 mM KH2PO4/K2HPO4, pH 6.8, 75 mM KCl, 25 mM NaCl, 3.3 mM MgCl2, 5% paraformaldehyde;
Activity Staining for ß-Galactosidase
Ovaries were dissected in PBS and fixed in 1% glutaraldehyde in PBS for 20 min at room temperature. After three to four washes in PBS, the tissue was incubated in prewarmed reaction solution (10 mM sodium phosphate, pH 7.2, 150 mM NaCl, 1 mM MgCl2, 3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6, 0.1% X-Gal) for at least 30 min at 37°C. The samples were extensively washed in PBS and mounted in mounting medium.
Sample Imaging
Imaging of immunofluorescently stained ovaries was done using an MRC1024 confocal microscope (Bio-Rad Laboratories). Imaging of ovaries stained for ß-galactosidase activity was done on a BX50 microscope (Olympus Corp.) equipped with a Dage/MTI CCD72T camera and DSP2000 signal processor, and were imported directly into a Power Macintosh 8100/80AV (Apple Computer) using a DT2255 frame grabber (Data Translations) controlled by the public domain program NIH Image (v1.61 available on the internet at http://rsb.info.nih.gov/nih-image). Images were contrast stretched as appropriate using Adobe Photoshop v4.0 (Adobe Systems, Inc.) and the figures assembled and annotated in Adobe Illustrator v6.0.
Morphometric Analysis
Images of sagittal optical sections of 81 wild-type and 318 karst mutant egg chambers at stage 9 or 10A (a comprehensive combination of kst1, kst2, kst14.1, and kst01318 alleles over one another and over Df(3L)1226), that had been stained for -spectrin, DE-cadherin, actin, or Notch, were acquired. The distances described in text and Figure 2 were then measured and analyzed using Excel 98 (Microsoft Corp.) and/or Deltagraph (DeltaPoint, Inc.).
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Measurement of Apical Surface Areas of Follicle Cells
To measure the follicle cell apical surfaces, we stained wild-type and kst01318 egg chambers for DE-cadherin to outline the apical contours. To ensure that only cells being viewed en face were measured, the following procedure was used. The z-axis motor on the confocal was stepped gradually out from the sagittal plane of the oocyte towards the apical domain of the upper follicle cell monolayer until the oocyte had just disappeared. Measurement of the apical surface areas was done using NIH Image software. Specifically, we used the Threshold option to isolate the cell outlines, after which the Measure option was used to quantify the number of pixels per apical surface per cell. Any breaks in the DE-cadherin staining, particularly in karst mutant egg chambers, were manually closed with straight, one pixel-wide lines before area measurement. The position of the center of each cell measured relative to the nurse cell/oocyte boundary was also recorded for each cell.
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Results |
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The Distribution of ßH during Drosophila Oogenesis
Oogenesis in flies takes place in ovaries formed of 12–16 ovarioles, each of which consists of an anterior structure called the germarium and several egg chambers sequentially ordered with regard to their developmental stage (for review, see
At the onset of stage 9, after all divisions have ceased, the majority of the follicle cell monolayer undergoes a concerted migration towards the posterior of the egg chamber onto the oocyte membrane. Those follicle cells that are left behind become squamous and continue to reside on the nurse cells. During this morphogenetic event, the migratory follicle cells undergo a change in cell shape from cuboidal to columnar, a process that is instrumental in accommodating them on the growing oocyte membrane. Concomitantly, a group of 6–10 anterior follicle cells called border cells round up (Figure 1 A, * in stage 9) and plunge in between the nurse cells to reach the anterior of the oocyte (Figure 1 A; * in stage 10). After the follicle cell monolayer and the border cells have reached the anterior of the oocyte, a specialized subset of follicle cells called centripetal cells migrate along the nurse cell–oocyte interface such that the follicle cells now completely surround the oocyte. Communication between nurse cells and the oocyte is maintained via ring canals that remain open until the nurse cells dump their cytoplasmic contents into the oocyte at stage 11.
Although two partial descriptions of the distribution of ßH during oogenesis have been published by other groups (
In the soma, ßH is strongly expressed at the very anterior tip of the germarium in the terminal filament and the cap cells that contact the germline stem cells (Figure 1A and Figure B). In close proximity to the 16 cell cysts in region 2, we detect high levels of ßH expression in the vicinity of the somatic stem cells and in their progeny, the follicle cells (Figure 1A and Figure B). As individual cysts become enveloped in follicle cells, ßH is slightly enriched in the cells that move in to segregate adjacent cysts (Figure 1 B). ßH continues to be strongly expressed here as these become the stalk cells that separate successive egg chambers along the ovariole (Figure 1 A, arrow). ßH is apically polarized in the follicle cells (Figure 1A and Figure B). ßH is downregulated in the migrating border cells at stage 9 (Figure 1 A, *), but is again expressed on the apical surface of these cells when they begin to secrete the micropyle after stage 10 (Figure 1C and Figure E, arrowheads). At stage 10, ßH is part of a prominent terminal web-like structure at the apical ends of the follicle cells that are secreting egg components (Figure 1 G). This structure appears to be anchored in the ZA by fine fibers of staining around its edge. ßH is also expressed on the apical surfaces of the follicle cells secreting the dorsal appendages (Figure 1 D, arrow) and the chorion (data not shown). ßH colocalizes with -spectrin in the germline and soma at all these locations (Figure 1); however, the latter is more widespread presumably through its association with the conventional ß isoform.
Border and Follicle Cell Migrations Are Uncoordinated in karst Mutant Egg Chambers
In mid–stage 9 egg chambers, the follicle cell monolayer and the border cells migrate in a concerted fashion (
The lack of coordination in cell migration makes it difficult to assess the progression of each chamber through stage 9. We therefore resorted to a morphometric approach. The four distances indicated in Figure 2 B were measured in sagittal optical sections of 81 wild-type and 318 karst mutant egg chambers. Since the nurse cells do not grow or shrink at this time, all these measurements were normalized against the anterior-posterior length of this cell cluster.
Pairwise comparisons of the parameters (FC/NC), (BC/NC), and [oocyte/(oocyte+NC)] (Figure 2, C–E) reveals the following defects in karst mutant egg chamber morphogenesis. (a) Most karst border cell clusters are migrating ahead of the follicle cell monolayer during stage 9 (Figure 2 C). This could arise either due to faster border cell migration or slower follicle cell migration. (b) Most karst mutant oocytes occupy a larger portion of the egg chamber than in the wild-type during follicle cell migration, while the oocyte exhibits no significant overgrowth at the completion of migration (Figure 2 D). This suggests that karst follicle cells are delayed in their migration relative to growth of the oocyte, or may respond more slowly to oocyte growth. (c) Similarly, most karst mutant oocytes occupy a larger portion of the egg chamber than in wild-type during border cell migration (Figure 2 E). This effect is not as strong as for the follicle cells, consistent with the observation that the border cells generally migrate ahead of the follicle cells in karst mutant egg chambers, but it does suggest that there is a slight delay in border cell migration. The most parsimonious model accounting for these data suggests that the karst mutation causes a significant disruption of follicle migration onto the oocyte membrane and a slight delay in border cell delamination or migration through the nurse cells.
Consistent with the hypothesis that the primary morphogenetic defect lies in follicle cell migration, some follicle cells in karst mutant egg chambers often remain in contact with the nurse cell membranes at stage 10A. These follicle cells still attempt to make the appropriate adhesive contacts with the oocyte membrane (
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Aberrant Follicle Cell Migration Results from a Failure in Apical Constriction
The failure of karst follicle cells to complete their migration onto the oocyte by the onset of stage 10B implies that the total apical surface area of the epithelium is greater than that of the oocyte membrane. Moreover, karst mutant follicle cells often appear to have a more cuboidal shape than in the wild-type (see Figure 5G and Figure H). Since there is no over-proliferation in the mutant monolayer (data not shown), this cannot arise due to an increase in cell number. However, an inability of karst follicle cells to properly change their cell shape or constrain their apical surface area at the appropriate size would explain this observation. We therefore compared the apical surface area of wild-type and mutant follicle cells during monolayer migration. This analysis reveals a sharp decrease in the apical surface area of the wild-type follicle cells as they approach and migrate onto the oocyte (Figure 4 A). In contrast, the majority of karst mutant follicle cells fail to apically constrict (Figure 4 A). The mean apical surface area of the mutant follicle cells is almost twice that of the wild-type (Figure 4 B). Moreover, comparison of the apical surface areas of mutant follicle cells in chambers during migration with those where migration has been completed reveals a slight increase (Table 1; P < 0.001). This suggests that, in addition to the constriction defect, the monolayer cannot withstand the forces exerted by the growing oocyte.
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The zonula adherens Is Disrupted in karst Mutant Follicle Cells
Examination of the follicle cell apices stained for DE-cadherin also reveals conspicuous disruptions in the staining pattern of DE-cadherin in karst mutant egg chambers (Figure 5, A–F). In the mildest cases, this staining is missing at three- or four-cell vertices, but we also see large breaks in the normally continuous belt of staining in more extreme cases. These observations are consistent with the hypothesis that the absence of ßH weakens the ZA, and that it breaks up as the apices attempt to constrict or accommodate the growth of the oocyte. However, the apicolateral polarization of the ZA is largely unaffected (Figure 5G and Figure H).
The Border Cell Cluster is Often Disrupted in karst Mutants
The border cell cluster delaminates from the follicle cell epithelium and migrates between the nurse cells to the anterior of the oocyte during stage 9. In ~10% of karst mutant egg chambers, we observe migratory cells that are well separated from, or trail behind, the main border cell cluster (Figure 6). The trailing cells upregulate -spectrin (Figure 6A and Figure B) and DE-cadherin (data not shown) in a manner that resembles wild-type clusters, suggesting that they are true border cells. To confirm that these cells have a border cell fate, we looked for the expression of the border cell marker slow border cells (slbo) by introducing the enhancer trapped LacZ gene associated with the slbo1 P-element allele (
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The Apical Spectrin Membrane Skeleton Is Absent in karst Mutant Follicle Cells
ßH is no longer detectable at the apical domain of the follicle cells in any allelic combination of karst alleles that we have examined (data not shown). The localization of ßH to the apical domain has been previously shown to be dependent on -spectrin (-spectrin is dependent on ßH for its localization to the apical domain, and to confirm that no apical spectrin function remains in karst mutants, we examined the distribution of -spectrin in karst follicle cells. While the lateral -spectrin distribution is unaffected by this mutation, apical -spectrin is no longer detectable by immunofluorescence (Figure 7C and Figure E). This indicates that the stable recruitment of -spectrin to the apical domain is dependent on ßH, and that there is thus a mutual interdependence between -spectrin and ßH. This further suggests that ßH-spectrin is recruited to the apical domain as a heterodimer or tetramer, or that following separate recruitment only the dimers or tetramers remain stably associated with the apical domain.
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Apicobasal Polarity Is Maintained in karst Mutant Follicle Cells
Spectrin associated with E-cadherin has been implicated in the apicobasal cell polarization pathway (-spectrin mutations cause a breakdown in monolayer polarity including the loss of apical ßH (
Given the significant similarity between ßH and ß-spectrin (
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Discussion |
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ßHeavy-spectrin is a member of the spectrin family of proteins that have been implicated in cadherin-mediated cell polarization (
ßH Is Polarized in Somatic Epithelia but Not in Germline Cells
We present here the first complete description of the distribution of ßH during Drosophila oogenesis (Figure 1). ßH is strongly expressed in the terminal filaments and cap cells that sit adjacent to the germline stem cells at the anterior end of the germarium. In the germline, it is first seen on the membrane in region 2, in the 8–16-cell cysts. ßH is not obviously polarized at any of these locations, and remains uniformly distributed in the nurse cells and oocyte. In the soma, ßH is prominently expressed in the vicinity of the somatic stem cells and their derivative, the follicle cell epithelium, where it is restricted to the apical domain. These results corroborate the previous partial descriptions of the distribution of ßH during oogenesis (
The downregulation of ßH in the anterior region of the germarium would suggest that ßH has no role in germ cell division and/or oocyte specification within the germline. However, ßH is strongly expressed in the terminal filament and cap cells that sit adjacent to the germline stem cells and early cystoblasts, the cellular activities of which are believed to regulate germline stem cell division and polarization (reviewed in
ßH is part of a prominent terminal web subtending the follicle cell brush border that forms as they migrate onto the oocyte at stage 9 and begin to secrete yolk protein. ßH is also expressed in the border cells once these cells begin to secrete the micropyle and in the follicle cells that are secreting chorion to form the dorsal appendages. The prominence of ßH in all these locations of high secretory activity suggests that there may be a role for the apical membrane skeleton in the targeting or delivery of secretory vesicles even though it is not required for overall apicobasal polarity (see below). A similar role has been proposed for the gut-specific terminal web ß-spectrin, TW260, in the chicken (
ßHeavy-Spectrin Is Required for Epithelial Morphogenesis
In karst mutant ovaries, the primary defect in follicle cell morphogenesis is a failure of this monolayer to complete migration onto the oocyte membrane by the end of stage 9 (Figure 2 and Figure 3). Specifically, these cells fail to undergo the normal apical contraction associated with the development of a more columnar shape upon contacting the oocyte membrane (Figure 4). Furthermore, staining for the ZA marker DE-cadherin revealed conspicuous breaks in the normally continuous belt of staining for this protein around the follicle cell apices (Figure 5).
Apical contraction is a well established process for the generation of form in an epithelium; however, the exact mechanism by which cells achieve this phenomenon is still unclear. ßH is associated with two actin-rich structures at the apical pole, the ZA and the terminal web subtending the microvillar brush border. F-actin at the ZA lies in a circumferential band of microfilaments of mixed orientation that can be induced to undergo a purse string–like contraction in vitro that is mediated by myosin II (
Alternatively, ßH may play a structural role in the follicle cell terminal web (
The fact that follicle cell migration does initiate and proceed to some extent in karst mutants indicates that not all motile forces are eliminated by this mutation. This may simply mean that not all intercellular tension generated by apical constriction is eliminated by the karst mutation. However, another possibility is that apical constriction is not the only force-generating mechanism responsible for monolayer migration. Indeed, the observation that hypomorphic mutations in the regulatory light chain of cytoplasmic myosin II do not prevent the migration of the follicle cell monolayer (
The failure of karst follicle cells to complete their migration onto the oocyte by the onset of stage 10B leads to aberrant centripetal cell migration in a small number of egg chambers. In such cases, the inwardly migrating cells find their way in between nurse cell membranes enclosing one of the latter along with the oocyte. Given the high frequency of defective migration, it is perhaps surprising that this latter defect is relatively rare and that oogenesis can often proceed to completion in the absence of ßH. We suspect that the ability of the centripetally migrating cells to seek out the nurse cell/oocyte interface by specific adhesion to the oocyte membrane (
ßHeavy-Spectrin Is Required for Border Cell Morphogenesis
The border cells delaminate from the follicular epithelium at the onset of stage 9 and migrate between the nurse cells to the anterior of the oocyte (
The presence of ßH at two or possibly three locations during normal border cell delamination and migration suggests a number of possible roles for this protein in border cell morphogenesis. Its presence at the nurse cell membranes on which the border cells migrate could contribute to the rigidity of this substratum or to inter-nurse cell adhesion. In this context, we note the similarity between the patchy distribution of ßH (Figure 1 A, arrowhead) along the border cell migration route and that of DE-cadherin, a molecule required for migration (
The normal boundary between the border cells that will delaminate and the surrounding follicle cells is marked by a dramatic decrease in the level of ßH in the border cells. In addition, ßH is also prominent at the follicle cell/border cell membrane interface during delamination. This boundary must have at least two properties. It must serve to allow the border cells to detach and it must allow the follicle cell monolayer to reseal the gap left by the departing cluster. We hypothesize that the presence of ßH in the surrounding follicle cells is part of a differential adhesion system that causes the surrounding, nonmigratory follicle cells to seek out one another to reseal the gap and in doing so to sacrifice contact with the border cells. Elimination of ßH in the nonmigrating cells would affect the precise physical boundary between groups of cells with different adhesive properties preventing this rearrangement of cell contacts, and thus proper detachment of the border cells. The precise role of ßH in generating this boundary remains open. ßH is localized in part at the ZA and its presence or absence could be responsible for modulating DE-cadherin–based adhesion. Such differential adhesion is clearly part of the mechanism by which the oocyte positions itself relative to the overlying follicle cells (
Apicobasal Polarity Does Not Require Apical Spectrin
The initial development of the follicular epithelium is essentially unaffected by the loss of either -spectrin (-spectrin mutants in Drosophila (-spectrin mutants reflects a requirement for the basolateral SBMS specifically, or is a synergistic consequence of losing both the apical and basolateral membrane skeletons. Resolution of this ambiguity awaits further characterization of ß-spectrin mutants. Although overall apicobasal polarity remains intact in karst mutants, it remains possible that specific proteins that are dependent on binding to the apical SBMS for delivery and/or retention in the apical membrane are depolarized by the absence of (ßH)2.
In keeping with the observation that different spectrin isoforms do not generally colocalize (
Current models for the origins of epithelial polarity suggest that spectrin plays a key role in establishing and/or maintaining the apicobasal axis (-spectrin (ßH)2] does not. It remains unclear what the precise mechanism is by which apical spectrin acts during morphogenetic events. ßH exhibits a close colocalization with the ZA, and its levels at this location are regulated in concert with DE-cadherin (ßH)2 is associated with the ZA; however, the contrasting behavior of the apical and basolateral SBMS during the emergence of apicobasal polarity (
Conclusions and Perspective
The results presented in this paper add to a growing body of evidence that apical spectrin is essential for epithelial morphogenesis. Moreover, we show that an apical SBMS is not required for establishing or maintaining apicobasal polarity, as seems to be the case for the basolateral SBMS. It is unknown at present whether or not ßH or any other ß-spectrin plays a similar role in morphogenesis in vertebrates. The observations that ßH is evolutionarily old (-spectrin is associated with the involution of tissues during embryogenesis (
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The authors thank Spyros Artavanis-Tsakonas, Dan Branton, Dan Kiehart, John Lee, Mark Mooseker, and Tadashi Uemura for gifts of antibodies, Denise Montell for supplying the slbo1 enhancer trap line, and Carol Gay for the use of her confocal microscope. We also thank Maggie de Cuevas and Bryce MacIver for useful discussions, as well as Susan Abmayr, Esther Siegfried, and the members of the Thomas lab for critically reading this manuscript.
This work was funded by National Institutes of Health grant GM52506 to G.H. Thomas.
Submitted: 10 June 1999
Revised: 4 August 1999
Accepted: 5 August 1999
1.used in this paper: ßH, ßHeavy-spectrin; SBMS, spectrin-based membrane skeleton; ZA, zonula adherens
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