karst

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

Karst transcript is not maternally loaded, but the protein (ß_H spectrin) is present in eggs. The karst gene shows no detectable transcript accumulation until stage 10 (fully extended germband) of embryogenesis, after which transcript accumulates through the remainder of embryogenesis. Karst transcripts are always confined to a specific subset of cells and tissues. The first transcripts accumulate at stage 10 in the primordia of the foregut and hindgut, and then in numerous mesodermal cells that appear in a segmentally repeated pattern. These labeled mesodermal cells subsequently increase in number and are probably somatic muscle precursor cells. Concomitant with this, Karst transcript also accumulates deeper in the embryo, in the splanchnopleura, which gives rise to the visceral mesoderm. Karst is thus expressed in much or all of the muscular tissue of the developing embryo at a time comparable to the transcription factors nautilus and Nk1/S59, which are the earliest known markers of muscle tissue. As the germband starts to retract, expression is also seen in the abdominal and thoracic epidermis, and, after dorsal closure, expression is seen in the trachea (Thomas, 1994).

Maternal Karst protein is closely associated with the membrane of the egg during the syncytial stages of embryogenesis, and shows its first dynamic involvement with the developmental process when it becomes associated with the transient mitotic furrows during nuclear cycles 11, 12 and 13. During cellularization, Karst protein is seen in two locations: some Karst remains apically localized in the cell as a circumferential band of staining on the lateral membrane (apicolateral), but staining is also seen to descend with the invaginating cell membranes (basal), becoming concentrated in a region adjacent to the acto-myosin ring. The basal staining disappears during the latter part of cellularization (towards the end of the fast phase) and prior to gastrulation, suggesting that it plays a specific role during the cellularization process itself. The basal distribution of Karst appears to be lost first from the ventral cells that invaginate 5-10 minutes ahead of the dorsal cells. Redistribution of Karst to an apical cap or plate in the cell anticipates cell sheet morphogenesis. Whereas most of the Karst protein at the cellular blastoderm stage is seen to be apicolaterally localized in all cells except the pole cells, certain regions that are undergoing contractile processes to produce morphogenetic movements (ventral and cephalic furrows, and the posterior midgut invagination), have an additional cap of Karst across the apical ends of the cells. In the case of posterior midgut invagination, for example, the redistribution of Karst to a cap, or plate, at the apical surface of the cell clearly anticipates any gross visible movement of the cell sheet. Dynamic redistribution of Karst throughout these early developmental stages is therefore correlated with changes in cell shape that are all probably occurring due to contractile forces generated by nonmuscle myosin. The source of Karst for the new apical domain could be either from a cytoplasmic pool or from a pre-existing membrane domain (Thomas, 1994).

Anti-Karst staining in embryos at later stages (stage 10), after zygotic transcription has initiated, recapitulates the transcript accumulation seen with the whole embryo hybridizations. There is, in addition, visible but diminishing staining in all cells, which can be attributed to the perdurance of the maternally loaded protein (Thomas, 1994).

Karst is closely associated with the plasma membrane of the somatic and visceral musculature: in stage 10-12 embryos, bright Karst staining is seen in the somatic and visceral muscle precursors. The somatic muscle precursors are visible in the ventral mesoderm just above the developing central nervous system (CNS), and appear in a segmentally repeated pattern. As the germband retracts these cells move to more lateral positions, and staining can ultimately be seen in the maturing somatic musculature. The visceral musculature originates in segmentally repeated clusters of cells that join together along the anterior-posterior axis and form the characteristic palisade cell structure of the splanchnopleura. Concomitant with the appearance of Karst in the somatic muscle precursors, the protein is also seen in the developing splanchnopleura, and can eventually be seen staining the mature visceral musculature around the midgut epithelium. The earliest sites of karst transcription are associated with the morphogenesis of both the foregut and the hindgut at the stomodeal and proctodeal invaginations, respectively. The former ultimately results in positive staining for Karst protein in both the pharyngeal musculature and esophagus. The latter early transcription site is found in a layer of cells on the inner surface of the proctodeum, which goes on to give rise to the visceral mesoderm of the hindgut. These cells stain with Karst antibodies and form a layer that stretches along most of the dorsoventral extent of this invagination. These cells are still evident at later stages of development and move in an anterior direction along the hindgut. In contrast to the other tissues, there is no obvious polarization of the cortex in muscle tissue that is reflected in the Karst distribution (Thomas, 1994).

Karst is apically localized in the trachea: as with other invaginations during embryogenesis, each of the 20 bilaterally symmetric tracheal placodes reorganize their membrane skeleton such that Karst is seen across the apices of the cells as they move into the embryo during stage 10. At this stage karst transcription is not detectable in the epidermis, so it is assumed that the protein being redistributed is maternal in origin. As the trachea mature, the sections of the longitudinal trunk connect, the transverse and ventral branches grow out, and transcription starts in this tissue. Although the staining is greatest at the abdominal spiracles, the protein can be detected at the lumenal surface of all the cells in the network, including the branches that penetrate the CNS (Thomas, 1994).

Karst is a prominent component of the cells in the epidermis and amnioserosa: Karst transcript accumulation begins in the epidermis as the germband retracts: this tissue stains intensely for Karst protein. With the exception of sites of invagination the protein is seen exclusively in an apicolateral distribution that was originally established with maternal Karst protein. The amnioserosa also stains very intensely for Karst after (but not before) germband retraction, and transcript accumulation has not been detected here using digoxigenin-labeled whole-embryo hybridization methods. Two possibilities most easily account for the increase in protein staining in the apparent absence of transcription. Either Karst is assembled into the membrane skeleton from a cytoplasmic Karst pool that is not visualized, or transcription is occurring but is not detected. The amnioserosa is a particularly delicate and exposed structure: failure to detect transcription may be due to excessive damage to this tissue during the proteolysis step of the hybridization procedure. It is unlikely that this is due to cross reaction with another protein (Thomas, 1994).

Karst is also localized in an apical cap or plate at later sites of invagination: staining at the apical ends of the cells at the salivary gland placodes is evident as soon as the organ invaginates, and continues to be present at a high level on the lumenal surface of the cells. It is not known exactly when transcription of the karst gene starts in the salivary gland, since whole-mount hybridization is less efficacious after dorsal closure and the start of cuticle deposition, and prior to that, the staining intensity of the epidermal layer is very high, making weak internal signals hard to visualize. The bright line of Karst staining along the ventral midline, which forms as the ventral furrow invaginates and cells on either side come into contact, persists for some time after the ventral furrow disappears and accumulation of Karst transcript starts in the ectoderm. This line of staining lies between the two rows of mesectodermal cells, which will later become part of the CNS at stage 12-13, approximately when the Karst staining disappears from the midline. Since this line appears prior to detectable transcript accumulation in the ectoderm, it is likely to be established with maternally supplied protein (Thomas, 1994).

The presence of Karst staining at sites where contraction is generated by cytoplasmic myosin raises the possibility that these two proteins colocalize in the cell. The basal staining for Karst during cellularization is very close to the region that contains the actomyosin contractile ring at the base of the forming cells. Cytoplasmic myosin has also been observed at the apices of the cells that form the midgut invagination. Double staining for both proteins simultaneously reveals that they are in distinct domains at cellularization but colocalize in the posterior midgut invagination. Thus, while Karst could be involved in the contractile process at the latter location it is unlikely that this is the case at cellularization (Thomas, 1994).

Larval

To investigate the origins of the karst phenotype, the distribution of ß_H protein was examined in both the eye/antennal and wing imaginal disc epithelia. ß_H2-spectrin is present in all cells of both discs where it exhibits an apicolateral localization. Double staining with Shotgun (DE-cadherin) indicates that this localization coincides with the adherens junctions. In the eye disc, there is also a greater concentration of ß_H at the adherens junctions in the photoreceptor preclusters that coincides with more prominent staining for Shotgun. These regions correspond to the type III adherens junctions observed by Takahashi (1996). ß_H also colocalizes with alpha-spectrin, however, the alpha-spectrin staining extends into the basolateral region of the cells, demonstrating that the ß_H distribution is apically restricted within the membrane skeleton. Conventional ß-spectrin presumably partners alpha-spectrin in the basolateral regions (Thomas, 1998).

The alpha-spectrin mutation disrupts the microvillar brush border lining on the apical surfaces of the cuprophillic cells of the larval midgut (Lee, 1993). This mutation presumably compromises the function of both alpha2-/ß2- and alpha2-/ß_H2-spectrins. The midgut brush border was therefore examined for the presence of ß_H in wild-type flies and for any discernible effects of the karst mutation to see if the specific absence of alpha2/ßH2 spectrin would compromise this structure. ßH is found beneath the brush border in both adults and larvae. Since there is some concerned about the perdurance of maternal product, the adult midgut was examined for defects in karst mutants where maternal contribution is not a problem. Here the brush border is well ordered without any consistent irregularities of the cell surface or variations in microvillar length. During the development of a brush border, the microvilli emerge before a terminal web is visible, so it may not be surprising that ßH is not necessary for the assembly of microvilli (Thomas, 1998)

karst mutant flies are very infertile, precluding the maintenance of viable homozygous karst stocks. A few mutant offspring from karst- crosses have been produced, suggesting that, in exceptional cases, individuals may develop in the complete absence of ß_H. alpha-spectrin has been shown to be important for oogenesis both in the follicle cells and in the germline. Staining of ovaries for ß_H reveals that it is primarily concentrated at the apical ends of the somatic follicle cells. Fixed karst mutant egg chambers were stained with rhodamine phalloidin, but this did not reveal any conspicuous defects in the actin cytoskeleton (Thomas, 1998).

The simple cellular composition and array of distally pointing hairs has made the Drosophila wing a favored system for studying planar polarity and the coordination of cellular and tissue level morphogenesis. A gene expression screen was carried out to identify candidate genes that functioned in wing and wing hair morphogenesis. Pupal wing RNA was isolated from tissue prior to, during and after hair growth and used to probe Affymetrix Drosophila gene chips. 435 genes were identified whose expression changed at least 5 fold during this period and 1335 whose expression changed at least 2 fold. As a functional validation, 10 genes were chosen where genetic reagents existed but where there was little or no evidence for a wing phenotype. New phenotypes were found for 9 of these genes providing functional validation for the collection of identified genes. Among the phenotypes seen were a delay in hair initiation, defects in hair maturation, defects in cuticle formation and pigmentation and abnormal wing hair polarity. The collection of identified genes should be a valuable data set for future studies on hair and bristle morphogenesis, cuticle synthesis and planar polarity (Ren, 2005).

The expression karst gene, which encodes betaHeavy-spectrin, increased 5.5 fold from 32 to 40 hrs. Spectrin typically contains 4 chains, 2 alpha and 2 beta; these chains are known to link the actin cytoskeleton to the plasma membrane. Somewhat surprisingly, kst mutants are viable (at reduced levels) and female sterile due to defects in the follicular epithelium. Adult kst mutants have rough eyes and their wings often are cupped downward. kst wings were examined and an additional mutant phenotype was found that is nicely correlated with its expression profile. kst wing cells produce normal looking hairs but the hairs are often found on a small pedestal. The wing cell surface (that is not hair) is rough and at times remnants of cell outlines are visible. This phenotype can also be seen in mosaic clones. The clones can be recognized under the stereo microscope because they are often associated with a dimpling of the wing surface (Ren, 2005).

Effects of Mutation or Deletion

Mutations have been isolated in the gene encoding ß_Heavy-spectrin in Drosophila: this essential locus has been named karst. karst mutant individuals have a pleiotropic phenotype characterized by extensive larval lethality and, in adult escapers, rough eyes, bent wings, tracheal defects and infertility. Within karst mutant eyes, a significant number of ommatidia specifically lack photoreceptor R7 alongside more complex morphological defects. In descending order of penetrance karst mutant flies possess rough eyes, wings that typically curve downwards in an 'inverted spoon' shape and visible extrusion of material from the abdominal spiracles. These melanized protrusions appear most commonly in (but are not restricted to) abdominal segment 3: usually several spiracles can be seen to have material within them. It is presumed that this is coagulated hemolymph and reflects a loss of tracheal integrity. karst mutant escapers are very infertile and produce low numbers of eggs. It is unlikely that the karst mutation has a large effect on cell proliferation or viability in imaginal tissues, since neither the wings nor the eyes appear reduced in size. Immunoblots of karst mutant larvae indicate that, by the third instar, it is no longer possible to detect maternally contributed ß_H, so these phenotypes represent metamorphosis in the absence of detectable wild-type Karst protein (Thomas, 1998).

Immunolocalization of ß_Heavy-spectrin in wild-type eye-antennal and wing imaginal discs reveals that ß_Heavy-spectrin is present in a restricted subdomain of the membrane skeleton that colocalizes with Shotgun. A model is proposed where normal levels of Sevenless signaling are dependent on tight cell-cell adhesion facilitated by the ß_Heavy-spectrin membrane skeleton (Thomas, 1998).

Immunolocalization of ß_Heavy-spectrin in the adult and larval midgut indicates that it is a terminal web protein: no gross morphological defects are seen in the adult apical brush border in karst mutant flies. Similarly, rhodamine phalloidin staining of karst mutant ovaries reveals no conspicuous defect in the actin cytoskeleton or cellular morphology in egg chambers. This is in contrast to mutations in alpha-spectrin, the molecular partner of ß_Heavy-spectrin, which affect cellular structure in both the larval gut and adult ovaries (Thomas, 1998).

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. Apical ß_Heavy-spectrin (ß_H), 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 ß_H 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. The integrity of the migratory border cell cluster, a group of anterior follicle cells that delaminates from the follicle epithelium, is disrupted. Elimination of ß_H prevents the stable recruitment of alpha-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 (alphaß_H)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 (Zarnescu, 1999).

The distribution of ß_H was examined in developing wild-type ovarioles. ß_H expression in the germline is low to undetectable in the most anterior region of the germarium, and first appears in zone 2, at a time when 16 cell cysts become surrounded by a pool of follicle cells produced by asymmetric division of two to three somatic stem cells. In zone 3, fully formed stage 1 egg chambers emerge, ß_H is found uniformly along the nurse cell and oocyte membranes. Later, in mid-oogenesis, ß_H is slightly enriched on the outer edge of the ring canals. In the soma, ß_H 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. In close proximity to the 16 cell cysts in region 2, high levels of ß_H expression are detected in the vicinity of the somatic stem cells and in their progeny, the follicle cells. As individual cysts become enveloped in follicle cells, ß_H is slightly enriched in the cells that move in to segregate adjacent cysts. ß_H continues to be strongly expressed here because these become the stalk cells that separate successive egg chambers along the ovariole. ß_H is apically polarized in the follicle cells. ß_H is downregulated in the migrating border cells at stage 9, but is again expressed on the apical surface of these cells when they begin to secrete the micropyle after stage 10. At stage 10, ß_H is part of a prominent terminal web-like structure at the apical ends of the follicle cells that are secreting egg components. This structure appears to be anchored in the zonula adherens (ZA) by fine fibers of staining around its edge. ß_H is also expressed on the apical surfaces of the follicle cells secreting the dorsal appendages and the chorion. ß_H colocalizes with alpha-spectrin in the germline and soma at all these locations; however, the latter is more widespread, presumably through its association with the conventional ß isoform (Zarnescu, 1999).

In mid-stage 9 egg chambers, the follicle cell monolayer and the border cells migrate in a concerted fashion. However, in 73% of karst egg chambers the border cells migrate ahead of the follicle cell monolayer. The degree to which the border cells migrate ahead of the follicle cells during stage 9 is quite variable in keeping with the variable expressivity of the karst mutation. Occasionally egg chambers are found where the border cells are retarded relative to the follicle cells; however, this extreme situation probably arises from a combination of weak expression of the follicle cell phenotype combined with strong expression of the border cell phenotype. The lack of coordination in cell migration makes it difficult to assess the progression of each chamber through stage 9. A morphometric approach was therefore taken. The following defects are seen in karst mutant egg chamber morphogenesis. (1) Most karst border cell clusters are migrating ahead of the follicle cell monolayer during stage 9. This could arise either due to faster border cell migration or slower follicle cell migration. (2) 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. 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. (3) Similarly, most karst mutant oocytes occupy a larger portion of the egg chamber than in wild-type during border cell migration. 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 (Zarnescu, 1999).

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, pulling the oocyte membrane towards them and grossly distorting the nurse cells/oocyte interface. In most cases, the subsequent inward migration of the follicle cell layer at stage 10B proceeds along the nurse cell/oocyte interface in a relatively normal fashion. However, in rare, extreme cases, the centripetally migrating cells penetrate between nearby nurse cell membranes and cause one or more nurse cells to become included within the egg, along with the oocyte (Zarnescu, 1999).

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. Since there is no over-proliferation in the mutant monolayer, 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. The apical surface area of wild-type and mutant follicle cells has therefore been compared 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. In contrast, the majority of karst mutant follicle cells fail to apically constrict. The mean apical surface area of the mutant follicle cells is almost twice that of the wild-type. 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. This suggests that, in addition to the constriction defect, the monolayer cannot withstand the forces exerted by the growing oocyte (Zarnescu, 1999).

Examination of the follicle cell apices stained for Drosophila E-cadherin (Shotgun) also reveals conspicuous disruptions in the staining pattern of Shotgun in karst mutant egg chambers. In the mildest cases, this staining is missing at three- or four-cell vertices, but large breaks are also seen in the normally continuous belt of staining in more extreme cases. These observations are consistent with the hypothesis that the absence of ß_H 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. 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, migratory cells are observed that are well separated from, or trail behind, the main border cell cluster. The trailing cells upregulate alpha-spectrin and Shotgun in a manner that resembles wild-type clusters, suggesting that they are true border cells. These data suggest that the normal number of border cells is specified in karst mutants, but that the cluster is unable to remain together as a unit (Zarnescu, 1999).

ß_H was no longer detectable at the apical domain of the follicle cells in any allelic combination of karst alleles examined. The localization of ß_H to the apical domain is dependent on alpha-spectrin. To see if alpha-spectrin is dependent on ß_H for its localization to the apical domain, and to confirm that no apical spectrin function remains in karst mutants, the distribution of alpha-spectrin was examined in karst follicle cells. While the lateral alpha-spectrin distribution is unaffected by this mutation, apical alpha-spectrin is no longer detectable by immunofluorescence. This indicates that the stable recruitment of alpha-spectrin to the apical domain is dependent on ß_H, and that there is thus a mutual interdependence between alpha-spectrin and ß_H. This further suggests that alphaß_H-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 (Zarnescu, 1999).

Spectrin associated with Shotgun has been implicated in the apicobasal cell polarization pathway, and fly alpha-spectrin mutations cause a breakdown in monolayer polarity including the loss of apical ß_H. However, karst mutants form a follicle epithelium that appears to have a well polarized morphology. Given the significant similarity between ß_H and ß-spectrin, it is also possible that polarity is retained in karst mutant epithelia because of functional redundancy between these two proteins. Moreover, the karst mutant phenotype exhibits variable expressivity despite genetic evidence that the alleles are null, and this variable expressivity might also result from functional redundancy. Such redundancy would predict that conventional ß-spectrin would be recruited to the apical domain in the absence of ß_H. However, this is not the case, confirming that ß_H is unnecessary for apicobasal polarity and indicating that the source of variability in the karst phenotype must be sought elsewhere (Zarnescu, 1999).

Current models for the origins of epithelial polarity suggest that spectrin plays a key role in establishing and/or maintaining the apicobasal axis. This model is largely based on the behavior of molecules involved in establishing the basolateral domain. The combined observations on the alpha-spectrin and karst mutant phenotypes suggests that the basolateral membrane skeleton may be playing such a key role; however, these results indicate that apical spectrin [i.e., (alphaß_H)₂] does not. It remains unclear what the precise mechanism is by which apical spectrin acts during morphogenetic events. ß_H exhibits a close colocalization with the ZA, and its levels at this location are regulated in concert with Shotgun. Furthermore, mild disruptions of the ZA are observed in eye/antennal imaginal discs. None of the results to date reveal how closely (alphaß_H)₂ is associated with the ZA; however, the contrasting behavior of the apical and basolateral spectrin-based membrane skeleton during the emergence of apicobasal polarity, combined with the phenotypic data presented in this paper, strongly suggest that these two cytoskeletal structures have somewhat distinct rather than identical roles in their respective domains, at least when it comes to the generation and/or maintenance of apicobasal polarity (Zarnescu, 1999).

The results presented in this paper add to a growing body of evidence that apical spectrin is essential for epithelial morphogenesis. Moreover, it has been shown 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 ß_H or any other ß-spectrin plays a similar role in morphogenesis in vertebrates. The observations that ß_H is evolutionarily old, that there is a homolog in C. elegans encoded by the sma1 gene, and that the chicken brush border protein TW260 has a similar size and contour length as does ß_H (350 kD), all strongly suggest that there is a vertebrate homologue of ß_H that has yet to be cloned. The karst phenotype bears close resemblance to the phenotype exhibited by C. elegans embryos mutant at the sma1 locus that encodes the worm homolog of ß_H. In sma1 mutants, the worms fail to elongate at the wild-type rate during embryogenesis, resulting in a smaller embryo. Moreover, this phenotype seems to arise from a defective contraction of the embryonic epithelium. Thus, while the geometry of this developmental event is quite different from follicle cell migration in the fly, ß_H appears to be playing a similar role in the two organisms. In vertebrates, the apical accumulation of spectrin has been correlated with the initiation of neurulation in mice, and, in the sea urchin embryo, an apical accumulation of alpha-spectrin is associated with the involution of tissues during embryogenesis. This suggests that apical spectrin has a general role in the morphogenesis of epithelia mediated by apical contraction (Zarnescu, 1999).

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date revised: 20 December 2006

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