The genetic programs that control patterning along the gut dorsoventral (DV) axis have remained largely elusive. The activation of the Notch receptor occurs in a single row of boundary cells that separates dorsal from ventral cells in the Drosophila hindgut. rhomboid, which encodes a transmembrane protein, and knirps/knirps-related, which encode nuclear steroid receptors, are Notch target genes required for the expression of crumbs, which encodes a transmembrane protein involved in organizing apical-basal polarity. Notch receptor activation depends on the expression of its ligand Delta in ventral cells, and localizing the Notch receptor to the apical domain of the boundary cells may be required for proper signaling. The analysis of gene expression mediated by a Notch response element suggests that boundary cell-specific expression can be obtained by cooperation of Suppressor of Hairless and the transcription factor Grainyhead or a related factor. These results demonstrate that Notch signaling plays a pivotal role in determining cell fates along the DV axis of the Drosophila hindgut. The finding that Notch signaling results in the expression of an apical polarity organizer, one which, in turn, may be required for apical Notch receptor localization, suggests a simple mechanism by which the specification of a single cell row might be controlled (Fusse, 2002).
The regionalization of the hindgut tube involves the formation of three major subregions: the small intestine, which localizes to the anterior end of the hindgut; the large intestine, which represents the middle part and the rectum, the posterior part. The formation of the small intestine and the rectum has been shown to depend on Hedgehog and Wingless activities, which coordinate morphogenesis and cell differentiation in the hindgut. The steroid receptor-encoding genes knirps and knirps-related, which are expressed in banded expression domains in the small intestine and the rectum, are target genes of the Hedgehog and Wingless signaling pathways required for restricting endoreduplication cycles to the middle part of the hindgut, the large intestine (Fusse, 2002).
While studying the role of the kni and knrl genes which act redundantly during hindgut development, it was observed that both genes are also coexpressed in the large intestine, from germ band extension stage onward in two rows of lateral cells (20 ± 1) on each side of the tube. This expression is maintained until late stage 16. In the lateral cell rows, kni and knrl are coexpressed with the rhomboid (rho) gene that encodes a transmembrane protein involved in epidermal growth factor receptor (Egfr) signaling. rho gene expression in the lateral cells appears slightly earlier than kni/knrl gene expression and is also maintained until late stage 16. At the transitions to the small intestine and the large intestine, rho is expressed in two circular expression domains. The transmembrane protein and apical polarity determinant Crumbs (Crb) becomes strongly upregulated in the lateral cell rows after germ band extension stage and displays an unusual cellular distribution. This contrasts with the dorsal and ventral cells of the hindgut, where Crb is located at the apical cell margins -- Crb is localized to the entire apical domain of the lateral cell rows, and expression is maintained until the end of embryogenesis. Similarly, Discs lost (now redefined as Drosophila Patj), another apical polarity organizer, is located to the entire apical cell surface in these cells (Fusse, 2002).
Using various cell shape and cell polarity markers, such as the septate junction markers Fas III, Neurexin IV, and Discs lost, it was determined that the cells of the lateral cell rows show a flat and long-shaped morphology and that these cells separate homogenous cell populations in the dorsal and the ventral halves of the large intestine. The cells of the lateral cell rows can thus be considered boundary cells separating dorsal from ventral cells in the large intestine. The dorsal cells, which are big and columnar, express the homeodomain protein Engrailed (En) from extended germ band stage onward until late embryogenesis. In contrast, the ventral cells, which are small and cuboidal, display expression of Delta from extended germ band stage onward until late embryogenesis. Double immunostainings reveal that En expression in the dorsal half of the large intestine is adjacent and nonoverlapping to the kni/knrl/rho expression domains in the boundary cells. Similarly, the Delta expression domain in the ventral half is adjacent to the boundary cells, although coexpression at a low level in the boundary cells cannot be excluded. In summary, dorsal cells express En; boundary cells kni/knrl, rho, crb, and ventral cells express Delta (Fusse, 2002).
To investigate the role of the genes expressed in the large intestine, lack- and gain-of-function studies were performed. In amorphic Notch and Delta mutant embryos, kni/knrl, rho, and high levels of Crb expression on the apical plate are absent in the large intestine, and the boundary cell fate is not established. In contrast, ventral cell morphologies are normal in Notch or Delta mutant embryos, and En expression and dorsal cell fates are unchanged. This indicates that Notch signaling is required to establish the boundary cells but not for dorsal or ventral cell fates. To further test this, gain-of-function experiments were performed using the UAS/Gal4 system. As driver lines, the G445.2 or the 14-3-fkhGal4 strains were used -- they mediate ubiquitous gene expression in the developing hindgut from the extended germ band stage onward until late stage 16. In order to ectopically activate the Notch signaling pathway, flies carrying the Notch intracellular domain fragment, Nicd, under the control of UAS sequences were used. Expressing Nicd ubiquitously in the hindgut results in an ectopic induction of kni and of rho. In addition, the cellular localization of the Crb protein is affected in these embryos. In dorsal and ventral cells of the large intestine of wild-type embryos, Crb is localized to the apical cell margins, whereas it is localized to the entire apical plates of the boundary cells. In the embryos, in which Nicd is ectopically expressed, Crb protein is found on the apical plates of all the hindgut cells; in addition, it is found in high concentrations in vesicles, especially on the baso/lateral sides of the cells. A similar but less intensive ectopic expression of Crb can also be induced if both Kni and Rho are coexpressed in all the hindgut cells, suggesting that crb may be a downstream effector gene of Kni/Knrl and Rho activities. This is consistent with the analysis of rho7M; Df(3L) riXT1 mutants [Df(3L) riXT1 is a deficieny encompassing the kni and knrl transcription units] in which the expression of crb in the boundary cells is strongly reduced. In summary, these results suggest that rho, kni/knrl, and Crb are target genes which are activated in response to Notch signaling in the boundary cells (Fusse, 2002).
To investigate the relationship between rho and kni/knrl in the boundary cells, the expression of the genes in the respective mutants was examined. rho expression is still present in kni mutants and Df(3L) riXT1 mutants. Similarly, kni and knrl expression are maintained in amorphic rho7M mutants or EGF receptor mutants, such as faint little ball (flb). In flbIK35 mutants, the hindgut tube is much shortened due to a reduction of the cell number. However, banded expression of both genes is found in the small intestine and the rectum along the AP axis of the hindgut; expression is also found in a few cells in the large intestine region. Ectopic expression of rho using the corresponding UAS-effector line combined with a driver line that mediates ubiquitous expression in the hindgut does not result in ectopic kni/knrl gene expression and vice versa. This points toward rho and kni/knrl being regulated independently of one another (Fusse, 2002).
To study whether En, which is expressed in the adjacent dorsal cells, contributes to the boundary cell fate, the expression of kni/knrl, rho, and crb was examined in en mutants and in en; invected double mutants (enE), since en and invected are known to act redundantly. Whereas the expression of the Notch target genes remains unchanged in en mutants, it is absent in the large intestine of en; invected double mutants. Morphological studies indicate that the dorsal and the boundary cell fates are not established in these mutants, and the large intestine seems to consist entirely of the ventral cell fates. To investigate the cause for this effect, the expression of Delta was studied in these mutants and it was found to be expressed ubiquitously in the large intestine. These data indicate that a boundary between Delta expressing and nonexpressing cells is required for Notch receptor activation. Ectopic expression of En in the large intestine using the 14-3 fkh driver and UAS-En effector lines results in a repression of kni/knrl and rho gene expression. This indicates that En bears the potential to act as a negative regulator of Notch target genes. Upon ectopic activation of Notch signaling in the entire hindgut by expressing Nicd, En is repressed on the dorsal side of the large intestine, thus allowing ectopic activation of Notch target genes (Fusse, 2002).
In order to investigate whether Notch signaling in the large intestine of wild-type embryos is activated beyond the boundary cells but actively repressed dorsally and ventrally, flies that carry the chimeric Notch receptor/transcription factor fusion construct N-Gal4/VP16 were used and the range of Notch signaling was determined. Upon heat shock, this fusion protein, which is membrane bound, becomes ubiquitously expressed in the embryo. In cells in which the Notch receptor is activated by ligand binding, the intracellular Gal4-VP16 transcription factor moiety is cleaved off and is able to subsequently activate reporter gene expression in cells that carry a UAS-lacZ construct. The ß-Gal expression pattern of such embryos reflects the range of Notch signaling. When anti-ß-Gal stainings of embryos that were heat shocked and carried the N-Gal4/VP16 and UAS-lacZ constructs was performed, ß-Gal expression was observed exclusively in the lateral boundary cells of the large intestine, demonstrating that Notch signaling is restricted to the boundary cells only. To further test this, flies were used carrying a lacZ-reporter construct in which multiple Su(H) binding sites from the Enhancer of Split m8 gene were combined with binding sites for the transcription factor Grainyhead (Grh). In cells, in which Notch signaling is active and Grh is expressed, Su(H) cooperates with Grh to yield high levels of reporter gene expression, whereas reporter gene expression is repressed in cells in which Notch is inactive. Determining the activity pattern of this construct in the hindgut using anti-ß-Gal antibody stainings demonstrates that activation of the reporter gene occurs exclusively in the boundary cells of the large intestine, consistent with the N-Gal4/VP16 data (Fusse, 2002).
In the Drosophila wing imaginal disc, the Notch receptor is activated along the border between dorsal and ventral cells, leading to the specification of cells that express Wingless and organize wing growth and patterning. The range of Notch signaling is determined by the spatial and temporal expression pattern of its ligands, Delta and the transmembrane protein Serrate (Ser), and by the activity of the glycosyltransferase Fringe (Fng). Fng controls ligand selectivity of Notch and plays a major role in the Notch-dependent positioning of sharp compartment boundaries. Fng has been shown to modify the glycosylation state of the receptor in the Golgi complex, thereby lowering its sensitivity to Ser and raising its sensitivity to Delta. To investigate whether Fng or Ser are also taking part in restricting Notch signaling to the boundary cells in the hindgut, expression studies and lack- and gain-of-function analyses were performed. In situ hybridization using a Ser antisense probe or ß-Gal expression studies of a Ser-lacZ enhancer trap line show that Ser is not expressed in the large intestine of the hindgut, and Ser mutants show a normal hindgut. Furthermore, ectopically expressing Ser in all the hindgut cells has no effect on Notch target gene expression. In contrast, Fng is expressed in boundary and dorsal cells as shown by double immunostainings of Fng and En. However, in amorphic fng80 mutants, misspecification of boundary cells occurs only at a low frequency, and ectopic expression of Fng using the 14-3 fkh driver and UAS-Fng flies does not induce an ectopic activation of the kni/knrl and rho genes in the hindgut. These results indicate that Ser seems not to be required, and fng may play only a minor role in restricting Notch signaling to the boundary cells (Fusse, 2002).
These results suggest that the activation of the Notch receptor in the boundary cells of the hindgut is triggered by the binding of Delta, which is expressed at high levels in adjacent ventral cells. If Delta levels are uniform and this boundary condition is lost, as in enE mutants, Notch signaling fails to occur. To further obtain insight into how the spatial control of Notch receptor activation is mediated, the localization of the receptor was determined using antibody stainings to Notch. In ventral and dorsal cells, Notch is expressed in the apical cell margins, as can be demonstrated using coimmunostainings with Neurexin IV. However, in the boundary cells, the Notch receptor is positioned to the entire apical plate where it is colocalized with Crb or Discs-lost. To test whether the apical localization of the receptor is necessary for its signaling activity, amorphic crb mutants were studied in which the sorting of proteins to the apical domain of the cells is affected. In these mutants, a strong reduction of the number of boundary cells was found, although hindgut morphogenesis is only slightly affected. In addition, the remaining boundary cells are mislocalized, and two rows of cells are often found instead of a single row as is found in wild-type. Anti-Notch/anti-Kni double immunostainings of crb mutants demonstrate the reduction of apical Notch receptor localization in crb mutants. Furthermore, in cells in which the Notch receptor is not localized along the apical plate of the cells, the activation of Notch target genes fails to occur. These results indicate that apical localization of the receptor may be important for boundary cell fate determination (Fusse, 2002).
Thus a single row of boundary cells forms in between En-expressing dorsal cells and Delta-expressing ventral cells and is determined by Notch signaling. Unlike in wing imaginal disc development, Ser seems not involved, and the glycosyltransferase Fringe plays only a minor role for the proper positioning of the DV boundary in the large intestine. These results rather suggest two major determinants that control where Notch signaling can occur in the hindgut: (1) the localization of Delta, which is expressed in ventral cells at high levels and not expressed (or at very low levels) in the adjacent boundary cells in which Notch signaling is eventually activated; (2) the Crb-dependent transport of the Notch receptor to the apical membrane domain of the boundary cells. How the initial En and Dl expression domains are set up in the large intestine is not known. It is noted, however, that the En expression domain in the hindgut primordium is initially broader in early stage 7 embryos and only subsequently refines to the dorsal cells, whereas Dl expression is confined to ventral cells from early stage 7 onward. It is thus possible that Notch signaling in the boundary cells leads to a cell-autonomous repression of en expression, consistent with the repression of en upon Nicd overexpression. Immunhistological studies show that the proper specification of boundary cells in crb mutants correlates with the apical localization of the Notch receptor. The finding that Crb expression in the boundary cells depends on Notch signaling suggests the possibility of a feedback loop that ensures proper receptor localization required for establishing the competence of the boundary cells to receive the Delta signal. It cannot be excluded, however, that the failure of Notch signaling in crb mutants may also be caused by the mislocalization of other localized proteins. It is noteworthy that the apical side of the boundary cells faces the lumen of the hindgut. Activating Notch receptors along the entire apical plate of the boundary cells would therefore require also a secreted form of Delta. The extracellular domain of Delta has been found as a soluble product in the supernatant of Drosophila cultured cells and in embryonic extracts, and has been shown to arise by a proteolytic activity of the ADAM metalloprotease Kuzbanian. Both soluble forms of Delta and Serrate are able to act as antagonists and agonists of the Notch pathway in vivo. It is possible that such a form of Delta and/or additional apically localized factors are involved in binding and activating the Notch receptor locally in the boundary cells of the hindgut (Fusse, 2002).
These results further demonstrate that Notch signaling induces the expression of the rho and kni/knrl genes and that both components are required, in turn, for the expression of Crb. It has been suggested recently that Su(H) functions as a core of a molecular switch by which the transcription of Notch target genes is regulated. In the absence of Notch signaling, Su(H) functions as a repressor, and, in the presence of Notch signaling, Su(H) can cooperate synergistically with other transcriptional activators to induce transcription of target genes. The finding that boundary cell-specific reporter gene expression can be induced in the hindgut by using a model Notch response element [composed of binding sites for Su(H) and the widely expressed activator Grainyhead] suggests the possibility that the localized activation of the rho and kni/knrl genes could rely on the same factors and the same molecular switch mechanism that has recently been proposed for this element and for Notch-dependent atonal and single minded expression. In evolutionary terms, the gut is most likely one of the most ancient organs that evolved in multicellular organisms. Consistently, the morphological processes involved in the development of the gastrointestinal tract of animals are highly similar. It remains to be shown whether or not the evolutionarily conserved regulators of the Notch signaling cascade also determine dorsoventral aspects of gut development in other animals, including vertebrates (Fusse, 2002).
These results provide evidence that Notch signaling in the Drosophila hindgut controls the fate of a single row of boundary cells separating the dorsal and ventral halves of the gut tube. Activation of the Notch receptor in the boundary cells is mediated by its ligand Delta that is expressed in adjacent ventral cells. The induction of Notch target genes activate the expression of the apical polarity organizer Crb, which may be required, in turn, for apical Notch receptor localization. These findings identify a simple mechanism that controls the specification of a single row of DV boundary cells in an animal gut (Fusse, 2002).
Among the diverse cellular processes taking place during oogenesis, the delamination and migration of border cells (BCs), a group of anterior follicle cells, represent a powerful model to study cell invasion in a normal tissue. During stage 9 of oogenesis, BCs detach from the outer epithelium to invade the germline cyst compartment. The BC cluster contains two centrally located polar cells surrounded by approximately six outer border cells and undergoes a nearly 6-hour long posteriorward migration to reach the anterior part of the growing oocyte. Together with centripetal cells, they assemble the micropyle, a specialized structure required for sperm entry. domeless was isolated in a screen to identify genes essential in epithelial morphogenesis during oogenesis. The level of dome activity is critical for proper border cell migration and is controlled in part through a negative feedback loop. In addition to its essential role in border cells, dome is required in the germarium for the polarization of follicle cells during encapsulation of germline cells. In this process, dome controls the expression of the apical determinant Crumbs. In contrast to the ligand Upd, whose expression is limited to a pair of polar cells at both ends of the egg chamber, dome is expressed in all germline and follicle cells. However, Dome protein is specifically localized at apicolateral membranes and undergoes ligand-dependent internalization in the follicle cells. dome mutations interact genetically with JAK/STAT pathway genes in border cell migration and abolish the nuclear translocation of Stat92E in vivo. dome functions downstream of upd and both the extracellular and intracellular domains of Dome are required for JAK/STAT signaling. Altogether, the data indicate that Dome is an essential receptor molecule for Upd and JAK/STAT signaling during oogenesis (Ghiglione, 2002).
The dramatic, early follicle cell phenotype contrasts with the essentially normal phenotype of dome mutant cells observed in later stage egg chambers. In this case, follicle cells are viable and divide normally. A similar, dual phenotype has been reported in crumbs mutant chambers. After initial polarization of the follicle cells in the germarium, Crumbs is no longer required and its loss has no visible effects. Importantly, dome controls Crumbs expression in follicle cells, thus providing a novel link between the JAK/STAT signaling pathway and epithelial polarity (Ghiglione, 2002).
In addition to its early function in the germarium, dome is required for the normal expression of several follicle cell markers, including DE-cadherin and Fas3. It is important to note that despite a clear defect in the expression of these markers, dome mosaic egg chambers are morphologically normal. However, because completely mutant egg chambers cannot be obtained because of the early effect of dome in the germarium, one cannot rule out the possibility that large mutant clones would lead to abnormal development of egg chambers (Ghiglione, 2002).
The pattern of epithelial markers in dome mutant cells indicates that the JAK/STAT pathway is active in all follicle cells, a notion that is reinforced by the wide expression of nuclear Stat92E. How is Dome activated during egg chamber development and does this activation follow the same profile at all stages? Given the restricted pattern of upd expression in the egg chamber and its dramatic effect upon overexpression, it is unlikely that Upd is able to signal long distances in the follicular epithelium of late stage egg chambers. Rather, a model by which the JAK/STAT pathway plays a pre-patterning function is favored, acting early during egg chamber development to activate DE-cadherin and Crumbs expression. This view is consistent both with the expression pattern of upd and the distribution of Dome-containing vesicles described in this study. The formation of endogenous vesicles can be promoted by Upd, and a gradient of such vesicles is present around polar cells. Strikingly, these vesicles, which likely indicate active signaling through Dome, are widespread at early stages and become more restricted later on. It is proposed that during early development, the Upd signal produced by anterior and posterior polar cells contributes to the differentiation of all follicle cells. At this stage, Upd would be more diffusible than later, as suggested by the pattern of Dome intracellular vesicles. The study of the mechanisms controlling Dome activation and Upd activity will require additional tools to directly detect Upd, as, for example, Upd-GFP fusion proteins (Ghiglione, 2002).
This study has revealed several new findings about the function of dome and the JAK/STAT pathway during oogenesis. Future work will help to understand how Upd and Dome initially interact at the cell surface and transduce the signal to downstream JAK/STAT pathway members (Ghiglione, 2002).
Formation of tubes of the correct size and shape is essential for viability of most organisms, yet little is understood of the mechanisms controlling tube morphology. A new allele of hairy has been identified in a mutagenesis screen. hairy mutations cause branching and bulging of the normally unbranched salivary tube, in part through prolonged expression of huckebein (hkb). Hkb controls polarized cell shape change and apical membrane growth during salivary cell invagination via two downstream target genes, crumbs (crb), a determinant of the apical membrane, and klarsicht (klar), which mediates microtubule-dependent organelle transport. In invaginating salivary cells, crb and klar mediate growth and delivery of apical membrane, respectively, thus regulating the size and shape of the salivary tube (Myat, 2002).
Shark is a single protein that possesses a tyrosine kinases, ankyrin repeat, and Src homology 2 domains. During Drosophila embryogenesis, shark is expressed exclusively by ectodermally derived epithelia and is localized preferentially to the apical surface of these cells. This apical localization persists, even as tissues undergo complex invaginations, moving from the external surface of embryos to form internal structures, but expression is lost when cells lose their polarity. This pattern closely mimics the expression of Crumbs. Shark's structure and localization pattern suggest that it functions in a signaling pathway for epithelial cell polarity, possibly transducing the Crumbs signal (Ferrante, 1995).
Cell interactions mediated by Notch family receptors have been implicated in the specification of tissue boundaries. Tightly localized activation of Notch is crucial for the formation of sharp boundaries. In the Drosophila wing imaginal disc, the Notch receptor is expressed in all cells. However, Notch activity is limited to a narrow stripe of cells along the dorsal-ventral compartment boundary, where it induces the expression of target genes. How a widely expressed protein becomes tightly regulated at the dorsal-ventral boundary in the Drosophila wing is not completely understood. This study shows that the transmembrane protein Crumbs is involved in a feedback mechanism used by Notch to refine its own activation domain at the Drosophila wing margin. Crumbs reduces the activity of the gamma-Secretase complex, which mediates the proteolytic intracellular processing of Notch. These results indicate a novel molecular mechanism of the regulation of Notch signal, and also that defects in Crumbs might be involved in similar abnormal gamma-Secretase complex activity observed in Alzheimer's disease (Herranz, 2006; full text of article).
Crumbs associates with the Stardust and DPATJ proteins through its short cytoplasmic tail to establish apical–basal cell polarity in the embryo. Expression of this cytoplasmic tail in a mutant background for crb is sufficient to partially rescue the failure in apical–basal cell polarity, indicating that the large extracellular domain of Crb is dispensable for this process. Three different observations indicate that the extracellular domain of Crb is required to attenuate Notch signalling and that the intracellular domain is dispensable. Mutant clones for a null allele of stardust (sdtXP96) are able to cover large areas of the wing without any overt phenotype when abutting the DV boundary or when running along the longitudinal veins. The crb mutant wing phenotype can be rescued when simultaneously expressing either full-length Crb or a truncated form of Crb lacking the whole intracellular tail (Crb-Extra-TM). Overexpression of Crb-Extra-TM leads to a mild downregulation of the Notch signalling pathway. In the adult wing, veins are thicker, resembling a Notch loss-of-function phenotype. In the wing imaginal disc, Crb-Extra-TM overexpression reduces the expression levels of Cut at the DV boundary, a target of Notch that requires high levels of Notch activity. Wg expression is not affected (Herranz, 2006).
Signalling centres along compartment boundaries are required to organize the growth and pattern of the surrounding tissue. However, too much of a signal has deleterious effects. The Notch signalling center organizes the growth and pattern of the developing wing primordium, partially through the secreted protein Wingless. Wingless activity contributes to limit Notch activity to cells immediately adjacent to the DV boundary. This study presents evidence that Notch also contributes to the refinement of its activation domain through its target gene crumbs. Crumbs attenuates Notch signalling by repressing the activity of the gamma-Secretase complex. Many loss-of-function mutations in the human homologue of Crumbs, CRB1, cause recessive retinal dystrophies, including retinitis pigmentosa. Given the fact that the gamma-Secretase complex also mediates the intracellular cleavage of the transmembrane protein APP, leading to accumulation of the Aβ peptide in plaques in AD, it is postulated that Crumbs may also be involved in modulating AD pathogenesis. The analysis indicates a role for the extracellular part of the Crb protein in this process. It is interesting to note that many mutations that give rise to retinal dystrophies are missense mutations that affect different EGF or LG domains of CRB1. Thus, molecular interactions mediated by the extracellular domain of Crb may be crucial in both types of disease (Herranz, 2006).
The polarized architecture of epithelial tissues involves a dynamic balance between apical and basolateral membrane domains. This study shows that epithelial polarity in the Drosophila embryo requires the exocyst complex subunit homolog Exo84. Exo84 activity is essential for the apical localization of the Crumbs transmembrane protein, a key determinant of epithelial apical identity. Adherens junction proteins become mislocalized at the cell surface in Exo84 mutants in a pattern characteristic of defects in apical, but not basolateral, components. Loss of Crumbs from the cell surface precedes the disruption of Bazooka and Armadillo localization in Exo84 mutants. Moreover, Exo84 mutants display defects in apical cuticle secretion that are similar to crumbs mutants and are suppressed by a reduction in the basolateral proteins Dlg and Lgl. In Exo84 mutants at advanced stages of epithelial degeneration, apical and adherens junction proteins accumulate in an expanded recycling endosome compartment. These results suggest that epithelial polarity in the Drosophila embryo is actively maintained by exocyst-dependent apical localization of the Crumbs transmembrane protein (Blankenship, 2007).
Epithelial cells in the Drosophila embryo generate molecularly distinct apical and basolateral surfaces that provide structural integrity to the developing embryo. Specialized cell surface domains are separated by intercellular adherens junctions that initiate as diffuse apicolateral accumulations and subsequently coalesce to form a discrete apical band called the zonula adherens. The spatial organization of mature adherens junctions is actively maintained by input from both apical and basolateral proteins. The Crumbs EGF-repeat transmembrane protein and its cytoplasmic binding partners Stardust and PATJ localize to the apical cell surface and are required for epithelial structure and adherens junction morphology. In addition, overexpression of Crumbs leads to a selective expansion of the apical cell surface, demonstrating that Crumbs is necessary and sufficient for apical identity. The localization of mature adherens junctions also requires the basolateral PDZ-domain proteins Discs large (Dlg) and Scribble (Scrib) and the WD40-domain protein Lethal giant larvae (Lgl) Epithelial defects caused by disruption of apical Crumbs activity can be rescued by a simultaneous reduction in the activity of basolateral proteins, indicating that apical and basolateral domains function in opposition to maintain epithelial polarity (Blankenship, 2007 and references therein).
Misregulation of Crumbs activity can have severe effects on cell and tissue function and is associated with human retinal diseases. Multiple mechanisms contribute to Crumbs localization, stability and activity to precisely control its function. The basolateral proteins Dlg, Lgl and Scrib oppose Crumbs activity and restrict its localization in the Drosophila embryo, and the Yurt FERM-domain protein associates with the Crumbs cytoplasmic domain and negatively regulates Crumbs activity at the apicolateral cell surface. Endocytosis of Crumbs protein is also required for tissue morphology; mutations in the Avalanche syntaxin or the Rab5 GTPase lead to Crumbs accumulation and wing imaginal disc overgrowth. In addition, a complex containing the Rich1 Cdc42 GAP protein and the angiomotin scaffolding protein associates with cytoplasmic binding partners of Crumbs and provides a potential link between the Crumbs complex and the endocytic machinery. However, the mechanisms that govern the delivery of Crumbs protein to the cell surface are not known (Blankenship, 2007).
The targeting of transmembrane proteins to specific destinations at the cell surface is a widely used mechanism for establishing cell polarity. The spatial specificity of vesicle trafficking is thought to occur at a late step in this process through the tethering of exocytic vesicles at defined membrane sites by the eight-subunit exocyst (or Sec6/8) complex. Exocyst components were originally identified based on their role in polarized secretion in Saccharomyces cerevisiae and were subsequently shown to form a complex that is highly conserved from yeast to mammals. In multicellular organisms, exocyst components are required for multiple developmental processes including epithelial polarity, membrane integrity, photoreceptor morphogenesis, cell fate determination and synapse formation. These diverse functions demonstrate that polarized exocytosis is a fundamental mechanism for regulating cell morphology (Blankenship, 2007).
This study provided evidence that the Drosophila homolog of the Exo84 exocyst complex subunit is essential for epithelial polarity and apical protein localization in the Drosophila embryo. In Exo84 mutants, adherens junction proteins become mislocalized along the apical-basal axis in a manner reminiscent of cells lacking the Crumbs apical determinant. Loss of Crumbs from the apical surface is the earliest defect detected in Exo84 mutants. Exo84 mutants at advanced stages of epithelial degeneration display defects in trafficking apical and junctional proteins from the recycling endosome to the cell surface. These results demonstrate that the Drosophila homolog of the exocyst complex subunit Exo84 plays an essential role in epithelial polarity by regulating the localization of the Crumbs apical determinant (Blankenship, 2007).
It is concluded that epithelial polarity in the Drosophila embryo is actively maintained by the Exo84-dependent localization of the Crumbs transmembrane protein to the apical surface. Exo84 mutants display an aberrant distribution of junctional proteins that resembles the phenotype of crumbs mutants, and depletion of Crumbs from the apical surface is the earliest defect detected in Exo84 mutants. In addition, the onset of epithelial disruption at stage 9 in Exo84 mutants is comparable with the timing of the crumbs mutant defects, and the Crumbs protein still aggregates in Exo84 embryos with greatly reduced E-cadherin. Exo84 is likely to function as part of the exocyst complex in the Drosophila embryo, in light of the genetic interactions observed between Exo84 and the Sec5 and Sec6 exocyst subunits and the common defects in recycling endosome morphology caused by exocyst disruption in multiple cellular contexts. These results suggest a role for exocyst-dependent membrane trafficking in the maintenance of apical epithelial identity in the Drosophila embryo (Blankenship, 2007).
In contrast to the relatively specific mislocalization of Crumbs in stage 9 Exo84 mutant embryos, by late stage 10 these embryos display defects in the delivery of multiple proteins to the cell surface. Epithelial polarity and the distribution of apical and junctional proteins are established correctly in Exo84 mutants, either because these processes occur independently of Exo84 or because of residual Exo84 activity in this mutant background. The earliest defect observed in Exo84 mutants is a loss of Crumbs from the apical surface during epithelial maturation. As a likely consequence of the loss of cell-surface Crumbs localization, adherens junction proteins become mislocalized to varying positions along the basolateral cell membrane. Mutant embryos at later stages display a cytoplasmic accumulation of apical and adherens junction proteins in an expanded Rab11 recycling endosome compartment, consistent with a defect in vesicular transport to the cell surface. The failure to deliver junctional proteins to the cell surface is unlikely to result from a defect in Crumbs localization, because the cytoplasmic accumulation of junctional proteins does not occur in crumbs mutants. These results indicate that disruption of exocyst-dependent membrane trafficking ultimately results in the failure to deliver both apical and junctional proteins from the recycling endosome to the cell surface. The mislocalization of apical and junctional proteins in Exo84 mutant embryos is associated with a loss of columnar morphology, demonstrating that Exo84 activity is essential for epithelial organization (Blankenship, 2007).
A precise balance between apical and basolateral determination is essential for epithelial integrity and the placement of the zonula adherens in the Drosophila embryo. This balance is actively maintained by Exo84-dependent localization of the Crumbs transmembrane protein to the apical cell surface. Loss of apical or basolateral identity leads to distinct patterns of junctional protein distribution, suggesting that the apical and basal limits of the zonula adherens are defined by different mechanisms. In crumbs mutants, DE-cadherin and Armadillo are restricted to focused puncta at varying locations at the cell surface. By contrast, in embryos defective for the basolateral proteins Dlg and Lgl, junctional proteins are dispersed along the plasma membrane rather than aggregating at a single site. A basolateral expansion of the apical Crumbs domain has also been reported in dlg and lgl mutants. These results suggest that basolateral proteins create a nonpermissive barrier to adherens junction expansion, whereas apical proteins may play a positive role in recruiting or stabilizing junctions at the apical cell surface. Consistent with this possibility, the apical Crumbs domain is closely apposed to the zonula adherens, and it was found that Bazooka and Armadillo colocalize at the cell surface and in the cytoplasm of Exo84 mutant embryos. Exocyst-dependent trafficking of Crumbs to the apical surface may reinforce the apical epithelial domain and stabilize the apicolateral localization of the zonula adherens (Blankenship, 2007).
The recycling endosome is the primary vesicular compartment affected in embryos mutant for the exocyst subunit homolog Exo84, while Golgi, early endosomal and late endosomal compartments remain largely intact. Exocyst proteins are required for recycling endosome morphology in several epithelial and sensory cell types and the Rab11 recycling endosome protein can associate directly with the exocyst subunits Sec5 and Sec15. It was found that Rab11 vesicles in maturing embryonic epithelia are enriched in the apical cytoplasm, where they preferentially accumulate in the plane of the adherens junctions. Conversely, a basal expansion of recycling endosomes during cellularization correlates with a basal bias in membrane addition. These results suggest that there is a spatial correlation between the sites of recycling endosome accumulation and the surface destinations of proteins trafficked through recycling endosomes. The redistribution of the recycling endosome compartment to the apical cytoplasm accompanies the transition from basolateral to apical membrane insertion and may reflect the onset of a critical requirement for Crumbs activity during epithelial maturation (Blankenship, 2007).
The requirement for Exo84 in apical protein localization in the Drosophila embryo is distinct from exocyst functions in other epithelia, in which exocyst components are required for the localization of basolateral or junctional proteins. The results indicate that Exo84 is also required for delivery of DE-cadherin to the cell surface in the embryo, consistent with the demonstrated roles for Sec5, Sec6 and Sec15 in DE-cadherin trafficking in the pupal epithelium. However, although the mislocalization of the apical Crumbs protein is a primary defect of Exo84 mutant embryos, exocyst mutations do not appreciably affect Crumbs localization in pupal epithelial and photoreceptor cells. These results are consistent with a model in which distinct cargo proteins are trafficked by the exocyst complex in different cellular contexts. Alternatively, DE-cadherin and Crumbs may be delivered to the cell surface in an exocyst-dependent fashion in multiple cell types, but undergo different rates of turnover. For example, Crumbs may be dynamically trafficked in the embryo but stably maintained at the surface of pupal epithelial cells. Differential effects on specific target proteins are not atypical of exocyst function, because loss of Sec6 activity in Drosophila photoreceptor cells disrupts the localization of the rhabdomere proteins Chaoptin and Rhodopsin1, whereas the apical localization of Crumbs and DE-cadherin occurs normally. The Drosophila embryonic epithelium undergoes pronounced changes in structure and organization during development that rely on a balance between apical and basolateral surface domains. A requirement for exocyst-dependent Crumbs trafficking during this process may facilitate the dynamic remodeling of epithelial polarity during morphogenesis (Blankenship, 2007).
Discs lost (Dlt) is expressed abundantly in ovaries. To assess the consequences of the loss of maternal Dlt in embryos, the dominant female sterile technique was used. However, females that lay eggs could not be recovered, even though more than 50% of the control females produce eggs. Hence, Dlt is essential for oogenesis. To circumvent the inability to produce eggs with reduced maternal Dlt, dsRNA interference was carried out. This process had previously been shown to be effective in C. elegans and Drosophila. The effects of dsRNA treatment soften the effects of mutation in other systems. dlt dsRNA-injected embryos express Dlt at very low levels, or not at all. More importantly, 3- to 4-hr-old injected embryos lack the columnar cell morphology. Instead, the epithelium consists of small cuboidal cells that are about one-fourth the length of the same cells in control embryos. These cells have completed cellularization, since low levels of Dlt and Dlg are present in the basal area. However, unlike control embryos where Dlg is localized laterally, Dlg is localized apically and laterally. These observations, combined with the expression pattern of Dlt, indicate that Dlt is required to establish epithelial polarity during cellular blastoderm formation. Since Crumbs is expressed after cellular blastoderm, at the onset of gastrulation, injected embryos were aged to germband extension (stage 11) and immunostained with anti-Dlt and anti-CRB. dlt dsRNA-injected embryos fail to express Dlt but do express CRB. At higher magnification, control-injected embryos show normal cell morphology and localization of cell junction-specific proteins, but dlt dsRNA-injected embryos display a completely aberrant Crb localization and embryo morphology. Thus, removal of Dlt prior to cellularization causes a complete lack of polarity based on morphological features and Crb localization (Bhat, 1999).
To determine whether ectopic dlt expression affects epithelial polarity, dlt was expressed using a heat shock-GAL4/UAS-dlt bipartite system. Four- to seven-hour-old embryos that received a 1 hr heat shock at 37.5 degrees C do not survive, whereas heat-shocked control embryos showed no lethality. The distribution of Dlt and Crb in ectodermally derived epithelia of control embryos is not altered by heat shock alone, and both proteins are localized apically. However, overexpression of dlt leads to an irregular epithelial cell layer with abnormally shaped cells that cannot maintain an epithelial context. A redistribution of Crb localization to basal and baso-lateral areas was also observed in some cells that overexpress Dlt. These data indicate that epithelial polarity is at least partially lost in the presence of additional Dlt protein, possibly because of the aberrant distribution of Crb (Bhat, 1999).
The apical localization of Dlt and Crb suggests that Dlt may interact with Crumbs. Previous studies have shown that deletion of 23 carboxy-terminal amino acids of the Crb protein leads to a null mutant phenotype (Wodarz, 1993 and Wodarz, 1995); therefore, an investigation was carried out to see whether the two proteins interact using a GST-Crb fusion protein containing the 37 cytoplasmic amino acids of CRB. GST pull down experiments show that full-length T7-tagged Dlt interacts with the cytoplasmic domain of Crb (GST-CRB). However, Dlt that lacks the first PDZ domain fails to bind GST-CRB, suggesting that the first PDZ domain is required for Crb binding. In addition, Dlt from adult extracts also binds to GST-Crb, indicating that both proteins bind to one another in vivo (Bhat, 1999).
To determine whether loss of crb had any effect on the localization of Dlt, crb mutant embryos were double labeled with anti-Crb and anti-Dlt. Dlt fails to localize to the apical side in the absence of Crb protein and is distributed over the entire cell membrane and cytoplasm in embryos that lack Crb. Hence, Dlt depends on Crb for its proper subcellular localization at later developmental stages (Bhat, 1999).
Analysis of the mechanisms that control epithelial polarization has revealed that cues for polarization are mediated by transmembrane proteins that operate at the apical, lateral, or basal surface of epithelial cells. Whereas for any given epithelial cell type only one or two polarization systems have been identified to date, the follicular epithelium in Drosophila ovaries uses three different polarization mechanisms, each operating at one of the three main epithelial surface domains. The follicular epithelium arises through a mesenchymal-epithelial transition. Contact with the basement membrane provides an initial polarization cue that leads to the formation of a basal membrane domain. Moreover, mosaic analysis was used to show that Crumbs (Crb) is required for the formation and maintenance of the follicular epithelium. Crb localizes to the apical membrane of follicle cells that is in contact with germline cells. Contact to the germline is required for the accumulation of Crb in follicle cells. Discs lost (Dlt), a cytoplasmic PDZ domain protein that has been shown to interact with the cytoplasmic tail of Crb, overlaps precisely in its distribution with Crb, as shown by immunoelectron microscopy. Crb localization depends on Dlt, whereas Dlt uses Crb-dependent and -independent mechanisms for apical targeting. The cadherin-catenin complex is not required for the formation of the follicular epithelium, but only for its maintenance. Loss of cadherin-based adherens junctions caused by armadillo mutations results in a disruption of the lateral spectrin and actin cytoskeleton. Also Crb and the apical spectrin cytoskeleton are lost from armadillo mutant follicle cells. Together with previous data showing that Crb is required for the formation of a zonula adherens, these findings indicate a mutual dependency of apical and lateral polarization mechanisms (Tanentzapf, 2000).
Drosophila ovaries are composed of ovarioles, each representing an anterior-posterior series of follicles of increasing age. The assembly of follicles takes place in the germarium that is located at the anterior tip of each ovariole. During follicle formation, ~30 follicle cells form an epithelial monolayer that surrounds a cluster of 16 germline cells. Whereas the germline cells increase in size but not in number, the cells in the follicular epithelium (FE) proliferate so that the FE in a mature follicle contains ~650 cells. A second population of follicle cells that does not establish direct contact with germline cells forms short stacks by cell intercalation, giving rise to the interfollicular stalk (Tanentzapf, 2000).
Crb is initially expressed in all follicle cells at stage 1 of oogenesis, but its expression becomes rapidly restricted to the cells of the FE, and is not maintained in the interfollicular stalk. Crb is found in the apical membrane of the cells of the FE. Dlt shows a temporal and spatial distribution similar to Crb in follicle cells, suggesting that both proteins might interact physically in follicle cells as they do in embryonic epithelia. Dlt also associates in vitro with Neurexin IV, a transmembrane component of the septate junction. The septate junction, which blocks paracellular diffusion similar to the chordate tight junction, is located basally to the ZA. To reconcile these data, it has been proposed that Dlt interacts with Crb apically to the ZA, with the ZA itself, and with Neurexin IV basally to the ZA. To clarify the subcellular localization of Dlt, the distribution of Dlt in the FE and in the embryonic ectoderm and epidermis was determined by interference electron microscopy (IEM). IEM reveals that Dlt, similar to Crb, is confined to the apical membrane. Within the apical membrane, Dlt accumulates at the marginal zone, an area of cell-cell contact apical to the ZA, and shows a lower concentration throughout the apical surface. The signal for Dlt at the ZA or basally to it does not exceed background levels, and no signal is observed at septate junctions. These findings suggest that Dlt does not physically interact with Neurexin IV at the septate junction, and further corroborates the notion that Dlt and Crb form a complex at the apical membrane of epithelial cells (Tanentzapf, 2000).
Crb and Dlt are also expressed in the germline, although the distribution of both proteins overlaps only partially. Dlt and Crb colocalize at the membrane of the nurse cells during early and mid oogenesis. High levels of Crb are seen in the plasma membrane of the oocyte, whereas Dlt is not detectable. Dlt accumulates transiently at the ring canals that connect nurse cells. During late stages of oogenesis, when the content of the nurse cells is rapidly transferred to the oocyte, Dlt is found at the nuclear membrane in close association with actin filaments that connect the nuclei of the nurse cells with the plasma membrane. Previous germline clone experiments did not reveal a function for crb in the germline. In contrast, maternal expression of dlt is essential for egg production, suggesting that dlt plays an important role in germline development. The function of dlt in germline development requires more detailed study (Tanentzapf, 2000).
The follicle cells are generated by two stem cells that are located in the middle of the germarium at the boundary of region 2a and 2b. Offspring of these stem cells establish contact with the basement membrane that surrounds the germarium and the follicles. Analysis of agametic ovaries has shown that the contact between follicle cells and germline cells is required for the formation of the FE. Follicle cells continue to proliferate in agametic ovaries and form a column that is two to three cells wide. To determine whether, and to what extent, contact between follicle cells and the basement membrane contributes to the polarization of the FE, follicle cells were examined in agametic ovaries. In agametic ovaries, ßPS-integrin localizes to the basal membrane of follicle cells as in wild type. Markers that normally localize to the lateral membrane (Fasciclin III and N-cadherin), to the lateral and apical membranes (E-cadherin and Armadillo), or to the apical membrane (ßHeavy-spectrin) are excluded from the basal cell pole. Apical and lateral markers show an overlapping distribution at the non-basal cell surface, but are concentrated at the cell pole that opposes the basal membrane. These findings suggest that contact to the basement membrane causes a partial polarization of follicle cells. The basal membrane is established and an asymmetric distribution of apical and lateral markers is observed, but the apical and lateral membrane domains are not clearly demarcated. As the follicle cells express the cadherin-catenin complex constitutively, it appears that basal polarization cues together with the activity of the cadherin-catenin complex are insufficient to fully polarize follicle cells. Interestingly, the presence of germline cells is required for the accumulation of Crb in follicle cells, since Crb is not detected in follicle cells in agametic ovaries (Tanentzapf, 2000).
To study the role of crb and dlt in the development of the FE, homozygous mutant follicle cell clones were generated. Clones were induced for the two protein-negative crb null alleles crb11A22 and crbB82, for the dlt protein negative null allele dltMY10, and the hypomorphic protein-positive allele dltdre1. Two types of mutant follicle cell clones were examined. Clones were generated either before the formation of the FE by inducing mitotic recombination in the follicle stem cells or their immediate offspring. Alternatively, clones were induced after the formation of the FE by inducing mitotic recombination in region 2b or stage 1 follicles. Early induced clones can be distinguished from late induced clones by their larger size, since all follicle cells derive from only two stem cells, and follicle cells continue to proliferate during early stages of follicle development. Clones are considered as induced early (early clones) if they comprise 15% or more of the cells of the FE of an individual follicle. Early clones can make up the entire FE of a single follicle. In contrast, late-induced clones (late clones) typically comprise less than 10 cells at mid to late stages of oogenesis (Tanentzapf, 2000).
A variable phenotype is observed when crb mutant follicle cells are generated before the FE forms. Many experimental follicles show an incomplete FE with areas in which the germline cells are not covered by follicle cells, a defect that is never seen in wild-type follicles. The area of a follicle not covered by a FE varies greatly in size and in some cases comprises most of the follicle. These gaps in the FE are likely caused by the failure of crb mutant follicle cells to integrate into the FE. The missing follicle cells could not be followed and it is presumed that they degenerate within the germarium. In contrast, some crb mutant follicle cells can form a FE with apparently normal morphology. The distribution of various markers was analyzed in crb mutant follicle cells that were part of the FE. Such crb mutant follicle cells retain apical Dlt, although the level of Dlt associated with the apical membrane is reduced in these cells. The level of ßHeavy-spectrin is also slightly reduced compared with wild-type follicle cells. These findings indicating that the apical localization of Dlt and ßHeavy-spectrin depends on Crb, but also on other mechanisms. No significant alteration was noticed in the level or distribution of Arm (Tanentzapf, 2000).
dltMY10 and dltdre1 mutant follicle cell clones, if generated before the formation of the FE, display gaps of variable size in the FE similar to those caused by crb mutations. Since dltdre1 is a protein-positive allele, it could not be determined whether some dltdre1 mutant follicle cells become part of the FE. In contrast to crb mutant follicle cell clones, no dltMY10 mutant follicle cells were found that participate in the formation of the FE in early clones. dltMY10 is a deletion that removes the genes encoding cdc37 and alpha-spectrin, in addition to dlt. The loss of cdc37 is compensated for by a transgene. Thus, dltMY10 mutant clones lack dlt and alpha-spectrin, raising the possibility that the observed mutant phenotype is the consequence of a synergistic effect between dlt and alpha-spectrin mutations, although it has been shown that alpha-spectrin is not required for the formation of the FE. These findings suggests that Dlt is required, and may be essential for the formation of the FE (Tanentzapf, 2000).
Large crb mutant clones within the FE that survive until mid-oogenesis often form a multilayered epithelium indicating that Crb is important for the maintenance of the FE. The multilayering of follicle cells in crb mutant clones is limited to posterior follicle cells that cover the oocyte. To determine whether removal of Crb and Dlt from cells of the FE disrupts the integrity of the FE, small crb or dlt mutant follicle cell clones were examined that were induced after the FE had been established. Small crb mutant cell clones develop normally until late in oogenesis, although they show a substantial reduction in the level of Dlt. Cells in small dlt mutant clones develop with apparently normal morphology, and show a normal distribution of F-actin and Arm. Crb is lost from most dlt mutant clones, indicating that Dlt is important for maintaining apical Crb. Some dlt mutant clones retain Crb, suggesting that a mechanism other than binding to Dlt can contribute to maintaining Crb at the apical membrane. Crb was also undetectable in many cell clones mutant for dltdre1. Dlt protein in dltdre1 mutant cells forms a 'cap' in the center of the apical membrane rather than being distributed throughout. Together, these findings suggest that the dltdre1 allele may carry a mutation that specifically disrupts the interaction of Dlt and Crb, but not the interaction of Dlt with other apical factors (Tanentzapf, 2000).
The consequences of Crb overexpression in the FE were examined to analyze the response of the FE to mislocalization of Crb, and to further study the interactions between Crb and Dlt. Overexpression of Crb in embryonic epithelia causes a severe disruption of epithelial integrity that includes an extension of the apical cell surface and multilayering of epithelial tissues. A truncated form of Crb that lacks all EGF and laminin G domains (UAS>crbintra), was expressed. In embryos, this construct causes a similar phenotype as overexpression of full-length Crb, but, as it is not recognized by the anti-Crb antibody, the effect of the expression of crbintra on the distribution of endogenous Crb can be examined. Expression of UAS>crbintra causes a strong reduction in the level of endogenous Crb, suggesting that crbintra acts competitively with Crb for interaction to binding partners that are important for maintaining Crb in the plasma membrane. Overexpression of full-length Crb leads to misdistribution of Crb into the lateral membrane, at levels that are similar to the apical membrane. In most of these cell clones, no significant mislocalization of Dlt was seen, suggesting again that the apical localization of Dlt is at least in part Crb independent. A fraction of the Crb overexpressing follicle cells show mislocalization of Dlt, a thinning of the epithelium, and a strong reduction of lateral markers such as Arm and Fasciclin III. These findings indicate that overexpression of Crb disrupts the lateral membrane in follicle cells (Tanentzapf, 2000).
The cadherin-catenin complex plays a major role in epithelial polarization because cadherin-mediated adhesive contacts cause the assembly of the lateral surface domain. Consequently, lack of cadherin activity compromises epithelial integrity in many tissues or epithelial cell culture lines. Removal of E-cadherin (Shotgun) from follicle cells causes only mild defects in the development of the FE. In follicle cells that lack E-cadherin, some Arm is retained at adherens junctions, raising the possibility that the FE coexpresses a second cadherin that interacts with Arm. Indeed, N-cadherin is expressed in follicle cells in a pattern that overlaps with E-cadherin in early- to mid-oogenesis. N-cadherin disappears from the FE at stage 10 of oogenesis, whereas E-cadherin is expressed throughout oogenesis. In contrast to E-cadherin, N-cadherin is not expressed in the cells of the germline. However, Arm appears to be the only ß-catenin homolog in Drosophila, in contrast to vertebrates, where ß-catenin can be functionally replaced by plakoglobin in the cadherin-catenin complex. Thus, to effectively remove the cadherin-catenin complex from follicle cells, clones were generated that lack Arm, which is known to interact with both E- and N-cadherin (Tanentzapf, 2000).
Clones were induced for three different mutant arm alleles that carry premature stop codons in the 6th (armYD35), 7th (armXK22), and 10th (armXP33) arm repeat. armXP33 is an intermediate hypomorph; armXK22 is a strong hypomorph, and armYD35 is believed to be a null allele. Embryos derived from germline clones mutant for armXP33 show a dramatic disruption of epithelial morphology that occurs at the onset of gastrulation and is substantially more severe than the defects in epithelial structure seen in crb null mutant embryos. Moreover Crb is needed for the formation of the ZA in embryonic epithelia. If the failure to form the ZA is the major consequence of compromising Crb or Dlt activity in the FE, the lack of the cadherin-catenin complex would be expected to cause similar defects as seen in crb and dlt mutant follicle cells; that is, a failure to form a FE. Surprisingly, follicle cells mutant for any of the three arm alleles form a FE. No follicles were observed in these experiments that show epithelial discontinuities, as seen in crb and dlt mutant follicles. arm mutant follicle cells often show an irregular morphology at early stages of follicle development. The irregularities in epithelial structure increase in severity until the FE is compromised and the follicle degenerates at mid- to late-oogenesis. To determine whether adherens junctions were effectively disrupted in arm mutant follicle cells, the expression of E- and N-cadherin was examined in those cells. Neither E- nor N-cadherin are detected in follicle cells mutant for any of the three arm alleles studied. Taken together, these findings suggest that cadherin-based adherens junctions are not essential for the formation of the FE, but are important for maintaining its epithelial structure (Tanentzapf, 2000).
Advantage was taken of the fact that arm mutant cells in the FE are maintained for several days and their molecular architecture was examined. armXP33 mutant follicle cells, which in most cases have a normal cuboidal to columnar shape, show a decrease of F-actin and alpha-spectrin at the lateral membrane, and an accumulation of these molecules at the apical cell pole. In contrast, the apical marker ßHeavy-spectrin shows a normal distribution in armXP33 mutant cells, suggesting that the apical spectrin cytoskeleton is intact. Follicle cells mutant for armXK22 or armYD35 often develop a squamous cell morphology or show a multilayered structure. alpha-Spectrin remains associated with the narrow lateral membranes in squamous arm mutant cells. ßHeavy-Spectrin, in contrast, is lost from these follicle cells, suggesting that the apical spectrin cytoskeleton is disrupted. To further examine the apical surface domain of arm mutant follicle cells, the distribution of Crb and Dlt was examined in these cells. armXP33 mutant follicle cells typically show a normal apical localization of Crb and Dlt. In contrast, Crb is lost from the apical membrane of follicle cells mutant for strong arm alleles, whereas apical Dlt is retained in these cells. Similar to dltdre1 mutant cell clones, Dlt forms a cap in the center of the apical membrane of arm mutant follicle cells. These observations suggest that the disruption of adherens junctions leads to a breakdown of the lateral membrane domain, as expected, but that this also compromises the apical surface domain. The differential behavior of Crb and Dlt in strong arm mutant cell clones again emphasizes that Dlt can rely on a Crb-independent apical targeting mechanism, and shows that apical Dlt can be retained in the absence of an apical spectrin cytoskeleton (Tanentzapf, 2000).
stardust (sdt) encodes multiple PDZ domain MAGUK (membrane-associated guanylate kinase) proteins that are expressed in all primary embryonic epithelia from the onset of gastrulation. The polarized architecture of these epithelial cells depends on the highly stereotypic distribution of cellular junctions and other membrane-associated protein complexes. In epithelial cells of the Drosophila embryo, three distinct domains subdivide the lateral plasma membrane. The most apical domain comprises the subapical complex (SAC). It is followed by the zonula adherens (ZA) and, further basally, by the septate junction. A core component of the SAC is the transmembrane protein Crumbs, whose cytoplasmic domain recruits the PDZ-protein Discs Lost into the complex. stardust provides an essential component of the SAC. Cells lacking crumbs or the functionally related gene stardust fail to organize a continuous ZA and to maintain cell polarity. Thus, Stardust and Crumbs are mutually dependent in their stability, localization and function in controlling the apicobasal polarity of epithelial cells. Stardust proteins colocalize with Crumbs and bind to the carboxy-terminal amino acids of the Crumbs cytoplasmic tail. Nevertheless, Stardust and Crumbs expression patterns in sensory organs differ. The polarity of neuroblasts requires the function of Bazooka, but not of Stardust or Crumbs. In conclusion, the Stardust proteins represent versatile candidates as structural and possibly regulatory constituents of the SAC, a crucial element in the control of epithelial cell polarity (Buchmann, 2001; Hong, 2001).
The sdt mutant phenotype is characterized by the loss of epithelial integrity and cell shape. sdt mutant embryos fail to concentrate the scattered spot adherens junctions into a continuous ZA. As a consequence, cells lose contact and the epithelium becomes multilayered. The assembly of the ZA requires a functional SAC. Formation of the SAC depends on an intact cytoplasmic tail of Crumbs, which recruits the PDZ protein Discs Lost (Dlt) into the complex. In sdt mutant embryos, the SAC components Crumbs (Crb) and Dlt fail to localize apically from stage 8 onwards. Because Dlt is correctly associated with the ingrowing plasma membrane during cellularization in sdt embryos, it is suggested that sdt is, similarly to crb, essential for maintenance, but not for initiation of Dlt localization. The transmembrane protein Stranded at Second is lost from the apical pole, whereas the basolateral marker Neurotactin, initially retained at the lateral membranes, is delocalized in later stages. Lack of sdt also affects localization of the PDZ protein Bazooka (Baz) in epithelial cells. Baz forms, together with the PDZ protein DmPar-6 and Atypical protein kinase C (DaPKC), another apical protein complex. This complex is essential for the polarity of epithelial cells and neuroblasts, the progenitors of the central nervous system. In the CNS Baz controls spindle orientation and localization of determinants. Lack of sdt function does not affect Baz localization in neuroblasts, showing that sdt, unlike baz, is not required for the apicobasal polarity of neuroblasts (Buchmann, 2001).
Crumbs, a transmembrane protein with EGF-like repeats, is an apical determinant for establishing apicobasal polarity in embryonic epithelia. The sdt and crb mutants exhibit nearly identical defects in epithelial apicobasal polarity, and crb and sdt function in a common genetic pathway. Precise colocalization of Sdt and Crb throughout embryonic development was found in all cells examined except for those in sensory organs: Sdt but not Crb is at the distal dendrites of sensory neurons. In epithelial cells Crb and Sdt are mutually dependent for their localization and stability. There is a marked reduction of the protein levels of Crb in sdt mutants and of Sdt in crb mutants after gastrulation. In both cases, instead of the normal apicolateral distribution, these proteins are found in sparse and discrete random spots. Dlt, which interacts with the Crb intracellular domain, is distributed similarly to Crb in sdt mutants, although its early expression and localization during cellularization is not affected even in sdt germline clone embryos. Conversely, overexpression of the Crb intracellular domain (Crb-intra) is sufficient to cause a 'dominant' phenotype, disrupting cell polarity, expanding the apical domain, and displacing endogenous Crb into cytoplasm. In such embryos a large fraction of Sdt, together with Crb and Crb-intra, is also displaced away from the membrane into the cytoplasm (Hong, 2001).
To test whether the mutual dependence of Sdt and Crb is due to their physical interaction, a GST::Crb-intra pull-down assay was carried out using in vitro translated 35S-labelled Sdt. Both SdtA and SdtB bind specifically to Crb-intra. Two domains of Crb-intra are known: the C-terminal domain containing the EERLI motif, and the amino-terminal domain containing amino acids, such as Y10 and E16, that are conserved in Crb and its C. elegans homologs. Mutation in either domain abolishes the ability of Crb-intra to rescue the crb phenotype, but only the EERLI motif is required for inducing the dominant overexpression phenotype of Crb-intra. In the in vitro binding assay, binding of Sdt to Crb-intra is nearly abolished by removal of the C-terminal EERLI motif, but is not affected by Y10A and/or E16A mutations, indicating that Crb binds Sdt with its C-terminal EERLI motif (Hong, 2001).
The same C-terminal EERLI is also required for Dlt to bind to Crb. It will be of interest to determine whether Sdt and Dlt bind Crb cooperatively to form a tertiary complex, or whether they compete with each other for binding to the Crb intracellular domain. No direct binding between Dlt and Sdt was detected using the same in vitro GST pull-down assay. However, MAGUK proteins frequently show intramolecular auto-inhibition between different binding motifs so it remains possible that Sdt binds Dlt only after it first binds to Crb. Thus, whereas the Crb-Sdt (and possibly Dlt) pathway controls the epithelial but not neuroblast polarity, the Baz-DmPar-6-aPKC complex affects both epithelial and neuroblast polarity. Proteins in both pathways are expressed in epithelial cells with identical apico-lateral localization patterns but only Baz-DmPar-6-aPKC are expressed in neuroblasts. In epithelia, these two pathways are probably partially redundant in controlling apicobasal polarity. Their role in regulating ZA formation seems to be the key to their epithelial and cuticle phenotypes. Sdt and Crb colocalize in epithelia and together serve as apical determinants, but only partially overlap and are likely to serve different functions in sensory organs. A mutation in a human Crb homolog has been implicated as the cause of one form of retinitis pigmentosa, suggesting a potential role for the Crb family of proteins in the localization of a sensory transduction complex. The localization of Sdt to the dendritic tip and scolopale cells, the probable sites for sensory transduction, raises the possibility that Sdt may have a role in the development or function of a sensory transduction apparatus (Hong, 2001).
The apical transmembrane protein Crumbs is necessary for both cell polarization and the assembly of the zonula adherens (ZA) in Drosophila epithelia. The apical spectrin-based membrane skeleton (SBMS) is a protein network that is essential for epithelial morphogenesis and ZA integrity, and exhibits close colocalization with Crumbs and the ZA in fly epithelia. These observations suggest that Crumbs may stabilize the ZA by recruiting the SBMS to the junctional region. Consistent with this hypothesis, it is reported that Crumbs is necessary for the organization of the apical SBMS in embryos and Schneider 2 cells, whereas the localization of Crumbs is not affected in karst (ßHeavy-spectrin) mutants that eliminate the apical SBMS. The data indicate that specifically the 4.1 protein/ezrin/radixin/moesin (FERM) domain binding consensus, and in particular, an arginine at position 7 in the cytoplasmic tail of Crumbs is essential to efficiently recruit both the apical SBMS and the FERM domain protein, Moesin-like. Crumbs, Discs lost, ßHeavy-spectrin, and Moesin-like are all coimmunoprecipitated from embryos, confirming the existence of a multimolecular complex. It is proposed that Crumbs stabilizes the apical SBMS via Moesin-like and actin, leading to reinforcement of the ZA and effectively coupling epithelial morphogenesis and cell polarity (Médina, 2002).
The Crumbs-Discs lost-Stardust pathway is essential for polarity and has been shown to be a major apical signal for establishing the ZA at the apical-lateral boundary. The observation that mutations affecting ßH and Crumbs both cause a junctional phenotype, along with the close colocalization of both proteins in the marginal zone of epithelial cells, suggested a possible connection between the activities of these two proteins. Crumbs can indeed recruit apical ßH together with the FERM domain protein Moesin-like and actin. The data are in good agreement with the hypothesis that polarity cues are used to organize the SBMS, but this is the first time that this has been shown for an apical determinant (Médina, 2002).
Several lines of evidence indicate that Crumbs can recruit ßH into its complex: (a) ßH is mislocalized in embryos mutant for the truncation allele crumbs 8F105, in which the mutant Crumbs protein itself is mislocalized; (b) ßH mislocalization can be induced by overexpression of the Crumbs transmembrane and cytoplasmic domains in vivo; (c) ßH is recruited to Crumbs protein clusters in an S2 cell cocapping assay; (d) Crumbs can be coimmunoprecipitated with ßH; and (e) the protein-null allele crumbs11A22 acts as a dominant enhancer of hypomorphic karst alleles, strongly indicating that a reduction in the normal amount of Crumbs reduces the level of partially functional ßH at the membrane. Moreover, because the karst mutant alleles all produce COOH-terminally truncated proteins, these results further suggest that the Crumbs-ßH interaction site lies in the NH2-terminal portion of the latter. Finally, loss of Crumbs has been shown (Pellikka, 2002b) to eliminate ßH from the stalk membrane of photoreceptors in the adult eye (Médina, 2002).
Current evidence indicates that ßH can be recruited to the membrane in several additional ways. First, it can associate with the specialized basal adherens junctions during cellularization in a Crumbs-independent manner. Second, it is found in the terminal web subtending brush borders in the midgut epithelium that does not express Crumbs. Finally, it has also been shown that ßH is only partially reduced in crumbs11A22 mutant follicle cell clones, indicating that in this Crumbs-expressing epithelium there are multiple mechanisms to recruit ßH. These data provide a compelling explanation for the modest nature of the karst-crumbs genetic interaction. By reducing Crumbs, only one of these pathways is affected. The observation that the karst1 allele produces readily detectable quantities of truncated product, most of which is not recruited to the membrane in any of these epithelia, suggests that there is a general and essential role of the COOH-terminal half of ßH in its stable membrane localization. Together, the above data are consistent with the multifunctional nature of spectrin membrane skeletons and with the idea that specific pathways recruit the SBMS to establish spatially distinct polarized membrane domains, whereas general COOH-terminal membrane association domains permit tight membrane association and network integration (Médina, 2002).
Partial rescue of crumbs mutants has been attained by the crumbsmyc-intra construct. This suggested that the transmembrane and cytoplasmic domains of Crumbs might be sufficient to concentrate ßH to some areas of the apical membrane. This result has been confirmed and extended, showing that the critical region for recruiting ßH is just 9 amino acids from position 6 through 14 of the cytoplasmic domain in the putative FERM domain binding site. Within this region, a conserved tyrosine residue at cytoplasmic domain position 10 (crucial for Crumbs function in vivo) and an arginine at position 7 are both required for this activity. It is worth noting that all Crumbs genes cloned so far contain a charged amino acid residue at position 7 in the cytoplasmic domain, suggesting that this is an evolutionarily conserved interaction site (Médina, 2002).
FERM domains are found in the protein 4.1 family of proteins that link the SBMS to cell-surface receptors as well as several other proteins which organize the cortical actin (ezrin/radixin/moesin). The founding member of this group, protein 4.1, was originally identified as a major component of the erythrocyte SBMS where it facilitates the interaction of spectrin with actin and the transmembrane protein Glycophorin C. Therefore, the presence of a conserved FERM binding domain in the Crumbs cytoplasmic domain suggests that Crumbs may bind to ßH via a FERM domain protein (Médina, 2002).
In Drosophila, the FERM domain family includes the proteins Coracle, Merlin, Moesin-like, and Expanded. Of these four proteins, Coracle is an unlikely candidate to bind to the Crumbs juxtamembrane domain since it is localized to the septate junctions basal to the ZA. However, the Merlin, Moesin-like, and Expanded proteins are localized in part or in whole at the ZA region in epithelia, and could thus be involved in the interaction between Crumbs and ßH. The fact that none of protein 4.1 family members known in Drosophila contains a spectrin-binding domain as defined by the archetypal protein 4.1 does not necessarily abrogate this hypothesis. ßH-spectrin is clearly recruited to the membrane by different mechanisms than its basolateral counterpart, and this specificity would likely be reflected in divergent interaction domains. In this work, it has been found that ßH and Moesin-like can both coimmunoprecipitate Crumbs. Furthermore, the capping assay and embryo expression evidence provide in vivo support for this result. Not only will Moesin-like cocap with the Crumbs cytoplasmic domain, but it is also dependent on exactly the same sequences that recruit ßH. These results, together with the existence of the consensus binding site for a FERM domain protein in Crumbs, strongly support the hypothesis that Moesin-like forms a bridge between Crumbs and the SBMS. A functional test of this relationship must wait until mutations in the Moesin-like locus become available. Thus, the current data, although highly suggestive, do not formally distinguish between the possibility of a Moesin-like bridge between Crumbs and the SBMS, and the existence of two separate complexes with direct interaction between Crumbs and ßH or Moesin-like in each. Significantly, actin does not cap consistently with Crumbs in S2 cells and is not present in immunoprecipitates. This suggests that other components present in epithelial cells are necessary for stabilization of the actin skeleton around the Crumbs complex. It also indicates that ßH is specifically recruited to the proposed complex and is not merely a passive arrival along with bulk actin (Médina, 2002).
These results indicate that Crumbs interacts with at least two different protein networks, a Moesin-like/Spectrin/actin-based network and a PDZ protein scaffold (Dlt/Sdt). However, it is unclear at present whether Dlt/Sdt and Moesin-like/ßH/actin coexist in the same complex with Crumbs. In the erythrocyte model, glycophorin C is linked to spectrin via a ternary complex containing protein 4.1 and the PDZ domain protein p55 bound to a topologically similar pair of binding sites to the two functional regions identified in the Crumbs cytoplasmic domain. If such a ternary complex forms in association with Crumbs, then the observation that the Sdt-binding domain of Crumbs is not required for the interaction between Crumbs and ßH, would indicate that the latter cannot be dependent on Dlt/Sdt for association with Crumbs in such a complex or that both interactions can coexist (Médina, 2002).
Because both the crumbs and karst phenotypes disrupt the ZA, it is hypothesized that Crumbs promotes the accumulation of ßH to the apicolateral region during gastrulation to orchestrate the fusion of spot adherens junctions and/or to stabilize the ZA. Moreover, the observation that karst mutants exhibit morphogenetic defects without any loss of epithelial polarity, whereas dlt mutants exhibit a strong polarity phenotype, suggests that the polarization and junction building functions of Crumbs are separate and parallel pathways. In support of this hypothesis, the FERM domain binding region of Crumbs is indeed required (Izaddoost, 2002) for correct organization of the ZA (Médina, 2002).
The loss of ßH function causes defects in cell shape change that are associated with apical contraction driven by an apically located actomyosin contractile ring. In this context the discovery that this spectrin isoform is complexed with Moesin-like is particularly provocative, since the activity of the latter is strongly correlated with modulation of cell shape and the actin cytoskeleton. Furthermore, the activity of moesin is modulated by phosphorylation in response to activation of Rho-associated kinase (ROK) in parallel with myosin II. Both Moesin and myosin light chain are activated by ROK phosphorylation and by ROK mediated inhibition of the myosin/moesin phosphatase. Therefore, it is speculated that ßH is part of the cytoskeletal network that facilitates such cell shape changes, and that in organizing spectrin at the membrane, Crumbs would appear to be acting as a molecular coordinator of polarity and morphogenesis. Furthermore, the finding that in human, mutations in CRB1 lead to pathologies such as retinitis pigmentosa (RP12) emphasizes the importance of deciphering the molecular networks associated with Crumbs in Drosophila. The human orthologue of ßH, ßV-spectrin, is strongly expressed in photoreceptor cells. This raises the exciting possibility that a similar interaction between CRB1 and ßV-spectrin exists in these cells. This will be examined in future work (Médina, 2002).
Apicobasal cell polarity is crucial for morphogenesis of photoreceptor rhabdomeres and adherens junctions (AJs) in the Drosophila eye. Crumbs (Crb) is specifically localized to the apical membrane of photoreceptors, providing a positional cue for the organization of rhabdomeres and AJs. The Crb complex consisting of Crb, Stardust (Sdt) and Discs-lost (Dlt) colocalizes with another protein complex containing Par-6 and atypical protein kinase C (aPKC) in the rhabdomere stalk of photoreceptors. Loss of each component of the Crb complex causes age-dependent mislocalization of Par-6 complex proteins, and ectopic expression of Crb intracellular domain is sufficient to recruit the Par-6 complex. The absence of Par-6 complex proteins results in severe mislocalization and loss of Crb complex. Dlt directly binds to Par-6, providing a molecular basis for the mutual dependence of the two complexes. These results suggest that the interaction of Crb and Par-6 complexes is required for the organization and maintenance of apical membranes and AJs of photoreceptors (Nam, 2003).
The strong dependence of Crb localization on Sdt and Dlt suggests that Crb may be destabilized or may not be targeted to the membrane in the absence of Sdt or Dlt. It is intriguing that Sdt and Dlt are lost only partially in the absence of Crb. The findings of a direct interaction between Dlt and Par-6 suggest that Sdt-Dlt can still be targeted to the membrane in the absence of Crb through the binding of Dlt to the Par-6 complex. However, it is important to note that Dlt is essentially lost in sdt mutant clones and vice versa. This raises an intriguing possibility that Dlt or Sdt are dependent on each other in vivo to be targeted to the apical membrane via binding to either Crb or Par-6. This mutual dependency between Dlt and Sdt may explain why Dlt and Sdt are lost in the absence of the other, rather than being associated with the Par-6 complex (Nam, 2003).
The interaction between the Crb and Par-6 complexes is mediated by the PDZ3 region of Dlt and the N-terminal domain of Par-6. The N-terminal domain of Par-6 is also used for binding aPKC. Therefore, a potential function of Dlt is to bind Par-6 in competition with aPKC or to facilitate the interaction of Par-6 with aPKC or other Par-6 binding proteins. Mutant analysis indicates that loss of Dlt and Sdt in sdt- clones causes mislocalization of both Crb and Par-6 complex proteins. This suggests that Sdt-Dlt interaction provides a scaffold to recruit Crb complex to the Par-6 complex and enhance the stability of these two complexes rather than functioning as a competitor for aPKC (Nam, 2003).
Proteins in Crb and Par-6 complexes consist of multiple functional domains which may be involved in diverse protein-protein interactions. A recent study has shown that in mammalian cell culture systems the PDZ domain of Par-6 binds not only Par-3 but also the N terminus of Pals1. These results suggest that the crosstalk between the Crb and Par-6 complexes is mediated by multiple domain-specific interactions. Evidence from genetic analysis using mutants suggests that the crosstalk between the two complexes is mutually required for normal organization of apical membranes and AJs in vivo, and also provides a basis for partial redundancy of these complexes in the organization of photoreceptor cell polarity. Interestingly, when either Crb or Sdt is lost, mislocalization or elimination of other associated components including Par-6 complex proteins becomes more severe in the age-dependent manner. This suggests that the Crb complex may be required for the maintenance rather than the formation of the Par-6 complex. The age-dependent degenerative phenotype may be related to the requirement of extensive apical membrane growth to make rhabdomeres and AJs along the growing axis of photoreceptors during pupal stage. Loss of any one component of the Crb complex is likely to be increasingly more detrimental as the process of membrane reorganization proceeds. In crb- or sdt- mutants, significant fractions of Par-6 complex proteins remain in the membrane despite the age-dependent and progressive mislocalization of apical markers. By contrast, loss of Par-6 or aPKC results in mislocalization of Dlt from the apical membrane. This suggests that the Par-6 complex plays essential functions for membrane localization of Crb complex proteins. Furthermore, both Par-6 and aPKC seem to be important for survival and/or proliferation of retinal cells because mutant clones were very small compared with adjacent twin spots and often completely disrupted, probably due to cell death. This is consistent with the findings of frequent apoptosis in aPKC- or par-6- embryos (Nam, 2003).
An important distinction of Par-6 complex in the photoreceptors from other epithelia is the localization of Baz. Baz localizes with Crb complex in the subapical membrane or both the subapical region and AJ in the Drosophila embryonic epithelia. Vertebrate Par-3 also localizes to the apical tight junction in vertebrate epithelial cells. By contrast, Baz in the photoreceptors is specifically positioned in the AJs basal to the all other proteins in the Crb/Par-6 complexes. Baz and Arm are recruited together to ectopic membrane sites by misexpression of CrbJM, suggesting that Baz is an integral component of AJ. However, Baz is not recruited by CrbPBM, whereas Par-6 and aPKC can be ectopically recruited by CrbPBM rather than CrbJM. Therefore, Baz appears to be recruited to AJ independently of Par-6/aPKC (Nam, 2003).
Intriguingly, despite its specific localization to AJs, loss of Baz results in most severe disruption of AJ as well as the more apical Dlt domain. It has been proposed that the Par-6/aPKC cassette is recruited to the site of cell-cell contact and then moves along the most apical zone of the developing cell-cell contact. In this process, an important step for cell polarity formation is to tether the cytoplasmic Par-6/aPKC complex to the site of cell-cell contact at the membrane, which is mediated by the interaction of Par-3 and a membrane protein JAM. Therefore, the results that baz mutation causes loss of Dlt and AJs support the crucial role of Baz in the initial step of cell polarization. However, the distinct localization of Baz from Par-6 and aPKC in the photoreceptors suggests that the mode of Baz localization varies in different systems. In photoreceptors, Baz may be targeted to the membrane with Par-6 but be sorted out from Par-6 in subsequent steps of polarization to remain in the AJs, whereas Par-6-aPKC-Baz cassette remains together in the complex in other epithelia. In contrast to Baz, aPKC localizes to both rhabdomere stalk and AJ, suggesting that Baz and Par-6 are completely separated during polarization while aPKC is not sorted from both Par-6 and Baz. The critical function of Baz in the localization of Crb complex in the rhabdomere stalk is consistent with the requirement of Baz for Crb localization in embryonic epithelia. However, the requirement of Baz in the embryo appears to be dependent on the stage of development since Crb distribution in the absence of Baz becomes normal in late embryos. On the contrary, such stage-dependent recovery of Crb complex localization has not been observed in baz- photoreceptor cells (Nam, 2003).
Recent studies have shown that mutations in human CRB1 cause RP12 and LCA, severe recessive retinal diseases, emphasizing the importance of Crb family proteins in the eyes of mammals including humans. The Drosophila Crb and human CRB1 are localized in analogous subcellular membrane domains of photoreceptors, the rhabdomere stalk and the inner segment in Drosophila and human photoreceptors, respectively. Besides similar subcellular localization, Crb and human CRB1 are functionally conserved. Age-dependent photoreceptor defects in the crb mutant also provide analogy to age-dependent retinal degeneration in RP12/LCA patients. These studies here imply that hCRB1 may function as a protein complex with homologs of Sdt and Dlt and such a complex may interact with a homologous Par-6 complex. Whether such homologous human genes are the targets of inherited retinal diseases such as RP remains to be studied (Nam, 2003).
Formation of multiprotein complexes is a common theme to pattern a cell, thereby generating spatially and functionally distinct entities at specialised regions. Central components of these complexes are scaffold proteins, which contain several protein-protein interaction domains and provide a platform to recruit a variety of additional components. There is increasing evidence that protein complexes are dynamic structures and that their components can undergo various interactions depending on the cellular context. However, little is known so far about the factors regulating this behaviour. One evolutionarily conserved protein complex, which can be found both in Drosophila and mammalian epithelial cells, is composed of the transmembrane protein Crumbs/Crb3 and the scaffolding proteins Stardust/Pals1 and DPATJ/PATJ, respectively, and localises apically to the zonula adherens. In vitro analysis shows that, similar as in vertebrates, the single PDZ domain of Drosophila Par-6 can bind to the four C-terminal amino acids (ERLI) of the transmembrane protein Crumbs. To further evaluate the binding capability of Crumbs to Par-6 and the MAGUK protein Stardust, analysis of the PDZ structural database and modelling of the interactions between the C-terminus of Crumbs and the PDZ domains of these two proteins were performed. The results suggest that both PDZ domains bind Crumbs with similar affinities. These data are supported by quantitative yeast two-hybrid interactions. In vivo analysis performed in cell cultures and in the Drosophila embryo show that the cytoplasmic domain of Crumbs can recruit Par-6 and DaPKC to the plasma membrane. The data presented here are discussed with respect to possible dynamic interactions between these proteins (Kempkens, 2006).
Homology modelling and energy calculations indicate that the PDZ domains of Sdt and Par-6 both show a high affinity for Crb. According to a classification of PDZ domains, the PDZ domains of Par-6 and Sdt fall into different categories: n (neutral) – h (hydrophic) for Par-6 and Sp (small and polar) – h (hydrophobic) for Sdt. The distinction is based on the nature of the amino acids in two critical positions, those immediately following the second β-strand and at the beginning of the second α-helix. However, this classification, based exclusively on the amino acids at two positions, does not adequately describe the complexity of the PDZ family and is not sufficient to predict the specificity of binding. This is borne out by the results presented in this study (Kempkens, 2006).
The crystal structure used as a template in this study (D. melanogaster Par-6, 1RZX.PDB) includes a canonical type I ligand (ESLV), and this can easily be replaced in the structure by the C-terminal tetrapeptide of Crb (ERLI). Although better binding of the Crb C-terminus to Par-6 than to Sdt is predicted, this conclusion should be treated with caution, since the predicted structure of the Crb-Sdt complex is based solely on homology modelling. The analysis of interactions between Crb and PDZ domains showed minor van der Waals clashes (<0.3 kcal/mol), as well as strong polar and hydrophobic ligand/PDZ-domain interactions. An important component of the interaction is electrostatic. This could favour fast association (Kon) and dissociation rates (Koff), which would facilitate flexible regulation of complex formation. These values, which are slightly better for Par-6, could account for the affinity and specificity observed in the experimental interaction studies. The predicted Kd for interaction between the domains considered in this study fall in the usual range for experimental determinations of PDZ-ligand binding affinities reported by many authors. The theoretical determination of binding interactions by FoldX has been tested for many domain-peptide interactions (i.e. SH3, SH2, PDZ, Ras-Rab, etc.), is validated by available experimental data, and shows a high degree of accuracy. In this sense, the theoretical cut-off for non-binders can be reasonably approximated to that used for experimental procedures, normally 1E−4M. Thus, all putative ligands with interaction energies below 1E−4M are considered to be non-binders (Kempkens, 2006).
It has been shown that the PDZ domain of vertebrate Par-6 can bind not only to the C-terminus of CRB3, but also to the C-terminus of the N-type Ca2+ channel and the C-terminus of neurexin, and that the PDZ domains of vertebrate and Drosophila Par-6 proteins can interact with an internal region in the N-terminal portion of Pals1/Sdt. These data support the view that PDZ domains are promiscuous and can bind different ligands, thanks to the plasticity of the carboxylate-binding loop. This conformational flexibility can be modulated by interaction with other proteins. For example, binding of Cdc42 to Par-6 significantly enhances its affinity for the C-terminal ligand, while it has no effect on the interaction with the internal binding sequence of Pals1. To what extent other components facilitate loop rearrangement to accommodate internal ligands to the Par-6 PDZ binding pocket in vivo remains to be determined. Interestingly, although the PDZ domains of both Par-6 and Sdt bind to the C-terminus of Crb, only the PDZ domain of Par-6 interacts with the internal sequence in Sdt (Kempkens, 2006).
Since the binding affinities of the PDZ domains of Sdt and Par-6 for the C-terminus of Crb are in the same range, and all three proteins are expressed in the same cells, other factors must be considered that might influence their interactions. These could include, for example, (1) temporal differences in expression, (2) protein modifications, and (3) interactions with other factors. Par-6 is present in the embryo before Sdt and Crb appear, since it is expressed maternally. In the genetic hierarchy controlling cell polarity and ZA assembly, components of the Baz/Par-6/DaPKC complex appear to form the top tier, with Baz acting very early during cellularisation to establish the apical domain in an adherens junction-independent manner. At this stage, Par-6 is diffusely distributed in the cytoplasm. It becomes localised apically only after cellularisation is complete. Localisation of Par-6 requires an interaction with the monomeric GTPase Cdc42, which, like its vertebrate homologue, binds Par-6 via a conserved CRIB (Cdc42/Rac interactive binding) domain. Thus, overexpression of a dominant-negative form of Cdc42 prevents apical localisation of Par-6, and a mutant version of Par-6 that cannot interact with Cdc42 (Par-6-ΔP) remains diffusely distributed in the cytoplasm (Kempkens, 2006).
Binding of the PDZ domain of Sdt to the C-terminus of Crb may also depend on additional contacts mediated by the other interaction domains of Sdt. Sdt is a member of the MAGUK protein family, with two L27 domains, one SH3, one PDZ and one guanylate kinase (GUK) domain. The L27 domains of Pals1 and Sdt bind to PATJ/DPATJ and mLin-7/DLin-7. No partner(s) for the SH3 and GUK domains of Sdt have been described so far. Recently, a direct interaction was shown to occur between the SH3/Hook domain of MPP5/Pals1 and the GUK domain of another MAGUK family member, MPP4, in mouse photoreceptor cells. Furthermore, yeast two-hybrid experiments have uncovered an interaction between the SH3 and the Hook/GUK domains of Sdt. In other members of the MAGUK family, such as human Discs large (hDlg), CASK and p55, these two domains participate in intra- as well as intermolecular interactions, with the former being preferred, at least in vitro. Currently, the data do not allow a decision as to whether the observed SH3-Hook/GUK interaction is intra- or intermolecular, nor to what extent this might influence the capacity of the PDZ domain to bind to Crb in vivo. Similarly, at present it is only possible to speculate on the possible influence on the binding specificity/activity of the phosphorylation of the intracellular domain of Crb, which has been demonstrated by in vitro assays, but also occurs under certain in vivo conditions. Future experiments will contribute to the elucidation of the dynamic interactions among these proteins within the cell and help in the understanding of how they establish and maintain the polarised phenotype (Kempkens, 2006).
The Crumbs (Crb) complex is a key regulator of epithelial cell architecture where it promotes apical membrane formation. Binding of the FERM protein Yurt to the cytoplasmic domain of Crb is part of a negative-feedback loop that regulates Crb activity. Yurt is predominantly a basolateral protein but is recruited by Crb to apical membranes late during epithelial development. Loss of Yurt causes an expansion of the apical membrane in embryonic epithelia and photoreceptor cells similar to Crb overexpression and in contrast to loss of Crb. Analysis of yurt crb double mutants suggests that these genes function in one pathway and that yurt negatively regulates crb. The mammalian Yurt orthologs YMO1 and EHM2 bind to mammalian Crb proteins. It is proposed that Yurt is part of an evolutionary conserved negative-feedback mechanism that restricts Crb complex activity in promoting apical membrane formation (Laprise, 2006).
The formation and maintenance of cell polarity is essential for epithelial morphogenesis. Patj (the Drosophila homolog of mammalian Patj) is multi-PDZ domain protein that localizes to the apical cell membrane and form a protein complex with cell polarity proteins, Crumbs (Crb) and Stardust (St). Whereas Crb and Sdt are known to be required for the organization of adherens junctions (AJs) and rhabdomeres in differentiating photoreceptors, the in vivo function of Dpatj as a member of the Crb complex in developing eye has been unclear due to the lack of loss-of-function mutations specifically affecting the patj gene. Genetic analysis of hypomorph, null, and RNA inerference reveals distinct dual functions of Patj in developing and mature photoreceptors. The C-terminal region (PDZ domains 2-4) of Patj is not essential for development of the animal but is required to prevent late-onset photoreceptor degeneration. In contrast, the N-terminal region of Patj is essential for animal viability and photoreceptor morphgenesis during development. The localization and maintenance of Crb and Sdt in the apical photoreceptor membrane are strongly affected by reduced level of Patj. Patj is necessary for proper positioning of AJs and the integrity of photoreceptors in the developing retina as well as for the maintenance of adult photoreceptors. This study provides evidence that Drosophila Patj has domain-specific early and late functions in regulating the localization and stability of the Crb-Sdt complex in photoreceptor cells (Nam, 2006).
Dynamic changes take place in developing photoreceptors to reorganize the apical cell membrane during pupal stage. Crb and Sdt are required for growth and maintenance of rhabdomeres and AJs during this time of photoreceptor morphogenesis. Patj binds Sdt to form a conserved heterotrimeric Crb complex (Roh, 2002), but its function in vivo has been unclear. In this study, a key question whether Patj is an essential member of the Crb complex was addressed in vivo (Nam, 2006).
This study made three new observations that demonstrate important functions of Patj: (1) hypomorphic patj shows severe late-onset degeneration of photoreceptor cells in adult eye although the mutant eyes develop relatively normally; (2) analysis of patj null mutant patj RNAi demonstrate that Patj is essential for early development of the animal and for morphogenesis of AJ and apical membrane domains of photoreceptor cells during pupal development (Nam, 2006).
Consistent with these results on the late-onset degeneration of photoreceptor cells in hypomorphic adult eyes, (3) degeneration of adult photoreceptor cells was found. However, despite the findings on the phenotypes of the hypomorphic mutant, it is worth noting that there are important differences between these two studies. First, a second study on patj function (Richard, 2005) is limited to the analysis of a viable hypomorphic condition patj that shows no obvious defects in the pattern of AJs and rhabdomeres in the eyes until approximately 70% late pupal development. In contrast, the current study with the newly generated null mutant and RNAi reveals important developmental functions of Patj in the eye as well as animal viability. Second, the hypomorpic mutant is not only deleted in the PDZ2-4 domain portion of the dpatj gene but also deficient for JTBR and partially CG32327 that are adjacent to the 3' end of patj. In this study, it was shown that patj RNAi causes similar phenotypes of patj null mutant in the eye, suggesting that JTBR and CG32327 do not affect eye development and, thus, have no detectable influence on analysis of hypomorphic and null mutants (Nam, 2006).
The complete loss of patj causes several significant defects in early pupal eyes at approximately 45% pupal development. (1) Both Crb and Sdt are strongly reduced or mislocalized in the absence of Patj, although the loss of patj shows slightly weaker phenotypes than those of crb and sdt mutants. Furthermore, and in contrast to the hypomorphic mutant, (2) null clones or the eyes expressing patj RNAi reveal striking basolateral displacement or expansion of AJ markers such as Arm and Baz. These results suggest that the N- and C-terminal region of Patj protein may have distinct functions in the photoreceptor cells. Because the hypomorphic mutant shows relatively normal development of photoreceptors until later pupal stage or eclosion, the PDZ2-4 domains are not required during early photoreceptor development but are necessary for the maintenance of fully differentiated photoreceptors during very late pupal and adult stage. Of interest, a significant level of Crb and Sdt are apically localized in 45% pupal photoreceptors in hypomorphic mutant eyes (Richard, 2005) but are lost later in adult eyes (Richard, 2005), suggesting that PDZ2-4 domains are required to maintain the Crb complex in late pupal and adult stages. In contrast, the N-terminal region of Patj containing MRE and PDZ1 is crucial for morphogenesis of AJ and rhabdomere during the first half of pupal stage. The MRE motif of Patj interacts with the L27 domain of Sdt. Thus, it is likely that the N-terminal region expressed in hypomorphic mutants might be sufficient for interaction with Sdt, allowing normal development. Conversely, Sdt cannot bind Patj in the null mutant, resulting in the failure of normal development as in crb or sdt mutants (Nam, 2006).
Recently, it has been shown that reduction of Patj by RNAi in MDCK epithelial cell culture leads to delayed tight junction formation and defects in cell polarization. Similarly, mammalian Patj is required for localizing tight junction proteins and stabilizing the Crb3 complex in human intestinal cells. Thus, the findings on the important function of Drosophila Patj in developing photoreceptors are consistent with the results from these mammalian studies, and further provide evidence for the developmental function of Patj in the organization of apical cell membrane in the in vivo animal model. Mouse Patj is closely related to Drosophila Patj, but it has 10 PDZ domains in contrast to the presence of 4 PDZ domains in Drosphila Patj. It will be interesting to see whether the mammalian Patj also show distinct functions of the N-terminal MRE and the multiple PDZ domains in organizing cell polarity and maintaining the stability of the Crb complex, respectively (Nam, 2006).
Mutations in the human CRB1 gene cause retinal diseases such as retinitis pigmentosa 12 (RP12) and Leber congenital amaurosis. The current results that Patj has dual functions in photoreceptor morphogenesis and maintenance in developing and adult animals, respectively, suggest that like CRB1, human Patj may be a target for early- and late-onset retinal diseases. It has been shown that the extracellular domain of Crb is required for preventing photoreceptor degeneration in ageing adult fly eyes. Results from this study and Richard (2005) indicate that PDZ2-4 domains of Drosophila Patj are required for blocking late-onset photoreceptor degeneration. It is currently unknown whether the requirement of Crb extracellular domain and the intracellular function of Drosophila Patj PDZ2-4 domains are related events in the maintenance of adult photoreceptors (Nam, 2006).
The establishment of apicobasal polarity in epithelial cells is a prerequisite for their function. Drosophila photoreceptor cells derive from epithelial cells, and their apical membranes undergo elaborate differentiation during pupal development, forming photosensitive rhabdomeres and associated stalk membranes. Crumbs (Crb), a transmembrane protein involved in the maintenance of epithelial polarity in the embryo, defines the stalk as a subdomain of the apical membrane. Crb organizes a complex composed of several PDZ domain-containing proteins, including Patj (formerly known as Discs lost). Taking advantage of a Patj mutant line in which only a truncated form of the protein is synthesized, Patj was demonstrated to be necessary for the stability of the Crb complex at the stalk membrane and is crucial for stalk membrane development and rhabdomere maintenance during late pupal stages. Moreover, Patj protects against light-induced photoreceptor degeneration (Richard, 2006).
This study presents evidence to show that Patj plays an important role in stabilizing the Crb complex in photoreceptor cells (PRCs). A truncated form of Patj, consisting of the N-terminal L27 and the first PDZ domain, is produced in Patj mutant eyes. It is further shown that the truncated Patj protein fails to stabilize Crb and Sdt at the stalk membrane during late pupal development and in adult eyes. It has been demonstrated that Crb is required for proper localization of Sdt and Patj at the stalk membrane of PRCs and that, in sdtXP96, the maintenance of Crb and Patj is compromised. These data together with the results presented in this study support the view that lack of any component of the Crb complex leads to mislocalization and/or dysfunction of the whole complex in the Drosophila eye (Richard, 2006).
To understand how the Crb complex may ensure a proper morphogenesis of photoreceptor cells, two major events during photoreceptor development have to be considered. In the first half of pupal development, stabilization of the ZAs is essential to maintain adhesion between PRCs during the tremendous cell shape changes that take place later when the cells undergo elongation. Furthermore, from 37% pupal development onward, the apical membrane differentiates into the rhabdomere and the stalk (Richard, 2006).
In crb and sdt mutants, the rhabdomeres are shorter and thicker, suggesting a failure to stabilize adhesion in early stages of pupal development, which in turn prevents proper elongation. This interpretation is consistent with the observation that, in eyes lacking crb function, the continuity of the ZA is interrupted at early stages of pupal development. Patj mutants do not exhibit any obvious defects in ZA development or PRC elongation, which can be explained by the fact that components of the Crb complex are still correctly localized until 70% pupal development. This timing contrasts with crb or sdt mutant PRCs, in which the integrity of the Crb complex is lost at an early stage of pupal development. Several explanations may account for this different behavior of Patj mutant PRCs. (1) The N-terminal portion of Patj may still retain some function during early pupal development, which stabilizes the Crb complex and, hence, the ZA. (2) Alternatively, Patj does not play a major function for the stability of the complex at early stages. (3) Finally, additional factors may interact with Patj and stabilize the Crb complex at early stages of development, and these interactions still occur with a truncated Patj. In fact, recent in vitro studies have suggested direct interactions between Patj and either Drosophila Par-6 or Drosophila PKC, two members of the other apically localized protein complex, which is essential for epithelial cell polarity in the embryo. However, it can be excluded that the suggested interaction between the third PDZ domain of Patj and the N-terminal domain of DmPar-6 plays any role in the stabilization of the complex during the first half of pupal development. The truncated Patj protein studied here lacks the third PDZ domain, yet it remains localized at the apical membrane at this stage (Richard, 2006).
The other major aspect of PRC maturation -- the differentiation of the apical membrane into rhabdomere and stalk -- is affected in crb, sdtXP96, and Patj mutant eyes, suggesting that all three components are necessary for this process. These mutations result in a shortening of the stalk membrane. The weaker phenotype of the Patj mutant relative to that of crb mutants is probably due to the hypomorphic nature of the former. Separation of the apical membrane of PRCs into two distinct domains, the rhabdomere and the stalk, becomes manifest at approximately 55% pupal development and coincides with the restriction of Crb and its associated proteins to this region. No other mutant affecting the length of stalk membrane has been described to date, although some mutants affect individual aspects of the crb or Patj morphogenetic phenotype, displaying thicker (bifocal; DSec61), malformed (Glued; WASp) or missing (overexpression of amphiphysin) rhabdomeres. Thus, the regulation of stalk membrane development seems to be a unique function for members of the Crb complex (Richard, 2006).
One phenotype of Patj mutants observed in this study, the progressive resorption of rhabdomeric microvilli, has not been described to date for any other mutant of the Crb complex. This raises the question whether Patj is involved in other processes in addition to those that are controlled by crb and sdt. The rhabdomere is composed of microvilli, each of which is supported by actin filaments. Rhabdomere morphogenesis and integrity depend on constant renewal of the membrane and on a highly organized actin cytoskeleton. Thus, it is not surprising that mutations in proteins involved in endo- or exocytosis, such as dynamin, Rab1, Rab6, Rab11, Sec6, Sec61, or Sunglasses, affect the integrity of the rhabdomere. It has been suggested that the addition of new membrane occurs at the base of the rhabdomere in Drosophila, while shedding occurs at the distal tip in tipulids. The further analysis of the function of these genes, the subcellular distribution of the respective proteins and their possible interactions with members of the Crb complex will be required to determine any involvement of the Crb complex in these processes. Rhabdomere integrity is also affected in eyes lacking proteins involved in actin structure and remodeling, such as NinaC, Chaoptin, Glued, Moesin or Rac1 but also in mutants for rhodopsin itself, which plays a structural role in addition to its function in signal transduction. In this scenario, Patj could help to stabilize the cytoskeleton and thereby maintain the integrity of the rhabdomere. Alternatively, the four PDZ domains in Patj may mediate the assembly of additional proteins. The identification and functional characterization of these proteins will shed light on the process by which Patj controls the stability of PRCs (Richard, 2006).
At present, the possibility that the defects observed in pigment cells in Patj mutant eyes contribute to the mutant phenotype observed in PRCs cannot be excluded. In vertebrate eyes, the pigment epithelium plays an active role in the renewal of rhodopsin, and defects in the pigment epithelium can lead to degeneration of PRCs. It is not yet known whether pigment cells in the Drosophila eye have a comparable function, although they certainly serve to insulate the PRCs of individual ommatidia from the light impinging on their neighbors. The accumulation and fusion of pigment granules in Patj mutant eyes may point to a defect in vesicular biogenesis and/or secretion. Whether this defect also affects interactions with the PRCs, in other words, whether pigment cells play an active role in the maintenance of the rhabdomeres or PRC function, and if so, whether Patj is involved in this process, is not known (Richard, 2006).
Finally, the results of this study demonstrate that Patj, like Crb, protects PRCs from the deleterious effects of excess light. The degeneration of PRCs observed in Patj mutant eyes may be a direct consequence of the failure to stabilize the components of the Crb complex at the stalk membrane. Previously published data have shown that the absence of crb in Drosophila eyes leads to retinal degeneration under similar lighting conditions. Similarly, mice deficient for CRB1 display signs of retinal degeneration upon exposure to light, which are reminiscent of defects seen in patients bearing mutations in the CRB1 gene. However, the penetrance of degeneration observed in crb eyes is much higher than that observed in Patj eyes, although the cellular features of degeneration observed in both mutants are similar. Taking into account that the crb clones were produced in a white background, whereas Patj eyes are red (due the transgenes that were introduced), it is not unlikely that the pigments could play a protective role in the latter case, as also shown previously for white mutants. Preliminary experiments suggest that the presence of pigments in crb mutant ommatidia indeed slows down the light-dependent degeneration (Richard, 2006).
Taken together, these results extend the knowledge of the genes involved in controlling retinal morphogenesis and preventing light-dependent PRC degeneration in the fly. Mutations in human CRB1 lead to RP12 and LCA, two severe forms of retinal dystrophy, raising the question whether mutations in the homologues of the other members of the complex might result in similar phenotypes. Understanding the molecular mechanisms leading to the mutant phenotype in the fly will certainly contribute to unraveling the pathogenesis of these retinal dystrophies in humans (Richard, 2006).
Lack of abstrakt function does not compromise the ability of cells to undergo their differentiation program or cellular behaviour typical of their differentiated state. Thus, neurite outgrowth still occurs, and pole cells are able to migrate over large distances, but both types of cells appear to fail to recognize or react to the cues that direct their morphogenetic behavior in the spatially appropriate manner. One reason for this defect might be a loss of subcellular order or polarity. Various aspects of cell polarity were indeed affected in abs mutants. The first abnormality detected was in the localization of mRNAs in the blastoderm. In the wild-type blastoderm, the mRNAs of several genes are distributed unevenly in the cell. For example, the transcripts of short gastrulation (sog) and crumbs (crb) are tightly apposed to the apical cell surface. In abs14B mutants, CRB mRNA is not seen apically but predominantly at the level of the nuclei, while SOG mRNA forms a gradient from the apical towards the basal side of the cell. In the case of crb, the mislocalization of RNA is also seen at later stages and in other tissues. For example, the tight apical localization in the hindgut epithelium is disturbed in abs mutants. Remarkably, the Crumbs protein is synthesized properly and targeted to the apical cell surface, showing that apical-basal cell polarity in the epithelial cells is correctly established and maintained in abs mutants (Irion, 1999).
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