The Crumbs protein is exclusively expressed on the apical membrane of all ectodermally derived epithelia concentrated at the borders between cells. Later, the protein can be detected on the apical and basolateral membranes (Knust, 1993 and Tepass, 1990).
The Crumbs mRNA is likewise concentrated at the apical periplasm. The expression of crumbs is correlated with epithelium with the exception of the peripheral nervous system. It is detectable at the blastoderm stage, and found in the stomatogastric nervous system as long as it displays an epithelial organization. The only non-epithelial organs expressing crumbs are the external sensory organs and the chordotonal organs. CRB is detectable for a short time in cells of the anterior and posterior midgut primordia (Tepass, 1990).
The Drosophila hindgut develops three morphologically distinct regions along its anteroposterior axis: small intestine, large intestine and rectum. Single-cell rings of 'boundary cells' delimit the large intestine from the small intestine at the anterior, and the rectum at the posterior. The large intestine also forms distinct dorsal and ventral regions; these are separated by two single-cell rows of boundary cells. Boundary cells are distinguished by their elongated morphology, high level of both apical and cytoplasmic Crb protein, and gene expression program. During embryogenesis, the boundary cell rows arise at the juxtaposition of a domain of Engrailed- plus Invected-expressing cells with a domain of Delta (Dl)-expressing cells. Analysis of loss-of-function and ectopic expression phenotypes shows that the domain of Dl-expressing cells is defined by En/Inv repression. Further, Notch pathway signaling, specifically the juxtaposition of Dl-expressing and Dl-non-expressing cells, is required to specify the rows of boundary cells. This Notch-induced cell specification is distinguished by the fact that it does not appear to utilize the ligand Serrate and the modulator Fringe (Iwaki, 2002).
At its anterior, the hindgut joins the posterior midgut; at its posterior, it forms the anus. Along this AP axis, the hindgut of the mature embryo consists of three morphologically distinct domains: the wide, looping small intestine, the long and narrow large intestine, and the tapered rectum. Beginning at stage 13, these domains are demarcated at their junctions by rings of unusually high accumulation of the apical surface protein Crumbs (Crb). The ring at the small intestine/large intestine junction is designated the anterior boundary cell ring, and the ring at the large intestine/rectum junction is designated the posterior boundary cell ring (Iwaki, 2002).
Patterning of the hindgut in the DV axis is detected at stage 10 (germ band extension) when the hindgut develops an interiorly directed (dorsal) convexity. The side of the hindgut closest to the interior of the embryo is dorsal and expresses both En and Inv; that closest to the exterior is ventral and expresses dpp. By the completion of germ band retraction, the convexity at the anterior of the hindgut has shifted toward the left side of the embryo. Thus at the anterior of the hindgut, the initially dorsal, En- and Inv-expressing side comes to lie on the outer (left-facing) curve, while the initially ventral, Dpp-expressing side of the hindgut comes to lie on the inner (right-facing) curve; the DV relationship is retained at the posterior connection to the rectum. These initially DV patterned domains of the large intestine persist to the end of embryogenesis and into the larval stages; they are referred to as large intestine dorsal (li-d) and large intestine ventral (li-v). At each of the two boundaries between li-d and li-v, there is a single row of cells with high levels of Crb expression running the length of the large intestine, from the anterior boundary cell ring to the posterior boundary cell ring. These are designated the 'boundary cell rows'. In addition to their high level of Crb expression, the boundary cell rows and rings express the nuclear protein Dead ringer (Dri). Double antibody staining reveals that boundary cell rows at the border of the En/Inv-expressing li-d domain and the Dpp-expressing li-v domain express Dri in their nuclei and have strong Crb expression at their apical surfaces (Iwaki, 2002).
Interestingly, the Dri- and Crb-expressing boundary cells delimit both AP and DV boundaries in the hindgut. The rings form borders at the anterior and posterior ends of the large intestine, while the rows form borders between the dorsal (li-d) and ventral (li-v) regions of the large intestine. This study focusses primarily on the establishment and characteristics of the boundary cell rows (Iwaki, 2002).
Staining with both anti-Crb and anti-ßHEAVY Spectrin shows that the boundary cell rows are significantly more elongated along the AP axis than other hindgut epithelial cells. Staining of bynapro/+ embryos (containing a P-element insert in byn) with anti-ß-Gal antibody reveals that the nuclei of the cells of the boundary rows (identified by strong staining with anti-Crb) are also elongated in the AP axis (Iwaki, 2002).
The dramatically higher level of Crb expression in the boundary cells (both rings and rows) suggests that their apical surface may differ from that of other hindgut epithelial cells, and/or that, in the boundary cells, Crb may be present in cellular compartments in addition to the apical surface. Both of these expectations are borne out by a higher magnification examination of the boundary cells. In cross-sections of the large intestine viewed by electron microscopy, short microvilli on the apical surfaces of two cells on opposite sides of the hindgut lumen were observed; these cells most likely correspond to the boundary cell rows. The microvilli of the presumed boundary cell rows appear more organized and parallel than the irregular protrusions on the surfaces of the other cells of the hindgut epithelium. Because of their apical microvilli, the presumed boundary cell rows have a larger apical membrane surface and are expected to be labeled more strongly with anti-Crb. Consistent with this, cross-sections of anti-Crb-stained embryos viewed by light microscopy reveal two cells on opposite sides of the large intestine lumen with a higher level of Crb on their apical surfaces. In addition to their stronger apical labeling with anti-Crb, these presumed boundary cell rows also display an accumulation of Crb in their cytoplasm; this is strongest apical to the nucleus. The cytoplasmic accumulation of Crb suggests that Crb is produced at a higher level, or is more stable, in the boundary cells (Iwaki, 2002).
In conclusion, differences in gene expression demonstrate that the boundary cells are a separately patterned (fated) group of cells in the large intestine. The unique fate of the boundary cells is manifested both molecularly, in their expression of Dri and high cytoplasmic accumulation of Crb, and morphologically, in their marked AP elongation and development of apical microvilli (Iwaki, 2002).
The boundary cell rows form at the junction of the li-d and li-v domains, which express different genes. To investigate whether the spatially restricted gene expression observed in these domains is essential for establishment of boundary cell rows, embryos homozygous for loss-of-function alleles of en, inv, dpp, dri, Dl, Ser, Notch, or fng were examined. The presence or absence of boundary cells was assessed by anti-Crb staining, since this delineates their characteristic morphology, and also detects one of their unique differentiated features (i.e. the cytoplasmic accumulation of Crb) (Iwaki, 2002).
The data presented here support the following model. En/Inv is expressed in li-d and represses Dl in that domain; Dl expression is thereby restricted to the li-v domain. At the li-v/li-d transition, the Dl-expressing cells induce, by Notch signaling, a row of Dl-non-expressing cells to become a boundary cell row. Since En/Inv is not detected in differentiated boundary cells, Notch activation likely represses En/Inv expression. Notch activation also leads to Dri expression and an upregulation of Crb expression. While all of these transcriptional changes could be mediated by Su(H), they could also be further downstream (Iwaki, 2002).
The insertion of Crumbs into the plasma membrane is both necessary and sufficient to confer apical character on a membrane domain. Overexpression of crumbs results in an enormous expansion of the apical plasma membrane and the concomitant reduction of the basolateral domain. This is followed by the redistribution of beta Heavy-spectrin, a component of the membrane cytoskeleton, and by the ectopic deposition of cuticle and other apical components into these areas. Strikingly, overexpression of the membrane-bound cytoplasmic portion of crumbs alone is sufficient to produce this dominant phenotype (Wodarz, 1995).
A mutation in crb deleting a small cytoplasmic region, behaves as a null allele. In embryos homozygous for crb8F105, the Crumbs protein is diffusely distributed in the cytoplasm instead of being apically localized as in wild-type; this mislocation occurs before any morphologically detectable cellular phenotype becomes manifest, suggesting that apical targeting of proteins is affected in crb mutant embryos. This resulet suggests that the polarizing ativity of Crumbs resides in its cytoplasmic domain (Wodarz, 1993).
Mutations in crumbs lead to severe disruptions in the organization of ectodermally derived epithelia and in some cases to cell death (see Reaper) in these tissues (Tepass, 1990).
Loss-of-function mutations in the Drosophila genes crumbs and stardust are embryonic lethal and cause a breakdown of ectodermally derived epithelia during organogenesis, leading to formation of irregular cell clusters and extensive cell death in some epithelia. The mutant phenotype develops gradually and affects, to different extents, the various epithelia. Mutations in stardust produce a phenotype nearly identical to that associated with crumbs mutations, suggesting that both genes are functionally related. Double mutant combinations and gene dosage studies suggest that both genes are part of a common genetic pathway, in which stardust acts downstream of crumbs. The gene function is completely abolished by a crumbs mutation that causes production of a protein with a truncated cytoplasmic domain. Instead of being apically localized as in wild-type, the mutant Crumbs protein is diffusely distributed in the cytoplasm. This occurs before any morphologically detectable cellular phenotype is visible, suggesting that targeting of proteins is affected in crumbs mutant embryos. (Knust, 1993).
In sdt mutant embryos CRB is present only during gastrulation, but becomes undetectable during germ band extension [Image]; the protein is again visible during early organogenesis, at the time when the sdt mutant phenotype becomes apparent. In sdt mutant embryos, CRB is associated with the apical membrane only in well-differentiated epithelial cells, but it is expressed diffusely in the cytoplasm of cells which have lost epithelial morphology. Mosaic experiments suggest that sdt is required cell autonomously, in contrast to the crb requirement, which appears to be non-cell-autonomous. It seems that sdt acts downstream of crb and is activated by the latter (Tepass, 1993).
Deletion of reaper protects embryos from apoptosis caused by x-irradiation and developmental defects. Mutation of the gene crumbs leads to widespread defects in the development of the epithelial tissues, followed by massive cell death during embryogenesis. reaper deletion blocks the massive ectopic death seen in crumbs mutant embryos (White, 1994).
The mutations bazooka and sdt belong to a group in which mutant embryos show severe abnormalities in the differentiation of the larval cuticles, including the genes crumbs (crb) and shotgun (shg). Although the similarity in the late phenotypes of these mutants shows that the respective genes are all required for the same process, i.e., epithelial differentiation, it is difficult to determine whether all these genes act in a common pathway. Nevertheless, the genes crb and sdt show an interesting genetic interaction. Using chromosomal duplications, it has been shown that the phenotype of crb (null) embryos can be rescued by an additional copy of sdt but not vice versa (Tepass, 1993). Based on these findings, a model has been proposed that positions sdt downstream of crb in a regulatory hierarchy (Tepass, 1993). This model is complicated by the fact that sdt regulates Crb protein distribution (Tepass, 1993). A more attractive model might be that sdt functions in a parallel pathway, and, in sufficient dosage, bypasses the requirement for crb (Muller, 1996).
Loss of cell polarity and tissue architecture are characteristics of malignant cancers derived from epithelial tissues. Cells in epithelial sheets are characterized by columnar or cuboidal shape, strong cell-cell adhesion, and pronounced apicobasal polarity. However, tumors of epithelial origin lose these characteristics as they progress from benign growth to malignant carcinoma, and this loss is associated with poor clinical prognosis. Evidence is provided that a group of membrane-associated proteins act in concert to regulate both epithelial structure and cell proliferation. Scribbled (Scrib) is a cell junction-localized protein required for polarization of embryonic and imaginal disc and follicular epithelia. The tumor suppressor scrib was isolated in a screen for maternal effect mutations that disrupt aspects of epithelial morphogenesis such as cell adhesion, shape and polarity. scrib encodes a multi-PDZ (PSD-95, Discs-large and ZO-1) and leucine-rich-repeat protein. The structure of the embryonic cuticle was used to reflect the organization of the underlying epithelial epidermis that secretes it. The wild-type cuticle forms a smooth, continuous sheet, but embryos that are maternally and zygotically mutant for scrib produce a corrugated cuticular surface that is riddled with holes, hence the name scribbled (Bilder, 2000).
To place Scrib within the known pathway for Drosophila epithelial polarity determination, the effect of scrib mutations on Crumbs (Crb) was examined. Crb is an apically localized transmembrane protein that is necessary and sufficient to confer apical character on plasma membrane. In scrib embryos, Crb shows unrestricted localization in both apical and basolateral regions. Whether scrib mutants are identical to a gain-of-function crb phenotype was examined by comparing them with embryos in which GAL4-driven Crb is present throughout the cell membrane. Ectopic Crb that is produced in this manner is sufficient to phenocopy several aspects of scrib embryos, including mislocalization of apical proteins and the cuticle pattern. These data indicate that a major function of Scrib in epithelial polarity is to exclude Crb from the basolateral domain. Since ectopic Crb does not cause the epithelial morphology and multilayering defects seen in scrib embryos, Scrib may be required for the localization of additional epithelial determinants as well (Bilder, 2000).
Analysis of the morphological and polarization phenotypes exhibited by scrib embryos shows that Scrib is a critical component of epithelial architecture in the Drosophila ectoderm, and suggests that its function is closely linked to that of Crb. Scrib is not required for the early localization of basal Discs lost (now redefined as Drosophila Patj) or apical Armadillo during blastoderm formation, and scrib mutants do not exhibit the defective cellularization or precipitous loss of cell adhesion seen when Discs lost or Armadillo, respectively, are depleted in the embryo. The increasingly severe cell shape, polarity and epithelial organization defects of scrib embryos are first manifested after gastrulation, coincident with the onset of defects in crb and stardust embryos. Loss of Crb results in loss of apical proteins from the plasma membrane and a failure to consolidate early adherens junction material into an apical band of zonula adherins (ZAs), while in scrib embryos early adherens junctions become misdistributed basolaterally. Together with the similarities between scrib loss-of-function and crb gain-of-function phenotypes, these data place Scrib and Crb in a pathway required for the progression from the initially differentiated blastoderm membrane domains into a fully polarized epithelium with mature junctions (Bilder, 2000).
These results show that the junctional protein Scrib specifically restricts apical membrane determinants to the apical cell surface. This restriction allows the proper segregation of apical and basolateral domain components, and the appropriate placement of the adherens junction, resulting in full epithelial polarization. How does Scrib, a putative scaffolding protein whose localizaton bounds the apical domain, dictate the proper confinement of apical proteins? Two models suggest themselves. Scrib could assemble a diffusion barrier that physically separates apical and basolateral compartments, similar to the 'fence' function proposed for the vertebrate tight junction. To date, such a barrier has been shown to exist only for lipid diffusion in the outer leaflet of the plasma membrane. If scrib mutations disrupt such a mechanical barrier, then secondary retention systems must serve to maintain basolateral protein restriction from the apical cell surface. An alternative is that Scrib has a role in the polarized targeting of transport vesicles carrying apical proteins. The junctional complex, and in particular the tight junction, has been proposed to be a key sorting site for a subset of Golgi-derived vesicles. In this model, Scrib might interact with the 'exocyst', a secretory targeting apparatus localized to the tight junction and involved in polarized segregation of transmembrane proteins. PDZ domain proteins have been implicated at several different sites of the protein trafficking pathway, and occasional punctate intracellular staining of Scrib is reminiscent of vesicles. Distinction between these models will rely on the identification of binding partners for Scrib (Bilder, 2000).
The asymmetry of neuroblast cell divisions might arise from neuroblast-specific expression of the proteins required for asymmetric division. Alternatively, both neuroblasts and neuroepithelial cells could be capable of dividing asymmetrically, but in neuroepithelial cells other polarity cues might prevent asymmetric division. By disrupting adherens junctions the symmetric epithelial division of epidermal cells can be changed into asymmetric division. The adenomatous polyposis coli (APC) tumor suppressor protein is recruited to adherens junctions, and both APC and microtubule-associated EB1 homologs are required for the symmetric epithelial division along the planar axis. These results indicate that neuroepithelial cells have all the necessary components to execute asymmetric division, but that this pathway is normally overridden by the planar polarity cue provided by adherens junctions (Lu, 2001).
Drosophila neuroblasts delaminate from a polarized epithelial layer in the ventral neuroectoderm and divide asymmetrically along the apical-basal axis to produce larger apical neuroblasts and smaller basal ganglion mother cells. Inscuteable (Insc) as a central protein in organizing neuroblast division. Insc provides positional information that couples mitotic spindle orientation with the basal localization of cell-fate determinants such as Numb and Prospero together with their respective adaptor proteins Partner of Numb (Pon) and Miranda (Lu, 2001 and references therein).
The apical localization of Insc involves both a Baz-dependent initiation step and a maintenance step that requires Baz and Partner of Inscuteable (Pins). The expression of Baz and Pins in both neuroblasts and neuroepithelial cells suggests that these cells share certain apical-basal polarity information. Consistent with this notion is the observation that, when Pon is expressed ectopically in epithelial cells it is localized to the basal cortex, as in neuroblasts. Unlike neuroblasts, however, epithelial cells divide symmetrically along the planar axis and segregate ectopic Pon equally between the two daughter cells. These observations raise further questions: do epithelial cells have the ability to couple spindle orientation with protein localization, and segregate proteins asymmetrically between two unequally sized daughter cells? If so, what prevents them from executing this asymmetric division (Lu, 2001)?
To characterize epithelial division by monitoring it in live embryos, transgenic embryos expressing Pon and tau proteins fused with green fluorescent protein (GFP) were used. During epithelial cell cycle, tau-GFP-labelled mitotic spindle is formed along the planar axis of the embryo, and Pon-GFP is initially uniformly associated with the cortex and then localized to a basal crescent. The mitotic spindle remains oriented along the planar axis throughout mitosis. After cytokinesis, the Pon-GFP crescent is bisected by the cleavage furrow and is equally distributed between two equally sized daughter cells. This in vivo analysis shows that the machinery for basal protein localization is intact in epithelial cells, but it is uncoupled from spindle orientation (Lu, 2001).
Double-stranded (ds) CRB RNA was injected into transgenic embryos expressing Pon-GFP and tau-GFP. In about 70% (n = 200) of crb(RNAi) embryos, the organization of the ectodermal epithelium is disrupted, with epithelial cells losing their columnar shape, adopting rounded morphology, and becoming separated from each other. Live imaging of epithelial divisions in these embryos reveals that nearly all the epithelial cells show a tight coupling between the positioning of Pon-GFP crescents and the orientation of the mitotic spindle. Pon-GFP crescents were found at basal and lateral positions and less frequently at apical positions on the cell cortex, and one of the spindle poles was positioned underneath the Pon-GFP crescent (Lu, 2001).
After cytokinesis, Pon-GFP was segregated to one of the two similarly sized daughter cells. Asymmetric segregation of Pon-GFP to one of two similarly sized daughter cells was also observed in crb zygotic mutant embryos. Immunostaining of crb(RNAi) embryos with antibodies against Asense, Prospero and Insc indicates that epithelial cells do not express these neuronal markers, suggesting that the ability of these cells to undergo asymmetric division is not a result of cell-fate change (Lu, 2001).
Overexpression of the membrane-bound cytoplasmic tail of Crb (Crb-intra) causes similar disorganization of the epithelium as seen in crb mutants. The effect of overexpressing Crb-intra on epithelial division was examined. As observed in crb(RNAi) embryos, epithelial cells overexpressing Crb-intra show coupling of the mitotic spindle with the Pon-GFP crescent and asymmetric segregation of Pon-GFP to one of the daughter cells. Thus, when the formation of the adherens junction is disrupted, epithelial cells switch from a symmetric to an asymmetric division pattern (Lu, 2001).
In addition to its function in localizing Insc and regulating division axis in the neuroblasts, Baz is also required for the formation of adherens junction and the maintenance of epithelial polarity. The function of Baz in epithelial division was examined. The baz(RNAi) embryos showed overall disruption of epithelium organization similar to that observed in crb(RNAi) embryos. Unlike in crb(RNAi) embryos, however, epithelial cells in baz(RNAi) embryos divide in a symmetric fashion, with Pon-GFP distributed uniformly around the cell cortex throughout mitosis and the mitotic spindle orients in random directions. After cytokinesis, two equally sized daughter cells are produced and Pon-GFP is equally distributed between them (Lu, 2001).
Daughter cell size asymmetry in neuroblast division is largely unaffected in baz(RNAi) embryos. In crb(RNAi) epithelial cells Baz can still be localized into a crescent but the crescent is mispositioned and Pon-GFP is always localized to the opposite side of the Baz crescent. This suggests that, although mispositioned, Baz is still functional in directing Pon-GFP localization in crb(RNAi) embryos. To test whether the coupling of Pon-GFP localization with spindle orientation observed in crb(RNAi) embryos is Baz dependent, double RNAi was performed by co-injecting a mixture of baz and crb dsRNAs. Epithelial divisions in the co-injected embryos appeared similar to baz single-injected embryos, with Pon-GFP segregated equally between two equally sized daughter cells. It is therefore concluded that epithelial cells depend on Baz to couple spindle orientation with protein localization when the adherens junction is disrupted (Lu, 2001).
The apical transmembrane protein Crumbs is a central regulator of epithelial apical-basal polarity in Drosophila. Loss-of-function mutations in the human homolog of Crumbs, CRB1 (RP12), cause recessive retinal dystrophies, including retinitis pigmentosa. Crumbs and CRB1 localize to corresponding subdomains of the photoreceptor apical plasma membrane: the stalk of the Drosophila photoreceptor and the inner segment of mammalian photoreceptors. These subdomains support the morphogenesis and orientation of the photosensitive membrane organelles -- rhabdomeres and outer segments, respectively. Drosophila Crumbs is required to maintain zonula adherens integrity during the rapid apical membrane expansion that builds the rhabdomere. Crumbs also regulates stalk development by stabilizing the membrane-associated spectrin cytoskeleton, a function mechanistically distinct from its role in epithelial apical-basal polarity. It is proposed that Crumbs is a central component of a molecular scaffold that controls zonula adherens assembly and defines the stalk as an apical membrane subdomain. Defects in such scaffolds may contribute to human CRB1-related retinal dystrophies (Pellikka, 2002a).
The rhabdomere of photo-receptor cells (PRCs) of Drosophila is a rod-shaped array of microvilli that contains the photopigment. The rhabdomere is surrounded by the stalk membrane that connects it to the zonula adherens (ZA), which separates apical and basolateral membranes. The architecture of PRCs is established during metamorphosis when the apical membranes of PRCs turn by 90o and undergo a dramatic distal-to-proximal expansion as the rhabdomere and stalk are formed. The apical membranes of mammalian PRCs show a similar subdivision into the outer segment, which contains the photopigment, and the surrounding inner segment, which connects to the ZA and the basolateral membrane (Pellikka, 2002a).
Human CRB1 and Crb exhibit a conserved domain architecture and their cytoplasmic domains show 54% identity. Alignment of the cytoplasmic domains of Crb, CRB1, a Caenorhabditis elegans Crb homolog, and two additional putative human Crb-like proteins (CRB2 and CRB3) reveals two conserved regions, including a carboxy-terminal PDZ-domain binding motif (ERLI), through which Crb interacts with the PDZ domain proteins Discs lost (Dlt) and Stardust (Sdt). This study investigated the distribution of Crb and CRB1 in PRCs and the function of Crb in PRC morphogenesis (Pellikka, 2002a).
This analysis identified two roles of Crb in PRC morphogenesis. (1) Crb is required to maintain the integrity of the ZA during its rapid 100-fold expansion in PRCs. In early embryos, Crb is needed for the final step of ZA formation when patches of adherens junction material fuse into a continuous band. Crb probably functions similarly in embryonic ZA formation and during the expansion of the ZA in PRCs. It is speculated that Crb establishes a mechanism at the transition zone of apical and lateral membranes that anchors adherens junction material and thereby supports ZA formation. ZA integrity in crb mutant embryos and PRCs shows recovery at later stages, and crb mutant cells of the imaginal disc display a normal ZA, suggesting that other mechanisms, which functionally overlap with Crb, contribute to ZA formation and maintenance. The Bazooka complex, composed of Bazooka (the Drosophila homolog of C. elegans Par-3 and mammalian ASIP), Drosophila Par-6 and atypical protein kinase 3, is a candidate for such a role because it co-localizes with Crb at the apical membrane and is required for ZA formation in early embryos. Defects in ZA integrity could contribute to retinal dystrophy in patients carrying CRB1 mutations. Functional redundancies among the mechanisms that stabilize cellular architecture may explain the slow progression of human CRB1-associated retinal degeneration (Pellikka, 2002a).
(2) Crb is a central regulator of stalk membrane biogenesis. Several arguments suggest that this role of Crb is independent from its role in ZA formation. (1) Within the stalk membrane, Crb does not localize immediately adjacent to the ZA as in undifferentiated epithelial cells. (2) Overexpression of Crb enlarges the stalk membrane of PRCs but does not transform basolateral membrane to apical membrane or disrupt the ZA as seen with overexpression of Crb in undifferentiated epithelia. (3) The gain-of-function effect that results from overexpression of Crb can be reproduced by expression of CrbextraTM-GFP but not by expression of Crbintra. In contrast, overexpression of Crb and Crbintra but not of CrbextraTM-GFP in undifferentiated epithelia such as the larval eye imaginal disc compromises apical-basal polarity and ZA integrity. (4) karst;crb (karst codes for ßH-Spectrin) double mutants have dramatically reduced stalk membranes but normal ZAs. These findings suggest that in supporting stalk membrane formation, Crb has adopted a role in PRC morphogenesis that is mechanistically distinct from its role in epithelial polarity and ZA formation. These results also reveal for the first time a mechanism that subdivides the apical membrane of epithelial cells into two different domains that support distinct structures and physiologies (Pellikka, 2002a).
The membrane-associated spectrin cytoskeleton can determine membrane topology and stability. These findings demonstrate such a role for the spectrin cytoskeleton of PRCs, because the lack of ßH-Spectrin causes shortening and reduced folding of the stalk membrane. Loss of ßH-Spectrin may destabilize the stalk membrane causing rapid endovesiculation, as is seen when the spectrin cytoskeleton of red blood cells is compromised. Recent work has shown that coated pit budding is accompanied by a local loss of spectrin, and blocking spectrin remodelling results in the retention of coated pits at the cell surface. Coated pits often associate with the folds within the stalk membrane of PRCs, indicating that these folds are the site of frequent endocytosis. It is hypothesized that a rate increase of endocytosis in response to lack of ßH-Spectrin could remove the membrane folds and thus straighten and shorten the stalk membrane. Conversely, stabilization of the apical spectrin cytoskeleton by Crb overexpression may prevent budding of coated pits and thus enlarge the stalk membrane. Crb must also interact with factors other than ßH-Spectrin to support stalk formation, because the loss of Crb leads to a much greater reduction in stalk size than the loss of ßH-Spectrin, and because the kst null mutant phenotype is dominantly enhanced by a crb mutation. Moreover, because the extracellular domain of Crb promotes stalk formation independently of the cytoplasmic domain, Crb is likely to interact with other integral membrane or extracellular factors that in turn can stabilize the stalk membrane. It is proposed therefore that Crb and ßH-Spectrin are components of an extensive molecular scaffold that integrates extracellular, integral membrane and cytoplasmic factors, and specifies topology and size of the stalk membrane (Pellikka, 2002a).
This analysis reveals a function for the extracellular part of the Crb protein. The dominant negative effect of Crbextra expression on stalk formation suggests that the inter-rhabdomeral space contains an important ligand that interacts with the extracellular part of Crb and is titrated out by Crb extra. Although Crb needs to be anchored in the stalk membrane to support stalk formation, cytoplasmic interactions are apparently not essential for this activity, as demonstrated by the effect of CrbextraTM-GFP expression. The critical activity that supports stalk formation resides in the extracellular part of Crb that includes thirty EGF and four LG domains, which represent protein and carbohydrate interaction domains. Also the extracellular domain of human CRB1 may mediate critical interactions, because many mutations that give rise to retinal dystrophies are missense mutations that affect different EGF or LG domains of CRB1. The localization of CRB1 in the inner segment supports the notion that the Drosophila stalk and the inner segment of vertebrate PRCs are homologous plasma-membrane domains that rely on similar mechanisms to support their structural integrity. The localization of CRB1 in the periphery of the cone outer segments is reminiscent of the localization of the tetraspanins peripherin/RDS and ROM1 in rod and cone PRCs. Like these tetraspanins, CRB1 may contribute to the maintenance of cone outer segments (Pellikka, 2002a).
A role for CRB1 in colour vision may have been masked by its function in general retinal maintenance, which is apparent in CRB1-related pathologies. Further analysis of Crb and CRB1 and their interacting factors will shed light on the mechanisms that organize epithelial surface domains, and should help to understand the mechanisms of retinal differentiation and maintenance in humans (Pellikka, 2002a).
Dorsal closure of the Drosophila embryo involves morphological changes in two epithelia, the epidermis and the amnioserosa, and is a popular system for studying the regulation of epithelial morphogenesis. The small GTPase Rac1 has been implicated in the assembly of an actomyosin contractile apparatus, contributing to cell shape change in the epidermis during dorsal closure. Evidence is presented that Rac1 and Crumbs, a determinant of epithelial polarity, are involved in setting up an actomyosin contractile apparatus that drives amnioserosa morphogenesis by inducing apical cell constriction. Expression of constitutively active Rac1 causes excessive constriction of amnioserosa cells and contraction of the tissue, whereas expression of dominant-negative Rac1 impairs amnioserosa morphogenesis. These Rac1 transgenes may be acting through their effects on the amnioserosa cytoskeleton, since constitutively active Rac1 causes increased staining for F-actin and myosin, whereas dominant-negative Rac1 reduces F-actin levels. Overexpression of Crumbs causes premature cell constriction in the amnioserosa, and dorsal closure defects are seen in embryos homozygous for hypomorphic crumbs alleles. The ability of constitutively active Rac1 to cause contraction of the amnioserosa is impaired in a crumbs mutant background. It is proposed that amnioserosa morphogenesis is a useful system for studying the regulation of epithelial morphogenesis by Rac1 (Hardin, 2002).
Expression of dominant negative Drac1N17 in the amnioserosa slows morphogenesis of this tissue which remains as a squamous epithelium for a longer period than in wild-type embryos. In Drac1N17-expressing embryos, where amnioserosa morphogenesis is lagging, the movement of the epidermis is also slowed, and the embryos have a larger dorsal hole than wild-type embryos of similar age. It is thought that the impaired movement of the epidermis in such embryos is caused by lack of morphogenesis in the amnioserosa. These results are strong evidence that active cell shape changes in the amnioserosa are required for normal dorsal closure. Examination of wild-type embryos has shown that this cell shape change in the amnioserosa begins with apical constriction of cells at the anterior and posterior ends of the amnioserosa. These cells have elevated levels of myosin, F-actin and phosphotyrosine, suggesting that an apically localized actomyosin contractile apparatus is driving their constriction. Early in dorsal closure, the middle cells in between the two clusters of apically constricted cells do not show elevated levels of F-actin or myosin but do change shape, losing their original elongation perpendicular to the A-P axis of the embryo. The middle cells may be stretching passively, in response to tension from the cell constrictions occurring at both ends of the amnioserosa. By the end of dorsal closure, the middle cells are both elongated along the A-P axis and apically constricted, and it is conceivable that late in dorsal closure they undergo an active cell shape change as their neighbors did earlier (Hardin, 2002).
Excessive Drac1 activity induces a dramatic contraction of the amnioserosa such that it shrinks to occupy less than half the dorsal hole, and this is accompanied by elevated levels of myosin, F-actin, and phosphotyrosine in this tissue. It is thought that Drac1V12 is driving premature and excessive amnioserosa cell constriction through its effects on the cytoskeleton. It is proposed that Drac1 participates in amnioserosa morphogenesis by driving the assembly of an apical actomyosin contractile apparatus that constricts the amnioserosa cells, first at the ends of the tissue and possibly later in the middle. Contraction of an apical actomyosin belt has been implicated in diverse types of epithelial morphogenesis including Drosophila gastrulation, which shows some similarity to amnioserosa morphogenesis in that both processes involve apical construction of a monolayer of cells that then invaginates (Hardin, 2002).
Cell ablation has been used to address the contributions of the epidermis and amnioserosa to dorsal closure. This work has demonstrated that the amnioserosa is under tension, since ablation of cells in the amnioserosa causes the tissue to recoil away from the wound site, and the leading edge is pushed back away from the dorsal midline. It is concluded that there is active cell shape change in the amnioserosa that contributes to dorsal closure, rather than the tissue being simply compressed by the movement of the leading edge. The finding that the recoiling of the amnioserosa after wounding pushes back the leading edge is consistent with the result that impairing amnioserosa morphogenesis through Drac1N17 expression hinders leading edge migration (Hardin, 2002).
Overexpression of Crb in the amnioserosa leads to contraction of the tissue and failure of dorsal closure. This phenotype was examined in more detail; excessive Crb activity induces a premature constriction of cells at the ends of the amnioserosa. Five P-element-induced crb alleles were identified that are hypomorphic mutations, causing defects in dorsal closure and germband retraction. One of these crb mutations, crbS010409, was characterized in detail. Embryos homozygous for crbS010409 show a dorsal closure defect similar to that seen with expression of Drac1N17 in the amnioserosa: amnioserosa morphogenesis is impaired, but the leading edge cytoskeleton is intact. In contrast to amorphic crb alleles, the epidermis is not disorganized in crbS010409 mutants and it secretes cuticle. Amnioserosa morphogenesis and germband retraction may be particularly sensitive to the level of Crb activity. It is thought that Crb activity in the amnioserosa is required for amnioserosa morphogenesis, although the possibility cannot be excluded that loss of Crb activity elsewhere in the embryo is affecting this process (Hardin, 2002).
Drac1 may act through Crb in regulating the cytoskeleton, since the constitutively active Drac1V12-induced phenotype of excessive contraction of the amnioserosa is weakened in a crbS010409 mutant background. This weaker Drac1V12 phenotype of premature constriction of the end cells of the amnioserosa is very similar to that caused by Crb overexpression. There may be sufficient Crb in the crbS010409 mutant embryos for Drac1V12 to be able to prematurely constrict cells at the ends of the amnioserosa but not to excessively contract the tissue. Crb overexpression does not appear to require Drac1 to cause premature constriction of amnioserosa cells, since it can achieve this in the presence of Drac1N17. The excessive contraction of the amnioserosa caused by Drac1V12 expression in embryos with wild-type Crb activity, and the dumbbell-shaped amnioserosa induced by Crb overexpression, could both result from excessive constriction of amnioserosa cells to produce a tissue that only occupies a fraction of the dorsal hole. Such excessive constriction may be driven by ectopic accumulation of a normally apically localized actomyosin contractile apparatus. A role for Crb in defining the location of the actomyosin contractile apparatus is consistent with the idea that Crb defines the range of the apical membrane cytoskeleton. The actin-crosslinking protein ßHeavy(ßH)-spectrin normally has an apicolateral distribution, but upon overexpression of Crb is also found at the basolateral membrane, indicating a redistribution of the membrane cytoskeleton. ßH-spectrin is required for apical constriction of follicle cells during Drosophila oogenesis and may participate in organization of an actomyosin contractile apparatus. It is conceivable that the ectopic localization of (ßH)-spectrin domain following Crb overexpression could be accompanied by an ectopic accumulation of F-actin and myosin. Future goals in studying Drac1-Crb function in amnioserosa morphogenesis will include addressing the nature of the interaction between the two proteins and defining which portion(s) of the Crb protein are required. The short cytoplasmic domain of Crb appears sufficient to execute all Crb functions studied to date. No definitive role has been found for the large extracellular domain, although there is evidence that the Drosophila and human Crb proteins have non-cell-autonomous functions (Hardin, 2002).
Although Drac1 and Crb both generate premature contraction of the amnioserosa when their activity is experimentally upregulated in this tissue, their phenotypic effects are not identical. Drac1V12 expression drives constriction of all amnioserosa cells early in closure, whereas, at the same stage, Crb overexpression only promotes constriction of the end cells. A plausible explanation for this is that constriction of the middle cells requires Drac1 to activate Crb-independent processes and that Crb function is necessary but not sufficient for middle cell constriction. Crb overexpression in the amnioserosa causes disruption of the leading edge cytoskeleton and a failure of cell shape change in the epidermis, suggesting that a signal from the amnioserosa required for dorsal closure is disrupted. That communication between the amnioserosa and the epidermis is a component of dorsal closure is demonstrated by the observation that JNK signaling in the amnioserosa is required for phosphotyrosine accumulation at the leading edge and dorsalward migration of the epidermis and by the observation that leading edge cells are specified wherever an interface of amnioserosa and dorsal epidermis occurs. Drac1V12 expression in the amnioserosa does not disrupt the leading edge cytoskeleton or prevent closure of the epidermis, and this result suggests that Drac1V12 cannot activate a function of Crb that influences communication between the amnioserosa and the epidermis (Hardin, 2002).
Mutations in the human transmembrane protein CRB1 are associated with severe forms of retinal dystrophy, retinitis pigmentosa 12 (RP12), and Leber's congenital amaurosis (LCA). The Drosophila homolog, crumbs, is required for polarity and adhesion in embryonic epithelia and for correct formation of adherens junctions and proper morphogenesis of photoreceptor cells. Mutations in Drosophila crumbs have been shown to result in progressive, light-induced retinal degeneration. Degeneration is prevented by expression of p35, an inhibitor of apoptosis, or by reduction of rhodopsin levels through a vitamin A-deficient diet. In the dark, rhabdomeres survive but exhibit morphogenetic defects. It is the extracellular portion of the Crumbs protein that is essential to suppress light-induced programmed cell death, while proper morphogenesis depends on the intracellular part. It is concluded that human and Drosophila Crumbs proteins are functionally conserved to prevent light-dependent photoreceptor degeneration. This experimental system is now ideally suited to study the genetic and molecular basis of RP12- and LCA-related retinal degeneration (Johnson, 2002).
The pattern of Crumbs expression in the adult eye was analyzed. Immunohistochemistry reveals that Crumbs protein is found in a circumferential stripe at the apical sides of the photoreceptor cells, bordering the ZA, which is visualized as a circumferential belt by anti-Armadillo staining. Within the apical domain, Crumbs is localized to the apical stalk membrane, which connects the ZA with the rhabdomeres. The rhabdomeres are also apical derivatives containing highly pleated stacks of microvilli, rich in F-actin, that carry the photosensitive pigment rhodopsin and the phototransduction complexes (Johnson, 2002).
Since, in Drosophila, hereditary retinal degenerations are light dependent in several cases, mosaic eyes containing large crb mutant clones of flies kept under constant illumination were examined. The Drosophila eye is composed of about 800 ommatidia, cylindrical, barrel-like structures, containing eight photoreceptor cells in their center that are arranged in a stereotypic manner. When flies carrying crb11A22 mosaic eyes are kept in constant light for 7 days, the retina shows massive degeneration. This phenotype strictly depends on continous exposure to light, since, in flies kept under standard laboratory conditions, i.e., in artificial low light, no degeneration of photoreceptors occurs. crb mosaic flies raised under these conditions show a slightly variable, mutant phenotype. Their rhabdomeres are thicker and shorter compared to wild-type and are often found in close contact with other rhabdomeres of the same ommatidium. Serial cross-sections and horizontal sections stained with FITC-phalloidin, which highlights the F-actin bundles of the rhabdomeres, reveal that the rhabdomeres fail to reach the basal lamina and extend from the distal pole near the lens to only about one third of the normal length. In addition, the stalk membrane is reduced in length. However, the tightly stacked 'semicristalline' internal structure of the rhabdomere is unaffected. The catacomb-like rhabdomere base is also unaffected in mutant photoreceptor cells. In many ommatidia, ZAs are visible in the distal regions of the cells. Control of morphogenesis seems to be cell autonomous, since ommatidia composed of wild-type and mutant cells only exhibit defects in the mutant cells (Johnson, 2002).
To analyze the temporal course of degeneration, eyes carrying mutant clones were sectioned at different time points after light exposure. Eyes kept for only 1 day in constant illumination do not show any sign of photoreceptor degeneration but exhibit only the morphogenetical defects described above. After 5 days in constant light, each ommatidium contains one or two photoreceptor cells with signs of degeneration. In ommatidia exposed to constant light for 7 days, most of the photoreceptor cells are degenerating. Signs of degeneration include the devolution of the highly pleated microvillar rhabdomere structure, concomitant with the loss of the catacomb-like rhabdomere base. Affected nuclei round up, and the nucleolus, nucleoplasm, and cytoplasm appear condensed. These features are indicative of PCD. Assays were conducted to determine whether PCD was the mechanism involved: would overexpression of the baculovirus-derived p35 survival protein inhibit light-induced photoreceptor cell death in crb11A22 mutant ommatidia? It is well established that PCD involves a conserved cascade of cysteine proteases, the caspases, many of which can be inhibited by the ectopic expression of p35 protein. During Drosophila eye development, all stages of endogenous, pattern-forming PCD can be inhibited by using a transgene expressing p35. Similarly, age-related and light-induced retinal degeneration in flies mutant for ninaERH27 (rhodopsin1), rdgC306 (rhodopsin phosphatase), norpAEE5 (phospholipase C), or arr2(P261S) (arrestin) can be prevented by overexpressing p35 survival protein. Overexpression of p35 protein rescues light-induced degeneration of crb11A22 photoreceptor cells, prevents devolution of the rhabdomeres, and preserves the rhabdomere bases. Even when exposed to constant illumination for 14 days, the rescue by p35 expression is similar, implicating PCD as the downstream consequence of crb-induced retinal degeneration. Together these results suggest that crb mediates two functions in photoreceptor cells. One function controls the proper morphogenesis of the rhabdomeres and allows the formation of highly elongated (>100 μm) cells. The other function is required after eclosion for survival of photoreceptor cells when exposed to light (Johnson, 2002).
Since Crumbs is not expressed in the rhabdomere, it cannot directly participate in the signal transduction process, which raises the question of how Crumbs controls cell survival. Recently, a mechanism has been put forward that explains retinal degeneration in a subset of retinal degeneration mutations (arr2, norpA, rdgB, and rdgC) as a result of abnormally stable, light-induced meta-rhodopsin/arrestin complexes. In mutant eyes, these stable complexes are internalized by clathrin-mediated endocytosis. The accumulation of internal meta-rhodopsin/arrestin complexes triggers PCD through an unknown mechanism. In these mutants, PCD is prevented if internalization is inhibited in a shibire (D-dynamin) mutant background or when larvae are raised on a vitamin A-deficient medium. The depletion of vitamin A reduces the amount of rhodopsin to about 3% of its normal amount, which leads to the development of normal, though smaller, rhabdomeres in wild-type flies (Johnson, 2002).
To test whether light-induced degeneration in crb mutant eyes is based on a similar mechanism, crb11A22 mosaic flies were raised on this vitamin A-deficient medium and exposed to continuous room light. Mutant photoreceptor cells show smaller, thinner rhabdomeres due to the reduced amount of rhodopsin. They also show morphogenetic defects similar to those of crb11A22 ommatidia raised on standard medium and kept in the dark. However, only minor signs of photoreceptor degeneration were found. This finding is consistent with the possibility that PCD in illuminated crb11A22 photoreceptor cells is due to the increased internalization of meta-rhodopsin/arrestin complexes. Proof of this will require a direct determination of the number of complexes formed, their localization, and the possible involvement of endocytosis in crb-mediated degeneration (Johnson, 2002).
For the embryo, it is well documented that the short intracellular domain of Crumbs is crucial for proper function since its truncation leads to a complete loss of function phenotype. The cytoplasmic domain recruits the MAGUK protein Stardust and the four PDZ-domain protein Discs Lost into a subapical protein scaffold (SAC). Overexpression of the membrane-bound cytoplasmic domain (Crumbsintra) is sufficient to achieve a partial rescue of crb embryos, and the degree of rescue is comparable to that obtained by overexpression of the full-length Crumbs protein. In order to determine which part of the Crumbs protein is necessary to prevent retinal degeneration, clones were induced that were homozygous mutant for the crb8F105 allele. The crb8F105 allele encodes a protein that lacks the C-terminal 23 amino acids of the cytoplasmic domain and is completely nonfunctional in the embryo. The morphogenetic phenotype observed in mosaic eyes of crb8F105 is comparable to that of the crb11A22 null allele kept in the dark. In contrast to crb11A22, however, crb8F105 mutant ommatidia do not show major signs of degeneration, even after 14 days of exposure to light. This indicates that either the extracellular domain or the N-terminal portion of the cytoplasmic tail of Crumbs prevents light-induced degeneration. To discriminate between these two possibilities, crb8F105 and crb11A22 mutant ommatidia were analyzed in the presence of a transgene expressing the membrane-bound cytoplasmic domain of Crumbs. This transgene largely rescues the morphogenetic defects in mosaic eyes of both alleles under low-light conditions. This is manifested by the fact that many rhabdomeres are elongated to reach the basal lamina. In contrast, crb11A22 photoreceptor cells exposed to light still undergo retinal degeneration despite the expression of the cytoplasmic domain. It is concluded that the intracellular domain of Crumbs is not sufficient to prevent light-induced photoreceptor degeneration and suggest a function for the extracellular domain in the prevention of PCD. Interestingly, all mutations mapped to the CRB1 gene in RP12 or LCA patients have been localized to the extracellular portion of the protein and lead either to amino acid exchanges, frame shifts, or stop codons. While this may simply reflect the fact that the intracellular domain is a small target for mutagenesis, the large number of RP12 cases showing exclusive amino acid exchanges extracellularly indicate a requirement for an intact extracellular CRB1 domain (Johnson, 2002).
The results in Drosophila indicate that the depletion of rhodopsin through a vitamin A-deficient diet prevents the light-induced crb11A22 degeneration of the retina. A randomized study of ungenotyped RP patients, given high-dose oral vitamin A supplementation, suggested a modest slowing of the disease progression. This finding may indicate that, at least for CRB1-induced RP12 and LCA cases, a high-dose vitamin A supplementation might be counterproductive rather than beneficial in slowing disease progression. Both assumptions on the progression of the disease should be tested in a vertebrate animal model, i.e., a knockout mouse. The Drosophila system, however, can now be exploited to unravel the underlying mechanism of crb-dependent retinal degeneration (Johnson, 2002).
The apicobasal polarity of epithelia depends on the integrated activity of apical and basolateral proteins, and is essential for tissue integrity and body homeostasis. Yet these tissues are frequently on the move as they are sculpted by active morphogenetic cell rearrangements. How does cell polarity survive these stresses? This question was analyzed in the renal tubules of Drosophila, a tissue that undergoes dramatic morphogenetic change as it develops. This study shows that whereas the Bazooka and Scribble protein groups are required for the establishment of tubule cell polarity, the key apical determinant, Crumbs, is required for cell polarity in the tubules only from the time when morphogenetic movements start. Strikingly, if these movements are stalled, polarity persists in the absence of Crumbs. Similar rescue of the ectodermal phenotype of the crumbs mutant when germ-band extension is reduced suggests that Crumbs has a specific, conserved function in stabilising cell polarity during tissue remodelling rather than in its initial stabilisation. A requirement was also identified for the exocyst component Exo84 during tissue morphogenesis, which suggests that Crumbs-dependent stability of epithelial polarity is correlated with a requirement for membrane recycling and targeted vesicle delivery (Campbell, 2009).
Epithelial cell rearrangements are accompanied, and may be driven by, changes in the spatial arrangement of their intercellular contacts. Contacts with new neighbours are engineered by the reorganisation of intercellular junctions through vesicle recycling; zonula adherens (ZAs) shrink as the interface disappears between cells that lose contact and expand as borders develop between new neighbours. These changes involve dynamin-dependent endocytosis of ZA components, rab-mediated vesicle trafficking and the targeted recycling of vesicles to specific membrane domains (Classen, 2005; Langevin, 2005). This study shows that Crb is specifically required to maintain apicobasal polarity and the integrity of ZAs as these cell activities occur. If cell rearrangements are halted or reduced in crb mutants, there is extensive rescue of epithelial integrity. This leads to proposal of a model in which the Crb complex is dispensable for the establishment of cell polarity in embryonic epithelia but that as soon as morphogenetic cell rearrangements start, the Crb complex acts both to stabilise apical proteins and to restrict the spread of basolateral proteins (Campbell, 2009).
The requirement for E-cadherin in different tissues shows a similar dependence on the degree of morphogenetic activity. It has been shown that the zygotic Drosophila E-cadherin mutant phenotype can be rescued in dynamic tissues, for example in the neurectoderm and Malpighian tubules, by suppressing morphogenetic cell movements (Campbell, 2009).
It is tempting to speculate that Crb acts by targeting recycling vesicles of ZA components in order to maintain junctional integrity in the elongating renal tubules. Without Crb ZAs are lost and membrane domains no longer remain distinct, leading to the collapse of cell polarity. Alternatively, lack of Crb could result in loss of cell polarity in morphogenetically active tissues and, as a consequence, ZAs cannot be maintained. In this case the primary requirement for Crb during cell movement would be to maintain the apical localisation of Baz/Par-6/aPKC, thereby also ensuring the normal distribution of basolateral proteins (Campbell, 2009).
The exo84 mutant phenotype in remodelling tissues could be explained by a depletion of apical Crb caused by reduced exocyst-mediated delivery, resulting in a phenotype reminiscent of crb mutant embryos. This view is supported by recent findings showing that expression of dominant-negative Cdc42 in stage 9-11 embryos results in loss of Crb and other proteins from the apical membrane and disruption of cadherin-based adhesion in the morphogenetically active ventral neuroectoderm. Furthermore, mutations in crb enhance the phenotype induced by dominant-negative Cdc42 expression in this tissue (Harris, 2008). That study suggests that Cdc42 normally acts to repress endocytosis of apical proteins including Crb, so that inactivation of Cdc42 results in defects in cell polarity, leading to the loss of ZAs and tissue disruption. Whether Crb ensures ZA plasticity during cell rearrangements by restricting excessive endocytosis of apical proteins indirectly via stabilisation of Baz and/or Par-6 and Cdc42, or in a more direct way by regulating the recycling of junctional proteins remains to be determined. Similar defects in ZA integrity were also observed in the epithelium of the developing adult dorsal thorax upon loss of Cdc42 function. This study has shown that Cdc42-Par-6-aPKC control endocytosis by the cytoskeletal regulators Arp2/3 and Cip4 and WASP. These results complement and support recent data showing that Cdc42, together with PAR-3, PAR-6 and PKC, are required for membrane trafficking in C. elegans coelomocytes and human HeLa cells (Campbell, 2009).
Members of the Crb complex also play a critical role in ZA stability and apical membrane delivery or stabilisation during photoreceptor development, when the ZAs enlarge and the apical domain selectively expands as the rhabdomere forms. These morphogenetic events require the targeted delivery and retention of large amounts of membrane. Here too, it is not yet clear whether Crb acts directly on the stability of ZA components or indirectly, by controlling other polarity proteins. Although Drosophila Par-6 is delocalised in crb mutant photoreceptor cells, other data suggest that the Crb complex regulates ZA integrity and trafficking of apical membrane via stabilisation of the membrane-associated cytoskeleton, including βH-spectrin (Campbell, 2009).
Vertebrate homologues of members of the fly Crb complex appear to have conserved roles in the control of epithelial integrity. Loss of oko meduzy, one of the five zebrafish crb orthologues, of the sdt orthologue nagie oko or of mosaic eyes, a regulator of Crb, perturbs polarity and morphogenesis of the retinal neuroepithelium and the heart. In addition, RNAi-mediated knock down of the mammalian Sdt orthologue, Pals1, in Madin Darby canine kidney (MDCK) cells in culture prevents proper delivery of E-cadherin to the cell surface, a phenotype strikingly similar to that of crb or sdt mutant epithelia in Drosophila (Campbell, 2009).
These observations suggest that, as in the fly, vertebrate epithelia affected by the loss of Crb are those that undergo morphogenetic reorganisation, including cell shape change. Loss of human CRB1 is associated with retinitis pigmentosa and Leber congenital amaurosis, resulting in retinal degeneration and blindness, a phenotype with striking similarity to flies with crb mutant photoreceptor cells, which exhibit light-dependent retinal degeneration. In mice mutant for Crb1, photoreceptor cells develop normally but later their ZAs degenerate so that photoreceptors in certain areas of the retina are displaced, followed by cell death. Further experiments will elucidate whether the defects observed in morphogenetically active epithelia and photoreceptor cells are based on a common cell biological function of the Crb complex in these two cell types (Campbell, 2009).
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date revised: 20 December 2009
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