Protein expression was not detectable on whole mount embryos before stage 12, just at the onset of germ band retraction. Coracle appears simultaneously in the epidermis, hindgut and foregut. As the germ band retracts, expression increases in these tissues and also appears in the tracheal branches and salivary glands. Coracle is not expressed within the CNS although it is expressed within some peripheral nervous system sensory neural cells such as the scolopidia of the pentascolopidial chordotonal organ. Once established, this pattern of expression is maintained throughout embryogenesis. Differential distribution of the different transcripts are not observed (Fehon, 1994).

Coracle is closely associated with the cell membrane in all expressing cells. In tall columnar epithelia, such as hindgut, there is an obvious polarity of Coracle expression toward the apicolateral epithelial surface (Fehon, 1994).


Imaginal disc cells show intense staining in regions of cell contact just below the apical epithelial surface, just as was observed in embryonic epithelia. Filamentous actin is largely localized to the most apical region of the cell, while the majority of Coracle localization is clearly more basal. In addition, there is a significant amount of filamentous actin in the basolateral domain of these cells, but only background levels of Coracle. Antibodies against Drosophila Spectrin have shown that Spectrin has a similar apical and basal location to that of actin, although the basolateral component is more predominant for spectrin. Thus, the Coracle protein does not appear to colocalize to a great extent with either filamentous actin or spectrin in the apical-basal axis of the cell (Fehon, 1994).

The distribution of proteins in the apico-lateral cell junctions in Drosophila imaginal discs was examined. The subcellular distribution of these proteins in normal and mutant proliferating cells was analyzed with marker antibodies and confocal microscopy. Antibodies to phosphotyrosine (PY), Armadillo (Arm) and Drosophila E-cadherin (DE-cad) as well as FITC phalloidin marking filamentous actin, labeled the site of the adherens junction, whereas antibodies to Discs large (Dlg), Fasciclin III (FasIII) and Coracle (Cor) labeled the more basal septate junction. The junctional proteins labeled by these antibodies underwent specific changes in distribution during the cell cycle. A loss-of-function dlg mutation, which causes neoplastic imaginal disc overgrowth, leads to loss of the septate junctions and the formation of what appear to be ectopic adherens junctions (Woods et al., 1996).

The current study was expanded to examine the effects of mutations in other genes that also cause imaginal disc overgrowth. Based on staining with PY and Dlg antibodies, the apico-lateral junctional complexes appear normal in tissue from the hyperplastic overgrowth mutants fat facets, discs overgrown, lethal (2) giant discs and warts. However, imaginal disc tissue from the neoplastic overgrowth mutants dlg and lethal (2) giant larvae show abnormal distribution of the junctional markers including a complete loss of apico-basal polarity in loss-of-function dlg mutations. These results support the idea that some of the proteins of apico-lateral junctions are required both for apico-basal cell polarity and for the signaling mechanisms controlling cell proliferation, whereas others are required more specifically in cell-cell signaling (Woods, 1997).

Invasive cell behavior during Drosophila imaginal disc eversion is mediated by the JNK signaling cascade

Drosophila imaginal discs are monolayered epithelial invaginations that grow during larval stages and evert at metamorphosis to assemble the adult exoskeleton. They consist of columnar cells, forming the imaginal epithelium, as well as squamous cells, which constitute the peripodial epithelium and stalk (PS). A new morphogenetic/cellular mechanism for disc eversion has been uncovered. Imaginal discs evert by apposing their peripodial side to the larval epidermis and through the invasion of the larval epidermis by PS cells, which undergo a pseudo-epithelial-mesenchymal transition (PEMT). As a consequence, the PS/larval bilayer is perforated and the imaginal epithelia protrude, a process reminiscent of other developmental events, such as epithelial perforation in chordates. When eversion is completed, PS cells localize to the leading front, heading disc expansion. The JNK pathway is necessary for PS/larval cells apposition, the PEMT, and the motile activity of leading front cells (Pastor-Pareja, 2004).

One hallmark of epithelial cells is their distinct apico-basal cell polarity. This polarity depends on a set of intercellular connections, which encircle epithelial cells at the border of the apical and basal-lateral membrane domains. The cells in insect epithelial tissues are interconnected by zonula adherens (ZAs), which function in both cellular adhesion and signaling. DE-cadherin is the major constituent of the ZAs in a complex with Armadillo (Arm, ß-catenin) and Dalpha-catenin. In addition, epithelia of flies and other invertebrates exhibit septate junctions, which are located basally to the ZAs. Septate junctions prevent diffusion through the pericellular space and are functionally equivalent to vertebrate tight junctions (Pastor-Pareja, 2004).

All imaginal disc cells at the third instar larval stage presented ZAs in an apical belt. During disc eversion, however, it was found that ZAs components delocalize from the free edges of the PS cells, remaining cytoplasmic at the edges of the perforations arising through the PS/larval bilayer and in those PS cells leading the spreading of the discs over the larval tissues. As a consequence, ZAs are lost in these cells. Moreover, septate junction components, such as Coracle and Disc Large are also found to be missing from the membranes of leading front cells (Pastor-Pareja, 2004).

The loss of apico/basal polarity and adhesion of the PS cells during disc eversion is reminiscent of an epithelial-mesenchymal transition (EMT), as described for mesoderm and neural crest cells in vertebrates, and for the acquisition of the invasive phenotype in carcinomas (Pastor-Pareja, 2004).

In summary, the evagination of imaginal disc can be divided into the following sequential steps: (1) an overall positional change of the imaginal discs leading to the confrontation and apposition of the PS and the larval epidermis; (2) a regulated modulation (PEMT) of PS cells, which involves the downregulation of their cell-cell adhesion systems and allows them to move into their local neighborhood and invade the larval epithelium; (3) the fenestration of the peripodial/larval bilayer and the formation of an unbound peripodial leading front, which will direct imaginal spreading by planar cell intercalation, and (4) a bulging of the imaginal tissue (Pastor-Pareja, 2004).

Once the hole is opened, the planar intercalation of PS cells ensures that, first in the hole and later in the leading front, all four dorsal, ventral, anterior, and posterior compartments of the wing disc are represented. This mechanism also guarantees the maintenance of a continuous epithelial barrier (Pastor-Pareja, 2004).

Transient apical polarization of Gliotactin and Coracle is required for parallel alignment of wing hairs

In Drosophila, wing hairs are aligned in a distally oriented, parallel array. The frizzled pathway determines proximal-distal cell polarity in the wing; however, in frizzled pathway mutants, wing hairs remain parallel. How wing hairs align has not been determined. A novel role for the septate junction proteins Gliotactin (Gli) and Coracle (Cora) in this process has been demonstrated. Prior to prehair extension, Gli and Cora are restricted to basolateral membranes. During pupal prehair development, Gli and Cora transiently form apical ribbons oriented from the distal wing tip to the proximal hinge. These ribbons are aligned beneath prehair bases and persisted for several hours. During this time, Gli was lost entirely from the basolateral domain. A Gliotactin mutation alters the apical polarization Gli and Cora and induces defects in hair alignment in pupal and adult stages. Genetic and cell biological assays demonstrate that Gli and Cora function to align hairs independently of frizzled. Taken together, these results indicate that Gli and Cora function as the first-identified members of a long-predicted, frizzled-independent parallel alignment mechanism. A model is proposed whereby the apical polarization of Gli and Cora functions to stabilize and align prehairs relative to anterior-posterior cell boundaries during pupal wing development (Venema, 2004).

The Gliotactin null phenotype is paralysis and death due to an open blood-nerve barrier at the end of embryogenesis. No adult-viable mutant Gli genotypes have been reported. In order to extend the analysis of Gliotactin function postembryogenesis, a mutagenesis screen was performed and a number of novel, ethylmethane-sulfonate (EMS)-induced Gli alleles were isolated that failed to complement the null allele GliAE2Δ45. One novel allele, Glidv5, was homozygous viable. Sequencing of this allele revealed a single, nonconservative mutation in the extracellular serine esterase-like domain. Several of the novel and previously identified homozygous-lethal Gli alleles generated adult escapers in trans to Glidv5, resolving these alleles into an allelic series. Structural changes underlying this allelic series were determined by sequencing each allele. GliRAR77 was found to contain a nonsense mutation in the intracellular carboxyl-terminal region. This homozygous-lethal allele generated more adult escapers in trans to Glidv5 than Glidv5 itself, indicating that the extracellular and intracellular domains of Gli can function together when on separate proteins. The Glidv1 allele results from a premature stop codon within the serine esterase like domain; this allele escaped in trans to Glidv5 at a low frequency. Two alleles (GliP34 and Glidv3) rarely escaped in trans to Glidv5; both alleles are premature stop codons early in the Gli open reading frame (Venema, 2004).

The availability of Gli genotypes that survive to adult stages allowed an examination of adult epithelia for mutant phenotypes. Adult wings of Gli mutants were examined for defects. Gli mutants display an increasing severity of wing hair orientation defects that parallels the Gli allelic series. The primary Gli wing hair phenotype was disruption of parallel alignment between neighboring hairs such that adjacent hairs converge in a chevron pattern. This phenotype was present in Gli wings not mounted under a coverslip, as well as in Gli wings examined using scanning electron microscopy, and as such is not a mounting artifact. To quantify the Gli phenotype, veins were used to divide the wing into seven main regions, 20 wings of each genotype were scored, and the number of regions that contained patches of nonparallel hairs were counted. The Gli+ chromosome used for mutagenesis had the wild-type pattern of parallel, distally oriented hairs. In weak Gli genotypes, only small numbers of hairs are affected, resulting in small, infrequent mutant patches. Random samples of 20 Glidv5/GliRAR77 or Glidv5/Glidv5 wings ranged from wings with disrupted hairs in every wing region to completely wild-type wings. On average, Glidv5/GliRAR77 wings had 2.9 regions with altered alignment compared to 3.4 for Glidv5/Glidv5 wings. In stronger Gli genotypes, mutant patches were more frequent and larger. All 20 Glidv5/Glidv1 wings sampled had at least one region of disrupted alignment, with an average of 4.1 regions affected. In the severe Glidv5/Glidv3 genotype, mutant patches covered large portions of the wing and were consistently present. In a random sample of 20 Glidv5/Glidv3 wings, eight had an area of disrupted alignment in six wing regions; the remaining 12 wings had disrupted alignment in all seven intervein areas, giving an average of 6.7 disrupted regions per wing. Despite this consistent phenotype, hairs with correct alignment remained in even the most severely affected Glidv5/Glidv3 wings. In addition to nonparallel alignment, patches of deformed hairs were occasionally observed in all adult Gli genotypes, often at the anterior-posterior boundary. Deformed hairs were thinner than normal and were often bent. A third phenotype, blistering at the distal margin, was observed in the strongest Gli genotype, Glidv5/Glidv3. In a sample of 20 Glidv5/Glidv3 wings, 7 contained blisters. Such blisters were always in contact with the distal margin (Venema, 2004).

In Drosophila, septate junctions are initially formed during embryogenesis: these septate junctions persist in epithelia derived from the epidermis, such as imaginal structures. In all previously examined tissues, septate junctions, once formed, appear static in the absence of cell division. In contrast, Gli and Cora are extensively remodeled during wing development at several time points in postmitotic cells. The initial Gli/Cora pattern in the wing before prehair extension is identical to that of the embryonic epidermis. Indeed, Gli and Cora remain stable until after prehair extension at approximately 32 h APF. Beginning at 35 h APF, Gli and Cora are apically polarized at the wing margins. Althought the initial stages of Cora polarization have been noted previously, previous studies did not assay Cora localization past 35 h APF or examine mutant cora genotypes. By 37 h APF, Gli and Cora colocalize to continuous apical ribbons across the pupal wing. These ribbons run along anterior-posterior cell boundaries and between proximal and distal vertices, passing just basal to prehair bases. At this stage, Gli is absent from basolateral membranes, yet Cora retains its basolateral pattern. This pattern remains stable until at least 40 h APF. By 47 h APF, Cora has lost its apical polarization and is present only around the basolateral cell periphery. In contrast, Gli is lost apically and basolaterally with only minimal Gli present on basolateral membranes, which is not predominantly tricellular at 47 h APF. It may be that the weak tricellular staining of Gli at this time point represents the start of relocalizing Gli to the tricellular septate junction; examination of Gli at stages after 47 h APF is underway to test this possibility (Venema, 2004).

The dynamic rearrangement of these junction components is without precedent in the literature. While septate junction components such as Discs-large, Coracle, Neurexin IV, Neuroglian, and the Na+/K+ ATPase are expressed in wing discs, these studies have not examined the localization of septate junction components past 30 h APF nor examined mutant genotypes for wing hair or adhesion phenotypes. Thus, it is an open question whether these septate junction proteins also are dynamic during later stages of wing development. It remains to be seen whether these components are recruited to the apical Gli/Cora ribbon and if their basolateral localization at septate junctions is modified during Gli and Cora polarization (Venema, 2004).

Loss of Dlg function in wing discs causes an overgrowth phenotype that includes loss of apical-basal polarity. In contrast to Dlg, cora null clones do not survive to adult stages. Additionally, clones for null alleles of Nrg and Nrv2 do not survive, consistent with the results that null Gli clones are cell lethal. Given the essential nature of the septate junction for both proliferation control and cell viability, it will not be possible to assess the adult wing phenotype for complete loss of septate-junction function. Attempts to induce null Gli clones after 30 h APF have not proved successful since cell division in the wing ceases around this stage. Null clones induced during the last round of cell division did not survive long enough to examine the start of Gli/Cora polarization at 35 h APF (Venema, 2004).

The polarization of Gli and Cora to apical ribbons is compromised in Gli mutant wings, which in turn leads to unstable prehairs later in development. The close proximity of the Gli ribbon to the prehair base, and the orientation of the ribbon along the axis the hairs will align to, is compelling evidence for a physical connection between prehairs and Gli/Cora ribbons that is required for hair alignment. In Gli mutant wings, the polarization of apical Gli and Cora is incomplete; additionally, it does not persist for the correct length of time. In wild-type wings, Gli and Cora remain polarized until at least 40 h APF; in contrast, in Gli mutant wings Gli is lost altogether by 40 h APF and Cora is no longer polarized. Thus, the Gli nonparallel phenotype seems to result from either a lack of apically polarized Gli/Cora or an insufficient time span of their polarization, or perhaps a combination of both. In either case, the nonparallel phenotype seems to arise from a failure to properly polarize apical Gli and Cora, which in turn leads to prehair instability later in development (Venema, 2004).

Though apical Gli and Cora define the distal-to-proximal axis in the wing, how they are connected to the prehair for its stabilization remains unresolved. The separation between the prehair base and the apical ribbon of Gli and Cora passing beneath it suggests an intermediate link between the two structures. A possible candidate to connect the Gli/Cora ribbon to the prehair pedestal is the cytoskeleton. Physical and genetic links between septate junction components and the cytoskeleton have previously been reported. Both subunits of the Na+/K+ ATPase are localized to the septate junction and physically interact with ankyrin, which acts as an adapter between transmembrane proteins and the spectrin/F-actin network. In Drosophila, the subcellular location of the Na+/K+ ATPase is disrupted in β spectrin mutants. Additionally, Neuroglian clustering assembles ankyrin at cell-cell contacts through physical association. Interactions through either component could physically connect the Gli/Cora ribbon to the F-actin prehair through spectrin; it remains to be seen, however, if the Na+/K+ ATPase and/or Neuroglian are apically polarized in conjunction with Gli and Cora. Previous studies in the wing have demonstrated that actin polymerization is necessary for prehair formation and cell viability. Disruption of F-actin regulation also causes wing hair phenotypes: perturbation of RhoA and its downstream effector Drok causes multiple hair phenotypes. Application of F-actin-disrupting drugs to pupal wings also causes a multiple hair phenotype. The Gli hair phenotype does not include multiple hairs per cell, suggesting that prehair F-actin is correctly regulated in Gli mutants (Venema, 2004).

The tubulin cytoskeleton is also a potential candidate for connecting the septate junction to the apical prehair. Microtubules form a web that contacts the cell periphery at the level of Cora staining at 30 h APF; additionally, microtubules are present inside the prehair after elongation. Experiments are underway to determine if the tubulin cytoskeleton is polarized to prehair bases in conjunction with Gli and Cora (Venema, 2004).

A relationship between septate junction components and the cytoskeleton may also explain the Gli/septate-junction marginal adhesion defects. Adhesion between wing layers is dependent on integrins at basal-basal contacts between wing layers and on the transalar cytoskeleton connecting them. Interestingly, this dependence does not extend to the wing margin: Clones lacking β-integrin adjacent to the wing margin do not cause blisters, even when the clone spans the wing margin, placing cells lacking β-integrin into basal-basal contact. Gli mutations are the first described that blister specifically at the wing margin. Adhesion in this area is thus dependent on Gli and independent of integrins. While the ultrastructure of the transalar cytoskeleton in this region has not been reported, the results suggest the possibility that the septate junction may be an alternative to integrin-based focal adhesions in this area of the wing (Venema, 2004).

When a mechanism functioning to align wing hairs in parallel was first suggested, it was predicted to be frizzled independent. The results have borne this prediction out. Gli and Cora are the first factors necessary for hair orientation shown to function independently of frizzled signaling: The localization and apical polarization of Gli and Cora does not require fz function; conversely, the fz pathway is unaffected by mutations in Gli. Gli and Cora are also the first factors shown to polarize in line with the distal-proximal axis in the pupal wing, the converse pattern of the fz pathway components. Thus, it appears that proximal-to-distal hair polarity is controlled by factors polarized in line with the anterior-posterior axis, while parallel alignment is controlled by factors polarized in line with the proximal-distal axis. Additionally, the Gli nonparallel phenotype appears to arise from unstable prehairs as a result of compromised, but partially functional, apically polarized Gli and Cora. The formation of the final wing hair pattern thus has two phases: the specification of the site of prehair emergence by the fz signaling pathway, and subsequent alignment and stabilization of extended prehairs through apically polarized septate junction components. Future experiments are necessary to determine the precise link between the apical Gli/Cora ribbon and the prehair pedestal; however, the demonstrated interactions between F-actin/ankyrin and the septate junction markers Neuroglian and the Na+/K+ ATPase are likely candidates for further investigation (Venema, 2004).

Effects of Mutation or Deletion

The primary effect of the two known truncation mutations in the coracle gene is a failure in Dorsal closure. Both alleles cause recessive embryonic lethaity, with the stronger allele causing abnormalities in head involution. Other Drosophila genes whose mutants are defective in dorsal closure include basket (also known as JNK), hemipterous, neurexin, Rac1, and zipper (also known as Myosin II). In addition, coracle mutations dominantly suppress Ellipse, a hypermorphic allele of the Drosophila EGF-receptor homolog. (Fehon, 1994).

Although extensively studied biochemically, members of the Protein 4.1 superfamily have not been as well characterized genetically. A phenotypic analysis of coracle has been carried out. Screens for new coracle alleles confirm the null coracle phenotype of embryonic lethality and failure in dorsal closure, and they identify additional defects in the embryonic epidermis and salivary glands. Hypomorphic coracle alleles reveal functions in many imaginal tissues. Analysis of coracle mutant cells indicates that Coracle is a necessary structural component of the septate junction required for the maintenance of the transepithelial barrier but is not necessary for apical-basal polarity, epithelial integrity, or cytoskeletal integrity. In addition, coracle phenotypes suggest a specific role in cell signaling events. Complementation analysis suggests that the conserved amino terminal region (CNTR) of Coracle constitutes a functional domain and that sequences C-terminal to this domain may also be functionally significant (Lamb, 1998).

Several studies have suggested that coracle and some of the other genes required for dorsal closure may play a role in epithelial morphogenesis. To better assess the functions of coracle in the embryonic epidermis, phenotypes of null and strongly hypomorphic mutant embryos have been examined. Cuticle preparations of coracle mutant embryos display characteristic epidermal phenotypes in addition to dorsal closure defects. In coracle mutant embryos the cuticle appears to be thinner than normal and often appears to split into two layers in cuticle preparations. The cuticular thinning is most obvious on the ventral surface where the denticle belts appear faint in cuticle preparations. Although the overall segmental pattern is normal, denticle belts contain fewer than normal denticles, and there are correspondingly fewer hairs on the dorsal side. Ultrastructural examination of the epidermis and cuticle reveals that the apparent delamination of the cuticle results from a failure of the epicuticle to adhere to the procuticle. All of the embryonic lethal alleles also display salivary gland defects, which are apparent as necrotic material that remains in cuticle preparations. This salivary gland defect is observed in embryos that have been aged beyond the stage at which wild-type embryos hatch as larvae, suggesting that this necrosis is a very late effect (Lamb, 1998).

The salivary glands express coracle at high levels. To study the effects of loss of coracle function at the cellular level in epithelial cells, the morphology of salivary gland epithelia in cor5 mutant embryos was studied using several molecular markers for the plasma membrane. These experiments were performed in midembryogenesis (stage 14), before the salivary gland necrosis described earlier becomes apparent. Markers for the adherens junction (Armadillo and Notch) and the apical membrane (Crumbs) all displayed normal subcellular localizations in cor5 mutant cells. It is important to note that these embryos have no detectable Coracle protein and that coracle is not expressed maternally. These results indicate that the apical-basal polarity of epithelial cells is not grossly affected by loss of Coracle function (Lamb, 1998).

It has been reported that in cor5 mutant embryos, Neurexin is mislocalized, raising the possibility that the integrity of the septate junction is compromised in coracle mutant embryos. To investigate this idea, an ultrastructural examination of the epidermis and internal epithelia of late stage 17 mutant embryos was carried out. Similar to the observations regarding loss of Neurexin, the septate junction in cor5 mutant epithelia is disrupted. Although the adherens junction appears unaffected, the individual septae that characterize the pleated septate junction are always absent in cor5 mutant tissue. To examine the functional consequences of this lack of an organized septate junction, the transepithelial barrier function of coracle mutant epithelia was carried out using a 10-kDa rhodamine-labeled dextran. This dye was injected into the hemocoel of stage 16 embryos. Although in wild-type embryos the dextran is confined to the hemocoel even 1 h after injection, in cor5 mutant embryos the dextran rapidly crosses the salivary gland epithelium and fills the luminal space. The epidermis, hindgut, and tracheae are similarly compromised. Taken together, these results demonstrate the essential requirement for coracle in the integrity of the transepithelial barrier function of the septate junction (Lamb, 1998).

To examine further the effects of loss of coracle function in epithelial cells, somatic mosaic analysis was used to generate cor minus cells in the imaginal epithelium. cor4 somatic clones were generated ~76 h after egg laying using the FLP recombinase/FRT target system. coracle mutant clones were observed in late third instar wing imaginal discs using anti-Coracle antibodies to identify mutant cells. The cor4 allele was chosen for this analysis because it is a strong allele that disrupts the ability of Coracle to associate with the plasma membrane. Cells within the mutant clones are contiguous with the rest of the imaginal epithelium and appear normally shaped. Loss of coracle function in imaginal epithelial cells results in disruption of Neurexin localization. In addition, Neurexin protein is not readily detectable in the center of cor- clones, suggesting that in imaginal epithelia Neurexin is not stable in the absence of coracle function. To assess apical-basal polarity and cytoskeletal organization in cor minus cells, imaginal discs were stained with antibodies for Notch, betaH-Spectrin, and Moesin. As in the embryonic epidermis, these markers for the apical junctional region are localized normally in mutant cells. In addition, the distribution of filamentous actin was examined using rhodamine phalloidin and also found to be normal. These results indicate that coracle function is not necessary for overall apical-basal polarity or cytoskeletal organization in imaginal tissues; however, when adult flies were examined for the presence of cor minus tissue, no mutant clones could be observed either in the eye or in the thorax, indicating that cor minus cells do not persist to the adult stage (Lamb, 1998).

The majority of coracle alleles are embryonic lethal; however, some weak alleles and several hypomorphic allelic combinations produce adult escapers. For example, cor7 is mostly embryonic lethal, but it produces 7% of expected viable, fertile adults and can be maintained as a homozygous stock at 25°C. cor14 is almost exclusively larval lethal, but 0.3% of the expected homozygous class survived to viable adults. cor15 is completely viable, with 100% of the expected homozygotes surviving to adulthood. All of the coracle alleles that produced adult escapers display a similar range of adult defects, although the severity and penetrance of the phenotypes varies with genotype. All of these alleles are also cold sensitive (Lamb, 1998).

All coracle escapers display some degree of a rough eye phenotype. These eye defects range from a slight roughening of the eye, especially across the equator, to eyes in which part of the eye tissue in the anterior equatorial region is replaced by head cuticle. The penetrance of severe phenotypes increases with the strength of the coracle allele, to include up to 60% of the mutant adults. Histological sections show that the roughened appearance of coracle mutant eyes is caused by abnormally shaped, fused, improperly spaced, and occasionally misoriented ommatidia, rather than abnormal photoreceptor cell differentiation. Essentially all of the ommatidia in the mutant eyes display normal numbers and positions of photoreceptors. Interommatidial bristles are also frequently lost in the roughened area. Another very penetrant phenotype seen in coracle mutant animals is partial or total loss of the ocelli and associated bristles. In addition to the eye defects, coracle hypomorphic escapers display a range of other pleiotropic defects. These include wing vein phenotypes (interrupted cross veins and truncated fifth veins); rotational defects in the male genital apparatus; kinked or curved bristles, and leg abnormalities. Also both male and female escapers displayed partial or complete sterility (Lamb, 1998).

The lateral mobility of cell adhesion molecules is highly restricted at septate junctions in Drosophila

A complex of three cell adhesion molecules (CAMs) Neurexin IV (Nrx IV), Contactin (Cont) and Neuroglian (Nrg) is implicated in the formation of septate junctions between epithelial cells in Drosophila. These CAMs are interdependent for their localization at septate junctions. For example, null mutation of nrx IV or cont induces the mislocalization of Nrg to the baso-lateral membrane. These mutations also result in ultrastructural alteration of the strands of septate junctions and breakdown of the paracellular barrier. Varicose (Vari) and Coracle (Cora), that both interact with the cytoplasmic tail of Nrx IV, are scaffolding molecules required for the formation of septate junctions. Photobleaching experiments were conducted on whole living Drosophila embryos to analyze the membrane mobility of CAMs at septate junctions between epithelial cells. GFP-tagged Nrg and Nrx IV molecules were shown to exhibit very stable association with septate junctions in wild-type embryos. Nrg-GFP is mislocalized to the baso-lateral membrane in nrx IV or cont null mutant embryos, and displays increased mobile fraction. Similarly, Nrx IV-GFP becomes distributed to the baso-lateral membrane in null mutants of vari and cora, and its mobile fraction is strongly increased. The loss of Vari, a MAGUK protein that interacts with the cytoplasmic tail of Nrx IV, has a stronger effect than the null mutation of nrx IV on the lateral mobility of Nrg-GFP. It is concluded that the strands of septate junctions display a stable behavior in vivo that may be correlated with their role of paracellular barrier. The membrane mobility of CAMs is strongly limited when they take part to the multimolecular complex forming septate junctions (see Organization of adhesion complexes at epithelial cell contacts in the wild-type and mutant embryos). This restricted lateral diffusion of CAMs depends on both adhesive interactions and clustering by scaffolding molecules. The lateral mobility of CAMs is strongly increased in embryos presenting alteration of septate junctions. The stronger effect of vari by comparison with nrx IV null mutation supports the hypothesis that this scaffolding molecule may cross-link different types of CAMs and play a crucial role in stabilizing the strands of septate junctions (Laval, 2008. Full text of article).

Epithelial polarity proteins regulate Drosophila tracheal tube size in parallel to the luminal matrix pathway

The integrity of polarized epithelia is critical for development and human health. Many questions remain concerning the full complement and the function of the proteins that regulate cell polarity. This study reports that the Drosophila FERM proteins Yurt (Yrt) and Coracle (Cora) and the membrane proteins Neurexin IV (Nrx-IV) and Na+,K+-ATPase are a new group of functionally cooperating epithelial polarity proteins. This 'Yrt/Cora group' promotes basolateral membrane stability and shows negative regulatory interactions with the apical determinant Crumbs (Crb). Genetic analyses indicate that Nrx-IV and Na+,K+-ATPase act together with Cora in one pathway, whereas Yrt acts in a second redundant pathway. Moreover, it was shown that the Yrt/Cora group is essential for epithelial polarity during organogenesis but not when epithelial polarity is first established or during terminal differentiation. This property of Yrt/Cora group proteins explains the recovery of polarity in embryos lacking the function of the Lethal giant larvae (Lgl) group of basolateral polarity proteins. It was also found that the mammalian Yrt orthologue EPB41L5 (also known as YMO1 and Limulus) is required for lateral membrane formation, indicating a conserved function of Yrt proteins in epithelial polarity (Laprise, 2009).

To clarify the mechanisms of epithelial polarization, the function was examined of the FERM-domain protein Yrt, which was shown to act as a negative regulatory component of the Crb complex in both Drosophila and vertebrates. Crb regulates epithelial apical basal polarity by promoting apical membrane and apical junctional complex formation. Crb also controls the growth of the apical membrane at late stages of epithelial differentiation. Yrt binds to the cytoplasmic tail of Crb and restricts Crb activity in apical membrane growth. However, Yrt is predominantly a basolateral protein that is recruited into the Crb complex only at late stages of epithelial differentiation. Embryos completely lacking maternal and zygotic Yrt (yrtM/Z) displayed polarity defects before Yrt is recruited into the Crb complex at late organogenesis. This raises the question whether Yrt has a function in epithelial organization as a basolateral protein (Laprise, 2009).

yrtM/Z mutants and double-mutant combinations of yrt and genes encoding basolateral proteins were examined for synergistic genetic interactions that could indicate functional cooperation. yrtM/Z embryos showed clear polarity defects at post-gastrula stages of development, as indicated by the basolateral mislocalization of Crb. In contrast, zygotic yrt mutants demonstrated only minor polarity defects in the trunk ectoderm. This indicates that yrt is required for polarity, and that zygotic yrt mutants could provide a sensitized background to reveal genetic interactions. Within the basolateral membrane, Yrt is enriched at the septate junction together with the Na+,K+-ATPase and other septate junction components from stage 14 onwards. Marked genetic interactions were found between yrt and Atpα (which encodes the α-subunit of Na+,K+-ATPase) and between yrt and Nrx-IV, but not between yrt and six other genes that encode septate junction transmembrane proteins. In contrast to wild type and single mutants, apical markers were mislocalized in yrt Atpα and yrt Nrx-IV double mutants similar to yrtM/Z embryos. Enhancement of the Nrx-IV null phenotype by yrt indicates that Nrx-IV and yrt have overlapping functions and do not operate in a linear pathway. The pathway relationship between yrt and Atpα remains uncertain as embryos completely devoid of Na+,K+-ATPase function cannot be analysed. These findings reveal previously unrecognized functions of the Na+,K+-ATPase and Nrx-IV in epithelial polarity (Laprise, 2009).

The developmental timing of the yrtM/Z and the yrt Atpα and yrt Nrx-IV polarity phenotypes is notable. These mutants show no polarity defects during cellularization when epithelial cells first form or during gastrulation when the apical junctional belt is assembled. Polarity defects are first seen at stage 11 or 12, and are most prominent at embryonic stage 13. In contrast to wild type, Crb and other apical markers are found throughout the plasma membrane and colocalize with basolateral markers such as Discs large (Dlg, also known as Dlg1) and Fasciclin 3 in yrtM/Z, yrt Atpα and yrt Nrx-IV mutants. During late embryogenesis, apical markers become restricted again to the apical membrane, which, however, remains abnormally extended and dome-shaped. Thus, Yrt, Na+,K+-ATPase and Nrx-IV cooperate and have critical functions in maintaining epithelial polarity during early organogenesis (stages 11-13), well before septate junction assembly is observed, and Yrt is recruited to the apical membrane at stage 14 (Laprise, 2009).

Na+,K+-ATPase and Nrx-IV are required for septate junction assembly. Because Yrt also accumulates at septate junctions, it was asked whether Yrt has a role in septate junction formation or function. Septate junctions are basal to the adherens junction and, like vertebrate tight junctions, prevent diffusion of solutes between cells. Dye injection assays show that paracellular barriers in epithelia of yrtM/Z embryos are compromised. However, yrtM/Z embryos showed a normal complement of septa when examined ultrastructurally whereas Atpα and Nrx-IV mutants lack septa. Immunoprecipitation experiments demonstrated FERM-domain-dependent interactions between Yrt and the septate junction transmembrane protein Neuroglian (Nrg), and the enrichment of Yrt at septate junctions was less pronounced in Nrg mutants. However, Nrg or Nrg yrt mutant embryos did not display overt defects in epithelial polarity. These findings indicate that Yrt is a bona fide septate junction component that has a function distinct from Nrx-IV and Na+,K+-ATPase because it is not required for septa formation but essential for barrier function (Laprise, 2009).

Next, the analysis extended to cytoplasmic adaptor proteins associated with septate junctions. yrt does not show genetic interactions with dlg or lgl, genes encoding conserved basolateral polarity proteins required for septate junction formation and apical/basal polarity. varicose (vari), which encodes a membrane-associated guanylate kinase, and cora, which encodes a FERM protein that is the Drosophila orthologue of mammalian erythrocyte protein band 4.1 and its paralogues, were examined. vari and cora null mutant embryos did not show defects in apical basal polarity. No functional interactions were seen between yrt and vari. In contrast, yrt cora double-mutant embryos displayed marked apicalization defects, with strongly expanded apical membranes and reduced basolateral membranes in all ectodermal epithelia including the epidermis. This phenotype is significantly stronger than the yrtM/Z or cora null mutant phenotypes indicating that Yrt and Cora have redundant functions in promoting epithelial polarity (Laprise, 2009).

Similar to yrtM/Z mutants, yrt cora double mutants did not show polarity defects during gastrulation. By stage 11, polarity defects were more prominent in yrt cora mutants than in yrtM/Z embryos, which correlates with Cora being expressed from stage 11 onwards. In yrt cora mutants, the most severe polarity defects characterized by overlapping distributions of apical and basolateral markers occurred during stage 13. However, by late embryogenesis, apical and basolateral markers were segregated, although expanded apical membranes and severe defects in tissue organization persisted. Interestingly, the yrt cora apicalization phenotype is very similar to the phenotype that results from high-level overexpression of Crb in both timeline and severity. The segregation of apical and basolateral markers in late-stage yrt cora mutants is also observed in late-stage yrtM/Z, yrt Atpα and yrt Nrx-IV embryos. Thus, polarization mechanisms are at play in late embryos that are not dependent on Yrt and Cora. To address the possibility that this repolarization is the result of redundancy between Yrt/Cora and Lgl-group proteins, which include Scribble (Scrib) as well as Lgl and Dlg, embryos lacking Yrt and Scrib (yrtM/Z scribM/Z) were examined. Apical and basolateral markers segregated in yrtM/Z scribM/Z embryos indicating that a basolateral polarity mechanism exists in late embryos that is independent of Yrt/Cora and Lgl-group proteins (Laprise, 2009).

Together, these findings suggest that Yrt, Cora, Nrx-IV and the Na+,K+-ATPase are a new functionally cooperating group of basolateral epithelial polarity proteins, which is refered to as the ‘Yrt/Cora group’. In contrast to the enhancement of the cora and Nrx-IV null phenotypes by yrt mutations, no phenotypic enhancement was seen in cora Nrx-IV or cora Atpα double mutants, indicating that the Yrt/Cora group is composed of two functionally overlapping pathways. Cora, Nrx-IV and the Na+,K+-ATPase belong to one pathway, which is consistent with previously documented biochemical and genetic interactions between these proteins, whereas Yrt defines a second redundant pathway (Laprise, 2009).

One critical aspect of polarity regulation is that apical and basolateral proteins act antagonistically to set up mutually exclusive membrane domains. In gastrulating Drosophila embryos, this interaction occurs between apical factors and basolateral Lgl-group proteins. Similar to lgl, dlg and scrib mutations, yrt mutations partially suppress the crb mutant phenotype. Because Yrt can bind Crb directly, it is suggested that Yrt negatively regulates Crb function as a component of the apical Crb complex in both apical basal polarity and the control of apical membrane size. The data reported in this study argue that Yrt opposes Crb function also as a basolateral polarity protein. To test this hypothesis further it was asked whether the loss of other Yrt/Cora-group genes could suppress the crb mutant phenotype. It was found that mutations in Atpα, Nrx-IV and cora ameliorate the epithelial defects of crb mutants to an extent similar to that of yrt mutations. Remarkably, yrt cora crb triple-mutant embryos not only showed a suppression of the crb mutant phenotype, which was much stronger than the one observed in yrt crb or cora crb double mutants, but also caused a strong suppression of the apicalization effect observed in yrt cora mutants. These findings further support the conclusion that Yrt and Cora act redundantly, and indicate that mutual competition of basolateral Yrt/Cora-group proteins and apical Crb organizes epithelial membrane domains (Laprise, 2009).

Epithelial differentiation during Drosophila embryogenesis can be subdivided into four phases that are characterized by distinct mechanisms governing epithelial polarity. (1) Initial cues for polarity are given before or during cellularization. (2) Fully polarized epithelial cells are established during gastrulation through the interplay of apical Par and Crb complexes and basolateral Lgl-group proteins. (3) The Yrt/Cora group acts during organogenesis to promote basolateral polarity and counteracts apical determinants, thereby functionally replacing the Lgl group. (4) The function of Yrt/Cora and Lgl groups does not account for the polarization of epithelial cells in late embryos, because normalization of polarity is observed in the absence of these factors. This implies the existence of a yet unknown mechanism that stabilizes basolateral polarity. Septate junctions form well after Lgl-group and Yrt/Cora-group proteins have contributed to epithelial polarity, indicating that polarity and septate junction formation are independent functions of these proteins. The identification of a novel group of polarity factors that acts in a discrete time window during development highlights the temporal complexity of the regulation of the epithelial phenotype and further explains why the loss of individual polarity proteins does not completely compromise polarity (Laprise, 2009).

The vertebrate Yrt orthologues Mosaic eyes (Moe, also known as Epb41l5) in zebrafish and EPB41L5 (also known as YMO1 and Limulus) and EPB41L4B (also known as EHM2) in mammals bind to Crb proteins and contribute to epithelial organization. However, as in Drosophila, vertebrate Yrt homologues are predominantly associated with the basolateral membrane and seem to be recruited to the apical membrane only at later stages of epithelial development. To test whether EPB41L5 is required for basolateral differentiation, RNA interference was used in MDCK cells. Transient depletion of EPB41L5 using shRNA resulted in a notable cell flattening and an expansion of the cell perimeter, indicating that lateral membranes were strongly reduced and apical and basal membranes were enlarged. Consistently, it was found that the lateral markers Na+,K+-ATPase, Scrib and E-cadherin were reduced or lost from the plasma membrane. The same effects were observed using siRNA oligonucleotides targeted to a distinct region of EPB41L5. MDCK cells were established that were stably transfected with EPB41L5 short hairpin RNA (shRNA), and lateral membrane formation was examined after Ca2+-switch. After re-addition of Ca2+, EPB41L5 shRNA cells displayed significant delays in lateral membrane formation and recruitment of E-cadherin to cell-cell contacts. At 8 h after Ca2+-switch, E-cadherin levels appeared similar in control and knockdown cells although experimental cells remained slightly flatter. Interestingly, by 24 h after Ca2+-switch, E-cadherin levels at the plasma membrane had decreased again and appeared lower than in control cells but similar to EPB41L5 shRNA cells before Ca2+-switch. EPB41L5 knockdown cells ultimately established normal cell shape. Whether the formation of a normal lateral membrane in EPB41L5 shRNA cells is due to residual EPB41L5 expression, or reflects a transient requirement of EPB41L5 for cell polarization as in Drosophila epithelia, remains unclear. It is concluded that the function of Yrt as a basolateral polarity protein is conserved in mammalian epithelial cells (Laprise, 2009).

The loss of basolateral membrane in Drosophila embryos that lack Yrt/Cora-group function and in MDCK cells depleted of EPB41L5 is reminiscent of the loss of basolateral membrane in MDCK cells depleted of the phosphoinositide PtdIns(3,4,5)P3 and of human bronchial epithelial cells depleted of β2-spectrin or ankyrin-G. Cora/band 4.1 and the Na+,K+-ATPase can associate with either spectrin or the spectrin adaptor ankyrin, and loss or ectopic expression of EPB41L5 can cause defects in basolateral actin. This raises the possibility that Yrt/Cora-group proteins act by stabilizing the lateral actin/spectrin membrane cytoskeleton. Analysis of vertebrate development in the absence of the function of the Yrt orthologues Moe in zebrafish and EPB41L5 in mice revealed defects in epithelial organization that reflect Crb-dependent and probably also Crb-independent functions of Yrt proteins. For example, the abnormal cell shape and multi-layering defects seen in the developing neuroepithelium of mutant mouse embryos could result from defects in basolateral polarity. Moreover, mouse embryos lacking EPB41L5 show defects in epithelial-mesenchymal transition of mesodermal cells during gastrulation. These surprising findings indicate that Yrt proteins are core regulators of animal tissue organization that can enhance epithelial or mesenchymal cell differentiation (Laprise, 2009).

Epithelial polarity proteins regulate Drosophila tracheal tube size in parallel to the luminal matrix pathway

Regulation of epithelial tube size is critical for organ function. However, the mechanisms of tube size control remain poorly understood. In the Drosophila trachea, tube dimensions are regulated by a luminal extracellular matrix (ECM). ECM organization requires apical (luminal) secretion of the protein Vermiform (Verm), which depends on the basolateral septate junction (SJ). This study shows that apical and basolateral epithelial polarity proteins interact to control tracheal tube size independently of the Verm pathway. Mutations in yurt (yrt) and scribble (scrib), which encode SJ-associated polarity proteins, cause an expansion of tracheal tubes but do not disrupt Verm secretion. Reducing activity of the apical polarity protein Crumbs (Crb) suppresses the length defects in yrt but not scrib mutants, suggesting that Yrt acts by negatively regulating Crb. Conversely, Crb overexpression increases tracheal tube dimensions. Reducing crb dosage also rescues tracheal size defects caused by mutations in coracle (cora), which encodes an SJ-associated polarity protein. In addition, crb mutations suppress cora length defects without restoring Verm secretion. Together, these data indicate that Yrt, Cora, Crb, and Scrib operate independently of the Verm pathway. These data support a model in which Cora and Yrt act through Crb to regulate epithelial tube size (Laprise, 2010).

The transmembrane protein Crumbs (Crb) acts as an apical determinant during establishment of epithelial apical-basal polarity. During later stages of epithelial differentiation, Crb promotes apical membrane growth independently of its role in apical-basal polarity. Crb activity is counteracted by different groups of basolateral polarity proteins including the Yrt/Cora group, which is composed of Yurt (Yrt), Coracle (Cora), Na+/K+-ATPase, and Neurexin IV (Nrx-IV). Loss of Yrt results in Crb-dependent apical membrane growth during late stages of epithelial cell maturation in Drosophila. Thus, the equilibrium between the activities of these polarity proteins is important to define the size of the apical domain. Precise control of the apical surface of tracheal cells is crucial to define epithelial tube size in the Drosophila respiratory system, suggesting a potential role for polarity proteins in tube morphogenesis. However, the contribution of polarity regulators to the regulation of epithelial tube shape and size is poorly understood. Controlling length and diameter of the lumen is important for organ function, as illustrated by the deleterious tubule enlargements that occur in polycystic kidney disease (Laprise, 2010).

To better understand the role of polarity regulators and apical membrane growth in epithelial tube morphogenesis, the role of Yrt was investigated in the formation of the Drosophila respiratory system, a network of interconnected tubules that delivers oxygen throughout the body. Yrt is mainly associated with the lateral membrane in tracheal cells and is enriched at septate junctions (SJs), as shown by its colocalization with the SJ marker Nrx-IV. Tracheal development was characterized in zygotic yrt mutants or yrt null embryos devoid of both maternal and zygotic yrt (yrtM/Z). Segmental tracheal placodes invaginated and established a normal branching pattern of tracheal tubes with intersegmental connections in both yrt and yrtM/Z mutants. yrtM/Z embryos display apical-basal polarity defects and irregularities in the tracheal epithelium at midembryogenesis. However, apical-basal polarity normalizes during terminal differentiation. In contrast, zygotic yrt mutants have only minor, if any, polarity defects in tracheal cells. The most apparent defect in yrt mutant trachea was the presence of excessively long and convoluted dorsal trunks compared to the straight dorsal trunks seen wild-type. The average dorsal trunk length was 417 ± 12 μm in wild-type embryos, whereas it was 476 ± 22 μm in yrt and 470 ± 23 μm in yrtM/Z mutant embryos. Similar but milder tube length defects were observed in other tracheal branches. In addition, the diameter of dorsal trunks in yrt and yrtM/Z mutants was uniform, but wider than in wild-type embryos. The average dorsal trunk diameter was 9.1 ± 0.6 μm in tracheal segment seven of wild-type embryos compared to 12.2 ± 0.9 μm in yrt and 12.7 ± 0.6 μm in yrtM/Z mutant embryos. In smaller branches, some diameter expansions were also apparent. In addition, the smaller tracheal branches in yrtM/Z embryos showed frequent interruptions indicating either breaks or a failure in the luminal accumulation of the 2A12 antigen. These findings indicate that Yrt regulates the size of tracheal tubes and supports the integrity of segmental tracheal branches. Remarkably, despite the prominent differences in the apical-basal polarity defects between yrt and yrtM/Z mutant embryos, both mutants exhibit similar dorsal trunk elongation and diameter defects. This finding suggests that the transient loss of apical-basal polarity in yrtM/Z embryos is not the cause of dorsal trunk size defects and that Yrt therefore has distinct functions during early and late stages of tracheal morphogenesis (Laprise, 2010).

The enlargement of the tracheal tube lumen observed in yrt mutants could be caused by an increase in cell number or an increase in the dimension of the apical surface of tracheal cells that surround the lumen. To address this question, the number of dorsal trunk cells in yrt mutants and wild-type embryos was counted. No significant differences in cell numbers between wild-type and yrt mutant embryos were found, indicating that the enlargement of tracheal tubes observed in yrt and yrtM/Z mutants must be accompanied by an increase in the dimension of the apical surface of tracheal cells (Laprise, 2010).

Several other mutants display enlarged dorsal trunks similar to yrt. One group of genes required for limiting tube length encodes components of the SJ, including the Na+/K+-ATPase (α and β subunits), Cora, Nrx-IV, Scribble (Scrib), Lachesin (Lac), Sinuous (Sinu), Megatrachea (Mega), and Varicose (Vari). Among these SJ proteins, Na+/K+-ATPase, Cora, Nrx-IV, and Scrib also play a role as basolateral polarity proteins. In Drosophila and other invertebrates, SJs appear as a ladder-like group of septa basal to the cadherin-based adherens junctions. SJs have functions analogous to vertebrate tight junctions, because they provide a transepithelial diffusion barrier. Yrt is not required for normal septa formation or localization of SJ components such as Cora but is essential for the barrier function of SJs. Zygotic yrt mutants show only minor defects in paracellular barrier function, whereas barrier function is fully compromised in yrtM/Z embryos. This observation supports the notion that transepithelial barrier function and the regulation of tube dimension are independent functions of Yrt because yrt and yrtM/Z mutant embryos show similar tube size defects. This conclusion is consistent with previous findings suggesting that the regulation of tracheal tube elongation and the regulation of the paracellular diffusion barrier are distinct roles of SJ proteins (Laprise, 2010).

A second class of mutants exhibiting abnormally long tracheal tubes are defective in the genesvermiform (verm) and serpentine (serp), which encode enzymes predicted to modify the chitin-based luminal extracellular matrix (ECM), and mutants of which show structural defects in the luminal ECM. The chitin matrix filling the tracheal lumen is transiently present during lumen morphogenesis and is critical for determining lumen diameter and length. Interestingly, all of the mutations affecting SJ components tested so far are associated with a failure to secrete Verm into the tracheal lumen. This suggests that SJ proteins control tube size by regulating apical secretion and remodeling of the apical chitin matrix. Although Yrt is required for the barrier function of SJs, it was found that luminal secretion of Verm and Serp was normal in yrt and yrtM/Z mutants. In contrast, low but detectable levels of Verm were observed in cora mutants. This finding suggests that Yrt regulates tracheal tube length through a pathway that is independent of Verm and Serp (Laprise, 2010).

During late stages of epithelia maturation, Yrt is known to restrict apical membrane growth in epidermal and photoreceptor cells by limiting Crb activity. This interplay between Yrt and Crb governs apical membrane size in stage 14 and later embryos when tracheal tube size is defined. This raises the possibility that Crb-dependent apical membrane growth is responsible for dorsal trunk expansion in yrt mutant embryos. To test this hypothesis, crb dosage was reduced in yrt mutant embryos by introducing one copy of a crb null allele into a yrt mutant background. Loss of one copy of crb suppressed the dorsal trunk elongation defects seen in yrt mutants, because the dorsal trunks appeared similar to wild-type in yrt crb/yrt + mutants. In addition, moderate Crb overexpression increased dorsal trunk length and diameter without interfering with the integrity of the tracheal epithelium or the secretion of Verm or Serp. These results show that Crb is required for promoting the expansion of tracheal tubes at late stages of embryogenesis. It was previously suggested that Crb also acts during early tracheal branch outgrowth in addition to its role in apical-basal polarity. Therefore, Crb plays a critical role at several steps of tracheal development. Together, these findings indicate that the antagonistic interactions between Yrt and Crb determine tracheal tube size (Laprise, 2010).

Verm levels in yrt crb/yrt + and in yrt/yrt mutants were indistinguishable, indicating that the minor reduction in Verm levels sometimes seen in yrt mutants are not the cause of the tracheal elongation defects. Accordingly, reduction of crb dosage in a verm or serp mutant background did not suppress tube size defects. Similarly, loss of one copy of crb in sinu mutant embryos, which fail to secrete Verm, had no impact on the length of dorsal trunks that remained enlarged as in sinu single mutants. These findings suggest that the apical secretion of matrix-modifying enzymes such as Verm and the control of Crb activity by Yrt are two independent and nonredundant modes of tracheal tube size regulation. The data also establish that epithelial tube size control by SJ-associated proteins involves Verm-dependent and Verm-independent mechanisms (Laprise, 2010).

To further characterize the function of SJ-associated polarity proteins in the regulation of tracheal tube size, tube elongation, the integrity of SJs, and the secretion of Verm in scrib, lethal giant larvae (lgl), and discs large (dlg) mutant embryos were examined. Zygotic loss of scrib, lgl, or dlg resulted in excessively long dorsal trunks, indicating that these genes are critical for tube size control. Zygotic loss of lgl expression caused fully penetrant defects in SJ paracellular barrier function, whereas zygotic scrib or dlg mutants did not have compromised transepithelial barriers. Luminal Verm deposition was not detected in lgl mutants but appeared near normal in dlg and in scrib mutants. Thus, Scrib and Dlg act like Yrt by controlling tracheal tube size through mechanisms distinct from Verm secretion (Laprise, 2010).

Further analysis concentrated on Scrib and it was asked whether this protein, like Yrt, controls tracheal tube size by negatively regulating Crb activity. Scrib together with Lgl and Dlg shows antagonistic interactions with Crb to regulate apical-basal epithelial polarity in early Drosophila embryos. However, the tracheal tube defects were not ameliorated in scrib crb/scrib + embryos compared to scrib single mutants. Thus, in contrast to Yrt, Scrib does not seem to limit tube length by restricting Crb activity. Because Yrt and Scrib appear to control tracheal tube size through different mechanisms, tests were performed to see whether yrt scrib double homozygous mutant embryos had a more severe phenotype. The double mutants had Verm levels that were lower compared to yrtM/Z and scrib mutants. Moreover, the tracheal defects also appeared more severe in yrt scrib mutants than in yrt null mutant embryos, particularly in the smaller-diameter branches. The defects in small-diameter branches of the yrt scrib double mutants are not likely caused by the reduction in Verm secretion, because the complete loss of Verm has only a mild effect on smaller branches. The enhanced severity of the yrt scrib double-mutant tracheal defects compared to the defects seen in yrt null embryos and the differences in the genetic interactions of scrib and yrt with crb suggest that Scrib and Yrt act in separate pathways to regulate the size of tracheal tubes and that Scrib does not act by modulating Crb activity. It is possible that Scrib acts through other proteins, such as proteins of the Par complex, that promote apical domain formation. Therefore, SJ-associated polarity proteins use at least two Verm-independent mechanisms to restrict the dimension of tracheal tubes (Laprise, 2010).

Cora is an SJ-associated protein required for optimal secretion of Verm. This suggests that Cora may control tracheal tube length through a Verm-luminal matrix pathway. However, Cora is also a basolateral polarity protein restricting the activity of Crb, which promotes Verm-independent expansion of the dorsal trunk. This led to an investigation of the functional relationship between Cora and Crb in tracheal morphogenesis. In the epidermis, a striking redundancy between yrt and cora is observed in the regulation of apical-basal polarity. Similarly, it was found that tracheal cells in yrt cora double mutants show severe apicalization defects characterized by a broad expansion of the surface distribution of Crb. The antigen recognized by the monoclonal antibody 2A12 was found surrounding tracheal cells and was not confined to the luminal cavity. In addition, the 2A12 antigen was associated not only with tracheal cells but also with epidermal cells. These tracheal defects seen in cora yrt mutant embryos mimic defects that result from high levels of Crb overexpression. This observation argues that the tracheal defects observed in cora yrt double-mutant embryos result from strong Crb overactivation, which is associated with a loss of basolateral polarity and an expansion of apical membrane character. Because epidermal cells did not acquire expression of the tracheal cell marker Tango, it is unlikely that epidermal cells adopt a tracheal cell fate in cora yrt mutants. The association of 2A12 with epidermal cells is therefore presumably due to the apicalization of tracheal cells, which would consequently secrete the 2A12 antigen not only on the luminal side but all around their cell surface, allowing the 2A12 antigen to diffuse and bind to surrounding cells. Accordingly, cuticle deposition, taking place at the apical membrane, was seen at both luminal and abluminal sides of tracheal cells overexpressing Crb (Laprise, 2010).

The data indicate that Yrt and Cora cooperate to control apical-basal polarity of tracheal cells by limiting Crb to the apical cell pole, but they do not reveal whether Cora and Crb interact to control the length of tracheal tubes. To address this question, cora crb/cora + embryos were examined for a suppression of the tracheal size defects seen in cora single mutants. Reduction of crb dosage suppresses tube overelongation defects resulting from the loss of Cora. This restriction of dorsal trunk elongation does not result from the restoration of Verm secretion, because the level of Verm present in the dorsal trunk lumen was as low in cora crb/cora + embryos as in single cora mutants. Together, these data suggest that Crb overactivation is the primary cause of epithelial tube length defects observed in the absence of Cora. Thus, Cora and Yrt act independently from each other to counteract Crb activity and maintain the appropriate size of epithelial tubes. Because the reduction of crb dosage does not rescue the verm mutant phenotype, it is concluded that the residual amount of Verm found in cora mutants is sufficient to maintain Verm pathway activity (Laprise, 2010).

This analysis suggests that basolateral proteins that are enriched at SJs have several critical functions in determining the size of epithelial tubes in the Drosophila tracheal system. This study shows that the increase in tube size is not caused by an increase in cell number and therefore must be accompanied by an increase in the apical surface area of individual tracheal cells. Given that Crb is a well-known regulator of apical membrane size, these findings suggest that the interplay between Yrt, Cora, and Crb modulates the dimensions of the apical surface of tracheal cells to control tracheal tube size. Moreover, this mechanism acts independently of and in parallel to a previously proposed pathway depending on the apical secretion of the matrix-modifying enzymes Verm and Serp, which requires several SJ-associated proteins. Yet another mechanism is revealed by the results demonstrating that scrib mutants also have long trachea with normal Verm levels but that, in contrast to cora and yrt, tracheal defects in scrib mutants are not suppressed by loss of one copy of crb. Together, these findings suggest that basolateral proteins utilize at least three distinct mechanisms to regulate tube size in the Drosophila tracheal system. Unexpectedly, these mechanisms involve functional interactions between polarity proteins that appear to be different from those involved in establishing apical-basal polarity at earlier stages of development. For example, in promoting apical-basal polarity, Yrt and Cora act redundantly so that cora mutants show polarity defects only in a yrt mutant background, and polarity defects in yrt mutants are strongly enhanced by removal of Cora. In contrast, both cora and yrt single mutants show similar strong tracheal size defects. Furthermore, Scrib and Crb display antagonistic functional interactions during establishment of apical-basal polarity, but not during tracheal elongation. An important challenge for future investigations will be to uncover the adaptations in the molecular pathways that allow polarity proteins to contribute to different aspects of epithelial development (Laprise, 2010).


An, X. L., et al. (1996). Modulation of band 3-ankyrin interaction by protein 4.1. Functional implications in regulation of erythrocyte membrane mechanical properties. J Biol Chem 271 (52): 33187-33191. PubMed Citation: 8969174

Baklouti, F., et al. (1996). Asynchronous regulation of splicing events within protein 4.1 pre-mRNA during erythroid differentiation. Blood 87 (9): 3934-3941. PubMed Citation: 8611723

Baklouti, F., et al. (1997). Organization of the human protein 4.1 genomic locus: new insights into the tissue-specific alternative splicing of the pre-mRNA. Genomics 39 (3): 289-302. PubMed Citation: 9119366

Baumgartner, S., et al. (1996). A Drosophila Neurexin is required for septate junction and blood-nerve barrier formation and function. Cell 87: 1059-68. PubMed Citation: 8978610

Boedigheimer, M. J., Nguyen, K. P., and Bryant, P. J. (1997). Expanded functions in the apical cell domain to regulate the growth rate of imaginal discs. Dev. Genet. 20 (2): 103-110. PubMed Citation: 9144921

Bogdanik, L., et al. (2008). Muscle dystroglycan organizes the postsynapse and regulates presynaptic neurotransmitter release at the Drosophila neuromuscular junction. PLoS ONE 3(4): e2084. PubMed Citation: 18446215

Chasis, J. A., et al. (1996). Differential use of protein 4.1 translation initiation sites during erythropoiesis: implications for a mutation-induced stage-specific deficiency of protein 4.1 during erythroid development. Blood 87 (12): 5324-5331. PubMed Citation: 8652848

Chen, K., Merino, C., Sigrist, S. J. and Featherstone, D. E. (2005). The 4.1 protein coracle mediates subunit-selective anchoring of Drosophila glutamate receptors to the postsynaptic actin cytoskeleton. J. Neurosci, 25: 6667-6675. PubMed Citation: 16014728

Conboy, J. G., et al. (1993). An isoform-specific mutation in the protein 4.1 gene results in hereditary elliptocytosis and complete deficiency of protein 4.1 in erythrocytes but not in nonerythroid cells. J Clin Invest 91 (1): 77-82. PubMed Citation: 8423235

D'Amico, L., Scott, I. C., Jungblut, B. and Stainier, D. Y. (2007). A mutation in zebrafish hmgcr1b reveals a role for isoprenoids in vertebrate heart-tube formation. Curr. Biol. 17: 252-259. PubMed Citation: 17276918

David, N. B., et al. (2005). Drosophila Ric-8 regulates Gαi cortical localization to promote G?i-dependent planar orientation of the mitotic spindle during asymmetric cell division. Nat. Cell Biol. 7: 1083-1090. PubMed Citation: 16228010

De Carcer, G., Lallena, M. J. and Correas, I. (1995). Protein 4.1 is a component of the nuclear matrix of mammalian cells. Biochem J 312 ( Pt 3): 871-877. PubMed Citation: 8554533

Discher, D., et al. (1993). Mechanochemistry of the alternatively spliced spectrin-actin binding domain in membrane skeletal protein 4.1. J. Biol. Chem. 268: 7186-95. PubMed Citation: 8463254

Discher, D. E., et al. (1995). Mechanochemistry of protein 4.1's spectrin-actin-binding domain: ternary complex interactions, membrane binding, network integration, structural strengthening. J Cell Biol 130 (4): 897-907. PubMed Citation: 7642705

Edison, R. J. and Muenke, M. (2005). Gestational exposure to lovastatin followed by cardiac malformation misclassified as holoprosencephaly. N. Engl. J. Med. 352: 2759. PubMed Citation: 15987932

Evans, C. J., Olson, J. M., Ngo, K. T., Kim, E., Lee, N. E., Kuoy, E., Patananan, A. N., Sitz, D., Tran, P., Do, M. T., Yackle, K., Cespedes, A., Hartenstein, V., Call, G. B. and Banerjee, U. (2009). G-TRACE: rapid Gal4-based cell lineage analysis in Drosophila. Nat Methods 6: 603-605. PubMed ID: 19633663

Fairchild, M.J., Yang, L., Goodwin, K. and Tanentzapf, G. (2016). Occluding junctions maintain stem cell niche homeostasis in the fly testes. Curr Biol [Epub ahead of print]. PubMed ID: 27546574

Fehon, R. G., Dawson, A. I. and Artavanis-Tsakonas, S. (1994). A Drosophila homologue of membrane-skeleton protein 4.1 is associated with septate junctions and is encoded by the coracle gene. Development 120: 545-57. PubMed Citation: 8162854

Genova, J. L. and Fehon, R. G. (2003). Neuroglian, Gliotactin, and the Na+/K+ ATPase are essential for septate junction function in Drosophila. J. Cell Biol. 161: 979-989. 12782686

Gu, M. and Majerus, P. W. (1996). The properties of the protein tyrosine phosphatase PTPMEG. J Biol Chem 271 (44): 27751-27759. PubMed Citation: 8910369

Hemming, N. J., et al. (1995). Identification of the membrane attachment sites for protein 4.1 in the human erythrocyte. J. Biol. Chem. 270: 5360-6. PubMed Citation: 7890649

Huang, J. P., et al. (1993). Genomic structure of the locus encoding protein 4.1. Structural basis for complex combinational patterns of tissue-specific alternative RNA splicing. J. Biol. Chem. 268: 3758-66. PubMed Citation: 8429050

Irion, U. (2012). Drosophila muscleblind codes for proteins with one and two tandem zinc finger motifs. PLoS One 7: e34248. Pubmed: 22479576

Krauss, S. W., et al. (1997). Structural protein 4.1 in the nucleus of human cells: dynamic rearrangements during cell division. J. Cell Biol. 137: 275-289. PubMed Citation: 9128242

Kraut, R., and Campos-Ortega, J. A. (1996). inscuteable, a neural precursor gene of Drosophila, encodes a candidate for a cytoskeletal adaptor protein. Dev. Biol. 174: 65-81. PubMed Citation: 8626022

Lallena, M. J. and Correas, I. (1997). Transcription-dependent redistribution of nuclear protein 4.1 to SC35-enriched nuclear domains. J Cell Sci 110 ( Pt 2): 239-247. PubMed Citation: 9044054

Lamb, R. S., et al. (1998). Drosophila coracle, a member of the protein 4.1 superfamily, has essential structural functions in the septate junctions and developmental functions in embryonic and adult epithelial cells. Mol. Biol. Cell 9: 3505-3519. PubMed Citation: 9843584

Laprise, P., et al. (2009). Yurt, Coracle, Neurexin IV and the Na(+),K(+)-ATPase form a novel group of epithelial polarity proteins. Nature 459: 1141-1145. PubMed Citation: 19553998

Laprise, P., Paul, S. M., Boulanger, J., Robbins, R. M., Beitel, G. J. and Tepass, U. (2010). Epithelial polarity proteins regulate Drosophila tracheal tube size in parallel to the luminal matrix pathway. Curr. Biol. 20(1): 55-61. PubMed Citation: 20022244

Laval, M., Bel, C. and Faivre-Sarrailh, C. (2008). The lateral mobility of cell adhesion molecules is highly restricted at septate junctions in Drosophila. BMC Cell Biol. 9: 38. PubMed Citation: 18638384

Lue, R. A., et al. (1994). Cloning and characterization of hdlg: the human homologue of the Drosophila discs large tumor suppressor binds to protein 4.1. Proc Natl Acad Sci U S A 91 (21): 9818-9822. PubMed Citation: 7937897

Lue, R. A., et al. (1996). Two independent domains of hDlg are sufficient for subcellular targeting: the PDZ1-2 conformational unit and an alternatively spliced domain. J. Cell Biol. 135 (4): 1125-1137. PubMed Citation: 8922391

Marfatia, S. M., et al. (1995). Identification of the protein 4.1 binding interface on glycophorin C and p55, a homologue of the Drosophila discs-large tumor suppressor protein. J. Biol. Chem. 270: 715-719. PubMed Citation: 7822301

Marfatia, S. M., et al. (1996). Modular organization of the PDZ domains in the human discs-large protein suggests a mechanism for coupling PDZ domain-binding proteins to ATP and the membrane cytoskeleton. J. Cell Biol. 135 (3): 753-766. PubMed Citation: 8909548

Medioni, C. et al. (2008). Genetic control of cell morphogenesis during Drosophila melanogaster cardiac tube formation. J. Cell Biol. 182: 249-261. PubMed Citation: 18663140

Morris, M. B. and Lux, S. E. (1995). Characterization of the binary interaction between human erythrocyte protein 4.1 and actin. Eur J Biochem 231 (3): 644-650. PubMed Citation: 7649164

Pastor-Pareja, J. C., Grawe, F., Martín-Blanco, E. and García-Bellido, A. (2004). Invasive cell behavior during Drosophila imaginal disc eversion is mediated by the JNK signaling cascade. Dev. Cell 7: 387-399. 15363413

Schischmanoff, P. O., et al. (1995). Defining of the minimal domain of protein 4.1 involved in spectrin-actin binding. J Biol Chem 270 (36): 21243-21250. PubMed Citation: 7673158

Schischmanoff, P. O., et al. (1997). Cell shape-dependent regulation of protein 4.1 alternative pre-mRNA splicing in mammary epithelial cells. J Biol Chem 272 (15): 10254-10259. PubMed Citation: 9092575

Schwabe, T., et al. (2005). GPCR signaling is required for blood-brain barrier formation in Drosophila. Cell 123: 133-144. PubMed Citation: 16213218

Venema, D. R., Zeev-Ben-Mordehai, T., Auld, V. J. (2004). Transient apical polarization of Gliotactin and Coracle is required for parallel alignment of wing hairs in Drosophila. Dev. Biol. 275(2): 301-14. 15501220

Walensky, L. D., et al. (1998). Neurobehavioral deficits in mice lacking the erythrocyte membrane cytoskeletal protein 4.1. Curr. Biol. 8(23): 1269-72. PubMed Citation: 9822582

Ward, R. E., Lamb, R. S. and Fehon, R. G.. (1998). A conserved functional domain of Drosophila Coracle is required for localization at the septate junction and has membrane-organizing activity. J. Cell. Biol. 140(6): 1463-1473. PubMed Citation: 9508778

Ward, R. E., et al. (2001). The Protein 4.1, Ezrin, Radixin, Moesin (FERM) domain of Drosophila Coracle, a cytoplasmic component of the septate junction, provides functions essential for embryonic development and imaginal cell proliferation. Genetics 159: 219-228. 11560899

Winardi, R., et al. (1995). Evolutionarily conserved alternative pre-mRNA splicing regulates structure and function of the spectrin-actin binding domain of erythroid protein 4.1. Blood 86: 4315-4322. PubMed Citation: 7492792

Woods, D. F., et al. (1996). Dlg protein is required for junction structure, cell polarity, and proliferation control in Drosophila epithelia. J. Cell Biol. 134: 1469-82. PubMed Citation: 8830775

Woods, D. F., Wu, J. W. and Bryant, P. J. (1997). Localization of proteins to the apico-lateral junctions of Drosophila epithelia. Dev. Genet. 20 (2): 111-118. PubMed Citation: 9144922

Wu, V. M., et al. (2004). Sinuous is a Drosophila claudin required for septate junction organization and epithelial tube size control. J. Cell Biol. 164: 313-323. PubMed Citation: 14734539

Yi, P., Han, Z., Li, X. and Olson, E. N. (2006). The mevalonate pathway controls heart formation in Drosophila by isoprenylation of Gγ1. Science 313: 1301-1303. PubMed Citation: 16857902

Yi, P., Johnson, A. N., Han, Z., Wu, J. and Olson, E. N. (2008). Heterotrimeric G proteins regulate a noncanonical function of septate junction proteins to maintain cardiac integrity in Drosophila. Dev. Cell 15(5): 704-13. PubMed Citation: 19000835

coracle: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 20 February 2013

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