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First published online September 18, 2007
doi: 10.1242/10.1242/jcs.010850
Research Article |
Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK
e-mails: ng288{at}hermes.cam.ac.uk; ama11{at}cam.ac.uk
Accepted 26 June 2007
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Key words: Adhesion, Dynamic, Epithelia
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Introduction |
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; Martinez Arias, 1993) as some of the forces driving this morphogenetic movement have been unravelled (Hutson et al., 2003; Kiehart et al., 2000; Peralta et al., 2007), and many of the molecular components that are required for DC have been identified through genetic screens (Harden, 2002).
Half way through Drosophila embryogenesis the epidermis exhibits a discontinuity on the dorsal side that is bridged by the amnioserosa (AS), a flat epithelium which will not contribute to the larva. DC is the process that seals the epidermal gap through coordinated cell-shape changes in the AS and the epidermis (Jacinto et al., 2002b; Martinez Arias, 1993). This is achieved through the coordinated elongation of the epidermal cells in the dorsoventral (DV) axis and the contraction of the AS, which disappears inside the embryo and undergoes apoptosis. Throughout the process, the AS lies on top of a large cell, the yolk cell, which provides anchorage for this process and also promotes survival and prevents death of this cell type (Narasimha and Brown, 2004; Reed et al., 2004). The process is completed by the joining of the two epidermal sheets at the dorsal midline.
Interactions between epidermis and AS play an important role in DC. For example, there is evidence that myosin-mediated contraction of AS cells generates a force that drives some of the process by pulling the epidermal sheets together (Franke et al., 2005; Kiehart et al., 2000), and it is possible that this pulling contributes to the elongation of the epidermal cells. However, it appears that it is the epidermis that induces the contraction of the AS through the secretion of the secreted signaling molecule Dpp (Wada et al., 2007) (Garcia Fernandez, Analysis of the role of Dpp during the morphogenetic process of Dorsal Closure in Drosophila. PhD thesis. Instituto de Tecnologia Quimica e Biologia. Universidade Nova de Lisboa, 2007). In addition, the assembly of an actin-myosin cable along the interface between the epidermis and the AS – the leading edge (LE) – provides a purse-string force that contributes to DC (Edwards et al., 1997; Jacinto et al., 2002a; Kaltschmidt et al., 2002; Kiehart et al., 2000). Counteracting the positive action of these two forces, there is tension created from the lateral epidermis that opposes the pulling of the AS (Kiehart et al., 2000).
Cell adhesion systems are an essential constituent of epithelial cells responsible for the maintenance of tissue integrity (Gumbiner, 1996), which must be adaptable to accommodate the deformations that tissues experience during morphogenesis (Gumbiner, 2005). In epithelial cells, adherens junctions (AJs) are a major contributor to inter-cellular adhesion that also coordinate cell organization and movements within epithelia, and transmit information from the environment to the interior of the cell (Perez-Moreno et al., 2003; Tepass et al., 2001). The core element of AJs is E-cadherin, a transmembrane protein that mediates Ca2+-dependent homophilic interactions and, thus, provides adhesion between neighboring cells. The intracellular domain of E-cadherin mediates an interaction with the cystokeleton through two linker proteins: β-catenin, which binds directly to E-cadherin, and 
-catenin which links β-catenin to actin and other actin-binding proteins. This set of interactions has provided a structural basis to a view of E-cadherin as a linker of the actin cytoskeleton of the components of epithelia. Recent data have challenged this view by showing that -catenin is not able to bind to β-catenin and actin at the same time (Drees et al., 2005; Yamada et al., 2005). These experiments have a biochemical basis and their significance will await analysis in vivo.
Several studies have hinted at the importance of AJs and their regulation during DC (Bloor and Kiehart, 2002; Fox et al., 2005; Grevengoed et al., 2001; Magie et al., 2002; McEwen et al., 2000; Murray et al., 2006; Takahashi et al., 2005). For example, defects associated with genetic interactions between shotgun (shg), the gene encoding E-cadherin in Drosophila, and actin regulators such as the small GTPase Rho (Fox et al., 2005) and the non-receptor tyrosine kinases Src (Takahashi et al., 2005) or Abl (Grevengoed et al., 2001), suggest that some aspect of the AJs is crucial for DC. However, although some shg mutations that reach the DC stages (Tepass et al., 1996; Uemura et al., 1996), there has not been a full study of the role of AJs in this epithelial sheet movement. Here, we undertake this study and analyze the effects of the reduction in the levels of E-cadherin in DC as well as the importance of its linkage to the cytoskeleton. We find that AJs are essential at the LE to sustain and modulate the forces generated in the interactions between the AS and epidermal epithelia. Furthermore our experiments show that, during DC, interactions between E-cadherin and β-catenin are dynamic, adding to the biochemical observations for the interactions between -catenin and β-catenin. Altogether, our results outline a requirement for dynamic remodeling of AJs in tissue interactions during epithelial cell movements.
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-catenin (Fig. 1A). Interestingly, higher levels of these AJ proteins, particularly of Armadillo (Fig. 1Aiv), are observed at the LE. As DC progresses, the distribution of AJ proteins changes at the LE: E-cadherin and Armadillo become concentrated in the actin-nucleating centers (ANCs) and become less abundant at the LE of every cell (Fig. 1Bi,Biii,Biv), while -catenin, displays a more diffuse pattern at the cell surface and in the cytoplasm (Fig. 1Bv). As cytoskeletal activity develops at the LE (Jacinto et al., 2000; Jankovics and Brunner, 2006), low levels of E-cadherin can be found colocalizing with actin in the filopodia (Fig. 1C and supplementary material Movies S1 and S2). Later on, when the two epidermal sheets meet at the dorsal midline, the components of the AJs are relocalized to the plasma membrane at the interface between opposite epidermal cells (Fig. 1Biii,Biv).
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Septate junctions (SJs) are intercellular junctions found in invertebrate epithelia that are characterized by a ladder-like appearance in electron micrographs (Tepass et al., 2001). SJs lie basal to AJs and provide a barrier for the diffusion of solutes through the intercellular space. They also play a role in the maintenance of cell polarity and cell adhesion, and in the control of the size of tubular epithelial organs like the tracheal system in Drosophila (Tepass et al., 2001; Tonning et al., 2005; Wang et al., 2006; Wu and Beitel, 2004). Components of the SJs exhibit marked differences between epidermal and AS cells. At the ultrastructural level, SJs can first be detected at stage-14 embryos in all epithelia except the AS (Tepass and Hartenstein, 1994). In agreement with this we find that neurexin IV (NrxIV) and coracle, two proteins required for SJ formation (Baumgartner et al., 1996; Fehon et al., 1994), and fasciclin III (FasIII), a SJ associated protein (Woods et al., 1997), can be found at the lateral membrane of epidermal cells from the beginning of DC onwards but are not present in the AS (Fig. 2A,B and data not shown). However, elements of the Lgl-Dlg-Scrib basolateral complex that regulate epithelial polarity by defining the apical/basolateral boundary (Knust and Bossinger, 2002; Tepass et al., 2001) are expressed in the lateral membrane of both epidermis and AS (Fig. 2A,B).
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As DC progresses, the dorsal-most epidermal (DME) cells acquire a polarity in the plane of the epithelium, which manifests itself in the asymmetric localization of membrane proteins and cytoskeletal components (Kaltschmidt et al., 2002; Morel and Martinez Arias, 2004). At present, the function of this polarization is unclear but is not determined by the planar cell polarity pathway (Morel and Martinez Arias, 2004), which provides polarized organization to epithelial cells in other contexts (Eaton, 1997; Mlodzik, 2002). The differential distribution of SJ components in the AS and the epidermis and, in particular, the planar polarization of these proteins in the DME cells (Kaltschmidt et al., 2002; Morel and Martinez Arias, 2004), led us to analyze in more detail the topology of the lateral membrane at the LE (Fig. 2C,D). To do this, we studied thick transverse sections at different stages of development following the procedure of Narasimha and Brown (Narasimha and Brown, 2004). At the LE, the membrane shows expression of Dlg and Scrib (Fig. 2Cii,Dii,Div) but not FasIII nor Nrx (Fig. 2Civ and data not shown) indicating that SJ components show an asymmetric localization in DME cells, whereas components of the basolateral complex are distributed along all the lateral perimeter of DME cells. These observations indicate that some features of the polarization of DME (i.e. the planar polarized localization of SJ associated proteins) might be a consequence of the differential junctional organization between the epidermis and the AS. To test this, we used an ubiquitous driver (daGal4) to lead the expression of NrxIV both in the epidermis and the AS (Fig. 2E). In these embryos, NrxIV is found now at the membrane of both epidermal and AS cells, and is also present at the LE of some DME cells (Fig. 2Eiii). The localization of NrxIV at the membrane of AS cells does not drive the relocalisation of other SJ associated proteins such as FasIII and coracle (Fig. 2Eii and data not shown) to the membrane suggesting that NrxIV alone is not sufficient to assemble SJ in the AS. Moreover, these embryos undergo DC and do not show defects in cuticles (data not shown). These results suggest that the polarization of SJ associated proteins in the DME cells might be a consequence of the absence of SJs in the AS, reflecting the ability of these molecules to participate in homophilic interactions (i.e. the localization of NrxIV at the membrane of AS cells allows the relocalization of this protein to the LE). These observations suggest that there is a differential junctional organization in the epithelia that participate in DC and, furthermore, that at the LE AJs undergo dynamic changes, which correlate with the different phases of the process.
Functional analysis of the AJs during DC
To analyze the role of the AJs in the interactions between the epidermis and the AS we have studied first the effects of reductions in the levels of E-cadherin, during DC. Complete loss of shg function results in the loss of epithelial integrity early in development (Tepass et al., 1996; Uemura et al., 1996). However, zygotic mutants for null alleles of shg, for example shgR64a, contain enough maternally derived E-cadherin to initiate DC, even though they lack ventral epidermis (Tepass et al., 1996; Uemura et al., 1996). These mutants exhibit small dorsal holes and puckering in cuticle preparations (Table 1, see below). During DC these embryos exhibit reduced levels of E-cadherin in the epidermis but not in the AS (Fig. 3Ai,Aiii), probably because the AS cells do not divide during embryogenesis and thus maintain the maternal contribution. Armadillo and -catenin, which are found at the membrane associated to E-cadherin, show a similar distribution (Fig. 3Aiv,Av). For the most part the DME cells show normal DV elongation and initiate zippering, which proceeds in an ineffective manner. The LE assembles a weak F-actin cable (Fig. 3Aii) that seeds filopodia and lamellipodia (Fig. 3D, inset). Live imaging of shgR64 mutants expressing GFP-labelled actin (ActinGFP) in the engrailed domain reveals frequent mismatches between the two epidermal sheets that appear to arise from the inability of contralateral cells with the same positional identity to find each other (Fig. 3D and supplementary material Movie S3).
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One allele, shgg317, has been characterized as possibly interfering with the maternal contribution of shg expression and acting as a dominant negative (Tepass et al., 1996). Zygotic shgg317 mutants also display a loss of ventral epidermis but, in contrast with the null alleles, they display a highly penetrant dorsal hole (Fig. 3E, Table 1), consistent with the idea that the protein encoded by this allele acts as a dominant negative (see Materials and Methods). In these embryos, the levels of E-cadherin, Armadillo and -catenin at the membrane of epidermal cells are even more reduced than in the null allele but can still be detected in the AS (Fig. 3Bi). F-actin can be observed to accumulate in the apical domain of the epidermis and the AS of shgg317 embryos (Fig. 3Bii) but there is no cable assembled at the LE, which contains very small filopodia (Fig. 3E, inset). At the beginning of DC these embryos exhibit large tears between the epidermis and AS that correlate with a lack of elongation of epidermal cells (Fig. 3Bi). In vivo analysis of shgg317 mutants expressing a GFP-tagged Src construct (see Materials and Methods) in the AS using the UAS/GAC4 system (Brand and Perrimon, 1993) shows that these cells detach from the epidermis and do not reduce their apical surface efficiently when compared with a wild-type embryo (see supplementary material Movies S4 and S5, notice the wild-type embryo carries a GFP-tagged E-cadherin under the control of the ubiquitin promoter, ubiECadGFP).
Sequencing of shgg317 reveals a 7bp insertion in the cytoplasmic domain which leads to a premature stop codon that leaves the juxtamembrane domain intact but eliminates the Armadillo-binding region (Fig. 4Bi). This is likely to explain the depletion of Armadillo and hence of -catenin at the junctions of shgg317 mutant embryos. These results reveal a stringent requirement for E-cadherin at the LE for maintaining the adhesion between these two epithelia. When this adhesion fails, epidermal cells cannot elongate. This result is in agreement with the idea that AS contraction provides an important component of epidermal cell elongation. However, AS cells do not contract properly, suggesting that AS contraction is not autonomous but requires some input from the epidermis.
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; Morel and Martinez Arias, 2004). The relationship that we observe between Armadillo and E-cadherin in several processes in shgg317 mutant embryos led us to analyze more in detail the requirements for Armadillo in DC.
Zygotic mutants for the null allele of Armadillo, armYD35, show reduced levels of maternal protein at the onset of DC by the end of embryonic stage 12 (not shown) and exhibit minor defects in Wingless signaling. During embryonic stage 13, the epidermal cells of armYD35, embryos begin to elongate and polarize (Morel and Martinez Arias, 2004). This is consistent with the strong maternal input of Armadillo and with the observation that the main requirements for Wingless signaling in the patterning of the dorsal epidermis are complete by germ-band retraction (Bejsovec and Martinez Arias, 1991). Although epidermal cells begin to elongate at the onset of embryonic stage 13, shortly afterwards the AS of these embryos tears off and detaches from the epidermis (Fig. 3Ci). This is not a wingless-mutant phenotype but is reminiscent of shg mutants and must reflect a requirement for Armadillo in the formation of junctions (McEwen et al., 2000). There are, however, some differences with the shg mutant: in arm mutants, we observe that high levels of surface E-cadherin are maintained throughout DC (Fig. 3Ciii) and, surprisingly, there is an actin cable at the LE (Fig. 3Cii), which develops active filopodia (Fig. 3F, inset). However, in agreement with the requirement for the interaction between β-catenin and -catenin at the AJ, the latter is mainly cytoplasmic in armYD35 mutants (Fig. 3Cv).
In vivo analysis of arm mutants carrying an ubiECadGFP construct show that AS contraction is perturbed and the epidermis shows very little migration towards the dorsal midline (see supplementary material Movie S6). AS cells expand and contract rhythmically their apical membrane but do not show a net reduction of the apical surface. Sometimes, a detachment of the AS from the epidermis can be observed in these live embryos (data not shown). No differences in the cuticle phenotype of armYD35 and armYD35; ubiECadGFP embryos were observed (data not shown). Altogether these results highlight the role of Armadillo as a central component of AJs and highlight the importance of its interactions with other components of the AJs.
Dynamic interactions between E-cadherin and -catenin are required at the LE during DC
The sensitivity of the LE to the levels of E-cadherin and Armadillo led us to analyze further the interactions between E-cadherin and Armadillo in this domain. As shown above, in shgg317 embryos the LE is very weak and tears lead to a highly penetrant dorsal hole. We made use of these embryos to investigate the requirement of the interaction between E-cadherin and Armadillo at the LE using fusion proteins between E-cadherin and -catenin, which bypass Armadillo (Pacquelet et al., 2003; Pacquelet and Rorth, 2005). Ubiquitous expression of a fusion between E-cadherin and -catenin (UAS-ECadFL–-catenin; Fig. 4C) in shgg317 embryos rescues their LE and dorsal hole (data not shown). Surprisingly, expression of the same molecule in stripes under the control of engrailed also rescues the defect (Fig. 4E,Hi and Table 1), and suggests that strong adhesion at some anchor points suffices to maintain weak adhesion elsewhere and provides enough force to sustain the process.
The interpretation of the effects of these chimeras has been questioned recently because of the finding that -catenin does not bind simultaneously to β-catenin and actin (Drees et al., 2005; Yamada et al., 2005). It appears that different molecular conformations are associated with the binding state of -catenin; as a monomer it binds β-catenin and as an homodimer it binds actin. It has been suggested that these chimeras are able to rescue adhesion by simply increasing the levels of E-cadherin at the cell surface (Weis and Nelson, 2006). However, in the chimeras between E-cadherin and -catenin, because Armadillo is bypassed, dimers of endogenous -catenin and the fusion protein could occur (Weis and Nelson, 2006). This could provide sufficient for -catenin to regulate actin at the junctions and could explain the functional capabilities of these chimeras. Interestingly, if a rescue of the shg phenotype is performed with an E-cadherin full-length protein, the grade of rescue is significantly lower than with the fusion E-cadherin–-catenin (Fig. 4J and Table 1). This is probably not owing to a difference in the levels of both the full-length E-cadherin and the chimera, because increasing the levels of the first by performing the experiment at 29°C still does not lead to a rescue comparable to the one obtained with the chimera. Interestingly, the expression of the full-length form of E-cadherin in the shgg317 background recruits Armadillo and -catenin to the membrane (Fig. 4J,H).
The DC-defects of shgg317 embryos can also be rescued if the E-cadherin–-catenin chimera is expressed in the AS cells using the ASGal4 driver (Fig. 4F,Ii and Table 1). This result shows that providing strong adhesion from the AS is enough to maintain the integrity of the LE. Because exogenous E-cadherin is provided in the AS only, dorsal most epidermal cells do not form an actin cable but still are able to elongate (Fig. 4Ii) showing that the pulling of AS can drive epidermal cell elongation if adhesion between the two tissues is maintained. These results evidence the robustness of DC, i.e. individual cellular processes can be perturbed and DC still occurs because the remaining processes compensate this perturbation (Kiehart et al., 2000; Peralta et al., 2007) (Garcia Fernandez, PhD thesis, 2007).
Expression of an E-cadherin–-catenin fusion that lacks the Armadillo-binding site (UAS-ECad
β–-catenin; Fig. 5A) cannot rescue the adhesion between the epidermis and the AS (Fig. 5B,Ci and Table 1). Because this chimera lacks the Armadillo-binding domain, Armadillo is not recruited to the membrane in the cells in which this chimera is expressed (Fig. 5Cii), in contrast to what is observed when the full-length E-cadherin fused to -catenin is expressed (Fig. 4Hi). Expression of this chimera in the AS only cannot rescue DC either (data not shown). Surprisingly, a similar fusion restores cell-cell adhesion in certain contexts although it is not able to rescue cell migration within an epithelial sheet. This was interpreted as a requirement for β-catenin during junction remodeling. During cell migration cells need to attach and detach from their neighbors and β-catenin might be required for the downregulation of adhesion. The LE is a very dynamic structure and the requirement for the Armadillo-binding site to maintain the adhesion between the epidermis and the AS might reflect that dynamic interactions between junctional components at the LE are an essential element of epithelial interactions during morphogenesis.
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In agreement with this conclusion, the expression of the chimera of full-length E-cadherin and -catenin in an armYD35-mutant background, both ubiquitously (data not shown) or in stripes (Fig. 5D,F,G), does not rescue either the adhesion between the epidermis and the AS nor the dorsal hole of armYD35 mutants (Fig. 5E,F). However, this same chimera is able to partially rescue the DC-defects of a hypomorph allele of Armadillo, armXM19, when expressed ubiquitously (data not shown), presumably because it stabilizes the decaying maternal levels of these embryos. Altogether these results suggest that the adhesion between the epidermis and the AS is an essential element for the process of DC and that this is brought about by Armadillo, which in turn mediates interaction of E-cadherin with the cytoskeleton.
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Cell-cell junctional complexes and DC
Our observations reveal a stringent requirement for the function of the AJs at the LE. Reductions in the dosage of E-cadherin, as in zygotic mutants for null alleles of shg, Drosophila E-cadherin, result in sluggish DC, with ineffective zippering, epithelial fusion and segment matching. Further reductions in shg activity, through a dominant-negative zygotic mutant, fail to sustain DC, mainly due to weakened adhesion at the interface between the AS and the epidermis. In these mutants, tears arise between the two tissues, indicating that the LE is very sensitive to decreases in E-cadherin. Interestingly, in vivo imaging of these mutants (supplementary material Movie S5) shows that, as both tissues detach, the AS snaps dorsally, revealing the tension that underlies this tissue and that had been inferred from laser ablations (Hutson et al., 2003; Kiehart et al., 2000). An interpretation of these observations is that the AJs at the LE are key elements in the integration and transmission of the forces generated on the epidermis by the contraction of the AS. A similar role for the catenin-cadherin complex has been described during the elongation of C. elegans (Costa et al., 1998), where these proteins anchor actin-filament bundles to the membrane and allow the force of the bundle contraction to be translated into cell-shape changes.
Our experiments also show that in these mutants the tears are likely to result from the stresses generated by the forces during DC acting upon a weakened LE. Thus, restoring wild-type adhesion in epidermal stripes or in the AS in shg mutants rescues the process, probably by providing enough adhesion to absorb the forces, i.e. DC does not depend on a specific point or a particular structure but on the distribution of forces over a multicellular structure.
As DC progresses, AJs become restricted to the ANCs and E-cadherin can be observed in filopodia together with actin. This observation suggests rapid recycling of AJ components at the LE. The restriction of AJs components to periodic defined points probably accounts for the suggestion that in the late stages of DC there are no AJs between AS and epidermis (Wada et al., 2007), as such evenly spaced anchor points would only show in a detailed EM analysis of the process; such analysis remains to be performed. Our interpretation is that a weakening of the contribution of these junctions to the adhesion between AS and epidermis might explain the sensitivity of the LE. It is also possible that, in these late stages, the contribution of AJs is assisted by other types of adhesive structures, in particular integrin-mediated cell adhesion. The lateral domain of the epidermis/AS interface maintains a tight association through integrin mediated adhesion (Narasimha and Brown, 2004; Wada et al., 2007). Integrin mutants show rips at the LE although they appear at later stages of DC compared with the early detachments observed in shg strong mutants. One possibility is that, as AJs at the LE disassemble to allow formation of new junctions between the migrating epidermal sheets, integrin-mediated adhesion maintains the association of the two tissues. Modulation of cadherin-mediated adhesion through integrins has been observed in other systems (Chen and Gumbiner, 2006) and raises the interesting possibility that such a mechanism might be acting during late stages of DC.
Septate junctions are a third class of adhesive system in epithelia, We observe that, in agreement with ultrastructural studies, SJs do not assemble in the AS but in the epidermis, except at the LE. As many of these components are homophilic adhesion molecules we wondered whether the planar organization of these proteins in the epidermis does not reflect their absence in one of the tissues. Our experiments indicate that this is the case. They also raise the possibility that this organization is required for the activity of the LE. Further experiments will have to explore this. We noticed that, despite the absence of SJs, there is expression and membrane association of Lgl, Dlg and Scrib. As mutations in these proteins exhibit DC defects, this suggests that they must have phenotypes associated with other than SJs.
Dynamic requirements for AJ components
The dynamics of the AJs at the LE is highlighted by the observation that E-cadherin requires interactions with Armadillo even when it is directly linked to -catenin. Whereas a full-length E-cadherin molecule fused to -catenin can rescue DC in shg mutants, a similar fusion lacking the Armadillo-binding domain cannot. This indicates that the rescue of the full-length molecule is mediated, in part, by binding of Armadillo – a surprising result in the context of the classical model in which the function of Armadillo and β-catenin is to link cadherin to the actin cytoskeleton via -catenin. This view has recently been challenged by the observation that -catenin has two mutually exclusive states. As a monomer it can bind β-catenin, whereas as a dimer it can bind actin (Drees et al., 2005; Yamada et al., 2005). This observation could help explain our results. If interactions between -catenin and Armadillo/β-catenin are dynamic, β-catenin could act to bring -catenin to the junctions where it would dissociate to promote actin polymerization. In the absence of an Armadillo-binding site, the only junctional -catenin would be the one in the chimera which is clearly not sufficient for full function, probably because it cannot dissociate from cadherin. Whereas this provides an explanation for our results, like the biochemical experiments, it cannot explain how the cytoskeleton is linked to cadherin based at AJs. One possibility is that the link is not a static structure but rather it is maintained through a dynamic equilibrium based on the ability of β-catenin to recruit -catenin, which in turn can recruit actin polymers to the LE. A second possibility is that, within this context, there are other proteins that provide the stable link between E-cadherin, -catenin and actin. In either case it might be that dynamic structures such as the LE exploit these properties of the AJ components, because the chimera can rescue adhesion in other circumstances (Nagafuchi et al., 1994; Pacquelet and Rorth, 2005). It will be important to compare the dynamics and activity of the AJs in different contexts to gain some insight into how the regulatory properties of AJs are used in the modulation of morphogenetic processes.
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) were kindly provided by V. Hartenstein (UCLA, Los Angeles, CA). shgg317 showed a weaker phenotype when crossed over a shg deletion confirming that it behaves as a an antimorphic allele. armYD35 is a null allele that was analyzed in the context of DC (Peifer and Wieschaus, 1993) and armXM19 is a hypomorph allele. UAS lines were expressed using the Gal4 system (Brand and Perrimon, 1993). enGAL4 was used as an epidermal driver (although some AS cells could be seen expressing the corresponding reporter, indicating that this driver is leaky). ASGal4 (Gal4332.3) was used as an AS specific driver. As an ubiquitous driver, daGal4 was used. All of them were provided by the Bloomington stock center.
We also used strains bearing the following transgenes: ubiECadGFP (Oda and Tsukita, 2001), UAS-ECadGFP (provided by F. Schweisguth, Ecole Normale Supérieure, Paris, France), UAS-ActinGFP, UAS-ActinRFP (Simoes et al., 2006), UAS-SrcGFP (provided by N. Brown, Gurdon Institute, Cambridge, UK; Src is a non-receptor tyrosine kinase associated to AJs). UAS-ECadFL–-catenin and UAS-ECadβ–-catenin (Pacquelet and Rorth, 2005) were kindly provided by P. Rorth (EMBL, Heidelberg, Germany). For the overexpression of NrxIV, the Nrx-IVEP609 line was used (Gene Disruption Project).
To facilitate scoring of homozygous mutant embryos during inmunohistochemical analysis, all mutant chromosomes were placed over balancer chromosomes bearing ftz-LacZ reporter. For the in vivo experiments, the following crosses were set up:
For the rescue experiments, the following crosses were set up:
-catenin -catenin -catenin β–-catenin -catenin. For the sequencing of the mutant alleles, shgg317 homozygous embryos were selected under the fluorescence scope for DNA extraction. Using specific primers, the coding sequence of E-cadherin was amplified and sequenced by Geneservice DNA Sequencing, Cambridge.
Immunostaining
Embryos were fixed and stained as described previously (Kaltschmidt et al., 2002). For thick sections, the procedure described by Narasihma and Brown (Narasihma and Brown, 2004) was followed. The following primary antibodies were used: mouse monoclonal against Armadillo (1:50, Developmental Studies Hybridoma Bank (DSHB), University of Iowa, developed by E. Wieschaus), guinea pig antisera against coracle (1:2000, a gift from R. Fehon, University of Chicago, IL), rat monoclonal against E-cadherin (DCAD2, 1:20, DSHB, developed by T. Uemura, Kyoto University, Japan), rat monoclonal against -catenin (DCAT1, 1:50, developed by M. Takeichi, Riken Institute, Kobe, Japan), mouse monoclonal against FasIII (1:50, DSHB, developed by C. Goodman, University of California, Berkeley, CA), rabbit antisera against NrxIV (1:1000) (Baumgartner et al., 1996), rabbit antisera against Scrib (1:2000, a gift from C. Doe, University of Oregon, Eugene, OR), and rabbit antiserum against β-galactosidase (1:10000, Cappel). The following conjugated secondary antibodies were used at 1:200: Cy5 conjugated antibodies from Jackson ImmunoResearch, Alexa Fluor-488- and Alexa Fluor-568-conjugated antibodies from Molecular Probes. For phalloidin staining, embryos were fixed in 8% paraformaldehyde in PBS or BBS with the addition of phalloidin (1 unit/ml), de-vitellinized with ethanol 80%, stained as described above and then incubated with Texas-Red-conjugated phalloidin (phalloidin-TxR, Molceular Probes) together with the secondary antibodies. Fluorescently labeled embryos were mounted in Vectashield (Vector) and examined under a Nikon Eclipse E800 microscope coupled to a BioRad MRC1024 confocal unit.
Cuticle preparations
Embryos were collected from 24-hour-old eggs and then aged 24 hours at 25°C or 29°C. They were dechorionated in bleach and de-vitellinized with methanol. Embryos were mounted in acetic acid:Hoyers (1:1) and the slide was incubated overnight at 65°C.
Time-lapse image collection
The embryos were mounted as described in Wood and Jacinto (Wood and Jacinto, 2004). Time-lapse images were taken with a Nikon Eclipse E800 microscope coupled to a BioRad MRC1024 confocal unit, with a 40x or 60x oil immersion objective. For each time point, between 20 and 40 Z sections (spaced between 0.5 µm and 1 µm) were collected. Movies were assembled using ImageJ (NIH).
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-catenin and UAS-ECadβ–-catenin flies. This work was supported by a BBSRC Grant (RG38087). |
Footnotes |
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References |
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Baumgartner, S., Littleton, J. T., Broadie, K., Bhat, M. A., Harbecke, R., Lengyel, J. A., Chiquet-Ehrismann, R., Prokop, A. and Bellen, H. J. (1996). A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function. Cell 87, 1059-1068.[CrossRef][Medline]
Bejsovec, A. and Martinez Arias, A. (1991). Roles of wingless in patterning the larval epidermis of Drosophila. Development 113, 471-485.[Abstract]
Bloor, J. W. and Kiehart, D. P. (2002). Drosophila RhoA regulates the cytoskeleton and cell-cell adhesion in the developing epidermis. Development 129, 3173-3183.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415.[Abstract]
Chen, X. and Gumbiner, B. M. (2006). Crosstalk between different adhesion molecules. Curr. Opin. Cell Biol. 18, 572-578.[CrossRef][Medline]
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