2002 - signaling pathways directing the movement and fusion of epithelial sheets - harden

8/2/2019 2002 - Signaling Pathways Directing the Movement and Fusion of Epithelial Sheets - Harden

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Differentiation (2002) 70:181203 C Blackwell Verlag 2002

REVIEW

Nicholas Harden

Signaling pathways directing the movement and fusion of epithelialsheets: lessons from dorsal closure in Drosophila

Accepted in revised form: 30 April 2002

Abstract Wound healing in embryos and various devel-opmental events in metazoans require the spreading andfusion of epithelial sheets. The complex signaling path-ways regulating these processes are being pieced togetherthrough genetic, cell biological, and biochemical ap-proaches. At present, dorsal closure of the Drosophilaembryo is the best-characterized example of epithelialsheet movement. Dorsal closure involves migration of the lateral epidermal anks to close a hole in the dorsalepidermis occupied by an epithelium called the am-nioserosa. Detailed genetic studies have revealed a net-work of interacting signaling molecules regulating thisprocess. At the center of this network is a Jun N-ter-

minal kinase cascade acting at the leading edge of themigrating epidermis that triggers signaling by the TGF-b superfamily member Decapentaplegic and which inter-acts with the Wingless pathway. These signaling modulesregulate the cytoskeletal reorganization and cell shapechange necessary to drive dorsal closure. Activation of this network requires signals from the amnioserosa andinput from a variety of proteins at cell-cell junctions.The Rho family of small GTPases is also instrumental,both in activation of signaling and regulation of thecytoskeleton. Many of the proteins regulating dorsal clo-sure have been implicated in epithelial movement inother organisms, and dorsal closure has emerged as anideal model system for the study of the migration andfusion of epithelial sheets.

Key words Drosophila epithelial morphogenesis dorsal closure cytoskeleton signal transduction

N. Harden ( )Department of Molecular Biology and Biochemistry, SimonFraser University, 8888 University Drive, Burnaby, BC, V5A1S6, CanadaTel: 1 604 291 5644, Fax: 1 604 291 5583e-mail: nharden / sfu.ca

U.S. Copyright Clearance Center Code Statement: 03014681/2002/7004181 $ 15.00/0

IntroductionThe spreading and fusion of epithelial sheets occurs ina diverse range of morphogenetic events (reviewed inBard, 1990) such as epiboly, neural tube closure and em-bryonic wound healing in vertebrates, ventral enclosurein Caenorhabditis elegans , and dorsal closure (DC) of the Drosophila embryo. Drosophila DC represents thesingle most thoroughly characterized example of epi-thelial movement and fusion in metazoans. This is dueto the ease of identifying mutants in the process and theadvanced genetics and developmental biology availableto study them. The picture that is emerging is control of

DC by a complex network of signaling pathways mediat-ing the transcriptional responses and cytoskeletalchanges necessary to promote epithelial migration. Al-though other types of epithelial closure remain less wellcharacterized, it is becoming apparent that these pro-cesses require many of the same proteins as DC, andwhat is learned about DC is widely applicable (reviewedin Jacinto et al., 2001). In particular, DC has been pro-posed as a model system for the study of wound healing(Kiehart, 1999; Jacinto and Martin, 2001; Jacinto et al.,2001). The last few years has seen a tremendous increasein the number of proteins known to participate in DC,but there remains much to be done in terms of sortingthrough all these participants and determining their pre-cise roles and inter-relationships. In this review, I willsummarize what is presently known about DC and theproteins driving it, and attempt to pull scattered piecesof data together into a model (see Fig. 3).

What is dorsal closure?

Following germband retraction of the Drosophila em-bryo, a hole is left in the dorsal surface of the epidermisthat is occupied by the amnioserosa, an epithelium of


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Fig. 1 Dorsal closure (DC) of the Drosophila embryo. Panels show photyrosine antibodies to show closure of the epidermis over thedorsal views of progressively older embryos stained with anti-phos- large, at cells of the amnioserosa.

large, at cells. DC involves a dorsalward migration of the lateral epidermis from both sides of the embryo withthe epidermal anks meeting at the dorsal midline, com-pletely covering the amnioserosa and sealing the embryo(Campos-Ortega and Hartenstein, 1985) (Fig. 1). Nu-merous mutants have been identied in which DC fails,

leading to a dorsal hole in the larval cuticle (Jrgens etal., 1984; Nsslein-Volhard et al., 1984; Wieschaus et al.,1984; Perrimon et al., 1989). It is the genetic and mol-ecular characterization of the genes disrupted in thesemutants (the DC genes) that has lead to the emer-gence of DC as an excellent system for studying theregulation of epithelial morphogenesis (reviewed pre-viously in Knust, 1996; 1997; Mart n-Blanco 1997; Gob-erdhan and Wilson, 1998; Noselli, 1998; Noselli andAgne s, 1999; Jacinto and Martin, 2001; Jacinto et al.,2001).

During DC there is an accumulation of nonmusclemyosin-II (hereafter referred to as myosin) and F-actinat the leading edge (LE) of the advancing epidermis(Young et al., 1993; Mizuno et al., 2002) (Fig. 2A, B).This cytoskeletal band ringing the dorsal hole has beenproposed to draw the hole shut in what is known as thepurse-string model (Young et al., 1993). At the cellularlevel, the purse-string is composed of a polarized ac-cumulation of F-actin and myosin at the dorsal end of each LE cell that acts as a contractile apparatus, drivingconstriction of the cell in an anterior-posterior (AP)direction. The purse-string model is based on the obser-vation that, during DC, cells in the lateral epidermisshift from polygonal in shape to being elongated in the

DV direction. This elongation is rst seen in the LEcells and is then transmitted to more ventrally locatedcells. In the model, elongation of the LE cells is drivenby their constriction in the AP direction, whereaselongation of the more ventrally located cells in the lat-eral epidermis is a passive response to events at the LE.

The net result is stretching of the lateral epidermis upover the amnioserosa. The rst DC gene analyzed in de-tail was zipper (Nsslein-Volhard et al., 1984) which en-codes Drosophila nonmuscle myosin-II heavy chain(Young et al., 1993). In zipper mutant embryos decientin myosin, cell shape change in the epidermis is aberrantand DC is not completed (Young et al., 1993). That theF-actin purse-string is driving cell constriction is sup-ported by the observation that LE cells in which theactin cytoskeleton is decient tend to splay out in theAP direction (Harden et al., 1996; Grevengoed et al.,2001). In addition to F-actin and myosin, there is anaccumulation of phosphotyrosine-rich structures at theLE during DC (Harden et al., 1996). These structurestake the form of triangular nodes linking LE cells attheir dorsal end (Fig. 2C). The level of phosphotyrosinein these structures is linked to the integrity of the LEcytoskeleton, as losses of the cytoskeleton at pointsalong the LE are accompanied by losses of LE phospho-tyrosine at the same positions (Harden et al., 1996). Asdiscussed below, the triangular nodes appear to beadherens junctions contributing to organization of theLE cytoskeleton.

It has become apparent that the contractile purse-string is not the only mechanism driving closure of the


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Fig. 2 Views of the leading edge (LE) during DC. ( A) The bound- migrating epidermal anks have met up at the end of DC. Noteary between the amnioserosa ( top of gure ) and the epidermis in that cells anking segment borders (marked with bars ) are widerphalloidin-stained embryo showing accumulation of F-actin along than their neighbors. ( E ) Phosphotyrosine-stained kay mutant em-the LE, and extension of F-actin-rich lopodia from the am- bryo lacking DFos, showing loss of LE phosphotyrosine nodes andnioserosa and LE cells ( arrows ). (B) Accumulation of myosin along failure of cell elongation in the epidermis. ( F) Embryo expressingthe LE. ( C ) Accumulation of phosphotyrosine along the LE in dominant negative Dcdc42 showing bunched epidermis character-triangular nodes ( arrows ). (D ) View of the dorsal midline after the ized by ectopic adhesions between LE cells ( arrows ).

epidermis. A detailed mechanistic study using laser ab-lation and green uorescent protein (GFP) imaging of live embryos conrms the contribution of a contractilepurse-string but also indicates that DC is driven byother forces (Kiehart et al., 2000). Ablation of cells at apoint along the LE causes neighboring cells to recoil,indicating that the LE is under tension, as predicted bythe purse-string model. Embryos can close the epidermiseven when the LE is repeatedly wounded, although theclosed dorsal surface is often irregular in appearance.This nding is consistent with results showing that some

mutants with disruptions at the LE in both the cytoskel-eton and cell shape change manage to close, albeit in adistorted manner (McEwen et al., 2000; Grevengoed etal., 2001). Cell shape change driven by the LE cytoskel-eton is clearly not the sole force driving DC but appearsto be required for the perfect, scar-free closure seen inwild-type embryos.

What are the other forces driving DC? The potentialcontribution of the lateral epidermis has been addressedby laser ablation of cells ventral to the LE cells (Kiehartet al., 2000). This leads to an acceleration of the dor-


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salward movement of the LE, indicating that the lateralepidermis does not contribute to DC by pushing the LEforward, rather, there is tension in the lateral epidermisthat retards movement of the LE.

Phalloidin staining of embryos xed during DC oftenshows actin-rich lopodia-like structures at the LE(Harden et al., 1996) (Fig. 2A). Imaging of live embryosexpressing GFP-tagged actin has conrmed that the LEcells extend lopodia as well as lamellipodia during DC(Jacinto et al., 2000). Filopodia and lamellipodia are ac-tin-based structures that contribute to cell motility andare involved in neuronal growth cone guidance (reviewedin Mueller, 1999; Small et al., 1999). As the advancingepithelial sheets approach one another during DC, thelopodia extending from the LE cells appear to seekout cells on the opposing sheet and contribute to cellmatching, such that the dorsal hole is neatly closed withthe segments from the two sides of the embryo properlyaligned. This interpretation of lopodia function in DCcomes from the observation of wild-type live embryosand live embryos in which the LE lopodia have beenlost due to mutation or transgene expression (Jacinto etal., 2000).

Laser ablation studies also indicate that the am-nioserosa plays an active role in DC and is not simplycompressed by the migrating epidermal anks (Kiehartet al., 2000). The amnioserosa is under tension as dem-onstrated by the nding that amnioserosa cells recoilfrom a wound in the tissue. The contraction of the am-nioserosa may contribute to DC as wounds in the am-nioserosa that release tension lead to retraction of theLE away from the dorsal midline. During DC, the am-

nioserosa changes from an elliptically-shaped squamousepithelium to a tubular structure. This change in mor-phology is driven both by an apical constriction of cellsin the amnioserosa and the loss of cells from the tissue(Rugendorff et al., 1994; Kiehart et al., 2000). Trans-mission electron microscopy shows that the apically-constricted amnioserosa cells become elongated alongthe apical-basal axis and the tissue invaginates to adoptits nal morphology (Rugendorff et al., 1994). The ex-trusion of cells from the amnioserosa has been observedin live embryos, where cells drop out of the plane of theepithelium and adjoining cells come together over them(Kiehart et al., 2000). This seems to occur sufcientlyoften to make a signicant contribution to the contrac-tion of the tissue. Severe disruption of the amnioserosaduring DC does not prevent DC from proceeding tocompletion, suggesting that the advancing epidermisdoes not require the amnioserosa to crawl over and thatamnioserosa contraction may serve a permissive role inDC (Kiehart et al., 2000; Harden et al., 2002). Thus, thecontraction of the amnioserosa could largely serve toremove a physical impediment to migration of the epi-dermis. Below, I discuss an additional role for the am-nioserosa in DC- a source of signals determining LE cellfate.

A Jun N-terminal kinase cascade is a centralcomponent of the signaling controlling DC

From the molecular characterization of several genes in-volved in DC, it became apparent that a mitogen-acti-vated protein kinase (MAPK) cascade was involved inthe process. MAPK cascades, which lead to the phos-phorylation and activation of various trasncription fac-tors, have been the subject of intense study due to theirinvolvement in vital cellular processes such as cell cycleprogression and differentiation (reviewed in Widmann etal., 1999). At the core of MAPK pathways is a three-kinase module in which there is sequential activation of kinases by phosphorylation. The module is composedof a MAPK kinase kinase (MAPKKK), MAPK kinase(MAPKK) and MAPK. Numerous upstream activatorshave been identied for MAPK pathways with some of these being kinases themselves, i.e. MAPKKKKs. TheMAPKKKs are serine/threonine kinases that phos-phorylate and activate the MAPKKs, which are dual-specicity kinases that phosphorylate MAPK at Thr-X-Tyr motifs. MAPKs subsequently phosphorylate sub-strates on serine and threonine residues, with the ma- jority of substrates identied to date being transcriptionfactors. In mammalian cells, the MAPK pathways canbe divided into ve families based on the MAPK beingactivated at the end of the cascade: extracellular regu-lated kinase 1 and 2 (ERK1/2), Jun N-terminal kinase(JNK), p38, ERK3/4, and ERK5. As the name suggests,the JNK cascade leads to the phosphorlyation and acti-vation of Jun, a leucine zipper transcription factor.

Cloning of the DC gene hemipterous (hep) revealedthat it encodes a MAPKK most similar to mammalianJNKK, and this was the rst indication that a JNK cas-cade was required for DC (Glise et al., 1995). Sub-sequent work demonstrated that Drosophila JNK(DJNK) is encoded by the DC gene basket (bsk ) andDrosophila Jun (DJun) by the DC gene l(2)IA109(Nsslein-Volhard et al., 1984; Riesgo-Escovar et al.,1996; Sluss et al., 1996; Hou et al., 1997; Kockel et al.,1997; Riesgo-Escovar and Hafen, 1997a). The prevalentfunctional form of Jun is as a heterodimer with the Fosleucine zipper protein to form the AP-1 factor (reviewedin Kockel et al., 2001). Not surprisingly then, DrosophilaFos (DFos) is encoded by a DC gene, kayak (kay )(Jrgens et al., 1984; Riesgo-Escovar and Hafen, 1997b;Zeitlinger et al., 1997). The DC phenotypes of the JNKpathway mutants are similar to each other. Loss of JNKsignaling is characterized by an initial D-V elongationof LE cells which later revert to a polygonal shape, dis-ruption of the accumulation of F-actin and myosin atthe LE, and failure of DC to proceed to completion(Glise et al., 1995; Riesgo-Escovar et al., 1996; Sluss etal., 1996; Hou et al., 1997; Kockel et al., 1997; Riesgo-Escovar and Hafen, 1997a; 1997b; Zeitlinger et al., 1997;Ricos et al., 1999; Jasper et al., 2001; Stronach and Per-


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rimon, 2002) (Fig. 2E). Imaging of live hep mutant em-bryos demonstrates that the JNK cascade is also re-quired for the LE lopodia (Jacinto et al., 2000). Gen-etic and biochemical data indicate that the DrosophilaJNK pathway acts as a classic MAPK cascade leadingto activation of DJun (Riesgo-Escovar et al., 1996; Slusset al., 1996; Hou et al., 1997; Riesgo-Escovar and Hafen,1997a; Sluss and Davis, 1997; Mart n-Blanco et al.,1998; Stronach and Perrimon, 2002). The fact that theloss of DJun or DFos have a similar effect on DC asloss of kinases in the JNK cascade suggests that a majorroute of action of the JNK pathway in DC is activationof AP-1 and subsequent transcriptional responses in thenucleus. Indeed, expression of a constitutively active ver-sion of DJun can signicantly rescue the DC defects of embryos lacking one of the JNK pathway kinases (Houet al., 1997; Riesgo-Escovar and Hafen, 1997a; 1997b;Sluss and Davis, 1997; Stronach and Perrimon, 2002).

As transcriptional regulation of genes seems to becentral to how the JNK cascade exerts its effects duringDC, there has been great interest in determining whichgenes show expression changes in response to JNK sig-naling. Detailed studies have been done on two geneswhose expression in the LE cells is dependent on theJNK cascade: decapentaplegic (dpp) and puckered ( puc).

The decapentaplegic (dpp) locus encodes a member of the transforming growth factor- b (TGF- b) superfamilythat is expressed in the LE cells during DC (St. Johnstonand Gelbart, 1987; Jackson and Hoffmann, 1994). Asdiscussed below, components of a Dpp signaling cascadehave been picked up as DC genes, indicating that Dppsignaling is required for DC. The LE expression of Dpp

is dependent on the JNK cascade as it is lost in mutantslacking cascade components (Glise and Noselli, 1997;Hou et al., 1997; Riesgo-Escovar and Hafen, 1997b;Sluss and Davis, 1997; Zeitlinger et al., 1997; Stronachand Perrimon, 2002).

The puckered ( puc) gene was identied through a Pelement insertion that caused a mild DC defect (Ringand Martinez Arias, 1993). puc is strongly expressed inthe LE cells during DC, but this LE transcription isabolished by loss of the JNK cascade (Glise et al., 1995;Riesgo-Escovar et al., 1996). puc encodes a dual speci-city MAPK phosphatase that likely acts by downregul-ating DJNK/Bsk activity through dephosphorylation(Mart n-Blanco et al., 1998). Thus, Puc appears to me-diate a negative feedback loop regulating the JNK path-way (Mart n-Blanco et al., 1998). Overexpression of Pucmimics the phenotype of loss of the JNK pathway inthat there is a failure of DC accompanied by loss of both the F-actin/myosin contractile apparatus and dppexpression at the LE. A reduction in Puc throughheterozygosity for a puc allele can partially rescue theDC defects caused by reduction of DJNKK/Hep. Fur-thermore, the expression patterns of dpp and puc re-spond to changes in Puc levels. In puc mutant embryos,both dpp and a b-galactosidase reporter gene under con-

trol of puc locus enhancer sequences ( puc-lacZ , an en-hancer trap widely used to assess puc expression in DCstudies) are overexpressed at the LE, suggesting that ex-cessive signaling by the JNK cascade is occurring (Ringand Martinez Arias, 1993; Glise and Noselli, 1997; Mar-tn-Blanco et al., 1998).

Work in various systems indicates that numerous ki-nases can operate as JNKKKs (Widmann et al., 1999).The identity of the main JNKKK operating in theDrosophila JNK cascade at the LE remained uncertainuntil recently. The rst candidate described was theMAPKKK dTAK1. Mammalian TAK1 is capable of acting as a JNKKK in the JNK cascade (Shirakabe etal., 1997), and the potential role of dTAK1 in the LEJNK pathway has been addressed. Expression of kinase-dead forms of dTAK1 causes DC defects, and overex-pression of dTAK1 induces ectopic expression of dppand puc (Takatsu et al., 2000; Mihaly et al., 2001). How-ever, inhibition of dTAK1 through double-strandedRNA interference produces only a low frequency of DCdefects, and ies homozygous for dTAK1 loss-of-func-tion mutations survive to adulthood (Mihaly et al.,2001; Vidal et al., 2001). It is now apparent that themain or only JNKKK acting during DC is a mixed lin-eage kinase (MLK) encoded by the slipper (slpr ) locus(Stronach and Perrimon, 2002). Embryos bearing astrong loss-of-function allele of slpr show similar pheno-types to JNK pathway mutants in that they fail to main-tain elongation of epidermal cells during DC, do notshow dpp expression at the LE, and have large dorsalholes in their cuticles. Epistasis analysis indicates thatSlpr signals through the JNK cascade. The dorsal open

phenotype of slpr mutant embryos can be partially res-cued by expression of constitutively active DJun orthrough a reduction in Puc levels.

The JNK cascade is required for the integrity of theLE cytoskeleton during DC, indicating that signalingthrough this pathway at some point has to affect theassembly of F-actin. This could occur either through in-teractions between the cytoplasmic portion of the cas-cade and proteins regulating cytoskeletal assembly, or bysuch proteins being affected in some way by JNK cas-cade-dependent gene transcription. As discussed below,the major route of action for the JNK cascade appearsto be through the Dpp signaling pathway. However, arecent serial analysis of gene expression (SAGE) studyhas identied numerous genes encoding signaling mol-ecules, cytoskeletal regulators, and cell adhesion mol-ecules that are transcriptionally responsive to alterationsin JNK signaling in Drosophila (Jasper et al., 2001). Thestudy was done by comparing tag frequencies amongSAGE libraries generated from wild-type embryos, em-bryos expressing a dominant negative version of DJNK/Bsk, or embryos expressing a constitutively active ver-sion of DJNKK/Hep. Among the genes upregulated byactivation of the JNK cascade is chickadee (chic), whichencodes Drosophila prolin (Cooley et al., 1992). Prolin


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is an actin binding protein involved in F-actin poly-merization (reviewed in Wear et al., 2000) and may con-stitute a link between the JNK cascade and regulationof the cytoskeleton in LE cells. chic mutant embryosshow DC defects and lack lopodia at the LE, and thereis genetic interaction between chic and hep during DCin that heterozygosity for a loss-of-function chic alleleincreases the frequency and severity of DC defects in hepmutant embryos (Jasper et al., 2001). These data suggestthat the JNK cascade drives production of prolin inthe LE cells, which is subsequently involved in the pro-duction of lopodia required for DC.

JNKKKKs in DC

Genetic and biochemical analysis of the MAPK path-way mediating pheromone response in S. cerevisiae in-dicates that the kinase Ste20p functions as aMAPKKKK (reviewed in Widmann et al., 1999). ASte20 group of kinases has been dened whose mem-bers function as upstream regulators of MAPK cas-cades (reviewed in Dan et al., 2001). The Ste20 groupkinases can be divided into the p21-activated kinase(PAK) and germinal center kinase (GCK) familiesbased on the location of their kinase domains. TheDrosophila gene misshapen (msn) encodes a member of the GCK family that is required upstream of the JNKpathway during DC (Su et al., 1998; 2000). Msn ishighly homologous to mammalian Nck-interacting ki-nase (NIK), a known activator of JNK (Su et al.,1997). dpp expression is reduced at the LE in msn mu-

tant embryos, and the DC defects of these embryos canbe substantially rescued by expression of constitutivelyactive DJun. Embryos heterozygous for a msn loss-of-function mutation do not show DC defects, but em-bryos doubly heterozygous for either msn and DJNK/ bsk alleles or msn and DJNKK/hep alleles display dor-sal holes. Finally, transfection of cells with Msn leadsto a four- to ve-fold increase in JNK activation.

Another Ste20 group kinase, DPAK, a PAK familymember, is enriched at the LE during DC (Harden etal., 1996). Loss-of-function DPAK mutants survive em-bryogenesis (Hing et al., 1999), suggesting that DPAKdoes not have a major role in DC or that maternallycontributed DPAK is sufcient. A role for DPAK in ac-tivation of the JNK cascade has not been addressed todate.

Regulation of the JNK cascade by CKA, a potentialscaffolding molecule

Characterization of the connector of kinase to AP-1(cka) gene has revealed another component of JNK sig-naling during DC (Chen et al., 2002). Embryos lackingthe Cka protein show DC defects similar to loss of JNK

pathway components in that there is lack of cell elonga-tion in the epidermis and reduced dpp expression at theLE. Cka has multiple domains and can bind DJNKK/Hep, DJNK/Bsk, Djun, and DFos. Various results indi-cate that this binding to JNK pathway componentsmakes a contribution to functioning of the cascade. Areduction in Cka levels can worsen the DC defectscaused by reductions in DFos or DJNK/Bsk function,and the DC defects associated with complete loss of Ckaare signicantly rescued by expression of constitutivelyactive versions of DJun or DJNK/Bsk. Furthermore, incultured cells, Cka stimulates activation of DJNK/Bskand the subsequent phosphorylation and transcriptionalactivity of DJun and DFos. Cka may act as a scaffoldingmolecule similar to the yeast Ste5 protein in the phero-mone response MAPK cascade (reviewed in Widmannet al., 1999). Cka may bring DJNK/Bsk into contactwith its activator DJNKK/Hep and further act in thenucleus to promote activation of transcription by AP-1(Chen et al., 2002).

Nonreceptor tyrosine kinases functioning asupstream activators of the JNK cascade

Two recent studies indicate that nonreceptor tyrosine ki-nases are required for activation of the JNK cascadeduring DC. The Drosophila Src42A tyrosine kinase par-ticipates with the Src64 tyrosine kinase and the Src-re-lated kinase Tec29 in activation of the JNK cascade dur-ing DC (Tateno et al., 2000). Although embryos missingany one of these kinases do not have DC defects, Src42A

Tec29 double mutant embryos and Src42A Src64B double mutant embryos have dorsal holes. The LE cellsof Src42A Tec29 double mutant embryos show disrup-tion of F-actin accumulation as well as loss of dpp and puc-lacZ expression. This DC phenotype is very similarto that seen in JNK mutant embryos, and expression of constitutively active DJun can partially rescue the DCdefects of Src42A Tec29 double mutant embryos. Fi-nally, overexpression of Src42A in larvae leads to in-creased levels of phosphorylated DJNK/Bsk.

Embryos completely devoid of another nonreceptortyrosine kinase, Shark, are defective in DC, lack cellelongation in the epidermis, and show loss of dpp ex-pression at the LE (Fernandez et al., 2000). That acti-vation of JNK signaling is an important component of Shark function during DC is conrmed by the partialrescue of the DC defects in shark mutant embryos byexpression of constitutively active DJun.

Nonreceptor kinases such as Src have been implicatedin signaling from various kinds of transmembrane recep-tors (reviewed in Abram and Courtneidge, 2000). It islikely that the various nonreceptor tyrosine kinases re-quired for DC constitute a link between JNK-promotingsignals received at the cell surface (for example, from theamnioserosa, see below) and activation of the cascade.


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Signaling by small GTPases during DC

Various members of the Ras superfamily of smallGTPases have been investigated for roles in DC. Thesmall GTPases act as molecular switches that cycle be-tween a GDP-bound off state and a GTP-bound onstate, regulating a diverse array of cellular processes byrelaying extracellular and intracellular signals to down-stream effectors. With regard to DC, particular empha-sis has been placed on the study of members of the Rhofamily of small GTPases, proteins that have been shownin various systems to be key regulators of the actin cyto-skeleton and upstream activators of JNK and p38MAPK cascades (reviewed in Bishop and Hall, 2000).Included in the Rho family are the Rac, Cdc42 and Rhosubgroups. The Drosophila Rac proteins Drac1, Drac2,and Mtl, the Drosophila Cdc42 protein Dcdc42, and theDrosophila Rho protein RhoA (also called Rho1) areinvolved in DC.

Drac1, Drac2 and Mtl

The requirement for Drac1 during DC has been ad-dressed through the expression of a dominant negativemutant version of the protein, Drac1N17. Embryos inwhich Drac1N17 has been expressed show loss of theLE cytoskeleton, a lack of cell elongation in the epider-mis, and do not complete DC (Harden et al., 1995;1999). These phenotypes are similar to those caused byloss of the JNK pathway, and expression of constitut-

ively active DJun can partially rescue Drac1N17-in-duced DC defects, indicating that a component of sig-naling downstream of Drac1 in DC is the JNK cascade(Hou et al., 1997). This is in line with results in mam-malian cultured cells showing that Rac can activate theJNK cascade (Coso et al., 1995; Minden et al., 1995).Expression of a constitutively active version of Drac1,Drac1V12, induces ectopic expression of puc-lacZ anddpp in a hep-dependent manner, demonstrating thatDrac1 can act as an upstream activator of the JNK cas-cade (Glise et al., 1995). Activation of the JNK cascadedoes not, however, appear to be wholly channeledthrough Drac1 as Drac1N17-expressing embryos or em-bryos overexpressing a negative regulator of Drac1, Ro-tundRacGAP, maintain dpp and puc-lacZ expression atthe LE (Raymond et al., 2001).

The route(s) by which Drac1 activates the JNK cas-cade remains uncertain. There is some evidence fromwork in mammalian cells that Rac and Cdc42 can trig-ger JNK activation through the Rac/Cdc42-bindingPAK kinases (reviewed in Van Aelst and DSouza-Schor-ey, 1997). The LE enrichment of DPAK appears to bedependent on Drac1 function (Harden et al., 1996).However, as noted above, there is presently no direct evi-dence of a role for DPAK in DC. It is possible that

Drac1 activates the JNK cascade through direct contactwith the JNKKK, Slpr. Slpr, like other MLKs, containssequences with homology to the Cdc42/Rac interactingbinding (CRIB) motif (Burbelo et al., 1995; Pirone etal., 2001). The mammalian protein MLK3 can bind Racand Cdc42, and this binding may contribute to the acti-vation of JNK signaling by MLK3 (Teramoto et al.,1996; Bock et al., 2000). Drac1 may also contribute tothe activation of the JNK cascade by Msn, although notthrough direct interaction. Rac cannot bind Msn orNIK, but dominant negative Rac can impair JNK acti-vation by Msn or NIK (Su et al., 1998).

A likely upstream activator of Drac1 during DC is theproduct of the myoblast city (mbc) gene. Alleles of mbcwere picked up in a screen for suppressors of a rougheye phenotype induced by overexpression of Drac1 (Nol-an et al., 1998). The Mbc protein is highly homologousto the mammalian protein DOCK180 (Erickson et al.,1997), which is an activator of Rac. DOCK180 binds tothe nucleotide-free form of Rac and promotes formationof active, GTP-bound Rac (Kiyokawa et al., 1998a; Nol-an et al., 1998). DOCK180 does not appear to be aguanine nucleotide exchange factor (GEF) itself but maystabilize nucleotide-free Rac, which is an intermediate inthe activation of Rac by GEFs (Kiyokawa et al., 1998a).DOCK180 can trigger the JNK cascade, as assessed bythe level of Jun phosphorylation, but this JNK pathwayactivation is blocked by dominant negative Rac, RacN17(Kiyokawa et al., 1998a; Nolan et al., 1998). RacN17can also suppress DOCK180-induced membrane spread-ing in cultured cells (Kiyokawa et al., 1998a). These re-sults suggest that DOCK180 can regulate JNK signaling

and the actin cytoskeleton through its effects on Rac.Embryos decient in Mbc show DC defects and exhibitmild losses of LE cytoskeleton and occasional modestreductions in dpp expression at the LE (Erickson et al.,1997; Nolan et al., 1998).

In cultured cells, DOCK180 forms a complex with thefocal adhesion proteins CrkII and p130 cas after integrinstimulation and is a likely mediator of integrin signaling(Kiyokawa et al., 1998b). Interestingly, mutants in thegenes myospheroid (mys) and scab (scb), which encodethe Drosophila bPS and a PS3 integrin subunits, respec-tively, exhibit DC defects (Brown, 1994; Stark et al.,1997; Brown et al., 2000), raising the possibility thatintegrin signaling through Mbc leads to Drac1 acti-vation.

The inability of activated DJun to completely rescueDrac1N17-induced DC defects suggests that the JNKcascade is not the only route of Drac1 activity. Work oncultured cells indicates that there are JNK-independentlinks between Rac and regulation of the actin cytoskel-eton (reviewed in Bishop and Hall, 2000; Takenawa andMiki, 2001). A potential downstream target of Drac1 inregulation of the cytoskeleton is Pkn, a member of thePKN family of PKC-related kinases (Lu and Settleman,1999). Pkn mutant embryos exhibit DC failures ac-


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companied by a lack of cell elongation in the epidermis.The JNK pathway is not disrupted in Pkn mutants asexpression of dpp at the LE appears normal. Pkn bindsspecically to the active, GTP-bound versions of Drac1and Rho1, and this binding stimulates Pkn kinase activ-ity. The biological roles of the PKN kinases remainpoorly characterized, but ectopic expression of mam-malian PKN in broblasts causes reorganization of F-actin and membrane rufing, suggesting a function incytoskeletal regulation (Dong et al., 2000). PKN maycontribute to cytoskeletal regulation by phosphorylatingcytoskeletal components (Amano et al., 1996; Watanabeet al., 1996). Other links between Rac and actin poly-merization have been identied through work on cul-tured cells. The Wiskott Aldrich protein (WASP) fam-ily, which is divided into the WASP and WAVE groups,links small GTPases to the actin cytoskeleton (reviewedin Takenawa and Miki, 2001). In response to activationof small GTPases, WASP family proteins promote acti-vation of the Arp2/3 complex, a multimeric proteincomplex that plays a central role in de novo actin poly-merization. WASPs and WAVEs bind directly to prolin,which may contribute to rapid actin polymerization inconjunction with the Arp2/3 complex. Rac-induced for-mation of F-actin-based lamellipodia at the cell peri-phery is mediated through the WAVE2 protein. AWAVE/Scar protein and members of the Arp2/3 com-plex have been identied in ies (Goldstein and Guna-wardena, 2000; Zallen et al., 2002), and DrosophilaWAVE/Scar mutants have been isolated (Zallen et al.,2002), but to date, roles in DC have not been reported.However, Drac1-dependent assembly of the LE cytoskel-

eton likely does involve the prolin produced throughJNK cascade activation of chic transcription (Jasper etal., 2001).

In addition to its LE roles, Drac1 participates in mor-phogenesis of the amnioserosa during DC (Harden etal., 2002). Amnioserosa-specic expression of Drac1N17retards contraction of the amnioserosa and impedesmovement of the LE, whereas such expression of consti-tutively active Drac1 causes a premature and excessivecontraction of the tissue. Preliminary indications arethat Drac1 acts through Crumbs, a determinant of epi-thelial polarity (Tepass et al., 1990; Wodarz et al., 1995),in regulation of cell shape in the amnioserosa. These re-sults lend support to the idea, discussed above, that ac-tive morphogenesis of the amnioserosa is a componentof DC.

Recently, loss-of-function mutations in the threeDrosophila Rac genes have been described (Hakeda-Su-zuki et al., 2002; Ng et al., 2002). Analysis of these indi-cates that they have overlapping roles in epithelial mor-phogenesis but that Drac1 makes the greatest contri-bution to DC (Hakeda-Suzuki et al., 2002). Embryosmutant for any one of these Rac genes complete DC,but Drac1 Drac2 double mutant embryos or Drac1 Mtl double mutant embryos exhibit DC defects. The most

severe and frequent DC defects are seen in Drac1 Drac2Mtl triple mutant embryos, whereas Drac2 Mtl doublemutant embryos complete DC. On the basis of these re-sults, it is likely that the DC-disrupting expression of Drac1N17 affects Drac2 and Mtl function to a degree,in addition to impairing Drac1 activity. The LE cyto-skeleton has been examined in Drac1 Drac2 Mtl triplemutant embryos. There is little F-actin at the LE, andboth lopodia and lamellipodia are severely reduced innumber (Hakeda-Suzuki et al., 2002). The JNK pathwayhas not yet been examined in the Drosophila Rac mu-tants.

Dcdc42

Dcdc42 function during embryogenesis has been studiedusing loss-of-function Dcdc42 alleles and transgenes. Itis not possible to produce embryos completely devoid of both maternal and zygotic Dcdc42 function due to arequirement for Dcdc42 in oogenesis (Genova et al.,2000). However, females bearing heteroallelic combi-nations of weak and strong Dcdc42 alleles can be usedto produce Dcdc42 mutant embryos severely depleted inDcdc42 function that show DC defects (Genova et al.,2000). The DC defects in Dcdc42-decient embryos havenot yet been examined in detail with regard to cell shapeand the status of the actin cytoskeleton. Expression of adominant negative version of Dcdc42, Dcdc42N17,leads to DC defects that are phenotypically distinct fromthose caused by Drac1N17 expression (Riesgo-Escovaret al., 1996; Harden et al., 1999). Dcdc42N17-expressing

embryos exhibit partial losses of the LE cytoskeleton,suggesting that Dcdc42 may make a contribution to theestablishment of the LE cytoskeleton albeit less substan-tial than that of Drac1. In cultured cells, Cdc42 partici-pates in the formation of lopodia (Kozma et al., 1995;Nobes and Hall, 1995), and this appears to be the caseduring DC as expression of dominant negative forms of Dcdc42 causes loss of lopodia at the LE (Jacinto et al.,2000). Induction of lopodia by Cdc42 in mammaliancells involves the interaction of the Cdc42-binding pro-tein N-WASP with the Arp2/3 complex and prolin (re-viewed in Takenawa and Miki, 2001). Surprisingly, em-bryos completely decient for the only known WASPprotein in Drosophil a, Wsp, secrete normal cuticles, indi-cating that there can be no signicant defects in DC(Ben-Yaacov et al., 2001). Wsp may not operate inDcdc42 signaling, as Dcdc42 binding is not essential forWsp function (Tal et al., 2002). Nevertheless, Dcdc42may act through prolin in driving lopodia formationduring DC, as chic mutants lack lopodia at the LE(Jasper et al., 2001).

Complex phenotypes generated by Dcdc42 transgenessuggest that this small GTPase has multiple roles in DC.In addition to the losses of LE cytoskeleton and lo-podia described above, Dcdc42N17 expression can also


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result in a bunched LE phenotype similar to that seenwith mutants in the Dpp/TGF- b pathway required forDC (Harden et al., 1999; Ricos et al., 1999) (Fig. 2F).As discussed below, Dcdc42 appears to function in Dpp/TGF- b signaling, possibly via downregulation of the LEcytoskeleton. Other data support the idea that Dcdc42function can contribute to both establishment anddownregulation of the LE cytoskeleton during DC(Harden et al., 1999). In co-expression experiments,there is a temporal shift from Dcdc42N17 expressionworsening Drac1N17-induced DC defects toDcdc42N17 expression partially rescuing Drac1N17-in-duced DC defects. This result suggests that Dcdc42 mayinitially help Drac1 establish the LE cytoskeleton butlater contributes to its downregulation. In support of adownregulatory role, about 10% of embryos expressingconstitutively active Dcdc42 (Dcdc42V12) show losses of LE F-actin and myosin.

Dcdc42 transgene expression affects the distributionof the DPAK kinase at the LE (Harden et al., 1999).Dcdc42N17 expression causes loss of DPAK from theLE, whereas Dcdc42V12 upregulates LE DPAK levels tohigher than wild-type. However, the 10 % of Dcdc42V12-expressing embryos that lose the LE cytoskeleton alsolose LE DPAK.

Similar to Drac1, expression of constitutively activeDcdc42 causes ectopic activation of the JNK cascade(Glise and Noselli, 1997), but other results suggest thatDcdc42 does not play a major role in activating the JNKcascade during DC. Dcdc42 mutant embryos maintaindpp expression at the LE (Genova et al., 2000), as doDcdc42N17-expressing embryos which have also been

shown to have normal puc-lacZ expression (Raymond etal., 2001).

RhoA/Rho1

Loss-of-function mutations in the RhoA/Rho1 locus orexpression of a dominant negative RhoA/Rho1 transgenelead to defects in the dorsal cuticle of the embryo (Struttet al., 1997; Harden et al., 1999; Lu and Settleman, 1999;Magie et al., 1999). RhoA/Rho1 mutant embryos man-age to complete DC, but the closed dorsal surface is dis-organized (Magie et al., 1999). This irregular closure ap-pears to be due to uneven constriction of cells along theA-P axis at the LE, with some cells being excessivelyconstricted and others being splayed out (Magie et al.,1999). Embryos expressing a dominant negative versionof RhoA/Rho1 display a similar phenotype of unevenconstriction of cells at the LE, accompanied by disrup-tion of LE myosin (Harden et al., 1999). In RhoA/Rho1mutants, some of the excessively constricted cells formectopic contacts with their lateral neighbors, as if theneighboring cells are being perceived as though theywere the cells in the opposing LE that contact is nor-mally made with (Magie et al., 1999). RhoA/Rho1 is not

required for activation of the JNK cascade during DC,as RhoA/Rho1 mutant embryos show wild-type levels of dpp transcripts and puc-lacZ expression at the LE (Luand Settleman, 1999; Magie et al., 1999). Expression of dominant negative RhoA/Rho1 also does not disruptthe JNK cascade, as shown by dpp transcript distri-bution (Lu and Settleman, 1999).

RhoA/Rho1 may be required to regulate the contrac-tility of the LE cytoskeleton through effects on LE myo-sin. The RhoA/Rho1 locus interacts genetically with zipper, the gene encoding nonmuscle myosin-II heavy chain,indicating that RhoA/Rho1 regulates myosin functionduring Drosophila morphogenesis (Halsell et al., 2000).The Rho-associated kinases (ROKs), downstream effec-tors for Rho in mammalian cells, promote phosphoryla-tion of the regulatory light chain of myosin (MRLC)through both direct action on MRLC and indirectlythrough inactivation of myosin phosphatase by phos-phorylating its myosin-binding subunit (MBS) (reviewedin Bishop and Hall, 2000). Phosphorylation of MRLCstimulates the ATPase of myosin, promoting the assemblyand function of the actomyosin contractile apparatus (re-viewed in Bresnick, 1999). A recent study demonstratesthat similar regulation of MRLC occurs during DC (Mi-zuno et al., 2002). Embryos lacking Drosophila MBS(DMBS) exhibit DC defects, indicating that regulation of the phosphorylation state of MRLC is required for cor-rect DC. In DMBS mutant embryos, the level of phos-phorylated MRLC is substantially elevated at the LE andcell shape change in the epidermis is aberrant. Overex-pressionof a Drosophila ROK, Drok (Mizuno et al., 1999;Winter et al., 2001), causes similar DC defects (Mizuno

et al., 2002). Mizuno et al. (2002) have tested to see if thelethality caused by loss of DMBS or excessive Drok activ-ity is dueto hyperactivation of myosin. Reduction of myo-sin function through heterozygosity for a zip allele cansubstantially suppress the lethality of DMBS mutant em-bryos or embryos overexpressing Drok.

As described above, another potential route forRhoA/Rho1 regulation of the cytoskeleton is the Rho/Rac effector Pkn (Lu and Settleman, 1999).

Ras1

Expression of Ras1 transgenes causes DC phenotypeswith some similarities to the effects of Dcdc42 trans-genes (Harden et al., 1999). Dominant negative Ras1 in-duces partial losses of the LE cytoskeleton but DPAKlevels are unaffected. Expression of constitutively activeRas1 causes greater than normal accumulations of DPAK at the LE. Given that the Rho family proteinsare known to function downstream of Ras in the trans-formation of cultured cells (reviewed in Zohn et al.,1998), it is possible that Ras1 signals thorugh Rho fam-ily proteins such as Dcdc42 during DC, but this theoryhas not yet been addressed.


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A TGF-b signaling pathway is required for DC andacts downstream of the JNK cascade

Mutations in the genes thick veins (tkv ) and punt ( put),which encode type-I and type-II TGF- b receptors, re-spectively, cause DC defects, indicating that TGF- b sig-naling is required for DC (Childs et al., 1993; Affolteret al., 1994; Brummel et al., 1994; Nellen et al., 1994;Penton et al., 1994; Letsou et al., 1995; Ruberte et al.,1995). As discussed above, the JNK cascade drives ex-pression of Dpp, a TGF- b superfamily member, at theLE, and it is clear that a Dpp/TGF- b signaling cascadeis a major route of action for the JNK pathway duringDC. Overexpression of Dpp or expression of an acti-vated version of the Dpp receptor Tkv can substantiallyrescue the DC defects of embryos with impaired JNKsignaling (Hou et al., 1997; Riesgo-Escovar and Hafen,1997a; Sluss and Davis, 1997; Su et al., 1998; Chen etal., 2002).

TGF- b ligands such as Dpp signal through receptorcomplexes containing type I and type II receptors, andit is likely that Dpp secreted by the LE cells activatessignaling in epidermal cells via the Tkv and Put recep-tors during DC (Letsou et al., 1995; Ruberte et al.,1995). A requirement for Dpp in DC cannot be directlyaddressed due to a need for Dpp earlier in embryonicdevelopment (reviewed in Morisato and Anderson,1995). The TGF- b superfamily member Glass bottomboat (Gbb, also called 60A) appears to contribute tosignaling through the Tkv/Put receptor complex duringDC, possibly as a heterodimer with Dpp (Chen et al.,

1998). Embryos bearing a hypomorphic tkv allele, tkv6

are viable, but tkv 6 gbb double mutant embryos showDC defects. The complete details of signaling occurringdownstream of the activated Tkv/Put receptor complexduring DC remain to be determined, but it involves atranscriptional response. The DC gene schnurri (shn)(Nsslein-Volhard et al., 1984) has been shown genetic-ally to function downstream of dpp and encodes a zincnger protein (Arora et al., 1995; Grieder et al., 1995;Staehling-Hampton et al., 1995). Work in various sys-tems has revealed that the Smad family proteins func-tion downstream of receptors for ligands belonging tothe TGF- b superfamily. In Drosophila , the Tkv receptorphosphorylates Mothers against Dpp (Mad), a Droso- phila Smad, which translocates into the nucleus with theSmad protein Medea (Med) (reviewed in Affolter et al.,2001). Embryos decient in Mad show DC defects, andMad has been shown to interact with Shn in mediatingtranscriptional responses to Dpp signaling (Hudson etal., 1998; Dai et al., 2000; Udagawa et al., 2000). Re-cently, it has been shown that the LE expression of thezip gene during DC is lost in tkv mutant embryos (Arqu-ier et al., 2001). Thus, production of myosin in the LEcells may be dependent on Dpp/TGF- b signaling to thenucleus. Despite the lack of myosin synthesis at the LE

in tkv mutant embryos, the Dpp/TGF- b pathway is notrequired for the assembly of the LE cytoskeleton orelongation of the LE cells (Riesgo-Escovar and Hafen,1997a; Ricos et al., 1999). This seemingly paradoxicalresult is consistent with the analysis of zip mutant em-bryos (Young et al., 1993). Maternally produced myosinlocalizes to the dorsal end of LE cells during DC in zipmutant embryos and is sufcient for a contractile appar-atus that can achieve a fair degree of cell elongation andsignicant progression of DC. The myosin expressedfrom zip in the LE cells during DC appears to be re-quired in the late stages of DC when the maternal sup-ply is exhausted (Young et al., 1993).

In tkv, put , and shn mutant embryos, cells ventral tothe LE cells in the epidermis fail to elongate and thedorsal hole does not close (Riesgo-Escovar and Hafen,1997a; Ricos et al., 1999). There are a number of plaus-ible explanations for this phenotype. One interpretationis that cell shape change in the lateral epidermis is anactive, Dpp/TGF- b-dependent process and not simply apassive response to elongation of the LE cells (Riesgo-Escovar and Hafen, 1997a). In tkv and put mutant em-bryos, expression of DFos in the lateral epidermis is re-duced but its expression at the LE is maintained (Ries-go-Escovar and Hafen, 1997b). The expression of DFosthroughout the lateral epidermis may be required forDC, as restricted induction of a DFos transgene in thedorsalmost epidermal cells cannot rescue DC defects inembryos mutant for kay, the gene encoding DFos (Ries-go-Escovar and Hafen, 1997b). DFos may be part of aDpp/TGF- b pathway driving cell shape change in thelateral epidermis (Riesgo-Escovar and Hafen, 1997b).

Another reason why cell elongation does not occur inthe lateral epidermis of embryos defective in the Dpp/TGF- b pathway could be misdirected elongation of LEcells leading to a failure of passive stretching of moreventrally located cells. In tkv, put , and shn mutant em-bryos, the LE cells initially undergo an elongation anddorsalward migration, but as DC proceeds, these cellsget pulled together into a series of bunches, each of which contains several adjacent segments squeezed to-gether at their dorsal end (Ricos et al., 1999). Thisbunching may halt further dorsalward movement of theepidermis and disrupt the forces promoting elongationof the lateral epidermal cells. The bunching phenotypecould be due to loss of a TGF- b-dependent regulatorymechanism(s) required for uniform DC. Candidate cellsmediating such regulation are the cells anking the seg-mental grooves at the LE. These segment border cellsdiffer from the other LE cells in a number of ways, mostimportantly being that they show high levels of tkv tran-scripts (Affolter et al., 1994) and may be major pointsof TGF- b signaling during DC. The segment bordercells also show upregulation of DPAK, a potential effec-tor for the small GTPases Drac1 and Dcdc42, and donot constrict as extensively in the A-P direction as otherLE cells (Harden et al., 1996) (Fig. 2D). The less con-


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stricted morphology of the segment border cells may berequired for uniform closure of the dorsal hole and maybe caused by transient losses of the LE cytoskeleton oc-curring in these cells during DC (Harden et al., 1996).If Dpp/TGF- b signaling is required for the downregula-tion of the cytoskeleton in the border cells, its loss wouldrelease these brakes on LE contraction, causing ex-cessive contraction and the bunched phenotype (Ricos etal., 1999). As mentioned above, Dcdc42N17-expressingembryos exhibit a similar bunched phenotype. Expres-sion of constitutively active Dcdc42 can partially rescuethe DC defects of tkv mutant embryos, indicating thatDcdc42 is a component of Dpp/TGF- b signaling (Ricoset al., 1999). It is possible that Dcdc42 contributes todownregulation of the cytoskeleton in the segment bor-der cells through DPAK (Ricos et al., 1999).

Another possible explanation for the bunched pheno-type, not necessarily mutually exclusive from those de-scribed above, is that instead of migrating dorsally andcontacting LE cells from the opposite epidermal ank,the LE cells in Dpp/TGF- b pathway mutants migrate inan A-P direction and establish ectopic adhesions withtheir neighbors in the sameepidermal ank (these ectopicadhesions are also seen in RhoA/Rho1 mutant embryos,see above). Such aberrant migration could be due to dis-ruption of guidance cues used by the lopodia of LE cells.In live embryos, the LE lopodia are seen to wave aboutsampling for the correct binding partners and may beresponding to guidancecues in a manner analogous to thelopodia of axonal growth cones navigating via attract-ants and repellents (Jacinto et al., 2000; 2001). In the pro-cess of ventral enclosure in C. elegans , which shows paral-

lels to DC, the axonal repellent Semaphorin-2A is re-quired to prevent ectopic contacts being made betweenmigrating hypodermal cells (Roy et al., 2000). Similarguidancecues may be used to ensure correct cell matchingduring DC, and could be supplied by segment bordercells.For example, secretion of a repellent bysegment bor-der cells would prevent the migrating LE cells from stray-ing too far in the A-P direction.

A recent study has led to the positioning of anothercomponent of Dpp/TGF- b signaling in DC. The zinc-nger transcription factor Pannier, which lies down-stream of Dpp in early development, acts as an upstreamregulator of dpp expression during DC (Herranz andMorata, 2001). In pnr mutant embryos the LE expres-sion of dpp is lost, and ectopic Pnr activity results inectopic dpp expression. Pannier is not required for acti-vation of the JNK cascade, however, as JNK-dependent puc-lacZ expression at the LE is maintained in pnr mu-tant embryos. The results indicate that expression of dppat the LE requires independent inputs from the JNKpathway and Pnr (Herranz and Morata, 2001). The DCdefects of pannier mutant embryos are similar to thoseof mutants in Dpp/TGF- b signaling and include bunch-ing of the segments (Jrgens et al., 1984; Heitzler et al.,1996; Ricos et al., 1999).

The Wingless pathway is required for LE dpp expression and cell shape change during DC

Signaling by the Wnt/Wingless (Wg) family of secretedligands determines cell fates in insects and vertebrates(reviewed in Wodarz and Nusse, 1998). In the canonicalWg pathway operating in Drosophila , the reception of Wg signal by the Frizzled 2 (Fz2) receptor leads to theaccumulation of cytoplasmic Arm. In the absence of Wgsignal, cytoplasmic Arm is targeted for degradation bya complex that includes the Zeste white-3 (Zw3) kinase.During Wg signaling, the activation of the Dishevelled(Dsh) protein downstream of Fz2 leads to inactivationof Zw3 and subsequent stabilization of Arm. Arm thenpromotes transcription of Wg-responsive genes by actingas a co-activator for the transcription factor dTCF.

A recent study demonstrates that the Wg pathway isan essential player in DC (McEwen et al., 2000). Em-bryos decient in the Wg pathway components Wg,Dsh, or Arm, show DC defects in cuticle preparationsand reductions in dpp and puc-lacZ expression at theLE. Expression of an N-terminally truncated form of dTCF, dTCF DN, which acts as a constitutive repressorof Wg target genes, also causes DC defects and reducedLE dpp expression. As a further indication that Wg sig-naling drives dpp expression, the Wg pathway was ec-topically activated by various means leading to an ex-pansion of dpp expression beyond the LE. Cell shapechange during DC has been evaluated in wg and armmutant embryos. In wg mutant embryos, the LE cellsbecome elongated in the AP axis, but most embryos

manage to close up in an distorted manner. In arm mu-tant embryos, there is elongation of cells in the D-V axis,but it is irregular, and the nal degree of closure tendsto be less than that seen in wg mutants. The Wg pathwaycomponent that has been studied in the greatest detailwith regard to DC is Arm. The DC defects caused byloss of Arm can be suppressed by stimulation of theJNK pathway (via a puc allele or expression of dTAK1)or by activation of Dpp signaling. This suggests that amajor role for the Wg pathway in DC is expression of dpp at the LE. Wg pathway-driven expression of dppappears to be JNK-dependent as constitutive activationof the Wg pathway cannot promote dpp expression inkay mutant embryos lacking DFos. Thus, Wg signalingmust collaborate with JNK signaling at some point inDC and McEwen et al. (2000) have proposed a numberof models to explain how this may occur. The Wg path-way may feed directly into activation of the JNK cas-cade at the level of Dsh. Work on epithelial planar po-larity in Drosophila has indicated that Dsh can activatethe JNK cascade through Rho family small GTPases(reviewed in Boutros and Mlodzik, 1999). In parallel tothis activation of JNK, Dsh may also trigger the rest of the canonical Wg pathway during DC, as indicated bythe requirement for Arm. Another possibility considered


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by McEwen et al. is that the Wg and JNK pathwaysconverge at the level of dTCF, which, in addition tobeing a positive effector for the Wg pathway, can actwith Groucho as a repressor of Wg signaling. In the ab-sence of Wg signal, dTCF could act as a repressor of dppexpression. Activation of the Wg pathway would thenfunction as a permissive signal, displacing Groucho withArm and releasing this repression.

As discussed below, a likely source of signals promot-ing the LE cell fate is the amnioserosa. Expression of Wg in the amnioserosa has not been reported, and theDC defects seen in wg mutant embryos are not as severeas those seen with embryos decient in other DC com-ponents. Wg may not be a key activator of DC but ap-pears to function as an important regulator of dpp ex-pression at the LE.

It should be noted here that experiments using Armto study the contribution of Wg signaling to DC mustbe interpreted with caution. Arm may contribute to DCin a Wg-independent fashion through it role in adherens junctions (McEwen et al., 2000), as detailed below.

Additional negative regulation of the JNKcascade during DC

Anterior open/Yan

In addition to Puc, several other proteins function as anegative regulators of the JNK cascade during DC. TheETS-domain protein Anterior open (Aop)/Yan is a gen-eral inhibitor of differentiation whose activity is down-

regulated in response to phosphorylation by the MAPKencoded by the rolled gene (Rebay and Rubin, 1995).Aop/Yan is also phosphorylated by DJNK/Bsk, and aopmutant embryos exhibit DC defects (Riesgo-Escovarand Hafen, 1997a). aop mutant embryos show ectopicdpp expression, whereas expression of a constitutivelyactive version of Aop/Yan that cannot be phosphoryl-ated by MAPK causes a reduction in dpp levels at theLE. A model has been proposed in which, in the absenceof JNK signaling, Aop/Yan represses the transcriptionof DJun-responsive genes (Riesgo-Escovar and Hafen,1997a). When the JNK cascade is activated, Aop/Yanbecomes phosphorylated and inactivated, releasing therepressed genes for DJun-dependent transcription.

DRal

Expression of a constitutively active version of theDrosophila Ral small GTPase, DRal, causes dorsal holesin the embryonic cuticle (Sawamoto et al., 1999). Ral isof interest with regard to Ras signaling, as the GEFsthat activate Ral are direct targets of Ras (reviewed inWolthuis and Bos, 1999). The DC phenotype caused byconstitutively active DRal expression has not been

studied in detail but might reect a disruption of theJNK cascade. DRal appears to be a negative regulatorof the JNK pathway as hair and bristle defects causedby dominant negative DRal expression can be sup-pressed by hep and bsk mutations, and constitutively ac-tive DRal inhibits the phosphorylation of JNK in cul-tured S2 cells (Sawamoto et al., 1999).

Notch

Embryos completely devoid of the Notch (N) receptor,an important mediator of cell fate decisions during de-velopment, exhibit holes in the dorsal cuticle, and afunction for N in DC has been characterized (Zecchiniet al., 1999). In N mutant embryos, dpp levels at the LEare transiently higher than in wild-type embryos, andthere is ectopic expression of dpp and puc-lacZ in theepidermis. This result suggests that the JNK pathway isoveractive in N mutant embryos, and indeed, extractsfrom these embryos have a greater ability to phosphoryl-ate JNK and Jun than wild-type extracts. That N is anegative regulator of JNK signaling is further indicatedby the nding that loss of N can suppress the DC defectscaused by reductions in JNK pathway function. Thebest-characterized pathway of N signaling is during theprocess of lateral inhibition in which cells that haveadopted a particular fate suppress the adoption of thesame fate by surrounding cells (reviewed in Artavanis-Tsakonas et al., 1995). Interaction between N and theligand Delta results in cleavage of the N intracellulardomain which translocates to the nucleus, where it binds

the Suppressor of Hairless (Su(H)) protein and activatesthe expression of transcriptional repressors. This path-way is not utilized by N during DC, as neither Su(H)nor cleavage of N participate in negative regulation of the JNK pathway. How might N negatively regulate theJNK cascade? One possibility is that N antagonizes theactivation of JNK signaling by Wg, as N has beenshown to inhibit Wg signaling in a Su(H)-independentmanner (Brennan et al., 1999a; 1999b; Lawrence et al.,2001). Another route may be through interaction withthe product of the DC gene canoe (cno) (Jrgens et al.,1984) at the adherens junction (see below). N localiz-ation at the adherens junction has been described and N interacts genetically with cno (Fehon et al., 1991; Miya-moto et al., 1995).

Raw

Embryos decient in the novel protein Raw fail to un-dergo DC (Nsslein-Volhard et al., 1984; Blake et al.,1998; Byars et al., 1999), and this is likely due to a lackof cell elongation in epidermis (Blake et al., 1998). Thedisruption of cell shape change in the epidermis in rawmutants is probably due to an uneven accumulation of


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myosin along the LE (Blake et al., 1998), as the raw DCdefect can be partially rescued by a zip mutation reduc-ing LE myosin (Blake et al., 1999). The cause of theuneven LE myosin levels in raw mutant embryos may bemisregulation of the JNK cascade. In raw mutant em-bryos, dpp and puc-lacZ expression spreads beyond theLE cells to more ventrally located epidermal cells, sug-gesting an increase in the size of the JNK expressiondomain (Byars et al., 1999). The ectopic expression of dpp in raw mutant embryos is lost along with the normalLE expression of dpp when embryos are made doublymutant for a loss-of-function DJun allele. The Raw pro-tein constitutes another negative regulator of JNK sig-naling but contains no recognizable motifs, and it is notknown where in the cell it operates or what proteins itinteracts with.

Importance of cell-cell junctions in DC

A number of proteins associated with adherens junctionsand septate junctions are required for DC, indicatingthat these cell-cell adhesions play a role in this process.

Adherens junction proteins and DC

As noted above, triangular nodes of phosphotyrosine-rich proteins are present along the LE during DC. Phos-photyrosine staining in Drosophila epithelia is largely lo-calized to adherens junctions (Woods and Bryant, 1993;Woods et al., 1997). Drosophila E-cadherin (DE-cadher-

in), Drosophila a -catenin (D a -catenin), and Arm, threeproteins known to be components of adherens junctions,localize to the triangular nodes at the LE, suggestingthat these structures are specialized cell-cell junctionsparticipating in DC (Jacinto et al., 2000; Grevengoed etal., 2001). Adherens junctions are found at the apical/lateral interface in epithelial cells and are organizedaround what is known as the cadherin-catenin-complex(CCC) (reviewed in Tepass et al., 2001). In Drosophila ,the CCC consists of the transmembrane protein DE-cadherin, encoded by the shotgun (shg) gene, D a -caten-in, Arm ( Drosophila b-catenin), and Dp120ctn. The onlycomponent of the CCC for which a role in DC has beendirectly demonstrated is Arm. The interpretation of Arms role in DC is complicated by the fact that theprotein is found both at adherens junctions and in thecytoplasm (McEwen et al., 2000). As described above,cytoplasmic Arm is used in Wg signaling, a pathwayparticipating in DC (McEwen et al., 2000). The largerdorsal holes seen in arm mutant embryos compared towg mutant embryos suggest that Arm has a role in DCindependent of Wg signaling, and this is likely to be atthe adherens junction. shg mutant embryos lacking zy-gotic DE-cadherin have a normal dorsal epidermis (Te-pass et al., 1996; Uemura et al., 1996). It is not possible

to make embryos completely devoid of DE-cadherin dueto a requirement for DE-cadherin in oogenesis (Tepasset al., 1996; Uemura et al., 1996). However, germlineclones of weak shg alleles show loss of dorsal cuticle(Tepass et al., 1996), although it is not known if this isdue to a DC defect. Mutations in the genes encodingD a -catenin and Dp120ctn have not yet been reported.In addition to the CCC, a variety of other proteins arefound to localize to adherens junctions, and several of these are required for DC. Two such molecules are thePDZ domain proteins Cno and Polychaetoid (Pyd)/ZO-1. The PDZ domain is named for a motif found in theMAGUK (membrane-associated guanylate kinase) pro-teins PSD-95, Discs-large (Dlg, see below), and ZO-1(reviewed in Harris and Lim, 2001). Pyd/ZO-1 appearsto be the Drosophila ortholog of ZO-1, a mammaliantight junction protein (Takahisa et al., 1996; Wei andEllis, 2001), whereas Cno is homologous to the mam-malian adherens junction protein Afadin (reviewed byTepass et al., 2001). Cno and Pyd/ZO-1 interact genetic-ally and biochemically and may form a complex at theadherens junction that acts as an activator of the JNKcascade (Takahashi et al., 1998). Cno localizes to thetriangular junctions at the LE, and cno mutant embryosshow a failure of DC characterized by a lack of cellelongation in the epidermis. Loss of JNK signaling incno mutant embryos is indicated by loss of dpp and puc-lacZ expression at the LE. Genetic interaction studiesconrm that a major route of action for Cno is the JNKpathway. Overexpression of DJNK/Bsk can partially res-cue DC defects in cno mutant embryos, whereas reduc-tion in DJNK/Bsk or DJNKK/Hep levels leads to DC

failure in cno partial loss-of-function embryos that nor-mally show no DC defects. Cno has been proposed toact upstream of, or in parallel with, Drac1 in its regula-tion of the JNK cascade as Drac1V12 is capable of driv-ing puc-lacZ expression at the LE in cno mutant em-bryos. Pyd/ZO-1 appears to collaborate with Cno in itsregulation of the JNK cascade during DC. Embryoshomozygous for the weak hypomorphic cno allelecnomis1 develop normally, as do embryos homozygousfor the hypomorphic pyd allele pyd tam1 , but embryosdoubly homozygous for cnomis1 and pyd tam1 show DCdefects. Pyd/ZO-1 localizes to the triangular junctions atthe LE and binds to Cno in in vitro binding assays andusing the yeast two-hybrid system. How Cno and Pyd/ZO-1 contribute to activation of the JNK cascade re-mains unknown, but a number of interesting possibilitiescan be considered. One is that they contribute to theassembly of a localized receptor complex that picks upa signal(s) promoting JNK activation (Takahashi et al.,1998; Noselli and Agne `s, 1999). There are precedents forPDZ domain proteins contributing to the formation of signaling complexes. For example, in C. elegans vulvaldevelopment the PDZ proteins LIN-2/LIN-7/LIN-10form a complex mediating basolateral membrane local-ization of the receptor tyrosine kinase LET-23 in vulval


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precursor cells (reviewed in Harris and Lim, 2001). Thisenables localized response to an EGF-like protein (LIN-3) secreted into the basal extracellular space by theneighboring anchor cell. By analogy, in the Drosophilaembryo receptors may be localized to the dorsal end of each LE cell at the interface with the amnioserosa. Asdescribed below, the amnioserosa is a likely source of signal(s) required for DC, and limiting receptor com-plexes to the dorsal end of the LE cells would allow alocalized response involving just the rst row of epider-mal cells. A membrane-anchored signal on the surfaceof an amnioserosa cell could interact with a receptorfound at the dorsal end of a neighboring LE cell, similarto the interaction of the Bride of Sevenless protein onthe surface of the R8 cell with the Sevenless receptortyrosine kinase on the neighboring R7 cell during Droso- phila photoreceptor specication (reviewed in Raabe,2000).

Cno may also regulate JNK signaling by acting onproteins regulating the cascade. Cno interacts geneticallywith the DC participants Arm, Mys, Ras1, and N, al-though these interactions have not been tested in thecontext of DC (Miyamoto et al., 1995; Matsuo et al.,1997). Furthermore, it has been shown that Cno bindsRas1, so there may be a direct link between Ras1 sig-naling and Cno function (Kuriyama et al., 1996; Matsuoet al., 1997). As discussed in the section on Wg sig-naling, Arm makes a positive contribution to activationof the JNK pathway (McEwen et al., 2000). N, on theother hand, is a negative regulator of the JNK pathway,whereas Mys and Ras1 have been implicated in DC butnot directly assessed for a role in the cascade (Brown,

1994; Harden et al., 1999; Zecchini et al., 1999). It willbe of interest to see if Cno interacts genetically with anyof these proteins during DC.

a -catenin in the adherens junction binds to F-actinand various proteins interacting with the actin cytoskel-eton, suggesting that it organizes a multiprotein complexregulating the actin cytoskeleton (reviewed in Vasioukh-in and Fuchs, 2001). The triangular junctions at the LEmay participate in the assembly of the actomyosin con-tractile apparatus, and Cno and Pyd/ZO-1 could have arole in this as the mammalian orthologs of these proteinsassociate with the actin cytoskeleton (Takahashi et al.,1998).

A further indication that adherens junctions contrib-ute to assembly of the LE cytoskeleton comes from astudy of the Drosophila Abelson nonreceptor tyrosinekinase, Abl (Grevengoed et al., 2001), a protein enrichedat adherens junctions (Bennett and Hoffmann, 1992).Embryos completely devoid of Abl show defects in epi-thelial morphogenesis, including DC. There is a lack of coordinated cell shape change in the epidermis of abl mutant embryos, and this is accompanied by an unevendistribution of F-actin along the LE, with some areasshowing excessive F-actin and other areas being de-cient. Filopodial extensions are maintained at the LE

in Abl mutant embryos and actually appear to be morenumerous than wild-type in late-stage mutant embryos.DC is slowed in abl mutant embryos, and the LE tendsto fold under the lateral epidermis. As the actin regu-lator Ena is a known target of Abl (reviewed in Lanierand Gertler, 2000), Ena was evaluated in the context of Abl function during DC. Ena localizes to adherens junc-tions, including the triangular junctions at the LE, andits distribution in abl mutant embryos is disrupted simi-larly to F-actin. Thus, areas where F-actin is lost tendto show reduced Ena staining, whereas areas where F-actin is increased show elevated Ena staining. An inter-action between Abl and Ena during DC is further sup-ported by the nding that heterozygosity for an ena al-lele suppresses the embryonic lethality of abl mutant em-bryos. The presence of Ena at adherens junctionsprompted an investigation of the interactions betweenAbl and Ena and adherens junction components. Muta-tions in ena increase the severity of DC defects of armmutant embryos, whereas shg alleles enhance the abl phenotype. Furthermore, in abl mutant embryos, thelevels of Arm and a catenin are reduced at adherens junctions. On the basis of these data, Grevengoed et al.(2001) speculate that Abl may translate extracellular sig-nals into regulation of the actin cytoskeleton through itsaction on Ena at adherens junctions. Its is tempting tospeculate that Abl may act through Drac1 in its regula-tion of the LE cytoskeleton as Trio, a guanine nucleotideexchange factor that can activate Rac proteins, interactsgenetically with Abl during CNS development (reviewedin Lanier and Gertler, 2000).

Involvement of Septate Junction Proteins in DC

The insect septate junction, which serves some of thesame roles as the vertebrate tight junction, is found justbasal to the adherens junction in mature epithelia (re-viewed in Tepass et al., 2001). Three proteins requiredfor the integrity of the septate junction, Coracle (Cor),Neurexin IV (Nrx), and Discs-large (Dlg), are essentialfor DC. Coracle is a member of the Protein 4.1 super-family of proteins that is localized to the cytoplasmicface of the septate junction (Fehon et al., 1994). Em-bryos lacking cor show disruption of the septate junc-tion, but apical-basal polarity and epithelial integrity isunaffected (Lamb et al., 1998). Nrx is a transmembrane,septate junction protein belonging to a family of neur-onal receptors, and embryos lacking Nrx show septate junction defects similar to those seen in cor mutants(Baumgartner et al., 1996). The cytoplasmic domain of Nrx is homologous to glycophorin C, a binding partnerfor Protein 4.1 in erythrocytes, and co-immunoprecipita-tion and in vitro binding studies indicate that Nrx inter-acts directly with Cor (Baumgartner et al., 1996; Wardet al., 1998). Another indication of the close relationshipbetween Cor and Nrx is that they are dependent on each


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other for their correct localization (Baumgartner et al.,1996; Ward et al., 1998). The rst septa of the pleatedseptate junctions in ectodermally-derived epithelia ap-pear in the embryo at stage 14, after DC commences,and additional septa are added to junctions throughoutthe rest of embryogenesis (Tepass and Hartenstein,1994). If the DC defects in cor and nrx mutant embryosare due to disruption of septate junction formation, theyare unlikely to reect a failure to initiate DC but rathera later event. The cuticles of nrx and cor mutant em-bryos have relatively small dorsal holes and do not ex-hibit the wide-open dorsal surface seen with loss of someother DC participants (Fehon et al., 1994; Baumgartneret al., 1996; Lamb et al., 1998; Ward et al., 1998). Ananalysis of embryonic morphology during DC has notbeen reported for nrx and cor mutants, but the cuticlephenotypes suggest that a considerable amount of clo-sure does take place. Cor antibody staining of wild-typeembryos indicates that the protein is not present alongthe dorsal side of the LE cells (Fehon et al., 1994) or inthe amnioserosa during DC (the amnioserosa does notcontain septate junctions, Tepass and Hartenstein,1994). The erythrocyte Protein 4.1 links transmembraneproteins with the spectrin/actin cytoskeleton, but thisfunction may not be conserved in Cor (Fehon et al.,1994). The spectrin/actin binding domain of Protein 4.1is not conserved in Cor, and Cor does not co-localizesubstantially with either F-actin or spectrin along theapical-basal axis of the cell. Furthermore, clones of cormutant cells in imaginal tissues show a normal distri-bution of F-actin (Lamb et al., 1998). The present dataon Cor and Nrx suggest that the septate junction does

not function in the organization of the cytoskeleton atthe LE during DC. Further support for this comes fromthe observation that the homophilic adhesion moleculeFasciclin III (Fas III) (Snow et al., 1989), a proteinknown to associate with septate junctions (Woods et al.,1997), is not present at the dorsal end of LE cells duringDC but is present on their other surfaces (Martinez-Ari-as, 1993). A potential role for the septate junction couldbe in establishing cell-cell adhesion between LE cellsfrom opposite sides of the embryo when they meet atthe dorsal midline (see below), and the DC defects of corand nrx mutants may be due to loss of such adhesion.

The third septate junction protein required for DC isDlg, which has a more extensive role in this process thanCor or Nrx. Loss of Dlg can lead to a complete failure of epidermal migration during DC, as indicated by cuticlepreparations and scanning electron microgaphs of em-bryos (Perrimon, 1988). Dlg is a MAGUK tumor sup-pressor that, similar to Cor and Nrx, is required for sep-tate junction formation (Woodsand Bryant, 1991; Woodset al., 1996). However, unlike Cor, lack of Dlg also leadsto disruption of apical-basal polarity in epithelia (Woodset al., 1996). Dlg functions together with two other pro-teins, Lethal giant larvae (Lgl) andScribble (Scrib), in theregulation of epithelial polarity (Bilder et al., 2000). Lgl is

a tumor suppressor protein required for DC (Manfruelliet al., 1996; Arquier et al., 2001).Embryos decient in Lglshow a strong DC phenotype characterized by a lack of cell shape change in the epidermis and a failure of epider-mal migration. Scrib is a PDZ domain tumor suppressorprotein, the complete lack of which causes extensive dis-organization of embryonic epithelia (Bilder and Perri-mon, 2000). This phenotype precludes a direct evaluationof a role for Scrib in DC, however, a genetic interactionbetween Scrib and Lgl suggests that Scrib function is re-quired. Embryos containing maternal Scrib but lackingzygotic Scrib hatch into larvae but when made hetero-zygous for an allele of dlg die as embryos with DC defects(Bilder et al., 2000). Scrib and Dlg co-localize throughoutdevelopment, and following gastrulation remain at theapical margin of the lateral membrane (ALM) at a posi-tion that becomes the septate junction (Woods and Bry-ant, 1991; Woods et al., 1996; Bilder et al., 2000; Bilderand Perrimon, 2000). Lgl shows a more dispersed distri-bution and is not restricted to the plasma membrane butdoes overlap with Scrib and Dlg at the ALM (Strand etal., 1994b; Bilder et al., 2000). Scrib, Dlg, and Lgl are mu-tually dependent for their localization (Bilder et al., 2000)and likely function as an interacting group of proteinsthat has been called the Lgl complex (Tepass et al., 2001).In the early embryo, the Lgl complex regulates epithelialpolarity, and among the effects of disrupting the complexare defects in adherens junction formation and mislocal-ization of the adherens junction protein Arm (Bilder etal., 2000; Bilder and Perrimon, 2000). Later in embryo-genesis, the Lgl complex may contribute to septate junc-tion fomation by acting as a scaffold upon which proteins

such as Cor and Nrx can assemble (Tepass et al., 2001).The DC defects associated with impairment of the Lglcomplex are likely partly due to disruption of septate junction formation. However, these DC defects are moresevere than those resulting from loss of Cor or Nrx, andthere are other requirements for the Lgl complex in DC.Given the participation of adherens junctions proteinssuch as Arm in DC, the disruption of adherens junctionsand Arm localization through loss of the Lgl complex isalmost certainly going to affect DC.

The Lgl protein further regulates DC through its ef-fects on LE myosin and Dpp/TGF- b signaling (Arquieret al., 2001). Lgl associates with the zip product non-muscle myosin-II heavy chain (Strand et al., 1994a), andthe lgl and zip genes interact genetically in the determi-nation of neuroblast polarity (Ohshiro et al., 2000; Penget al., 2000). The nature of this genetic interaction indi-cates that Lgl is a negative regulator of myosin function.Consistent with this, loss-of-function lgl alleles suppressthe DC phenotype in zip mutant embryos. Lgl also in-directly makes a positive contribution to myosin func-tion during DC as it is required for the transcription of zip in the LE cells. There is evidence that Lgl is requiredfor emission of Dpp by the various cells producing thisligand (Arquier et al., 2001). The lack of zip transcrip-


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tion at the LE in lgl mutant embryos may be due to lossof activation of the Tkv/Put pathway, which is requiredfor the LE expression of zip (see section on Dpp/TGF-b signaling). The positive contribution of Lgl to zygoticzip transcription would be irrelevant in a zip mutant em-bryo, and the suppression of the zip mutant DC pheno-type by loss of Lgl may be due to a reduced negativeregulation of the function of maternally suppliedmyosin.

Signaling from the amnioserosa establishesLE cell identity

We have seen that theLE cells duringDC exhibit complexsignaling driving cytoskeletal organization, cell shapechanges, and gene expression. It is clear that the LE cellshave a distinct identity from other epidermal cells and area crucial component of DC. A key issue in understandingDC, therefore, is how the LE cell fate is specied, and sev-eral recent studies demonstrate that signaling from theneighboring amnioserosa is involved. Stronach and Perri-mon (2001) genetically altered the position of the inter-face between the amnioserosa and dorsal epidermis usingmutations affecting DV patterning and found that theLE fate always arose as a single row of cells at the inter-face. They used two criteria to dene LE fate: puc-lacZ expression, which indicates the presence of JNK sig-naling, and asymmetrical staining for Fas III. The speci-cation of LE cells at the physical juxtaposition of am-nioserosa and dorsal epidermis suggests that there is in-ductive signaling between the two tissues during DC.

The U-shaped group of genes, consisting of u-shaped (ush), hindsight (hnt ), serpent (srp ), and tail-up (tup ), isrequired for amnioserosa maintenance, germband re-traction, and DC (Strecker et al. 1995; Frank and Rush-low, 1996; Reed et al., 2001; Stronach and Perrimon,2001). The DC defects in embryos mutant for hnt , a geneencoding a Zinc-nger protein, can be suppressed by re-ducing the dose of DJNK with a bsk allele, indicatingthat JNK signaling is upregulated by loss of Hnt func-tion (Reed et al., 2001). In wild-type embryos, there isJNK activity in the amnioserosa prior to DC, but bythe commencement of DC, this has been downregulated(Reed et al., 2001). In both hnt mutant embryos and pucmutant embryos, JNK signaling persists in the am-nioserosa beyond the onset of DC (Reed et al., 2001;Stronach and Perrimon, 2001) and Reed et al. (2001)have shown that this persistence of amnioserosa JNKsignaling underlies defects at the LE. In hnt mutant em-bryos, phosphotyrosine staining in the triangular junc-tions at the LE is missing. The LE phosphotyrosine canbe restored in hnt mutant embryos by downregulatingJNK signaling in the amnioserosa through expression of transgenes of either puc or dominant-negative DJNK/ Bsk . Phosphotyrosine accumulation at the LE occurs in-dependently of JNK signaling at the LE, as the JNK

pathway is intact in the LE cells of hnt mutant embryos(Reed et al., 2001; Stronach and Perrimon, 2001).

The existing data indicate that there are at least twodistinct signaling events operating between the am-nioserosa and the dorsal epidermis contributing to theLE cell fate. One is required for JNK pathway activationin the LE cells and requires the juxtaposition of am-nioserosa with dorsal epidermis (Stronach and Perri-mon, 2001). A potential contributor to activation of thissignaling from the amnioserosa is ush , a gene encodinganother Zinc-nger protein, as ush mutant embryos lackdpp expression at the LE (Stronach and Perrimon, 2001)and exhibit dorsal holes in cuticle preparations (Streckeret al., 1995; Frank and Rushlow, 1996). A second signal,which requires Hnt- and Puc-dependent downregulationof the JNK cascade in the amnioserosa, is needed forthe accumulation of phosphotyrosine in the triangular junctions at the LE. Hnt function in amnioserosa cellsabutting the dorsal epidermis establishes a germband re-traction signal detected by the Drosophila insulin recep-tor (Inr) in the epidermis (Lamka and Lipshitz, 1999).It is possible that the Hnt-dependent signal from the am-nioserosa to dorsal epidermis required for DC is convey-ed in the same manner as that required for germbandretraction, i.e. through Inr. In addition to germband re-traction failure, DC defects have been reported in Inrdecient embryos (Fernandez et al., 1995). The lack of phosphotyrosine staining in hnt mutant embryos sug-gests that the specialized adherens junctions at the LEare disrupted. As discussed above, these structures haveroles in cytoskeletal organization and consistent withthis, hnt mutant embryos show disruption of F-actin at

the LE (Reed et al., 2001).In addition to signaling via protein cascades, the am-nioserosa may also provide mechanical signals thatcontribute to establishment of the LE during DC. Alter-ations in the tension applied to cultured epithelial cellscan promote reorganization of the actin cytoskeleton;wounding a tissue creates a similar mechanical cue thatmay contribute to initiation of the wound healing process(reviewed in Jacinto et al., 2001). It is conceivable thatgermband retraction of the Drosophila embryo prior toDC and/or alterations in adhesion between the am-nioserosa and the LE will change the forces being appliedto the LE cells and contribute to cytoskeletal reorganiza-tion. Consistent with this idea, ablationof LE cells,whichalters the tension applied to the epidermis, is rapidly fol-lowed by assembly of a contractile apparatus in moreventrally located epidermal cells (Kiehart et al., 2000).

Other players in DC

A number of DC participants have been identied whichdont as yet t into any of the various pathways orgroupings laid out above. These are discussed briey inthis section.


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Ribbon

Embryos bearing mutations in the ribbon (rib ) locusshow defects in the dorsal cuticle ranging from smallholes and puckers to a large dorsal hole (Nsslein-Vol-hard et al., 1984; Bradley and Andrew, 2001). rib mutantembryos show loss of LE myosin and a reduction in LEF-actin and epidermal cell elongation is impaired (Blakeet al., 1998). These embryos also show some ectopic dppexpression in the epidermis, but no attempt has beenmade to look for genetic interactions with JNK pathwaycomponents (Bradley and Andrew, 2001). Rib containsa BTB/POZ motif, a protein-protein interaction domainfound in a diverse collection of proteins (Bradley andAndrew, 2001). Some of the phenotypes of rib mutantembryos are suppressed by zip alleles, suggesting a rolefor Rib in cytoskeletal regulation (Blake et al., 1999).

N-myristoyltransferaseEmbryos lacking Drosophila N-myristoyltransferase(dNMT), an enzyme that catalyzes the addition of thefatty acid myristic acid to an N-terminal glycine residueof various proteins, have DC defects (Ntwasa et al.,2001). The DC defects in dNMT mutant embryos arelikely due to disrupted function of myristoyl proteins re-quired for DC (Ntwasa et al., 2001). Two such proteinsare Src42A and Src64, which are both normally myris-toylated (Tateno et al., 2000; Ntwasa et al., 2001).

Steroid hormones

Ecdysteroids regulate many developmental events inDrosophila , and recent work on the disembodied (dib)gene, which encodes a cytochrome P450 enzyme in-volved in embryonic ecdysteroid biosynthesis, implicatesthese hormones in regulation of DC (Cha vez et al.,2000). Many dib mutant embryos fail to complete DCand show reduced titers of the ecdysteroids ecdysoneand 20-hydroxyecdysone. Another indication for steroidhormone participation in DC comes from a study ad-dressing the effects of the glucocorticoid, dexametha-sone, on Drosophila embryonic development. Embryosexposed to dexamethasone show germband retractiondefects and holes in the dorsal cuticle, a phenotype simi-lar to mutants in the U-shaped genes (Strecker et al.,1995).

Drosophila Winged-helix nude

The Drosophila Winged-helix nude (Whn)-like transcrip-tion factor, Dwhn, is required for DC, and Dwhn mutantembryos show impaired elongation of epidermal cellsand some loss of dpp expression at the LE (Sugimura et

al., 2000). Ectopic overexpression of Dwhn, however,does not cause ectopic dpp expression, nor does Dwhnappear to interact genetically with hep, bsk , or tkv.

Sealing the dorsal hole

As the migrating epidermal anks meet up at the dorsalmidline during DC, new cell-cell adhesions must formto seal the dorsal surface. The lopodia extending fromeach LE interdigitate at the dorsal midline and appearto prime the formation of adherens junctions betweenthe two rows of LE cells (Jacinto et al., 2000; 2001).Newly formed septate junctions are also used to seal thedorsal hole. The septate junction proteins Fas III andCor, which are excluded from the dorsal end of each LEcell during DC, appear at this position after the op-posing epidermal

2002 - signaling pathways directing the movement and fusion of epithelial sheets - harden

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