Differentiation (2002) 70:181203

C Blackwell Verlag 2002

REVIEW

Nicholas Harden

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

Accepted in revised form: 30 April 2002

Abstract Wound healing in embryos and various developmental events in metazoans require the spreading and fusion of epithelial sheets. The complex signaling pathways regulating these processes are being pieced together through genetic, cell biological, and biochemical approaches. At present, dorsal closure of the Drosophila embryo is the best-characterized example of epithelial sheet movement. Dorsal closure involves migration of the lateral epidermal anks to close a hole in the dorsal epidermis occupied by an epithelium called the amnioserosa. Detailed genetic studies have revealed a network of interacting signaling molecules regulating this process. At the center of this network is a Jun N-terminal kinase cascade acting at the leading edge of the migrating epidermis that triggers signaling by the TGFb superfamily member Decapentaplegic and which interacts with the Wingless pathway. These signaling modules regulate the cytoskeletal reorganization and cell shape change necessary to drive dorsal closure. Activation of this network requires signals from the amnioserosa and input 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 the cytoskeleton. Many of the proteins regulating dorsal closure have been implicated in epithelial movement in other organisms, and dorsal closure has emerged as an ideal model system for the study of the migration and fusion of epithelial sheets. Key words Drosophila epithelial morphogenesis dorsal closure cytoskeleton signal transduction
N. Harden ( ) Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, V5A 1S6, Canada Tel: 1 604 291 5644, Fax: 1 604 291 5583 e-mail: nharden/sfu.ca
U. S. Copyright Clearance Center Code Statement:

Introduction
The spreading and fusion of epithelial sheets occurs in a diverse range of morphogenetic events (reviewed in Bard, 1990) such as epiboly, neural tube closure and embryonic wound healing in vertebrates, ventral enclosure in Caenorhabditis elegans, and dorsal closure (DC) of the Drosophila embryo. Drosophila DC represents the single most thoroughly characterized example of epithelial movement and fusion in metazoans. This is due to the ease of identifying mutants in the process and the advanced genetics and developmental biology available to study them. The picture that is emerging is control of DC by a complex network of signaling pathways mediating the transcriptional responses and cytoskeletal changes necessary to promote epithelial migration. Although other types of epithelial closure remain less well characterized, it is becoming apparent that these processes require many of the same proteins as DC, and what is learned about DC is widely applicable (reviewed in Jacinto et al., 2001). In particular, DC has been proposed 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 increase in the number of proteins known to participate in DC, but there remains much to be done in terms of sorting through all these participants and determining their precise roles and inter-relationships. In this review, I will summarize what is presently known about DC and the proteins driving it, and attempt to pull scattered pieces of data together into a model (see Fig. 3).

What is dorsal closure?

Following germband retraction of the Drosophila embryo, a hole is left in the dorsal surface of the epidermis that is occupied by the amnioserosa, an epithelium of

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Fig. 1 Dorsal closure (DC) of the Drosophila embryo. Panels show dorsal views of progressively older embryos stained with anti-phos-

photyrosine antibodies to show closure of the epidermis over the large, at cells of the amnioserosa.

large, at cells. DC involves a dorsalward migration of the lateral epidermis from both sides of the embryo with the epidermal anks meeting at the dorsal midline, completely covering the amnioserosa and sealing the embryo (Campos-Ortega and Hartenstein, 1985) (Fig. 1). Numerous mutants have been identied in which DC fails, leading to a dorsal hole in the larval cuticle (Jrgens et al., 1984; Nsslein-Volhard et al., 1984; Wieschaus et al., 1984; Perrimon et al., 1989). It is the genetic and molecular characterization of the genes disrupted in these mutants (the DC genes) that has lead to the emergence of DC as an excellent system for studying the regulation of epithelial morphogenesis (reviewed previously in Knust, 1996; 1997; Martn-Blanco 1997; Gob erdhan and Wilson, 1998; Noselli, 1998; Noselli and Agnes, 1999; Jacinto and Martin, 2001; Jacinto et al., ` 2001). During DC there is an accumulation of nonmuscle myosin-II (hereafter referred to as myosin) and F-actin at 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 been proposed to draw the hole shut in what is known as the purse-string model (Young et al., 1993). At the cellular level, the purse-string is composed of a polarized accumulation of F-actin and myosin at the dorsal end of each LE cell that acts as a contractile apparatus, driving constriction of the cell in an anterior-posterior (AP) direction. The purse-string model is based on the observation that, during DC, cells in the lateral epidermis shift from polygonal in shape to being elongated in the

DV direction. This elongation is rst seen in the LE cells and is then transmitted to more ventrally located cells. In the model, elongation of the LE cells is driven by their constriction in the AP direction, whereas elongation of the more ventrally located cells in the lateral epidermis is a passive response to events at the LE. The net result is stretching of the lateral epidermis up over the amnioserosa. The rst DC gene analyzed in detail was zipper (Nsslein-Volhard et al., 1984) which encodes Drosophila nonmuscle myosin-II heavy chain (Young et al., 1993). In zipper mutant embryos decient in myosin, cell shape change in the epidermis is aberrant and DC is not completed (Young et al., 1993). That the F-actin purse-string is driving cell constriction is supported by the observation that LE cells in which the actin cytoskeleton is decient tend to splay out in the AP direction (Harden et al., 1996; Grevengoed et al., 2001). In addition to F-actin and myosin, there is an accumulation of phosphotyrosine-rich structures at the LE during DC (Harden et al., 1996). These structures take the form of triangular nodes linking LE cells at their dorsal end (Fig. 2C). The level of phosphotyrosine in these structures is linked to the integrity of the LE cytoskeleton, as losses of the cytoskeleton at points along the LE are accompanied by losses of LE phosphotyrosine at the same positions (Harden et al., 1996). As discussed below, the triangular nodes appear to be adherens junctions contributing to organization of the LE cytoskeleton. It has become apparent that the contractile pursestring 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 boundary between the amnioserosa (top of gure) and the epidermis in phalloidin-stained embryo showing accumulation of F-actin along the LE, and extension of F-actin-rich lopodia from the amnioserosa and LE cells (arrows). (B) Accumulation of myosin along the LE. (C) Accumulation of phosphotyrosine along the LE in triangular nodes (arrows). (D) View of the dorsal midline after the

migrating epidermal anks have met up at the end of DC. Note that cells anking segment borders (marked with bars) are wider than their neighbors. (E) Phosphotyrosine-stained kay mutant embryo lacking DFos, showing loss of LE phosphotyrosine nodes and failure of cell elongation in the epidermis. (F) Embryo expressing dominant negative Dcdc42 showing bunched epidermis characterized by ectopic adhesions between LE cells (arrows).

epidermis. A detailed mechanistic study using laser ablation and green uorescent protein (GFP) imaging of live embryos conrms the contribution of a contractile purse-string but also indicates that DC is driven by other forces (Kiehart et al., 2000). Ablation of cells at a point along the LE causes neighboring cells to recoil, indicating that the LE is under tension, as predicted by the purse-string model. Embryos can close the epidermis even when the LE is repeatedly wounded, although the closed 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 cytoskeleton and cell shape change manage to close, albeit in a distorted manner (McEwen et al., 2000; Grevengoed et al., 2001). Cell shape change driven by the LE cytoskeleton is clearly not the sole force driving DC but appears to be required for the perfect, scar-free closure seen in wild-type embryos. What are the other forces driving DC? The potential contribution of the lateral epidermis has been addressed by laser ablation of cells ventral to the LE cells (Kiehart et al., 2000). This leads to an acceleration of the dor-

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salward movement of the LE, indicating that the lateral epidermis does not contribute to DC by pushing the LE forward, rather, there is tension in the lateral epidermis that retards movement of the LE. Phalloidin staining of embryos xed during DC often shows actin-rich lopodia-like structures at the LE (Harden et al., 1996) (Fig. 2A). Imaging of live embryos expressing GFP-tagged actin has conrmed that the LE cells extend lopodia as well as lamellipodia during DC (Jacinto et al., 2000). Filopodia and lamellipodia are actin-based structures that contribute to cell motility and are involved in neuronal growth cone guidance (reviewed in Mueller, 1999; Small et al., 1999). As the advancing epithelial sheets approach one another during DC, the lopodia extending from the LE cells appear to seek out cells on the opposing sheet and contribute to cell matching, such that the dorsal hole is neatly closed with the segments from the two sides of the embryo properly aligned. This interpretation of lopodia function in DC comes from the observation of wild-type live embryos and live embryos in which the LE lopodia have been lost due to mutation or transgene expression (Jacinto et al., 2000). Laser ablation studies also indicate that the amnioserosa plays an active role in DC and is not simply compressed by the migrating epidermal anks (Kiehart et al., 2000). The amnioserosa is under tension as demonstrated by the nding that amnioserosa cells recoil from a wound in the tissue. The contraction of the amnioserosa may contribute to DC as wounds in the amnioserosa that release tension lead to retraction of the LE away from the dorsal midline. During DC, the amnioserosa changes from an elliptically-shaped squamous epithelium to a tubular structure. This change in morphology is driven both by an apical constriction of cells in the amnioserosa and the loss of cells from the tissue (Rugendorff et al., 1994; Kiehart et al., 2000). Transmission electron microscopy shows that the apicallyconstricted amnioserosa cells become elongated along the apical-basal axis and the tissue invaginates to adopt its nal morphology (Rugendorff et al., 1994). The extrusion of cells from the amnioserosa has been observed in live embryos, where cells drop out of the plane of the epithelium and adjoining cells come together over them (Kiehart et al., 2000). This seems to occur sufciently often to make a signicant contribution to the contraction of the tissue. Severe disruption of the amnioserosa during DC does not prevent DC from proceeding to completion, suggesting that the advancing epidermis does not require the amnioserosa to crawl over and that amnioserosa contraction may serve a permissive role in DC (Kiehart et al., 2000; Harden et al., 2002). Thus, the contraction of the amnioserosa could largely serve to remove a physical impediment to migration of the epidermis. Below, I discuss an additional role for the amnioserosa in DC- a source of signals determining LE cell fate.

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

From the molecular characterization of several genes involved in DC, it became apparent that a mitogen-activated protein kinase (MAPK) cascade was involved in the process. MAPK cascades, which lead to the phosphorylation and activation of various trasncription factors, have been the subject of intense study due to their involvement in vital cellular processes such as cell cycle progression and differentiation (reviewed in Widmann et al., 1999). At the core of MAPK pathways is a threekinase module in which there is sequential activation of kinases by phosphorylation. The module is composed of a MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK) and MAPK. Numerous upstream activators have been identied for MAPK pathways with some of these being kinases themselves, i.e. MAPKKKKs. The MAPKKKs are serine/threonine kinases that phosphorylate and activate the MAPKKs, which are dualspecicity kinases that phosphorylate MAPK at Thr-XTyr motifs. MAPKs subsequently phosphorylate substrates on serine and threonine residues, with the majority of substrates identied to date being transcription factors. In mammalian cells, the MAPK pathways can be divided into ve families based on the MAPK being activated at the end of the cascade: extracellular regulated 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 activation of Jun, a leucine zipper transcription factor. Cloning of the DC gene hemipterous (hep) revealed that it encodes a MAPKK most similar to mammalian JNKK, and this was the rst indication that a JNK cascade was required for DC (Glise et al., 1995). Subsequent work demonstrated that Drosophila JNK (DJNK) is encoded by the DC gene basket (bsk) and Drosophila 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 prevalent functional form of Jun is as a heterodimer with the Fos leucine zipper protein to form the AP-1 factor (reviewed in Kockel et al., 2001). Not surprisingly then, Drosophila Fos (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 JNK pathway mutants are similar to each other. Loss of JNK signaling is characterized by an initial D-V elongation of LE cells which later revert to a polygonal shape, disruption of the accumulation of F-actin and myosin at the LE, and failure of DC to proceed to completion (Glise et al., 1995; Riesgo-Escovar et al., 1996; Sluss et al., 1996; Hou et al., 1997; Kockel et al., 1997; RiesgoEscovar 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 embryos demonstrates that the JNK cascade is also required for the LE lopodia (Jacinto et al., 2000). Genetic and biochemical data indicate that the Drosophila JNK pathway acts as a classic MAPK cascade leading to activation of DJun (Riesgo-Escovar et al., 1996; Sluss et al., 1996; Hou et al., 1997; Riesgo-Escovar and Hafen, 1997a; Sluss and Davis, 1997; Martn-Blanco et al., 1998; Stronach and Perrimon, 2002). The fact that the loss of DJun or DFos have a similar effect on DC as loss of kinases in the JNK cascade suggests that a major route of action of the JNK pathway in DC is activation of AP-1 and subsequent transcriptional responses in the nucleus. Indeed, expression of a constitutively active version of DJun can signicantly rescue the DC defects of embryos lacking one of the JNK pathway kinases (Hou et al., 1997; Riesgo-Escovar and Hafen, 1997a; 1997b; Sluss and Davis, 1997; Stronach and Perrimon, 2002). As transcriptional regulation of genes seems to be central to how the JNK cascade exerts its effects during DC, there has been great interest in determining which genes show expression changes in response to JNK signaling. Detailed studies have been done on two genes whose expression in the LE cells is dependent on the JNK cascade: decapentaplegic (dpp) and puckered (puc). The decapentaplegic (dpp) locus encodes a member of the transforming growth factor-b (TGF-b) superfamily that is expressed in the LE cells during DC (St. Johnston and Gelbart, 1987; Jackson and Hoffmann, 1994). As discussed below, components of a Dpp signaling cascade have been picked up as DC genes, indicating that Dpp signaling is required for DC. The LE expression of Dpp is dependent on the JNK cascade as it is lost in mutants lacking cascade components (Glise and Noselli, 1997; Hou et al., 1997; Riesgo-Escovar and Hafen, 1997b; Sluss and Davis, 1997; Zeitlinger et al., 1997; Stronach and Perrimon, 2002). The puckered (puc) gene was identied through a P element insertion that caused a mild DC defect (Ring and Martinez Arias, 1993). puc is strongly expressed in the LE cells during DC, but this LE transcription is abolished by loss of the JNK cascade (Glise et al., 1995; Riesgo-Escovar et al., 1996). puc encodes a dual specicity MAPK phosphatase that likely acts by downregulating DJNK/Bsk activity through dephosphorylation (Martn-Blanco et al., 1998). Thus, Puc appears to me diate a negative feedback loop regulating the JNK pathway (Martn-Blanco et al., 1998). Overexpression of Puc mimics the phenotype of loss of the JNK pathway in that there is a failure of DC accompanied by loss of both the F-actin/myosin contractile apparatus and dpp expression at the LE. A reduction in Puc through heterozygosity for a puc allele can partially rescue the DC defects caused by reduction of DJNKK/Hep. Furthermore, the expression patterns of dpp and puc respond 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 enhancer trap widely used to assess puc expression in DC studies) are overexpressed at the LE, suggesting that excessive signaling by the JNK cascade is occurring (Ring and Martinez Arias, 1993; Glise and Noselli, 1997; Martn-Blanco et al., 1998). Work in various systems indicates that numerous kinases can operate as JNKKKs (Widmann et al., 1999). The identity of the main JNKKK operating in the Drosophila JNK cascade at the LE remained uncertain until recently. The rst candidate described was the MAPKKK dTAK1. Mammalian TAK1 is capable of acting as a JNKKK in the JNK cascade (Shirakabe et al., 1997), and the potential role of dTAK1 in the LE JNK pathway has been addressed. Expression of kinasedead forms of dTAK1 causes DC defects, and overexpression of dTAK1 induces ectopic expression of dpp and puc (Takatsu et al., 2000; Mihaly et al., 2001). However, inhibition of dTAK1 through double-stranded RNA interference produces only a low frequency of DC defects, and ies homozygous for dTAK1 loss-of-function mutations survive to adulthood (Mihaly et al., 2001; Vidal et al., 2001). It is now apparent that the main or only JNKKK acting during DC is a mixed lineage kinase (MLK) encoded by the slipper (slpr) locus (Stronach and Perrimon, 2002). Embryos bearing a strong loss-of-function allele of slpr show similar phenotypes to JNK pathway mutants in that they fail to maintain elongation of epidermal cells during DC, do not show dpp expression at the LE, and have large dorsal holes in their cuticles. Epistasis analysis indicates that Slpr signals through the JNK cascade. The dorsal open phenotype of slpr mutant embryos can be partially rescued by expression of constitutively active DJun or through a reduction in Puc levels. The JNK cascade is required for the integrity of the LE cytoskeleton during DC, indicating that signaling through this pathway at some point has to affect the assembly of F-actin. This could occur either through interactions between the cytoplasmic portion of the cascade and proteins regulating cytoskeletal assembly, or by such proteins being affected in some way by JNK cascade-dependent gene transcription. As discussed below, the major route of action for the JNK cascade appears to be through the Dpp signaling pathway. However, a recent serial analysis of gene expression (SAGE) study has identied numerous genes encoding signaling molecules, cytoskeletal regulators, and cell adhesion molecules that are transcriptionally responsive to alterations in JNK signaling in Drosophila (Jasper et al., 2001). The study was done by comparing tag frequencies among SAGE libraries generated from wild-type embryos, embryos expressing a dominant negative version of DJNK/ Bsk, or embryos expressing a constitutively active version of DJNKK/Hep. Among the genes upregulated by activation of the JNK cascade is chickadee (chic), which encodes Drosophila prolin (Cooley et al., 1992). Prolin

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is an actin binding protein involved in F-actin polymerization (reviewed in Wear et al., 2000) and may constitute a link between the JNK cascade and regulation of the cytoskeleton in LE cells. chic mutant embryos show DC defects and lack lopodia at the LE, and there is genetic interaction between chic and hep during DC in that heterozygosity for a loss-of-function chic allele increases the frequency and severity of DC defects in hep mutant embryos (Jasper et al., 2001). These data suggest that the JNK cascade drives production of prolin in the LE cells, which is subsequently involved in the production of lopodia required for DC.

JNKKKKs in DC
Genetic and biochemical analysis of the MAPK pathway mediating pheromone response in S. cerevisiae indicates that the kinase Ste20p functions as a MAPKKKK (reviewed in Widmann et al., 1999). A Ste20 group of kinases has been dened whose members function as upstream regulators of MAPK cascades (reviewed in Dan et al., 2001). The Ste20 group kinases can be divided into the p21-activated kinase (PAK) and germinal center kinase (GCK) families based on the location of their kinase domains. The Drosophila gene misshapen (msn) encodes a member of the GCK family that is required upstream of the JNK pathway during DC (Su et al., 1998; 2000). Msn is highly homologous to mammalian Nck-interacting kinase (NIK), a known activator of JNK (Su et al., 1997). dpp expression is reduced at the LE in msn mutant embryos, and the DC defects of these embryos can be substantially rescued by expression of constitutively active DJun. Embryos heterozygous for a msn loss-offunction mutation do not show DC defects, but embryos doubly heterozygous for either msn and DJNK/ bsk alleles or msn and DJNKK/hep alleles display dorsal holes. Finally, transfection of cells with Msn leads to a four- to ve-fold increase in JNK activation. Another Ste20 group kinase, DPAK, a PAK family member, is enriched at the LE during DC (Harden et al., 1996). Loss-of-function DPAK mutants survive embryogenesis (Hing et al., 1999), suggesting that DPAK does not have a major role in DC or that maternally contributed DPAK is sufcient. A role for DPAK in activation of the JNK cascade has not been addressed to date.

pathway components in that there is lack of cell elongation in the epidermis and reduced dpp expression at the LE. Cka has multiple domains and can bind DJNKK/ Hep, DJNK/Bsk, Djun, and DFos. Various results indicate that this binding to JNK pathway components makes a contribution to functioning of the cascade. A reduction in Cka levels can worsen the DC defects caused by reductions in DFos or DJNK/Bsk function, and the DC defects associated with complete loss of Cka are signicantly rescued by expression of constitutively active versions of DJun or DJNK/Bsk. Furthermore, in cultured cells, Cka stimulates activation of DJNK/Bsk and the subsequent phosphorylation and transcriptional activity of DJun and DFos. Cka may act as a scaffolding molecule similar to the yeast Ste5 protein in the pheromone response MAPK cascade (reviewed in Widmann et al., 1999). Cka may bring DJNK/Bsk into contact with its activator DJNKK/Hep and further act in the nucleus to promote activation of transcription by AP-1 (Chen et al., 2002).

Nonreceptor tyrosine kinases functioning as upstream activators of the JNK cascade

Two recent studies indicate that nonreceptor tyrosine kinases are required for activation of the JNK cascade during DC. The Drosophila Src42A tyrosine kinase participates with the Src64 tyrosine kinase and the Src-related kinase Tec29 in activation of the JNK cascade during DC (Tateno et al., 2000). Although embryos missing any 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 cells of Src42A Tec29 double mutant embryos show disruption of F-actin accumulation as well as loss of dpp and puc-lacZ expression. This DC phenotype is very similar to that seen in JNK mutant embryos, and expression of constitutively active DJun can partially rescue the DC defects of Src42A Tec29 double mutant embryos. Finally, overexpression of Src42A in larvae leads to increased levels of phosphorylated DJNK/Bsk. Embryos completely devoid of another nonreceptor tyrosine kinase, Shark, are defective in DC, lack cell elongation in the epidermis, and show loss of dpp expression at the LE (Fernandez et al., 2000). That activation of JNK signaling is an important component of Shark function during DC is conrmed by the partial rescue of the DC defects in shark mutant embryos by expression of constitutively active DJun. Nonreceptor kinases such as Src have been implicated in signaling from various kinds of transmembrane receptors (reviewed in Abram and Courtneidge, 2000). It is likely that the various nonreceptor tyrosine kinases required for DC constitute a link between JNK-promoting signals received at the cell surface (for example, from the amnioserosa, see below) and activation of the cascade.

Regulation of the JNK cascade by CKA, a potential scaffolding molecule

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

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Signaling by small GTPases during DC

Various members of the Ras superfamily of small GTPases have been investigated for roles in DC. The small GTPases act as molecular switches that cycle between a GDP-bound off state and a GTP-bound on state, regulating a diverse array of cellular processes by relaying extracellular and intracellular signals to downstream effectors. With regard to DC, particular emphasis has been placed on the study of members of the Rho family of small GTPases, proteins that have been shown in various systems to be key regulators of the actin cytoskeleton and upstream activators of JNK and p38 MAPK cascades (reviewed in Bishop and Hall, 2000). Included in the Rho family are the Rac, Cdc42 and Rho subgroups. The Drosophila Rac proteins Drac1, Drac2, and Mtl, the Drosophila Cdc42 protein Dcdc42, and the Drosophila Rho protein RhoA (also called Rho1) are involved in DC.

Drac1, Drac2 and Mtl The requirement for Drac1 during DC has been addressed through the expression of a dominant negative mutant version of the protein, Drac1N17. Embryos in which Drac1N17 has been expressed show loss of the LE cytoskeleton, a lack of cell elongation in the epidermis, and do not complete DC (Harden et al., 1995; 1999). These phenotypes are similar to those caused by loss of the JNK pathway, and expression of constitutively active DJun can partially rescue Drac1N17-induced DC defects, indicating that a component of signaling downstream of Drac1 in DC is the JNK cascade (Hou et al., 1997). This is in line with results in mammalian cultured cells showing that Rac can activate the JNK cascade (Coso et al., 1995; Minden et al., 1995). Expression of a constitutively active version of Drac1, Drac1V12, induces ectopic expression of puc-lacZ and dpp in a hep-dependent manner, demonstrating that Drac1 can act as an upstream activator of the JNK cascade (Glise et al., 1995). Activation of the JNK cascade does not, however, appear to be wholly channeled through Drac1 as Drac1N17-expressing embryos or embryos overexpressing a negative regulator of Drac1, RotundRacGAP, maintain dpp and puc-lacZ expression at the LE (Raymond et al., 2001). The route(s) by which Drac1 activates the JNK cascade remains uncertain. There is some evidence from work in mammalian cells that Rac and Cdc42 can trigger JNK activation through the Rac/Cdc42-binding PAK kinases (reviewed in Van Aelst and DSouza-Schorey, 1997). The LE enrichment of DPAK appears to be dependent on Drac1 function (Harden et al., 1996). However, as noted above, there is presently no direct evidence of a role for DPAK in DC. It is possible that

Drac1 activates the JNK cascade through direct contact with the JNKKK, Slpr. Slpr, like other MLKs, contains sequences with homology to the Cdc42/Rac interacting binding (CRIB) motif (Burbelo et al., 1995; Pirone et al., 2001). The mammalian protein MLK3 can bind Rac and Cdc42, and this binding may contribute to the activation of JNK signaling by MLK3 (Teramoto et al., 1996; Bock et al., 2000). Drac1 may also contribute to the activation of the JNK cascade by Msn, although not through direct interaction. Rac cannot bind Msn or NIK, but dominant negative Rac can impair JNK activation by Msn or NIK (Su et al., 1998). A likely upstream activator of Drac1 during DC is the product of the myoblast city (mbc) gene. Alleles of mbc were picked up in a screen for suppressors of a rough eye phenotype induced by overexpression of Drac1 (Nolan et al., 1998). The Mbc protein is highly homologous to the mammalian protein DOCK180 (Erickson et al., 1997), which is an activator of Rac. DOCK180 binds to the nucleotide-free form of Rac and promotes formation of active, GTP-bound Rac (Kiyokawa et al., 1998a; Nolan et al., 1998). DOCK180 does not appear to be a guanine nucleotide exchange factor (GEF) itself but may stabilize nucleotide-free Rac, which is an intermediate in the activation of Rac by GEFs (Kiyokawa et al., 1998a). DOCK180 can trigger the JNK cascade, as assessed by the level of Jun phosphorylation, but this JNK pathway activation is blocked by dominant negative Rac, RacN17 (Kiyokawa et al., 1998a; Nolan et al., 1998). RacN17 can also suppress DOCK180-induced membrane spreading in cultured cells (Kiyokawa et al., 1998a). These results suggest that DOCK180 can regulate JNK signaling and the actin cytoskeleton through its effects on Rac. Embryos decient in Mbc show DC defects and exhibit mild losses of LE cytoskeleton and occasional modest reductions in dpp expression at the LE (Erickson et al., 1997; Nolan et al., 1998). In cultured cells, DOCK180 forms a complex with the focal adhesion proteins CrkII and p130cas after integrin stimulation and is a likely mediator of integrin signaling (Kiyokawa et al., 1998b). Interestingly, mutants in the genes myospheroid (mys) and scab (scb), which encode the Drosophila bPS and aPS3 integrin subunits, respectively, exhibit DC defects (Brown, 1994; Stark et al., 1997; Brown et al., 2000), raising the possibility that integrin signaling through Mbc leads to Drac1 activation. The inability of activated DJun to completely rescue Drac1N17-induced DC defects suggests that the JNK cascade is not the only route of Drac1 activity. Work on cultured cells indicates that there are JNK-independent links between Rac and regulation of the actin cytoskeleton (reviewed in Bishop and Hall, 2000; Takenawa and Miki, 2001). A potential downstream target of Drac1 in regulation of the cytoskeleton is Pkn, a member of the PKN 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 as expression of dpp at the LE appears normal. Pkn binds specically to the active, GTP-bound versions of Drac1 and Rho1, and this binding stimulates Pkn kinase activity. The biological roles of the PKN kinases remain poorly characterized, but ectopic expression of mammalian PKN in broblasts causes reorganization of Factin and membrane rufing, suggesting a function in cytoskeletal regulation (Dong et al., 2000). PKN may contribute to cytoskeletal regulation by phosphorylating cytoskeletal components (Amano et al., 1996; Watanabe et al., 1996). Other links between Rac and actin polymerization have been identied through work on cultured cells. The Wiskott Aldrich protein (WASP) family, which is divided into the WASP and WAVE groups, links small GTPases to the actin cytoskeleton (reviewed in Takenawa and Miki, 2001). In response to activation of small GTPases, WASP family proteins promote activation of the Arp2/3 complex, a multimeric protein complex that plays a central role in de novo actin polymerization. WASPs and WAVEs bind directly to prolin, which may contribute to rapid actin polymerization in conjunction with the Arp2/3 complex. Rac-induced formation of F-actin-based lamellipodia at the cell periphery is mediated through the WAVE2 protein. A WAVE/Scar protein and members of the Arp2/3 complex have been identied in ies (Goldstein and Gunawardena, 2000; Zallen et al., 2002), and Drosophila WAVE/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 cytoskeleton likely does involve the prolin produced through JNK cascade activation of chic transcription (Jasper et al., 2001). In addition to its LE roles, Drac1 participates in morphogenesis of the amnioserosa during DC (Harden et al., 2002). Amnioserosa-specic expression of Drac1N17 retards contraction of the amnioserosa and impedes movement of the LE, whereas such expression of constitutively active Drac1 causes a premature and excessive contraction of the tissue. Preliminary indications are that Drac1 acts through Crumbs, a determinant of epithelial polarity (Tepass et al., 1990; Wodarz et al., 1995), in regulation of cell shape in the amnioserosa. These results lend support to the idea, discussed above, that active morphogenesis of the amnioserosa is a component of DC. Recently, loss-of-function mutations in the three Drosophila Rac genes have been described (Hakeda-Suzuki et al., 2002; Ng et al., 2002). Analysis of these indicates that they have overlapping roles in epithelial morphogenesis but that Drac1 makes the greatest contribution to DC (Hakeda-Suzuki et al., 2002). Embryos mutant 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 Drac2 Mtl triple mutant embryos, whereas Drac2 Mtl double mutant embryos complete DC. On the basis of these results, 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 cytoskeleton has been examined in Drac1 Drac2 Mtl triple mutant embryos. There is little F-actin at the LE, and both lopodia and lamellipodia are severely reduced in number (Hakeda-Suzuki et al., 2002). The JNK pathway has not yet been examined in the Drosophila Rac mutants.

Dcdc42 Dcdc42 function during embryogenesis has been studied using loss-of-function Dcdc42 alleles and transgenes. It is not possible to produce embryos completely devoid of both maternal and zygotic Dcdc42 function due to a requirement for Dcdc42 in oogenesis (Genova et al., 2000). However, females bearing heteroallelic combinations of weak and strong Dcdc42 alleles can be used to produce Dcdc42 mutant embryos severely depleted in Dcdc42 function that show DC defects (Genova et al., 2000). The DC defects in Dcdc42-decient embryos have not yet been examined in detail with regard to cell shape and the status of the actin cytoskeleton. Expression of a dominant negative version of Dcdc42, Dcdc42N17, leads to DC defects that are phenotypically distinct from those caused by Drac1N17 expression (Riesgo-Escovar et al., 1996; Harden et al., 1999). Dcdc42N17-expressing embryos exhibit partial losses of the LE cytoskeleton, suggesting that Dcdc42 may make a contribution to the establishment of the LE cytoskeleton albeit less substantial than that of Drac1. In cultured cells, Cdc42 participates in the formation of lopodia (Kozma et al., 1995; Nobes and Hall, 1995), and this appears to be the case during 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 mammalian cells involves the interaction of the Cdc42-binding protein N-WASP with the Arp2/3 complex and prolin (reviewed in Takenawa and Miki, 2001). Surprisingly, embryos completely decient for the only known WASP protein in Drosophila, Wsp, secrete normal cuticles, indicating that there can be no signicant defects in DC (Ben-Yaacov et al., 2001). Wsp may not operate in Dcdc42 signaling, as Dcdc42 binding is not essential for Wsp function (Tal et al., 2002). Nevertheless, Dcdc42 may act through prolin in driving lopodia formation during DC, as chic mutants lack lopodia at the LE (Jasper et al., 2001). Complex phenotypes generated by Dcdc42 transgenes suggest that this small GTPase has multiple roles in DC. In addition to the losses of LE cytoskeleton and lopodia described above, Dcdc42N17 expression can also

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result in a bunched LE phenotype similar to that seen with mutants in the Dpp/TGF-b pathway required for DC (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 LE cytoskeleton. Other data support the idea that Dcdc42 function can contribute to both establishment and downregulation of the LE cytoskeleton during DC (Harden et al., 1999). In co-expression experiments, there is a temporal shift from Dcdc42N17 expression worsening Drac1N17-induced DC defects to Dcdc42N17 expression partially rescuing Drac1N17-induced DC defects. This result suggests that Dcdc42 may initially help Drac1 establish the LE cytoskeleton but later contributes to its downregulation. In support of a downregulatory role, about 10 % of embryos expressing constitutively active Dcdc42 (Dcdc42V12) show losses of LE F-actin and myosin. Dcdc42 transgene expression affects the distribution of the DPAK kinase at the LE (Harden et al., 1999). Dcdc42N17 expression causes loss of DPAK from the LE, whereas Dcdc42V12 upregulates LE DPAK levels to higher than wild-type. However, the 10 % of Dcdc42V12expressing embryos that lose the LE cytoskeleton also lose LE DPAK. Similar to Drac1, expression of constitutively active Dcdc42 causes ectopic activation of the JNK cascade (Glise and Noselli, 1997), but other results suggest that Dcdc42 does not play a major role in activating the JNK cascade during DC. Dcdc42 mutant embryos maintain dpp expression at the LE (Genova et al., 2000), as do Dcdc42N17-expressing embryos which have also been shown to have normal puc-lacZ expression (Raymond et al., 2001).

RhoA/Rho1 Loss-of-function mutations in the RhoA/Rho1 locus or expression of a dominant negative RhoA/Rho1 transgene lead to defects in the dorsal cuticle of the embryo (Strutt et al., 1997; Harden et al., 1999; Lu and Settleman, 1999; Magie et al., 1999). RhoA/Rho1 mutant embryos manage to complete DC, but the closed dorsal surface is disorganized (Magie et al., 1999). This irregular closure appears to be due to uneven constriction of cells along the A-P axis at the LE, with some cells being excessively constricted and others being splayed out (Magie et al., 1999). Embryos expressing a dominant negative version of RhoA/Rho1 display a similar phenotype of uneven constriction of cells at the LE, accompanied by disruption of LE myosin (Harden et al., 1999). In RhoA/Rho1 mutants, some of the excessively constricted cells form ectopic contacts with their lateral neighbors, as if the neighboring cells are being perceived as though they were the cells in the opposing LE that contact is normally 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 (Lu and Settleman, 1999; Magie et al., 1999). Expression of dominant negative RhoA/Rho1 also does not disrupt the JNK cascade, as shown by dpp transcript distribution (Lu and Settleman, 1999). RhoA/Rho1 may be required to regulate the contractility of the LE cytoskeleton through effects on LE myosin. The RhoA/Rho1 locus interacts genetically with zipper, the gene encoding nonmuscle myosin-II heavy chain, indicating that RhoA/Rho1 regulates myosin function during Drosophila morphogenesis (Halsell et al., 2000). The Rho-associated kinases (ROKs), downstream effectors for Rho in mammalian cells, promote phosphorylation of the regulatory light chain of myosin (MRLC) through both direct action on MRLC and indirectly through inactivation of myosin phosphatase by phosphorylating its myosin-binding subunit (MBS) (reviewed in Bishop and Hall, 2000). Phosphorylation of MRLC stimulates the ATPase of myosin, promoting the assembly and function of the actomyosin contractile apparatus (reviewed in Bresnick, 1999). A recent study demonstrates that similar regulation of MRLC occurs during DC (Mizuno et al., 2002). Embryos lacking Drosophila MBS (DMBS) exhibit DC defects, indicating that regulation of the phosphorylation state of MRLC is required for correct DC. In DMBS mutant embryos, the level of phosphorylated MRLC is substantially elevated at the LE and cell shape change in the epidermis is aberrant. Overexpression of 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 the lethality caused by loss of DMBS or excessive Drok activity is due to hyperactivation of myosin. Reduction of myosin function through heterozygosity for a zip allele can substantially suppress the lethality of DMBS mutant embryos or embryos overexpressing Drok. As described above, another potential route for RhoA/Rho1 regulation of the cytoskeleton is the Rho/ Rac effector Pkn (Lu and Settleman, 1999).

Ras1 Expression of Ras1 transgenes causes DC phenotypes with some similarities to the effects of Dcdc42 transgenes (Harden et al., 1999). Dominant negative Ras1 induces partial losses of the LE cytoskeleton but DPAK levels are unaffected. Expression of constitutively active Ras1 causes greater than normal accumulations of DPAK at the LE. Given that the Rho family proteins are known to function downstream of Ras in the transformation of cultured cells (reviewed in Zohn et al., 1998), it is possible that Ras1 signals thorugh Rho family proteins such as Dcdc42 during DC, but this theory has not yet been addressed.

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A TGF-b signaling pathway is required for DC and acts 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, respectively, cause DC defects, indicating that TGF-b signaling is required for DC (Childs et al., 1993; Affolter et 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 expression of Dpp, a TGF-b superfamily member, at the LE, and it is clear that a Dpp/TGF-b signaling cascade is a major route of action for the JNK pathway during DC. Overexpression of Dpp or expression of an activated version of the Dpp receptor Tkv can substantially rescue the DC defects of embryos with impaired JNK signaling (Hou et al., 1997; Riesgo-Escovar and Hafen, 1997a; Sluss and Davis, 1997; Su et al., 1998; Chen et al., 2002). TGF-b ligands such as Dpp signal through receptor complexes containing type I and type II receptors, and it is likely that Dpp secreted by the LE cells activates signaling in epidermal cells via the Tkv and Put receptors during DC (Letsou et al., 1995; Ruberte et al., 1995). A requirement for Dpp in DC cannot be directly addressed due to a need for Dpp earlier in embryonic development (reviewed in Morisato and Anderson, 1995). The TGF-b superfamily member Glass bottom boat (Gbb, also called 60A) appears to contribute to signaling through the Tkv/Put receptor complex during DC, possibly as a heterodimer with Dpp (Chen et al., 1998). Embryos bearing a hypomorphic tkv allele, tkv6 are viable, but tkv6 gbb double mutant embryos show DC defects. The complete details of signaling occurring downstream of the activated Tkv/Put receptor complex during DC remain to be determined, but it involves a transcriptional response. The DC gene schnurri (shn) (Nsslein-Volhard et al., 1984) has been shown genetically to function downstream of dpp and encodes a zinc nger protein (Arora et al., 1995; Grieder et al., 1995; Staehling-Hampton et al., 1995). Work in various systems has revealed that the Smad family proteins function downstream of receptors for ligands belonging to the TGF-b superfamily. In Drosophila, the Tkv receptor phosphorylates Mothers against Dpp (Mad), a Drosophila Smad, which translocates into the nucleus with the Smad protein Medea (Med) (reviewed in Affolter et al., 2001). Embryos decient in Mad show DC defects, and Mad has been shown to interact with Shn in mediating transcriptional responses to Dpp signaling (Hudson et al., 1998; Dai et al., 2000; Udagawa et al., 2000). Recently, it has been shown that the LE expression of the zip gene during DC is lost in tkv mutant embryos (Arquier et al., 2001). Thus, production of myosin in the LE cells may be dependent on Dpp/TGF-b signaling to the nucleus. Despite the lack of myosin synthesis at the LE

in tkv mutant embryos, the Dpp/TGF-b pathway is not required for the assembly of the LE cytoskeleton or elongation of the LE cells (Riesgo-Escovar and Hafen, 1997a; Ricos et al., 1999). This seemingly paradoxical result is consistent with the analysis of zip mutant embryos (Young et al., 1993). Maternally produced myosin localizes to the dorsal end of LE cells during DC in zip mutant embryos and is sufcient for a contractile apparatus that can achieve a fair degree of cell elongation and signicant progression of DC. The myosin expressed from zip in the LE cells during DC appears to be required in the late stages of DC when the maternal supply is exhausted (Young et al., 1993). In tkv, put, and shn mutant embryos, cells ventral to the LE cells in the epidermis fail to elongate and the dorsal hole does not close (Riesgo-Escovar and Hafen, 1997a; Ricos et al., 1999). There are a number of plausible explanations for this phenotype. One interpretation is that cell shape change in the lateral epidermis is an active, Dpp/TGF-b-dependent process and not simply a passive response to elongation of the LE cells (RiesgoEscovar and Hafen, 1997a). In tkv and put mutant embryos, expression of DFos in the lateral epidermis is reduced but its expression at the LE is maintained (Riesgo-Escovar and Hafen, 1997b). The expression of DFos throughout the lateral epidermis may be required for DC, as restricted induction of a DFos transgene in the dorsalmost epidermal cells cannot rescue DC defects in embryos mutant for kay, the gene encoding DFos (Riesgo-Escovar and Hafen, 1997b). DFos may be part of a Dpp/TGF-b pathway driving cell shape change in the lateral epidermis (Riesgo-Escovar and Hafen, 1997b). Another reason why cell elongation does not occur in the lateral epidermis of embryos defective in the Dpp/ TGF-b pathway could be misdirected elongation of LE cells leading to a failure of passive stretching of more ventrally located cells. In tkv, put, and shn mutant embryos, the LE cells initially undergo an elongation and dorsalward migration, but as DC proceeds, these cells get pulled together into a series of bunches, each of which contains several adjacent segments squeezed together at their dorsal end (Ricos et al., 1999). This bunching may halt further dorsalward movement of the epidermis and disrupt the forces promoting elongation of the lateral epidermal cells. The bunching phenotype could be due to loss of a TGF-b-dependent regulatory mechanism(s) required for uniform DC. Candidate cells mediating such regulation are the cells anking the segmental grooves at the LE. These segment border cells differ from the other LE cells in a number of ways, most importantly being that they show high levels of tkv transcripts (Affolter et al., 1994) and may be major points of TGF-b signaling during DC. The segment border cells also show upregulation of DPAK, a potential effector for the small GTPases Drac1 and Dcdc42, and do not constrict as extensively in the A-P direction as other LE cells (Harden et al., 1996) (Fig. 2D). The less con-

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stricted morphology of the segment border cells may be required for uniform closure of the dorsal hole and may be caused by transient losses of the LE cytoskeleton occurring in these cells during DC (Harden et al., 1996). If Dpp/TGF-b signaling is required for the downregulation of the cytoskeleton in the border cells, its loss would release these brakes on LE contraction, causing excessive contraction and the bunched phenotype (Ricos et al., 1999). As mentioned above, Dcdc42N17-expressing embryos exhibit a similar bunched phenotype. Expression of constitutively active Dcdc42 can partially rescue the DC defects of tkv mutant embryos, indicating that Dcdc42 is a component of Dpp/TGF-b signaling (Ricos et al., 1999). It is possible that Dcdc42 contributes to downregulation of the cytoskeleton in the segment border cells through DPAK (Ricos et al., 1999). Another possible explanation for the bunched phenotype, not necessarily mutually exclusive from those described above, is that instead of migrating dorsally and contacting LE cells from the opposite epidermal ank, the LE cells in Dpp/TGF-b pathway mutants migrate in an A-P direction and establish ectopic adhesions with their neighbors in the same epidermal ank (these ectopic adhesions are also seen in RhoA/Rho1 mutant embryos, see above). Such aberrant migration could be due to disruption of guidance cues used by the lopodia of LE cells. In live embryos, the LE lopodia are seen to wave about sampling for the correct binding partners and may be responding to guidance cues in a manner analogous to the lopodia of axonal growth cones navigating via attractants and repellents (Jacinto et al., 2000; 2001). In the process of ventral enclosure in C. elegans, which shows parallels to DC, the axonal repellent Semaphorin-2A is required to prevent ectopic contacts being made between migrating hypodermal cells (Roy et al., 2000). Similar guidance cues may be used to ensure correct cell matching during DC, and could be supplied by segment border cells. For example, secretion of a repellent by segment border cells would prevent the migrating LE cells from straying too far in the A-P direction. A recent study has led to the positioning of another component of Dpp/TGF-b signaling in DC. The zincnger transcription factor Pannier, which lies downstream of Dpp in early development, acts as an upstream regulator of dpp expression during DC (Herranz and Morata, 2001). In pnr mutant embryos the LE expression of dpp is lost, and ectopic Pnr activity results in ectopic dpp expression. Pannier is not required for activation of the JNK cascade, however, as JNK-dependent puc-lacZ expression at the LE is maintained in pnr mutant embryos. The results indicate that expression of dpp at the LE requires independent inputs from the JNK pathway and Pnr (Herranz and Morata, 2001). The DC defects of pannier mutant embryos are similar to those of mutants in Dpp/TGF-b signaling and include bunching 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 secreted ligands determines cell fates in insects and vertebrates (reviewed in Wodarz and Nusse, 1998). In the canonical Wg pathway operating in Drosophila, the reception of Wg signal by the Frizzled 2 (Fz2) receptor leads to the accumulation of cytoplasmic Arm. In the absence of Wg signal, cytoplasmic Arm is targeted for degradation by a complex that includes the Zeste white-3 (Zw3) kinase. During Wg signaling, the activation of the Dishevelled (Dsh) protein downstream of Fz2 leads to inactivation of Zw3 and subsequent stabilization of Arm. Arm then promotes transcription of Wg-responsive genes by acting as a co-activator for the transcription factor dTCF. A recent study demonstrates that the Wg pathway is an essential player in DC (McEwen et al., 2000). Embryos decient in the Wg pathway components Wg, Dsh, or Arm, show DC defects in cuticle preparations and reductions in dpp and puc-lacZ expression at the LE. Expression of an N-terminally truncated form of dTCF, dTCFDN, which acts as a constitutive repressor of Wg target genes, also causes DC defects and reduced LE dpp expression. As a further indication that Wg signaling drives dpp expression, the Wg pathway was ectopically activated by various means leading to an expansion of dpp expression beyond the LE. Cell shape change during DC has been evaluated in wg and arm mutant embryos. In wg mutant embryos, the LE cells become elongated in the AP axis, but most embryos manage to close up in an distorted manner. In arm mutant embryos, there is elongation of cells in the D-V axis, but it is irregular, and the nal degree of closure tends to be less than that seen in wg mutants. The Wg pathway component that has been studied in the greatest detail with regard to DC is Arm. The DC defects caused by loss of Arm can be suppressed by stimulation of the JNK pathway (via a puc allele or expression of dTAK1) or by activation of Dpp signaling. This suggests that a major role for the Wg pathway in DC is expression of dpp at the LE. Wg pathway-driven expression of dpp appears to be JNK-dependent as constitutive activation of the Wg pathway cannot promote dpp expression in kay mutant embryos lacking DFos. Thus, Wg signaling must collaborate with JNK signaling at some point in DC and McEwen et al. (2000) have proposed a number of models to explain how this may occur. The Wg pathway may feed directly into activation of the JNK cascade at the level of Dsh. Work on epithelial planar polarity in Drosophila has indicated that Dsh can activate the JNK cascade through Rho family small GTPases (reviewed in Boutros and Mlodzik, 1999). In parallel to this activation of JNK, Dsh may also trigger the rest of the canonical Wg pathway during DC, as indicated by the requirement for Arm. Another possibility considered

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by McEwen et al. is that the Wg and JNK pathways converge at the level of dTCF, which, in addition to being a positive effector for the Wg pathway, can act with Groucho as a repressor of Wg signaling. In the absence of Wg signal, dTCF could act as a repressor of dpp expression. Activation of the Wg pathway would then function as a permissive signal, displacing Groucho with Arm and releasing this repression. As discussed below, a likely source of signals promoting the LE cell fate is the amnioserosa. Expression of Wg in the amnioserosa has not been reported, and the DC defects seen in wg mutant embryos are not as severe as those seen with embryos decient in other DC components. Wg may not be a key activator of DC but appears to function as an important regulator of dpp expression at the LE. It should be noted here that experiments using Arm to study the contribution of Wg signaling to DC must be interpreted with caution. Arm may contribute to DC in a Wg-independent fashion through it role in adherens junctions (McEwen et al., 2000), as detailed below.

studied in detail but might reect a disruption of the JNK cascade. DRal appears to be a negative regulator of the JNK pathway as hair and bristle defects caused by dominant negative DRal expression can be suppressed by hep and bsk mutations, and constitutively active DRal inhibits the phosphorylation of JNK in cultured S2 cells (Sawamoto et al., 1999).

Notch Embryos completely devoid of the Notch (N) receptor, an important mediator of cell fate decisions during development, exhibit holes in the dorsal cuticle, and a function for N in DC has been characterized (Zecchini et al., 1999). In N mutant embryos, dpp levels at the LE are transiently higher than in wild-type embryos, and there is ectopic expression of dpp and puc-lacZ in the epidermis. This result suggests that the JNK pathway is overactive in N mutant embryos, and indeed, extracts from these embryos have a greater ability to phosphorylate JNK and Jun than wild-type extracts. That N is a negative regulator of JNK signaling is further indicated by the nding that loss of N can suppress the DC defects caused by reductions in JNK pathway function. The best-characterized pathway of N signaling is during the process of lateral inhibition in which cells that have adopted a particular fate suppress the adoption of the same fate by surrounding cells (reviewed in ArtavanisTsakonas et al., 1995). Interaction between N and the ligand Delta results in cleavage of the N intracellular domain which translocates to the nucleus, where it binds the Suppressor of Hairless (Su(H)) protein and activates the expression of transcriptional repressors. This pathway 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 the JNK cascade? One possibility is that N antagonizes the activation of JNK signaling by Wg, as N has been shown to inhibit Wg signaling in a Su(H)-independent manner (Brennan et al., 1999a; 1999b; Lawrence et al., 2001). Another route may be through interaction with the product of the DC gene canoe (cno) (Jrgens et al., 1984) at the adherens junction (see below). N localization at the adherens junction has been described and N interacts genetically with cno (Fehon et al., 1991; Miyamoto et al., 1995).

Additional negative regulation of the JNK cascade during DC

Anterior open/Yan In addition to Puc, several other proteins function as a negative regulators of the JNK cascade during DC. The ETS-domain protein Anterior open (Aop)/Yan is a general inhibitor of differentiation whose activity is downregulated in response to phosphorylation by the MAPK encoded by the rolled gene (Rebay and Rubin, 1995). Aop/Yan is also phosphorylated by DJNK/Bsk, and aop mutant embryos exhibit DC defects (Riesgo-Escovar and Hafen, 1997a). aop mutant embryos show ectopic dpp expression, whereas expression of a constitutively active version of Aop/Yan that cannot be phosphorylated by MAPK causes a reduction in dpp levels at the LE. A model has been proposed in which, in the absence of JNK signaling, Aop/Yan represses the transcription of DJun-responsive genes (Riesgo-Escovar and Hafen, 1997a). When the JNK cascade is activated, Aop/Yan becomes phosphorylated and inactivated, releasing the repressed genes for DJun-dependent transcription.

DRal Raw Expression of a constitutively active version of the Drosophila Ral small GTPase, DRal, causes dorsal holes in the embryonic cuticle (Sawamoto et al., 1999). Ral is of interest with regard to Ras signaling, as the GEFs that activate Ral are direct targets of Ras (reviewed in Wolthuis and Bos, 1999). The DC phenotype caused by constitutively active DRal expression has not been Embryos decient in the novel protein Raw fail to undergo DC (Nsslein-Volhard et al., 1984; Blake et al., 1998; Byars et al., 1999), and this is likely due to a lack of cell elongation in epidermis (Blake et al., 1998). The disruption of cell shape change in the epidermis in raw mutants is probably due to an uneven accumulation of

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myosin along the LE (Blake et al., 1998), as the raw DC defect can be partially rescued by a zip mutation reducing LE myosin (Blake et al., 1999). The cause of the uneven LE myosin levels in raw mutant embryos may be misregulation of the JNK cascade. In raw mutant embryos, dpp and puc-lacZ expression spreads beyond the LE cells to more ventrally located epidermal cells, suggesting an increase in the size of the JNK expression domain (Byars et al., 1999). The ectopic expression of dpp in raw mutant embryos is lost along with the normal LE expression of dpp when embryos are made doubly mutant for a loss-of-function DJun allele. The Raw protein constitutes another negative regulator of JNK signaling but contains no recognizable motifs, and it is not known where in the cell it operates or what proteins it interacts with.

Importance of cell-cell junctions in DC

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

Adherens junction proteins and DC As noted above, triangular nodes of phosphotyrosinerich proteins are present along the LE during DC. Phosphotyrosine staining in Drosophila epithelia is largely localized to adherens junctions (Woods and Bryant, 1993; Woods et al., 1997). Drosophila E-cadherin (DE-cadherin), Drosophila a-catenin (Da-catenin), and Arm, three proteins known to be components of adherens junctions, localize to the triangular nodes at the LE, suggesting that these structures are specialized cell-cell junctions participating in DC (Jacinto et al., 2000; Grevengoed et al., 2001). Adherens junctions are found at the apical/ lateral interface in epithelial cells and are organized around what is known as the cadherin-catenin-complex (CCC) (reviewed in Tepass et al., 2001). In Drosophila, the CCC consists of the transmembrane protein DEcadherin, encoded by the shotgun (shg) gene, Da-catenin, Arm (Drosophila b-catenin), and Dp120ctn. The only component of the CCC for which a role in DC has been directly demonstrated is Arm. The interpretation of Arms role in DC is complicated by the fact that the protein is found both at adherens junctions and in the cytoplasm (McEwen et al., 2000). As described above, cytoplasmic Arm is used in Wg signaling, a pathway participating in DC (McEwen et al., 2000). The larger dorsal holes seen in arm mutant embryos compared to wg mutant embryos suggest that Arm has a role in DC independent of Wg signaling, and this is likely to be at the adherens junction. shg mutant embryos lacking zygotic DE-cadherin have a normal dorsal epidermis (Tepass et al., 1996; Uemura et al., 1996). It is not possible

to make embryos completely devoid of DE-cadherin due to a requirement for DE-cadherin in oogenesis (Tepass et al., 1996; Uemura et al., 1996). However, germline clones of weak shg alleles show loss of dorsal cuticle (Tepass et al., 1996), although it is not known if this is due to a DC defect. Mutations in the genes encoding Da-catenin and Dp120ctn have not yet been reported. In addition to the CCC, a variety of other proteins are found to localize to adherens junctions, and several of these are required for DC. Two such molecules are the PDZ domain proteins Cno and Polychaetoid (Pyd)/ZO1. The PDZ domain is named for a motif found in the MAGUK (membrane-associated guanylate kinase) proteins PSD-95, Discs-large (Dlg, see below), and ZO-1 (reviewed in Harris and Lim, 2001). Pyd/ZO-1 appears to be the Drosophila ortholog of ZO-1, a mammalian tight junction protein (Takahisa et al., 1996; Wei and Ellis, 2001), whereas Cno is homologous to the mammalian adherens junction protein Afadin (reviewed by Tepass et al., 2001). Cno and Pyd/ZO-1 interact genetically and biochemically and may form a complex at the adherens junction that acts as an activator of the JNK cascade (Takahashi et al., 1998). Cno localizes to the triangular junctions at the LE, and cno mutant embryos show a failure of DC characterized by a lack of cell elongation in the epidermis. Loss of JNK signaling in cno mutant embryos is indicated by loss of dpp and puclacZ expression at the LE. Genetic interaction studies conrm that a major route of action for Cno is the JNK pathway. Overexpression of DJNK/Bsk can partially rescue DC defects in cno mutant embryos, whereas reduction in DJNK/Bsk or DJNKK/Hep levels leads to DC failure in cno partial loss-of-function embryos that normally show no DC defects. Cno has been proposed to act upstream of, or in parallel with, Drac1 in its regulation of the JNK cascade as Drac1V12 is capable of driving puc-lacZ expression at the LE in cno mutant embryos. Pyd/ZO-1 appears to collaborate with Cno in its regulation of the JNK cascade during DC. Embryos homozygous for the weak hypomorphic cno allele cnomis1 develop normally, as do embryos homozygous for the hypomorphic pyd allele pydtam1, but embryos doubly homozygous for cnomis1 and pydtam1 show DC defects. Pyd/ZO-1 localizes to the triangular junctions at the LE and binds to Cno in in vitro binding assays and using the yeast two-hybrid system. How Cno and Pyd/ ZO-1 contribute to activation of the JNK cascade remains unknown, but a number of interesting possibilities can be considered. One is that they contribute to the assembly of a localized receptor complex that picks up a signal(s) promoting JNK activation (Takahashi et al., 1998; Noselli and Agnes, 1999). There are precedents for ` PDZ domain proteins contributing to the formation of signaling complexes. For example, in C. elegans vulval development the PDZ proteins LIN-2/LIN-7/LIN-10 form a complex mediating basolateral membrane localization of the receptor tyrosine kinase LET-23 in vulval

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precursor cells (reviewed in Harris and Lim, 2001). This enables localized response to an EGF-like protein (LIN3) secreted into the basal extracellular space by the neighboring anchor cell. By analogy, in the Drosophila embryo receptors may be localized to the dorsal end of each LE cell at the interface with the amnioserosa. As described below, the amnioserosa is a likely source of signal(s) required for DC, and limiting receptor complexes to the dorsal end of the LE cells would allow a localized response involving just the rst row of epidermal cells. A membrane-anchored signal on the surface of an amnioserosa cell could interact with a receptor found at the dorsal end of a neighboring LE cell, similar to the interaction of the Bride of Sevenless protein on the surface of the R8 cell with the Sevenless receptor tyrosine kinase on the neighboring R7 cell during Drosophila photoreceptor specication (reviewed in Raabe, 2000). Cno may also regulate JNK signaling by acting on proteins regulating the cascade. Cno interacts genetically with the DC participants Arm, Mys, Ras1, and N, although these interactions have not been tested in the context of DC (Miyamoto et al., 1995; Matsuo et al., 1997). Furthermore, it has been shown that Cno binds Ras1, so there may be a direct link between Ras1 signaling and Cno function (Kuriyama et al., 1996; Matsuo et al., 1997). As discussed in the section on Wg signaling, Arm makes a positive contribution to activation of the JNK pathway (McEwen et al., 2000). N, on the other hand, is a negative regulator of the JNK pathway, whereas Mys and Ras1 have been implicated in DC but not directly assessed for a role in the cascade (Brown, 1994; Harden et al., 1999; Zecchini et al., 1999). It will be of interest to see if Cno interacts genetically with any of these proteins during DC. a-catenin in the adherens junction binds to F-actin and various proteins interacting with the actin cytoskeleton, suggesting that it organizes a multiprotein complex regulating the actin cytoskeleton (reviewed in Vasioukhin and Fuchs, 2001). The triangular junctions at the LE may participate in the assembly of the actomyosin contractile apparatus, and Cno and Pyd/ZO-1 could have a role in this as the mammalian orthologs of these proteins associate with the actin cytoskeleton (Takahashi et al., 1998). A further indication that adherens junctions contribute to assembly of the LE cytoskeleton comes from a study of the Drosophila Abelson nonreceptor tyrosine kinase, Abl (Grevengoed et al., 2001), a protein enriched at adherens junctions (Bennett and Hoffmann, 1992). Embryos completely devoid of Abl show defects in epithelial 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 uneven distribution of F-actin along the LE, with some areas showing excessive F-actin and other areas being decient. Filopodial extensions are maintained at the LE

in Abl mutant embryos and actually appear to be more numerous than wild-type in late-stage mutant embryos. DC is slowed in abl mutant embryos, and the LE tends to fold under the lateral epidermis. As the actin regulator Ena is a known target of Abl (reviewed in Lanier and Gertler, 2000), Ena was evaluated in the context of Abl function during DC. Ena localizes to adherens junctions, including the triangular junctions at the LE, and its distribution in abl mutant embryos is disrupted similarly to F-actin. Thus, areas where F-actin is lost tend to show reduced Ena staining, whereas areas where Factin is increased show elevated Ena staining. An interaction between Abl and Ena during DC is further supported by the nding that heterozygosity for an ena allele suppresses the embryonic lethality of abl mutant embryos. The presence of Ena at adherens junctions prompted an investigation of the interactions between Abl and Ena and adherens junction components. Mutations in ena increase the severity of DC defects of arm mutant embryos, whereas shg alleles enhance the abl phenotype. Furthermore, in abl mutant embryos, the levels 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 signals into regulation of the actin cytoskeleton through its action on Ena at adherens junctions. Its is tempting to speculate that Abl may act through Drac1 in its regulation of the LE cytoskeleton as Trio, a guanine nucleotide exchange factor that can activate Rac proteins, interacts genetically with Abl during CNS development (reviewed in Lanier and Gertler, 2000).

Involvement of Septate Junction Proteins in DC The insect septate junction, which serves some of the same roles as the vertebrate tight junction, is found just basal to the adherens junction in mature epithelia (reviewed in Tepass et al., 2001). Three proteins required for the integrity of the septate junction, Coracle (Cor), Neurexin IV (Nrx), and Discs-large (Dlg), are essential for DC. Coracle is a member of the Protein 4.1 superfamily of proteins that is localized to the cytoplasmic face of the septate junction (Fehon et al., 1994). Embryos lacking cor show disruption of the septate junction, but apical-basal polarity and epithelial integrity is unaffected (Lamb et al., 1998). Nrx is a transmembrane, septate junction protein belonging to a family of neuronal 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 partner for Protein 4.1 in erythrocytes, and co-immunoprecipitation and in vitro binding studies indicate that Nrx interacts directly with Cor (Baumgartner et al., 1996; Ward et al., 1998). Another indication of the close relationship between 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 pleated septate junctions in ectodermally-derived epithelia appear in the embryo at stage 14, after DC commences, and additional septa are added to junctions throughout the rest of embryogenesis (Tepass and Hartenstein, 1994). If the DC defects in cor and nrx mutant embryos are due to disruption of septate junction formation, they are unlikely to reect a failure to initiate DC but rather a later event. The cuticles of nrx and cor mutant embryos have relatively small dorsal holes and do not exhibit the wide-open dorsal surface seen with loss of some other DC participants (Fehon et al., 1994; Baumgartner et al., 1996; Lamb et al., 1998; Ward et al., 1998). An analysis of embryonic morphology during DC has not been reported for nrx and cor mutants, but the cuticle phenotypes suggest that a considerable amount of closure does take place. Cor antibody staining of wild-type embryos indicates that the protein is not present along the dorsal side of the LE cells (Fehon et al., 1994) or in the amnioserosa during DC (the amnioserosa does not contain septate junctions, Tepass and Hartenstein, 1994). The erythrocyte Protein 4.1 links transmembrane proteins with the spectrin/actin cytoskeleton, but this function may not be conserved in Cor (Fehon et al., 1994). The spectrin/actin binding domain of Protein 4.1 is not conserved in Cor, and Cor does not co-localize substantially with either F-actin or spectrin along the apical-basal axis of the cell. Furthermore, clones of cor mutant cells in imaginal tissues show a normal distribution of F-actin (Lamb et al., 1998). The present data on Cor and Nrx suggest that the septate junction does not function in the organization of the cytoskeleton at the LE during DC. Further support for this comes from the observation that the homophilic adhesion molecule Fasciclin III (Fas III) (Snow et al., 1989), a protein known to associate with septate junctions (Woods et al., 1997), is not present at the dorsal end of LE cells during DC but is present on their other surfaces (Martinez-Arias, 1993). A potential role for the septate junction could be in establishing cell-cell adhesion between LE cells from opposite sides of the embryo when they meet at the dorsal midline (see below), and the DC defects of cor and nrx mutants may be due to loss of such adhesion. The third septate junction protein required for DC is Dlg, which has a more extensive role in this process than Cor or Nrx. Loss of Dlg can lead to a complete failure of epidermal migration during DC, as indicated by cuticle preparations and scanning electron microgaphs of embryos (Perrimon, 1988). Dlg is a MAGUK tumor suppressor that, similar to Cor and Nrx, is required for septate junction formation (Woods and Bryant, 1991; Woods et al., 1996). However, unlike Cor, lack of Dlg also leads to disruption of apical-basal polarity in epithelia (Woods et al., 1996). Dlg functions together with two other proteins, Lethal giant larvae (Lgl) and Scribble (Scrib), in the regulation of epithelial polarity (Bilder et al., 2000). Lgl is

a tumor suppressor protein required for DC (Manfruelli et al., 1996; Arquier et al., 2001). Embryos decient in Lgl show a strong DC phenotype characterized by a lack of cell shape change in the epidermis and a failure of epidermal migration. Scrib is a PDZ domain tumor suppressor protein, the complete lack of which causes extensive disorganization of embryonic epithelia (Bilder and Perrimon, 2000). This phenotype precludes a direct evaluation of a role for Scrib in DC, however, a genetic interaction between Scrib and Lgl suggests that Scrib function is required. Embryos containing maternal Scrib but lacking zygotic Scrib hatch into larvae but when made heterozygous for an allele of dlg die as embryos with DC defects (Bilder et al., 2000). Scrib and Dlg co-localize throughout development, and following gastrulation remain at the apical margin of the lateral membrane (ALM) at a position that becomes the septate junction (Woods and Bryant, 1991; Woods et al., 1996; Bilder et al., 2000; Bilder and Perrimon, 2000). Lgl shows a more dispersed distribution and is not restricted to the plasma membrane but does overlap with Scrib and Dlg at the ALM (Strand et al., 1994b; Bilder et al., 2000). Scrib, Dlg, and Lgl are mutually dependent for their localization (Bilder et al., 2000) and likely function as an interacting group of proteins that has been called the Lgl complex (Tepass et al., 2001). In the early embryo, the Lgl complex regulates epithelial polarity, and among the effects of disrupting the complex are defects in adherens junction formation and mislocalization of the adherens junction protein Arm (Bilder et al., 2000; Bilder and Perrimon, 2000). Later in embryogenesis, the Lgl complex may contribute to septate junction 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 Lgl complex are likely partly due to disruption of septate junction formation. However, these DC defects are more severe than those resulting from loss of Cor or Nrx, and there are other requirements for the Lgl complex in DC. Given the participation of adherens junctions proteins such as Arm in DC, the disruption of adherens junctions and Arm localization through loss of the Lgl complex is almost certainly going to affect DC. The Lgl protein further regulates DC through its effects on LE myosin and Dpp/TGF-b signaling (Arquier et al., 2001). Lgl associates with the zip product nonmuscle myosin-II heavy chain (Strand et al., 1994a), and the lgl and zip genes interact genetically in the determination of neuroblast polarity (Ohshiro et al., 2000; Peng et al., 2000). The nature of this genetic interaction indicates that Lgl is a negative regulator of myosin function. Consistent with this, loss-of-function lgl alleles suppress the DC phenotype in zip mutant embryos. Lgl also indirectly makes a positive contribution to myosin function during DC as it is required for the transcription of zip in the LE cells. There is evidence that Lgl is required for emission of Dpp by the various cells producing this ligand (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 loss of activation of the Tkv/Put pathway, which is required for the LE expression of zip (see section on Dpp/TGFb signaling). The positive contribution of Lgl to zygotic zip transcription would be irrelevant in a zip mutant embryo, and the suppression of the zip mutant DC phenotype by loss of Lgl may be due to a reduced negative regulation of the function of maternally supplied myosin.

Signaling from the amnioserosa establishes LE cell identity

We have seen that the LE cells during DC exhibit complex signaling driving cytoskeletal organization, cell shape changes, and gene expression. It is clear that the LE cells have a distinct identity from other epidermal cells and are a crucial component of DC. A key issue in understanding DC, therefore, is how the LE cell fate is specied, and several recent studies demonstrate that signaling from the neighboring amnioserosa is involved. Stronach and Perrimon (2001) genetically altered the position of the interface between the amnioserosa and dorsal epidermis using mutations affecting DV patterning and found that the LE fate always arose as a single row of cells at the interface. They used two criteria to dene LE fate: puc-lacZ expression, which indicates the presence of JNK signaling, and asymmetrical staining for Fas III. The specication of LE cells at the physical juxtaposition of amnioserosa and dorsal epidermis suggests that there is inductive 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), is required for amnioserosa maintenance, germband retraction, and DC (Strecker et al. 1995; Frank and Rushlow, 1996; Reed et al., 2001; Stronach and Perrimon, 2001). The DC defects in embryos mutant for hnt, a gene encoding a Zinc-nger protein, can be suppressed by reducing the dose of DJNK with a bsk allele, indicating that JNK signaling is upregulated by loss of Hnt function (Reed et al., 2001). In wild-type embryos, there is JNK activity in the amnioserosa prior to DC, but by the commencement of DC, this has been downregulated (Reed et al., 2001). In both hnt mutant embryos and puc mutant embryos, JNK signaling persists in the amnioserosa 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 JNK signaling underlies defects at the LE. In hnt mutant embryos, phosphotyrosine staining in the triangular junctions at the LE is missing. The LE phosphotyrosine can be restored in hnt mutant embryos by downregulating JNK signaling in the amnioserosa through expression of transgenes of either puc or dominant-negative DJNK/ Bsk. Phosphotyrosine accumulation at the LE occurs independently 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 two distinct signaling events operating between the amnioserosa and the dorsal epidermis contributing to the LE cell fate. One is required for JNK pathway activation in the LE cells and requires the juxtaposition of amnioserosa with dorsal epidermis (Stronach and Perrimon, 2001). A potential contributor to activation of this signaling from the amnioserosa is ush, a gene encoding another Zinc-nger protein, as ush mutant embryos lack dpp expression at the LE (Stronach and Perrimon, 2001) and exhibit dorsal holes in cuticle preparations (Strecker et al., 1995; Frank and Rushlow, 1996). A second signal, which requires Hnt- and Puc-dependent downregulation of the JNK cascade in the amnioserosa, is needed for the accumulation of phosphotyrosine in the triangular junctions at the LE. Hnt function in amnioserosa cells abutting the dorsal epidermis establishes a germband retraction signal detected by the Drosophila insulin receptor (Inr) in the epidermis (Lamka and Lipshitz, 1999). It is possible that the Hnt-dependent signal from the amnioserosa to dorsal epidermis required for DC is conveyed in the same manner as that required for germband retraction, i.e. through Inr. In addition to germband retraction failure, DC defects have been reported in Inr decient embryos (Fernandez et al., 1995). The lack of phosphotyrosine staining in hnt mutant embryos suggests that the specialized adherens junctions at the LE are disrupted. As discussed above, these structures have roles in cytoskeletal organization and consistent with this, hnt mutant embryos show disruption of F-actin at the LE (Reed et al., 2001). In addition to signaling via protein cascades, the amnioserosa may also provide mechanical signals that contribute to establishment of the LE during DC. Alterations in the tension applied to cultured epithelial cells can promote reorganization of the actin cytoskeleton; wounding a tissue creates a similar mechanical cue that may contribute to initiation of the wound healing process (reviewed in Jacinto et al., 2001). It is conceivable that germband retraction of the Drosophila embryo prior to DC and/or alterations in adhesion between the amnioserosa and the LE will change the forces being applied to the LE cells and contribute to cytoskeletal reorganization. Consistent with this idea, ablation of LE cells, which alters the tension applied to the epidermis, is rapidly followed by assembly of a contractile apparatus in more ventrally located epidermal cells (Kiehart et al., 2000).

Other players in DC
A number of DC participants have been identied which dont as yet t into any of the various pathways or groupings laid out above. These are discussed briey in this section.

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Ribbon Embryos bearing mutations in the ribbon (rib) locus show defects in the dorsal cuticle ranging from small holes and puckers to a large dorsal hole (Nsslein-Volhard et al., 1984; Bradley and Andrew, 2001). rib mutant embryos show loss of LE myosin and a reduction in LE F-actin and epidermal cell elongation is impaired (Blake et al., 1998). These embryos also show some ectopic dpp expression in the epidermis, but no attempt has been made to look for genetic interactions with JNK pathway components (Bradley and Andrew, 2001). Rib contains a BTB/POZ motif, a protein-protein interaction domain found in a diverse collection of proteins (Bradley and Andrew, 2001). Some of the phenotypes of rib mutant embryos are suppressed by zip alleles, suggesting a role for Rib in cytoskeletal regulation (Blake et al., 1999).

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

Sealing the dorsal hole

As the migrating epidermal anks meet up at the dorsal midline during DC, new cell-cell adhesions must form to seal the dorsal surface. The lopodia extending from each LE interdigitate at the dorsal midline and appear to prime the formation of adherens junctions between the two rows of LE cells (Jacinto et al., 2000; 2001). Newly formed septate junctions are also used to seal the dorsal hole. The septate junction proteins Fas III and Cor, which are excluded from the dorsal end of each LE cell during DC, appear at this position after the opposing epidermal anks meet up (Martinez-Arias, 1993; Fehon et al., 1994). Interestingly, embryos in which epidermal migration fails due to loss of various DC proteins often show accumulation of Fas III at the dorsal end of LE cells (Ring and Martinez Arias, 1993; Harden et al., 1995; Manfruelli et al., 1996; Erickson et al., 1997; Lu and Settleman, 1999; Magie et al., 1999). This suggests that the accumulation of Fas III at the end of DC is not mediated by contact between the two LEs, but by a signal that persists even when DC fails.

N-myristoyltransferase Embryos lacking Drosophila N-myristoyltransferase (dNMT), an enzyme that catalyzes the addition of the fatty acid myristic acid to an N-terminal glycine residue of various proteins, have DC defects (Ntwasa et al., 2001). The DC defects in dNMT mutant embryos are likely due to disrupted function of myristoyl proteins required for DC (Ntwasa et al., 2001). Two such proteins are Src42A and Src64, which are both normally myristoylated (Tateno et al., 2000; Ntwasa et al., 2001).

Pulling it all together

Fig. 3 is a schematic diagram of proposed signaling pathways operating in the epidermis during DC, which will be outlined briey here. In response to signals from the amnioserosa, and possibly elsewhere, the LE cells are induced to adopt their unique identity which includes an active JNK cascade and a polarized accumulation of Factin and myosin at the dorsal end of the cell. Proteins associated with cell-cell junctions are instrumental in establishing these characteristics. The JNK cascade is positively and negatively regulated by numerous proteins, including members of the Wg pathway. The JNK cascade leads to Dpp secretion by the LE cells, which triggers signaling in both LE and lateral epidermal cells through the Tkv/Put receptor complex. The consequences of this signaling remain unclear but may include regulation of the LE cytoskeleton and promotion of cell shape change in the lateral epidermal cells. The LE cytoskeleton promotes DC in several ways. The contractile apparatus at the dorsal end of each LE cell drives cell elongation through constriction, while the F-actin-based lamellipodia and lopodia that extend from the LE guide the movement of the epidermal sheet and ensure uniform closure. The assembly and function of the LE cytoskeleton is regulated by numerous proteins including the Rho family of small GTPases, and its integrity is dependent on transcriptional regulation downstream of

Steroid hormones Ecdysteroids regulate many developmental events in Drosophila, and recent work on the disembodied (dib) gene, which encodes a cytochrome P450 enzyme involved in embryonic ecdysteroid biosynthesis, implicates these hormones in regulation of DC (Chavez et al., 2000). Many dib mutant embryos fail to complete DC and show reduced titers of the ecdysteroids ecdysone and 20-hydroxyecdysone. Another indication for steroid hormone participation in DC comes from a study addressing the effects of the glucocorticoid, dexamethasone, on Drosophila embryonic development. Embryos exposed to dexamethasone show germband retraction defects and holes in the dorsal cuticle, a phenotype similar to mutants in the U-shaped genes (Strecker et al., 1995).

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

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199 Fig. 3 Schematic diagram summarizing signaling occurring during DC. Note that some of the interactions depicted remain speculative and not all known DC participants are shown. The diagram is centered on the JNK cascade (highlighted in red) in a LE cell migrating towards the top right hand corner of the gure. The LE accumulation of F-actin and myosin and extensions of lopodia and lamellipodia are shown in red. The triangular adherens junctions at the dorsal end of the LE cell are shown in green. Dpp/ TGF-b signaling events (highlighted in blue) in LE cells and a more ventrally located epidermal cell are also summarized. See text for details. Arquier, N., Perrin, L., Manfruelli, P. and Semeriva, M. (2001) The Drosophila tumor suppressor gene lethal(2)giant larvae is required for the emission of the Decapentaplegic signal. Development 128:22092220. Artavanis-Tsakonas, S., Matsuno, K. and Fortini, M.E. (1995) Notch signaling. Science 268:225232. Bard, J. (1990) Morphogenesis: The Cellular and Molecular Processes of Developmental Anatomy, rst edn. Cambridge University Press, Cambridge, U.K.. 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 87:10591068. Ben-Yaacov, S., Le Borgne, R., Abramson, I., Schweisguth, F. and Schejter, E.D. (2001) Wasp, the Drosophila Wiskott-Aldrich syndrome gene homologue, is required for cell fate decisions mediated by Notch signaling. J Cell Biol 152:113. Bennett, R.L. and Hoffmann, F.M. (1992) Increased levels of the Drosophila Abelson tyrosine kinase in nerves and muscles: subcellular localization and mutant phenotypes imply a role in cellcell interactions. Development 116:953966. Bilder, D., Li, M. and Perrimon, N. (2000) Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. Science 289:113116. Bilder, D. and Perrimon, N. (2000) Localization of apical epithelial determinants by the basolateral PDZ protein Scribble. Nature 403:676680. Bishop, A.L. and Hall, A. (2000) Rho GTPases and their effector proteins. Biochem J 348(Pt 2):241255. Blake, K.J., Myette, G. and Jack, J. (1998) The products of ribbon and raw are necessary for proper cell shape and cellular localization of nonmuscle myosin in Drosophila. Dev Biol 203:177188. Blake, K.J., Myette, G. and Jack, J. (1999) ribbon, raw, and zipper have distinct functions in reshaping the Drosophila cytoskeleton. Dev Genes Evol 209:555559. Bock, B.C., Vacratsis, P.O., Qamirani, E. and Gallo, K.A. (2000) Cdc42-induced activation of the mixed-lineage kinase SPRK in vivo. Requirement of the Cdc42/Rac interactive binding motif and changes in phosphorylation. J Biol Chem 275:1423114241. Boutros, M. and Mlodzik, M. (1999) Dishevelled: at the crossroads of divergent intracellular signaling pathways. Mech Dev 83:27 37. Bradley, P.L. and Andrew, D.J. (2001) ribbon encodes a novel BTB/ POZ protein required for directed cell migration in Drosophila melanogaster. Development 128:30013015. Brennan, K., Baylies, M. and Martinez Arias, A. (1999a) Repression by Notch is required before Wingless signalling during muscle progenitor cell development in Drosophila. Curr Biol 9:707710. Brennan, K., Tateson, R., Lieber, T., Couso, J.P., Zecchini, V. and Martinez Arias, A. (1999b) The abruptex mutations of Notch disrupt the establishment of proneural clusters in Drosophila. Dev Biol 216:230242. Bresnick, A.R. (1999) Molecular mechanisms of nonmuscle myosin-II regulation. Curr Opin Cell Biol 11:2633. Brown, N.H. (1994) Null mutations in the aPS2 and bPS integrin subunit genes have distinct phenotypes. Development 120:1221 1231. Brown, N.H., Gregory, S.L. and Martin-Bermudo, M.D. (2000) Integrins as mediators of morphogenesis in Drosophila. Dev Biol 223:116. Brummel, T.J., Twombly, V., Marques, G., Wrana, J.L., Newfeld, S.J., Attisano, L., Massague, J., OConnor, M.B. and Gelbart, W.M. (1994) Characterization and relationship of Dpp receptors encoded by the saxophone and thick veins genes in Drosophila. Cell 78:251261. Burbelo, P.D., Drechsel, D. and Hall, A. (1995) A conserved binding motif denes numerous candidate target proteins for both Cdc42 and Rac GTPases. J Biol Chem 270:2907129074.

the JNK cascade, for example upregulation of transcription from the chic and zip genes. Many of the proteins participating in DC are involved in other epithelial closure events in both invertebrates and vertebrates. Wound healing in Drosophila requires the JNK cascade (Rmet et al., 2002), while thorax closure in Drosophila requires both JNK and Dpp/TGFb signaling (Zeitlinger et al., 1997; Simin et al., 1998; Takahashi et al., 1998; Agnes et al., 1999; Zeitlinger and ` Bohmann, 1999; Fernandez et al., 2000; Martn-Blanco et al., 2000; Tateno et al., 2000). TGF-b signaling and Rho family small GTPase function have been implicated in wound healing in vertebrates (reviewed in Jacinto et al., 2001), and various adherens junction proteins participate in ventral enclosure in C. elegans (reviewed in (Simske and Hardin, 2001). It will be of interest to see how many more parallels with DC emerge as these other processes are better characterized.
Acknowledgements I thank B. Reed and M. Ricos for numerous discussions about dorsal closure, and E. Verheyen, C. Langmann, and B. Reed for their comments on the manuscript. Work in my laboratory is supported by grants from the Canadian Institutes of Health Research, the National Cancer Institute of Canada, and the Natural Sciences and Engineering Research Council of Canada.

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