Roles of the JNK signaling pathway in Drosophila morphogenesis

Roles of the JNK signaling pathway in Drosophila morphogenesis

466 Roles of the JNK signaling pathway in Drosophila Stbphane NoseHi*? and Fraqois Agnkst Epithelial cell differentiation and morphogenesis many a...

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Roles of the JNK signaling pathway in Drosophila Stbphane NoseHi*? and Fraqois Agnkst Epithelial

cell differentiation

and morphogenesis

many aspects of metazoan studies in Drosophila have amino-terminal kinase epithelial morphogenesis dorsal



development. revealed that



in the control


of planar


in Genetics


studies have linked the and Frizzled pathways degree of integrative



Addresses *Department of Genetics, Harvard Medical School, Avenue, Boston, Massachussetts 02115, USA; e-mail: [email protected] fCentre de Biologie du Developpement, UMR5547 Narbonne, 31062 Toulouse Cedex, France Current


(JNK) signaling pathway regulates during the process of embryonic

in several tissues. Importantly, these JNK pathway to the decapentaplegic these processes, suggesting a high signaling

are crucial

Recent genetic the conserved Jun

& Development


200 Longwood

1 16, route de 9:466-472 0 Elsevier



Abbreviations DC dorsal closure decapentaplegic dw JNK Jun amino-terminal MAPK mitogen-activated mbc myoblast city LE leading edge


kinase protein kinase

Introduction The study of signal transduction pathways has provided nice models of cell communication controlling differentiation, patterning and development. Among the known signaling cascades, the mitogen-activated protein kinase (MAPK) pathways have rapidly attracted a wide interest for several reasons [l]. First, at least three related but different MAPK pathways -the extracellular regulated kinase [ERK], Jun amino-terminal kinase and p38 -can be distinguished in metazoa that mediate specific cell and developmental responses. Second, MAPK pathways are versatile: for example, the JNK pathway can transduce signals of a very diverse nature, including unrelated extracellular stresses as well as developmental signals [l-3]. Finally, the rapid accumulation of information from molecular cloning of components of MAPK pathways in several species has revealed a strong evolutionary conservation, which may ultimately provide a wide view of the biology of these pathways. DrosopMa can be used as a good genetic model to study the role of MAPK pathways in development, an approach that is well supported by the existence in flies of homologous signaling cascades [4,5,6’]. In this review, we discuss the most recent studies on the role of JNK signaling in dorsal closure and also new emerging roles of this pathway in epithelial morphogenesis.

The Drosophila dorsal closure


JNK cascade


During the first half of embryonic development, the dorsal part of the embryo is occupied by a stretched epithelium (the amnioserosa), which by mid-embryogenesis is progressively covered by lateral ectodermal cells undergoing dorsal closure (DC): these epithelial cells elongating dorsally and moving in concert toward the dorsal midline where they fuse at the end of the process [2,7-91. Several mutations collectively known as the ‘dorsal open’ group affect the process of dorsal closure (Table l), which represent a unique collection of genes with specific functions in morphogenesis. A major goal is to determine the molecular and genetic interactions organizing these functions in development. An important initial step in DC is the differentiation within each lateral epithelium of a line of cells forming a leading edge (LE). These cells, which contact the amnioserosa, are the site of JNK activity during DC [Z]. The JNK pathway is best viewed as a linear cascade, comprising the hemipterous/DJNKK [lo], baskef/DJNK [11,12] and Djun [13-151 genes (Figure 1). When mutated, these genes lead to a typical ‘dorsal open’ phenotype reflecting a failure of the lateral ectoderm to move dorsally. These defects are accompanied by aberrant accumulation of a number of membrane-associated products, including F-actin, nonmuscle myosin, fasciclin III and proteins containing phosphotyrosines. In addition, LE-specific expression of puckered and decapentapbgic (dpp), two targets of the JNK pathway (see below), is abolished [10,14-171. In vitro studies have shown that the Hemipterous (DJNKK) protein can phosphorylate Basket (DJNK) which, in turn, can activate Djun by phosphorylation [11,12,17]. In addition to Djun, two other transcription factors have been identified as essential for DC -the Drosophila homolog of Fos, Dfos protein anterior open [18,191, and the ETS-domain (aop)/yan [18]. Both Djun and Dfos act as positive regulators, probably through the formation of heterodimers, whereas aop functions as a negative regulator in the process. Overexpression of aop provokes DC defects, whereas expression of an activated form of Djun can rescue the DC defect associated with basket mutations [13,15]. These data, together with recent knockouts in mouse [20,21], clearly demonstrate that the JNK pathway and&n have essential functions during normal development. More recently, new upstream components have been identified. midapen encodes a steZO-related kinase of the SPS-1 family [ZZ’], with homologs in vertebrates (NIK, Nck-inceracting kinase) and the nematode worm Caenorhabdits elegans (,mig-15). In contrast to other steZO-related molecules like DPAK [23], misshapen does not have a rac/cdc4Zbinding domain indicating that the two kinases function differently. Zygotic loss of misshapen function induces a dorsal-open



of the JNK signaling


in Drosophila







The Drosophila JNK pathway controls dorsal closure and planar polarity. During dorsal closure, the JNK signaling pathway is activated in the LE by an unknown signal, and one important outcome of this signaling activity is the induction of drop (the product of which is a TGF-6 homolog) expression in these cells. This coupling of JNK and dpp signaling pathways is proposed to control morphogenesis of the more lateral ectodermal cells by the dpp pathway. In addition to JNK signal transducers, membrane-associated proteins participate in the elaboration of normal JNK and cytoskeletal activities to realize a concerted movement. In planar polarity, the coupling of frizzledldishevelled and JNK activities signals the correct arrangement of cells within the plane in the eye imaginal disc. Arrows indicate activation and lines ending in a bar represent repressor functions. bsk, basket; Cno, Canoe; Cor, Coracle; Dlg, Discs large; DSH, dishevelled; Fz, Frizzled; hep, hemipterous; nrx, neurexin; msn, misshapen; put, puckered, put, punt; shn, schnurri; tkv, thickveins.


? t


DRacl DrhoA




Cor nrx IV

Dracl MEKK

t hep/DJNKK

t hep/DJNKK

t bsk/DJNK

t bsk/DJNK


Cno 20-l

t Djun

4 tkv put shn Dfos




closure Current

phenotype and a reduction of dpp LE expression in some embryos. Genetic epistasis experiments are consistent with misshapen acting upstream of HEP, probably activating an as yet unknown MEKK [ZZ’]. In a two-hybrid screen using misshapen as a bait, a Drosophila TNF-receptor-associated factor (DTRAFl) has been identified [24’]. These proteins are proposed to link TNF receptors to the JNK pathway in inflammation. In mouse TRAFZ homozygous mutant cells, activation of the JNK pathway following exposure to TNF is abolished [25]. Database searches have revealed the presence of another TRAF gene in Drosophil’a (DTRAFZ), raising the possibility that several of these molecules may act in DC or that the JNK pathway may be activated by different TRAFs in different tissues [W]. However, a demonstration that DTRAFs act in the Drosophila JNK pathway awaits the isolation of mutations in these genes. In vertebrate cells, a role for the small GTPases of the Rho family in JNK activation and in the regulation of the actin cytoskeleton has been well documented [26,27]. In Drosophila, expression of dominant negative Dracl, Dcdc42 or DrhoA in the ectoderm leads to DC phenotypes [ 11,28,29’,30’] and embryos mutant for DRhoA have a mild dorsal-open phenotype [31]. Their role as potent JNK pathway activators in Drosophila is shown by the ability of activated DraclVlZ and Dcdc42VlZ to induce the expres-


in Genetics

& Development

sion of puckered and dpp outside the LE, an activity that depends on the downstream activity of /zemipterow [16]. Interestingly, the effects of both molecules are slightly different, suggesting that they play only partly redundant roles in the process. Further support for this view is provided by monitoring the formation of the actin-myosin cytoskeleton when negative forms of Dracl, D&42 and DrhoA are differentially expressed in the LE: DraclNl7 disrupts actin and myosin assembly in the LE more efficiently than Dcdc4ZN17 [29’]. Dcdc42N17 also affects accumulation of DPAK, a potential Dracl/Dcdc42 effector, to downregulate cytoskeleton assembly. Surprisingly, the effects of DrhoAN19 on cytoskeletal assembly are restricted to cells flanking the segment borders. It will be interesting to confirm this result using DrhoA mutants and test the possibility that local changes along the AP axis may have a role in establishing a functional LE. Signaling at a border The accurate regulation of JNK activity in the LE is important to ensure proper morphogenesis, as illustrated by loss- and gain-of-function JNK pathway mutants. How is this activity regulated during DC? Although an activating signal for this pathway has not yet been identified in Drosophda, the phenotypic and molecular analysis ofpuckered revealed an important mechanism whereby JNK




and developmental


activity is downregulated [16,32’]. In puckered embryos, closure proceeds almost normally and the main defect is a puckering of the dorsal midline [33]. This phenotype suggests that puckered is not essential for the spreading of the ectoderm. Interestingly, when the activity of the pathway is monitored using dpp and puckered itself, a novel phenotype is observed which is opposite to the one observed in hemipterous, basket or Djun mutants: both dpp and puckered are overexpressed in the LE [16,32’]. These observations indicate that puckered is a repressor of the JNK pathway in the LE. Consistent with this, cloning of the puckered gene revealed that it encodes a MAPK phosphatase of the VH-1 family [32’]. In extracts prepared from puckered mutant embryos, the activity of the DJNK/BSK protein is specifically elevated relative to wild type, whereas activity of the rolled/ERK MAPK remained constant. As expected of a negative regulator, overexpression of puckered leads to a strong dorsal-open phenotype [32’]. The existence of a puckered-dependent negative feedback loop controlling the level of JNK signaling activity in the LE may serve two important roles: the first one is to turn off signaling to stop the accompanying movement of lateral cells at the end of the process, thus promoting contact inhibition and suture of the LEs. Constitutive morphogenetic activity at the time of suture probably leads to a LE conflict at the midline and the resulting puckering. Second, the forces that drive movement need to have their strength regulated for coordinating lateral ectoderm’s pace, and a gradient of dpp, a Drosophila TGF-B homolog, is proposed to control these morphogenetic parameters remotely [Z]. Thus, coupling dpp expression to JNK activity in the LE is a nice mechanism that may create diversity in several related morphogenetic movements. Dorsal closure and the membrane Another class of mutants described in this section exhibit a clear dorsal open phenotype. In contrast to the JNK pathway genes described above, they affect only moderately if at all, the expression of dpp and puckered. These genes may therefore correspond to components acting dowstream of or parallel to the JNK pathway, although a direct regulatory link between JNK activation and the membrane is still lacking. An emerging view is that some membrane-associated proteins may provide a specialized environment for appropriate JNK and/or dpp signaling, as well as regulating the assembly of cytoskeletal complexes. The cell-shape changes by membrane and (Table l), like the two 20-l that link the [34,35’]. PDZ-domain tion that are generally where they appear to example by clustering synapse [36].

accompanying DC are mediated cytoskeletal effector proteins PDZ-domain proteins Canoe and membrane and JNK signaling proteins mediate complex formabound to the plasma membrane, assemble signaling activities, for receptors at the neuromuscular

Table 1 Dorsal closure

genes. Protein

Gene JNK pathway DrhoA Dracl Dcdc42 DPAK Dtrafl and 2 misshapen hemipterous basket Dfos Djun anterior open (AoplYan) puckered




Small GTPase Small GTPase Small GTPase p21 -activated kinase TNF-receptor associated factor stelO-related kinase Jun amino-terminal kinase kinase Jun amino-terminal kinase AP-1 complex AP-1 complex ETS-domain Transcription factor Dual specificity MAPK Phosphatase (VH-1)


EWI [281 1231 124’1 122’1 1101 [11,121

118,191 [13-151 1151 [32’1


d/w thickveins punt schnurri

TGF-5 TGF-jT Transcription

Membrane junctions canoe polychaetoid coracle neurexin IV dig mbc myospheroid scab Cytoskeleton actin zipper lethal(2)giant Others DCgl ribbon raw


AF6 homolog (PDZ domain) homolog (MAGUK, PDZ domain) 4-l protein Caspr homolog MAGUK (PDZ domain) Dock1 80 lntegrin j3 subunit lntegrin Q subunit

Non larvae

CTG F-$1 (type I) receptor (type II) receptor factor (zinc finger)

Filamentous actin muscle myosin heavy Pioneer protein


IV ? ?


[14-161 [6 l-651 [65-671 168-701

[34,35’1 135’1

[42,431 141

[411 [39,40-l [711


191 [731

[741 1751


Different classes of DC genes can be distinguished: genes involved signaling in the leading edge (JNK pathway) and genes that are proposed to respond to dpp in the lateral ectoderm (dpp pathway); and genes encoding membrane-associated proteins that are components of the cytoskeleton and/or the adherens and septate junctions. Genes in italics are candidate DC genes for which mutations have not yet been described that affect this process. Guestion marks indicate that the gene has not been cloned. References have been selected for those describing the role of the corresponding genes in DC.


Canoe and 20-l are co-localized at the adherens junctions and interact both genetically and biochemically [35’]. Homozygous canoe as well as a synthetic lethal canoe, polyckaetoid (pyd, the gene encoding Drosophdia 20-l) combination lead to dorsal-open embryos. Evidence that canoe, but also pyd, participate in JNK signaling is supported by genetic interactions with Aemipterous and basket in embryos and adults. Furthermore, packered and dpp expression is reduced but not abolished in canoe mutants. A proposed model is that a Canoe-20-l complex may assemble or cluster


of the JNK signaling

signaling molecules (receptor[s]?) at the adherens junction that promote JNK activity [35’]. DOCKl80, an SH3-containing protein, has been implicated in the regulation of cell morphology [37,38]. Myoblast city (mbc), a DrosopMa homolog of DOCK180, affects DC and a proportion of mbcmutant embryos adopt a rounded shape and express Fasciclin III abnormally [39]. In addition, these embryos accumulate lessfilamentous actin than the wild type. These cytoskeletal changes are thought to be mediated specifically by DRacl, asmbc strongly suppresses Dracl-mediated dominant phenotypes in the adult fly eye and specifically binds to Dracl in vitro [40’]. dpp expression in mbc mutant embryos is only moderately affected, suggesting that mbccontributes to JNK activation, possibly via Dracl. In support of this function, DOCK180 has been shown to activate JNKl specifically and induce an increase in c-Jun phosphorylation in a Racl-dependent manner [37]. Thus, mbc may contribute to the dual function of Dracl in DC, that is, by regulating both the cytoskeleton and JNK. Interestingly, the subcellular localization of mbc/DOCK180 to membrane ruffles suggestsa model in which compartmentalization of Dracl/mbc in separatepools may convey the two complementary functions [40’]. The septate junctions in insects are thought to be functionally equivalent to the tight junctions in vertebrate epithelial cells. Interestingly, three proteins that are associated with the septate junction, discs-large (dlg; [41]), coracle [42,43] and neurexin IV (nrx; [44]), cause DC defects when the corresponding gene is mutated. Both a’~ and wx are required for correct subcellular localization of coracle [45,46]. In vitro studies show that nrx and coracle interact and it is likely that dlg and coracle associate,as do their mammalian homologs [47,48]. These results suggest that maintenance of epithelium integrity via the septate junctions is crucial for morphogenesis. However, phenotypic analysis indicates that coracle, although an integral component of the septate junction, is not necessaryfor apical-basal polarity and epithelial integrity; rather, it is proposed that dpp signaling from the LE requires a unique apical environment, provided at least in part by coracle proteins [43]. JNK signaling

and tissue


Planar or tissue polarity is characterized by the orientation of hairs or cells perpendicular to the apical-basal axis [49,50]. In DrosopMa, tissue polarity is controlled by the seven-passfrizzled receptor [Sl] and, recently, several members of the JNK signaling pathway have been identified downstream of this protein. DRhoA, a small GTPase required in DC, was shown to control the rotation of ommatidia in eyes and the orientation of wing hairs in adult flies [31]. Furthermore, genetic interactions have indicated a role for hemipterozls, basket and Djun in mediating ftixded signaling for eye polarity. Indeed, the multi-domain dsh protein was identified in flies and



Drosophila morphogenesis





vertebrates as a crucial adapter linking frizded and the JNK pathway in tissue polarity [.52,53’,54’,55]. Interestingly, the DEP domain of a’i.rhrnelLed is specifically required for tissue polarity but not for wingless signaling, as demonstrated by structure-function analysis [53’,54’]. Moreover, the DEP domain was shown to target dishevelled to the membrane, suggesting that translocation is important for tissue polarity function and further cytoskeletal orientation [54’]. In Drosophda, d&?ee)eLedl,a mutation displaying tissue polarity defects only, interacts genetically with several members of the JNK pathway and maps in the DEP domain. In vitro, dishevelledl is no longer able to activate Djun phosphorylation, nor does a truncated form of Dvl (the mouse ortholog of dishevelled) in COS-7 cells [53’,55]. As /iemipterovs or basket mutants only have subtle tissue polarity phenotypes, it is proposed that d&eveLr’edcan activate several related JNK and/or p38 MAPKs that may act redundantly in tissue polarity [53’]. Strikingly, planar polarity is also observed in the LE, as reflected by the asymmetric localization of several proteins along the dorsal-ventral axis (dorsal accumulation of filamentous actin, myosin, DrosopMa p21-activated kinase, or dorsal exclusion of Fasciclin III and coracle; [8,9,23,42]). In JNK pathway mutants, like Djun andpuckered, asymmetric localization in the LE is lost [14,33] but aspuckered mutant embryos can still close the ectoderm, it is not clear whether asymmetric development is essential for LE function and DC. Interestingly, the fact that at least some members of the JNK pathway mediate Frizzled signaling raises the possibility that Frizzled or a Frizzled-like receptor may activate the DC pathway.


and perspectives

In conclusion, the studies of DC and tissue polarity illustrate JNK signaling versatility and how a single pathway may regulate different aspectsof epithelial morphogenesis. However, the JNK pathway is not essential for all epithelia in DrosopAda, indicating that other mechanisms remain to be discovered. One important future direction will certainly be the quest for signal(s) and receptor(s) acting in DC. On the basis of our current understanding of the JNK pathway in DrosopMa, it is tempting to speculate that related processes - that is, those morphogenetic movements employing epithelia with ‘free’ or differentiated edges [56], including metamorphosis (F Agnes et al., unpublished data), ventral enclosure in Caenor~abditus elegans [57], epiboly and wound-healing in vertebrates [58,59] - may also involve JNK signaling.

Acknowledgements We wish to thank D Gibbs and B Mathey-Prevost for critically reading the manuscript. S Noselli thanks N Perrimon for discussions. F Aants is supported by a fellowship from Ligue Nationale Contre le Can&. This work is supported by the Centre National de la Recherche Scientifique (CNRS), thd North Atlantic Treaty Organisation (NATO), Association pour la Recherche sur le Cancer and Ligue Contre le Cancer.




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