basket/JNK

Embryonic

Early DJNK staining is detected in cleavage stage embryos and appears to represent expression of maternal mRNA. Zygotic DJNK staining is present in all germ layers during germ-band extension. During germ-band retraction, the same intense staining remains in the epidermis and the central nervous system. When dorsal closure begins, Basket/JNK staining is increased in the CNS and is present in the lateral epidermal region extending up to the amnioserosa. At stage 15, DJNK stains in the ventral nerve cord, the brain and some peripheral neurons (Sluss, 1996).

In head midline structures, in particular the optic lobe and stomatogastric nervous system, there may be a late phase of EGFR signaling (as assayed by the expression of aos and activated ERK) whose significance is not yet known. EGFR signaling could be involved in modifying the inhibitory feed-back loop between neurogenic and proneural genes that exists in other neurectoderm cells. In the head midline neurectoderm, regulation of proneural and neurogenic genes has to be different. Thus, instead of a short burst of proneural gene expression in proneural clusters that is resolved into expression in individual neuroblasts, proneural genes are expressed for a long period of time; at the same time, the expression is never restricted to single neuroblasts. Since genes of the E(spl) complex are expressed in the same cells that express lísc, the inhibitory loop between E(spl)-C and proneural genes must be interrupted at some level. It is possible that Egfr signaling is causing the interruption of this inhibitory loop. Based on genetic studies of Notch and Egfr signaling in the compound eye, it has been speculated that one of the consequences of Egfr activation (which ultimately is required for all ommatidial cell types to differentiate) is to inhibit N signaling, since constitutively active N inhibits ommatidial cell differentiation by preventing response to differentiative signals. However, the same effect could be achieved if Egfr signaling, similar to what is proposed here for the midline neurectoderm, interrupts the inhibition of proneural genes by E(spl). Although this would not prevent N signaling, it would cancel the effect of N signaling on downregulating proneural genes and thereby keep cells in a state of competency to respond to signals (Dumstrei, 1998).

During dorsal closure in Drosophila melanogaster, cells of the lateral epidermis migrate over the amnioserosa to encase the embryo. At least three classes of dorsal-open group gene products are necessary for this morphogenetic movement. Class I genes code for structural proteins that effect changes in epidermal cell shape and motility, including zipper, coracle, canoe and myospheroid. Class II and III genes code for regulatory components of closure: Class II genes encode Drosophila Jun amino (N)-terminal kinase (DJNK) signaling molecules, including misshapen, hemipterous, basket, Jun-related antigen, kayak, anterior open/yan and puckered, and Class III genes encode Decapentaplegic-mediated signaling molecules. All characterized dorsal-open group gene products function in the epidermis. Reported here is a molecular and genetic characterization of raw, a newly defined member of the Class II dorsal-open group genes. The novel protein encoded by raw is required for restriction of DJNK signaling to leading edge epidermal cells as well as for proper development of the amnioserosa. Taken together, these results demonstrate a role for Raw in restriction of epidermal signaling during closure and suggest that this effect may be mediated via the amnioserosa (Byars, 1999).

The distribution of raw transcripts, which is uniform in early embryos, becomes more refined as development proceeds. In gastrulating embryos, raw transcripts are evident in the midgut rudiments as well as in the ventral furrow and cephalic and dorsal folds. In germband-extended-stage embryos, raw transcripts appear limited to the gut primordia and ventral neurogenic region. This spatial restriction continues in germband-retracted-stage embryos where transcription is most conspicuous in the central nervous system (CNS) and midgut. Although raw is transcribed throughout the CNS in later stages, its pattern of expression is refined in the midgut. Here, raw transcription is limited primarily to the second midgut constriction. The patterns of raw transcription observed correlate well with documented roles for raw in the nervous system and intestinal tract; however, they provide little insight into how Raw might affect dorsal closure. All previously characterized dorsal-open group genes are expressed in the epidermis or in the epidermis and amnioserosa, and raw could not be detected in either of these tissues. Reports that amnioserosal gene transcription is sometimes recalcitrant to staining in standard in situ staining protocols led the authors to evaluate raw expression by other means. More notably, reporter expression allowed for detection of beta-galactosidase in the amnioserosa (Byars, 1999).

Raw function could affect dorsal closure at any of several steps in the morphogenetic pathway. It could play a structural role in either the amnioserosa or the epidermis. As examples, Raw might be required for the physical interaction of the opposing tissues during closure or for the restructuring of the cytoskeleton that is associated with changes in epidermal cell shape during closure. Alternatively, Raw could play a signaling role, providing either an amnioserosal or epidermal cue that regulates signaling in the epidermis during closure. In an effort to distinguish between these structural and instructive models and to understand better the role of Raw in dorsal closure, epidermal morphology and signaling was examined Epidermal cell elongation, a hallmark of dorsal closure, is thought to be driven by the movement of a myosin motor with a filamentous actin (F-actin) substrate in leading edge cells. Several lines of evidence indicate that this initiating event of closure occurs normally in raw mutant embryos. Proper F-actin localization has been documented in raw mutant embryos by staining with phalloidin. The fact that F-actin accumulates normally in leading edge epidermal cells and that these cells can elongate in animals harboring either hypomorphic or amorphic raw alleles indicates that dorsal closure initiates properly in raw mutants. Moreover, these data suggest that the DJNK signaling pathway is operative in the epidermal leading edge of raw mutant embryos (Byars, 1999).

To more directly test DJNK activation in raw mutants, expression of dpp and puc, the two known transcriptionally regulated targets of DJNK signaling during closure, were examined. In wild-type embryos, epidermal expression of both dpp and puc is dependent upon DJNK signaling and is confined to leading edge cells. Transcription of these targets is abolished in leading edge epidermal cells in hep (DJNKK), bsk (DJNK) and Jra (DJun) mutant embryos, and expanded in embryos overexpressing either activated c-Jun or wild-type DJun. As seen in embryos with ectopic DJun function, the domains of dpp and puc transcription in the epidermis of raw mutant embryos are markedly expanded beyond their normal ranges. It was also noted that puc transcription, as assayed by an enhancer reporter, expands to a greater lateral distance in raw mutants than in puc mutants. These data demonstrate that Raw is required for restriction of dpp and puc to the leading edge of the dorsal epidermis, and point to an upstream role for Raw in negatively modulating DJNK signaling during closure (Byars, 1999).

To establish a regulatory link between raw and Jra, their epistatic relationship was determined. Embryos doubly mutant for raw and Jra were scored for the appearance of alternative dpp expression phenotypes (either missing from leading edge epidermal cells, as in Jra mutants or ectopic epidermal expression, as in raw mutants). The finding that dpp is not expressed in leading edge epidermal cells in raw;Jra double mutants defines Jra as epistatic to raw and confirms the hypothesis that raw functions upstream of the DJNK signaling pathway (Byars, 1999).

In summary, distinct features of functionality define raw as unique. The raw gene is the first of the dorsal-open group for which defects in gene expression have been documented in the amnioserosa. More notably, raw represents the first of the dorsal-open group mutants to show gross defects in dorsal closure that are attributable to a gain in DJNK signaling rather than a loss of DJNK signaling. The characterization of raw as a novel upstream component of the dorsal closure pathway represents an important first step in understanding the mechanism of regulating DJNK signaling during closure (Byars, 1999).

Additional clues as to the function of raw come from a second analysis of the role of raw in dorsal closure. Mutations in two genes, rib and raw cause defects in the morphology of a number of tissues in homozygous mutant embryos. A variety of tubular epithelial tissues adopt a wide, round shape in mutants and dorsal closure fails. Cells of the normal tubular epithelia are columnar and wedge-shaped, and cells of the epidermis become elongated dorsoventrally as dorsal closure occurs. However, the cells of mutants are round or cuboidal in all of the tissues with mutant phenotypes, consistent with the hypothesis that the products of these genes are required for proper cell shape. Cytoskeletal defects, in particular, defects in myosin-driven contraction of the cortical actin cytoskeleton, could be responsible for the lack of specific cell shapes in mutant embryos. This possibility is supported by the observation that the intracellular localization of nonmuscle myosin to the leading edge of the dorsally closing epidermis is absent or reduced in rib and raw mutant embryos. In contrast, the band of actin that is also located at the leading edge is neither eliminated nor interrupted by either rib or raw mutations. Furthermore, mutations of zipper, the gene encoding the nonmuscle myosin heavy chain, exhibit mutant phenotypes in most of the same tissues affected by rib and raw, and many of the phenotypes are similar to those of rib and raw. Therefore, the products of rib and raw may be required for proper myosin-driven contraction of the actin cytoskeleton (Blake, 1998).

Various lines of evidence from mammalian tissue culture suggest that Cdc42 functions in regulating the JNK signaling cascade. In Drosophila, the JNK pathway plays an integral role in dorsal closure, a morphogenetic process involving cell shape changes and local signaling events that occurs late in embryogenesis. One demonstrated function of the JNK pathway is to promote expression of the morphogen Decapentaplegic in cells at the leading edge of the lateral epidermis during dorsal closure. Consistent with this notion, previous studies have shown that dpp expression in the leading-edge epidermal cells is disrupted in embryos carrying mutations in members of the JNK signaling pathway. Reduction but not complete loss of maternally contributed hemipterous results in complete loss of dpp expression while loss of a negative regulator, puckered, results in increased dpp expression (Genova, 2000 and references therein).

Thus dpp expression at the leading edge is sensitive to the level of JNK pathway function. Previous studies using ectopic expression of dominant Cdc42 alleles have suggested that Cdc42 is necessary for dorsal closure and functions upstream of the JNK pathway at the leading edge. If this is so, then loss of Cdc42 function should disrupt JNK signaling and therefore dpp expression in these cells. To test this hypothesis, in situ hybridization to dpp mRNA was performed on embryos derived from Cdc42⁴/Cdc42⁶ mothers. Although ~70% of embryos produced by these females displayed epithelial defects and lethality, normal levels of dpp expression were observed in all embryos that developed to the onset of dorsal closure, including those that had arrested development due to insufficient levels of Cdc42 function. Thus, unlike known upstream components of the JNK pathway, reduction in Cdc42 function has no apparent effect on dpp expression by leading-edge cells at the time of dorsal closure (Genova, 2000).

Shark (SH2 domain ankyrin repeat kinase) is a Drosophila nonreceptor tyrosine kinase that contains from amino to carboxyl terminus, a Src homology 2 (SH2) domain (N-SH2), five ankyrin repeats, a second SH2 domain (C-SH2), a proline-rich and basic region, and a tyrosine kinase domain. Analysis of the phenotypes associated with a shark loss-of-function mutation demonstrate that Shark activity is essential for the migration of the dorsolateral epidermis of the embryo during dorsal closure (DC). Shark kinase functions in DC upstream of Dpp expression by leading edge (LE) cells (Fernandez, 2000).

Because no obvious genetic interactions were obtained between Shark and mutations of the JNK pathway, tests were performed to see whether constitutive activation of the JNK or the Dpp pathway could rescue the shark¹ DC phenotype. When shark¹ GLCs were generated in the background of flies carrying a shark¹ chromosome with an inserted transposon expressing an activated form of c-Jun (hs-SEjunAsp), shark¹ DC defects were completely rescued, in some cases, as determined by the decreased penetrance of embryonic lethality (~10% lower than the fully penetrant 50% observed without the expression of hs-SEjunAsp) and by the complete or partial enclosure observed in unhatched embryos. These results are consistent with the action of Shark upstream of bsk (JNK) in the JNK pathway in LE cells (Fernandez, 2000).

scribble mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth in Drosophila: Involvement of JNK pathway in apoptosis

Cancer is a multistep process involving cooperation between oncogenic or tumor suppressor mutations and interactions between the tumor and surrounding normal tissue. This study is the first description of cooperative tumorigenesis in Drosophila, and uses a system that mimics the development of tumors in mammals. The MARCM system was used to generate mutant clones of the apical-basal cell polarity tumor suppressor gene, scribbled, in the context of normal tissue. scribbled mutant clones in the eye disc exhibit ectopic expression of cyclin E and ectopic cell cycles, but do not overgrow due to increased cell death mediated by the JNK pathway and the surrounding wild-type tissue. In contrast, when oncogenic Ras or Notch is expressed within the scribbled mutant clones, cell death is prevented and neoplastic tumors develop. This demonstrates that, in Drosophila, activated alleles of Ras and Notch can act as cooperating oncogenes in the development of epithelial tumors, and highlights the importance of epithelial polarity regulators in restraining oncogenes and preventing tumor formation (Brumby, 2003).

A clonal approach, more closely resembling the clonal nature of mammalian cancer, was used to analyze the effects of removing Scrib function on tumor formation. This analysis indicates that Drosophila scrib^- tumors: (1) lose tissue architecture, including apical-basal cell polarity; (2) fail to differentiate properly; (3) exert non-cell-autonomous effects upon the surrounding wild-type tissue; (4) upregulate cyclin E and undergo excessive cell proliferation; (5) are restrained from overgrowing by the surrounding wild-type tissue via a JNK-dependent apoptotic response, and (6) show strong cooperation with oncogenic alleles of Ras and Notch to produce large amorphous tumors. These conclusions are summarized in a model for tumor development in Drosophila. It is suggested that the role of epithelial cell polarity regulators in restraining oncogenes is likely to be of general significance in mammalian tumorigenesis (Brumby, 2003).

The model suggests that the wild-type larval eye disc is a monolayered columnar epithelium, in which cell proliferation is tightly regulated. Cell architecture is maintained by the formation of adherens junctions, the apical localization of Scribbled, and adhesion to the basement membrane. Mutation of scrib results in loss of apical-basal polarity, leading to multilayering and rounding up of cells. scrib^- tissue also shows impaired differentiation, and ectopic cyclin E expression (by an unknown mechanism) leads to ectopic cell proliferation. Unrestrained overgrowth and tumor formation of scrib^- cells is held in check by compensatory JNK-mediated apoptosis, dependent upon the presence of surrounding wild-type cells. Secondary mutations are required to avoid this apoptotic fate. If JNK activity is blocked within scrib^- cells, by expressing a dominant-negative form of JNK, apoptosis is prevented, resulting in tissue overgrowth and lethality. Even more aggressive overgrowth results from the addition of activating oncogenic alleles of Ras or Notch. In addition to promoting cell survival, these oncogenes must also promote tumor cell proliferation; however, it is proposed that other downstream effectors of these oncogenes are likely also to be important, since it was not possible to mimic the cooperative overgrowth effects of Ras^ACT or N^ACT on scrib^- tissue by simply blocking apoptosis and enhancing cell proliferation (Brumby, 2003).

Overproliferation of scrib^- clones in the eye disc is compensated for by JNK-mediated apoptosis. Blocking JNK pathway activity in scrib^- eye clones greatly increases the proportion of clonal tissue, and results in lethality to the host. Since downregulating JNK pathway activity in otherwise wild-type clones of tissue does not induce increased cell proliferation, it is suggested that JNK pathway activity in scrib^- clones induces apoptosis. This is consistent with previous reports on the pro-apoptotic effects of the JNK pathway in the Drosophila eye and the current observations. Recent studies in mammals would also suggest that activation of the JNK pathway can limit the growth of tumors in situ, possibly by increasing apoptosis (Brumby, 2003).

How JNK-mediated apoptosis is induced in scrib^- clones is not known. While Scrib could play a direct role in repressing JNK pathway activity, it is also possible that the JNK pathway is activated indirectly, in response to other cellular defects. In the wing disc, removal of cells by JNK-mediated apoptosis is linked to discontinuities in a cell's response to morphogen gradients, most notably the antero-posterior patterning regulator, Dpp, in a process probably related to cell competition, with the purpose of eliminating aberrant or slow growing cells. Although this form of compensatory JNK-mediated apoptosis has not yet been demonstrated within the eye disc, the observation that the surrounding wild-type tissue context plays an important role in limiting the overgrowth of scrib^- tissue argues against a simple cell-intrinsic apoptotic response of scrib^- cells to a loss of cell polarity, and is more consistent with an integrative response mediated by both the tumor cells and the surrounding wild-type cells, as exemplified by cell competition. Whether this is dependent on a failure of scrib^- cells to transduce Dpp signaling is not known; however, other interesting possibilities also warrant further investigation. Notable is the recent identification of a tumor necrosis factor-induced apoptotic signaling pathway involving the JNK pathway. It is also important to keep in mind the involvement of the JNK pathway in orchestrating cell shape changes during the morphogenetic movements of dorsal and thorax closure and wound healing. clones of scrib^- tissue expressing Bsk^DN (JNK^DN) appear morphologically different from those expressing the apoptosis inhibitor p35; most notably, the clones are generally larger and less rounded than those expressing p35. This would imply either that p35 is not as effective as Bsk^DN in preventing cell death, or that there are other effectors of the JNK pathway that are important in the inhibition of scrib^- tumor overgrowth. The possibility that JNK activation could play a role in eliminating scrib^- tissue from the epithelium in a process reminiscent of wound healing is currently being investigated (Brumby, 2003).

dlg and lgl mutant clones also show poor viability, suggesting that JNK-mediated apoptosis could be a common response to the loss of cell polarity and overproliferation induced by the absence of these tumor suppressors. Indeed, while other regulators of epithelial cell polarity, such as Crumbs and E-cadherin, apparently do not act as tumor suppressors in Drosophila, the effects of these mutations on cell proliferation when cell death is blocked warrant further examination. Interestingly, in mammalian systems, the polarized nature of epithelia is also important in protecting cells from an apoptotic response, and this acts as a brake on tumor development when polarity is disrupted (Brumby, 2003).

Integrin-dependent apposition of Drosophila extraembryonic membranes promotes morphogenesis and prevents JNK-pathway dependent anoikis

Two extraembryonic tissues form early in Drosophila development. One, the amnioserosa, has been implicated in the morphogenetic processes of germ band retraction and dorsal closure. The developmental role of the other, the yolk sac, is obscure. By using live-imaging techniques, intimate interactions are reported between the amnioserosa and the yolk sac during germ band retraction and dorsal closure. These tissue interactions fail in a subset of myospheroid (mys: ßPS integrin) mutant embryos, leading to failure of germ band retraction and dorsal closure. The Drosophila homolog of mammalian basigin (EMMPRIN , CD147) -- an integrin-associated transmembrane glycoprotein -- is highly enriched in the extraembryonic tissues. Strong dominant genetic interactions between basigin and mys mutations cause severe defects in dorsal closure, consistent with basigin functioning together with ßPS integrin in extraembryonic membrane apposition. During normal development, JNK signaling is upregulated in the amnioserosa, as midgut closure disrupts contact with the yolk sac. Subsequently, the amnioserosal epithelium degenerates in a process that is independent of the reaper, hid, and grim cell death genes. In mys mutants that fail to establish contact between the extraembryonic membranes, the amnioserosa undergoes premature disintegration and death. It is concluded that intimate apposition of the amnioserosa and yolk sac prevents anoikis of the amnioserosa. Survival of the amnioserosa is essential for germ band retraction and dorsal closure. It is hypothesized that during normal development, loss of integrin-dependent contact between the extraembryonic tissues results in JNK-dependent amnioserosal disintegration and death, thus representing an example of developmentally programmed anoikis (Reed, 2004).

Physical interaction of the amnioserosa and yolk sac has been shown to play a crucial role in both germ band retraction and dorsal closure of the embryo. βPS integrin mediates extraembryonic membrane interactions that are required for survival of the amnioserosa. Anoikis of the amnioserosa occurs during normal development after closure of the midgut disrupts integrin-dependent apposition of the amnioserosa and yolk sac. In mys mutants, failure to establish apposition of extraembryonic membranes leads to premature anoikis of the amnioserosa. A possible role for JNK signaling and the reaper/hid/grim cell death genes in amnioserosal anoikis during normal development was investigated (Reed, 2004).

It is possible to visualize a subset of the amnioserosal cells as acridine orange positive either before they leave the tube or shortly thereafter. Both acridine orange staining and engulfment by hemocytes are hallmarks of dying cells. To determine whether death of amnioserosal cells might be reaper dependent, it was asked whether reaper expression could be visualized in the amnioserosal cells prior to or after extrusion. No reaper-expressing cells were detected. To further test whether amnioserosal cell death might be reaper dependent, the H99 deficiency [Df(3L)H99] was used; this deficiency removes the reaper, head involution defective (hid), and grim genes, and the amnioserosa with anti-HNT antibody was visualized. If amnioserosal death were reaper dependent, one would expect HNT-positive cells to persist in H99 mutants when compared with wild-type. Such persistence does not occur. While it is conceivable that HNT expression is downregulated in a persistent amnioserosa, the simplest interpretation of these data is that death of the amnioserosa is reaper independent. This conclusion is consistent with the recent suggestion that Drosophila embryos have a caspase-independent cell engulfment system, which is still operative in H99 mutants (Reed, 2004).

It has been shown that loss of integrin-dependent contact between cells and the extracellular matrix leads to cell death, a process referred to as anoikis. Anoikis is promoted by the Jun amino-terminal kinase (JNK) pathway. Previous analyses have shown that JNK signaling in the amnioserosa is downregulated prior to dorsal closure. In those analyses, puckered-lacZ expression was used as a read-out of JNK signaling, and it was shown that relocation of JUN and FOS proteins from the nucleus to the cytoplasm of amnioserosal cells correlates with downregulation of JNK signaling. While JNK signaling is downregulated in the amnioserosa prior to dorsal closure, JNK signaling is upregulated in this tissue as dorsal closure approaches completion. Thus, reactivation of JNK signaling in the amnioserosa follows loss of integrin-dependent apposition of the amnioserosa and yolk sac membrane and precedes amnioserosal disintegration and death. These data are consistent with the hypothesis that midgut closure disrupts integrin-dependent apposition of the amnioserosa and yolk sac, thus inducing JNK signaling in the amnioserosa and its subsequent anoikis (Reed, 2004).

It remains to be determined whether disintegration and death of the amnioserosa during normal development is caused solely by loss of contact with the yolk sac (i.e., is nonautonomously induced) versus whether signals from cell types other than the yolk -- or even an amnioserosa-autonomous program -- also play a role. For example, it is possible that upregulation of JNK signaling in the amnioserosa is independent of loss of contact with the yolk sac. Analysis of mutants lacking a midgut provide a test of this possibility: if disintegration and death of the amnioserosa occur even when apposition with the yolk sac is maintained, signals from other cell types or amnioserosa-autonomous processes would be implicated (Reed, 2004).

The specific role of JNK signaling in amnioserosal anoikis is difficult to assess because downregulation of JNK signaling in the amnioserosa and up-regulation of JNK signaling in the leading edge of the epidermis are required for dorsal closure. Thus JNK pathway mutants stall morphogenesis prior to dorsal closure, making it impossible to assess a possible later role. Expression of dominant-negative JNK specifically in the amnioserosa only later in development, when closure is almost complete, will be necessary to rigorously test the role of JNK activation in amnioserosal anoikis (Reed, 2004).

It is concluded that the extraembryonic tissues of Drosophila play a crucial role in directing embryonic morphogenesis. Close apposition of the yolk sac membrane and the basal cell membranes of the amnioserosa is dependent on βPS integrin. This intimate membrane association is required to promote survival and to prevent anoikis of the amnioserosa. The amnioserosa then directs germ band retraction and dorsal closure through physical contacts and/or signaling. Disintegration and death of the amnioserosa after closure of the epidermis and midgut correlates with upregulation of JNK signaling in the amnioserosa, is independent of reaper/hid/grim function, and is likely to represent the first example of developmentally programmed anoikis in Drosophila (Reed, 2004).

Apoptotic cells can induce compensatory cell proliferation through the JNK and the Wingless signaling pathways

In many metazoans, damaged and potentially dangerous cells are rapidly eliminated by apoptosis. In Drosophila, this is often compensated for by extraproliferation of neighboring cells, which allows the organism to tolerate considerable cell death without compromising development and body size. Despite its importance, the mechanistic basis of such compensatory proliferation remains poorly understood. Apoptotic cells are shown to express the secretory factors Wingless and Decapentaplegic. When cells undergoing apoptosis were kept alive with the caspase inhibitor p35, excessive nonautonomous cell proliferation is observed. Significantly, Wg signaling is necessary and, at least in some cells, also sufficient for mitogenesis under these conditions. Finally, evidence is provided that the DIAP1 antagonists reaper and hid can activate the JNK pathway and that this pathway is required for inducing wg and cell proliferation. These findings support a model where apoptotic cells activate signaling cascades for compensatory proliferation (Ryoo, 2004).

To investigate how the inhibition of diap1 may lead to mitogen expression, attention was focused on Dronc and the Jun N-terminal Kinase (JNK) pathway. Dronc has been implicated in compensatory proliferation, and its activity can be inhibited by the expression of dronc^DN. In addition, the JNK signaling pathway was considered as a candidate, since its activity is known to correlate with many forms of stress-provoked apoptosis, including disruption of morphogens, cell competition, and rpr expression. In Drosophila, the JNK pathway can be effectively blocked by the expression of puckered (puc), which encodes a phosphatase that negatively regulates JNK (Ryoo, 2004).

To induce patches of undead cells, wing imaginal discs were generated with mosaic clones expressing hid and p35. 48 hr after induction, these imaginal discs contained hid-expressing clones that autonomously induced wg. Using this experimental setup, it was asked whether additional expression of either dronc^DN or puc would block wg induction in undead cells. When dronc^DN was coexpressed, a subset of the hid-expressing population was still able to induce wg. In contrast, when puc was coexpressed, wg induction by hid was almost completely blocked. These results provide evidence that the JNK pathway is required for wg induction under these conditions but fail to uncover a similar requirement for Dronc (Ryoo, 2004).

To independently investigate the role of puc and dronc^DN in compensatory proliferation, the size of wing discs harboring undead cells was measured and they were compared with those of the sibling controls. Under the experimental conditions, wing discs harboring hid- and p35-expressing clones were on average 53% larger than their sibling controls. Coexpression of puc within these undead clones significantly limited growth, resulting in only a small increase in wing disc size that was not statistically significant. In contrast, coexpression of dronc^DN did not limit growth. Wing size measurements also correlated with the degree of wg induction. The larger size of discs harboring hid- and p35-expressing cells is not due simply to extra cell survival: (1) these undead cells are derived from the normal lineage; (2) the size of wing discs expressing hid, p35, and puc serves as a control. In this case, although a large number of undead cells were generated, no significant increase in disc size was observed, in stark contrast to the discs expressing hid and p35 only. It is concluded that the JNK pathway is required for the nonautonomous growth promoting activity of the undead cells (Ryoo, 2004).

To confirm a role of puc in imaginal disc growth, rpr and p35 werecoexpressed in wild-type and puc^−/+ imaginal discs. Like hid, rpr is a DIAP1 antagonist, but with a weaker cell killing activity when overexpressed in imaginal disc cells. In a puc^+/+ background, a small amount of ectopic wg expression was observed, indicative of rpr's weaker DIAP1 inhibiting activity. In contrast, ectopic wg expression was strongly enhanced in puc^−/+ discs. Because the puc allele used, puc^E69, also acts as a lacZ reporter, JNK pathway induction could be monitored simultaneously. wg induction in undead cells correlates very well with puc-lacZ expression, with a stronger induction at the center of the wing pouch. These results further support the role of JNK in the induction of wg (Ryoo, 2004).

Next to be tested was whether the reduction of puc had an effect on apoptosis-induced cell proliferation. Whereas puc^−/+ discs expressing only p35 had BrdU incorporation similar to wild-type discs, coexpression of rpr and p35 in puc^−/+ led to a significant increase in BrdU incorporation. Also, the size of these discs were on average 41% larger than those coexpressing rpr and p35 in a puc^+/+ background. Taken together, these results show that diap1 inhibition leads to JNK activation and that JNK activity promotes wg induction and cell proliferation (Ryoo, 2004).

To directly test if JNK signaling can activate wg and dpp expression, hep^CA, a constitutively active form of hemipterous (hep), the Drosophila JNK kinase was conditionally expressed. Expression of hep^CA causes induction of wg-lacZ within 22 hr and to a lesser extent also dpp-lacZ. These ß-gal-expressing cells shifted basally and were apoptotic as assayed by anti-active caspase-3 antibody labeling. Hid protein levels were also elevated in these cells. Significantly, since p35 was not use to block apoptosis in this experiment, this demonstrates that wg and dpp can be induced not only in undead cells, but also in 'real' apoptotic cells (Ryoo, 2004).

This study provides evidence that the central apoptotic regulators can control the activity of mitogenic pathways. In particular, inhibition of DIAP1, either via expression of Reaper and Hid or by mutational inactivation, leads to the induction of the putative mitogens wg and dpp. When apoptosis was initiated through DIAP1 inhibition but cells were kept alive by blocking caspases, the resulting 'undead cells' exhibited strong mitogenic activity and stimulated tissue overgrowth. Inhibiting wg signaling with a conditional TCF^DN blocked cell proliferation in imaginal discs, indicating that wg has an essential mitogenic function. Finally, evidence was provided that the JNK pathway mediates mitogen expression and imaginal disc overgrowth in response to rpr and hid. Based on these results, it is proposed that apoptotic cells actively signal to induce compensatory proliferation. DIAP1 inhibits both caspases as well as dTRAF1. According to this model, when DIAP1 is inhibited in response to cellular injury, the JNK pathway is activated and wg/dpp are induced in apoptotic cells. Secretion of these factors stimulates growth of proliferation-competent neighboring cells and leads to compensatory proliferation (Ryoo, 2004).

Highwire restrains synaptic growth by attenuating a MAP kinase signal

Highwire is an extremely large, evolutionarily conserved E3 ubiquitin ligase that negatively regulates synaptic growth at the Drosophila NMJ. Highwire has been proposed to restrain synaptic growth by downregulating a synaptogenic signal. This study identifies such a downstream signaling pathway. A screen for suppressors of the highwire synaptic overgrowth phenotype yielded mutations in wallenda, a MAP kinase kinase kinase (MAPKKK) homologous to vertebrate DLK and LZK. wallenda is both necessary for highwire synaptic overgrowth and sufficient to promote synaptic overgrowth, and synaptic levels of Wallenda protein are controlled by Highwire and ubiquitin hydrolases. highwire synaptic overgrowth requires the MAP kinase JNK and the transcription factor Fos. These results suggest that Highwire controls structural plasticity of the synapse by regulating gene expression through a MAP kinase signaling pathway. In addition to controlling synaptic growth, Highwire promotes synaptic function through a separate pathway that does not require Wallenda (Collins, 2006).

JNK signaling affects many cellular processes, often by regulating transcription factor activity that leads to changes in gene expression. A common downstream effector of JNK-mediated changes in gene expression is the AP-1 complex of Fos and Jun transcription factors, which can regulate synaptic growth at the Drosophila NMJ. To investigate whether Drosophila Fos or Jun (known as D-fos and D-jun, respectively) are required for highwire-dependent synaptic overgrowth, each was inhibited by expressing dominant-negative transgenes that contain the DNA binding and dimerization domains of Fos and Jun but lack the transcriptional activation domains. Expression of these dominant-negative transgenes in postmitotic neurons allowed circumvention of early embryonic requirements for D-fos and D-jun (Collins, 2006).

When Fos^DN and Jun^DN are neuronally expressed in a wild-type background, there is a modest trend toward inhibition of synaptic growth. When expressed in a highwire mutant background, the Fos^DN transgene confers dramatic suppression of the highwire synaptic phenotype, reducing bouton number and branching (42%) and increasing the intensity of staining for synaptic vesicle markers at the synapse. The reduction in highwire-dependent synaptic overgrowth is much greater than the reduction of growth in a wild-type background. In contrast, Jun^DN does not suppress the highwire phenotype. This suggests the existence of a pathway that is separate from AP-1, consistent with results in Drosophila demonstrating that D-Fos can act independently of D-Jun. The requirement for D-Fos in highwire synaptic overgrowth suggests that the highwire phenotype involves changes in gene expression rather than exclusively local changes to the synapse (Collins, 2006).

If Fos^DN acts downstream of Wallenda to inhibit synaptic overgrowth, it should also suppress the synaptic overgrowth caused by overexpressing wallenda. Indeed, when Fos^DN was coexpressed with UAS-wnd in neurons, Fos^DN could suppress the wallenda gain-of-function phenotype, leading to a 38% reduction in synaptic bouton number, a 52% reduction in synaptic branching, a 54% increase in bouton size, and a 3.8-fold increase in the intensity of staining of synaptic vesicle markers. This is consistent with D-Fos acting downstream of Wallenda to promote synaptic growth. Therefore, the synaptic overgrowth phenotypes caused by loss of highwire and by overexpression of wallenda are similar in their requirements for the transcription factor D-Fos (Collins, 2006).

Current models suggest that Highwire functions as an E3 ubiquitin ligase to downregulate a signaling pathway that promotes synaptic growth. This study identified a MAPKKK, Wallenda, whose protein levels are controlled by Highwire and the activity of ubiquitin hydrolases. Wallenda is both necessary for highwire-dependent synaptic overgrowth and sufficient to promote synaptic growth. Downstream of Wallenda, the MAP kinase JNK and transcription factor Fos are required for highwire-dependent synaptic overgrowth. It is proposed that Highwire restrains synaptic growth by downregulating the MAPKKK Wallenda, thereby inhibiting signaling through the JNK MAP kinase and the Fos transcription factor. In the absence of highwire, this signaling pathway is overactive, leading to changes in gene expression that result in excessive synaptic growth (Collins, 2006).

The regulation of the MAPKKK Wallenda is conserved in Drosophila and C. elegans (Nakata, 2005). In both organisms, the synaptic phenotype of highwire/rpm-1 requires the Wallenda/DLK-1 MAPKKK and downstream MAPK signaling. However, the downstream MAPK pathways diverge: in C. elegans, the rpm-1 phenotype requires a p38 MAP kinase (Nakata, 2005), while the highwire phenotype requires JNK signaling. This suggests that regulation of the specific MAPKKK Wallenda/DLK-1, rather than a particular downstream MAP kinase pathway, is a fundamental activity of Highwire and its orthologs (Collins, 2006).

Since Highwire functions as an E3 ubiquitin ligase to restrain synaptic growth, Wallenda is a compelling candidate target for the following reasons: (1) wallenda functions downstream of highwire and is essential for the synaptic overgrowth in highwire mutants; (2) increasing the levels of Wallenda by overexpression is sufficient to confer synaptic overgrowth; (3) Highwire regulates Wallenda protein levels through a posttranscriptional and most likely posttranslational mechanism. Each of the points above is conserved in C. elegans (Nakata, 2005 ). (4) Wallenda protein levels are regulated by ubiquitination in vivo, since inhibiting ubiquitination by overexpressing ubiquitin hydrolases increases the levels of Wallenda protein. (5) The RING domain of the C. elegans homolog rpm-1 can interact with the Wallenda homolog DLK-1 (Nakata, 2005) and stimulate its ubiquitination when both are overexpressed in 293T cells (Collins, 2006).

Targeting a MAPKKK, which sits at the top of a MAP kinase signaling pathway, is an attractive mechanism for spatially and temporally controlling a synaptogenic signal without affecting downstream components shared by multiple MAPK signaling cascades. Restraining MAP kinase signaling is essential for controlling diverse cellular processes, including cell proliferation, differentiation, and apoptosis. The targeting of MAPKKKs by specific ubiquitin ligases may be a powerful and general mechanism for regulating MAP kinase signals (Collins, 2006).

While Wallenda is an essential mediator of the highwire mutant phenotypes in both Drosophila and C. elegans, an endogenous synaptic function for Wallenda has not yet been identified in either organism: the wallenda mutants have surprisingly normal synapse morphology and function. This may be due to another pathway that compensates for the loss of wallenda function. Such redundancy would obscure the role of wallenda. A second possibility is that wallenda functions in an aspect of synaptic growth that is not detected or required under laboratory culture conditions. For instance, wallenda could promote synaptic growth as part of a structural plasticity program that responds to unknown experience-dependent stimuli. A third possibility is that Wallenda does not normally function at synapses, but its upregulation in highwire mutants causes a neomorphic phenotype. In this scenario, the regulation of Wallenda by Highwire is required for normal synaptic development, but endogenous Wallenda would not itself regulate the synapse. The neuropil and synaptic localization of Wallenda and the vertebrate homolog DLK (Hirai, 2005) is, however, consistent with a synaptic function (Collins, 2006).

As an activator of MAP kinase signaling, Wallenda and its homologs might also control other processes beyond the synapse. Functional studies in vertebrates suggest that DLK and JNK signaling regulate neuronal migration and axon outgrowth in the developing cortex (Hirai, 2002). Outside of the nervous system, DLK influences keratinocyte differentiation, and LZK is highly expressed in the pancreas, liver, and placenta. In Drosophila, wallenda mutants are female sterile. It is predicted that the regulation of DLK and LZK is conserved from worms and flies to vertebrates. Therefore, the vertebrate homologs of Highwire might regulate some of these neuronal and/or extraneuronal developmental processes (Collins, 2006 and references therein).

Highwire is a large, multidomain protein that, in addition to acting as an E3 ubiquitin ligase, has been shown to inhibit adenylate cyclase, influence TSC signaling and pteridine biosynthesis, and interact with the myc oncogene and the co-SMAD Medea. It is remarkable that throughout millions of years of evolution, members of the Highwire family have retained an exceptionally large size and complex domain structure. An attractive explanation for this conservation is that this molecule could serve as an intersection point for multiple signaling pathways, integrating MAP kinase and other signals during neural development (Collins, 2006).

The ubiquitin ligase activity alone could be responsible for regulating more than one downstream target. Interactions with components of TSC (tuberin/hamartin) and TGF-β signaling pathways suggest that Highwire might target either or both of these pathways. The model that Highwire regulates TGF-β signaling through interaction with the co-SMAD Medea has received considerable attention. Since the TGF-β pathway regulates synaptic growth at the NMJ, it has been proposed that synaptic overgrowth of highwire mutants is caused by overactivity of this pathway. Null alleles of wit, which completely disrupt TGF-β signaling at the NMJ, can partially suppress the highwire phenotypes: they partially suppress the increase in bouton number, but show little or no suppression of the reduced bouton size and the reduced intensity for synaptic vesicle markers. This partial suppression of highwire by wit is consistent with the model that overactive TGF-β signaling contributes to the highwire phenotype. However, the data are also consistent with the alternate model that TGF-β signaling and Highwire act in parallel pathways. An assay for the activity of TGF-β signaling is to stain for phosphorylated-MAD (phospho-MAD), the major transducer of BMP signals in Drosophila, in motoneuron nuclei. No change was detected in the levels of phospho-MAD staining in highwire mutants compared to wild-type. This assay is sensitive to changes in pathway activity—neuronal expression of the constitutively active type I receptor thick veins leads to a 40% increase in phospho-MAD staining. Interestingly, this increase in TGF-β signaling does not lead to excess synaptic growth. Combining a highwire mutant with expression of constitutively active thick veins does cause excess growth, but it does not lead to any further increase in phospho-MAD staining. These data are consistent with highwire and TGF-β signaling acting in parallel pathways (Collins, 2006).

Whether or not Highwire regulates TGF-β signaling, it is likely to target an additional pathway. Highwire not only restrains synaptic growth, but also promotes synaptic function. Synaptic function requires the ubiquitin ligase activity of Highwire and is sensitive to the levels of the ubiquitin hydrolase fat facets. This study demonstrates that this regulation of neurotransmitter release does not require Wallenda. Therefore, Highwire must regulate at least two distinct molecular pathways. If Wallenda is a substrate whose downregulation is essential for restraining synaptic growth, there is likely another substrate for Highwire whose downregulation promotes neurotransmitter release (Collins, 2006).

Downstream of Wallenda, the JNK MAP kinase and Fos transcription factor are required for the highwire synaptic morphology phenotype. Therefore, Highwire attenuates a JNK signaling pathway that presumably controls gene expression to regulate synaptic growth. Previous studies have implicated JNK-dependent transcriptional control in activity-dependent growth of the Drosophila NMJ. However, this previously described pathway is probably distinct from the JNK signal that is controlled by Highwire and activated by Wallenda. The previously described role for JNK requires AP-1, a heterodimer of Fos and Jun transcription factors; inhibiting either D-Fos or D-Jun disrupts this pathway. In contrast, highwire-induced overgrowth requires D-Fos, but not D-Jun. The Wallenda pathway could therefore involve a homodimer of D-Fos or another transcription factor that interacts with Fos. Such D-Jun-independent functions of D-Fos have been described previously in Drosophila. The differential requirement for transcription factors suggests that the output of Wallenda signaling cannot simply be activation of JNK, but instead activation of JNK in a particular spatial or temporal context, such as in the presence of cofactors that influence downstream signaling (Collins, 2006).

In addition to transcription factors, substrates for activated JNK include components of the cytoskeleton. Because the NMJ is distant from the motoneuron nucleus, and because vertebrate DLK colocalizes with tubulin in axonal regions of the brain, it was initially expected that the Highwire/Wallenda/JNK pathway would influence synaptic morphology through local action upon the synaptic cytoskeleton. Instead, a requirement was identified for a transcription factor and presumably changes in gene expression. However, this does not exclude an interaction with the cytoskeleton or local changes at the synapse. It is possible that Highwire regulates the Wallenda signal in the cell body. However, the observation that Wallenda accumulates in the synapse-rich neuropil and at the NMJ when Highwire is absent suggests that Wallenda could become activated at the synapse. This would imply the need for a mechanism to transport the activated JNK signal back to the nucleus. In addition, cell-wide changes in gene expression must then be translated into localized growth at the synapse. Activated Wallenda at the synapse is an attractive candidate to integrate changes in gene expression with regulation of the synaptic cytoskeleton to control synaptic growth (Collins, 2006).

Larval

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

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

One of the processes that best exemplify the dramatic changes that shape organisms is insect metamorphosis. In Drosophila and other holometabolous insects, most of the larval structures are replaced with new tissues that will give rise to the adult or imago. In particular, the adult epidermis with the exception of the abdominal structures develops from imaginal epithelial discs. During larval stages, the primordia of imaginal discs, set during embryogenesis, invaginate and grow to become flattened sacs arranged in a monolayer epithelium connected to the larval epidermis by a stalk. The mature discs contain two populations of cells, a columnar epithelium that will give rise to most of the adult structures and a thinner and more squamous peripodial epithelium (PE) with a reduced contribution to adult tissues. Upon metamorphosis, the imaginal discs undergo striking morphological changes, everting, expanding, and fusing to ipsilateral and contralateral adjacent discs generating the adult exoskeleton (Pastor-Pareja, 2004).

The process of movement and sealing of imaginal discs and, in general, epithelial sheets can be subdivided into three sequential steps: (1) leading cells are specified and brought into position; (2) cells execute coordinated forward movements by changing shape and/or migrating over a substratum, and (3) sheets merge and fuse. Most recent work on disc morphogenesis has focused on the cellular and molecular events underlying their late expansion and fusion, while the mechanisms involved in disc eversion have been poorly explored in vivo (Pastor-Pareja, 2004).

In late third instar larvae, the steroid molting hormone 20-hydroxyecdysone is believed to coordinate the almost simultaneous eversion of all discs by inducing a contraction of the PE. This is thought to drive movement of the appendages to the outside of the larval epidermis through relaxed and widened disc stalks. This classical view is supported by in vitro studies showing that treatment of cultured discs with ecdysone is sufficient to induce eversion and that contraction of an intact PE is necessary to achieve this goal. These descriptive reports have led to the proposition that cell shape changes (longitudinal contraction in the PE and circumferential elongation at the disc stalks) are sufficient for imaginal disc eversion. However, there are as yet no data to confirm that this mechanism exists in vivo and no convincing explanation on how a stalk of no more than ten cells in diameter could achieve the width required to allow the entire disc (more than 60,000 cells) to pass through. Further, this accepted view neglects earlier proposals suggesting a different eversion mechanism mediated by the rupture of the PE. A model supported by fate maps has been developed for the PE of Calliphora, a related dipteran, imaginal wing discs (Pastor-Pareja, 2004).

Several studies have revealed a requirement for cytoskeletal components and a number of signal transduction molecules for imaginal disc morphogenesis during the first hours of metamorphosis. The latter include the Drosophila AP-1 transcription factors, D-Jun and D-Fos (Kayak [Kay]), and an upstream kinase cascade homologous to the Jun-NH2-terminal kinase (JNK) pathway in mammals. The core of this cascade is formed by the stress-activated kinases JNKK and JNK. In Drosophila, JNKK and JNK homologs are encoded by the genes hemipterous (hep) and basket (bsk). JNK signaling mutant larvae do not spread their discs in the process of thorax closure. This phenotype is accompanied by a loss of puckered (puc) expression in the disc stalk and the PE. Puc is a dual-specificity phosphatase that selectively inactivates Bsk and, thus, is thought to act in a negative feedback loop. JNK activity is necessary to maintain the adhesion of the imaginal leading edge cells to their larval substrate and to promote actin dynamics (lamellipodia and filopodia formation). It has been shown that this signaling cascade also regulates the process of embryonic dorsal closure, where the embryonic epidermis fuses along the dorsal midline. Based on these similarities, it has been suggested that a conserved mechanism regulates the spreading and fusion of epithelial sheets (Pastor-Pareja, 2004).

A new morphogenetic/cellular mechanism has been uncovered for disc eversion based on histological sections and direct observation of imaginal morphogenesis in vivo. At the onset of metamorphosis, imaginal discs coordinately appose their peripodial sides and stalks (PS cells) to the larval epidermis. Then, eversion proceeds through the progressive invasion of the larval epidermis by PS cells undergoing a pseudo-epithelial-mesenchymal transition (PEMT). Multiple perforations in the peripodial/larval bilayer are thus generated: these coalesce with the disc stalk into a single hole, widening the gap and allowing disc evagination. When eversion is complete, the PS cells localize to the leading front of the discs, spearheading their expansion over larval cells. The roles of the JNK pathway at discrete steps of disc morphogenesis progression have been analyzed. The JNK cascade functions to promote the apposition of PS and larval cells, to determine the degree of PEMT and the motility of leading edge/PS cells, and to maintain the adhesion between the larval and imaginal tissue. It is proposed that this molecular mechanism can be relevant to morphogenetic processes of perforation of transient epithelia in different phyla (Pastor-Pareja, 2004).

The current view of imaginal disc eversion asserts that the externalization of appendage primordia proceed through widened discs' stalks during early pupal development. However, a detailed analysis of PS cell markers appears to challenge this simple inversion mechanism (Pastor-Pareja, 2004).

In early third instar imaginal wing discs, the gene puc is expressed at high levels in stalk cells and some PE cells. This expression evolves through the third instar until all PS cells (about 700 in the mature wing disc) express puc at the white prepupa stage (0-1 hr hours after puparium formation [APF]). These dynamic changes of puc expression are also observed in leg, haltere, and eye discs. The PS expression of puc strictly depends on JNK activity, and it is abolished from mutant hep (JNKK) larvae or after Puc overexpression. Thus, a JNK signaling feedback loop, first described during embryonic dorsal closure, is shared by PS cells at the onset of the eversion and closure of the discs. During wing disc eversion, only cells found at the edge of the hole through which the disc everts and at the leading front mediating fusion to adjacent prothoracic, mesothoracic, and metathoracic discs show puc expression, and hence JNK activity. Importantly, marking all cells that have expressed puc as well as their descendants shows that puc-expressing cells do not change their identity, nor do they die or get excluded from the epithelium until the end of the disc fusion process, when most of these cells are lost. Hence, all PS cells are recruited to the front edge during disc eversion (Pastor-Pareja, 2004).

These findings lead to a topological dilemma. In order to reach their final position at the leading front, the PS cells would need to reposition themselves within the epithelia. Although this rearrangement just could be achieved through a massive constriction of the PE, a complementary mechanism has been uncovered, which involves larval epidermis perforation and PE cells intercalation (Pastor-Pareja, 2004).

At third instar larval stages, the wing disc obliquely hangs from the larval epidermis, which is separated from the peripodial surface of the disc at the notum level by several larval muscles and tracheal tubules. The disc and the larval epidermis are isolated by their corresponding extracellular basal lamina. During late third instar stages and the first hours APF, the notum-wing side of the disc folds progressively to acquire the adult organ shape. At the initiation of pupariation, the disc affixes to the larval epidermis through its peripodial side. At 3 hr APF, the PS cells lose their squamous shape to adopt a more rounded one and are found in close contact with the larval epidermal cells via their basal surfaces. Multiple actin-rich protrusions lead this apposition. At this step, the basal lamina in between both layers degrades, leading to an intimate adhesion (Pastor-Pareja, 2004).

Once imaginal discs appose the larval epidermis, PS cells, mostly around the disc stalk, invade the larval epithelium, gradually replacing the larval cells at the pupal surface without compromising the integrity of the peripodial sheet. Several holes are opened in the peripodial/larval bilayer, which within a few minutes converge with the original stalk into a single aperture. Interfering with apoptosis by overexpressing the P35 cell death inhibitor in imaginal and larval tissues does not affect epithelial perforation and disc eversion (Pastor-Pareja, 2004).

Following coalescence, the progressive widening of the hole by intercalation of PS cells at the leading front was observed. A cell lineage analysis was performed and multiple clones of PS cells were found, that remain compact up to the third instar larval stage in the PE and lose cohesion during eversion. Thus, PS cells appear to change neighbors, become extremely active, and emit and retract filopodia and lamellipodia at their front and rear ends. They squeeze in between themselves and the rest of the epithelium (planar intercalation), migrating to and expanding the front of the disc, and leading the migration over the larval tissue (Pastor-Pareja, 2004).

Simultaneous to wing disc eversion, all legs and haltere discs evert using the same mechanism (Pastor-Pareja, 2004).

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

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

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

The JNK signaling cascade dictates the expression of puc in all PS cells but their early specification appears not to be affected by lowering the level of JNK activity, since the complete absence of Hep function did not alter either their number or morphology in third instar larval discs. However, several mutant phenotypes have provided strong evidence for a leading role of the JNK pathway in imaginal disc fusion and disc eversion; e.g., hep mutants occasionally show uneverted wing discs lying inside the body of the pupa. When and how is JNK signaling needed? Transversal semi-thin sections, at 6 ± 1 hr APF, long after closure is completed in wild-type, of hep^r75 (a strong hypomorphic mutation) pupae show a range of phenotypic defects (classes I to III). Class I corresponds to a complete failure of PS/larval apposition (40% of individuals); in class II, discs apposed to the larval tissue but did not complete their eversion (50%); the mildest condition, class III, refers to discs that everted completely and advanced to some extent but were unable to fuse (10%). By the complete inactivation of the JNK signaling cascade through the ubiquitous overexpression of puc (from 48-60 hr before puparium formation onward), a fully penetrant failure was found of disc apposition to the larval epidermis (class I phenotype). A delayed or reduced (in a puc heterozygous background) overexpression of puc produced less severe class II and III phenotypes. Thus, the JNK cascade appears to be essential for PS and larval cell apposition and, as suggested by the observed phenotypic progression, may also be involved in later steps during eversion (Pastor-Pareja, 2004).

JNK activity levels also affect the degree of PEMT in PS cells. ZAs are absent from leading front cells and the membrane localization of DE-cadherin and Arm is progressively lost, as PS cells moved closer to the free edge. However, in hep^r75 mutant pupae (class III), the cells at the leading front of the disc do not delocalize either Arm or DE-cadherin in the free edge, suggesting that partial loss of JNK signaling blocks the correct transition of these cells from immotile epithelial to migrating and invading leading front cells. Further, a surplus of JNK activity in PS cells in puc^E69F-GAL4 mutants conveyed the transition of an excess of PS cells to a mesenchymatic phenotype. Hence, an adequate balance of JNK activity is key to control the level of PEMT. Too few mesenchymal-like PS cells restrain the ability of discs to evert and spread, while too many transformed cells affect the ability of discs to appropriately fuse. Further, puc^E69F-GAL4 mutants also showed enhanced cell motility and massive cell detachment from the free edges of the epithelium. These cells adopted a rounded shape but remained in close proximity, establishing transient contacts. Conversely, leading cells in hep^r75 mutants do not show any migratory activity. Hence, the JNK pathway regulates not only the adhesive properties of PS cells, but also their motility (Pastor-Pareja, 2004).

In summary, the JNK signaling cascade participates in four key steps in the process of disc eversion: (1) the expression of puc in PS cells; (2) the apposition of PS and larval cells; (3) the regulation of the adhesive and motile properties of PS cells as they undergo PEMT, and (4) the maintenance of the adhesion between the larval and imaginal tissue (Pastor-Pareja, 2004).

Thus, within the first 5 hr after puparium formation, the precursors of the adult structures evert. Multiple evidences show that eversion is mediated by actin microfilaments contraction, which modulate a general change of morphology of PS and larval cells driving the inside-out eversion of the disc. Several observations, however, suggest that other morphogenetic mechanisms are also involved (Pastor-Pareja, 2004).

(1) An imaginal disc is a rigidly determined primordium, which allows the construction of fate maps. Surprisingly, peripodial fate maps of Calliphora, a related diptera, show that adjacent territories develop into nonadjacent adult pleural structures, suggesting that the peripodial layer splits during metamorphosis (Pastor-Pareja, 2004).

(2) PS cells expressing puc relocate during eversion to the leading front. Thus, intercalation of PS cells appears to be concurrent to eversion (Pastor-Pareja, 2004).

(3) Pupal serial sectioning shows that, at eversion, imaginal discs appose to the larval epidermis through their peripodial side. Just before eversion, PS cells lose their basal lamina and detach from the extracellular matrix (Pastor-Pareja, 2004).

(4) Preceding disc eversion, in vivo time-lapse reveals the opening of larval/peripodial gaps, which are the outcome of the invasive behavior and planar intercalation (PEMT) of PS cells (Pastor-Pareja, 2004).

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

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

Suppression of Polycomb group proteins by JNK signalling induces transdetermination in Drosophila imaginal discs

The PcG proteins function through cis-regulatory elements called PcG response elements (PREs), which enable them to bind and to maintain the state of transcriptional silencing over many cell divisions. PcG proteins operate in two key evolutionarily conserved chromatin complexes, and reduced expression of these complexes, as found in PcG mutants, results in the derepression of PRE-controlled genes. To determine whether PcG silencing is modulated in regenerating tissue, the FLW-1 line, which contains a lacZ reporter gene under the control of the Fab7 PRE, was used. Prothoracic leg discs silent for lacZ expression were fragmented and transplanted into the abdomen of host flies. Flies were fed with 5-bromodeoxyuridine (BrdU) to mark the regenerated tissue (the blastema). In uncut discs, there was little proliferation and expression of lacZ was undetectable. On fragmentation, however, lacZ was expressed in the blastema. To confirm that this derepression was due to a reduction in PcG silencing and not simply to massive proliferation at the wound site, the line LW-1 was used; this line lacks the Fab7 PRE and is normally silent, but it can be activated by induction of GAL4. Neither uncut nor cut leg discs of the LW-1 line showed expression of lacZ after transplantation (Lee, 2005).

To show that transdetermination takes place only in cells with downregulated PcG function, fragmented leg discs of the FLW-1 line were stained for lacZ expression and for Vg in order to visualize the transdetermination to wing fate. It was consistently observed that the Vg staining lay within the lacZ expression domain, suggesting that PcG genes are downregulated in the blastema, enabling PRE-silenced genes to be reactivated according to new morphogenetic cues (Lee, 2005).

To investigate direct targets of PcG regulation that, when reactivated, might contribute to transdetermination, the PREs predicted at the wg and vg genes were tested and both were found to be controlled by PcG proteins. The fact that both the transgenic vg-lacZ reporter construct (which lacks the PRE) and the endogenous vg gene were upregulated in the blastema suggests that PcG proteins may affect vg expression both indirectly (for example, through wg) and directly by means of the vg PRE (Lee, 2005).

JNK signalling in Drosophila is crucial for wound healing and is implicated in many different developmental processes, such as dorsal and thorax closure. hemipterous encodes the JNK kinase (JNKK) that activates the Drosophila JNK Basket. Products of DJun and kayak (the Drosophila homologue of Fos) form the AP-1 transcription factor. A downstream target of JNK signalling is puckered (puc), which encodes a phosphatase that selectively inactivates Basket and thus functions in a negative feedback loop. The expression of puc thus mirrors JNK activity. Because wound healing takes place after fragmentation, it was reasoned that activation of the JNK pathway might be causing the downregulation of PcG proteins in the blastema. The puc^E69 line, which carries a P(lacZ) insertion at the puc locus, was used to monitor JNK activity. During the third-instar larval stage puc is not expressed and thus JNK signalling was not activated in leg discs. As expected, however, puc was expressed on fragmentation in all cells at the annealing cut edge (Lee, 2005).

To check whether cells that have activated the JNK pathway also show transdetermination, fragmented leg discs of flies carrying the puc-lacZ reporter and vg^BE-Gal4; UAS-GFP constructs were transplanted. In these flies, cells that adopted a wing fate were identified by their expression of green fluorescent protein (GFP). Two days after fragmentation, weak residual puc-lacZ staining was still visible in the central region of the disc. puc-lacZ staining is known to decline rapidly after wound healing is completed. It was found that stronger staining was visible along the cut site, probably owing to ongoing wound healing. On comparison of puc-lacZ staining and GFP fluorescence, JNK-active cells showed a substantial overlap with transdetermined cells; thus, it is concluded that JNK signalling is activated in cells that undergo transdetermination (Lee, 2005).

JNK signalling affects the transcription of numerous genes, including those encoding chromatin regulating factors. Therefore whether JNK signalling can downregulate the PcG proteins required for transdetermination was examined. A constitutively active form of hep was overexpressed in UAS-hep^act; hsGal4 flies by a heat-shock pulse. Activating the JNK pathway caused a downregulation of some PcG genes, such as Pc, ph-p and E(Pc). No downregulation of these genes was observed in wild-type larvae before and after heat shock, indicating that this was not an unspecific heat-shock response. Expression was examined of two genes of the Trithorax group (ash1 and brm) that function antagonistically to PcG proteins, but found no upregulation on JNK induction (Lee, 2005).

To show further that JNK has a specific effect on PcG proteins, the analogous experiment was carried out in mammalian cells. The JNK pathway can be activated in mouse embryonic fibroblasts by exposing the cells to ultraviolet light. The expression of MPh2 (mouse polyhomeotic2) was examined because this mammalian PcG gene is expressed in these cells. The expression of MPh2 was decreased on JNK induction, but after treatment with a specific JNK inhibitor it was partially restored. In addition, to show that the downregulation of PcG genes is directly controlled by AP-1, chromatin immunoprecipitation was carried out using antibodies against Fos on chromatin from UAS-hep^act; hsG4 and kay¹ mutant flies. Enrichment of Fos on the promoter region of ph-p was observed, but no enrichment in chromatin from flies lacking Fos. This finding suggests that AP-1 binds directly to this region to regulate negatively the transcription of ph-p (Lee, 2005).

If activation of JNK signalling in the blastema indeed leads to a downregulation of PcG genes, then impairment of the JNK pathway should result in reduced efficiency of transdetermination. The transdetermination behaviour of wild-type discs was compared with that of discs bearing mutations in the JNKK hep. The transdetermination events were classified into three categories: large regions, small regions, and no regions of transdetermination. In wild-type discs only large regions were detected. In males hemizygous for hep¹ (a weak hypomorphic allele), most transplanted leg discs had large transdetermined regions; however, a substantial proportion showed only small regions of transdetermination and a few showed no transdetermination event. In flies heterozygous for hep^r75 (a null allele which is hemizygous lethal), most discs showed no or only small regions of transdetermination, and large regions were rarely seen. The morphology of the regenerated discs seemed unaffected in these mutants, indicating that the decline of transdetermination efficiency was not due to inefficient wound healing (Lee, 2005).

This study has shown that PcG genes are downregulated by JNK signalling. Because many developmental regulators need to be switched, the role of PcG downregulation may be to render the cells susceptible to a change in cell identity by shifting the chromatin to a reprogrammable state. Transdetermination has been ascribed to the action of ectopic morphogens, which induce cells to activate incorrect gene cascades. Without doubt, wg and decapentaplegic signalling must be crucially involved in this process, because transdetermination does not result from any random cut but occurs preferentially when cuts are made through particular regions of the disc called 'weak points', which are regions of high morphogen. Inappropriate or overextreme downregulation of the PcG system by JNK in sensitive cells of the weak points thus may create such aberrant local patterns. Indeed, the data indicate that at least the two patterning genes, wg and vg, may be direct targets of the PcG. Notably, hyperactive Wnt signalling can also induce a switch in lineage commitment in mammals, implying that signalling pathways are a potent inducer of cell fate changes in many organisms (Lee, 2005).

Another study has shown that regenerating and transdetermining cells in the blastema have a distinct cell-cycle profile in contrast to the surrounding normal disc cells. It has been proposed that this change in cell-cycle regulation is a prerequisite for the change in cell fate. Indeed, PcG targets include genes involved in cell-cycle regulation, suggesting that this initial step is part of the complete reprogramming cascade required for the regenerating cells to achieve multipotency. Downregulation of PcG silencing by JNK seems to be a fundamental, evolutionarily conserved mechanism of cell fate change and thus may also have implications for studies of stem cell plasticity and tissue remodelling (Lee, 2005).

A signaling network for patterning of neuronal connectivity in the Drosophila brain

The precise number and pattern of axonal connections generated during brain development regulates animal behavior. Therefore, understanding how developmental signals interact to regulate axonal extension and retraction to achieve precise neuronal connectivity is a fundamental goal of neurobiology. This question was investigated in the developing adult brain of Drosophila. Extension and retraction is regulated by crosstalk between Wnt, fibroblast growth factor (FGF) receptor, and Jun N-terminal kinase (JNK) signaling, but independent of neuronal activity. The Rac1 GTPase integrates a Wnt-Frizzled-Disheveled axon-stabilizing signal and a Branchless (FGF)-Breathless (FGF receptor) axon-retracting signal to modulate JNK activity. JNK activity is necessary and sufficient for axon extension, whereas the antagonistic Wnt and FGF signals act to balance the extension and retraction required for the generation of the precise wiring pattern (Srahna, 2006).

Based on the observation that blocking Fz2 results in decreased numbers of dorsal cluster neuron (DCN) axons in the medulla, it was reasoned that Fz2 could be a receptor for a putative stabilization signal. Since Fz2 and Fz are partially redundant receptors for the canonical Wnt signaling pathway, expression of the canonical Wnt ligand Wingless (Wg) was investigated in the brain during pupation. However, no Wg expression was detected in the pupal optic lobes, suggesting that Wg is unlikely to be involved in regulating DCN axon extension. Therefore, the expression of Wnt5, which has been shown to be involved in axon repulsion and fasciculation in the embryonic CNS, was investigated. Anti-Wnt5 staining revealed widely distributed Wnt5 expression domains beginning at PF and lasting throughout pupal development and into adult life. Wnt5 is strongly expressed in the distal medulla and is also present on axonal bundles crossing the second optic chiasm.The number of DCN axons crossing to the medulla was examined in wnt5 mutant flies. The number of DCN axons crossing the optic chiasm is reduced from 11.7 to 7.9 in the absence of wnt5, suggesting that it may play a role in stabilizing DCN axons (Srahna, 2006).

Next, the requirement of the Wnt signaling adaptor protein Dsh was tested. In animals heterozygous for dsh⁶, a null allele of dsh, the average number of DCN axons crossing between the lobula and the medulla is reduced from 11.7 to 7.6 with 78.5% showing less than eight axons crossing. Signaling through Dsh is mediated by one of two domains. Signaling via the DIX (Disheveled and Axin) domain is thought to result in the activation of Armadillo/β-Catenin. DEP (Disheveled, Egl-10, Pleckstrin) domain-dependent signaling results in activation of the JNK signaling pathway by regulation of Rho family GTPase proteins during, for example, convergent extension movements in vertebrates. To uncover which of these two pathways is required for DCN axon extension the dsh¹ mutant, deficient only in the activity of the DEP domain, was tested. Indeed, in brains from dsh¹ heterozygous animals the number of extending axons was reduced from 11.7 to 7.4. In flies homozygous for the dsh¹ allele the average number of axons crossing was further reduced to 4.7, with all the samples having less than six axons crossing. In contrast, the DCN-specific expression of Axin, a physiological inhibitor of the Wnt canonical pathway, did not affect the extension of DCN axons. Similarly, expression of a constitutively active form of the fly β-Catenin Armadillo also had no apparent effect on DCN extension. Finally, whether Wnt5 and Dsh interact synergistically was tested. To this end, wnt5, dsh¹ trans-heterozygous animals were generated. These flies show the same phenotype as flies homozygous for dsh¹, suggesting that Wnt5 signals through the Dsh DEP domain (Srahna, 2006).

To determine if dsh is expressed at times and places suggested by its genetic requirement in DCN axon outgrowth, the distribution of Dsh protein during brain development was examined. Dsh protein is ubiquitously expressed during brain development. High expression of Dsh is detected in the distal ends of DCN axons at about 15% PF shortly before they extend across the optic chiasm toward the medulla. In general, higher levels of Dsh were observed in the neuropil than in cell bodies (Srahna, 2006).

In summary, these data indicate that the stabilization of DCN axons is dependent on the Dsh protein acting non-canonically via its DEP domain. Importantly, the axons that do cross in dsh mutant brains do so along the correct paths. This suggests that, like JNK signaling, Wnt signaling regulates extension, but not guidance, of the DCN axons (Srahna, 2006).

Wnt signaling to Dsh requires the Fz receptors. To examine if the effect of Wnt5 on DCN axon extension is also mediated by Fz receptors, the number of DCN axons crossing the optic chiasm in was counted fz, fz2, and fz3 mutants. There was no significant change in the number of axons crossing in the brain of fz3 homozygous animals. In contrast, in brains heterozygous for fz and fz2, the number of the axons crossing was reduced from 11.7 to 6.6 (fz) and 6.9 (fz2), with 71% and 85.7%, respectively, showing less than eight axons crossing. These data suggest that DCN axons respond to Wnt5 using the Fz and Fz2 receptors, but not Fz3. To determine whether the Fz receptors act cell-autonomously in individual DCNs, single-cell clones doubly mutant for fz and fz2 were generated and the number of DCN axons crossing the optic chiasm was counted. In contrast to wild-type cells, where 37% of all DCN axons cross, none of the fz, fz2 mutant axons reach the medulla. To test whether wnt5, fz, and fz2 genetically interact in DCNs, flies trans-heterozygous for wnt5 and both receptors were examined. Flies heterozygous for both wnt5 and fz mutations show a strong synergistic loss of DCN axons (11.7 to 3.7) and in fact have a phenotype very similar to that of flies homozygous for dsh¹. Flies doubly heterozygous for wnt5 and fz2 also show a significant decrease in DCN axons (5.7), compared with either wnt5 (~8) or fz2 (8.5) mutants. These data indicate that the genetic interaction between wnt5 and fz is stronger than the interaction between wnt5 and fz2 (Srahna, 2006).

Examination of the expression domains of Fz and Fz2 in the developing brain supports the possibility that they play roles in stabilizing DCN axons. Both Fz and Fz2 are widely expressed in the developing adult brain neuropil. In addition, Fz is expressed at higher levels in DCN cell bodies (Srahna, 2006).

The observation that the wnt5 null phenotype can be enhanced by reduction of Fz, Fz2, or Dsh suggests that another Wnt may be partially compensating for the loss of Wnt5. To test this possibility, flies heterozygous for either wnt2 or wnt4 were examined. wnt2 heterozygotes display a reduction of DCN axon crossing from 11.7 to 7.3, whereas no phenotype was observed for wnt4. Thus, wnt2 and wnt5 may act together to stabilize the subset of DCN axons that do not retract during development. In summary, these results support the model that Wnt signaling via the Fz receptors transmits a non-canonical signal through Dsh resulting in the stabilization of a subset of DCN axons (Srahna, 2006).

Data is provided that supports the hypothesis that the regulation of JNK by Rac1 modulates DCN axon extension. As such attempts were made to determine how Wnt signaling might interact with Rac1 and JNK. The opposite phenotypes of dsh and Rac1 loss-of-function suggest that they might act antagonistically. To determine if Rac1 is acting upstream of, downstream of, or in parallel to Dsh in DCN axon extension, dominant-negative Rac1 was expressed in dsh¹ mutant flies. If Rac1 acts upstream of Dsh, the dsh¹ phenotype (i.e., decreased numbers of axons crossing the optic chiasm) is expected. If Rac1 acts downstream of Dsh, the Rac1 mutant phenotype (i.e., increased number of axons crossing) would be expected If they act in parallel, an intermediate, relatively normal phenotype is expected. Increased numbers of axon crossing were observed, suggesting that Rac1 acts downstream of Dsh during DCN axon extension and that Dsh may repress Rac1 (Srahna, 2006).

Next, whether Dsh control of DCN axon extension is mediated by the JNK signaling pathway acting downstream of Wnt signaling was tested, as the similarity of their phenotypes suggests. If this were the case, activating JNK signaling should suppress the reduction in Dsh levels. Conversely, reducing both should show a synergistic effect. Therefore the JNKK hep was expressed in dsh¹ heterozygous flies and it was found that the hep gain-of-function is epistatic to dsh loss-of-function. Furthermore, reducing JNK activity by one copy of BSK-DN in dsh¹ mutant animals results in a synergistic reduction of extension to an average of 0.8 axons with 60% showing no axons crossing and no samples with more than three axons. In summary, the results of genetic analyses suggest that Wnt signaling via Dsh enhances JNK activity through the suppression of Rac1 (Srahna, 2006).

Dsh appears to promote JNK signaling and to be expressed in DCN axons prior to their extension toward the medulla early in pupal development. Since JNK signaling is required for this initial extension, it may be that Dsh also plays a role in the early extension of DCN axons. To test this possibility, DCN axon extension was examined at 30% pupal development in dsh¹ mutant brains. In wild-type pupae, essentially all (~40) DCN axons extend toward the medulla. In contrast, in dsh¹ mutant pupae, a strong reduction in the number of DCN axons crossing the optic chiasm between the lobula and the medulla was observed (Srahna, 2006).

Although the genetic data indicate that Dsh- and Rac-mediated signaling have sensitive and antagonistic effects on the JNK pathway, they do not establish whether the Dsh-Rac interaction modulates JNK's intrinsic activity. To test this, the amount of phosphorylated JNK relative to total JNK levels in fly brains was evaluated by Western blot analysis using phospho-JNK (P-JNK) and pan-JNK specific antibodies. Then it was determined if Dsh is indeed required for increased levels of JNK phosphorylation. Dsh¹ mutant brains showed a 25% reduction in P-JNK consistent with a stimulatory role for Dsh on JNK signaling. The reduction caused by loss of Dsh function is reversed, when the amount of Rac is reduced by half, consistent with a negative effect of Rac on JNK signaling downstream of Dsh. These data support the conclusion that Dsh and Rac interact to regulate JNK signaling by modulating the phosphorylated active pool of JNK (Srahna, 2006).

Taken together, these data suggest that during brain development DCN axons extend under the influence of JNK signaling. A non-canonical Wnt signal acting via Fz and Dsh ensures that JNK signaling remains active by attenuating Rac activity. In contrast, activation of the FGFR activates Rac1 and suppresses JNK signaling. These data support a model whereby the balance of the Wnt and FGF signals is responsible for determining the number of DCN axons that stably cross the optic chiasm. To test this model, FGFR levels were reduced, using the dominant-negative btl transgene, in dsh¹ heterozygous flies. It was found that simultaneous reduction of FGF and Wnt signaling restored the number of axons crossing the optic chiasm to almost wild-type levels (10.2, with 33% of the samples indistinguishable from wild-type, suggesting that the two signals in parallel, act to control the patterning of DCN axon connectivity (Srahna, 2006).

These data suggest the following model of DCN axon extension and retraction. DCN axons extend due to active JNK signal. These axons encounter Wnt5 and probably Wnt2 as well, resulting in activation of Disheveled. Disheveled, via its DEP domain, has a negative effect on the activity of the Rac GTPase, thus keeping JNK signaling active. After DCN axons cross the second optic chiasm they encounter a spatially regulated FGF/Branchless signal that activates the FGFR/Breathless pathway. Breathless in turn activates Rac, which inhibits JNK signaling in a subset of axons. These axons then retract back toward the lobula. The wide expression of the different components of these pathways and the modulation of JNK phosphorylation by Dsh and Rac in whole-head extracts strongly suggests that this model may apply to many neuronal types (Srahna, 2006).

Oogenesis

Notch signaling through tramtrack bypasses the mitosis promoting activity of the JNK pathway in the mitotic-to-endocycle transition of Drosophila follicle cells

The follicle cells of the Drosophila egg chamber provide an excellent model in which to study modulation of the cell cycle. During mid-oogenesis, the follicle cells undergo a variation of the cell cycle, endocycle, in which the cells replicate their DNA, but do not go through mitosis. Previously, it was shown that Notch signaling is required for the mitotic-to-endocycle transition, through downregulating String/Cdc25, and Dacapo/p21 and upregulating Fizzy-related/Cdh1. In this paper, it is shown that Notch signaling is modulated by Shaggy and temporally induced by the ligand Delta, at the mitotic-to-endocycle transition. In addition, a downstream target of Notch, tramtrack, acts at the mitotic-to-endocycle transition. It is also demonstrated that the JNK pathway is required to promote mitosis prior to the transition, independent of the cell cycle components acted on by the Notch pathway. This work reveals new insights into the regulation of Notch-dependent mitotic-to-endocycle switch (Jordan, 2006).

Notch controls the mitotic-to-endocycle transition in follicle epithelial cells; Notch pathway activity arrests mitotic cell cycle and promotes endocycles by downregulating string/cdc25 and dacapo/p21, and upregulating fzr/Cdh1. This study identified components regulating this transition, Delta, Shaggy, and Tramtrack. Shaggy and Delta are required for the activation of Notch protein. However, Delta is sufficient to activate Notch in this process, since premature expression of Delta in the germline stops mitotic division of the follicle cells. This study identified Tramtrack as a connection between Notch and the cell cycle regulators stg, fzr, and dap. Loss of Tramtrack function phenocopies the Notch and Su(H) phenotypes; overproliferation and misregulation of cell cycle components. However, high FAS3 expression, indicative of differentiation defects in Notch clones, is not observed in ttk clones, suggesting that Tramtrack might regulate a branch of the Notch pathway specific for cell cycle control. It was also shown that the JNK-pathway is a critical mitosis promoting pathway in follicle cells. Loss of JNK(bsk) or JNKK(hep) activities stop follicle cell mitotic cycles, while loss of JNK promotes premature endocycles. In addition, loss of the negative regulator of the pathway, the phosphatase Puckered, results in a lack of endocycles. However, the Notch-responsive cell cycle targets that, in combination, can induce the mitotic-to-endocycle transition, stg, fzr, and dap, are not regulated by the JNK-pathway (Jordan, 2006).

Notch signaling is highly regulated throughout development. The Notch receptor can be regulated by glycosylation of the extracellular domain, as well as by endocytosis and degradation of the intracellular domain, thus affecting the activity of the pathway. Shaggy has been shown to phosphorylate and thus affect the stability of Notch protein. Normal processing and clearing of Notch protein from the apical surface of follicle cells upon Notch activation does not occur in shaggy clones, indicating that Notch is not normally activated and therefore regulation of the downstream targets does not take place (Jordan, 2006).

In many organisms and tissues the Notch ligands are ubiquitously expressed and thus not likely to regulate Notch pathway activation. However, at the mitotic to endocycle transition, Delta is upregulated in the germline, making ligand expression a likely candidate for regulation of Notch activity. Premature expression of Delta in the germline can cause mitotic division to stop at least one stage earlier than in control ovarioles. Nonetheless, this effect is seen in only half of the ovarioles. Therefore, it is possible that yet another process is regulating Notch activity at the transition in addition to Delta expression. Further testing will determine if endocytosis of Notch might also regulate Notch activity at the mitotic-to-endocycle transition. One possible protein is Numb, which regulates Notch in human mammary carcinomas, indicating that Numb may have a more general role in cell cycle control than just the division of the sensory organ precursors (Jordan, 2006).

The fact that Notch overrides the mitotic activity of the JNK pathway by acting on cell cycle regulators that can induce the mitotic-to-endocycle transition puts further demand on understanding the connection between Su(H) and cell cycle regulators. One such component, the transcription factor Tramtrack, has been identified. Two Tramtrack proteins exist, Ttk69 and Ttk88, both of which are affected by the allele used in these studies. However, staining with antibodies specific to the two forms reveals that only Ttk69 is detectable in the follicle cells and downregulated in Notch clones (Jordan, 2006).

Ttk69 can control proliferation in glial cells, strengthening its candidacy for a critical component between Notch and cell cycle controllers in follicle epithelial cells. In addition, the Ttk-like BTB/POZ-domain zinc-finger transcription repressor in humans is Bcl-6, a protein associated with B-cell lymphomas (Jordan, 2006).

Ttk function in the follicle cell mitotic-to-endocycle transition was analyzed and it has been shown that the Notch-responsive cell cycle components stg, dap, and fzr are responsive to Ttk function. Interestingly, Ttk69 controls the string promoter in the Drosophila eye discs. In the future, it will be important to determine whether Ttk DNA binding sites are found in the Notch-responsive stg promoter as well. In addition, the binding sites of transcription factors that can interact with Ttk will be of interest, since Ttk can act as a DNA binding or non-binding repressor (Jordan, 2006).

Previous work revealed that the JNK pathway is closely connected to cell cycle control. For example, in fibroblasts the JNK pathway is critical for cdc2 expression and G2/M cell cycle progression. In the case of the follicle cell mitotic-to-endocycle transition, it was shown that the JNK pathway is a critical positive controller of the mitotic cycles. Lack of JNK activity leads to a block in mitosis and initiation of premature endocycles. Conversely, lack of the negative regulator of the JNK-pathway, the phosphatase Puckered, results in a loss of endocycles. However, puc mutant clones do not consistently support extra divisions but might induce apoptosis as shown recently in disc clones (Jordan, 2006).

These data are interesting in light of the results showing that the JNK pathway does not control the same cell cycle targets as the Notch pathway, and could be explained by the following hypothesis: the JNK-pathway positively regulates the mitotic cycles prior to stage 6 in follicle epithelial cells. This positive action on mitotic cycles is negatively short-circuited by the direct control of cell cycle regulators by the Notch pathway at stage 6 in oogenesis, resulting in the mitotic-to-endocycle transition. Premature termination of the JNK pathway is sufficient to induce mitotic-to-endocycle transition. However, prolonged JNK activity, while disrupting endocycles, cannot maintain mitotic cycling efficiently, due to Notch action on string, dacapo, and fzr (Jordan, 2006).

What then terminates JNK-pathway activity at stage 6 in oogenesis? Prolonged JNK activity (puc mutant clones) affects endocycles and the expression of pJNK and Puc subsides at stages 6–7; results that both suggest the downregulation of JNK activity at the mitotic-to-endocycle transition. One possibility is that Notch activity downregulates the JNK pathway. However, at least Su(H)-dependent Notch activity does not regulate the JNK pathway, since no effect on puckered expression was observed in Su(H) mutant clones. It is plausible that Su(H)-independent Notch activity regulates the JNK pathway in this context, as has been shown to be the case in dorsal closure. Interestingly, Deltex might play a role in this Su(H)-independent Notch activity (Jordan, 2006).

An important question in analyzing the developmental control of cell cycle is whether the same signaling pathways control both differentiation and cell cycle, and if so, how the labor is divided. The Notch-dependent mitotic-to-endocycle transition is an example of such a question; Notch action in stage 6 follicle cells is critical for the cell cycle switch and for at least some aspects of differentiation. This work reports the first component that separates Notch dependent cell cycle regulation from Fas3 marked differentiation; Ttk. In the ttk mutant clones, upregulation of FAS3, characteristic for Notch clones, is not observed. Therefore, Ttk constitutes a branch of Notch activity that might be solely required for cell cycle control in this context. However, Ttk's independent function cannot yet be rule out. In the future, it will be important to understand whether signaling pathways in general show a clear separation of differentiation and cell cycle control on the level of downstream transcription factors. Importantly, these and previous results have revealed the essential cell cycle regulators and their roles in controlling the Notch-dependent mitotic-to-endocycle switch (Jordan, 2006).

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