Djun

DEVELOPMENTAL BIOLOGY

Embryonic

DJun appears to be uniformly expressed at a low level in all cell types (Perkins, 1990).

Larval

The expression of the transcription factor DJun in the eye imaginal disc correlates temporally and spatially with the determination of neuronal photoreceptor fate. Expression of dominant negative forms of DJun in photoreceptor precursor cells results in dose-dependent loss of photoreceptors in the adult fly. Conversely, localized overexpression of DJun in the eye imaginal disc can induce the differentiation of additional photoreceptor cells. The transformation of nonneuronal cone cells into R7 neurons elicited by constitutively active forms of sevenless, Ras1, Raf, and MAP kinase is relieved in the presence of DJun mutants. These results demonstrate a requirement of DJun downstream of the sevenless/ras signaling pathway for neuronal development in the Drosophila eye (Bohmann, 1994).

A good example of the function of EGF-R in regulating cell development is found by examining the role of EGF-R in midline glia maturation. The midline glial cells are required for correct formation of the axonal pattern in the embryonic ventral nerve cord. Initially, six midline cells form an equivalence group with the capacity to develop as glial cells. By the end of embryonic development three to four cells are singled out as midline glial cells. Midline glia development occurs in two steps, both of which depend on activation of the EGF-Receptor and subsequent Ras1/Raf-mediated signal transduction (See Drosophila Ras1) (Scholz, 1997).

Egf-r mutants show a reduced number of midline glial cells and argos mutants, which possibly exhibit an increased activation of Egf-r in the midline, show an increased number of midline glial cells. Furthermore, expression of activated ras1 (or activated raf) in the midline results in the appearance of extra midline cells. This model suggests that activation of ras1 signaling in the entire midline glial equivalence group promotes survival of all cells in this cluster. Thus, in wild-type flies, about 2-3 cells in each group down-regulate Egf-r signaling and are destined for cell death.

Another factor appears to promote midline glial cell survival: a signal appears to be conveyed via contact with commissural axons. In mutants that lack commissural axons, the midline glial cells die. One can bypass the requirement of axonal contact for midline glia survival by the expression of activated Drosophila jun. Expression of activated Drosophila jun results in missing commissural axon tracts (Scholz, 1997).

Effects of Mutation or Deletion

DJun in the embryo is a downstream target of the Jnk signal transduction pathway during dorsal closure formation, and the function of the JNK/DJun pathway is to control the localized expression of decapentalegic. In contrast to previous observations, both in the embryo and during photoreceptor cell determination, DJun is not regulated by a pathway that involves MAPK. Clones of cells homozygous for DJun show no detectable phenotype, indicating that DJun plays no role in photoreceptor cell differentiation in the eye. This evidence contradicts earlier results (Bohmann, 1994, Treier, 1995, Peverali, 1996 and Kockel, 1997). A simple explanation is that the previous experiments were performed under nonphysiological conditions. The dominant negative and constitutively activated forms of DJun employed in these experiments may bind to factors regulated by the MAPK pathway and block their normal functions, or the modified Jun proteins may compete with factors in the MAPK pathway for binding to the promoter elements of dowstream target genes. Alternatively, there may be another jun gene in the Drosophila genome (Hou, 1997).

Embryos lacking Jun activity exhibit a dorsal closure phenotype, very similar to that of basket and hemipterous mutants, indicating that Jun is a target of Hep/Bsk signaling. In eye and wing development Jun participates in a separate signaling pathway comprised of Ras, Raf, and the ERK-type kinase Rolled. In contrast to the strict requirement for Jun in dorsal closure, its role in the eye is redundant but can be uncovered by mutations in other signaling components. The removal of Jun function in the eye by mutation shows only minor defects. Occasionally, only one or two photoreceptors are lost in mutant ommatidia. Nevertheless, gain- and loss-of-function forms of Jun interfere specifically with the endogenously expressed wild-type protein, and Jun interacts genetically with the Sev/Ras/Raf/ERK signal transduction pathway. For example, when one copy of DJun is removed from transgenic lines expressing gain of function sevenless, ras and rolled mutations, a clear suppression of the mutant extra photoreceptor phenotype can be observed. The redundant function of Jun in eye development may contribute to the precision of photoreceptor differentiation and ommatidial assembly. Analysis of DJun mutants in the wing does not reveal any phenotypic defect characteristic of the Ras pathway. Nevertheless, removal of one copy of DJun suppresses the wing phenotypic defects of Ellipse gain-of-function alleles of the Epidermal growth factor receptor. It is concluded that DJun plays a role both in wing and eye development. It is suggested that the role of DJun in the wing and eye is not essential since other systems maintain proper morphogenesis in the absence of DJun. It is also concluded that DJun is a target of both JNK and MAP kinase in Drosophila (Kockel, 1997).

Frizzled family proteins have been described as receptors of Wnt signaling molecules. In Drosophila, the two known Frizzled proteins are associated with distinct developmental processes. Genesis of epithelial planar polarity requires Frizzled, whereas Dfz2 affects morphogenesis by wingless-mediated signaling. Dishevelled is required in both signaling pathways. Genetic and overexpression assays have been used to show that Dishevelled activates JNK cascades. In contrast to the action of wingless-pathway components, mutations in rhoA, hemipterous, basket, and jun as well as deficiencies removing the Rac1 and Rac2 genes show a strong dominant suppression of a Dishevelled overexpression phenotype in the compound eye. In an in vitro assay, expression of Dsh has been shown to induce phosphorylation of Jun, indicating that Dsh is a potent activator of the JNK pathway. Whereas the PDZ domain of Dsh, known to be required in the transduction of the wingless signal, is dispensable for signal-independent induction of Jun phosphorylation, the C-terminal DEP domain of Dsh is found to be essential. The planar polarity-specific dsh1 allele is found to be mutated in the DEP domain. These results indicate that different Wnt/Fz signals activate distinct intracellular pathways, and Dishevelled discriminates among them by distinct domain interactions (Boutros, 1998).

How can Fz/Dsh signaling be linked to small GTPase and JNK/MAPK pathways? Recent studies provided evidence that links G protein-coupled receptors, which share structural features with Fz proteins, to MAPK signaling through heterotrimeric G proteins and PI-3 kinases. It is intriguing to speculate that a subset of Fz proteins might signal through a similar pathway. It was also shown recently that XWnt5A and rFz2, in a heterologous assay, increase intracellular calcium via G proteins and phosphoinositol signaling. A mutation in the beta-subunit of a heterotrimeric G protein in C. elegans prevents correct spindle orientation, a process that is believed to be dependent on a Wnt and a Fz receptor, but not on Arm. Further studies regarding a possible involvement of PI-3K and G proteins in planar polarity signaling may provide additional insight to the diversity of Fz-related signaling pathways (Boutros, 1998 and references).

A test of a constitutively active form of Jra (DJun) determined it could rescue the dorsal open phenotype in misshapen msn mutant embryos. Previous studies have shown that activated Djun rescues the bsk phenotype, indicating that one of the main functions of JNK is to phosphorylate and activate Jun. A constitutively active form of Jra was made by replacing the JNK phosphorylation sites with acidic residues. To test whether this activated Djun rescues the dorsal open phenotype in msn mutant embryos, it was expressed under the control of the hsp70 heat shock promoter in the msn mutant background. Expression of activated Djun rescues the dorsal open phenotype in most of the msn mutant embryos; heat shock decreased the number of embryos with a dorsal open phenotype from about 50%. In addition, expression of an activated form of tkv, tkv^Q253D, also rescues the dorsal open phenotype in msn mutant embryos. GAL4 driven by the ectoderm-specific promoter at 69B was used to direct the expression of UAS-tkv^Q253D in msn mutant embryos. This expression of activated tkv partially rescues the dorsal open phenotype caused by msn; it also has a dorsalizing effect on the ventral ectoderm of the embryos related to the earlier function of dpp in establishing the dorsoventral axis, which served to mark embryos expressing activated tkv. Thus, these findings provide genetic evidence that msn functions upstream of the JNK MAP kinase module in leading edge cells (Su, 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).

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).

Jun acts as a signal-regulated transcription factor in many cellular decisions, ranging from stress response to proliferation control and cell fate induction. Genetic interaction studies have suggested that Jun and JNK signaling are involved in Frizzled (Fz)-mediated planar polarity generation in the Drosophila eye. However, simple loss-of-function analysis of JNK signaling components does not show comparable planar polarity defects. To address the role of Jun and JNK in Fz signaling, a combination of loss- and gain-of-function studies has been used. Like Fz, Jun affects the bias between the R3/R4 photoreceptor pair that is critical for ommatidial polarity establishment. Detailed analysis of jun^- clones reveals defects in R3 induction and planar polarity determination, whereas gain of Jun function induces the R3 fate and associated polarity phenotypes. Affecting the levels of JNK signaling by either reduction or overexpression leads to planar polarity defects. Similarly, hypomorphic allelic combinations and overexpression of the negative JNK regulator Puckered causes planar polarity eye phenotypes, establishing that JNK acts in planar polarity signaling. The observation that Delta transcription in the early R3/R4 precursor cells is deregulated by Jun or Hep/JNKK activation, reminiscent of the effects seen with Fz overexpression, suggests that Jun is one of the transcription factors that mediates the effects of fz in planar polarity generation (Weber, 2000).

Jun, as a member of the AP-1 family, is activated by many distinct extracellular stimuli and acts downstream of several signaling pathways. Besides its involvement in stress response, Jun has been implicated in the control of proliferation, apoptosis, morphogenesis and cell fate induction. In Drosophila, Jun is critical for the process of dorsal closure in embryogenesis acting downstream of the JNK module. It has also been implicated in cell fate induction downstream of Ras/ERK signaling in the eye. This analysis has shown that Jun also acts downstream of Fz in planar polarity signaling in the eye. It is the first transcription factor implicated in Fz/planar polarity signaling. Fz signaling also requires a JNK (or related kinase) module, and thus in the eye imaginal disc Jun acts downstream of both ERK and JNK. How does Jun achieve a specific response in this context? The S/T residues that are phosphorylated in Jun are the same for both ERK and JNK. Thus, although differences in phosphorylation level and/or preference for any of the serine/threonine target residues cannot be excluded in vivo, differential phosphorylation is unlikely to create specificity. A potential mechanism for specificity might be provided by other transcription factors that cooperate with Jun in the different processes. This is supported by the observation that the sev-Jun^Asp (expression of a constitutively active Jun) phenotype is a composite of two events, photoreceptor recruitment and ommatidial polarity generation. These two effects can, however, be separated by the reduction of specific interacting partners. In the process of Ras/ERK signaling in photoreceptor induction Jun interacts and synergizes with the ETS domain transcription factor Pointed (Pnt). Pnt has been characterized as a target of the ERK/Rl kinase in Drosophila in all ERK-dependent processes analyzed. However, it has not been linked to any JNK-mediated process. Removing one dose of pnt strongly suppresses the Ras/ERK-related extra photoreceptor phenotype of sev-Jun^Asp, whereas the polarity defects persist and thus are more prominent. This observation indicates that, in the absence of normal Pnt levels, sev-Jun^Asp specifically affects polarity, suggesting that the interaction with Pnt is important for its role in the ERK-mediated induction. It is likely that for its planar polarity function other specific transcription factors provide the specificity cues (Weber, 2000).

Although all components of the JNK module tested genetically interact with sev-Fz and sev-Dsh, analysis of existing loss-of-function mutants did not show defects in planar polarity establishment, suggesting a redundant role. Even null alleles of the Drosophila homolog of JNKK hep have no effects on planar polarity (Weber, 2000).

However, expression of a dominant negative (kinase dead) isoform of Bsk interferes with planar polarity, giving rise to typical polarity phenotypes, implying that Bsk and JNK signaling are important in this process. Consistently, homozygous mutant clones of the deficiency Df(2R)flp170B that removes bsk and other neighboring loci (a deficiency considered to be a true null for bsk), show a mild polarity phenotype in the eye, including the presence of symmetrical ommatidia (Weber, 2000).

What are the redundant kinases in this process? Genetic interaction analysis with sev-Msn (Misshapen expressed in a Sevenless pattern) has shown that, besides hep and bsk, deficiencies affecting other MKKs and the Drosophila p38a and p38b loci suppress the sev-Msn phenotype. This suggested that the p38 kinase module [related to JNK and has been shown to have (at least partially) overlapping phosphorylation targets] might be responsible for the redundancy in this process. The analysis with the dominant negative (DN) Bsk isoform and the respective deficiencies suggests that the p38 kinase(s) are contributing to this redundancy, because they enhance the DN-Bsk phenotype in a manner very similar to that of the bsk deficiency. The identification of specific mutant alleles of p38a/b and double mutant analysis with bsk will be necessary to further clarify this issue (Weber, 2000).

The available results indicate that the level of JNK/p38 signaling in planar polarity establishment is important, but that the removal of a single kinase does not significantly affect this level. In support, the observation that an allelic combination of hep and puc hypomorphic alleles can give rise to planar polarity eye phenotypes suggests that the balance between negative and positive regulators of JNK and related kinases is critical. Similarly, overexpression of the negative JNK regulator Puc, a dual specificity phosphatase, causes typical polarity defects similar to those of fz or dsh mutants. It is likely that this phosphatase negatively regulates all JNK-related kinases and thus reduces the overall signaling more than the lack of a single kinase (Weber, 2000).

In summary, these data indicate that the transcriptional events downstream of Fz in R3 specification and chirality establishment (e.g. regulation of Dl) are mediated by Jun. The factors with which Jun is redundant in the imaginal discs are not yet identified. It is possible that other members of the AP-1 family are also involved in planar polarity signaling, since they are related to Jun and could dimerize with it via the leucine-zipper motif. A potential candidate is Fos, because like Jun, Fos is required downstream of JNK in the process of dorsal closure in the embryo. Similarly, the ETS domain protein Yan acts as a negative regulator in dorsal closure and is inactivated by JNK in the process. However, these factors do not show informative planar polarity phenotypes in clones and thus their involvement in this process remains unclear. Although AP-1 and ETS family members are attractive candidates, transcription factors belonging to other families cannot be excluded in this context (Weber, 2000).

An examination was carried out to see whether directed overexpression of TGF-ß activated kinase 1 in the eye imaginal disc of third instar larvae (at the time of planar polarity Fz/JNK signaling) can interfere with the correct establishment of planar polarity. To this end UAS-Tak1 was expressed in photoreceptor precursors R3/R4 in the eye imaginal disc (under the sev-enhancer GAL4 driver: sev>Tak1). This type of overexpression creates specific eye planar polarity phenotypes with Fz, Dsh and other components of planar polarity signaling. Weak Tak1 expression (by rearing the flies at 18°C) causes a specific phenotype reminiscent of that caused by the components of planar polarity signaling, with polarity defects affecting both rotation and chirality, and also some loss of photoreceptors. This phenotype is already evident with the appropriate markers (e.g. svp-lacZ) at the time of planar polarity establishment in the third instar eye disc, indicating that it is a primary defect in polarity establishment, and not due to late differentiation defects (Mihaly, 2001).

The GOF sev>Tak1 phenotype provides a tool to test for genetic interactions with mutations in components of the Fz/planar polarity pathway and other signaling cascades. In such genetic interaction assays, it was found that reducing the dosage of the JNK signaling components (hep, bsk and D-jun) causes a strong suppression of the sev>Tak1 phenotype. These results are consistent with Tak1 acting upstream of the JNK module in polarity signaling, and support the notion that Tak1 can act generally upstream of JNK signaling (Mihaly, 2001).

During Drosophila oogenesis, the formation of the egg respiratory appendages and the micropyle require the shaping of anterior and dorsal follicle cells. Prior to their morphogenesis, cells of the presumptive appendages are determined by integrating dorsal-ventral and anterior-posterior positional information provided by the epidermal growth factor receptor (EGFR) and Decapentaplegic (Dpp) pathways, respectively. Another signaling pathway, the Drosophila Jun-N-terminal kinase (JNK) cascade, is essential for the correct morphogenesis of the dorsal appendages and the micropyle during oogenesis. Mutant follicle cell clones of members of the JNK pathway, including DJNKK/hemipterous (hep), DJNK/basket (bsk), and Djun, block dorsal appendage (DA) formation and affect the micropyle shape and size, suggesting a late requirement for the JNK pathway in anterior chorion morphogenesis. In support of this view, hep does not affect early follicle cell patterning as indicated by the normal expression of kekkon (kek) and Broad-Complex (BR-C), two of the targets of the EGFR pathway in dorsal follicle cells. Furthermore, the expression of the TGF-ß homolog dpp, which is under the control of hep in embryos, is not coupled to JNK activity during oogenesis. hep controls the expression of puckered (puc) in the follicular epithelium in a cell-autonomous manner. Since puc overexpression in the egg follicular epithelium mimics JNK appendages and micropyle phenotypes, it indicates a negative role of puc in their morphogenesis (Suzanne, 2001).

The making of a mature egg is a multistep process during which the oocyte differentiates, grows, acquires polarity, and is finally embedded into a shell secreted by the overlaying follicle cells. During this maturation process, the activities of several signaling cascades are required and coordinated, with some of them, like the Egfr pathway, being used reiteratively. The JNK pathway is required during late oogenesis for the morphogenesis of the DA and micropyle, thus adding a new player in the signaling machinery underlying egg formation. Since the other two MAPK pathways (ERK and p38) have also been shown to be involved in oogenesis, the Drosophila egg chamber represents a paradigm for the study of multiple MAPK signaling pathways during development (Suzanne, 2001).

The outer follicular epithelium surrounding each oocyte secretes the chorionic envelope to protect the mature egg from external aggressions. During the late stages of its development, the follicular epithelium undergoes extensive morphogenesis in its anterior region, resulting in the decoration of the egg with few stereotyped structures. These include the DA, the operculum, and the micropyle, which are all essential for the egg. The micropyle allows sperm entry and fertilization, the operculum provides an exit for the hatching larvae, and the two DA serve as floating and breathing devices when the egg is covered by liquid. Interestingly, the DA show an extreme variation in their shape and number in different Drosophila species and the Egfr pathway may provide the molecular basis for this variability (Suzanne, 2001).

The analysis of hep, bsk, and Djun mutant clones indicates that JNK pathway activity is crucial in the follicular epithelium for DA morphogenesis. The observation of a complete series of phenotypes, ranging from reduced, 'paddle-less' to completely nonelongated appendages, suggests that the JNK pathway plays a role in the elongation and shaping of these structures. As shown by the normal expression of two targets of the ERK pathway, kek and BR-C, hep does not affect the patterning or development of the appendages during early and midoogenesis. It is proposed that the JNK pathway plays a previously unknown role in late oogenesis for appropriate morphogenesis of the DA and micropyle. The unique phenotype of JNK pathway mutants may thus provide a link between pattern formation and morphogenesis in the egg chamber (Suzanne, 2001).

hep and the JNK pathway lie downstream of both the Egfr and Dpp pathways in DA formation during late oogenesis. One interesting question is whether or not JNK activation is directly mediated by the ERK and/or Dpp pathways. Since both the ERK and JNK pathways are required for appendage formation, it is tempting to speculate that they may converge and their inputs integrate at the molecular level. One good candidate for such an integrating element is the AP-1 (activating protein-1) transcription factor, whose levels of expression and activity are regulated by both the ERK and JNK pathways in vertebrate cells. As their vertebrate counterparts, the Drosophila Djun and Dfos homologs are also part of the JNK pathway, and these factors may also interact with the ERK cascade in the eye. Although the level of Dfos protein is normal in hep mutant follicle cells, analyzing the pattern of AP-1 activation in the egg chamber will be of particular interest to understand the relative contributions of the two MAPK pathways to appendage morphogenesis (Suzanne, 2001).

It has been observed that ectopic ERK activation in the posterior region of the egg can induce the formation of appendage-like material. However, this material does not fully elongate as normal appendages do, but remains very rudimentary, as observed in hep, bsk and Djun mutant clones. A possible explanation for this 'incompetence' to normally elongate is that the JNK pathway may not be activated or fully inducible in the posterior part of the egg, due to the lack of some component(s) of the JNK pathway. In this respect, it is worth noting that puc expression escapes hep control in the posterior part of the follicular epithelium (Suzanne, 2001).

The hep chorionic phenotype is accompanied by a loss of puc expression in the anterior stretched and main body follicle cells, suggesting that these cells are important for JNK-dependent morphogenesis of the appendages. This is supported by overexpression of puc in subsets of columnar follicle cells. The use of a slboGAL4 line allows for the exclusion of a role for centripetal cells in DA formation. These observations suggest that anterior main body follicle cells, including appendage precursor cells, and stretched cells, require hep function for DA elongation. Morphogenesis of the DA may thus require JNK activity in the two adjacent epithelia (stretched and columnar). For micropyle formation, the use of a slboGAL4 line has identified the centripetal cells as the ones requiring JNK activity for normal micropyle development. The absence of any obvious migratory defect in mutant border and centripetal cells excludes the possibility that micropyle shape defects are due to an early aberrant behavior of these two cell types. As for the DA, it is proposed that the micropyle is assembled in two steps: a hep-independent step requiring border and centripetal cells during early migration, and a hep-dependent step that takes place during late stages (stage 11 onward) of oogenesis (Suzanne, 2001).

Epithelia are components of many different tissues, which they shape and make functional through elaborate morphogenesis. Different cellular mechanisms underlie the movement of epithelia, including folding (gastrulation), branching (tracheal development), or migration of entire sheets (dorsal closure, imaginal discs morphogenesis, wound-healing). One important goal is to identify the molecular mechanisms underlying these different behaviors, and understand how these are modulated to contribute to the diversity encountered in developing animals. One way to understand the basis of cell movement diversity is to compare related processes controlled by a single signaling cascade, like the JNK pathway. The comparison of dorsal closure and imaginal disc morphogenesis, which both are controlled by the JNK pathway in flies, allows the proposal of a model for the morphogenesis of symmetrical epithelia containing 'free margins'. In this model, the morphogenesis or movement of bilateral epithelial sheets, like those taking place during dorsal closure, is driven by the activation of the JNK pathway in particular sites called margins. Interestingly, these margins are morphologically distinguishable, delineating two adjacent populations of cells: a columnar epithelium and a stretched one. In flies, several tissues show such an organization, including the lateral ectoderm in embryos and the imaginal discs. Strikingly, JNK activity in the egg chamber is essential for structures originating near such a boundary between a columnar (the main body or centripetal follicular epithelium) and a squamous epithelium (stretched cells), and may share several features with apparently different morphogenetic processes, like dorsal and thorax closures. Based on the current understanding of the JNK pathway in Drosophila, it is also tempting to speculate that every epithelium showing a discontinuity (i.e., the juxtaposition of a columnar and a stretched epithelium) may use the JNK pathway for its morphogenesis (Suzanne, 2001).

All the processes involving the JNK pathway also require a normal dpp activity, suggesting that these two pathways are intimately linked during epithelial morphogenesis in flies. During dorsal closure, but not during thorax closure, the JNK pathway controls the expression of dpp in leading edge cells. Interestingly, during oogenesis, dpp expression is not under the control of the JNK pathway, as it is during dorsal closure. Thus, based on the presence or the absence of a transcriptional coupling between JNK and dpp, it is possible to define two different types of epithelial morphogenesis. In this respect, the way the JNK and dpp pathways are set up in the ovary is more similar to the situation found in imaginal discs. The study of JNK signaling in these different but related processes in Drosophila thus represents a unique system to study the molecular origin of diversity in epithelial morphogenesis (Suzanne, 2001).

The phenotypic similarities between slipper and genes encoding the JNK signaling cascade, hep, bsk, and dJun, suggest that slpr may regulate JNK signaling. To further test whether slpr mutants diminish signaling through the JNK pathway, genetic epistasis tests were performed. Activation of positive components functioning downstream of slpr may be expected to alleviate the defect caused by slpr loss-of-function. Inducible expression of a constitutive active form of the Jun transcription factor that normally serves as a substrate for phosphorylation by Bsk significantly rescues the slpr mutant phenotype. Similarly, loss-of-function mutations in downstream negative components may augment residual signaling activity to functional levels. Consistent with this line of reasoning, slpr is dominantly suppressed by reducing the dosage of a negative regulator of JNK signaling, puc, encoding a JNK phosphatase. Heterozygosity at the puc locus significantly suppresses the severe cuticle phenotype of the strong slpr⁹²¹ allele, indicated by the clear reduction in size of dorsal cuticle holes. Moreover, loss of one copy of puc rescues embryos mutant for the weaker slpr^3P5 allele such that they develop to adulthood. Mutant male flies emerge but are weakly viable and show no gross morphological defects. Taken together, these data support a role for slpr in JNK signal transduction, upstream of bsk (Stronach, 2002).

Fos and Jun proteins homo- or hetero-dimerize to form functional AP-1 transcription factors. Drosophila mutants lacking either Jun or Fos display indistinguishable dorsal open phenotypes, indicating an essential function of both Jun and Fos for embryonic dorsal closure. Experiments were carried out to determine the basis for this dual requirement. By combining mutant alleles and transgenes expressing Fos and Jun variants with altered dimerization preferences, fly lines were generated in which only specifically defined dimer variants could form. Phenotypic analysis of these mutants reveals that homodimers of Fos or of Jun cannot replace the function of the heterodimeric complex. This defect is not explained by the lower stability of homodimers as compared to heterodimers, because 'pseudo-homodimers' which are as stable as native Jun-Fos heterodimers, cannot substitute for native Jun-Fos function. It is concluded that Jun and Fos play complementary roles and that both are required for signal transduction and gene activation during dorsal closure (Ciapponi, 2002).

To compare the role of Jun-Fos heterodimers and homodimers, two types of 'zipper swap mutants' were generated. The FJF mutant represents a version of D-Fos, in which the leucine zipper was precisely replaced with the corresponding domain of D-Jun. The complementary construct, dubbed JFJ, is a D-Jun mutant carrying the D-Fos leucine zipper. This design was chosen so that FJF would be able to form 'pseudo-homodimers' with wild-type Fos, which should have the same stability as Fos-Jun heterodimers. Conversely, FJF should dimerize with Jun only weakly with the affinity of a Jun homodimer. To confirm the expected dimerization characteristics of the chimeric proteins, they were analyzed in a GST pull-down assay. In vitro translated and ³⁵S-labeled FJF or JFJ proteins were incubated in various combinations with bacterially expressed Jun or Fos GST fusion proteins, or with GST alone as a negative control. Retention of radiolabeled JFJ and FJF proteins by GST proteins, which were immobilized on Sepharose beads, was visualized by autoradiography. The results of this experiment indicate that both homo- and hetero-dimeric complexes can form in vitro, with Fos-Fos homodimers being significantly less stable than Jun-Jun homodimers or Jun-Fos heterodimers. It is worth noting that dimerization occurred in the absence of AP-1 binding sites, and might be further stabilized when the dimeric complexes bind to DNA (Ciapponi, 2002).

Whether the zipper swap mutants could replace the function of endogenous Jun or Fos proteins during embryogenesis and rescue the respective mutants when expressed as transgenes was tested. The following mutant alleles were used. Animals homozygous for the jun² null allele express no D-Jun protein and are embryonic lethal. They can be rescued to adulthood by expression of a D-jun transgene. fos mutant alleles are designated kayak. kay¹ is a null allele causing a phenotype that is indistinguishable from that of jun² mutants. The dorsal closure phenotype of kay¹ homozygotes can be rescued by a D-fos transgene; however, the animals do not survive to adulthood due to the loss of one or more essential genes in addition to D-fos on the kay¹ chromosome. The kay² allele, while solely affecting D-fos, only represents a partial loss of function mutation. Occasionally, kay² homozygotes survive to adulthood and show a characteristic thorax cleft phenotype. kay¹/kay² transheterozygotes are strictly lethal. D-jun and D-fos mutant flies provide a background for in vivo complementation assays in which engineered forms of these proteins can be functionally tested in the developing organism (Ciapponi, 2002).

The overexpression of the wild-type D-Fos protein from a transgene in a jun homozygous mutant embryo or of wild-type D-Jun in a kay¹ mutant background is not sufficient to rescue the DC mutant phenotype. This indicates that neither D-Fos nor D-Jun homodimers alone are sufficient to direct the dorsal closure process, even when expressed at elevated levels (Ciapponi, 2002).

If it were the higher stability of the Fos-Jun heterodimer as compared to the two respective homodimers that was required to provide sufficient AP-1 function for the completion of DC, then 'pseudo-homodimers' of Jun and JFJ or of Fos and FJF held together by the Fos-Jun zipper interaction might be expected to rescue the dorsal open phenotype of kay or jun mutants, respectively. To test this possibility, Drosophila stocks carrying JFJ or FJF transgenes under the control of the heat shock promoter were recombined with the kay¹ or with the jun² mutant allele, respectively. In the animals of the hs FJF, jun² and the hs JFJ, kay¹ genotypes only stable Fos-FJF or Jun-JFJ 'pseudo-homodimers' but no Fos-Jun heterodimers can form. In both cases, no rescue could be observed, i.e. no viable flies of the observed genotype could be recovered, nor could either mutant carry out DC. This result indicates that Jun or Fos homodimers are not sufficient for DC to occur properly, even when the homodimer is held together by a more stable hetero-leucine zipper interaction (Ciapponi, 2002).

Next, flies were generated in which the only possible heterodimers are FJF-Jun or JFJ-Fos, respectively. Essentially, these are heterodimers that are nevertheless held together by the weak homotypic interaction between either two Jun or two Fos leucine zippers. These animals carry the hs FJF transgene in a homozygous kay¹ background or the JFJ transgene in flies that are homozygous for the jun² allele. Significantly, FJF and JFJ can rescue the mutant DC phenotypes and the lethality of fos and jun mutants, respectively, in both these combinations. Expression of the hs FJF transgene at least partially suppresses the completely penetrant DC phenotype of kay¹ mutants. Moreover, the strict lethality of kay¹/kay² transheterozygotes can be rescued to adulthood by the hs FJF transgene. Thus, in the different kay mutant backgrounds, FJF expression has the same rescuing potential as transgenic expression of a wild-type Fos protein. The similarity also extends to the adult phenotype of the kay¹/kay² flies that are rescued by FJF or by wild-type D-Fos expression. In both cases adults show a notum cleft phenotype, reminiscent of occasional homozygous escapers of the hypomorphic kay² stock. In line with this result, the JFJ transgene when expressed in a jun² null allele background significantly reverts the dorsal open phenotype (Ciapponi, 2002).

Several conclusions can be drawn from these experiments: (1) the data indicate that both Fos and Jun make non-redundant contributions to the regulation of dorsal closure independent of their leucine zippers, since stabilized homodimers cannot rescue Fos or Jun loss-of-function mutations, whereas destabilized heterodimers can do this. What could the complementary functions of Fos and Jun be? Recent results have indicated that both Fos and Jun represent primary recipients of JNK signaling which is essential for DC and can serve as substrates for the Drosophila JNK homolog, Basket. Both have transcription activation domains. Thus, the functional differences and the basis for the cooperation between Fos and Jun might be more specific. Either transcription factor may contribute distinct contacts to the initiation machinery or mediate separate contacts to other DNA-bound transcription factors in the assembly of regulatory complexes on target gene promoters and enhancers (Ciapponi, 2002).

(2) The results further indicate that homotypic interactions, mediated by two Jun or two Fos leucine zipper domains (such as between FJF and Jun) are in principle stable enough to assemble AP-1 dimers in the animal. Therefore, it is possible that in biological situations other than DC, Jun or Fos might act independently and that target genes exist that can be regulated by Jun or Fos homodimers. Indirect evidence indicates that Fos may have functions that are Jun-independent, possibly as a homodimer (Ciapponi, 2002).

Genetic interactions between Drosophila melanogaster menin and Jun/Fos

Menin is a tumor suppressor required to prevent multiple endocrine neoplasia in humans. Mammalian menin protein is associated with chromatin modifying complexes and has been shown to bind a number of nuclear proteins, including the transcription factor JunD. Menin shows bidirectional effects acting positively on c-Jun and negatively on JunD. Protein null alleles of Drosophila menin (mnn1) have been produced and the Mnn1 protein has been overexpressed. Flies homozygous for protein-null mnn1 alleles are viable and fertile. Localized over-expression of Mnn1 causes defects in thoracic closure, a phenotype that sometimes results from insufficient Jun activity. Complex genetic interactions are observed between mnn1 and jun in different developmental settings. These data support the idea that one function of menin is to modulate Jun activity in a manner dependent on the cellular context (Cerrato, 2006).

Human multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant cancer syndrome characterized by tumors occurring prevalently in endocrine tissues. Common features of most MEN1 tumors are low proliferation rates, well-differentiated morphology and excessive hormone secretion. Hereditary tumors arise in individuals heterozygous for a loss-of-function MEN1 allele followed by somatic loss of wild type alleles. Sporadic tumors also show bi-allelic loss of MEN1. The MEN1 locus encodes menin, a nuclear protein with two nuclear-localization sites at the C-terminal quarter of the protein, but no other overt sequence motifs. Menin is ubiquitously expressed, but shows a loss of heterozygosity phenotype in only a highly restricted set of cells. This context dependency suggests that regulated co-factors or modifiers act in conjunction with menin for cell-type specific function. Menin has also been found in a SET1-like histone methylation complex. The mouse menin gene is required for embryonic viability and, like in humans, inactivation of both alleles results in endocrine tumors. Therefore, menin is a classic tumor suppressor in the endocrine system. Interestingly, there is also recent evidence that menin is an oncogenic co-factor in Mixed Lineage Leukemia. The nature of this dual growth suppressing and enhancing role in the regulation of proper cell number and differentiation has not been clarified (Cerrato, 2006).

Multiple potential transcription factor partners for mammalian menin protein have been identified including JunD, which has been shown to interact directly with menin. It is unclear how these protein–protein interactions relate to menin in the SET-1 like histone methylation complex, although it is possible that menin association with many different nuclear proteins helps target the complex to appropriate regions of chromatin. Experiments performed in immortalized mouse embryo fibroblasts have shown that menin binding to JunD is necessary for JunD to act as a growth suppressor. Menin functions to reduce JunD activity and has been shown to inhibit the accumulation of active phosphorylated JunD or c-Jun. Even though menin does not directly bind c-Jun, it augments the transcriptional activity of this transcriptional factor. Thus, menin is strongly implicated in regulating Jun function. Interestingly, according to the potential roles of menin to promote or suppress tumorigenesis, menin can act in turn negatively on JunD or positively on c-Jun function (Cerrato, 2006).

The Drosophila melanogaster menin protein (Mnn1) is 47% identical to the human protein, including 69% of the amino acid residues that are required for tumor suppression in human endocrine tissues. The ongoing sequencing of multiple species in Drosophila reveals that menin is highly conserved among them. Despite this high degree of conservation, menin is not required for viability in D. melanogaster. Flies lacking mnn1 expression are viable and fertile. One report suggests that mnn1 is required for a wild type life span and some aspect of either chromosome stability or DNA repair (Busygina, 2004), while another report (Papaconstantinou, 2005) suggests that mnn1 is required for a robust response to various types of stress (Cerrato, 2006).

Two protein-null mnn1 alleles and have generated transgenic flies for the controlled over-expression of Drosophila Mnn1 protein. mnn1⁻ Drosophila is viable and fertile. Over-expression of Mnn1 results in pharate-adult phenotype, proboscis ablation and a cleft thorax. These over-expression phenotypes are modified by both gain-of-function and loss-of-function alleles of jun. Dominant-negative alleles of fos are enhanced by loss-of-function alleles of mnn1. The finding that both Drosophila and mammalian menin are capable of interacting with Jun suggests that an evolutionarily conserved menin function in normal development and disease is linked to the Jun/Fos family of transcriptional regulators. Interestingly, as in mammals, Drosophila menin shows bidirectional modulation of Jun function (Cerrato, 2006).

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