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).

Temporal coherency between receptor expression, neural activity and AP-1-dependent transcription regulates Drosophila motoneuron dendrite development

Neural activity has profound effects on the development of dendritic structure. Mechanisms that link neural activity to nuclear gene expression include activity-regulated factors, such as CREB, Crest (Ca2+-responsive transactivator, a syntaxin-related nuclear protein that interacts with CREB-binding protein and is expressed in the developing brain) or Mef2, as well as activity-regulated immediate-early genes, such as fos and jun. This study investigates the role of the transcriptional regulator AP-1, a Fos-Jun heterodimer, in activity-dependent dendritic structure development. Genetic manipulation, imaging and quantitative dendritic architecture analysis were combined in a Drosophila single neuron model, the individually identified motoneuron MN5. First, Dalpha7 nicotinic acetylcholine receptors (nAChRs) and AP-1 are required for normal MN5 dendritic growth. Second, AP-1 functions downstream of activity during MN5 dendritic growth. Third, using a newly engineered AP-1 reporter it was demonstrated that AP-1 transcriptional activity is downstream of Dalpha7 nAChRs and Calcium/calmodulin-dependent protein kinase II (CaMKII) signaling. Fourth, AP-1 can have opposite effects on dendritic development, depending on the timing of activation. Enhancing excitability or AP-1 activity after MN5 cholinergic synapses and primary dendrites have formed causes dendritic branching, whereas premature AP-1 expression or induced activity prior to excitatory synapse formation disrupts dendritic growth. Finally, AP-1 transcriptional activity and dendritic growth are affected by MN5 firing only during development but not in the adult. These results highlight the importance of timing in the growth and plasticity of neuronal dendrites by defining a developmental period of activity-dependent AP-1 induction that is temporally locked to cholinergic synapse formation and dendritic refinement, thus significantly refining prior models derived from chronic expression studies (Vonhoff, 2013).

By combining genetic and neuroanatomical tools with imaging in a single-cell model, the adult MN5 in Drosophila, this study demonstrates that: (1) AP-1 is transcriptionally active during all stages of postembryonic motoneuron dendritic growth, (2) AP-1 and excitatory cholinergic inputs are required for normal dendrite growth in MN5, (3) AP-1 transcriptional activity is enhanced via a CaMKII-dependent mechanism by increased neural activity during pupal development but not in the adult, and (4) both activity and AP-1 can promote or inhibit dendritic branching, depending on the developmental stage. AP-1 is required for normal MN5 dendrite growth downstream of activity and CaMKII (Vonhoff, 2013).

Although AP-1 has been thought to regulate dendrite development in an activity-dependent manner via global changes in gene expression, probably in a calcium-dependent manner as described for CREB or Crest, direct evidence for this hypothesis was sparse (Vonhoff, 2013).

This study demonstrated that excitatory cholinergic input to MN5 and AP-1 transcriptional activity were required for normal dendrite growth of MN5 during pupal life. MN5 total dendritic length and branch numbers were significantly reduced (~50%) by inhibition of AP-1 [by Jbz (a dominant-negative form of Jun) expression] and in Dα nAChR mutants. Conversely, overexpression of AP-1 or increased MN5 excitability as induced by potassium channel knockdown (by EKI) increased dendritic branching (Duch, 2008). Clearly, AP-1 acted downstream of activity as inhibition of AP-1 by Jbz completely attenuated EKI (electrical knock-in) mediated dendritic growth and branching (Vonhoff, 2013).

A new AP-1 reporter was employed to measure activity-induced AP-1 transcriptional activity by imaging, and to gain insight into the pathway that might connect MN5 activity to AP-1-dependent transcription. Although the detection threshold of this reporter might be too low to detect small changes in AP-1 activity, sensitivity was sufficient to reliably report increased AP-1 activity following overexpression of fos and jun, inhibition of AP-1 transcriptional activity by Jbz expression, and changes in AP-1 activity as induced by various manipulations of cellular signaling. Therefore, the reporter was deemed suitable for testing changes in AP-1 transcriptional activity in MN5 (Vonhoff, 2013).

Targeted expression of TrpA1 channels in MN5 allowed the induction of firing in vivo by temperature shifts during selected developmental periods. Activation of MN5 during pupal life for 36 hours (P9 to adult) or longer (P5 to adult) caused significant increases in AP-1-induced nuclear GFP fluorescence. By contrast, in adults neither similar nor longer durations of TrpA1 activation resulted in any detectable increase in AP-1 reporter-mediated nuclear GFP fluorescence in MN5. Similarly, live imaging in semi-intact adult preparations did not reveal any detectable AP-1 activity upon acute TrpA1 activation for various durations. This indicated that activity-dependent AP-1 activation was restricted to pupal life. However, whether AP-1 activation in the adult MN5 occurred upon patterned activity was not tested. Spaced stimuli that reflect endogenous activity patterns are required for insect motoneuron axonal and dendritic development and can regulate mammalian neuron dendritic morphology. However, during flight, MN5 fires tonically at frequencies between 5 and 20 Hz, a pattern that is well reflected by temperature-controlled TrpA1 channel activation. Therefore, adult flight behavior is unlikely to induce AP-1 activity, which is involved in dendrite and synapse development (Freeman, 2010). This is consistent with the assumption that dendritic structure is fairly stable in the adult (Vonhoff, 2013).

cAMP and Jun N-terminal kinase (Jnk) have been implicated as potential links between activity and AP-1 activation. Cell culture studies on Drosophila larval motoneurons and giant neurons demonstrate a role of calcium. This study showed that Dα7 nAChRs, which are highly permeable to calcium, were required for normal MN5 dendritic growth. Combining genetic manipulation of Dα7 nAChRs, AP-1 and CaMKII with imaging of AP-1 reporter activity revealed that CaMKII was required downstream of Dα7 nAChRs to cause AP-1-dependent transcription. These data show that activity-dependent calcium influx through nAChRs might activate AP-1 during pupal life via a CaMKII-dependent mechanism in vivo. Activity and AP-1 can promote or inhibit dendritic growth during pupal life, depending on timing (Vonhoff, 2013).

In larval motoneurons, AP-1 is required for dendritic overgrowth as induced by artificially increased activity (Hartwig, 2008). In MN5, AP-1 is required downstream of nAChRs and CaMKII for normal dendritic growth. By contrast, premature expression of AP-1 in MN5 inhibited dendritic growth. These data were consistent with the hypothesis that timing is the crucial factor. First, P103.3 and D42 both caused similar overgrowth but exhibited fairly different expression patterns. Second, C380-GAL4 and Dα7 nAChR-GAL4 both inhibited MN5 dendrite growth but expressed in largely different sets of neurons. Therefore, the common factor of C380 and Dα7 nAChR on the one hand and D42 and P103.3 on the other hand was timing. Third, shifting the timing of C380-GAL4-driven AP-1 expression to later stages prevented dendritic defects. Fourth, imposed activity prior to P5 by TrpA1 activation also inhibited dendritic branching. Dendritic defects as induced by imposed premature activity were rescued by inhibition of AP-1 via Jbz expression in MN5 (Vonhoff, 2013).

MN5 early dendritic growth starts at early pupal stage 5 (P5), and expression of Dα7 nAChRs begins 2.5 hours later, at mid stage P5. Similarly, Xenopus optic tectal and turtle cortical neurons receive glutamatergic and GABAergic inputs as soon as the first dendrites are formed. In vertebrates, early synaptic inputs and neurotransmitters play essential roles in dendrite development. The current data are consistent with the hypothesis that the endogenous expression of nAChRs caused increased activity throughout the developing motor networks, which, in turn, upregulated AP-1-dependent transcription and dendritic growth via a CaMKII-dependent mechanism. During zebrafish spinal cord development, activity is required for strengthening functional central pattern generator (CPG) connectivity. As dendrites are the seats of input synapses to motoneurons, an activity-dependent component in motoneuron dendritic growth that follows early synaptogenesis might function to refine dendrite shape during the integration into the developing CPG (Vonhoff, 2013).

Impaired Hippo signaling promotes Rho1-JNK-dependent growth

The Hippo and c-Jun N-terminal kinase (JNK) pathway both regulate growth and contribute to tumorigenesis when dysregulated. Whereas the Hippo pathway acts via the transcription coactivator Yki/YAP to regulate target gene expression, JNK signaling, triggered by various modulators including Rho GTPases, activates the transcription factors Jun and Fos. This study shows that impaired Hippo signaling induces JNK activation through Rho1. Blocking Rho1-JNK signaling suppressed Yki-induced overgrowth in the wing disk, whereas ectopic Rho1 expression promoted tissue growth when apoptosis was prohibited. Furthermore, Yki directly regulates Rho1 transcription via the transcription factor Sd. These results identify a novel molecular link between the Hippo and JNK pathways and implicate the essential role of the JNK pathway in Hippo signaling-related tumorigenesis (Ma, 2005).

Recent studies have revealed a complex interaction network between Hippo and other key signaling pathways, including TGF- β /SMAD and Wnt/β-catenin pathways, whereas its communication with JNK signaling remains elusive. This study provides genetic evidences that impaired Hippo signaling promotes overgrowth through Rho1-JNK signaling in Drosophila. First, loss of Hippo signaling induces JNK activation and its target gene expression. Second, Yki-induced overgrowth is suppressed by blocking Rho1-JNK signaling. Third, ectopic Rho1 expression phenocopies Yki-triggered overgrowth and proliferation when cell death is compromised (Ma, 2005).

Yki/YAP's ability in promoting tissue growth depends on transcription factors, including Sd/TEADs and SMADs. Consistent with this notion, this study found Sd, but not Mad, is essential for Yki-induced JNK activation, whereas ectopic Sd expression is sufficient to activate JNK signaling by itself. The Rho1 GTPase was further implicated as the critical factor that bridges the interaction between Hippo and JNK signaling. Rho1 not only mediates Yki-induced JNK activation and overgrowth, but also serves as a direct transcriptional target of Yki/Sd complex. Intriguingly, Rho1 activation was also found to promote nuclear translocation of Yki in wing discs, and reducing Yki activity significantly impeded Rho1 induced growth, implying the existence of a potential positive feedback loop to amplify Yki-induced overgrowth and to help maintain signaling in a steady state. Consistent with thi observation, recent studies reported that GPCRs could activate YAP/TAZ through RhoA in mammals, whereas elevated JNK signaling in Drosophila could stimulate Yki nuclear translocation during regeneration and tissue growth. Thus, these results provide the other side of the story about a novel cross-talk between Hippo and JNK signaling (Ma, 2005).

Intriguingly, it was found that ectopic Yki expression driven by ptc-Gal4 induced MMP1 activation, puc-LacZ expression, rho1 transcription, and Yki target gene transcription predominantly in the proximal region of wing disk, but not that of the dorsal/ventral boundary. This is consistent with a recently published paper showing that tension in the center region of Drosophila wing disk is lower than that in the periphery, which correlates with lower Yki activity. It is also worth noting that despite the requirement of JNK signaling in Yki-induced wing overgrowth, JNK was not activated strictly in an autonomous manner upon Yki overexpression. This could be caused by supercompetitive activity of Yki expression clones, or, alternatively, through a propagation of JNK signal into neighboring cells, which would be very interesting to study further (Ma, 2005).

Apart from its role in growth control, the Hippo pathway also regulates tumor invasion and metastasis. Similarly, JNK signaling plays a major role in modulating metastasis in both flies and mammals. Rho1 was also reported to cooperate with oncogenic Ras to induce large invasive tumors. Hence, it is likely that Rho1 also acts as the molecular link between Yki and JNK signaling in modulating metastasis as well (Ma, 2005).

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, tkvQ253D, 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-tkvQ253D 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-JunAsp (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-JunAsp, whereas the polarity defects persist and thus are more prominent. This observation indicates that, in the absence of normal Pnt levels, sev-JunAsp 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 slpr3P5 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).

A genome-wide RNAi screen reveals multiple regulators of caspase activation

A genome-wide RNA interference screen was performed to systematically identify regulators of apoptosis induced by DNA damage in Drosophila cells. Forty-seven double- stranded RNAs were identified that target a functionally diverse set of genes, including several with a known function in promoting cell death. Further characterization uncovers 10 genes that influence caspase activation upon the removal of Drosophila inhibitor of apoptosis 1. This set includes the Drosophila initiator caspase Dronc and, surprisingly, several metabolic regulators, a candidate tumor suppressor, Charlatan, and an N-acetyltransferase, ARD1. Importantly, several of these genes show functional conservation in regulating apoptosis in mammalian cells. These data suggest a previously unappreciated fundamental connection between various cellular processes and caspase-dependent cell death (Yi, 2007).

The genes that are specifically involved in caspase-dependent cell death were classified. Substantial induction of caspase activity was observed 8 h after treatment with a topoisomerase II inhibitor, doxorubicin (dox), to induce dose-dependent cell death. Any RNAi suppressing this activity implicates the target gene in early regulation of caspase activation. In addition to dcp-1 RNAi, knockdown of dronc and jra (the Drosophila homolog of c-Jun) significantly suppressed caspase-3/7-like activity in the presence of dox, whereas the negative control, RNAi against calpain A, a calcium-dependent cysteine protease, did not affect this pathway (Yi, 2007).

This analysis was expanded to all of the genes identified in the initial RNAi screen and 20 dsRNAs were discovered that suppressed caspase activation induced by DNA damage. Interestingly, 12 of these genes were found to be epistatic to diap1 (Yi, 2007).

diap1 epistatic analysis was performed to further categorize the genes. DIAP1, the fly orthologue of the mammalian inhibitors of apoptosis proteins, is a direct inhibitor of caspases, and deficiency in DIAP1 leads to rapid caspase activation and apoptosis in vivo. Thus, apoptosis induced by the loss of DIAP1 presents an alternative apoptotic assay independent of DNA damage. Silencing of genes that regulate activation of the core apoptotic machinery may provide protection against apoptosis induced by both DNA damage and the loss of DIAP1. RNAi against dcp-1 partially suppressed cell death induced by the depletion of DIAP1 in Kc cells. Also, dronc RNAi potently protected cells against apoptosis induced by deficiency in DIAP1. Altogether, 32 of the genes confirmed from the primary screen provided significant protection against cell death induced by the silencing of DIAP1 (Yi, 2007).

Interestingly, 12 dsRNAs suppressed caspase-3/7-like activity after dox treatment and protected against cell death induced by diap1 RNAi, suggesting that these genes are required for apoptosis induced by multiple stimuli. To confirm that these genes are necessary for the full activation of caspases, it was determined whether these dsRNAs could suppress spontaneous caspase activity induced by diap1 RNAi. Maximal induction of caspase activity by diap1 RNAi was observed after 24 h, and this effect was completely suppressed by dsRNA against dcp-1. Importantly, ablating 10/12 dsRNAs resulted in the significant suppression of caspase activity compared with diap1 RNAi only (Yi, 2007).

In addition to dronc RNAi, dsRNAs targeting chn and dARD1 provided the strongest suppression of spontaneous caspase activity. Consistent with the observation that RNAi against chn protects against DNA damage-induced cell death, the mammalian orthologue neuron-restrictive silencer factor (NRSF)/RE1-silencing transcription factor (REST) was recently identified as a candidate tumor suppressor in epithelial cells (Westbrook, 2005). Previous work indicates that Chn and NRSF/REST function as a transcriptional repressor of neuronal-specific genes (Chong, 1995; Schoenherr, 1995; Tsuda, 2006), suggesting that cellular differentiation may render cells refractory to caspase activation and apoptosis. Also, several metabolic genes, CG31674, CG14740, and CG12170, were identified that may be involved in the general regulation of caspase activation. It has been demonstrated that NADPH produced by the pentose phosphate pathway regulates the activation of caspase-2 in nutrient-deprived Xenopus laevis oocytes. Together with these results, these observations provide further evidence for an intimate link between the regulation of metabolism and induction of apoptosis (Yi, 2007).

To further explore the significance of these findings, whether silencing the mammalian orthologues of the fly genes identified from the RNAi screen confers protection against dox-induced cell death was investigated in mammalian cells. A set of mammalian orthologues was selected that are believed to be nonredundant. The list includes the orthologues of dMiro, which functions as a Rho-like GTPase; dARD1, which functions as an N-acetyltransferase; CG12170, which functions as a fatty acid synthase; and Chn, which functions as a transcriptional repressor (RHOT1, hARD1, OXSM, and REST, respectively; FlyBase). In addition, Plk3, a mammalian orthologue of Polo, was tested since dsRNA targeting polo potently protected against dox treatment (Yi, 2007).

The ability of siRNAs targeting a gene of interest to protect against DNA damage was tested in HeLa cells. As a positive control, cells were transfected with siRNAs targeting Bax or Bak, two central regulators of mammalian cell death. Indeed, silencing of Bax or Bak resulted in significant protection against dox- induced cell death. It was observed that plk3 RNAi provided partial protection against dox treatment, which is consistent with previous studies implicating Plk3 in stress-induced apoptosis. Interestingly, the knockdown of hARD1 dramatically enhanced cell survival in the presence of dox to levels similar to that of Bak. This protective effect was also evident at the morphological level. In cells transfected with a nontargeting control siRNA, dox treatment resulted in typical apoptotic morphology, including cell rounding and membrane blebbing. In direct contrast, cells transfected with siRNAs against hARD1 maintained a normal and healthy morphology and continued to proliferate in the presence of dox (Yi, 2007).

To examine whether the protection provided by siRNAs targeting hARD1 and plk3 is associated with the suppression of caspase activation, caspase activity was measured in these cells treated with dox. RNAi against plk3 provided partial suppression of caspase activity, again supporting the observed protection phenotype. Interestingly, the depletion of REST resulted in some suppression of caspase activity in the presence of dox even though the protection against cell death was not statistically significant. Consistent with the viability assay, complete suppression of caspase-3/7 activity was observed in cells transfected with hARD1 siRNA. These results indicate that hARD1 is required for caspase-dependent cell death induced by DNA damage. Furthermore, all four siRNAs targeting hARD1 were individually capable of providing robust protection against cell death, strongly suggesting that these siRNAs target hARD1 specifically (Yi, 2007).

Because the silencing of hARD1 dramatically suppressed activation of the downstream caspases, whether activation of the upstream caspases in response to dox treatment is also perturbed was also examined. Remarkably, hARD1 RNAi inhibited the cleavage of caspase-2 and -9 in cells treated with dox, whereas caspase cleavage was readily detected in control cells. Thus, it is proposed that hARD1 regulates the signal transduction pathway apical to the apoptotic machinery in the DNA damage response itself or the activation of upstream caspases (Yi, 2007).

Consistent with the results of the caspase-3/7 assay, silencing of hARD1 completely inhibited the appearance of activated caspase-3 induced by dox. This assay was used for a hARD1 complementation experiment to demonstrate the proapoptotic role of hARD1 in response to DNA damage. A new siRNA pool was used, targeting the 5' untranslated region of hARD1 (5'si); this treatment inhibited caspase-3 cleavage induced by dox treatment. Furthermore, caspase-3 cleavage was observed in reconstituted hARD1 knockdown cells. Because six out of six siRNAs against hARD1 provided strong protection against DNA damage-induced apoptosis and complementation of hARD1-sensitized cells to caspase activation, it is concluded that the functional role of ARD1 for dox-induced apoptosis is evolutionally conserved from Drosophila to mammals (Yi, 2007).

In summary, this study used an unbiased RNAi screening platform in Drosophila cells to identify genes involved in promoting DNA damage-induced apoptosis. Forty-seven dsRNAs were isolated that suppress cell death induced by dox. These genes encode for known apoptotic regulators such as Dronc, the Drosophila orthologue of the known proapoptotic transcriptional factor c-Jun, and an ecdysone-regulated protein, Eip63F-1, thereby validating the primary screen. Furthermore, this study implicates a large class of metabolic genes that were previously not suspected to have a role in modulating caspase activation and apoptosis, such as genes involved in fatty acid biosynthesis (CG11798), amino acid/carbohydrate metabolism (CG31674), citrate metabolism (CG14740), complex carbohydrate metabolism (CG10725), and ribosome biosynthesis (CG6712). These results support the proposal that the cellular metabolic status regulates the threshold for activation of apoptosis and thus plays a critical role in the decision of a cell to live or die (Yi, 2007).

Of particular interest is the identification of ARD1. Evidence is presented that RNAi against ARD1 provides protection against cell death and leads to the suppression of caspase activation induced by DNA damage in fly cells and HeLa cells. Furthermore, deficiency in dARD1 renders fly cells resistant to the spontaneous caspase activity and cell death associated with loss of Diap1. Importantly, substantial evidence is provided that hARD1 is required for caspase activation in the presence of DNA damage in mammalian cells. Cleavage of initiator and executioner caspases are suppressed in hARD1 RNAi cells treated with dox, suggesting that hARD1 functions further upstream of caspase activation, and the complementation of hARD1 knockdown cells restores caspase-3 cleavage. These data indicate that ARD1 is necessary for DNA damage-induced apoptosis in flies and mammals (Yi, 2007).

ARD1 functions in a complex with N-acetyltransferase to catalyze the acetylation of the Nα-terminal residue of newly synthesized polypeptides and has been implicated in the regulation of heterochromatin, DNA repair, and the maintenance of genomic stability in yeast. These studies suggest that ARD1 may be involved in regulating an early step in response to DNA damage. It is anticipated that future studies will focus on determining whether ARD1 functions in similar processes in mammals. The diversity of genes identified in the screen illustrates the complex cellular integration of survival and death signals through multiple pathways (Yi, 2007).

Overexpression screen in Drosophila identifies neuronal roles of GSK-3 beta/shaggy as a regulator of AP-1-dependent developmental plasticity

AP-1, an immediate-early transcription factor comprising heterodimers of the Fos and Jun proteins, has been shown in several animal models, including Drosophila, to control neuronal development and plasticity. In spite of this important role, very little is known about additional proteins that regulate, cooperate with, or are downstream targets of AP-1 in neurons. This paper outlines results from an overexpression/misexpression screen in Drosophila to identify potential regulators of AP-1 function at third instar larval neuromuscular junction (NMJ) synapses. First, >4000 enhancer and promoter (EP) and EPgy2 lines were used to screen a large subset of Drosophila genes for their ability to modify an AP-1-dependent eye-growth phenotype. Of 303 initially identified genes, a set of selection criteria were used to arrive at 25 prioritized genes from the resulting collection of putative interactors. Of these, perturbations in 13 genes result in synaptic phenotypes. Finally, one candidate, the GSK-3α-kinase homolog, shaggy, negatively influences AP-1-dependent synaptic growth, by modulating the Jun-N-terminal kinase pathway, and also regulates presynaptic neurotransmitter release at the larval neuromuscular junction. Other candidates identified in this screen provide a useful starting point to investigate genes that interact with AP-1 in vivo to regulate neuronal development and plasticity (Franciscovich, 2008).

The transcription factor AP-1 is a key regulator of neuronal growth, development, and plasticity, and in addition to cAMP response element binding (CREB) protein, it controls transcriptional responses in neurons during plasticity. Acute inhibition of Fos attenuates learning in mice and in invertebrate models such as Drosophila; AP-1 positively regulates developmental plasticity of motor neurons. Essential to the understanding of AP-1 activity in neurons is the knowledge of other proteins that influence AP-1 function or are downstream transcriptional targets. This study describes a forward genetic screen for modifiers of AP-1 in Drosophila (Franciscovich, 2008).

Using a conveniently scored AP-1-dependent adult-eye phenotype, 4307 EP and EPgy2 lines were screened for genes that modified this phenotype. Several advantages of this screen include: (1) the ease and rapidity of screening as compared to the neuromuscular junction, (2) immediate gene identification, (3) the potential to analyze in vivo phenotypes that arise from overexpression/misexpression, and finally (4) the scope for rapidly generating loss-of-function mutations through imprecise excision of the same P-element. A total of 249 known genes were isolated of which 73 can be directly implicated in eye development. The selection was prioritized using several criteria, to derive a short list of 13 final candidates that were then tested at the NMJ. Future work will focus on other predicted but as yet unstudied genes that are likely to have important functions at the NMJ (Franciscovich, 2008).

The prescreening strategy using the adult eye was successful because (1) almost all the genes selected did not result in eye phenotypes when expressed on their own, but selectively modified a Fbz dependent phenotype (Fbz is a dominant-negative transgenic construct that expresses the Bzip domain of Drosophila Fos); (2) several genes were identified that are known to interact with AP-1 in regulating synaptic phenotypes (these include ras and bsk); (3) multiple alleles of some genes were recovered confirming the sensitivity of the screening technique; (4) several genes involved in eye development were isolated (including cyclinB, which has been shown to be a downstream target of Fos in the regulation of G2/M transition in the developing eye); (5) a large number of putative interactors have connections with neural physiology and/or AP-1 function in other cell types; (6) some candidates with strong phenotypes have previously been shown to play important roles in motor neurons; and finally (7) the majority of candidates (but not all) isolated as enhancers or suppressors of Fbz in the eye exerted a similar effect on AP-1 at the synapse (Franciscovich, 2008).

Although the relative success and merits of a functional screen are considerable, there are a few disadvantages. First, the use of P-element transposons naturally excludes a large fraction of genes that are refractory to P-element transposition events. Second, insertions of EP elements within or in inverse orientation to the gene make it difficult to assign phenotypes to specific genes. Even in instances where overexpression was predicted, it has to be verified that this is indeed the case and also the phenotypes derive from hypomorphic mutations that result from the insertion of the P-element close to the target gene have to be tested. Third, although recover genes that play conserved roles in AP-1 biology is to be expected, those genes that specifically affect synaptic physiology and play no role in the eye will be excluded by this scheme. Finally, this screen will not discriminate between genes that function upstream or downstream of AP-1 in neurons. In spite of these deficiencies, it is believed that candidates identified in this screen provide strong impetus for the investigation of additional factors that are involved in the regulation of synaptic plasticity and development by AP-1 (Franciscovich, 2008).

Following their identification, it was found that several candidates had synaptic functions since several of these genes resulted in significant differences in synaptic size when compared to appropriate controls. This provided the first confirmation of the screening strategy. Next, experiments to determine genetic interaction with AP-1 showed that expression of four genes (pigeon, lbm, Cnx99A, and sty) suppressed the Fbz-dependent small synapse phenotype. Of these, sty had been isolated as an enhancer while the other three similarly suppressed the Fbz-derived eye phenotype, suggesting potentially conserved functions of these genes in the two tissues (Franciscovich, 2008).

Four genes isolated as enhancers, similarly enhanced an Fbz-mediated small synapse (cnk, pde8, fkbp13, and sgg). Notably, expression of these genes also suppressed an AP-1-dependent synapse expansion at the NMJ. These two lines of evidence indicate that these genes are negative regulators of AP-1 function in these neurons. Together with the fact that all four have previously described functions in the nervous system, these observations confirm the validity of the screen and highlight the utility of genetic screens to uncover novel molecular interactions. Further studies will provide a more comprehensive understanding of the interplay between these genes and AP-1 in the regulation of neuronal development and plasticity. For instance, more careful analysis needs to be carried out to discern whether synaptic phenotypes in each of these cases are due to overexpression or potential insertional mutagenesis of specific genes (Franciscovich, 2008).

Although GSK-3β-signaling has been implicated in several neurological disorders such as Alzheimer's disease, it is only recently that neuronal roles for this important kinase have come to light. For instance, several studies have demonstrated the role of GSK-3β in the regulation of long-term potentiation (LTP) in vertebrate hippocampal synapses (Hooper, 2007; Peineau, 2007; Zhu, 2007). In particular, these reports highlight the negative regulatory role of GSK-3β in the induction of LTP or in one case, the switching of long-term depression (LTD) into LTP. Interestingly, LTP induction leads to GSK-3β-inhibition thus precluding LTD induction in the same neurons. In flies, sgg mutations have defects in olfactory habituation, circadian rhythms and synaptic growth. These observations point to a conserved and central role for GSK-3β in neuronal physiology (Franciscovich, 2008).

GSK-3β-dependent modulation of transcriptional responses is widely acknowledged. Among several transcription factors that are known to be regulated by this kinase, are AP-1, CREB, NFAT, c/EBP, and NF-kappaB. In the context of neuronal function, for instance, RNA interference-based experiments in cultured rat cortical neurons have shown that GSK-3β-activity influences CREB and NF-kappaB-dependent transcription. Additionally, two other transcription factors, early growth response 1 and Smad3/4 have been identified in DNA profiling experiments in the same study. Significantly, GSK-3β is also a primary target of lithium, a drug used extensively to treat mood disorders. Lithium treatment has been reported to result in an upregulation of AP-1-dependent transcription, though a role for GSK-3β in this phenomenon has not been tested directly (Franciscovich, 2008).

In Drosophila, recent experiments have described the negative regulation of synaptic growth by the GSK3β-homolog shaggy (Franco, 2004). These studies demonstrate that sgg controls synaptic growth through the phosphorylation of the Drosophila MAP1B homolog futsch. The current studies suggest that Sgg-dependent regulation of synapse size occurs through the immediate-early transcription factor AP-1. GSK-3β is believed to inhibit transcriptional activity of AP-1 in cultured cells by direct inhibitory phosphorylation of c-Jun. Circumstantial evidence also suggests that GSK-3β provides an inhibitory input into AP-1 function in neurons (Franciscovich, 2008).

It was intriguing to find that Sgg inhibition leads to an expanded synapse with reduced presynaptic transmitter release, similar to highwire mutants. Given that in several instances, Sgg-dependent phosphorylation targets a protein for ubiquitination, and that Highwire encodes an E3 ubiquitin ligase, it is conceivable that sgg and hiw function in the same signaling pathway. Consistent with this hypothesis, both hiw and sgg function at the synapse seem to impinge on AP-1-dependent transcription through modulation of the JNK signaling pathway. Considering previous reports of GSK-3β-involvement in multiple signaling cascades, it will be interesting to study how sgg controls multiple aspects of cellular physiology to regulate neural development and plasticity, particularly in the context of brain function and action of widely used drugs such as lithium (Franciscovich, 2008).

Fos and Jun potentiate individual release sites and mobilize the reserve synaptic-vesicle pool at the Drosophila larval motor synapse

In all nervous systems, short-term enhancement of transmitter release is achieved by increasing the weights of unitary synapses; in contrast, long-term enhancement, which requires nuclear gene expression, is generally thought to be mediated by the addition of new synaptic vesicle release sites. In Drosophila motor neurons, induction of AP-1, a heterodimer of Fos and Jun, induces cAMP- and CREB-dependent forms of presynaptic enhancement. Light and electron microscopic studies indicate that this synaptic enhancement is caused by increasing the weight of unitary synapses and not through the insertion of additional release sites. Electrophysiological and optical measurements of vesicle dynamics demonstrate that enhanced neurotransmitter release is accompanied by an increase in the actively cycling synaptic vesicle pool at the expense of the reserve pool. Finally, the observation that AP-1 mediated enhancement eliminates tetanus-induced forms of presynaptic potentiation suggests: (1) that reserve-pool mobilization is required for tetanus-induced short-term synaptic plasticity; and (2) that long-term synaptic plasticity may, in some instances, be accomplished by stable recruitment of mechanisms that normally underlie short-term synaptic change (Kim, 2009).

Drosophila larval motor synapses show increased synaptic strength when AP-1 is overexpressed in motor neurons (Sanyal, 2002). This synaptic enhancement is accompanied by increases in the quantal content of neurotransmitter release, and increases in the number of presynaptic varicosities (Sanyal, 2002). This study asked whether AP-1 mediated synapse enhancement can be explained by increases in synapse number, Ca2+ influx, Ca2+ sensitivity of vesicle fusion or synaptic vesicle number. The observations support a model in which: (1) AP-1 induced synaptic enhancement occurs without an accompanying increase in synapse number; (2) AP-1 increases the size of the cycling synaptic vesicle pool through mobilization of the reserve pool; (3) that AP-1 causes persistent synaptic change by stably recruiting a cellular mechanism transiently used for posttetanic potentiation, a ubiquitous but poorly understood form of short-term synaptic facilitation (Kim, 2009).

Previous studies have shown AP-1 overexpression in Drosophila motor neurons enhances glutamate release from motor terminals in a manner that is accompanied by an increase in bouton number (Sanyal, 2002). These conclusions were confirmed using failure frequency analysis, which, under conditions of very low Ca2+, measures frequency of 'failure' to release even a single quantum of neurotransmitter. At 0.3 mM Ca2+, frequencies of failure events are reduced in C155/+;UAS Fos/+;UAS Jun/+ (hereafter referred to as 'AP-1') compared with control C155/+ hereafter 'control') synapses. Therefore, this analysis confirmed quantal content (m = ln [number of events/number failures]) is significantly increased in motor synapses from AP-1 animals. Similar results were obtained under nonfailure conditions where quantal content is calculated by m = EJP/mEJP. Because quantal amplitude is not increased by AP-1 (SF1Fig. S1), presynaptic mechanisms completely account for the measured synaptic strengthening. These observations, taken together with previous work (Sanyal, 2002), show that AP-1 increases quantal content of transmitter release at both low and physiological Ca2+ concentrations (Kim, 2009).

Although AP-1 overexpression increases the number of presynaptic boutons (Sanyal, 2002), the average bouton size is significantly reduced. For this reason, and because individual boutons contain multiple release sites, bouton number is not necessarily a reliable measure of synapse number. The following strategy was used to assess whether AP-1-terminals have more functional synapses, which is defined as presynaptic release sites apposed to postsynaptic receptor clusters. In wild-type neuromuscular junctions (NMJ), ~95% of GluR clusters are coupled to Bruchpilot (brp/CAST) immunopositive presynaptic puncta (Rasse, 2005). This fraction is not altered by AP-1 expression. Thus, the number of Brp-positive puncta provides a measure of synapse number in AP-1 synapses (Kim, 2009).

Since individual Brp spots are clearly resolved, they could be counted and analyzed with a spot-detection/analysis program. This method yielded values that were in good agreement with those derived from previous serial EM studies of wild-type NMJs. Surprisingly, total Brp positive puncta (per NMJ) decreased by 21% in AP-1 synapses. AP-1 induction did not detectably alter the distribution of T-Bar or synapse size assessed by quantitative fluoresence and electron microscopy respectively. Thus, it is concluded that although AP-1 increases total bouton number, the number of functional synapses is significantly reduced. Because the quantal content of neurotransmitter release is N × p (where N is synapse number and p is the average probability of vesicle release per synapse), these observations point to an increase in p at AP-1 terminals (Kim, 2009).

If AP-1 overexpression leads to changes in the probability of release, it was reasoned that forms of short-term plasticity, which also alter p, might be altered at these motor terminals. To test this idea, two separable forms of short-term plasticity observed at the Drosophila larval NMJ at low Ca2+ concentrations were measured. The first form, paired-pulse facilitation (PPF) is short-lived and decays within milliseconds. This is easily distinguished from longer-lived presynaptic plasticity, observed during and after tetanic stimulation, which decays more slowly (10s of seconds to minutes). Although multiple processes (e.g., augmentation and posttetanic potentiation) could contribute to this longer-lived form of plasticity, this phenomenon is referred by a single term, tetanus-induced potentiation (TIP) (Kim, 2009).

At interstimulus intervals (ISI) of 25 ms, 50 ms, 100 ms, and 1,000 ms, the paired pulse ratios exhibited by control and AP-1 motor terminals did not differ significantly. The site of action for residual Ca2+ during paired pulse facilitation (PPF) has been demonstrated in previous studies to be located in the Ca2+ microdomain immediately surrounding clustered Ca2+ channels and vesicle release sites (Blatow, 2003; Zucker, 2002). The observation that PPF is normal in AP-1 synapses suggests that Ca2+ dynamics in this microdomain are not significantly altered by AP-1 (Kim, 2009).

In contrast, TIP was strikingly altered by AP-1 expression. In control synapses, transmitter release increases during a 2-min train of 10-Hz stimulation, eventually reaching a plateau. Contributions from both facilitation and TIP processes underlie the potentiated response during delivery of the tetanic stimulus train. Facilitation, however, decays within a few hundred milliseconds. Thus, longer-lived components (TIP), which decay on the order of seconds to minutes, can be isolated in the potentiated response after the tetanic train ends. TIP is greatly reduced in AP-1 terminals compared with the control. The potentiation factor immediately after the tetanus is 2.53 ± 0.13 for control and 1.15 ± 0.10 for AP-1. This early potentiation decays with time but lasts for several minutes as evidenced by the values for PF2.75 measured 2.75 min after stimulation cessation, which are 1.54 ± 0.14 for control and 0.93 ± 0.11 for AP-1. Thus, in AP-1 appears to affect both PF0 (Kim, 2009).

The absence of TIP components in AP-1 synapses is consistent with a model where individual release sites are 'prepotentiated' in AP-1 motor terminals. Loss of TIP cannot be explained by postsynaptic receptor saturation, because EJPs of twice this magnitude can easily be detected at this motor synapse. The observation that one form of short-term plasticity (PPF) remains unaltered, whereas longer lived forms (TIP) are dramatically diminished argues that AP-1 acts through a selective and relatively specific mechanism normally used for tetanus-induced presynaptic plasticity (Kim, 2009).

To determine the underlying mechanism of synaptic enhancement by AP-1, three key parameters that influence the efficiency of neurotransmitter release were measured: (1) presynaptic Ca2+ entry; (2) sensitivity of the exocytotic machinery to Ca2+; and (3) the available pool of synaptic vesicles (Kim, 2009).

A simple mechanism for increasing the probability of exocytosis from an active zone is enhanced Ca2+ entry, e.g., because of a decreased presynaptic potassium conductance and/or an increased Ca2+ current. The highly comparable paired-pulse ratios in AP-1 and control terminals suggest presynaptic Ca2+ entry and, particularly, the molecular target of residual Ca2+ during PPF, is unchanged in AP-1 expressing motor neurons (Kim, 2009).

Direct Ca2+ imaging to support the above argument is difficult, because small changes in single-action potential induced Ca2+ entry potentially can account for the observed increase in quantal content. Using an indirect approach, it was instead asked whether summed Ca2+ entry during 40-Hz nerve stimulation was increased in AP-1 expressing animals (Kim, 2009).

In motor terminals expressing the genetically encoded Ca2+ indicator, GCaMP 1.6, fluorescence was imaged during sustained 40-Hz stimulation. Values for DF/F at a plateau reached in ~2 seconds were similar in AP-1 and control synapses. Unexpectedly, Ca2+ rise times in AP-1 terminals were slightly slower than in the control. This cannot be ascribed to faster Ca2+ extrusion as GCaMP signal does not decay any faster in AP-1 synapses after stimulation cessation. Instead, these data indicate that less Ca2+ enters AP-1 presynaptic terminals per action potential, at least during high-frequency stimulation. Although GCaMP imaging does not provide absolute measurement of presynaptic Ca2+ before and after stimulation, these data argue against increased evoked Ca2+ entry as being the primary mechanism for AP-1's effect on transmitter release (Kim, 2009).

Another mechanism to enhance transmitter release is to increase sensitivity of the exocytotic machinery to free Ca2+. Measurements, however, show Ca2+ cooperativity of transmitter release was not significantly altered by AP-1 expression (Kim, 2009).

The last major parameter that influences and often correlates with quantal content is the size of the active cycling vesicle pool (also referred to as exo-endo cycling pool, ECP) available for release (Murthy, 1999). At Drosophila motor synapses, the ECP contributes to transmitter release at low to moderate rates of nerve stimulation, e.g., 3 Hz. A second 'reserve' pool of vesicles (RP) poorly accessed at 3-Hz stimulation, is efficiently mobilized during high frequency stimulation >10 Hz. Two independent approaches, one electrophysiological and the other, optical allow the sizes of the cycling and total synaptic vesicle pools to be compared at the Drosophila NMJ (Kim, 2009).

ECP sizes were compared as follows. First, AP-1 and control synapses were stimulated continuously at 3 Hz in the presence of 1 μM bafilomycin A1, a drug that pharmacologically blocks the refilling of vesicles with neurotransmitter. Initial rates of synaptic depression under these experimental conditions largely reflect depletion of the cycling pool of vesicles. The later phase in the decay plot, after significant ECP depletion, represents vescles that arise from slow mixing between RP and ECP. The initial phase is extended in AP-1 compared with control, consistent with a larger ECP. To quantitatively estimate ECP size, Y-intercept values were determined by linear regression of the points from the later slow phase of depression in a cumulative plot. These ECP estimates were consistent with substantial enlargement of the ECP in AP-1 motor terminals. Because these estimates derive from fitting the observed curves to a specific (previously suggested) model (Delgado, 2000), a second and completely independent technique was used to estimate the ECP. In this technique, optical measurements of styryl dye uptake into individual varicosities were used. Consistent with predictions from electrophysiological measurements, varicosities at AP-1 synapses were more brightly labeled than control synapses when the ECP was loaded with FM1-43 dye by 3-Hz stimulation for 7 min, indicating a larger ECP (Kim, 2009).

To test whether this increased ECP in AP-1 synapses occurs at the expense of the reserve poolwe measured the total vesicle content in AP-1 and control terminals was measured by stimulating them to depletion at 10-Hz frequency in the presence of Bafilomycin. Total vesicle pool size was estimated by integrating the complete depression curve of quantal content versus stimulus number. This direct electrophysiological estimate showed a slightly smaller total pool size in AP-1 terminals. To independently assess the sizes of the total vesicle pool FM1-43 uptake into presynaptic boutons was measured after 7 min of 30-Hz stimulation, conditions that should label both ECP and RP. Remarkably, both control and AP-1 terminals were labeled to very similar levels under these conditions, with AP-1 showing slightly lower labeling. This indicates that the total number of synaptic vesicles is similar in control and AP-1 synapses. Thus, 2 independent approaches-electrophysiological and optical establish that AP-1 increases the actively cycling vesicle pool by partially mobilizing the reserve pool of synaptic vesicles. EM analyses of synaptic-vesicle density in AP-1 and control nerve terminals are also conistent with this conclusion (Kim, 2009).

Based on these observations, AP-1 synapses show 2 major differences from the wild-type. First, they have a larger fraction of actively cycling vesicles. Second, they exhibit highly reduced TIP. These 2 phenotypes can be linked if one proposes that mobilization from the reserve vesicle pool is required for TIP. In such a model, AP-1 synapses cannot be further potentiated because the RP has already been mobilized. It was therefore asked whether tetanus-induced potentiation requires RP mobilization (Kim, 2009).

Previous work has established that RP mobilization depends on activity of the myosin light chain kinase (MLCK) in Drosophila motor terminals (Verstreken, 2005). Blocking the activity of this enzyme results in failure to recruit vesicles from the inactive pool under high frequency stimulation (Verstreken, 2005). Strikingly, the MLCK inhibitor ML-7 also inhibited tetanus-induced potentiation; PF was not examined at later time points because in relevant control preparations, the small amount of DMSO required to dissolve MLCK increased the rate of decay of TIP]. Taken together, the above experiments indicate that (1) TIP requires synaptic-vesicle mobilization from the reserve pool; and, by inference, (2) AP-1 driven prepotentiation of transmitter release is accompanied by a stable expansion of the cycling pool of vesicles through reserve pool mobilization (Kim, 2009).

One important conclusion from this work is that Fos and Jun enhance synaptic strength, not by increasing synapse number, but rather by increasing the average probability of release from individual active zones. This conclusion is based on the following: (1) increases in synaptic strength in AP-1 motor terminals can be completely accounted for by increased transmitter release; and (2) light microscopic studies show no increases in the number of release sites. Thus, there is an increase in the average probability of synaptic vesicle fusion at release sites of AP-1 motor neurons. There are some caveats to this argument. First, the definition of functional synapses as Brp-positive puncta is based on the assumptions that Brp puncta: (1) mark the large majority of release sites; and (2) are mostly capable of transmitter release postsynaptic stimulation. These assumptions are supported by the tight colocalization of presynaptic Ca2+ channels and postsynaptic receptors with Brp puncta (Kim, 2009).

Increased vesicle-release probability from active zones could conceivably be explained by several different mechanisms. In AP-1 synapses, a large increase in the size of the actively cycling synaptic vesicle pool (ECP), which arises at the expense of the reserve pool (RP), was demonstrated. An increased ECP can account for the observed synaptic enhancement in AP-1 motor terminals if it increases the number of synaptic vesicles immediately available for fusion. RP mobilization has been associated with specific instances of short-term plasticity: e.g., cocaine-induced increases in dopamine release from rat striatal neurons. This study shows that this process can be initiated by nuclear gene expression (Kim, 2009).

How widely might RP mobilization be deployed for synaptic change in vivo? Studies of the reserve pool in hippocampal synapses do not easily support the idea that vesicle trafficking from this source controls synaptic vesicle availability within these small axonal terminals. However, RP mobilization can regulate the output from larger synapses such as neuromuscular junctions or the calyx of Held, where inhibition of the myosin light chain kinase required for vesicle mobilization has been shown to reduce the stability of the synaptic firing during repetitive stimulation (Verstreken, 2005). Because the actual mechanism of RP mobilization is poorly understood, more experiments will be required to understand exactly how Fos and Jun regulate this process. In one model, phosphorylation of synapsin, which tethers synaptic vesicles to an actin-based cytoskeleton in the central domain of synaptic boutons, may mobilize the reserve pool by triggering the dissociation of vesicles from the cytoskeleton, and their transport/diffusion to peripheral release sites. In Drosophila NMJs, mitochondria within the presynaptic terminal are required for sustained release of neurotransmitter under high frequency stimulation. One study suggests ATP production from mitochondria is required to fuel MLCK-activated, myosin-propelled transport of reserve vesicles from central to peripheral sites (Verstreken, 2005). Although such processes might be involved, it is also possible that different pathways are used for Fos and Jun mediated RP mobilization. For instance, smaller sized boutons, such as those observed in AP-1 animals, may be less efficient at holding a central pool of reserve vesicles. In such a scenario, bouton geometry, rather than a specific regulatory protein, may prove to be the relevant target of AP-1 activity (Kim, 2009).

The simplest interpretation of these observations is that stable reserve pool mobilization underlies the observed loss of tetanus-induced potentiation in AP-1 synapses. Other work at the Drosophila neuromuscular junction has associated mobilization of the reserve pool with the expression of TIP (Kim, 2009).

Cytosolic Ca2+ accumulation and signaling is required for the induction of TIP. However, links between Ca2+ signaling and the expression of TIP are poorly defined; indeed, tetanus induced potentiation could include multiple Ca2+-dependent processes including augmentation and posttetanic potentiation/PTP. If TIP expression requires RP mobilization, then it would be occluded in 1 of 2 ways. Either: (1) by preexisting mobilization of the reserve pool; or (2) by inhibition of reserve pool mobilization. Drosophila dnc mutants with enhanced cAMP signaling, and rut mutants with reduced cAMP signaling respectively illustrate these 2 different mechanisms of inhibition. Both mutants do not show tetanus-induced potentiation. Although dnc mutants show a greatly increased ECP and already enhanced transmitter release, rut mutants show a large, stable reserve pool that cannot be mobilized by tetanic stimulation. These data indicate that AP-1 synapses behave like dnc mutants in which reserve vesicles have already been mobilized (Kim, 2009).

If reserve pool mobilization is required for TIP, then mutations or drugs that inhibit reserve pool mobilization would also be expected to block TIP. Consistent with this prediction, application of an MLCK inhibitor, which blocks reserve pool mobilization, was shown to dramatically inhibit TIP induction in wild-type motor synapses. Thus, although alternative contributing mechanisms cannot be ruled out, this study shows that tetanus induced presynaptic potentiation is tightly linked to reserve pool mobilization (Kim, 2009).

It is possible that many different direct and indirect targets of AP-1 contribute to various observed AP-1 dependent neuronal phenomena: e.g., increased bouton number, reduced bouton size, increased dendritic growth, elevated evoked transmitter release and increased ECP size. In addition, AP-1 may have effects on some phenomena that are not yet measured, e.g., kinetic and spatial features of synaptic Ca2+ dynamics. Nonetheless, this work shows functions of Fos and Jun in neurons, and provides substantial evidence for a model in which transcription-dependent changes in synaptic function occur through stable recruitment of mechanisms used in short-term plasticity. Recent observations that short-term forms of presynaptic plasticity are altered following synaptic enhancements induced by either BDNF or postsynaptic PSD-95 overexpression suggest that this could be a viable strategy for long-term information storage in central synapses (Zakharenko, 2003). If long-term plasticity requires stable recruitment of short-term plasticity mechanisms, then the lability of long-term memory traces, as observed in studies of reconsolidation, may not require the elimination of stable synaptic connections representing the stored memory (Kim, 2009).

Genetic screen in Drosophila melanogaster uncovers a novel set of genes required for embryonic epithelial repair

The wound healing response is an essential mechanism to maintain the integrity of epithelia and protect all organisms from the surrounding milieu. In the 'purse-string' mechanism of wound closure, an injured epithelial sheet cinches its hole closed via an intercellular contractile actomyosin cable. This process is conserved across species and utilized by both embryonic as well as adult tissues, but remains poorly understood at the cellular level. In an effort to identify new players involved in purse-string wound closure a wounding strategy suitable for screening large numbers of Drosophila embryos was developed. Using this methodology, wound healing defects were observed in Jun-related antigen (encoding DJUN) and scab (encoding Drosophila alphaPS3 integrin) mutants and a forward genetics screen was performed on the basis of insertional mutagenesis by transposons that led to the identification of 30 lethal insertional mutants with defects in embryonic epithelia repair. One of the mutants identified is an insertion in the karst locus, which encodes Drosophila betaHeavy-spectrin. betaHeavy-spectrin (betaH) localizes to the wound edges where it presumably exerts an essential function to bring the wound to normal closure (Campos, 2010).

Using previously described DC or wound healing mutants a pilot screen was performed to validate the embryonic wounding strategy. The fact that a member of the DJNK pathway (Jra/DJun) was identified in the assay is in accordance with other reports that implicate this pathway in wound healing. Specifically, two mutations in components of the DJNK pathway, bsk/DJNK and kay/DFos, were previously shown to have defects in fly larval and adult wound closure, respectively. In addition, a reporter construct has been describes that requires consensus binding sites for the JUN/FOS complex to be activated upon wounding. Interestingly, treporter activation was still observed in Jra mutants, which suggests that additional signaling pathways are involved in wound closure (Campos, 2010).

An apparent discrepancy arose when the assay revealed a phenotype with Jra but not with puc mutants, another component of the same signaling pathway. This result might be explained by the fact that Jra and puc function in opposite directions in the DJNK signaling pathway. Puc functions as a pathway repressor, so in a puc mutant the JNK pathway should be less repressed and an opposite effect to a Jra mutation could be expected. In addition, activation of a puc-lacZ reporter has been shown to occur in larvae, wing imaginal discs, and adult wounds that take 18-24 hr to close, but it is only robustly detectable 4-6 hr postpuncture. Embryonic wounds are faster to heal, and even after inflicting a large laser wound on stage 14/15 embryos, no activation of the puc-lacZ reporter (assessed in open wounds 3 hr postwounding by immunofluorescence; data not shown) was detected. This observation suggests that, in rapidly healing epithelial wounds, the JNK pathway is not activated to high enough levels to trigger auto-inhibition (Campos, 2010).

The α-integrin scab was never before implicated in embryonic wound healing, but this mutant's phenotype comes as no great surprise. The first scab mutation was isolated due to its abnormal larval cuticle patterning. The scab gene encodes for Drosophila α-PS3 integrin, which is zygotically expressed in embryonic tissues undergoing invagination, tissue movement, and morphogenesis. Integrin proteins are involved in cell-matrix interactions and α-PS3 integrin regulation, in particular, mediates zipping of opposing epithelial sheets during DC. Similarly, the observation of a wound defect in scb5J38 mutants is consistent with a role for α-PS3 integrin in zipping of opposing epithelial cells during the healing process (Campos, 2010).

A previous study using confocal video microscopy has shown that Rho11B mutants take twice as long to close an epithelial wound when compared to wild type. Rho1 was confirmed in the assay to be important for wound healing, although with a weaker phenotype (22% of embryos had unclosed holes). This result shows nonetheless that the assay can be sensitive enough to pick up a 'weak' wound healing mutant such as Rho11B, which is still able to heal wounds albeit slower than wild type (Campos, 2010).

The genes identified in the screen represent a variety of functions indicating that wound healing is a complex mechanism that requires the participation of many cellular processes. A large class of the candidate mutants are involved in several aspects of gene expression, including factors that regulate chromatin remodeling (dUtx and Pc), elongation (dEaf), splicing (Glo and CG3294), and translation (CG33123). These factors are likely needed during wound healing for the induction of a repair transcriptome. Interestingly, JNK signaling-dependent Pc group (PcG) gene downregulation has been observed during imaginal disc regeneration. In addition, a recent study revealed that PcG methylases are downregulated during wound healing, while counteracting demethylases, Utx and Jmjd3, are upregulated. The results for the Pc and Utx mutants are consistent with these studies and highlight the importance of epigenetic reprogramming in the repair process (Campos, 2010).

Some of the genes such as arc-p20 and karst probably have a more direct role in the cell shape changes that drive the tissue morphogenetic movements during epithelial repair. The gene product of arc-p20 is a component of Arp2/3, a complex that controls the formation of actin filaments, and karst encodes a component of the spectrin membrane cytoskeleton. Also related to morphogenesis, CG12913 encodes an enzyme involved in the synthesis of chondroitin sulfate, which is usually found attached to proteins as part of a proteoglycan, suggesting a predictable contribution of the extracellular matrix in the tissue movements necessary for wound healing (Campos, 2010).

The epithelium is the first line of defense of the organism against pathogens and tissue integrity. It would thus seem plausible that genes involved in innate immunity could be identified with the screening protocol. Indeed, two of the genes (Ser12 and CG5198) seem to point to the involvement of the immune response in the healing of the laser-induced wounds. Ser12 is a member of the serine protease family, a class of proteins that has been shown to play a role in innate immunity. The CG5198 gene has no described function in Drosophila so far, but its homolog, CD2-binding protein 2, is involved in T lymphocyte activation and pre-RNA splicing. Another candidate that might represent a link to immunity is Atg2, a gene important for the regulation of autophagy, a process by which cells degrade cytoplasmic components in response to starvation. In Drosophila, autophagy has been linked to the control of cell growth, cell death, and, recently, to the innate immune response mechanism against vesicular stomatitis virus and listeria infection (Campos, 2010).

Isolation of an insertion in the stam gene points to the involvement of the JAK-STAT signaling cascade in this regenerative process. Interestingly, stam has been shown to be involved in Drosophila tracheal cell migration and is upregulated following Drosophila larvae infection by Pseudomonas entomophila (Campos, 2010).

One candidate could be involved in the uptake or export of some important wound signal (CG7627) as this gene encodes for a multidrug resistant protein (MRP), part of the ABC transporter superfamily, involved in drug exclusion properties of the Drosophila blood-brain barrier (Campos, 2010).

The kinase encoded by grapes is the Drosophila homolog of human Check1 (Chk1) involved in the DNA damage and mitotic spindle checkpoints. All the Chk1 literature has focused on its role during the cell cycle. However, the Drosophila late embryonic epithelium is a quiescent tissue, even after wounding. Understanding Grapes function in this context is a challenging task that could lead to new paradigms. One hypothesis is that Grapes is involved in tension sensing, as it is in the spindle checkpoint, or may uncover a cellular repair process that could help damaged cells 'decide' to either die by apoptosis or participate in the repair process (Campos, 2010).

The remaining genes with a putative function represent a wide range of general metabolic processes (aralar1, gs1-like, CG4389, CG9249, CG11089, and CG16833), suggesting that healing the epithelium is a highly demanding process (Campos, 2010).

Finally, a significant number of genes that have not yet been studied and do not contain identifiable protein domains (CG2813, CG31805, CG6005, CG6750, CG10217, CG15170, and CG30010) were selected. At the moment it is not possible to predict the role that these genes may play, but further study may help to identify novel wound healing regulatory mechanisms (Campos, 2010).

One of the mutants identified in the transposon screen was kstd11183, an insertion in the βH-spectrin locus. This mutation is likely producing a truncated protein terminating three amino acids into the P-element insertion. Other mutations identified in nearby segments 14 (kst14.1, kst2) and 16 (kst1) lead to the production of a detectable truncated protein so it is likely that karstd11183 mutation also gives rise to a truncated protein. These mutant forms of βH lack approximately half of the wild-type protein, including a COOH-terminal PH domain region, which is involved in targeting the protein to the membrane, thus producing a potential dominant negative form of βH. However, the karstd11183 mutant should still have maternally loaded wild-type protein, as previous studies describe a complete absence of maternal protein only by the third instar larval stage. This maternal contribution is likely the main reason that this mutant, as well as the other mutants isolated in the screen, does not have a fully penetrant wound healing phenotype (Campos, 2010).

βH-spectrin was shown to localize to the actomyosin purse string, a supracellular contractile cable that forms rapidly upon wound induction. Live imaging has demonstrated that actin and myosin can accumulate in this cable structure within minutes after wounding. Unfortunately, due to the size of the βH gene (>13 kb) cloning and tagging it for live imaging is not possible using standard methods, but the experiments in fixed tissue reveal that βH can accumulate very rapidly in this cable structure. βH accumulation was observed at the earliest time point technically feasible, 15 min postwounding. These observations are consistent with previous studies, also in fixed tissue, demonstrating rapid changes in βH localization during the process of cellularization in Drosophila embryos. Taken together, it is clear that at least the βH component of the membrane skeleton is not just a static structural scaffold as the name implies, but rather a dynamic protein capable of responding to or directing changes in cellular dynamics. The studies suggest that polarized redistribution of βH exerts an essential function to facilitate actin-based cellular responses, such as cable accumulation/maintenance and wound edge filopodia dynamics, which are necessary to properly close a wound (Campos, 2010).

βH has been previously observed in association with actin 'rings' during development of Drosophila and C. elegans. Arguably, C. elegans provides an example of actin ring function most analogous to the Drosophila wound edge purse string. During the final stages of C. elegans development, cortical arrays of actin in the outer epithelial cells, the hypodermis, dramatically reorganize to form parallel apically localized bundles of circumferencial supracellular actin rings. In this system, sma1, the C. elegans ortholog of βH, also localizes apically to these actin rings. In sma1 mutants the rings fail to productively contract and begin to disorganize, losing connection to the cell membranes. An additional phenotype observed in these mutants is the inability of cells to change their shape, a process normally 'directed' by these contractile rings, the end result being a short worm, a phenotype seen as functionally analogous to an unclosed wound in the Drosophila system (Campos, 2010).

In Drosophila, βH has been implicated in modulating cell shape changes during apical constriction of follicle cells (a process also involving actin rings) and has been proposed to function as a link between cross-linked actin networks/rings and the cell membrane. Further studies revealed that the C-terminal domain of βH has the ability to directly modulate the apical membrane area by regulating endocytosis, adding one more tantalizing piece of evidence pointing to the fact that βH could be a major player in cell shape changes, not only as a structural link but also by directly modulating the membrane area in response to cytoskeletal clues (or vice versa) (Campos, 2010).

Although it is known from previous studies that the actin cable is not absolutely required for wound closure, the process takes much longer without one. In Rho1 mutant embryos, cells lacking a cable are able to pull the wound closed using filopodia. The filopodial defect observed in karst mutants, adds another line of evidence to the absolute requirement of these structures for wound closure. In addition to the reduced actin cable accumulation and filopodial dynamics in karst mutants (which would lack the C-terminal domain responsible for membrane modulation), a lack of cell shape change is seen in the wound edge cells. Taken together, these data and the published work, introduce the intriguing possibility that βH could be serving as a link between wound edge dynamics and the coordinated cell shape changes usually observed in wild-type wound edge cells. The combination of the proposed ability of βH to modulate the apical membrane area as well as cross-link actin and act as an apical membranewide scaffold for other interactions, makes βH a good candidate to provide the physical link that would coordinate tissuewide actions, such as supracellular actin cable contraction, with the individual cellular responses, such as cell shape change and polarized filopodia activity (Campos, 2010).


DJun Biological Overview | Evolutionary Homologs | Regulation | Effects of Mutation | References

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