Fos-related antigen/kayak



Fra/Kayak protein is first detectable in the head mesoderm at stage 9, and from stage 11 onwards in additional tissues, including the amnioserosa and the ectoderm. FRA mRNA levels are elevated in the embryonic endoderm of the second gut lobe (Perkins, 1990). During stage 13, endodermal cells begin to show weak Fra staining, with a slightly higher level in a band of the forming midgut epithelium spanning the fusion junction of the two gut primordia. This band of elevated Fas expression becomes more and more prominent, and remains clearly visible from stage 15 onwards throughout late embryogenesis; it stretches throughout the second gut lobe, from the first to the second gut constriction (i.e. through approx. ps6-7). Fra is predominantly (if not exclusively) nuclear in all cell types observed. Fra protein accumulates in all endodermal cells in the second gut lobe. This contrasts with labial, which is expressed only in a subset of the endodermal cells in this lobe; lab expression is not detectable in the most posterior cells within this lobe; throughout the lobe, lab-expressing cells are interspersed with cells not expressing lab (Reise, 1997). Fra staining is strongest in the central region of the lobe, fading slightly towards both constrictions (Reise, 1997), whereas Lab staining shows a striking anteroposterior gradient of expression, with highest levels most posterior (Immergluck, 1990).

Fra-expressing neurons appear to be located near the lateral chordotonal cells and might be extrasensory or multiple dendritic neurons. At the same stages of development, FRA mRNA is localized to a portion of the ectoderm that corresponds with muscle attachment sites. It is also observed in part of the midgut and hindgut and in the anal pad (Perkins, 1990).


EGF signaling regulates the proliferation of intestinal stem cells in Drosophila

Precise control of somatic stem cell proliferation is crucial to ensure maintenance of tissue homeostasis in high-turnover tissues. In Drosophila, intestinal stem cells (ISCs) are essential for homeostatic turnover of the intestinal epithelium and ensure epithelial regeneration after tissue damage. To accommodate these functions, ISC proliferation is regulated dynamically by various growth factors and stress signaling pathways. How these signals are integrated is poorly understood. This study shows that EGF receptor signaling is required to maintain the proliferative capacity of ISCs. The EGF ligand Vein is expressed in the muscle surrounding the intestinal epithelium, providing a permissive signal for ISC proliferation. The AP-1 transcription factor FOS serves as a convergence point for this signal and for the Jun N-terminal kinase (JNK) pathway, which promotes ISC proliferation in response to stress. These results support the notion that the visceral muscle serves as a functional 'niche' for ISCs, and identify FOS as a central integrator of a niche-derived permissive signal with stress-induced instructive signals, adjusting ISC proliferation to environmental conditions (Biteau, 2011).

These findings establish a crucial role for EGF signaling in the regulation of ISC proliferation, and thus support the notion that the visceral muscle surrounding the intestinal epithelium has the characteristics of a functional niche. vein expression in the muscle maintains the competence of ISCs to enter rapid proliferation in responses to stress and JNK signaling, and is thus expected to regulate epithelial homeostasis. Interestingly, it was found that both the EGFR-mediated permissive signal and the JNK-derived inductive signal are relayed by FOS, establishing an integrated molecular mechanism for the control of ISC proliferation (Biteau, 2011).

Many stem cell populations are regulated by their microenvironments, and larval ISC progenitors are regulated by a transient niche (Mathur, 2010). However, ISCs in adult flies apparently lack such a closely associated cell population within the intestinal epithelium. By contrast, control of ISC maintenance by muscle-derived Wingless suggested this tissue as a potential functional niche for adult ISCs. The current results support and extend this idea by identifying a second growth factor derived from the visceral muscle that controls ISC proliferation. In its regulation of stem cell function through Wingless and Vein, and in the close association of ISCs and muscle cells, the muscle thus shares characteristics of stem cell niches in other systems, yet it also differs from these in important ways. In mammals, as well as in the Drosophila and C. elegans gonads, the niche of most stem cell populations maintains stem cell quiescence and prevents differentiation. The EGF signal originating from the muscle, however, maintains the capacity of ISCs to divide, allowing these cells to respond to stimulating signals while not affecting ISC differentiation. Interestingly, EGFR signaling has not been described so far as crucial for interactions between the niche and stem cell populations in other systems, and these findings raise the possibility that this signaling pathway might also regulate the function of other stem cell populations in both invertebrates and vertebrates (Biteau, 2011).

Whereas knocking down the expression of vein in the muscle partially affects the ability of ISCs to proliferate under normal conditions and in response to stress, the inhibition of EGFR completely abolishes stem cell division. This might reflect the inefficiency of the veinRNAi constructs used in this study, but might also suggest a contribution of other EGFR ligands to the regulation of ISC function. Accordingly, a genome-wide analysis of the transcriptional response of the adult intestine to bacterial infection suggests that expression of vein, as well as of two other genes encoding EGFR ligands, Keren and spitz, is increased after immune challenge. However, the potential role for these additional EGF-like ligands in regulating ISC function remains to be investigated and the cells expressing spitz and Keren in the adult intestine have yet to be identified (Biteau, 2011).

ISC function is regulated by systemic insulin-like peptides expressed by neurosecretory cells in the brain, muscle-derived vein and wingless, local unpaired cytokine expressed by ECs, and cell-intrinsic signals. These multiple signals are integrated in ISCs to adapt their proliferation rate and differentiation program to environmental and physiological challenges. To fully understand stem cell regulation in this high-turnover tissue, the molecular structure of this signaling network has to be unraveled. The findings of this study introduce the transcription factor FOS as a crucial regulator of ISC proliferation that integrates mitogenic and stress signals, and indicate that JNK and ERK regulate FOS activity directly by phosphorylation on distinct residues, controlling ISC proliferation in a combinatorial fashion. This signal-specific mode of FOS regulation by ERK and JNK in Drosophila had previously been described in the context of morphogenetic movements (in which FOS is regulated by JNK) and of eye and wing growth during development (in which it is regulated by ERK and JNK) (Biteau, 2011).

How FOS promotes ISC proliferation remains unclear. In developing imaginal discs, inhibition of FOS causes an accumulation of cells in the G2/M phase of the cell cycle, probably owing to a loss of Cyclin B expression, an essential regulator of the G2/M transition. Interestingly, in ISCs, expression of FosRNAi not only inhibits stress-induced accumulation of pH3+ cells, but also represses BrdU incorporation, indicating that FOS regulates entry into S phase. In these cells, FOS might thus regulate the transcription of essential S phase components. Further studies will be required to identify such ISC-specific FOS target genes (Biteau, 2011).

The maintenance of stem cells in a primed state, ready to respond to inductive mitogenic stress signals, is likely to be crucial for high-turnover tissues like the intestinal epithelium, which require rapid activation of stem cell division for an efficient regenerative response to tissue damage. At the same time, this enhanced mitogenic potential of ISCs might contribute to the loss of tissue homeostasis in the aging gut, and contribute to cancer formation in mammalian intestinal epithelia. Interestingly, a conserved role of AP-1 transcription factors and JNK signaling in the regulation of intestinal stem cell proliferation and intestinal cancer is emerging in mice. JNK activation is sufficient to induce cell proliferation in the intestinal crypt and increases tumor incidence and tumor growth in an inflammation-induced colon cancer model. These effects of JNK signaling are mediated by the FOS binding partner JUN, as shown by the requirement for JNK-mediated phosphorylation of JUN for APCmin/+-induced tumorigenesis. Strikingly, ISC-specific activation of WNT signaling, by mutating APC or expressing an active form of ß-catenin or wingless itself, is sufficient to induce the formation of tumor-like stem cell clusters in the fly intestine. A potential interaction of WNT signaling with JNK and JUN or FOS in ISCs remains to be tested in Drosophila. Interestingly, increased FOS activity has also recently been shown to be sufficient to promote hematopoietic stem cell self-renewal in mice, further illustrating the conserved function of FOS in the regulation of stem cell function (Deneault, 2009). AP-1 transcription factors are thus emerging as conserved essential regulators of stem cell function and the current findings provide an important starting point for further studies characterizing stem cell-specific signaling networks that integrate mitogenic, survival and stress signals to control stem cell maintenance, quiescence and proliferation, and thus influence the balance between regeneration and tumor suppression in high turnover tissues (Biteau, 2011).

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).

Effects of Mutation or Deletion

Drosophila kayak mutant embryos exhibit defects in dorsal closure, a morphogenetic cell sheet movement that takes place during embryogenesis. It is shown that kayak encodes D-Fos, the Drosophila homolog of the mammalian proto-oncogene product, c-Fos. D-Fos is shown to act in a similar manner to Drosophila Jun: in the cells of the leading edge it is required for the expression of the TGFbeta-like Decapentaplegic (Dpp) protein, which is believed to control the cell shape changes that take place during dorsal closure. The kayak expression domain include the cells of the amnioserosa and the lateral epidermis during the process of dorsal closure. At the onset of dorsal closure, elevated levels of D-Fos can be detected in the nuclei of leading-edge cells as they initiate elongation. Subsequently, elevated expression of Fos can also be observed in more ventrally located epidermal cells. Concurrently, cell elongation spreads laterally, until the two edges meet at the dorsal midline. At this stage, Fos is strongly expressed throughout the embryonic epidermis, with the highest levels remaining in the cells of the leading edge. This expression pattern is very similar to that of D-Jun and also correlates with the JNK-pathway-dependent stripe of dpp expression in the leading edge, which becomes apparent during the initiation phase of dorsal closure. Therefore, it is conceivable that Drosophila Fos acts in conjunction with Jun to regulate dorsal closure and dpp expression (Zeitlinger, 1997).

Defects observed in mutant embryos, and adults with reduced Fos expression, are reminiscent of phenotypes caused by 'loss of function' mutations in the Drosophila JNKK homologue, hemipterous. Mutant alleles of D-fos have not previously been described. Based on the potential involvement of D-Fos in the process of dorsal closure, known dorsal open mutants were examined for defects in D-fos. kay1 mutant embryos all die during embryogenesis with large dorsal and anterior holes that indicate failed dorsal closure and head involution. In kay2 embryos, dorsal holes are also observed, but at lower penetrance. Depending on the temperature and genetic background, up to ~1% of kay2 homozygotes even develop to adulthood, as seen after recessive markers had been removed from the kay2 mutant chromosome by recombination. The transheterozygous kay1/kay2 allelic combination displays an intermediate phenotype and embryonic, or early larval, lethality. These observations indicate that kay2 is a weaker allele than kay1, and thus is a hypomorph. To examine the cause of the kay defect at the cellular level, mutant and wild-type embryos were stained with an anti-Coracle serum, which outlines the epidermal cells. It was found that the kay mutant phenotype is caused by a failure of the lateral epidermal cells to elongate. As previously observed in the case of D-jun, leading-edge cells of zygotic kay1 mutants initiate elongation transiently, but fail to maintain it and subsequently resume the unelongated shape. The more lateral epidermal cells elongate to a very minor extent and resume the typical polygonal shape after the process has been terminated prematurely. Thus, the kay mutant phenotype closely resembles those described for hep, bsk and D-jun mutant embryos, also at the cellular level (Zeitlinger, 1997)

The phenotypic similarity between D-jun and kay mutants suggests that D-Fos and D-Jun act in concert to mediate dorsal closure. Thus, one may predict that D-fos/kay, like D-jun and the upstream signaling components bsk/JNK and hep/JNKK, is required for the expression of dpp in the leading edge cells. To test this idea, the expression of DPP mRNA was monitored in wild-type and kay-mutant backgrounds. Expression of dpp in the cells of the future leading edge is normally initiated when the germ band is fully extended and is maintained throughout dorsal closure. In contrast, in kay1 homozygous embryos, dpp expression is absent (or reduced in kay2 mutants) in cells of the leading edge. Significantly, other pattern elements of dpp expression are still present in the kay/D-fos mutants, including a more ventral stripe and midgut-specific expression, which have previously been shown to be independent of JNK signaling. Another downstream effect of Bsk signaling, the transcriptional activation of the puckered gene (puc) in the cells of the leading edge is also abrogated in kay1 mutant embryos. Taken together, the requirement of both D-Fos and D-Jun for dpp and puc expression in leading-edge cells suggests that the JNK signal is relayed by a heterodimeric transcription factor composed of D-Jun and D-Fos. These results indicate that D-Fos is required downstream of the Drosophila JNK signal transduction pathway, consistent with a role in heterodimerization with D-Jun, to activate downstream targets such as dpp (Zeitlinger, 1997).

Thorax closure in Drosophila: involvement of Fos and the JNK pathway

Dorsal closure, a morphogenetic movement during Drosophila embryogenesis, is controlled by the Drosophila JNK pathway, D-Fos and the phosphatase Puckered (Puc). To identify principles of epithelial closure processes, another cell sheet movement that can be termed thorax closure was studied: the joining of the parts of the wing imaginal discs that gives rise to the adult thorax during metamorphosis. The genes that are required for dorsal closure give rise to an interesting abnormal adult phenotype, suggesting that there is an additional requirement for these genes during later development: homozygous animals of mutant alleles of D-fos, hep, pannier (pnr) and components of the Dpp pathway show a cleft at the dorsal midline of the thorax and neighbouring bristles are abnormally parted to both sides. In thorax closure a special row of margin cells express puc and accumulate prominent actin fibers during midline attachment. Genetic data indicate a requirement of D-Fos and the JNK pathway for thorax closure, and a negative regulatory role of Puc. Furthermore, puc expression co-localizes with elevated levels of D-Fos; is reduced in a JNK or D-Fos loss-of-function background, and is ectopically induced after JNK activation. This suggests that Puc acts downstream of the JNK pathway and D-Fos to mediate a negative feed-back loop. Therefore, the molecular circuitry required for thorax closure is very similar to the one directing dorsal closure in the embryo, even though the tissues are not related. This finding supports the hypothesis that the mechanism controlling dorsal closure has been co-opted for thorax closure in the evolution of insect metamorphosis and may represent a more widely used functional module for tissue closure in other species as well (Zeitlinger, 1999).

In order to mark and visualize the dorsal parts of the wing imaginal discs that fuse during thorax closure, the UAS-Gal4 system was used to express green fluorescent protein in the expression domain of pnr, a gene encoding a GATA transcription factor whose expression is restricted to dorsal tissues throughout development. The prepupae were then dissected in a way that leaves the entire thorax complex intact and different stages were inspected by confocal microscopy. In addition, actin filaments were visualized by staining with phalloidin to monitor the behaviour of the cytoskeleton during this process. Phalloidin also stains three oblique muscles on each side, a useful marker during thorax closure. Already in third instar wing imaginal discs, pnr expression marks the dorsal part, the future medial notum. At around 6 hours after pupation (AP), after eversion, the dorsal parts of the two wing imaginal discs spread toward the dorsal midline, while the larval epidermis degenerates. When they subsequently meet and attach to each other at around 7 hours AP, filamentous actin becomes visible at the medial edge of the epithelium. These actin bundles at the dorsal midline are most abundant at 8 hours AP and are predominantly localized basally (Zeitlinger, 1999).

In summary, the process of thorax closure resembles embryonic dorsal closure at a tissue-morphological level: two epithelial sheets with a straight margin approach one another, meet at the dorsal midline, and attach. The actin organization seen along the margin of the epithelium is reminiscent of the accumulation of actin along the leading edge of the closing embryo. However, in contrast to the simple epithelial stretching of embryonic dorsal closure, the morphogenetic movements involved in thorax closure appear to be more complex: most cells are of polygonal shape and not obviously elongated along the dorsoventral axis. Furthermore, the tissue movements also include unfolding (as part of the eversion) and an anterior folding-in during midline fusion with subsequent back folding during head eversion (Zeitlinger, 1999).

Having established a system to monitor the progress of thorax closure, the tissue movements were monitored in a mutant background that gives rise to a cleft phenotype in adults. The hypomorphic mutation in D-fos, kay 2 was used in this experiment. It revealed that the dorsomedialward spreading of the epithelium is already abnormal at 6 hours AP in most kay2 prepupae. While, in a wild-type background, the pnr expression domain of the wing imaginal disc is found on top of the three oblique muscles and close to the degenerating larval epidermis, the corresponding epithelium in kay2 prepupae of this stage has failed to reach this position and is still located more laterally. At 8 hours AP, the spreading epithelium often appears to have retracted and fallen back into its original folded position found at earlier stages, although filamentous actin typical of this stage is detectable. These findings strongly argue that the defects observed in kay2 adult animals result from defects in thorax closure during prepupal stages (Zeitlinger, 1999).

The thoracic cleft phenotype observed with hypomorphic mutations in D-fos (kay2 ) and hep (hep1) suggests that D-Fos and the JNK pathway are involved in thorax morphogenesis. To confirm that the cleft phenotype is a result of a D-fos loss-of-function condition, a dominant negative form of D-fos (UAS-D-Fos bZIP) was expressed under the control of pnr-Gal4. This results in the appearance of a marked cleft in the thorax. A similar phenotype is obtained by overexpressing Puc (UAS-Puc) in the pnr domain. In the embryo, overexpression of Puc phenocopies loss-of-function mutations in the JNK pathway, consistent with the proposed function of Puc as a phosphatase that negatively regulates the JNK pathway by dephosphorylation of Basket. The fact that this is also true in thorax closure represents further evidence that the JNK pathway is involved in thorax closure. Next, a test was performed to see whether D-Fos genetically interacts with components of the JNK pathway during thorax closure. In contrast to the D-fos hypomorphic mutant kay2, kay1 represents a D-fos null allele (a deficiency). The heterozygous allelic combination (kay1 / kay2) is strictly lethal, but can be rescued by ubiquitous expression of D-Fos under a heterologous promoter. Strikingly, the lethality of kay1 / kay2 could also be rescued by eliminating one copy of the wild type puc gene. More than 50% of the expected Mendelian frequency could be recovered. Thus, pucE69 has a dominant effect in a kay mutant background, even though heterozygosity for pucE69 has no phenotypic effects in an otherwise wild-type fly. Furthermore, not only the lethality but also the thorax cleft phenotype of kay mutant flies could be dominantly rescued. The cleft phenotype of the rescued kay2 / kay1 puc flies ranges from strong to very mild. Heterozygous pucE69 in a kay2 homozygous background (kay2 / kay2 puc E69 ) gives rise to a stable stock in which most flies show a very mild or no thorax cleft at all. Therefore, the puc mutation has a dominant effect on thorax closure and two conclusions can be drawn: (1) Puc must be expressed during thorax closure; (2) as in dorsal closure, Puc negatively regulates the pathway in which D-Fos is acting during thorax closure (Zeitlinger, 1999).

The role of the Drosophila TAK homologue dTAK during development

TGF-ß activated kinase 1 is required during morphogenetic changes and the fusion of the epithelial wing disc cell layers that takes place in thoracic closure, acting in the context of JNK signaling. JNK signaling is required in thoracic closure. The notum of the adult animal is formed by tissue of the two collateral wing imaginal discs, which undergo extensive morphogenetic rearrangements during metamorphosis. LOF in hep/JNKK and kayak/D-Fos results in aberrant wing disc morphogenesis and failure of wing disc fusion, giving rise to a thoracic cleft along the dorsal midline in the adult. To test whether Tak1 can also act in this context, DN (kinase dead) forms of Tak1 (UAS-Tak1K46R or UAS-Tak1D159A) were overexpressed with ap-GAL4 and pnr-GAL4 in the thoracic parts of the wing discs. Examination of such flies shows incomplete closure of the thorax, giving rise to a mild thoracic cleft at the dorsal midline of the notum. Although this phenotype is relatively weak, it has a very high penetrance of 91% and is highly reminiscent of that observed in hypomorphic allelic combinations of either hep/JNKK or kay/d-fos. This is also in agreement with the phenotypic result of expressing Puckered, a negative regulator of JNK signaling at the time when wing disc fusion occurs. Puc overexpression driven by pnrGAL4 leads to the same phenotypic thorax cleft defects. These observations suggest that Tak1 is required during morphogenetic changes and the fusion of the epithelial wing disc cell layers, acting in the context of JNK signaling (Mihaly, 2001).

Top-DER- and Dpp-dependent requirements for the Drosophila fos/kayak gene in follicular epithelium morphogenesis

The Drosophila fos/kayak gene is a key regulator of epithelial cell morphogenesis during dorsal closure of the embryo and fusion of the adult thorax. It is also required for two morphogenetic movements of the follicular epithelium during oogenesis: (1) it is necessary for the proper posteriorward migration of main body follicle cells during stage 9; (2) it controls, from stage 11 onwards, the morphogenetic reorganization of the follicle cells that are committed to secrete the respiratory appendages. Egfr pathway activation and a critical level of Dpp signaling are required to pattern a high level of transcription of kayak at the anterior and dorsal edges of the two groups of cells that will give rise to the respiratory appendages. In addition, evidence is provided that, within the dorsal-anterior territory, the level of paracrine Dpp signaling controls the commitment of follicle cells towards either an operculum or an appendage secretion fate. kayak is required in follicle cells for the dumping of the nurse cell cytoplasm into the oocyte and the subsequent apoptosis of nurse cells. This suggests that in somatic follicle cells, kayak controls the expression of one or several factors that are necessary for these processes in underlying germinal nurse cells (Dequier, 2001).

The earliest requirement for kayak in egg chamber development occurs during stage 8/9. The Kayak protein is thus the first factor to be identified that controls the posteriorward migration of main body follicle cells at these stages. It has been suggested that this migratory process involves adhesion molecules, possibly integrins, located within the basal membrane of migrating main body follicle cells. In addition adhesion molecules may be involved in the establishment of a small region of strong adhesion between the posterior-most follicle cells and the posterior region of the oocyte. However, Shotgun (DE-Cadherin), which is necessary for the migration of both border cells and centripetally migrating follicle cells is not required for this migratory process. It could be postulated that the narrowing of main body follicle cells as they migrate posteriorly plays an active role in this process by creating a driving force for the migration of these main body follicle cells towards this posterior region of strong adhesion. In mosaic egg chambers with all follicle cells homozygous for the kay1 mutation, the migration of main body follicle cells initiates at the correct stage but stops rapidly (Dequier, 2001).

As a consequence, the morphology of the follicular epithelium at late stage 9 is indistinguishable from that of a wild-type egg chamber at early stage 9. This phenotype may reflect the requirement for kayak in the expression of the somatic components of adhesive complexes involved in the migration of main body follicle cells. Alternatively, this migratory defect may reveal its necessity for the reorganization of the shape of main body follicle cells. The data also suggest that the JNK encoding gene basket is also required for this morphogenetic movement. Interestingly, the migration of border cells, which occurs also during stage 9, neither requires kayak nor the JNK pathway. Therefore, these two morphogenetic movements, which are temporally co-ordinated, do not involve the same pathways (Dequier, 2001).

The results strongly suggest that the thin, 'paddleless' or shortened shapes of the respiratory appendages of eggs derived from kay2 or kay1351.3 mosaic egg chambers reflect the requirement for kayak in the reorganization and migration of the respiratory appendage secreting follicle cell (RASFC) prior to and during appendage secretion, and are not an indirect consequence of the partial 'dumpless' phenotype of these egg chambers. This leads to a proposal that the cells displaying the kayak columnar expression pattern together with those accumulating BR-C Z1 characterize a functional respiratory appendage secretion unit whose identity can be traced as early as stage 10B by the expression of these two genes. However, because of the present limitations of clonal analysis in the follicular epithelium, it was not possible to determine whether kayak is required for appendage morphogenesis in the entire unit or solely in the 'G-shaped' rows of columnar follicle cells (CFC) that first start expressing a high level of the gene at stage 10B (Dequier, 2001).

Close parallels can be drawn between the roles played by the kayak gene in migration of main body and respiratory appendage secretory follicle cells and in dorsal closure of the embryo. During dorsal closure, cells from the two lateral ectodermal sheets elongate, migrate dorsally and then fuse along the dorsal midline of the embryo. This process is controlled by the JNK signal transduction pathway and kayak. During embryonic stages 11 and 12, kayak is expressed at a high level in a single row of cells corresponding to the dorsal-most epidermal cells that form the leading edge of the lateral ectoderm, and at a lower level in cells located more ventrally. In kay1 mutant embryos, these groups of cells initiate a dorsalward stretching which they then fail to maintain and they subsequently resume an unelongated shape after premature termination of the migration process (Dequier, 2001).

In mosaic egg chambers with kay1 mutant follicular epithelium, the migration of main body follicle cells towards the posterior pole starts properly at early stage 9 but stops almost immediately. Moreover, during the reorganization of the RASFC territories that takes place from stage 10B/11 onwards, Kayak accumulates to a high level first in single rows of cells at the anterior and median edges of these territories, and expands to all RASFCs by stage 12/13. By analogy with the expression pattern of the kayak gene in leading-edge cells of the lateral epidermis during embryonic dorsal closure, it is proposed that expression of kayak in follicle cells located at the edges of the presumptive respiratory appendage territories controls the proper elongation and migration of these cells prior to and during secretion of the respiratory appendages (Dequier, 2001).

Mosaic egg chambers comprised of follicle cells homozygous for the hypomorphic kay2 or kay1351.3 mutations give rise to small and occasionally deflated eggs. In addition, the disappearance of nurse cell nuclei during the latest stages of oogenesis is delayed in these egg chambers. This is reminiscent of the phenotypes of mutations in 'dumpless' genes such as chickadee (chic), quail (qua) or singed (sn), which are required for the rapid transfer of the nurse cell cytoplasm into the oocyte during stages 10B and 11. It has been shown that 'dumpless' eggs display short and broad respiratory appendages. This is likely due to the inhibition of RASFC migration by residual nurse cell material. In contrast, the thin and 'paddleless' phenotype of respiratory appendages of eggs derived from kay2 or kay1351.3 follicular mosaic egg chambers is not the mere consequence of the dumping defect, because no correlation could be drawn between the strength of this defect, as determined by egg length or deflation, and the alterations in respiratory appendages (Dequier, 2001).

It has been shown that the dumping process is driven by nurse cell contractions induced by their subcortical actin filaments that form a dense network. This is in good agreement with the fact that the three 'dumpless' genes chic, qua and sn encode actin-binding proteins. Moreover, these three genes are required in germinal cells only, like the bullwinkle (bwk) gene, which is necessary for proper completion of the dumping process. In contrast, clonal analysis has demonstrated that the requirement for kayak in nurse cell dumping depends on its transcription in the follicular epithelium. This suggests that the transfer of the cytoplasm of nurse cells and the disappearance of their nuclei both involve one or several kayak-dependent somatic signals emanating from the nurse-cell-associated follicle cells. However, the precise role played by the kayak gene in this process remains unclear at present since neither the subcortical network of actin fibers nor the cytoplasmic actin bundles that anchor nurse cell nuclei at stage 10B display any detectable alterations in mosaic kay2 or kay1351.3 egg chambers. However, preliminary experiments show that overexpression of kayak in all CFC using the GAL4-UAS method induces large gaps in the subcortical actin network of the oocyte. This observation suggests that kayak expression in the nurse-cell-associated follicle cells controls subtle aspects of the organization of the actin cytoskeleton in underlying nurse cells (Dequier, 2001).

The data show that determination and localization of the kayak columnar expression pattern requires both Egfr pathway activation and a precise level of paracrine Dpp signaling. The alteration of kayak expression in mutants affecting different components of the Egfr pathway shows clearly that Grk-dependent Egfr activation and secondary Spitz- dependent Egfr amplification and refinement are necessary to determine the kayak columnar expression pattern. Nonetheless, colchicine feeding experiments demonstrate that Grk-dependent Egfr activation is not sufficient to induce kayak transcription in CFC, as is the case for the Egfr target gene kekkon. However, alteration of kayak expression resulting from either a reduction of the Dpp level or its overincrease throughout the columnar epithelium, provides direct evidence that this signaling process is also required for proper patterning of kayak expression (Dequier, 2001).

In C532-GAL4/UAS-dpp females grown at 18°C, a slight increase in the level of Dpp accumulation in CFC induces multiple patches of cells showing a pattern of BR-C Z1 and Kayak accumulation reminiscent of that of respiratory appendage secreting units in wild-type egg chambers. Strikingly, these patches are located at the lateral and posterior peripheries of the dorsal-anterior follicle cell territory, which is consistent with the hypothesis that the central-most CMFC are the localized source of a Dpp gradient. In addition, these results indicate that ectopically provided Dpp in FLP-out clones represses BR-C Z1 and Kayak accumulation in both dpp-expressing cells and those located within a radius of one to two cells, thus providing a direct evidence that Dpp acts in a paracrine manner to repress expression of the BR-C Z1 and kayak genes. The observation that the Dpp-dependent repression of BR-C Z1 is restricted to DAFC suggests that it is mediated by a component of the Dpp-signaling pathway, i.e., either a Dpp receptor or a Smad cofactor expressed differentially in DAFC. It has been shown that among the known Dpp receptors, Saxophone and Punt are ubiquitously expressed in CFC whereas Thick-vein is expressed in a row of anterior follicle cells. In a preliminary investigation of the pattern of expression of the Drosophila Smad genes in follicle cells, it has been observed that medea is expressed from stage 11 onwards in two patches of CFC that may correspond to RASFC. However, whereas the medea gene is required for kayak transcription in the main body follicle cells during stage 9, it appears to be fully dispensable for the kayak columnar expression pattern. Work is currently in progress to investigate the pathway involved in the restriction of the Dpp-dependent repression of BR-C Z1 to DAFC (Dequier, 2001).

JNK signaling pathway is required for efficient wound healing in Drosophila

Efficient wound healing including clotting and subsequent reepithelization is essential for animals ranging from insects to mammals to recover from epithelial injury. It is likely that genes involved in wound healing are conserved through the phylogeny and therefore, Drosophila may be a useful in vivo model system to identify genes necessary during this process. Furthermore, epithelial movement during specific developmental processes, such as dorsal closure (DC), resembles that seen in mammalian wound healing. Since puckered (puc) gene is a target of the JUN N-terminal kinase signaling pathway during DC, puc gene expression was investigated during wound healing in Drosophila. puc expression is induced at the edge of the wound in epithelial cells and Jun kinase is phosphorylated in wounded epidermal tissues, suggesting that the JUN N-terminal kinase signaling pathway is activated by a signal produced by an epidermal wound. In the absence of the Drosophila c-Fos homologue, puc gene expression is no longer induced. Finally, impaired epithelial repair in JUN N-terminal kinase deficient flies demonstrates that the JUN N-terminal kinase signaling is required to initiate the cell shape change at the onset of the epithelial wound healing. It is concluded that the embryonic JUN N-terminal kinase gene cassette is induced at the edge of the wound. In addition, Drosophila appears as a good in vivo model to study morphogenetic processes requiring epithelial regeneration, such as wound healing in vertebrates (Ramet, 2002).

In most cases, flies were anesthetized and then mechanically wounded with iridectomy scissors to cut adult abdomen vertically between the third and the seventh tergites. Semi-thin sections were used to examine the histology of wound healing at the cellular level. The first response to epithelial wound is the formation of a clot at the initial site. Subsequently, the clot becomes melanized making the location of the wound clearly visible. The clot appears to consist of an accumulation of melanin and by hemocytes that aggregated at the site of injury. Hemocytes may be involved also in the clearance of cellular debris and invading microbes (Ramet, 2002).

During the first 2 h after wounding no sign of epithelial cell movement can be seen. In most cases, the edges of the cut epidermis are found far away from the broken cuticle. As for the wounded embryonic epidermis, the adult epidermal layer may be submitted to an intrinsic isotropic epidermal tension that retracts it upon any break injury. By 4 h, the epithelial cells of the edge of the wound seem to shed from the disrupted cuticle. These cells appear larger than the epithelial cells lining the normal cuticle, and exhibit cytoplasmic protrusions. By 12 h, the protrusive cytoplasmic extensions extend from the cells of the edge of the wound and 'migrate' toward each other under the melanin clot, Subsequently, they cause the epidermis to form a suture. These cytoplasmic extensions suggest that adult epidermis is healed by the activity of dynamic lamellipodia or filipodia. Correspondingly, cytoskeleton reorganization has been previously described in wound healing model of cultured Madin-Darby canine kidney cell (MDCK) and during Drosophila DC. At this point, the epithelial cells are still enlarged but start to return to their initial shape. The suture of the epithelium is normally achieved within 18 h after injury. By this time, the wounded epithelium has healed, and cells have returned to their original shape (Ramet, 2002).

To ascertain the importance of melanin production in wound healing, the survival rate of Black cells (Bc) homozygous flies was measured after a transversal wound of the adult abdomen cuticle. Bc/Bc flies lack hemolymphatic phenoloxydase activity and therefore, do not produce melanin. By 24 h after wounding, wild type flies and Bc/+ heterozygous flies, present a dominant melanized crystal cell phenotype, but have wild-type phenoloxidase activity, both of which have about 20% mortality, suggesting an efficient wound repair. In contrast, the vast majority (91%) of wounded Bc/Bc mutants died. 50% mortality of Bc flies was already seen by 6 h, suggesting that phenoloxydase activity is essential early in the wound healing process. Similarly, lozenge (lz) mutants, which lack crystal cells and hence present a weak hemolymphatic phenoloxydase activity, exhibit a poor ability to recover from the injury (Ramet, 2002).

The wound clots differently in Bc flies compared to wild type. In wild type flies, a melanin deposit is observed as early as 10 min after wounding and it is still visible 6 h after injury. In contrast, there is no evidence of melanin formation in the wounded integument of Bc flies, indicating that the latter is of hemolymphatic origin. Furthermore, the two edges of the wound are found apart in Bc flies. This failure to keep the edge of the wound in close proximity leads to death due to bleeding. These results underlie the essential role of the phenoloxydase activity, or an associated phenomenon, when it comes to efficient clot formation and the prevention of bleeding (Ramet, 2002).

To ascertain that the Drosophila JNK pathway is activated in wounded epidermis, DJun N-terminal kinase activity was assayed using anti-phospho-JNK antibodies. Protein extracts from adult abdominal integument from control and wounded flies were assayed for anti-phospho-JNK reactivity. Phospho-JNK can be detected only in the protein extracts from the wounded epidermis, whereas the unphosphorylated form of DJNK is present in all of the samples. This indicates that DJNK is phosphorylated in response to wounding in epidermal cells, and therefore, suggests that the JNK signaling pathway is activated (Ramet, 2002).

In contrast to embryonic DC where only the most dorsal cell row at the leading edge is expressing puc gene, several rows of adult epidermal cells show a strong ß-galactosidase activity during wound healing. This result is consistent with high DJNK activity in the vicinity of the wound. Indeed, the extent of the area expressing puc-lacZ clearly depends on the size of the wound (up to 8 cell rows). Furthermore, puc gene expression showed a decreasing gradient from the edge of the wound towards healthy epithelium. This suggests that a newly formed signal emerges from the wound and diffuses through the epidermal layer (Ramet, 2002).

During embryonic DC, the DJNK pathway activates puc expression in the LE and as a negative feedback loop, puc itself down-regulates the DJNK. To further test if the DJNK pathway also mediates puc induction during wound healing, puc expression pattern in adult epidermis was analyzed in fly mutants of this pathway. In hep, a hypomorphic allele of hemipterous, (encoding the MAPKK), puc-lacZ expression is almost completely normal. This result is not totally unexpected since hep flies present no epidermal defects. Stronger hep allele mutations are lethal and could not be studied. However, a heteroallelic combination of kay mutations leads to viable flies. Since the incidence of dorsal thoracic cleft phenotype of this mutant is higher than that in hep, it is more likely to affect the regulation of puc expression. In kay1/kay2 animals, puc gene induction is drastically reduced at the site of injury compared to wild-type. This result demonstrates that puc regulation is dependent on the transcriptional activator DFos during wound healing. Interestingly, still 18 h after wounding, the mutant cells are separated from the edge of the wound comparably to that observed with wild type at 3 h after wounding. This suggests that the epidermal sheet has been unable to spread under the wound and that the process is blocked at the initiation stage. Since DFos, together with DJun, is the target of the DJNK pathway, and forms the transcription factor AP-1, it is likely that the DJNK pathway is switched on by an integument injury (Ramet, 2002).

To find out if DFos mutation has a cell autonomous effect, the UAS/GAL4 system was used to express a dominant negative form of DFos in the pannier (pnr) expression domain. In the pnr-Gal4 line, Gal4 protein is expressed in a large dorsal band of adult epidermis. A continuous wound was done to overlap this dorsal epidermal expression domain and the dorso-lateral and ventral epidermal domain. puc-lacZ expression was then assayed 12 h after wounding. In control flies, puc is expressed at the wounded epidermis independent of the location. When DFosbZip dominant negative form of DFos is expressed in the dorsal band, X-Gal staining shows a clear, albeit not total, reduction of puc expression at the expected places. In contrast, puc expression is induced normally outside of the pnr expression domain. This demonstrates that the puc gene induction is under the control of the DFos transcriptional factor in a cell-autonomous manner similar to that observed during dorsal and thorax closure (Ramet, 2002).

To ascertain the importance of the DJNK pathway in wound healing, the phenotype of kay mutant was investigated during the course of wound healing. As expected, the wounded epidermis from kay deficient flies fails to recover. The epithelial cells at the edge of the wound also fail to undergo any evident cell shape change or show any cytoplasmic protrusive extensions. Even at 18 h after injury, the wound is not repaired. Therefore, the transcriptional activator DFos appears necessary for a normal epithelial repair in adult Drosophila. Interestingly, over a period of 6 days, wounded mutant flies did not suffer any higher mortality compared to wounded wild type flies, suggesting that epithelial repair is not crucial for early survival (Ramet, 2002).

Hole-in-one mutant phenotypes link EGFR/ERK signaling to epithelial tissue repair in Drosophila

Epithelia act as physical barriers protecting living organisms and their organs from the surrounding environment. Simple epithelial tissues have the capacity to efficiently repair wounds through a resealing mechanism. The known molecular mechanisms underlying this process appear to be conserved in both vertebrates and invertebrates, namely the involvement of the transcription factors Grainy head (Grh) and Fos. In Drosophila, Grh and Fos lead to the activation of wound response genes required for epithelial repair. ERK is upstream of this pathway and known to be one of the first kinases to be activated upon wounding. However, it is still unclear how ERK activation contributes to a proper wound response and which molecular mechanisms regulate its activation. In a previous screen, mutants were isolated with defects in wound healing. This study describes the role of one of these genes, hole-in-one (holn1), in the wound healing process. Holn1 is a GYF domain containing protein that is required for the activation of several Grh and Fos regulated wound response genes at the wound site. Evidence is provided suggesting that Holn1 may be involved in the Ras/ERK signaling pathway, by acting downstream of ERK. Finally, it was shown that wound healing requires the function of EGFR and ERK signaling. Based on these data, it is concluded that holn1 is a novel gene required for a proper wound healing response. A model is proposed whereby Holn1 acts downstream of EGFR and ERK signaling in the Grh/Fos mediated wound closure pathway (Geiger, 2011).

Holn1 is not required for the initial rapid response to wound infliction, i.e. the formation of the actomyosin cable within minutes of wounding and the phosphorylation of ERK, which is also detectable soon after wounding. This observation is consistent with Holn1 playing an indirect role in the mechanics of wound closure by regulating the mRNA levels of genes required for this process, such as those involved in rapid and productive cable contraction. Interestingly, the actin cable was present in all the wound closure mutants isolated in the previous screen, suggesting that regulatory events downstream of cable formation dominate the wound closure process. In any case, it is clear that Holn1 is required to perform some additional function needed to sustain the closure process, as holn1 mutants take on average 1.5 times longer to close a wound compared to wild type embryos. A similar delay in wound closure was previously reported for rho1 GTPase mutants, which do not form an actin cable, but can still close small wounds, albeit 2 times slower than wild type embryos]. Aside from its possible role in the epithelial hole closure process, Holn1 could also be involved in cuticle repair. Grh and ERK activity are required for the re-establishment of the epithelial permeability barrier after injury. Thus, Holn1 might be involved in this process by regulating the ERK/Grh pathway (Geiger, 2011).

In the future, just as the holn1 mutation uncovered a connection with the EGF/Ras/ERK signaling pathway and wound healing, microarray analysis of wounded holn1 embryos would identify genes that are likely activated downstream of this wound closure pathway. Performing the same experiment using an alternative splicing array as in would further reveal if Holn1 plays a role in wound dependent splicing events (Geiger, 2011).

An essential function of AP-1 heterodimers in Drosophila development

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).

Highwire restrains synaptic growth by attenuating a MAP kinase signal

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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).

Normal dendrite growth in Drosophila motor neurons requires the AP-1 transcription factor

During learning and memory formation, information flow through networks is regulated significantly through structural alterations in neurons. Dendrites, sites of signal integration, are key targets of activity-mediated modifications. Although local mechanisms of dendritic growth ensure synapse-specific changes, global mechanisms linking neural activity to nuclear gene expression may have profound influences on neural function. Fos, being an immediate-early gene, is ideally suited to be an initial transducer of neural activity, but a precise role for the AP-1 transcription factor in dendrite growth remains to be elucidated. This study measured changes in the dendritic fields of identified Drosophila motor neurons in vivo and in primary culture to investigate the role of the immediate-early transcription factor AP-1 in regulating endogenous and activity-induced dendrite growth. The data indicate that (1) increased neural excitability or depolarization stimulates dendrite growth, (2) AP-1 (a Fos, Jun heterodimer) is required for normal motor neuron dendritic growth during development and in response to activity induction, and (3) neuronal Fos protein levels are rapidly but transiently induced in motor neurons following neural activity. Taken together, these results show that AP-1 mediated transcription is important for dendrite growth, and that neural activity influences global dendritic growth through a gene-expression dependent mechanism gated by AP-1 (Hartwig, 2009).

Like CREB, AP-1 (Fos and Jun dimer) is likely to be an important activity-dependent regulator of dendritic plasticity. Fos is upregulated rapidly in neuronal populations following activity and can generate a rapid genomic response to incoming stimuli since it is an immediate-early gene not requiring protein synthesis for its own induction. At the Drosophila NMJ AP-1 controls both structural and functional aspects of long-term plasticity (Hartwig, 2009).

To test if AP-1 plays a role in activity-dependent dendritic plasticity, experimental systems were established to study dendrites of the RP2 motor neuron in vivo in the larval CNS, and this was complemented with an in vitro culture system using a strategy to enrich for larval motor neurons. This dual analysis utilizes technical strengths of both systems and permits cross-validation of results. Results reported in this study support three main conclusions: (1) motor neuron dendrites in Drosophila show activity, or depolarization, dependent plasticity, (2) normal dendrite growth requires AP-1, and (3) activity or depolarization driven dendritic growth is gated by AP-1 (Hartwig, 2009).

It was important to initially establish that motor neuron dendrites in Drosophila display robust changes in response to neural activity. Both pharmacological and genetic manipulations were used to alter neural activity in vivo and in vitro. Maintaining cultured motor neurons in high K+ medium enhanced the growth of neurites that have the characteristics of dendrites. Expression of dominant-negative subunits of the potassium channels Eag and Sh, increased excitability and caused increased dendrite growth both in vivo and in vitro. These experiments suggest that a conserved mechanism of plasticity operates in these motor neurons to regulate dendritic growth. Additionally, by developing the first assay for activity-stimulated dendritic growth in Drosophila, a wide range of experiments were enabled to further dissect underlying signaling and cell biological pathways (Hartwig, 2009).

It is worth noting that the cell culture approach allowed the comparison of acute depolarization (high potassium) with the more chronic alteration of excitability caused by the expression of eag and shaker dominant-negative transgenes. Despite the interesting possibility of compensatory changes following long-term transgenic manipulations, the functional properties of larval motor neurons in vivo were stably altered by reducing potassium channel function. By contrast, the effects of elevated potassium reflect an acute manipulation after cells are placed in culture. In spite of these differences the effects of expression of a recombinant including both UAS-Sh(DN) and UAS-eag(DN) termed 'Electrical Knock In (EKI)' on cell growth in vitro were similar to those induced by elevated potassium. The lack of an effect on outgrowth by UAS-Sh(DN) and UAS-Sh[act] (EKO) expression may reflect a cellular compensatory mechanism. Although effects on cell physiology were detected, the difference in excitability measured in vivo was not significant. Two alternatives are that there is a basal level of normal growth that is not activity-sensitive, or that reduced activity had an influence on growth or branching that the analysis did not detect (Hartwig, 2009).

It is suggested that the depolarization by high potassium or the increased excitability caused by EKI expression would lead to an increase in calcium influx through voltage-dependent channels. The voltage-dependent properties of Eag and Shaker potassium channels suggest that EKI expression would not necessarily be expected to alter the resting membrane potential. Even without altering the resting membrane potential, however, the reduction in voltage-dependent potassium currents might allow increased calcium influx in response to spontaneous depolarization or calcium waves. This would be augmented, in vivo, by the higher action potential frequency evoked by depolarization of motor neurons in EKI larvae as compared to wild type. It is also possible that the expression of eag-DN alters a modulatory function of the potassium channel subunit. Consistent with this hypothesis, it was found that inhibiting PLTX-sensitive calcium channels prevents EKI mediated neurite outgrowth. A similar dependence of neuronal growth on calcium channels has been demonstrated convincingly in the Drosophila giant neuron culture system. Ultimately, it will be important to establish the specific parameters of activity and calcium flux that are essential for modulating the intracellular signals that mediate growth plasticity (Hartwig, 2009).

It has been shown previously that AP-1 regulates synaptic plasticity at Drosophila motor terminals. The present results uncover a role for AP-1 in regulating dendritic growth. Reduction of AP-1 activity decreases, while enhancement of AP-1 increases, dendritic outgrowth in vivo and in vitro. These conclusions are further strengthened by the fact that known and predicted loss-of-function alleles of kayak (DFos) also reduce dendrite volume significantly, when present in heterozygous combinations with wild-type alleles. It is to be noted that these mutations in Fos are homozygous lethal at early developmental stages, and transgenic strategies for tissue specific genetic manipulations are especially useful in these contexts. Additionally, multiple Fos isoforms have been reported, and using a dominant negative construct offers a good way to inhibit these multiple Fos proteins. In conclusion, the observations that (1) Fos is normally detectable in motor neuron nuclei, (2) Fbz, Jbz and Fos-RNAi inhibit and AP-1 increases dendrite growth, (3) loss-of-function alleles of kayak decrease dendrite volume, and (4) a variety of controls validate the genetic perturbations used in this study, strongly suggest that AP-1 functions physiologically to control dendrite growth (Hartwig, 2009).

To test if neural activity driven growth requires AP-1 dependent transcription, AP-1 function was inhibitied in a background of elevated neural activity. The results indicate that neural activity induced dendrite growth is completely abolished in vivo by the coexpression of Fbz. Further, in vitro experiments with High K+ induced dendrite growth show that expression of either the domainant negative bZip domain of either dFos or dJun (Fbz or Jbz) substantially reduces the extent of depolarization induced growth. The results also indicate that the extent of overgrowth seen through AP-1 induction cannot be increased any further by High K+ induced depolarization. Furthermore, calcium channel inhibition by PLTX toxin does not preclude AP-1 driven neurite growth, suggesting that AP-1 functions downstream of neural activity and calcium entry to enhance neurite growth. These results are consistent with the idea that AP-1 is a major contributor to activity-induced plasticity of dendrites (Hartwig, 2009).

Finally, to ascertain the mechanism by which neural activity might influence AP-1 function, expression of Fos protein was tested following acute induction of activity in cultured motor neurons. Fos protein levels are increased by ~35% as compared to uninduced controls. This induction is rapid, occurring within 4 h, but transient, since Fos levels return to baseline during 20 h of stimulation. Although modest, this change in cellular Fos levels is consistent with previous observations and suggests, though it does not prove, a model where neural activity recruits AP-1 possibly through synthesis of new Fos protein, to promote dendrite growth. Hence, the results confirm that AP-1, positively regulates plasticity in postsynaptic compartments, as has been demonstrated for presynaptic terminals, and establishes AP-1 as a key component in activity-dependent neuronal plasticity (Hartwig, 2009).

A key goal for future studies must be to determine how neural activity is translated into transcription factor activity. Calcium dependent signaling is arguably the most important pathway for the activation of both CREB and Mef2A, two transcription factors that influence plasticity in opposite ways, as well as AP-1. A second question is that of interaction between transcription factors. Transcriptional regulation of Fos by CREB (and vice versa) has been described in some detail, but the temporal sequence in which these proteins function or their relative importance in plasticity have not been assayed rigorously in the same preparation. Finally, further experiments to identify the downstream targets of these transcription factors, will enable the description of a composite transcription factor network that mediates protein synthesis during long-term neural plasticity (Hartwig, 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).

A new genetic model of activity-induced Ras signaling dependent pre-synaptic plasticity in Drosophila

Techniques to induce activity-dependent neuronal plasticity in vivo allow the underlying signaling pathways to be studied in their biological context. This study demonstrates activity-induced plasticity at neuromuscular synapses of Drosophila double mutant for comatose (an NSF mutant) and Kum (Calcium ATPase at 60A: a SERCA mutant), and presents an analysis of the underlying signaling pathways. comt; Kum (CK) double mutants exhibit increased locomotor activity under normal culture conditions, concomitant with a larger neuromuscular junction synapse and stably elevated evoked transmitter release. The observed enhancements of synaptic size and transmitter release in CK mutants are completely abrogated by: a) reduced activity of motor neurons; b) attenuation of the Ras/ERK signaling cascade; or c) inhibition of the transcription factors Fos and CREB. All of which restrict synaptic properties to near wild type levels. Together, these results document neural activity-dependent plasticity of motor synapses in CK animals that requires Ras/ERK signaling and normal transcriptional activity of Fos and CREB. Further, novel in vivo reporters of neuronal Ras activation and Fos transcription also confirm increased signaling through a Ras/AP-1 pathway in motor neurons of CK animals, consistent with results from the genetic experiments. Thus, this study: a) provides a robust system in which to study activity-induced synaptic plasticity in vivo; b) establishes a causal link between neural activity, Ras signaling, transcriptional regulation and pre-synaptic plasticity in glutamatergic motor neurons of Drosophila larvae; and c) presents novel, genetically encoded reporters for Ras and AP-1 dependent signaling pathways in Drosophila (Freeman, 2010).

This study describes a new model for activity-dependent pre-synaptic plasticity in Drosophila. In the double mutant combination of comt and Kum, sustained elevation of neural activity (potentially including seizure-like motor neuron firing under normal rearing conditions) results in the expansion of motor synapses with a concomitant increase in transmitter release. These synaptic changes are mediated by the Ras/ERK signaling cascade and the activity of at least two key transcription factors, CREB and Fos. In vivo reporter assays also directly demonstrate Ras activation and enhanced transcription of Fos in the nervous system. CK is the only genetic model of synaptic plasticity in Drosophila in which pre-synaptic plasticity has been correlated with the Ras/ERK signaling cascade. This result is especially relevant given the wide conservation of the Ras/ERK signaling cascade in plasticity and recent demonstrations of the involvement of this signaling cascade in learning behavior in flies (Godenschwege, 2004; Moressis, 2009). Significant insights into Ras mediated regulation of both synapse growth and transmitter release are also presented (Freeman, 2010).

Non-invasive methods to manipulate neural activity in select neurons continue to be an important experimental target in plasticity research. In Drosophila, combinations of the eag and Shaker potassium channel mutants have long been used to chronically alter neural activity and study downstream cellular events. In recent years, transgenic expression of modified Shaker channels has also been generated and used to alter excitability in both neurons and muscles. However, the CK model of activity-dependent plasticity was developed since in synaptic changes in CK were consistently more robust than eag Sh and core plasticity-related signaling components were activated in a predictable manner in CK mutants. Another advantage with CK is the option of acutely inducing seizures as has been used to identify activity-regulated genes. CK thus combines advantages of both eag Sh and seizure mutants, and as is shown in this study, leads to an activity-dependent increase in synaptic size and transmitter release. It is believed that this model will prove highly beneficial to the large community of researchers who investigate synaptic plasticity in Drosophila. The utility of more recent techniques (such as the ChannelRhodopsin or the newly reported temperature sensitive TrpA1 channel transgenes) to induce neural activity-dependent synaptic plasticity at Drosophila motor synapses has not been tested yet and it will be interesting to see if these afford greater experimental flexibility in the future (Freeman, 2010).

Signal transduction through the Ras cascade has been shown to affect both dendritic and pre-synaptic plasticity in invertebrate and vertebrate model systems. In mammalian neurons, Ras signaling has been linked to hippocampal slice LTP, changes in dendritic spine architecture and plasticity of cultured neurons. In this context, Ras signaling has been shown to impinge on downstream MAP kinase signaling, thus implicating a canonical signaling module already established as a mediator of long-term plasticity in vertebrates. In Drosophila, expression of a mutant constitutively active Ras that is predicted to selectively target ERK leads to synapse expansion and increased localized phosphorylation of ERK at pre-synaptic terminals. In light of these observations, tests were performed to see if Ras signaling os necessary and sufficient for synaptic plasticity in CK. The results suggest that synaptic changes in CK are driven by stimulated Ras/ERK signaling in Drosophila motor neurons, and these can be replicated by directly enhancing Ras signaling in these cells. Furthermore Ras activation was found to be sufficient to cause stable elevation in pre-synaptic transmitter release. Finally, evidence is provided to show that synaptic effects of Ras activation require the function of both Fos and CREB in motor neurons. The consistency of signaling events in CK with those observed in mammalian preparations makes this a more useful and generally applicable genetic model of synaptic plasticity (Freeman, 2010).

In vivo reporters of neural activity have been difficult to design but offer better experimental resolution and flexibility over standard immuno-histochemical or RNA in situ methods to detect changes in gene expression in the brain. Thus, a good reporter permits increased temporal and spatial resolution, the option of live imaging (for fluorescent reporters) and in the case of transcriptional reporters, better understanding of cis-regulatory elements that control activity-dependent gene expression. This paper describes two genetically encoded reporters with utility clearly beyond the current study; a Raf based reporter to detect Ras activation in neurons and an enhancer based reporter to detect transcription of Fos (Freeman, 2010).

The Ras binding domain of Raf has been used previously to detect Ras expression in yeast, mammalian cell lines, and recently in hippocampal neuron dendrites. This study used a similar strategy to model the reporter using the conserved Ras binding domain and the cysteine-rich domain (RBD + CRD) from Drosophila Raf, under the reasonable assumption that this would provide sensitive reporter activity in neurons. This is the first time that a Ras reporter has been utilized in an intact metazoan organism to measure changes in endogenous Ras activity. In addition to confirming Ras activation in CK brains, it is expected that this reporter will find widespread use in tracing Ras activation in multiple tissues through development and in response to signaling changes in the entire organism. Since the reporter is based on the GAL4-UAS system, it can be expressed in tissues of choice, limiting reporter activity to regions of interest. Indeed, the experiments with the eye-antennal imaginal disc illustrate the utility of this reporter in identifying regions of activated Ras signaling during eye development (Freeman, 2010).

The Fos transcriptional reporter is one of the very few activity-regulated reporters in existence in Drosophila and it should find broad acceptance as a tool to map neural circuits in the fly brain that show activity-dependent plasticity. The reporter believed to be reasonably accurate since it is expressed in expected tissue domains (embryonic leading edge cells, for instance), and also co-localizes extensively with anti-Fos staining in the larval brain. There are several recognizable transcription factor binding motifs that can be detected in this 5 kb region of DNA (including binding sites for CREB, Fos, Mef2 and c/EBP). Which of these transcription factors regulate activity-dependent Fos expression from this enhancer is currently unknown. However, future experiments that dissect functional elements in this large enhancer region are expected to refine and identify these regulatory elements. Such studies are likely to lead the way in the development of a new generation of neural activity reporters in the brain (Freeman, 2010).

Interplay among Drosophila transcription factors Ets21c, Fos and Ftz-F1 drives JNK-mediated tumor malignancy

This study defines TF network that triggers an abnormal gene expression program promoting malignancy of clonal tumors, generated in Drosophila imaginal disc epithelium by gain of oncogenic Ras (RasV12) and loss of the tumor suppressor Scribble (scrib1). Malignant transformation of the rasV12scrib1 tumors requires TFs of distinct families, namely the bZIP protein Fos, the ETS-domain factor Ets21c and the nuclear receptor Ftz-F1, all acting downstream of Jun-N-terminal kinase (JNK). Depleting any of the three TFs improves viability of tumor-bearing larvae, and this positive effect can be enhanced further by their combined removal. Although both Fos and Ftz-F1 synergistically contribute to rasV12scrib1 tumor invasiveness, only Fos is required for JNK-induced differentiation defects and Matrix metalloprotease (MMP1) upregulation. In contrast, the Fos-dimerizing partner Jun is dispensable for JNK to exert its effects in rasV12scrib1 tumors. Interestingly, Ets21c and Ftz-F1 are transcriptionally induced in these tumors in a JNK- and Fos-dependent manner, thereby demonstrating a hierarchy within the tripartite TF network, with Fos acting as the most upstream JNK effector. Of the three TFs, only Ets21c can efficiently substitute for loss of polarity and cooperate with Ras(V12) in inducing malignant clones that, like rasV12scrib1 tumors, invade other tissues and overexpress MMP1 and the Drosophila insulin-like peptide 8 (Dilp8). While rasV12ets21c tumors require JNK for invasiveness, the JNK activity is dispensable for their growth. In conclusion, this study delineates both unique and overlapping functions of distinct TFs that cooperatively promote aberrant expression of target genes, leading to malignant tumor phenotypes. (Kulshammer, 2015).

Genome-wide transcriptome profiling in the Drosophila epithelial tumor model has generated a comprehensive view of gene expression changes induced by defined oncogenic lesions that cause tumors of an increasing degree of malignancy. These data allowed discovery of how a network of collaborating transcription factors confers malignancy to RasV12scrib1 tumors (Kulshammer, 2015).

This study revealed that the response of transformed RasV12scrib1 epithelial cells is more complex in comparison to those with activated RasV12 alone with respect to both the scope and the magnitude of expression of deregulated genes. Aberrant expression of more than half of the genes in RasV12scrib1 tumors requires JNK activity, highlighting the significance of JNK signaling in malignancy. Importantly, the tumor-associated, JNK-dependent transcripts cluster with biological functions and processes that tightly match the phenotypes of previously described tumor stages. Furthermore, the RasV12scrib1 transcriptome showed significant overlap (27% upregulated and 15% downregulated genes) with microarray data derived from mosaic EAD in which tumors were induced by overexpressing the BTB-zinc finger TF Abrupt (Ab) in scrib1 mutant clones as well as with a transcriptome of scrib1 mutant wing discs. It is proposed that 429 misregulated transcripts (e.g. cher, dilp8, ets21c, ftz-f1, mmp1, upd), shared among all the three data sets irrespective of epithelial type (EAD versus wing disc) or cooperating lesion (RasV12 or Ab), represent a 'polarity response transcriptional signature' that characterizes the response of epithelia to tumorigenic polarity loss. Genome-wide profiling and comparative transcriptome analyses thus provide a foundation to identify novel candidates that drive and/or contribute to tumor development and malignancy while unraveling their connection to loss of polarity and JNK signaling (Kulshammer, 2015).

In agreement with a notion of combinatorial control of gene expression by an interplay among multiple TFs, this study identified overrepresentation of cis-acting DNA elements for STAT, GATA, bHLH, ETS, BTB, bZIP factors and NRs in genes deregulated in RasV12scrib1 mosaic EAD, implying that transcriptome anomalies result from a cross-talk among TFs of different families. Many of the aberrantly expressed genes contained binding motifs for AP-1, Ets21c and Ftz-F1, indicating that these three TFs may regulate a common set of targets and thus cooperatively promote tumorigenesis. This is consistent with the occurrence of composite AP-1-NRRE (nuclear receptor response elements), ETS-NRRE and ETS-AP-1 DNA elements in the regulatory regions of numerous human cancer-related genes, such as genes for cytokines, MMPs (e.g., stromelysin, collagenase) and MMP inhibitors (e.g., TIMP) (Kulshammer, 2015).

Interestingly, Drosophila ets21c and ftz-f1 gene loci themselves contain AP-1 motifs and qualify as polarity response transcriptional signature transcripts. Indeed, this study has detected JNK- and Fos-dependent upregulation of ets21c and ftz-f1 mRNAs in RasV12scrib1 tumors. While JNK-mediated control of ftz-f1 transcription has not been reported previously, upregulation of ets21c in the current tumor model is consistent with JNK requirement for infection-induced expression of ets21c mRNA in Drosophila S2 cells and in vivo. Based on these data, it is proposed that Ftz-F1 and Ets21c are JNK-Fos-inducible TFs that together with AP-1 underlie combinatorial transcriptional regulation and orchestrate responses to cooperating oncogenes. Such an interplay between AP-1 and Ets21c is further supported by a recent discovery of physical interactions between Drosophila Ets21c and the AP-1 components Jun and Fos (Rhee, 2014). Whether regulatory interactions among AP-1, Ets21c and Ftz-F1 require their direct physical contact and/or the presence of composite DNA binding motifs of a particular arrangement to control the tumor-specific transcriptional program remains to be determined (Kulshammer, 2015).

Importantly, some of the corresponding DNA elements, namely AP-1 and STAT binding sites, have recently been found to be enriched in regions of chromatin that become increasingly accessible in RasV12scrib1 mosaic EAD relative to control. This demonstrates that comparative transcriptomics and open chromatin profiling using ATAC-seq and FAIRE-seq are suitable complementary approaches for mining the key regulatory TFs responsible for controlling complex in vivo processes, such as tumorigenesis (Kulshammer, 2015).

The prototypical form of AP-1 is a dimer comprising Jun and Fos proteins. In mammals, the Jun proteins occur as homo- or heterodimers, whereas the Fos proteins must interact with Jun in order to bind the AP-1 sites. In contrast to its mammalian orthologs, the Drosophila Fos protein has been shown to form a homodimer capable of binding to and activating transcription from an AP-1 element, at least in vitro (Kulshammer, 2015).

The role of individual AP-1 proteins in neoplastic transformation and their involvement in pathogenesis of human tumors remain somewhat elusive. While c-Jun, c-Fos and FosB efficiently transform mammalian cells in vitro, only c-Fos overexpression causes osteosarcoma formation, whereas c-Jun is required for development of chemically induced skin and liver tumors in mice. In contrast, JunB acts as a context-dependent tumor suppressor. Thus, cellular and genetic context as well as AP-1 dimer composition play essential roles in dictating the final outcome of AP-1 activity in tumors (Kulshammer, 2015).

This study shows that, similar to blocking JNK with its dominant-negative form, Bsk, removal of Fos inhibits ets21c and ftz-f1 upregulation, suppresses invasiveness, improves epithelial organization and differentiation within RasV12scrib1 tumors and allows larvae to pupate. Strikingly, depletion of Jun had no such tumor-suppressing effects. It is therefore concluded that in the malignant RasV12scrib1 tumors, Fos acts independently of Jun, either as a homodimer or in complex with another, yet unknown partner. A Jun-independent role for Fos is further supported by additional genetic evidence. Fos, but not Jun, is involved in patterning of the Drosophila endoderm Ÿand is required for expression of specific targets, e.g., misshapen (msn) and dopa decarboxylase (ddc), during wound healing. Future studies should establish whether the JNK-responsive genes containing AP-1 motifs, identified in this study, are indeed regulated by Fos without its 'canonical' partner (Kulshammer, 2015).

The current data identify Fos as a key mediator of JNK-induced MMP1 expression and differentiation defects in RasV12scrib1 tumors. Only Fos inhibition caused clear suppression of MMP1 levels and restoration of neurogenesis within clonal EAD tissue, thus mimicking effects of JNK inhibition. Improved differentiation and reduced invasiveness are, however, not sufficient for survival of animals to adulthood, because interfering with Fos function in RasV12scrib1 clones always resulted in pupal lethality (Kulshammer, 2015).

The systems approach of this paper, followed by genetic experiments, identified Ets21c and Ftz-F1 as being essential for RasV12scrib1-driven tumorigenesis. It was further shown that mutual cooperation of both of these TFs with Fos is required to unleash the full malignancy of RasV12scrib1 tumors (Kulshammer, 2015).

TFs of the ETS-domain family are key regulators of development and homeostasis in all metazoans, whereas their aberrant activity has been linked with cancer. ets21c encodes the single ortholog of human Friend leukemia insertion1 (FLI1) and ETS-related gene (ERG) that are commonly overexpressed or translocated in various tumor types. While FLI1 is considered pivotal to development of Ewing's sarcoma, ERG has been linked to leukemia and prostate cancer. As for Ftz-F1 orthologs, the human liver receptor homolog-1 (LRH-1) has been associated with colonic, gastric, breast and pancreatic cancer, whereas steroidogenic factor 1 (SF-1) has been implicated in prostate and testicular cancers and in adrenocortical carcinoma. However, the molecular mechanisms underlying oncogenic activities of either the ERG/FLI1 or the SF-1/LRH-1 proteins are not well understood (Kulshammer, 2015).

This study shows that removal of Ftz-F1 markedly suppressed invasiveness of RasV12scrib1 tumors, restoring the ability of tumor-bearing larvae to pupate. Additionally, and in contrast to Fos, Ftz-F1 inhibition also partly reduced tumor growth in the third-instar EAD and allowed emergence of adults with enlarged, rough eyes composed predominantly of non-clonal tissue. The reduced clonal growth coincided with downregulation of the well-established Yki target, expanded, implicating Ftz-F1 as a potential novel growth regulator acting on the Hpo/Yki pathway. It is further speculated that reduced viability of RasV12scrib1ftz-f1RNAi clones and induction of non-autonomous compensatory proliferation by apoptotic cells during the pupal stage could explain the enlargement of the adult eyes. The precise mechanism underlying compromised growth and invasiveness of RasV12scrib1ftz-f1RNAi tumors and improved survival of the host remains to be identified (Kulshammer, 2015).

In contrast, effects of Ets21cLONG knockdown in RasV12scrib1 tumors appeared moderate relative to the clear improvement conferred by either Fos or Ftz-F1 elimination. ets21cLONG RNAi neither reduced tumor mass nor suppressed invasiveness, and pupation was rescued only partly. However, unlike ftz-f1RNAi, ets21cLONG RNAi significantly reduced expression of dilp8 mRNA. Based on abundance of Ets21c binding motifs in the regulatory regions of tumor-associated genes and the normalized expression of >20% of those genes upon removal of Ets21c, it is further suggested that Ets21c acts in RasV12scrib1 tumors to fine-tune the tumor gene-expression signature (Kulshammer, 2015).

Dilp8 is known to be secreted by damaged, wounded or tumor-like tissues to delay the larval-to-pupal transition. This study has corroborated the role of JNK in stimulating dilp8 expression in RasV12scrib1 tumor tissue, and has further implicated Ets21c and Fos as novel regulators of dilp8 downstream of JNK. However, the data also show that elevated dilp8 transcription per se is not sufficient to delay metamorphosis. Unlike the permanent larvae bearing RasV12scrib1 tumors, those with RasV12scrib1ftz-f1RNAi tumors pupated despite the excessive dilp8 mRNA. Likewise, pupation was not blocked by high dilp8 levels in larvae bearing EAD clones overexpressing Abrupt. As Dilp8 secretion appears critical for its function, it is proposed that loss of Ftz-F1 might interfere with Dilp8 translation, post-translational processing or secretion (Kulshammer, 2015).

Consistent with the individual TFs having unique as well as overlapping functions in specifying properties of RasV12scrib1 tumors, knocking down pairwise combinations of the TFs had synergistic effects on tumor suppression compared with removal of single TF. This evidence supports the view that malignancy is driven by a network of cooperating TFs, and elimination of several tumor hallmarks dictated by this network is key to animal survival. An interplay between AP-1, ETS-domain TFs and NRs is vital for development. For example, the ETS-factor Pointed has been shown to cooperate with Jun to promote R7 photoreceptor formation in the Drosophila adult eye. In mosquitoes, synergistic activity of another ETS-factor, E74B, with the ecdysone receptor (EcR/USP) promotes vitellogenesis. It is thus proposed that tumors become malignant by hijacking the developmental mechanism of combinatorial control of gene activity by distinct TFs (Kulshammer, 2015).

Despite the minor impact of ets21cLONG knockdown on suppressing RasV12scrib1 tumors, Ets21cLONG is the only one of the tested TFs that was capable of substituting for loss of scrib in inducing malignant clonal overgrowth when overexpressed with oncogenic RasV12 in EAD. While invasiveness of such RasV12ets21cLONG tumors required JNK activity, JNK signaling appeared dispensable for tumor growth. Importantly, the overgrowth of RasV12ets21cLONG tumors was primarily independent of a prolonged larval stage, because dramatic tumor mass expansion was detected already on day 6 AEL. How cooperativity between Ets21cLONG and RasV12 ensures sufficient JNK activity and the nature of the downstream effectors driving tumor overgrowth remain to be determined. In contrast, co-expression of either Ftz-F1 or Fos with RasV12 resulted in a non-invasive, RasV12-like hyperplastic phenotype (Kulshammer, 2015).

Why does Ets21cLONG exert its oncogenic potential while Fos and Ftz-F1 do not? Simple overexpression of a TF may not be sufficient, because many TFs require activation by a post-translational modification (e.g., phosphorylation), interaction with a partner protein and/or binding of a specific ligand. Full activation of Fos in response to a range of stimuli is achieved through hyperphosphorylation by mitogen-activated protein kinases (MAPKs), including ERK and JNK. Indeed, overexpression of a FosN-Ala mutated form that cannot be phosphorylated by JNK was sufficient to phenocopy fos deficiency, indicating that Fos must be phosphorylated by JNK in order to exert its oncogenic function. Consistent with the current data, overexpression of FosN-Ala partly restored polarity of lgl mutant EAD cells. It is therefore conclude that the tumorigenic effect of Fos requires a certain level of JNK activation, which is lacking in EAD co-expressing Fos with RasV12. Nevertheless, the absence of an unknown Fos-interacting partner cannot be excluded (Kulshammer, 2015).

Interestingly, MAPK-mediated phosphorylation also greatly enhances the ability of SF-1 and ETS proteins to activate transcription. Two potential MAPK sites can be identified in the hinge region of Ftz-F1, although their functional significance is unknown. Whether Ets21c or Ftz-F1 requires phosphorylation and how this would impact their activity in the tumor context remains to be determined. Genetic experiments demonstrate that at least the overgrowth of RasV12ets21cLONG tumors does not require Ets21c phosphorylation by JNK (Kulshammer, 2015).

In addition, previous crystallography studies revealed the presence of phosphoinositides in the ligand binding pocket of LHR-1 and SF-1 and showed their requirement for the NR transcriptional activity. Although developmental functions of Drosophila Ftz-F1 seem to be ligand independent, it is still possible that Ftz-F1 activity in the tumor context is regulated by a specific ligand. An effect of Ftz-F1 SUMOylation cannot be ruled out (Kulshammer, 2015).

In summary, this work demonstrates that malignant transformation mediated by RasV12 and scrib loss depends on MAPK signaling and at least three TFs of different families, Fos, Ftz-F1 and Ets21c. While their coordinated action ensures precise transcriptional control during development, their aberrant transcriptional (Ets21c, Ftz-F1) and/or post-translational (Fos, Ftz-F1, Ets21c) regulation downstream of the cooperating oncogenes contributes to a full transformation state. The data implicate Fos as a primary nuclear effector of ectopic JNK activity downstream of disturbed polarity that controls ets21c and ftz-f1 expression. Through combinatorial interactions on overlapping sets of target genes and acting on unique promoters, Fos, Ftz-F1 and Ets21c dictate aberrant behavior of RasV12scrib1 tumors. Although originally described in Drosophila, detrimental effects of cooperation between loss of Scrib and oncogenic Ras has recently been demonstrated in mammalian tumor models of prostate and lung cancer. This study and further functional characterization of complex TF interactions in the accessible Drosophila model are therefore apt to provide important insight into processes that govern cancer development and progression in mammals (Kulshammer, 2015).

Fos-related antigen/kayak: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

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