rolled/MAPK


Regulation

The Su(var)2-5 locus, an essential gene in Drosophila, encodes the heterochromatin-associated protein HP1. The Su(var)2-5 lethal period is late third instar. Maternal HP1 is still detectable in first instar larvae, but disappears by third instar, suggesting that developmentally late lethality is probably the result of depletion of maternal protein. Heterochromatic silencing of a normally euchromatic reporter gene is completely lost by third instar in zygotically HP1 mutant larvae, implying a defect in heterochromatin-mediated transcriptional regulation in these larvae. However, expression of the essential heterochromatic genes rolled and light is reduced in Su(var)2-5 mutant larvae, suggesting that reduced expression of essential heterochromatic genes could underlie the recessive lethality of Su(var)2-5 mutations. These results also show that HP1, initially recognized as a transcriptional silencer, is required for the normal transcriptional activation of heterochromatic genes (Lu, 2000).

Both the dominant and recessive phenotypes of mutations in HP1 were examined to look for an essential requirement for HP1 in development. It is proposed that reduced expression of one or more essential heterochromatic genes results in the recessive late larval lethality of Su(var)2-5. In support of this hypothesis, the essential heterochromatic genes rolled and light are misregulated in Su(var)2-5 mutants. rolled transcription at its normal chromosomal location is reduced in Su(var)2-5 mutant flies. Since no maternal Rolled protein is detectable in third instar larvae homozygous for rolled deficiencies, the RNA levels that are detected in mutant larvae and adults reflect zygotic gene expression. In the case of the heteroallelic mutant larvae, it should be emphasized that at the time the larvae were collected for Northern analysis, the Su(var)2-5 mutant larvae appeared healthy and would have lived on for several more days as third instar larvae before dying; indeed, a further decline in rolled RNA preceding larval death cannot be ruled out. Thus, reduced expression of rolled could contribute to the defects associated with loss of HP1. Of course, reduced expression of other heterochromatic genes probably also contributes to lethality due to HP1 deficiency (Lu, 2000 and references therein).

light also experiences variegated inactivation in Su(var)2-5 larval Malpighian tubules, and light transcripts are dramatically reduced overall in Su(var)2-5 mutant larvae. It is important to stress that the repressed light locus in these experiments is also in its normal chromosomal location. It is concluded that silencing of light in these experiments is a direct consequence of HP1 depletion, depriving the light locus of the heterochromatin context required for its normal expression. Several other genes reside in heterochromatin, and it will be interesting to see whether dependence on HP1 is a general attribute of gene expression in heterochromatin (Lu, 2000).

Mutations in rolled, like Su(var)2-5 mutations, lead to late larval or early pupal lethality with defective or missing imaginal discs. At the cytological level, rolled mutations cause defects in mitosis, including overcondensed and/or lagging anaphase chromosomes. Intriguingly, neuroblasts of larvae doubly mutant for hypomorphic alleles of rl and abnormal spindles (encodes a microtubule-associated protein) show telomeric stickiness and increased frequency of aneuploid mitotic figures. These phenotypes are also seen in neuroblasts of larvae heteroallelic for Su(var)2-5 mutations; indeed, the highest frequency of defects occurs in larvae heteroallelic for the Su(var)2-5205 allele, which is carried on a chromosome marked with a hypomorphic rl allele. Therefore, reduced expression of rolled caused by loss of HP1 could contribute to mitotic defects in HP1 mutant larval brains (Lu, 2000).

How can HP1 be required both for activation of heterochromatic genes and silencing of euchromatic genes? It has been proposed that certain heterochromatin-associated proteins function to support normal transcription of heterochromatic genes when those genes are at their normal chromosomal sites and that position effects result when heterochromatic genes are deprived of such essential heterochromatic proteins by displacement away from heterochromatin 'compartments' where such proteins are in high concentration. Such context-dependent regulatory activity has also been described for yeast RAP1 (repressor/activator protein 1); RAP1 is required for high-level expression of many ribosomal protein and glycolytic enzyme genes, but it promotes position-effect silencing at the HM silent mating type cassettes and telomeres. Genetic evidence suggests that RAP1 has distinct activator and silencing domains that could recruit or stabilize distinct chromosomal complexes at distinct chromosomal sites. Similarly, HP1 could interact with different proteins or protein complexes to promote silencing or activation in different chromosomal contexts. Another possibility is that HP1 may contribute to the formation of a particular chromatin structure that interferes with activation of euchromatic genes but to which heterochromatic genes have become adapted and dependent. Loss of HP1 would deplete the nucleus of this particular chromatin conformation, releasing silenced genes from repression while simultaneously depriving the resident heterochromatin genes of their functional context (Lu, 2000).

Line HS-2 of Drosophila, carrying a silenced transgene in the pericentric heterochromatin, was used to investigate in detail the chromatin structure imposed by this environment. Digestion of the chromatin with micrococcal nuclease (MNase) shows a nucleosome array with extensive long-range order, indicating regular spacing, and with well-defined MNase cleavage fragments, indicating a smaller MNase target in the linker region. The repeating unit is about 10 bp larger than that observed for bulk Drosophila chromatin. The silenced transgene shows both a loss of DNase I-hypersensitive sites and decreased sensitivity to DNase I digestion within an array of nucleosomes lacking such sites; within such an array, sensitivity to digestion by MNase is unchanged. The ordered nucleosome array extends across the regulatory region of the transgene, a shift that could explain the loss of transgene expression in heterochromatin. Highly regular nucleosome arrays are observed over several endogenous heterochromatic sequences, indicating that this is a general feature of heterochromatin. However, genes normally active within heterochromatin (rolled and light) do not show this pattern, suggesting that the altered chromatin structure observed is associated with regions that are silent, rather than being a property of the domain as a whole. The results indicate that long-range nucleosomal ordering is linked with the heterochromatic packaging that imposes gene silencing (Sun, 2001).

Over 30 genetic functions reside within D. melanogaster heterochromatin: those characterized require a heterochromatic environment for their proper expression, exhibiting a variegating phenotype or reduced expression when rearrangements place them adjacent to a breakpoint in euchromatin. Particularly striking is the observation that expression of the heterochromatic genes rolled and light in their endogenous heterochromatic position is reduced in larvae mutant for HP1, suggesting that proper maintenance of heterochromatin structure is required for expression of these genes. Thus, it was somewhat surprising to observe that light and rolled have nucleosome arrays similar to those observed for the euchromatic transgene, rather than the heterochromatic transgene. This suggests that the regulation of these heterochromatic genes by HP1 may not be based on the impact of HP1 on heterochromatin structure in general (which is correlated with silencing of transgenes such as hsp70-white) but may be the consequence of a context-dependent (positive or negative) activity, similar to that displayed by RAP1 in S. cerevisiae. Alternatively, the impact of HP1 on a heterochromatic gene may reflect packaging of the surrounding area, rather than the transcribed region. A more detailed analysis of the chromatin structure encompassing these genes and their regulatory regions will be required to resolve this question (Sun, 2001)

EGF receptor signaling regulates pulses of cell delamination from the Drosophila ectoderm: Activation of Rolled

Many different intercellular signaling pathways are known but, for most, it is unclear whether they can generate oscillating cell behaviors. Time-lapse analysis of Drosophila embryogenesis has been used to show that oenocytes delaminate from the ectoderm in discrete bursts of three. This pulsatile process has a 1 hour period, occurs without cell division, and requires a localized EGF receptor (EGFR) response. High-threshold EGFR targets are sequentially activated in rings of three cells, prefiguring the temporal pattern of delamination. Surprisingly, widespread misexpression of the relevant activating ligand, Spitz, is compatible with robust delamination pulses. A single chordotonal organ precursor (called C1) and its progeny provide the source of secreted Spi relevant for oenocyte induction. Although Spitz ligand becomes limiting after only two pulses, artificially prolonging its secretion generates up to six additional cycles, revealing a rhythmic underlying mechanism. These findings illustrate how intercellular signaling and cell movements can generate multiple cycles of a cell behavior, despite individual cells experiencing only one cycle of receptor activation (Brodu, 2004).

The induction of larval oenocytes in Drosophila has been used as a simple model system for investigating the developmental regulation of EGFR signaling. Oenocytes are induced from the dorsal ectoderm of abdominal segments by a fixed and highly restricted source of Spi. This triggers a local EGFR response within a ring of overlying dorsal ectodermal cells, termed a whorl, leading to the upregulation of numerous oenocyte-specification genes and subsequent cell delamination. The simple cell geometry of the oenocyte whorl, together with time-lapse microscopy, was used to explore the timing of Spi secretion, EGFR-target activation, early cell induction, and later cell delamination. These studies reveal that oenocytes delaminate in bursts of three and identify the cell-counting mechanism as an EGFR-dependent pulse generator converting the time window of Spi secretion into final oenocyte number. This represents the first example of a rhythmic cell behavior other than the cell cycle to be reported in the Drosophila embryo (Brodu, 2004).
Rather than delaminating from the ectoderm in a continuous stream, oenocyte precursors segregate in discrete well-separated bursts of three cells. Genetic backgrounds affecting the pattern of cell segregation but not early fate specification were used to show how these pulses are regulated by EGFR signaling. The signaling parameters regulating the time of onset, time of cessation, and in particular, the cyclical nature of cell delamination of oenocytes are discussed (Brodu, 2004).

Using a panel of markers for double- and single-ring stages, it was possible to place gene expression 'snapshots' in temporal order with the cell movements recorded in movies. Three generic EGFR targets (activated Rolled/ERK, Yan, and argos) and three oenocyte-specific EGFR targets (Sal, svplacZ, and svplacZΔ18) were analyzed. In wild-type embryos, the high-threshold EGFR outputs of argos and svplacZ expression, detectable Rolled activation, and strong Yan downregulation are all inner ring specific, whereas lower-threshold outputs such as Sal upregulation and svplacZΔ18 expression are present in both precursor rings. Delamination itself also appears to be a high-threshold EGFR response and is thus confined to the inner ring (Brodu, 2004).

Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling

Insulin and insulin growth factor have central roles in growth, metabolism and ageing of animals, including Drosophila melanogaster. In Drosophila, insulin-like peptides (Dilps) are produced by specialized neurons in the brain. This study shows that Drosophila short neuropeptide F (sNPF), an orthologue of mammalian neuropeptide Y (NPY), and sNPF receptor sNPFR1 regulate expression of Dilps. Body size was increased by overexpression of sNPF or sNPFR1. The fat body of sNPF mutant Drosophila had downregulated Akt, nuclear localized FOXO, upregulated translational inhibitor 4E-BP and reduced cell size. Circulating levels of glucose were elevated and lifespan was also extended in sNPF mutants. These effects are mediated through activation of extracellular signal-related kinase (ERK) in insulin-producing cells of larvae and adults. Insulin expression was also increased in an ERK-dependent manner in cultured Drosophila central nervous system (CNS) cells and in rat pancreatic cells treated with sNPF or NPY peptide, respectively. Drosophila sNPF and the evolutionarily conserved mammalian NPY seem to regulate ERK-mediated insulin expression and thus to systemically modulate growth, metabolism and lifespan (Lee, 2008).

Neuropeptides regulate a wide range of animal behaviours related to nutrition. In particular, mammalian NPY produced in the hypothalamus of the brain controls food consumption. NPY injection in the hypothalamus of rats produces hyperphagia and obese phenotypes. The Drosophila orthologue of NPY is sNPF. This peptide is expressed in the nervous system and it regulates food intake and body size; overexpression of sNPF produces bigger and heavier flies. Likewise, the G-protein-coupled receptor of sNPF (sNPFR1) is expressed in neurons and shows significant similarity with vertebrate NPY receptors. In mammals, however, little is known about how NPY and sNPF systemically modulate growth, metabolism and lifespan. This study shows that these neuropeptides control expression of insulin-like peptides and subsequently affect insulin signalling in target tissues (Lee, 2008).

Initially the effects of sNPF and sNPFR1 on body size were characterized by measuring the length of flies from head to abdomen. The body size of sNPF hypomorphic Drosophila mutants (sNPFc00448) was 23% of that of the wild-type, whereas overexpressing two copies of the sNPF in the sensory neurons and sensory structures of the nervous systems by MJ94-Gal4 (MJ94>2XsNPF) increased body size by 24%. Similar changes were seen in the overall size of adult wings, which resulted from changes in both cell size and number. Effects on body size were associated with sNPF expression levels: relative to wild type, sNPF levels were 3.5-fold higher in MJ94>2XsNPF and less than half of the wild type in sNPFc00448. In contrast to the effect of sNPF on body size, there was little effect on size from repression or overexpression of the sNPF receptor in MJ94-expressing cells (Lee, 2008).

Drosophila insulin-like peptides (Dilps) modulate growth and adult size; therefore, whether sNPF has a role in insulin-producing neurons was tested. For positive controls, Dilp2 was overexpressed in insulin-producing cells (IPCs) through Dilp2-Gal4, which increased body size, and the IPCs were ablated by expression of Dilp2>reaper to decrease body size. To investigate sNPF signalling, sNPFR1 was overexpressed in the IPCs and a 10% increase in body size was observed. Conversely, expression of the sNPFR1 dominant-negative mutant (Dilp2>sNPFR1-DN) reduced body size by 14%. Manipulation of the sNPF ligand with IPCs expressing Dilp2-Gal4, however, did not affect body size: flies overexpressing sNPF (Dilp2>2XsNPF) or in which sNPF was silenced by RNAi (Dilp2>sNPF-Ri) were of similar size to the wild type. Taken together, these results suggest that sNPF peptide may be secreted from MJ94-expressing sensory neurons and activate sNPFR1 of Dilp2-expressing IPCs (Lee, 2008).

To assess this model, the sNPF ligand and sNPFR1 receptor were visualized in the larval brain. Seven IPCs were detected in each brain hemisphere using the marker Dilp2-Gal4>nGFP. Neurons containing sNPF peptide in the axon terminal and cell body (sNPFnergic neurons) were stained adjacent to these IPCs. As expected, sNPFR1 receptors were localized in the plasma membrane of IPCs marked with Dilp2>DsRed. sNPFR1 was also localized in the neurons of the larval brain hemispheres, sub-oesophagus ganglion, ventral abdominal neurons and descending axons in the ventral ganglion (Lee, 2008).

To study genetic interactions between sNPFR1 and Dilps in IPCs, Dilp1 and Dilp2 interference mutants were generated in the sNPFR1 overexpression background. In contrast to the 10% body size increase by sNPFR1 overexpression in IPCs (Dilp2>sNPFR1), inhibition of Dilp1 and Dilp2 in IPCs (Dilp2>Dilp1-Ri and Dilp2>Dilp2-Ri) generated reduced body size by 10% and 15%, respectively. Inhibition of Dilp1 and Dilp2 with sNPFR1 overexpression in IPCs (Dilp2>sNPFR1+Dilp1-Ri and Dilp2> sNPFR1+Dilp2-Ri) also generated a reduction in body size of 8% and 13%, indicating that Dilp1 and Dilp2 are downstream genes of sNPFR1 in IPCs for regulating body size (Lee, 2008).

To test whether sNPF regulates Dilp expression in larval IPCs, expression of Dilp1, 2, 3 and 5 were assessed in sNPF mutants. Neuronal overexpression of sNPF (MJ94>2XsNPF) markedly increased expression of Dilp2 in IPCs; it also produced novel Dilp2 expression outside of these cells. As expected, reduction of sNPF by MJ94>sNPF-Ri inhibited expression of Dilp2. In common with Dilp2, the expression of Dilp1 was positively regulated by sNPF overexpression and reduced by sNPF hypomorphs (MJ94>sNPF-Ri and sNPFc00448). Consistent with the model, expression of Dilp1 and Dilp2 was increased more than fourfold with overexpression of the receptor in IPCs (Dilp2>sNPFR1) and decreased by half with inhibition of the receptor gene in IPCs (Dilp2>sNPFR1-DN). Larval IPCs also express Dilp3 and Dilp5. Expression of Dilp3 was reduced by sNPF hypomorphs (MJ94>sNPF-Ri and sNPFc00448) but expression of Dilp5 was not regulated by any sNPF mutants. There are few functions known to distinguish these various insulin-like peptides. Nutrition-dependent growth regulation is associated with expression of Dilp3 and Dilp5, but not with that of Dilp2. Recent reports show that Dilp2 is reduced in long-lived flies expressing dFOXO or Jun-N-terminal kinase (JNK), whereas Dilp5 is uniquely upregulated upon dietary restriction that increases lifespan (Lee, 2008).

To investigate how Drosophila sNPF regulates Dilp expression, the activation of Drosophila MAP kinase signalling, which includes the action of ERK (encoded by Rolled) and JNK, was measured. sNPF overexpression with MJ94-Gal4 increased phospho-activated pERK relative to basal ERK1/2. Expression of the receptor protein sNPFR1 in IPCs also increased pERK. There were no detectable changes in phospho-activated pJNK in these sNPF and sNPFR1 mutants. Next, whether ERK activation in IPCs was sufficient to induce Dilp expression was tested. Expression of a constitutively active ERK in IPCs (Dilp2>rolledSEM) increased expression of Dilp1 and Dilp2 more than threefold, and both transcripts were repressed less than half by the expression of an ERK inhibitory phosphatase DMKP-3 in IPCs (Dilp2>DMKP-3). In addition, the inhibition of ERK with the sNPFR1 overexpression in IPCs (Dilp2>sNPFR1+DMKP-3) also repressed expression of Dilp1 and Dilp2 compared with that of sNPFR1 overexpression in IPCs (Dilp2>sNPFR1). These results indicate that sNPF and sNPFR1 signalling regulate ERK activation in IPCs, which in turn modulates expression of Dilp1 and Dilp2 (Lee, 2008).

To further examine the effect of sNPF on Dilp, Drosophila CNS-derived neural BG2-c6 cells, which endogenously express sNPFR1 were treated with a synthetic sNPF peptide. Dilp1 and Dilp2 were induced within 15 min, and the elevated transcript persisted for 1 h. Concomitant with this gene expression, sNPF-treated cells activated ERK. Importantly, sNPF did not induce Dilp expression significantly when cells were treated with ERK-specific kinase MEK inhibitor PD98059. To compare the functional conservation of sNPF and NPY in the regulation of insulin expression, similar tests were conduced with rat insulinoma INS-1 cells, which express NPY receptors NPYR1 and NPRY2. When treated with the human NPY peptide, expression of insulin1 and insulin2 and ERK was activated within 15 min. Furthermore, treatment with the MEK inhibitor PD98059 and NPY abolished the induction of insulin1 and insulin2. Together, these findings suggest that the regulation of insulin expression by sNPF or NPY through ERK is evolutionarily conserved in Drosophila and mammals (Lee, 2008).

To verify that sNPF induction of Dilp expression has a physiological consequence, insulin signals at a target tissue, the Drosophila fat body were examined. Fat body cells in flies with neuronal overexpression of sNPF (MJ94>2XsNP) were 42% larger than in the control, whereas inhibition of sNPF by MJ94>sNPF-Ri and sNPFc00448 reduced cell size by 38% and 51% respectively. These differences in size correspond to changes in insulin signal transduction within the cells. Overexpression of sNPF (MJ94>sNPF and MJ94>2XsNPF) leads to phosphorylation and activation of Akt in the fat body, whereas the opposite effect was seen with neuronal inhibition of sNPF (MJ94>sNPF-Ri and sNPFc00448). Activated Akt represses the transcription factor dFOXO by phosphorylation and subsequent cytoplasmic localization. In wild-type flies, dFOXO localized equally in the cytoplasm and nucleus. As predicted, neuronal induction of sNFP (MJ94>2XsNPF) increased the cytoplasmic localization of dFOXO, whereas inhibition of sNPF (MJ94>sNPF-Ri and sNPFc00448) yielded fat body cells with dFOXO predominantly localized in the nucleus. Finally, dFOXO induces expression of the translational inhibitor d4E-BP, and, consistent with the current observations, expression of d4E-BP was elevated in animals where sNPF was inhibited (MJ94>sNPF-Ri and sNPFc00448) and reduced in animals where sNPF was overexpressed (MJ94>2XsNPF) (Lee, 2008).

Besides cell growth, Drosophila insulin-like peptides modulate aspects of metabolism and ageing. For instance, ablation of the IPCs reduces animal size, elevates the level of haemolymph carbohydrates.Therefore trehalose and glucose were assessed in sNPF mutant flies. As predicted, both carbohydrates were reduced upon sNPF overexpression, and both were elevated in sNPF hypomorphs. Also the lifespan of sNPF mutants was measured. As expected, inhibition of sNPF by MJ94>sNPF-Ri increased median lifespan by 16-21%, whereas sNPF overexpression (MJ94>2XsNPF) did not affect lifespan in flies (Lee, 2008).

Overall, the effects on Dilp1 and Dilp2 expression in IPCs regulated by sNPF are associated with cellular, carbohydrate and lifespan responses that are predicted to be caused by changes in the actual level of available insulin peptides. It is concluded that sNPF ultimately regulates insulin secretion from the IPC to affect target tissue insulin/dFOXO signalling and thus modulate growth, metabolism and lifespan (Lee, 2008).

Regulation of food consumption by neuropeptides is a critical step for interventions for managing obesity and metabolic syndromes. Mammalian NPY is known to positively regulate appetite and has thus been thought to promote weight gain primarily by affecting food intake. Thus study revealed a novel physiological role for NPY that is conserved by sNPF of Drosophila. These neuropeptides can affect growth, metabolism and lifespan by modulating ERK-regulated transcription of insulin-like peptides. In Drosophila, sNPFnergic and IPC neurons are adjacent in the brain. This study found, however, that pancreatic β-cells are also responsive to NPY, which is of hypothalamic origin. Although the hypothalamic neurosecretory cells and responding pancreatic endocrine cells are spatially distinct in mammals, recent developmental analysis suggests a parallel developmental pathway for hypothalamic neurosecretory cells and the IPCs of Drosophila, raising the possibility of a common molecular mechanism for β-cell formation. This would suggest that β-cells are not only evolutionarily tied to the hypothalamic neurosecretory cells but also that they retain their functional relationship to their hypothalamic origin by regulating insulin in response to the neuropeptide NPY (Lee, 2008).

Drosophila Abelson kinase mediates cell invasion and proliferation through two distinct MAPK pathways

The Abelson (Abl) family of non-receptor tyrosine kinases has an important role in cell morphogenesis, motility, and proliferation. Although the function of Abl has been extensively studied in leukemia, its role in epithelial cell invasion remains obscure. Using the Drosophila wing epithelium as an in vivo model system, this study shows that overexpression (activation) of Drosophila Abl (dAbl) causes loss of epithelial apical/basal cell polarity and secretion of matrix metalloproteinases, resulting in a cellular invasion and apoptosis. The in vivo data indicate that dAbl acts downstream of the Src kinases, which are known regulators of cell adhesion and invasion. Downstream of dAbl, Rac GTPases activate two distinct MAPK pathways: c-Jun N-terminal kinase signaling (required for cell invasion and apoptosis) and ERK signaling (inducing cell proliferation). Activated Abl also increases the activity of Src members through a positive feedback loop leading to signal amplification. Thus, targeting Src-Abl, using available dual inhibitors, could be of therapeutic importance in tumor cell metastasis (Singh, 2010).

This is the first study to provide in vivo evidence for the role of Abl in cell invasion. Cells expressing dAbl (in the dpp-domain) become invasive and migrate into the area of the posterior compartment, where they are located basally to the basement membrane. Although during this process many cells die, those that 'resist' cell death can be visualized by the presence of GFP at the base of the epithelium in either compartment. Furthermore, mechanistic evidence is provided for an Src-Abl signaling cascade and an Abl/Src signal amplification loop in epithelial cell invasion. Targeting both kinase types using dual Abl/Src inhibitors in cancer patients could thus be of clinical significance. It was also shown that increased cell proliferation associated with Abl can be separated from its cell invasion function by distinct downstream effectors. Different MAPKs are activated downstream of dAbl and Rac, and mediate the cell proliferation and cell invasion phenotypes, respectively (Singh, 2010).

Loss of cell polarity has been linked to tumor growth and cell invasion. The mechanism(s) by which dAbl downregulates cell adhesion/polarity genes like DE-Cadherin, β-Catenin/Armadillo and Dlg are not known. This could be a direct effect of dAbl on junctional complexes or a consequence of the cell invasive behavior. Downregulation of E-cadherin has been linked to several types of tumors. Furthermore, Src family members have been shown to increase the turnover of AJs, which in turn would cause an increase in cell mobility, a possible mechanism by which Abl can mediate loss of cell polarity. This hypothesis is further supported by the observation that overexpression of DE-cadherin suppresses the effects induced by Src upregulation (through Csk reduction, using UAS-dCsk-RNAi) in the retina. Consistent with this notion, overexpression of DE-Cadherin rescues the dAbl-induced cell invasion phenotype. Moreover, removing a genomic copy of mmp1 and mmp2 results in suppression of the dAbl cell invasion phenotype. On the basis of these data it is concluded that loss of cell polarity and MMP secretion are the key factors in contributing to cell invasive behavior of dAbl-expressing cells. However, the possibility of minor contributions of unknown factors in this process cannot be completely ruled out (Singh, 2010).

A complicated question is how dAbl causes cell proliferation in epithelial cells (dAbl lacks nuclear localization signals). The effect of dAbl expression results in cell-autonomous and non-autonomous cell proliferation. In Drosophila, cells destined to undergo apoptosis express specific growth factors (Wingless and Dpp; their upregulation is mediated by JNK activation), inducing non-autonomous compensatory proliferation in neighboring cells. This compensatory proliferation is important for maintaining proper tissue homeostasis and may also be relevant for the induction of tumor cell proliferation. As dAbl activation results in cell death in migrating cells, one argument could be that cell proliferation associated with dAbl activation is a consequence of compensatory proliferation. Interestingly, dAbl expression results in an increase in Wg expression, suggesting that compensatory proliferation takes place in response to dAbl. Taken together, these data suggest that at least some aspects of dAbl-mediated cell proliferation (mediated by activation of ERK) are cell-autonomous independent of such compensatory proliferation, as Bsk-DN co-expression in a dAbl overexpression background (which blocks JNK signaling and thus induction of Wg and Dpp expression), does not block excessive proliferation within the dAbl expression domain (Singh, 2010).

The cell invasion phenotype of dAbl overexpression is similar to Csk reduction (dCsk-RNAi) and the data indicate that dAbl acts downstream of dCsk. As Csk negatively regulates Abl/Src family kinases (SFKs), this suggested that Src mediates the effect of dCsk on dAbl. Previous studies have shown that Abl can act as a substrate of SFKs, though other studies have shown that the opposite can also be true. The data indicate that Src acts upstream of Abl and that Abl can feed back and amplify the signal through its positive effect on Src. How is the dAbl-Src feedback loop working mechanistically? From the in vivo experiments it is not possible to conclude whether dAbl acts directly on Src, dCsk, or unknown upstream components. dAbl does not co-immunoprecipitate either dCsk or the Src kinases in Drosophila S2 cells. Since binding between kinases can be of very transient nature, it is possible that even if dAbl would bind Src or dCsk in vivo, it may not be possible to detect it. However, in vivo data suggest that dCsk does not mediate the dAbl effect: if dAbl would act through dCsk (by inhibiting it), phospho-Src (pSrc) levels should be similar with dCsk-IR or dAbl expression, which is not the case. dAbl expression results in a much more robust activation Src with pSrc detected in all dAbl/GFP-positive cells, whereas dCsk-IR does not result in such strong activation. This observation suggests that dAbl does not act through dCsk in this process. Although the possibility cannot be excluded that dAbl could modulate an unknown component upstream of dCsk, the fact that co-expression of dCsk-IR and dAbl (dCsk-IR; UAS-Abl at 18°C) shows a synergistic effect (even at 18°C, where neither individual transgene has a phenotype on its own) suggests that dAbl and Csk act in parallel on Src. As Abl can phosphorylate Src kinases, a direct effect of dAbl on the Src kinases is favored (Singh, 2010).

JNK signaling is activated in response to environmental stress and by several classes of cell surface receptors, including cytokine receptors and receptor tyrosine kinases. In mammalian cells, JNK has been implicated in oncogenic transformation in fibroblasts and hematopoietic cells, and in cell invasion. In oncogenic transformation, JNK signaling can promote tumor growth, while it can also act as a tumor suppressor. It also functions in basement membrane remodeling during imaginal disc eversion and tumor invasion. This study provides evidence for a link between Src and JNK during cell invasion, mediated through dAbl. The cell invasion and apoptosis phenotypes induced by dAbl require JNK activity, whereas the cell proliferation function of dAbl appears to be mediated by ERK signaling. dAbl does not affect expression levels of JNK but instead causes an increase in active JNK (phospho-JNK). It is worth noting that removing a genomic copy of each of the Drosophila Rac genes suppresses all phenotypes associated with dAbl overexpression (cell invasion, death, and proliferation). These data are consistent with the study of BCR-Abl-mediated cell growth, which requires Rac function, suggesting a general relevance of Rac GTPases as Abl effectors (Singh, 2010).

It is not established how Abl mediates Rac activation. A possible link can be Crk, which primarily consists of SH2 and SH3 domains, serving as an adaptor. Crk-I can associate with and be phosphorylated by c-Abl. Furthermore, ectopic expression of Crk can result in JNK activation. As overexpression/activation of dAbl results in JNK activation, Crk may provide a missing link between dAbl and Rac for JNK activation. Another candidate to mediate an interaction between dAbl and Rac GTPases can be Trio, a guanine exchange factor. Trio has two putative Rac and Rho-binding domains. In Drosophila, Trio function has been studied extensively in the context of axon guidance where it has been shown to interact with dAbl. Interestingly, a recent report has identified Trio as one of the guanine exchange factors responsible for invasive behavior of glioblastoma. Thus, a potential role of Trio in the context of Abl-mediated cell invasion warrants further investigation (Singh, 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).

Occluding junctions maintain stem cell niche homeostasis in the fly testes

Stem cells can be controlled by their local microenvironment, known as the stem cell niche. The Drosophila testes contain a morphologically distinct niche called the hub, composed of a cluster of between 8 and 20 cells known as hub cells, which contact and regulate germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Both hub cells and CySCs originate from somatic gonadal precursor cells during embryogenesis, but whereas hub cells, once specified, cease all mitotic activity, CySCs remain mitotic into adulthood. Cyst cells, derived from the CySCs, first encapsulate the germline and then, using occluding junctions, form an isolating permeability barrier. This barrier promotes germline differentiation by excluding niche-derived stem cell maintenance factors. This study shows that the somatic permeability barrier is also required to regulate stem cell niche homeostasis. Loss of occluding junction components in the somatic cells results in hub overgrowth. Enlarged hubs are active and recruit more GSCs and CySCs to the niche. Surprisingly, hub growth results from depletion of occluding junction components in cyst cells, not from depletion in the hub cells themselves. Moreover, hub growth is caused by incorporation of cells that previously express markers for cyst cells and not by hub cell proliferation. Importantly, depletion of occluding junctions disrupts Notch and mitogen-activated protein kinase (MAPK) signaling, and hub overgrowth defects are partially rescued by modulation of either signaling pathway. Overall, these data show that occluding junctions shape the signaling environment between the soma and the germline in order to maintain niche homeostasis (Fairchild, 2016).

The hub regulates stem cell behavior in multiple ways. First, the hub physically anchors the stem cells by forming an adhesive contact with both germline stem cells (GSCs) and cyst stem cells (CySCs). The hub thus provides a physical cue that orients centrosomes such that stem cells predominantly divide asymmetrically, perpendicular to the hub. Following asymmetric stem cell division, one daughter cell remains attached to the hub and retains stem cell identity while the other is displaced from the hub and differentiates. Second, hub cells produce signals, including the STAT ligand Unpaired-1 (Upd), Hedgehog (Hh), and the BMP ligands Decapentaplegic (Dpp) and Glass-bottomed boat (Gbb), that signal to the adjacent stem cells to maintain their identity. As germ cells leave the stem cell niche, two somatic cyst cells surround and encapsulate them to form a spermatocyst. As spermatocysts move from the apical to the basal end of the testis, both somatic cyst cells and germ cells undergo a coordinated program of differentiation. Previous studies have shown that differentiation of encapsulated germ cells requires their isolation behind a somatic occluding junction-based permeability barrier. Specifically, a role was identified for septate junctions, which are functionally equivalent to vertebrate tight junctions, in establishing and maintaining a permeability barrier for each individual spermatocyst (Fairchild, 2016).

During analysis of septate junction protein localization, it was observed that some, notably Coracle, were expressed in both the hub and the differentiating cyst cells. Moreover, knockdown of septate junction components in the somatic cells of the gonad resulted in enlarged hubs. Based on these results, the role of septate junction components in regulating the number of hub cells was explored in detail. To this end, RNAi was used to knock down the expression of the core septate junction components Neurexin-IV (Nrx-IV) and Coracle (Cora) in both the hub and cyst cell populations and the number of hub cells counted in testes from newly eclosed and 7-day-old adults. RNAi was expressed using three tissue-specific drivers: upd-Gal4, expressed in hub cells; tj-Gal4, expressed weakly in hub cells and strongly in both CySCs and early differentiating cyst cells; and eyaA3-Gal4, expressed strongly in all differentiating cyst cells, weakly in CySCs, and at negligible levels in the hub. To visualize hub cells, multiple established hub markers, including upd-Gal4, upd-lacZ, Fasciclin-III (FasIII), and DN-cadherin (DNcad) were used. Surprisingly, it was found that knockdown of Nrx-IV or cora driven by upd-Gal4 gave rise to normal hubs. In comparison, knockdown of Nrx-IV or cora using tj-Gal4 or eyaA3-Gal4 led to large increases in the number of the hub cells. Hub growth was not uniform and varied between testes, but median hub cells numbers in Nrx-IV and cora knockdown testes grew by 30% and 55%, respectively, between 1 and 7 days post-eclosion (DPEs). However, in extreme cases, hubs contained up to five times the number of cells found in age-matched control testes. This result was confirmed using a series of controls that discounted the possibility that hub overgrowth was due to temperature or leaky expression of the RNAi lines. These results suggested that hub growth occurred as a result of knockdown of septate junction proteins in cyst cells rather than the hub. This was further supported using another somatic driver that is not thought to be expressed in the hub, c587-Gal4. However, analysis of c587-Gal4 was complicated by the fact this driver severely impacted fly viability when combined with Nrx-IV or cora RNAi lines (Fairchild, 2016).

Intriguingly, hub growth largely occurred after adult flies eclosed and not in earlier developmental stages. For example, when the driver eyaA3-Gal4 was used to knock down Nrx-IV or cora, hubs from 1-day-old adults were not larger than controls, but hubs from 7-day-old adults were significantly larger. Moreover, overgrowth phenotypes were recapitulated when temperature-sensitive Gal80 was used to delay induction of eyaA3-Gal4-mediated Nrx-IV knockdown until after eclosion. Hub growth manifested both in a higher mean number of hub cells per testis and by a shift in the distribution of hub cells per testis upward, toward larger hubs sizes. This distribution suggested a gradual, stochastic process of hub growth, resulting in a population of testes containing a range of hub sizes. These results reveal progressive hub growth in adults upon knockdown of septate junction components in cyst cells and suggest that this growth is not driven by events occurring in the hub itself but rather by events occurring in cyst cells (Fairchild, 2016).

Niche size has been shown in various tissues, including vertebrate hematopoietic stem cells and somatic stem cells in the fly ovary, to be an important factor in regulating the number of stem cells that the niche can support. In the fly testes, it has been shown that mutants having few hub cells could nonetheless maintain a large population of GSCs. To determine how a larger hub, containing more cells, affects niche function, the number of GSCs and CySCs was monitored after knockdown of septate junction components in cyst cells. Overall, the average number of germ cells contacting the hub grew substantially in Nrx-IV or cora knockdown testes between 1 and 7 DPEs. To confirm that the germ cells contacting the hub were indeed GSCs, spectrosome morphology was studied, and it was found to be to be consistent with that seen in wild-type GSCs. Moreover, in individual testes, there was a positive correlation between the number of hub cells and the number of GSCs. Similar growth was also observed in the number of CySCs, defined as cyst cells expressing Zfh1, but not the hub cell marker DNcad. Control testes (from tj-Gal4 x w1118 progeny) had on average 34.3 CySCs whereas Nrx-IV and cora knockdown testes had 53.4 and 50.2 CySCs, respectively. These results show the importance of maintaining a stable stem cell niche size, as enlarged hubs were active and could support additional stem cells, which may result in the excess production of both germ cells and cyst cells (Fairchild, 2016).

Next, it was of interest to determine the mechanism driving hub growth in adult flies upon knockdown of septate junction components in cyst cells. One possible mechanism that can explain this growth is hub cell proliferation. However, a defining feature of hub cells is that they are not mitotically active. Consistent with this, a large number of testes were stained for the mitotic marker phospho-histone H3 (pH3), and cells were never observed where upd-LacZ and pH3 were detected simultaneously. These results argue that division of hub cells is unlikely to explain hub growth in the adult Nrx-IV and cora knockdown testes. To determine the origin of the extra hub cells, the lineage of eyaA3-expressing cells was traced using G-TRACE (Evans, 2009). eyaA3 was chosed as both the expression pattern of septate junctions, and Nrx-IV or cora knockdown results suggested that hub growth involved differentiating eyaA3-positive cyst cells. The eyaA3-Gal4 driver utilizes a promoter region of the eya gene, which is required for somatic cyst cell differentiation and is expressed at very low levels in CySCs and at high levels in differentiating cyst cells. Using G-TRACE allows identification of both cells that previously expressed eyaA3-Gal4 (marked with GFP) and cells currently expressing eyaA3-Gal4 (marked with a red fluorescent protein [RFP]); additionally, the hub was identified using expression of upd-LacZ and FasIII. In control experiments at both 1 and 7 DPEs, few GFP-positive cells were observed in the hub. Those few GFP-positive cells could be explained by the transient expression of eya in the embryonic somatic gonadal precursor cells that form both hub and cyst cell lineages or extremely low levels of expression in adult hub cells. When G-TRACE was combined with knockdown of Nrx-IV, the results were strikingly different. Initially, 1 DPE, hubs were only slightly larger than controls and few GFP-positive hub cells were observed. In comparison, 7-DPE hubs contained on average more than twice as many cells compared to controls. Importantly, hub growth in Nrx-IV knockdowns was largely attributable to the incorporation of GFP-positive cells. Moreover, a population of upd-LacZ-labeled cells that were also RFP-positive was observed consistent with ongoing or recent expression of eyaA3-Gal4 in hub cells. These results suggest that knockdown of Nrx-IV or cora leads cyst cells to adopt hallmarks of hub cell identity and express hub-cell-specific genes (Fairchild, 2016).

To learn more about the differentiation state of non-endogenous hub cells in Nrx-IV and cora knockdown testes, various markers were used to label the stem cell niche. This analysis showed normal expression of hub cell markers, such as Upd, FasIII, DNcad, as well as Hedgehog (hh-LacZ), Armadillo (Arm), and DE-Cadherin (DEcad). It was asked how cells that were previously, and in some instances were still, eyaA3 positive could express multiple hub-cell fate markers. To answer this question, the signaling mechanisms that determine hub fate were investigated in Nrx-IV and cora knockdown testes. Hub growth phenotypes similar to those produced by Nrx-IV and cora knockdown have been described previously, most notably in agametic testes that lack germ cells, suggesting that the germline regulates the formation of hub cells. One specific germline-derived signal shown to regulate hub fate is the epidermal growth factor (EGF) ligand Spitz. In embryonic testes, somatic cells express the EGF receptor (EGFR), which, when activated, represses hub formation. EGFR-induced mitogen-activated protein kinase (MAPK) signaling, visualized by staining for di-phosphorylated-ERK (dpERK), was active in CySCs and spermatogonial-stage cyst cells. Quantifying dpERK-staining intensity in cyst cell nuclei showed that MAPK activity was lower in CySCs following knockdown of Nrx-IV or cora, suggesting reduced EGFR signaling. Moreover, the effect of Nrx-IV or cora knockdown on MAPK signaling was not restricted to CySCs, as lower dpERK staining was observed at a distance from the hub. To see whether disruption of EGFR signaling could underlie hub defects in Nrx-IV and cora knockdown testes, attempts were made to rescue these phenotypes by increasing EGF signaling. When a constitutively activated EGF receptor (EGFR-CA) was co-expressed in cyst cells along with Nrx-IV RNAi, hub growth was attenuated, resulting in a reduction in the average number of hub cells compared to expressing only Nrx-IV RNAi. Similar results were also observed in the growth of the GSC population, suggesting that reduced EGFR activation in cyst cells contributes to the overall growth of the stem cell niche caused by the knockdown of Nrx-IV or cora. Surprisingly, analysis of testes with loss-of-function mutations in the EGFR/MAPK pathway reveals different phenotypes than those observed: encapsulation is disrupted and CySCs are lost, but hub size is largely unaffected. This result shows that the partial reduction in EGFR/MAPK signaling seen in Nrx-IV and cora knockdown testes results in distinct phenotypes and highlights the complexity of EGFR signaling in the fly testis (Fairchild, 2016).

Another pathway that is documented to regulate hub cell fate is Notch signaling. Notch plays important roles in hub specification in embryos. The Notch ligand Delta is produced by the embryonic endoderm and acts to promote hub cell specification in the anterior-most somatic gonadal precursor cells. Whereas it has been suggested that Notch acts in the adult to regulate hub fate, such a role has not been clearly demonstrated. A reporter for the Notch ligand Delta (Dl-lacZ) was observed in hub cells of both control and Nrx-IV knockdown testes. Intriguingly, reducing Notch signaling efficiently rescued the hub overgrowth seen in adult Nrx-IV knockdown testes. When a dominant-negative Notch (Notch-DN) was co-expressed in the somatic cells, along with Nrx-IV RNAi, the growth of the hub was reduced compared to the expression of Nrx-IV RNAi alone. Growth in the GSC population was not significantly reduced by co-expression of Notch-DN, suggesting that the Notch pathways may modulate hub growth through a different mechanism compared to the EGFR pathway. Because Notch is well established to regulate hub growth in the embryo, temperature-sensitive Gal80 was used to delay expression of Notch-DN and confirm that the reduction in hub cells was due to disruption of post-embryonic Notch signaling. These results suggest that Notch signaling in cyst cells may contribute to the hub overgrowth phenotypes caused by septate junction knockdown in the adult testes (Fairchild, 2016).

In addition to Notch and EGFR, other signaling pathways that regulate hub size may contribute to the hub growth seen upon somatic knockdown of septate junction components. For example, it has been previously shown that the range of BMP signaling is expanded following Nrx-IV or cora knockdown in cyst cells. Constitutive activation of BMP signaling in the germline was shown to increase the size of the hub and the number of GSCs. Additionally, the relative expression levels of the genes drm, lines, and bowl regulate hub size in the adult. In particular, it is known that lines maintains a “steady state” in the testes by repressing expression of a subset of hub genes in the cyst cell population. Unlike lines mutants, Nrx-IV or cora knockdowns generally lack ectopic hubs. This may reflect the more gradual hub growth seen in septate junction knockdowns or, alternatively, highlight key mechanistic differences in how hub growth is achieved in each respective genetic background. The current work is consistent with the model whereby occluding junctions are required for proper soma-germline signaling in the fly testes. This signaling maintains stem cell niche homeostasis by preventing somatic cyst cells from adopting hub cell fate, which would lead to niche overgrowth. It is well established that, in embryonic testes, hub fate is both positively and negatively regulated by signals from the germline and the endoderm.The results, and recent findings about the genes lines and traffic jam, argue that, in the adult testes, hub fate is actively repressed in the cyst cell lineage. Failure to repress hub fate allows cyst cells to exhibit features of hub cells and act as a functional stem cell niche. However, these cyst-cell-derived hub cells are distinct from the true endogenous hub cells in that they show non-hub-cell features, including expression of the differentiating cyst cell markers eyaA3-Gal4 and β3-tubulin. The data suggest that, following disruption of septate junctions proteins, the signaling environment surrounding the somatic cells is altered such that cyst cells gradually begin expressing hub cell markers (Fairchild, 2016).

One major outstanding question is how eyaA3-Gal4-expressing cyst cells become incorporated into the endogenous hub. Previously, it was shown that a septate-junction-mediated permeability barrier forms by the four-cell spermatogonial-stage spermatocyst. The hub growth phenotypes induced by Nrx-IV and cora knockdowns may occur due to defects in cell-cell signaling, possibly involving EGFR and Notch, that manifest in these later spermatocysts. However, this model requires an explanation for how these cyst cells translocate back to and join the hub. Alternatively, signaling defects in these later spermatocysts are somehow instructing earlier cyst cells, such as CySCs, to join the hub. It is easier to envisage the latter model, as early cyst cells are spatially much closer to the hub, but the sequence of signaling events in such a case will be complex and require further elucidation. The ability of CySCs to convert into hub cells in wild-type testes is a controversial subject. However, the incorporation of CySCs into the hub does not necessitate complete conversion into hub cells but could rather involve simple de-repression or activation of genes that confer hub cell function, including regulators of the cell-cycle- and hub-cell-specific signaling ligands. Notably, the transition between CySC and hub cell fate is linked to the cell cycle (Fairchild, 2016).

Why would loss of the septate-junction-mediated somatic permeability barrier result in disruption of signaling between the soma and germline? There are many possible answers, but it is possible to speculate about two such mechanisms that explain hub overgrowth. One possibility is that germline differentiation, which is dependent on the permeability barrier, is required for the release of signals that maintain stem cell niche homeostasis. Another possibility is that the permeability barrier locally concentrates germline-derived signals that repress hub cell fate by trapping them in the luminal space between the encapsulating cyst cells and the germline. The latter scenario could explain the observation that activated EGFR signaling partially rescues hub overgrowth. In this model, septate junctions allow localized buildup of the EGF ligand Spitz, ensuring that sufficient signaling is available to repress hub fate. It is more difficult to draw strong conclusions about how Notch signaling is altered when septate junctions are disrupted, particularly as the Notch ligand Delta appears restricted to the hub. Overall, an unexpected role was found for an occluding-junction-based permeability barrier in mediating stem cell niche homeostasis. This work highlights how the architecture of the stem cell niche system in the fly testes, which is highly regular and contains a reproducible number of stem cells and niche cells, is in fact the result of an active and dynamic signaling environment (Fairchild, 2016).

MAPK/ERK signaling regulates insulin sensitivity to control glucose metabolism in Drosophila

The insulin/IGF-activated AKT signaling pathway plays a crucial role in regulating tissue growth and metabolism in multicellular animals. Although core components of the pathway are well defined, less is known about mechanisms that adjust the sensitivity of the pathway to extracellular stimuli. In humans, disturbance in insulin sensitivity leads to impaired clearance of glucose from the blood stream, which is a hallmark of diabetes. This study presents the results of a genetic screen in Drosophila designed to identify regulators of insulin sensitivity in vivo. Components of the MAPK/ERK pathway were identified as modifiers of cellular insulin responsiveness. Insulin resistance was due to downregulation of insulin-like receptor gene expression following persistent MAPK/ERK inhibition. The MAPK/ERK pathway acts via the ETS-1 transcription factor Pointed. This mechanism permits physiological adjustment of insulin sensitivity and subsequent maintenance of circulating glucose at appropriate levels (Zhang, 2011).

The insulin signal transduction pathway is regulated by cross-talk from several other signaling pathways. This includes input from the amino-acid sensing TOR pathway into regulation of insulin pathway activity by way of S6 kinase regulating IRS. Signaling downstream of growth factor receptors has also been linked to regulation of insulin signaling. The active form of the small GTPase Ras can bind to the catalytic subunit of PI3K and promote its activity. Expression of a form of PI3K that cannot bind Ras allows insulin signaling, but at reduced levels. The work reported in this study provides evidence for a second mechanism through which growth factor receptor signaling through the MAPK/ERK pathway modulates insulin pathway activity. Transcriptional control of inr gene expression by EGFR signaling may provide a means to link developmental signaling to regulation of metabolism. In this context, a statistically significant correlation wass noted between EGFR target gene sprouty and inr gene expression at different stages during Drosophila development (Zhang, 2011).

Several steps of the insulin pathway can be regulated by phosphorylation. Given that the MAPK/ERK pathway is a kinase cascade, a priori, the possibility of phosphorylation-based interaction between these pathways would seem likely. However, this appears not to be the case. Acute pharmacological inhibition of the MAPK/ERK pathway proved to have no impact on insulin pathway activity. Thus short-term changes in MAPK/ERK pathway activity do not seem to be used for transient modulation of insulin pathway activity. Instead, the MAPK/ERK pathway acts through the ETS-1 type transcription factor Pointed to control expression of the inr gene. Transcriptional control of inr suggests a slower, less labile influence of the MAPK pathway. Taken together with the earlier studies, these findings suggest that growth factor signaling can regulate insulin sensitivity by both transient and long-lasting mechanisms (Zhang, 2011).

Why use both short-term and long-term mechanisms to modulate insulin responsiveness to growth factor signaling? The use of direct and indirect mechanisms that elicit a similar outcome is reminiscent of feed-forward network motifs. Although these motifs are often thought of in the context of transcriptional networks, the properties that they confer are also relevant in the context of more complex systems involving signal transduction pathways. In multicellular organisms, feed-forward motifs are often used to make cell fate decisions robust to environmental noise. The findings suggest a scenario in which a feed-forward motif is used in the context of metabolic control, linking growth factor signaling to insulin responsiveness. In this scenario, growth factor signaling acts directly via RAS to control PI3K activity and indirectly via transcription of the inr gene to elicit a common outcome: sensitization of the cell to insulin. This arrangement allows for a rapid onset of enhanced insulin sensitization, followed by a more stable long-lasting change in responsiveness. Thus a transient signal can both allow for an immediate as well as a sustained response. The transcriptional response also makes the system stable to transient decreases in steady-state growth factor activity. It is speculated that this combination of sensitivity and stability allows responsiveness while mitigating the effects of noise resulting from the intrinsically labile nature of RTK signaling. As illustrated by the data, failure of this regulation in the fat body leads to elevated circulating glucose levels, likely reflecting impaired clearance of dietary glucose from the circulation by the fat body. Maintaining circulating free glucose levels low is likely to be important due to the toxic effects of glucose. In contrast, circulating trehalose, glycogen or triglyceride levels showed no significant change in animals with reduced InR expression, suggesting that these aspects of energy metabolism can be maintained through compensatory mechanisms in conditions of moderately impaired insulin signaling (Zhang, 2011).

Earlier studies have shown that the transcription of the inr gene is under dynamic control. Activation of FOXO in the context of low insulin signaling leads to upregulation of inr transcription, thus constituting a feedback regulatory loop. Thus, InR expression appears to be under control of two receptor-activated cues, which have opposing activities: inr expression is positively regulated by the EGFR-MAPK/ERK module, but negatively regulated by its own activity on FOXO. In the setting of this study, the cross-regulatory input from the MAPK/ERK pathway was found to dominate over the autoregulatory FOXO-dependent mechanism. If conditions exist in which the FOXO-dependent mechanism was dominant, a limited potential for crossregulation by the MAPK/ERK pathway would be expected. Whether Pointed and FOXO display regulatory cooperativity at the inr promoter is an intriguing question for future study (Zhang, 2011).

The exon junction complex controls the splicing of MAPK and other long intron-containing transcripts in Drosophila

Signaling pathways are controlled by a vast array of posttranslational mechanisms. By contrast, little is known regarding the mechanisms that regulate the expression of their core components. This study conducted an RNAi screen in Drosophila for factors modulating RAS/MAPK signaling and identified the Exon Junction Complex (EJC) as a key element of this pathway. The EJC binds the exon-exon junctions of mRNAs and thus far, has been linked exclusively to postsplicing events. The EJC was shown to be required for proper splicing of mapk transcripts by a mechanism that apparently controls exon definition. Moreover, whole transcriptome and RT-PCR analyses of EJC-depleted cells revealed that the splicing of long intron-containing genes, which includes mapk, is sensitive to EJC activity. These results identify a role for the EJC in the splicing of a subset of transcripts and suggest that RAS/MAPK signaling depends on the regulation of MAPK levels by the EJC (Ashton-Beaucage, 2010).

Exon junction complex subunits are required to splice Drosophila MAP kinase, a large heterochromatic gene

The exon junction complex (EJC) is assembled on spliced mRNAs upstream of exon-exon junctions and can regulate their subsequent translation, localization, or degradation. Mutations in Drosophila mago nashi (mago), which encodes a core EJC subunit, based on their unexpectedly specific effects on photoreceptor differentiation. Loss of Mago prevents epidermal growth factor receptor signaling, due to a large reduction in MAPK mRNA levels. MAPK expression also requires the EJC subunits Y14 and eIF4AIII and EJC-associated splicing factors. Mago depletion does not affect the transcription or stability of MAPK mRNA but alters its splicing pattern. MAPK expression from an exogenous promoter requires Mago only when the template includes introns. MAPK is the primary functional target of mago in eye development; in cultured cells, Mago knockdown disproportionately affects other large genes located in heterochromatin. These data support a nuclear role for EJC components in splicing a specific subset of introns (Roignant, 2010).

The exon junction complex (EJC) plays an important role in coupling nuclear and cytoplasmic events in gene expression; its recruitment allows nuclear pre-mRNA splicing to influence the subsequent fate of the spliced mRNAs. The EJC is assembled onto mRNAs during splicing, 20-24 bases upstream of each exon junction. The DEAD box RNA helicase eIF4AIII is the first subunit to associate with pre-mRNA through interactions with the intron-binding protein IBP160. eIF4AIII then recruits Magoh (known as Mago in Drosophila) and Y14. These three subunits constitute the pre-EJC; the fourth core subunit, MLN51 (Barentsz [Btz] in Drosophila). The EJC is best known for its role in nonsense-mediated decay (NMD), a surveillance mechanism that degrades mRNAs containing premature termination codons (PTCs). In mammals, NMD is greatly enhanced by the presence of a spliceable intron downstream of a PTC and is mediated by the EJC and accessory factors that include three up-frameshift (UPF) proteins. In Drosophila, the EJC has a role in mRNA localization; all four core EJC components are required to localize oskar mRNA to the posterior pole of the oocyte (Roignant, 2010).

This study isolated mutant alleles of mago based on their specific defects in epidermal growth factor receptor (EGFR)-dependent processes in eye development. Phosphorylation of mitogen-activated protein kinase (MAPK) is a critical step in signal transduction downstream of the EGFR and other receptor tyrosine kinases. Loss of mago strongly reduces the total level of the mRNA encoding Rolled (Rl), the Drosophila extracellular signal-regulated kinase (ERK)-related MAPK. Y14 and eIF4AIII, the other two subunits of the pre-EJC, also positively regulate MAPK transcript levels, but Btz does not. An intronless MAPK cDNA is independent of mago and can rescue photoreceptor differentiation in mago mutant clones; inclusion of the introns renders it Mago dependent. Mago does not affect MAPK transcription or mRNA stability but alters its splicing pattern. MAPK is a large gene located in heterochromatin; a genome-wide survey of Mago-regulated genes found that genes that shared these features were overrepresented. Based on these observations, it is proposed that the pre-EJC is essential to splice a specific set of transcripts that includes the critical signal transduction component MAPK (Roignant, 2010).

The EJC is thought to bind to all spliced mRNAs independently of their sequence, allowing them to be distinguished from unspliced transcripts in the cytoplasm. Despite these very general binding properties, this study found that loss of core EJC subunits causes surprisingly specific defects. Investigation of the basis for the effect of EJC subunits on one target gene, MAPK, has revealed a function of the pre-EJC during the splicing process (Roignant, 2010).

A genome-wide expression analysis found that loss of Mago reduces the transcript levels of only 7% of the genes expressed in S2R+ cells by 1.5-fold or more. The number of genes directly regulated by the pre-EJC is likely to be much smaller because transcript levels were measured after an extensive period of RNAi treatment that was necessary to eliminate the Mago protein. The ability of MAPK to rescue photoreceptor differentiation in mago mutant clones also suggests that many genes are downregulated as an indirect consequence of loss of MAPK. Similarly, many of the defects of mouse neuroepithelial stem cells heterozygous for Magoh are rescued by restoring the expression of a single gene, Lis1. Cytoplasmic functions of the EJC also show specificity; for instance, the EJC is required to localize oskar mRNA to the posterior of the oocyte but has no effect on the subcellular localization of other spliced mRNAs such as bicoid or gurken. This functional specificity might indicate that EJC components are, in fact, assembled on only a subset of spliced transcripts. Indeed, only the first intron in the oskar transcript contributes to its localization by the EJC. However, experiments in vertebrate and Drosophila cells have found no specific requirement for EJC assembly other than an upstream exon at least 20 bases long. Localization of EJC components to particular cytoplasmic regions in Drosophila oocytes and mammalian neurons may simply represent their selective retention on mRNAs that are translationally repressed (Roignant, 2010).

The importance of MAPK for receptor tyrosine kinase signaling has led to the evolution of multiple mechanisms to regulate its expression as well as its phosphorylation. Other vital targets for the pre-EJC may be found in the ovary. mago and Y14, but not btz, are required early in oogenesis for germline stem cell differentiation and oocyte specification. Because germline inactivation of the Ras pathway has no effect on oogenesis, these functions of Mago and Y14 may reflect a requirement for the pre-EJC to splice transcripts other than MAPK (Roignant, 2010).

The EJC has been shown to act on previously spliced mRNAs in the cytoplasm to increase their translation, direct their subcellular localization, or target them for degradation if they contain premature stop codons. However, none of these mechanisms could explain the strong reduction of MAPK mRNA levels in the absence of pre-EJC subunits. This study has provided several lines of evidence suggesting that the pre-EJC facilitates splicing of a specific subset of introns, including at least one present in the MAPK pre-mRNA. First, MAPK is not an indirect transcriptional target of the pre-EJC because MAPK pre-mRNA is not uniformly reduced in the absence of mago, and Mago is required for the expression of a MAPK genomic construct driven by a heterologous promoter. Second, the EJC-associated splicing factors RnpS1 and SRm160 contribute to maintaining normal MAPK levels, whereas Btz, the only core EJC subunit absent from the spliceosomal complex, is dispensable for MAPK expression. Third, an abnormally spliced MAPK product is detected in Mago-depleted cells. Finally, heterochromatic genes with large introns show an increased propensity for regulation by Mago. Previous experiments did not detect any positive function for the EJC in splicing; however, they were performed in vitro using short introns with strong splice sites and would therefore have missed a function specific to one class of introns (Roignant, 2010).

It will be interesting to determine what features of introns make their splicing dependent on the pre-EJC. The genome-wide analysis points to heterochromatic location and intron size as two characteristics that are likely to be important. Unlike mammalian genomes, the Drosophila genome contains primarily short introns. Large introns are most common in heterochromatic genes such as MAPK, where they are rich in repetitive DNA composed of transposons, retrotransposons, and satellite sequences. Production of endo-siRNAs from such repetitive elements or the presence of splice sites within these elements could interfere with the splicing of the introns they occupy. Chromatin structure might also directly influence splicing, as suggested by recent studies showing differences in nucleosome occupancy and histone modifications between exons and introns and recruitment of splicing regulators by chromatin-binding proteins (Roignant, 2010).

Recognition of splice sites over long distances poses a challenge to the splicing machinery. Splice sites for large introns are initially identified by an exon definition mechanism. The pre-EJC, which is assembled upstream of the 5' splice site during splicing, might interact with other factors across the exon to facilitate recognition of the upstream 3' splice site. Perhaps pre-EJC complexes deposited upstream of introns that can be easily detected due to their small size, strong splice sites, or other features contribute to the subsequent recognition of neighboring introns. Alternatively, because the pre-EJC is assembled prior to exon ligation, it might act during its own recruitment into the spliceosome to promote the second step of splicing. These alternatives cannot be distinguished at present because the measurements of 5' and 3' splice junctions in the MAPK pre-mRNA were made at steady state and thus reflect the balance between transcription, splicing, and degradation. The presence of recursive splice sites that allow large introns to be spliced in multiple steps makes genes less likely to require the EJC. Of interest, recursive splice sites are much less common in vertebrate introns than in Drosophila, suggesting that the EJC-dependent mechanism might be more widely used in higher organisms. The current data challenge the view that the EJC acts only as a marker that affects postsplicing events and suggest that this complex also functions within the nucleus to process a specific set of transcripts (Roignant, 2010).

Moleskin is essential for the formation of the myotendinous junction in Drosophila

It is the precise connectivity between skeletal muscles and their corresponding tendon cells to form a functional myotendinous junction (MTJ) that allows for the force generation required for muscle contraction and organismal movement. The Drosophila MTJ is composed of secreted extracellular matrix (ECM) proteins deposited between integrin-mediated hemi-adherens junctions on the surface of muscle and tendon cells. This paper identifies a novel, cytoplasmic role for the canonical nuclear import protein Moleskin (Msk) in Drosophila embryonic somatic muscle attachment. Msk protein is enriched at muscle attachment sites in late embryogenesis and msk mutant embryos exhibit a failure in muscle-tendon cell attachment. Although the muscle-tendon attachment sites are reduced in size, components of the integrin complexes and ECM proteins are properly localized in msk mutant embryos. However, msk mutants fail to localize phosphorylated focal adhesion kinase (pFAK) to the sites of muscle-tendon cell junctions. In addition, the tendon cell specific proteins Stripe (Sr) and activated mitogen-activated protein kinase (MAPK) are reduced in msk mutant embryos. Rescue experiments demonstrate that Msk is required in the muscle cell, but not in the tendon cells. Moreover, muscle attachment defects due to loss of Msk are rescued by an activated form of MAPK or the secreted epidermal growth factor receptor (Egfr) ligand Vein. Taken together, these findings provide strong evidence that Msk signals non-autonomously through the Vein-Egfr signaling pathway for late tendon cell late differentiation and/or maintenance (Liu, 2011).

In Drosophila, the formation of a stable myotendinous junction is essential to withstand the force of muscle contraction required for larval hatching. Proper formation of the MTJ requires proper integrin heterodimer formation at the junctions between both muscle cells and tendon cells for a permanent linkage to the ECM proteins deposited between these two cell types. The precise mechanism by which the muscle cells signal to the tendon cells to form and maintain the semi-adherens junctions that comprise the stable MTJ is still being elucidated. This study shows that the canonical nuclear import protein Msk is essential for Drosophila somatic muscle attachment. Moreover, a model is provided explaining how Msk may function in non-cell autonomously from the muscle to the tendon cell for proper MTJ maintenance. Though vein mRNA is produced in the myotubes, Vein protein is secreted and is restricted to the junctions at muscle-tendon attachment sites. Msk signals through the secreted Egfr ligand Vein to mediate cross-talk between the muscle and tendon cells. The binding of Vein to the tendon-expressed Egfr activates a signaling cascade through activated MAPK. Activated MAPK translocates to the nucleus and with SrA, activates downstream genes to induce terminal differentiation in the tendon cells. An inability of the tendon cells to maintain activated MAPK and Sr activity would affect the amounts of target proteins required to maintain stable muscle-tendon adhesion. For example, a decrease in Tsp deposition into the ECM would result in smaller attachment sites and an inability to maintain a tight integrin-ECM association (Liu, 2011).

Msk plays a general role in myogenesis as defects in msk mutant embryos were observed in all hemisegments and affected all muscle groups. The variable penetrance, which was classified as either major (Class I) or moderate (Class II) muscle detachment phenotypes, present in msk mutant embryos is likely due to the presence of maternal msk transcript. Attempts to further knockdown Msk levels by removal of maternal load resulted in non-viable egg chambers, consistent with a requirement for Msk function in cell viability. It is possible that a further decrease, but not complete loss in Msk function, could result in earlier defects in myogenesis, since muscle patterning defects, predominantly characterized by missing muscles, were observed in a subset of msk mutant embryos (Liu, 2011).

After cell fate determination is established in somatic muscle development, myoblast fusion and myotube migration begin to proceed simultaneously in stage 13 embryos until the final muscle pattern is completed. As the migrating myotubes approach their target tendon cells, the αPS2ΒPS integrin heterodimer begins to accumulate at the leading edge of the muscle. This integrin complex is required for at least two separate events: (1) to serve as a transmembrane link between the internal actin cytoskeleton and the ECM components Tsp and Tig; and (2) for the proper localization and/or accumulation of Vein. The accumulation of Vein at the sites of muscle-tendon interactions is necessary for activation of the Egfr pathway and subsequent late tendon cell differentiation. In these mature, muscle-linked tendon cells, SrB expression is positively regulated. SrB also turns on the downstream transcriptional target Tsp, resulting in more Tsp secretion and subsequent strengthening of the MTJ through integrin binding (Liu, 2011).

The results suggest that Msk affects the later stages of tendon cell maturation and MTJ formation and/or maintenance. (1) No obvious defects were observed in myoblast fusion or the guidance of muscles to their correct target tendon cell. (2) Msk protein expression, visualized by both antibody immunolocalization and a fluorescently-tagged Msk fusion protein, demonstrates that enrichment of Msk protein at the future muscle-tendon attachment sites occurs after stage 15. The appearance of Msk protein localization corresponds to the timing of MTJ junction formation, but it does not rule out the possibility that Msk has a role in earlier myogenic events. (3) The muscle detachment phenotypes observed in msk mutant embryos are consistent with the myospheroid phenotype observed for other genes, including myospheroid (βPS int), inflated (αPS2 int), and rhea (Talin), which are well-characterized for their role in embryonic muscle attachment. Finally, in all msk mutants examined, regardless of the severity of the muscle detachment phenotypes, MTJ formation occurred in the correct location. Although the muscle attachment sites were smaller in msk mutants than in WT embryos, they were initially formed correctly, but not capable of reaching their mature size. These data taken together indicate that the muscle-specific αPS2βPS integrin complex initially forms an attachment to the ECM proteins Tig and Tsp. However, as mature tendon cell induction is compromised in msk mutant embryos, whereby the tendon cells are not able to produce and secrete proper levels of Tsp protein. Thus, the size of the mature MTJ is reduced and results in a decreased affinity at the muscle-tendon junctions (Liu, 2011).

Sr is a key factor in tendon cell differentiation in the embryo and fly thorax. In the embryo, SrB is essential for early tendon cell induction and SrA is activated for later tendon cell maturation. Furthermore, ectopic expression of Sr can act as a guidance cue for migrating myotubes as ectopic expression of Sr in epidermal cells, the salivary glands, or the CNS results in muscle patterning defects where muscles take the incorrect route and/or become attached to ectopic cells expressing Sr. Even though the data shows that Msk is required for nuclear Sr in the tendon cells, ectopic expression of Msk is not sufficient for tendon cell induction based upon three lines of experimentation. First, ectopic Msk expression in either the muscle or epidermis resulted in aberrant muscle attachment, but not misguided myotubes. All muscles were found to be in the correct position, regardless of Msk expression in domains outside of the normal hemisegments. Second, ectopic Msk expression was not sufficient to induce either early or elevated Sr levels. Third, expression of Msk in the salivary glands or CNS did not result in misguided muscles toward these locations (Liu, 2011).

The tissue-specific rescue experiments show that Msk is required in the muscle cell for proper muscle-tendon attachment to occur. Thus, two mechanisms that are not mutually exclusive are proposed by which Msk may be functioning in the muscle cells to exert its effect on tendon cell maturation. First, Msk may be required directly and/or indirectly for the localization and/or accumulation of secreted Vein. As antibodies against Vein were not available for testing this possibility, it was shown that reintroducing Vein in msk mutants could rescue muscle attachment defects. Second, Msk may act via an integrin-dependent mechanism to modulate adhesion, which is explained in detail below (Liu, 2011).

From these studies, Msk localizes to the ends of muscles at the sites of muscle attachment. It is proposed that this localization of Msk recruits other proteins to the sites of muscle attachment to sequester proteins near the cell periphery and/or to modulate integrin affinity at the muscle attachment site. First, the absence of pFAK localization in msk mutants strongly suggests that Msk is essential for pFAK localization to the muscle-tendon attachment site. Surprisingly, mutations in FAK do not result in embryonic muscle attachment defects. However, pFAK localization is also lost in integrin mutants, suggesting that pFAK is involved in undefined events in myogenesis. If Msk serves as a scaffold protein to localize pFAK and/or other molecules to the sites of muscle-tendon cell attachment, these proteins may play an accessory role in integrin-mediated adhesion. One idea is that a Msk-pFAK complex may serve to limit the signaling function of integrins so its adhesive role predominates in MTJ formation. It is well-established that integrins play both adhesive and signaling roles in cell migration and development. Clustering of the cytoplasmic tails of βPS-integrins initiates a downstream signaling pathway that regulates gene expression in the Drosophila midgut, but is not sufficient to induce tendon cell differentiation in formation of the MTJ. This suggests that integrin-mediated adhesion is required to assemble ECM components and influence the ability of Vein to activate the Egfr pathway. While loss of FAK activity does not result in somatic muscle defects, overexpression of FAK does. Muscles that have detached from the epidermis as a result of FAK overexpression still retain βPS2 integrins at the muscle ends. As observed in mammalian systems, this raises the possibility that pFAK may play a role in integrin complex disassembly. Excess pFAK may either displace proteins that bind to the cytoplasmic domain of integrins or excessively phosphorylate proteins resulting in integrin complex turnover and a decrease in stable adhesion. Alternatively, pFAK may exhibit redundancy with another protein at the attachment sites. There is precedence for this in the fly as pFAK functions redundantly with the tyrosine kinase Src downstream of integrins in the larval neuromuscular junctions (NMJs) to restrict NMJ growth. Furthermore, in FAK mutants, phosphotyrosine signal is still observed at the sites of muscle attachment, supporting the idea that another tyrosine kinase is functional. A viable candidate may be Src42A, as it is also expressed at the sites of muscle attachment in the embryo (Liu, 2011).

The canonical role for Msk is to import proteins, such as activated MAPK into the nucleus. However, not activated MAPK or nuclear Msk were detected in the nuclei of muscle cells. As this study has shown, strong Msk immunolocalization is detected at the MTJ in stage 16 embryos, but not in the nuclei of developing muscles in stages 13-15. It cannot be ruled out that Msk is present at low levels in the muscle, and was not detectable. It is true that the YFP-Msk fusion protein was detected in the nucleus, but this may be due to the over-expression of the protein. While it is possible that nuclear Msk and MAPK in muscles are required for some aspects of myogenesis, this requires further analysis (Liu, 2011).

Future experiments will determine the precise mechanism by which Msk influences Vein secretion, localization, and/or accumulation at muscle-tendon cell attachment sites. Is it mediated through integrins? Is phosphorylation of Msk essential for activity? Msk is tyrosine phosphorylated in response to insulin and PS integrins, although the kinase remains unknown. FAK may be an example of a kinase that can phosphorylate Msk at the muscle attachment sites (Liu, 2011).

In mammalian studies, the canonical role for Importin-7 is in nuclear import. Other roles for cytoplasmic Importin-7 have not been examined. Thus, it will be interesting to uncover new roles for Importin-7, specifically in vertebrate muscle development (Liu, 2011).

Negative Regulation of EGFR/MAPK Pathway by Pumilio in Drosophila melanogaster

In Drosophila melanogaster, specification of wing vein cells and sensory organ precursor (SOP) cells, which later give rise to a bristle, requires EGFR signaling. This study shows that Pumilio (Pum), an RNA-binding translational repressor, negatively regulates EGFR signaling in wing vein and bristle development. Loss of Pum function yielded extra wing veins and additional bristles. Conversely, overexpression of Pum eliminated wing veins and bristles. Heterozygotes for Pum produced no phenotype on their own, but greatly enhanced phenotypes caused by the enhancement of EGFR signaling. Conversely, over-expression of Pum suppressed the effects of ectopic EGFR signaling. Components of the EGFR signaling pathway are encoded by mRNAs that have Nanos Response Element (NRE)-like sequences in their 3'UTRs; NREs are known to bind Pum to confer regulation in other mRNAs. This study shows that these NRE-like sequences bind Pum and confer repression on a luciferase reporter in heterologous cells. Taken together, the evidence suggests that Pum functions as a negative regulator of EGFR signaling by directly targeting components of the pathway in Drosophila (Kim, 2012).

In the absence of Pum, extra bristles and wing veins develop, while over-expression of Pum eliminates bristles and wing veins. Several lines of evidence show that the role of Pum is to negatively regulate development of wing veins and bristles. First, loss- and gain-of Pum function produced aberrant wing vein and bristle phenotypes that are inverse to those produced by altered EGFR signaling. Second, reduction of Pum activity greatly enhanced phenotypes associated with reduced EGFR signaling. Third, concomitant expression of Pum suppressed phenotypes associated with ectopic EGFR signaling. In support of the genetic conclusion, it was shown that Pum binds the NRE-like sequence of EGFR, Rl, Sos, and Drk mRNAs and represses translation of a reporter containing these sequences in heterologous cells, suggesting that Pum is a negative regulator of EGFR signaling (Kim, 2012).

To define Pum's role in the development of wing veins and bristles precisely, attempts were made to locate Pum protein and measure Pum activity through a GFP-NRE construct in the 3rd-instar larval and pupal wing imaginal discs where wing vein and SOP cells are specified. A low- level ubiquitous expression of Pum and broad Pum activity was obtained, suggesting that Pum might function as general attenuator of EGFR signaling (Kim, 2012).

This discovery of negative regulation of EGFR signaling by Pum is not confined to Drosophila somatic cells, since it has also been reported in germline cells of C. elegans, cultured human stem cells, and yeast cells. Thus, it is likely that Pum regulation of EGFR signaling is universal and involves diverse developmental contexts, ranging from C. elegans to Drosophila and humans (Kim, 2012).

beta amyloid protein precursor-like (Appl) is a Ras1/MAPK-regulated gene required for axonal targeting in Drosophila photoreceptor neurons

beta amyloid protein precursor-like (Appl), the ortholog of human APP, which is a key factor in the pathogenesis of Alzheimer's disease, was found in a genome-wide expression profile search for genes required for Drosophila R7 photoreceptor development. Appl expression was found in the eye imaginal disc and it is highly accumulated in R7 photoreceptor cells. The R7 photoreceptor is responsible for UV light detection. To explore the link between high expression of Appl and R7 function, Appl null mutants were analyzed and reduced preference for UV light was found, probably because of mistargeted R7 axons. Moreover, axon mistargeting and inappropriate light discrimination are enhanced in combination with neurotactin mutants. R7 differentiation is triggered by the inductive interaction between R8 and R7 precursors, which results in a burst of Ras1/MAPK, activated by the tyrosine kinase receptor Sevenless. Therefore, whether Ras1/MAPK is responsible for the high Appl expression was examined. Inhibition of Ras1 signaling leads to reduced Appl expression, whereas constitutive activation drives ectopic Appl expression. Appl was shown to be directly regulated by the Ras/MAPK pathway through a mechanism mediated by PntP2, an ETS transcription factor that specifically binds ETS sites in the Appl regulatory region. Zebrafish appb expression increased after ectopic fgfr activation in the neural tube of zebrafish embryos, suggesting a conserved regulatory mechanism (Mora, 2013).

Two main conclusions can be drawn from this work. First, Drosophila Appl is involved in R7 axonal targeting. Moreover, the finding that the Appl loss-of-function defects are enhanced when combined with Nrt heterozygous mutant suggest that Appl acts at the membrane of R7, where it interacts with other proteins such as Nrt. Second, Appl activation downstream of the RTK/Ras1 is independent of neural specification, occurs in vivo, and is mediated by direct binding of PntP2 to ETS sequences in the Appl regulatory region (Mora, 2013).

Together, these findings may provide insights into the pathogenesis of neurological disorders such as Alzheimer's disease. The β-amyloid peptides, which accumulate in the amyloid plaques found in the brain of Alzheimer's disease patients, are produced after APP proteolysis. However, Alzheimer's disease has not only been associated to the production of the primary component Aβ by proteolysis of APP, but also by transcriptional regulation. Increased APP transcription underlies the phenotype in some cases of familial Alzheimer's disease. In addition, overexpression of APP appears to be responsible for the early onset of Alzheimer's disease in individuals with Down syndrome. Thus, the current results open the possibility to explore whether in some cases of Alzheimer's disease a burst of RTK/Ras1/MAPK occurs and whether this signaling activity ends with high APP accumulation (Mora, 2013).

Amyloid β peptides are known to be involved in vision dysfunction caused by age-related retinal degeneration in mouse models. Thus, the current in vivo observations could be the basis for further research in mammalian models for neurodegenerative retinal disorders that share several pathological features with Alzheimer's disease (Mora, 2013).

Brinker possesses multiple mechanisms for repression because its primary co-repressor, Groucho, may be unavailable in some cell types

Transcriptional repressors function primarily by recruiting co-repressors, which are accessory proteins that antagonize transcription by modifying chromatin structure. Although a repressor could function by recruiting just a single co-repressor, many can recruit more than one, with Drosophila Brinker (Brk) recruiting the co-repressors CtBP and Groucho (Gro), in addition to possessing a third repression domain, 3R. Previous studies indicated that Gro is sufficient for Brk to repress targets in the wing, questioning why it should need to recruit CtBP, a short-range co-repressor, when Gro is known to be able to function over longer distances. To resolve this, genomic engineering was used to generate a series of brk mutants that are unable to recruit Gro, CtBP and/or have 3R deleted. These reveal that although the recruitment of Gro is necessary and can be sufficient for Brk to make an almost morphologically wild-type fly, it is insufficient during oogenesis, where Brk must utilize CtBP and 3R to pattern the egg shell appropriately. Gro insufficiency during oogenesis can be explained by its downregulation in Brk-expressing cells through phosphorylation downstream of EGFR signaling (Upadhyai, 2013).

A structure/function analysis of the transcriptional repressor Brk has been performed by replacing the endogenous brk gene with a ΦC31 bacteriophage attP site into which mutant forms of brk were introduced by integrase-mediated transgenesis. The goal was to generate mutations that disrupted the ability of Brk to recruit the CoRs Gro and CtBP and/or that deleted the less well characterized 3R repression domain and to test their activity in different tissues at different times of development to determine if and why they are required by Brk to repress transcription. Previous studies with Brk and other TFs that can recruit both CoRs indicated that Gro recruitment is essential for at least some of the activities of these TFs, but the reason for recruiting CtBP has proven more elusive. This study has confirmed that Gro recruitment is essential for Brk activity, but have also showed that Brk needs to recruit CtBP and to possess the 3R domain for full activity in some tissues, in particular during oogenesis (Upadhyai, 2013).

Lethality of the brkGM mutant reveals Gro recruitment is necessary for Brk activity. The brkΔ3RCM mutant, which utilizes Gro as its sole repressive activity, can progress from fertilization to an almost morphologically wild-type adult, indicating that Gro is close to sufficiency in this regard. However, brkΔ3RCM mutants often die as embryos and show defective oogenesis, with eggs having aberrant egg shell pattering, a characteristic of brk null mutants. The single mutants, brkΔ3R and brkCM, show less severe egg shell defects and reduced fertility, the latter probably relating to a defective micropyle, the structure through which sperm normally enter. The apparent inactivity of BrkΔ3RCM protein in follicle cells appears to be explained by active, unphosphorylated Gro being reduced there. The egg shell is patterned by the surrounding follicle cells, where Brk is expressed at high levels in the dorsal anterior. This coincides with high levels of EGFR signaling and previous studies have shown that Gro activity is attenuated following phosphorylation by MAPK downstream of EGFR signaling. As expected, lower levels of unphosphorylated or active Gro were found in the dorsal-anterior follicle cells. Consistent with the activity of BrkΔ3RCM being compromised by EGFR-dependent downregulation of Gro activity, upregulation of EGFR signaling in the wing disc of brkCM mutants results in derepression of the targets salE1 and ombZ (Upadhyai, 2013).

EGFR signaling also probably reduces the levels of active Gro available for Brk in other tissues, including the ventral ectoderm where Brk activity is required to ensure proper patterning of the denticle belts and where EGFR signaling is known play a key role. Many brkΔ3RCM mutants do not survive embryogenesis and demonstrate defects in denticle patterning similar to, but weaker than, those of null mutants. In addition, the VDB phenotype of brkGM mutants is less severe than in brkKO or brk3M mutants. Thus, CtBP and 3R appear to provide repressive activity in the ventral ectoderm (Upadhyai, 2013).

No Brk targets have been characterized in the follicle cells, but these would be expected to be partially derepressed in both brkCM and brkΔ3R mutants and possibly completely derepressed in brkΔ3RCM mutants based on the egg shell phenotypes, although there might be some differences between brkCM and brkΔ3R given the differences between CtBP and 3R just discussed. However, again, this would not imply that these targets are CtBP/3R specific, because the inability of Gro to participate in their repression is presumed to be due to its unavailability. Thus, although studies have indicated that TFs that have the ability to recruit both Gro and CtBP may only recruit one or other at specific targets, this might not reflect a CoR specificity for individual targets, but rather a cell-specific availability of CoRs (Upadhyai, 2013).

It is possible that if Gro were available in all cells then the CiM and 3R domain would be dispensable and so, at least for Brk, downregulation of Gro by MAPK phosphorylation could be considered inconvenient. This might be true for other TFs, including Hairy, Hairless and Knirps, which also function in multiple tissues, many of which are exposed to RTK signaling, and might explain why these TFs need to resort to recruiting CtBP as well as Gro. It should also be noted that Gro activity can be downregulated in other ways, including phosphorylation by Homeodomain-interacting protein kinase. This downregulation of Gro activity has been explained in terms of reducing the activity of specific repressors in specific tissues, such as E(Spl) factors during wing vein formation. This appears to be a somewhat illogical way to downregulate the activity of specific repressors, as there are almost certainly many other TFs utilizing Gro in the same cells and in other tissues exposed to RTK signaling and their activity might be compromised. There are no data indicating whether the downregulation of Gro activity in follicle cells serves any purpose and could simply be a consequence of the decision to downregulate Gro activity by this means in other tissues. However, this has serious implications for Brk and has required Brk to be versatile in its mechanisms of repression. Of course, the possibility has not been ruled out that downregulation of Gro activity does serve a purpose for Brk in follicle cells; for example, if Gro were available here it might provide Brk with too much activity or allow it to inappropriately repress a target that CtBP or 3R cannot. This might be tested by assessing egg shell phenotypes after driving unphosphorylatable Gro at physiological levels in a brkΔ3RCM mutant, but currently this is technically challenging (Upadhyai, 2013).

The idea that repressors need to be versatile in their repressive mechanisms because of variable CoR availability presumably extends beyond Brk and Hairless, Hairy and Knirps. In fact, other repressors in Drosophila possess both CtBP- and Gro-interaction motifs, including Snail. This might not be simply related to downregulation of Gro activity, as CtBP activity can also be modulated; for example, SUMOylation and acetylation of mammalian CtBPs is implicated in regulating their nuclear localization. In addition, other CoRs might similarly be available only in some cells; MAPK activity has been shown to phosphorylate and lead to the nuclear export and inactivation of the SMRT CoR complex. Finally, a further consideration raised by the present study is that care should be taken in assuming that a TF requires and can use a specific CoR to repress its targets in a particular tissue simply because it possesses an interaction motif for that CoR (Upadhyai, 2013).

The Drosophila Arf GEF Steppke controls MAPK activation in EGFR signaling

Guanine nucleotide exchange factors (GEFs) of the cytohesin protein family are regulators of GDP/GTP exchange for members of the ADP ribosylation factor (Arf) of small GTPases. They have been identified as modulators of various receptor tyrosine kinase signaling pathways including the insulin, the vascular epidermal growth factor (VEGF) and the epidermal growth factor (EGF) pathways. These pathways control many cellular functions, including cell proliferation and differentiation, and their misregulation is often associated with cancerogenesis. In vivo studies on cytohesins using genetic loss of function alleles are lacking, however, since knockout mouse models are not available yet. Recently studies have identified mutants for the single cytohesin Steppke (Step) in Drosophila, and an essential role of Step in the insulin signaling cascade has been demonstrated. The present study provides in vivo evidence for a role of Step in EGFR signaling during wing and eye development. By analyzing step mutants, transgenic RNA interference (RNAi) and overexpression lines for tissue specific as well as clonal analysis, it was found that Step acts downstream of the EGFR and is required for the activation of mitogen-activated protein kinase (MAPK) and the induction of EGFR target genes. It was further demonstrated that step transcription is induced by EGFR signaling whereas it is negatively regulated by insulin signaling. Furthermore, genetic studies and biochemical analysis show that Step interacts with the Connector Enhancer of KSR (CNK). It is proposed that Step may be part of a larger signaling scaffold coordinating receptor tyrosine kinase-dependent MAPK activation (Hahn, 2013).

The proper development of multicellular organisms requires the coordination of proliferation and differentiation, which is a particular challenge during the formation of the tissues and organs of the body. Numerous studies have shown that receptor tyrosine kinases such as the vascular growth factor receptor (VEGFR) epidermal growth factor receptor (EGFR) and insulin/insulin-like growth factor receptors (InR/IGF-Rs) play prominent roles in signaling cell proliferation and differentiation. Misregulation of both pathways is often causative for tumor development and progression through their effects on uncontrolled cell growth, inhibition of apoptosis, angiogenesis, and tumor-associated inflammation. Determining how growth and differentiation are coordinated by these pathways is thus essential to understanding normal development, as well as disease states such as cancer (Hahn, 2013).

Steppke (Step) has been identified as a new and essential component of the insulin signaling pathway in Drosophila. The insulin signaling cascade is conserved from flies to humans and was shown to regulate cell and organismal growth in response to extrinsic signals such as growth factors and nutrient availability. In Drosophila, activation of a unique insulin-like receptor (InR) stimulates a conserved downstream cascade that includes the Phosphatidylinositol-3-kinase (PI3K) and Protein Kinase B (PKB or AKT). AKT is involved in enhancement of glucose absorption and glycogen synthesis, and regulates the activity of the Forkhead box O (FoxO) transcription factor, a negative regulator of cell growth. Step acts downstream of the insulin receptor and upstream of PI3K in the insulin/IGF-like signaling (IIS) cascade (Fuss, 2006). Step is a member of the cytohesin family of guanine nucleotide exchange factors (GEFs) which regulate small GTPases of the ADP-ribosylation factor (ARF) family. Small ARF GTPases are involved in the regulation of many cellular processes including vesicle transport, cell adhesion and migration. Studies in mice have confirmed an evolutionary conserved role of cytohesin family members in IIS (Hahn, 2013 and references therein).

Whereas previous studies focused on the role of Step in IIS-dependent larval growth control, this study examined its function in the Drosophila wing, which develops from an epithelial sheet during larval and pupal stages. The wing is an ectodermal structure formed by a dorsal and ventral epithelium, interspersed with cuticular ectodermal tubes, the so called wing veins. Stereotypical arrangement of wing veins is determined in the imaginal wing disc in late larval and pupal stages by several signaling pathways including the EGFR cascade. EGFR activation by EGF-like ligands Spitz or Vein results in the activation of the small GTPase RAS by its loading with guanosine triphosphate (GTP), which as a result triggers the activation of a number of downstream effector proteins including the Ser/Thr-kinase RAF [mitogen-activated protein kinase (MAPK) kinase kinase]. Once activated, RAF phosphorylates and activates MEK (MAPK kinase), which in turn phosphorylates and activates MAPK/ERK. Phosphorylated MAPK exerts its role in the cytoplasm as well as in the nucleus, where it controls expression of EGFR target genes like pointed (pnt), argos (aos), rhomboid (rho) and ventral nervous system defective (vnd). The scaffolding protein Connector enhancer of KSR (CNK) has been described to facilitate RAS/RAF/MAPK signaling by providing a protein scaffold at the plasma membrane that integrates Src and RAS activities to enhance RAF and MAPK activation (Claperon, 2007). EGFR/MAPK signaling is crucial quite early during wing vein differentiation, where phosphorylation of MAPK determines the positioning of proveins and later during development for maintenance of longitudinal veins. In addition to patterning, both EGFR/RAS/MAPK signaling and IIS control general cell proliferation and cell growth during wing development. Thus, EGFR/RAS/MAPK signaling controls both cell fate (vein versus intervein) and general cell proliferation along with IIS at similar times within the wing tissue (Hahn, 2013).

Recent studies in human lung and breast adenosarcoma cancer cell lines indicated a function of cytohesins in ErbB (EGFR) signaling, where they facilitate signaling by stabilizing an asymmetric ErbB receptor dimer (Bill, 2010). This study provides the first in vivo model that the cytohesin Step, in addition to its previously characterized function as component of IIS, regulates EGFR signaling dependent wing growth and vein differentiation. Genetic, immunohistochemical and biochemical experiments indicate that Step acts downstream of the EGF receptor in the EGFR signaling cascade and is necessary and sufficient for MAPK activation and the induction of EGFR target genes. Whereas step transcription is negatively regulated by IIS (Fuss, 2006), it is induced by EGFR signaling. Evidences are further provided that Step might directly interact with the Connector Enhancer of KSR (CNK) protein that is part of a protein scaffold known to coordinate RAS-dependent RAF and MAPK signaling from tyrosine kinase receptors (Hahn, 2013).

This study demonstrates an in vivo function of the Arf GEF Step as an essential component of the EGFR signaling pathway which acts downstream of the EGFR. Step is necessary and sufficient for activation of MAPK and the induction of EGFR target genes in the Drosophila wing. Based on biochemical, immunohistochemical and the genetic data a mechanistic model is proposed in which Step and dCNK interaction is important for EGFR signaling. dCNK is the single member of the CNK protein family in Drosophila. CNK proteins are scaffolding proteins that have been linked with RAS, Rho, Rac, Ral and Arf GTPases and are proposed to act as general regulators of GTPase-mediated events downstream of receptor tyrosine kinases, including EGFR and InR/insulin-like growth factor receptors (Claperon, 2007). Together with the kinase suppressor of RAS (KSR), CNK was shown to assemble a signaling complex including RAF and MEK which promotes RAS-dependent RAF activation and the subsequent phosphorylation of MAPK. It is suggested that Step is a functional part of this scaffolding complex via its direct interaction with CNK. This is also consistent with recent data in HeLa and 393T cells showing that human CNK1 directly interacts with cytohesin-2 to coordinate PI3K/AKT signaling downstream of InR/IGF-R. It was proposed that CNK1 recruits cytohesin-2 to the plasma membrane, where activity of plasma membrane bound GTPases leads to a PIP2 rich microenvironment, which enhances IRS1 recruitment and hence facilitates PI3K/AKT signaling. Similarly, Drosophila cytohesin Step was shown to be required for PI3K activation. Together, several lines of evidence support a role of cytohesins and CNK in similar signaling contexts (RAS/RAF/MAPK and PI3K/AKT signaling), where a direct interaction of both proteins as part of a signaling platform might promote downstream signaling events like MAPK phosphorylation and PI3K activation. This does not exclude other functions of cytohesins, e.g. the stabilization of asymmetric ErbB (EGFR) dimers, as shown recently in human lung and breast adenosarcoma cancer cell lines. The data indicate, however, that a major function of the Drosophila cytohesin Step in EGFR signaling resides downstream of the EGFR and upstream of MAPK (Hahn, 2013).


rolled/MAPK: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.