InteractiveFly: GeneBrief

bantam: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

Growth of tissues and organs during animal development involves careful coordination of the rates of cell proliferation and cell death. Cell proliferation depends on signals to stimulate cell growth and cell division. In addition, cells compete for intercellular survival signals which are required to prevent them from undergoing apoptosis in response to growth stimuli. How these cellular processes are coordinated with pattern formation during animal development is a challenging question in developmental biology. The bantam gene of Drosophila has been found to encode a 21 nucleotide microRNA (miRNA) that promotes tissue growth. bantam expression is temporally and spatially regulated in response to patterning cues. bantam microRNA simultaneously stimulates cell proliferation and prevents apoptosis. The pro-apoptotic gene hid has been identified as a target for regulation by bantam miRNA, providing an explanation for bantam's anti-apoptotic activity (Brennecke, 2003).

Secreted signaling proteins of the Hedgehog, Wingless/Wnt, and Dpp/BMP families control spatial pattern formation during animal development. Evidence has also begun to accumulate implicating these signaling proteins in control of imaginal disc growth during Drosophila development. Hedgehog and Dpp signaling have been shown to stimulate cell proliferation in the imaginal discs of Drosophila. Dpp is also thought to provide a survival signal. Notch and Wingless signaling are required for tissue growth and cell survival, and in some circumstances direct exit from cell proliferation. At the cellular level, the functions of a number of genes involved in control of cell growth and division have been characterized in the imaginal discs, including components of the Insulin/PI3Kinase pathway, the Myc, Ras and E2F oncogenes, and Cyclin D/CDK4. In spite of this considerable progress, how intercellular signals coordinate pattern formation with cell proliferation and cell survival remains poorly understood (Brennecke, 2003 and references therein) (Brennecke, 2003).

The bantam locus of Drosophila was identified in a gain-of-function screen for genes that affect tissue growth (Hipfner, 2002). Evidence that the bantam gene encodes a 21 nucleotide microRNA (miRNA). miRNAs are small RNAs, typically of 21-23 nucleotides, that direct posttranscriptional regulation of gene expression. miRNAs are excised by the Dicer RNase complex from longer precursor RNAs that form imperfect hairpin structures. Typically only one arm of the hairpin is recovered as a mature product. Processing of miRNAs has much in common with the production of the short interfering RNAs that direct RNA-mediated interference (RNAi) (Brennecke, 2003 and references therein).

Two mechanisms for regulation of gene expression by miRNAs have been reported. Target RNAs containing sequences perfectly complementary to the miRNA are cleaved by ribonucleases in the RISC complex, as described recently for transcripts encoding scarecrow-like family transcription factors in plants (Llave, 2002). Target RNAs containing sequences imperfectly complementary to the miRNA can be subject to translational control. The let-7 and lin4 miRNAs of C. elegans (see Drosophila let-7) act in this manner to repress translation of several mRNAs, which control the transitions between larval stages. Hundreds of miRNAs have been identified in plants and animals. Many of these are conserved between closely related species, and some across phyla. Such numbers suggest that miRNAs have diverse and important regulatory roles in organisms. However, apart from the few exceptions mentioned above, their functions are unknown (Brennecke, 2003 and references therein). The findings of this study assign a role to the miRNA encoded by the bantam gene in control of cell proliferation and apoptosis during Drosophila development. Further, they provide a link between the mechanisms that control patterning and tissue growth during animal development (Brennecke, 2003).

Evidence has been found that the bantam gene of Drosophila encodes a miRNA that controls cell proliferation and apoptosis. bantam-induced tissue growth results from an increase in cell number due to an increase in the rate of cell proliferation, not from an increase in cell size (Hipfner, 2002). The anti-apoptotic effects of bantam are not sufficient to explain its effects on tissue growth. Expression of the caspase inhibitor P35 effectively blocks apoptosis in vivo but does not cause net tissue growth (Brennecke, 2003).

bantam's effects are distinguishable from those of Ras, Myc, and the insulin/PI3K pathway, which primarily affect cellular growth. The effects of bantam most closely resemble those caused by altered levels of cyclinD/CDK4 activity. Like cyclinD/CDK4, bantam controls cellular growth and cell cycle progression in a coordinated manner (Brennecke, 2003).

To understand how bantam miRNA promotes cell proliferation and prevents cell death, it will be necessary to identify the genes that it regulates. Using a computational method for predicting possible target genes of miRNAs, the pro-apoptotic gene hid has been identified as a direct target for regulation by bantam miRNA, suggesting one mechanism by which bantam contributes to controlling cell death. bantam target sequences were not found in any of the following genes: ras, myc, dE2F, dDP, cyclin D, CDK4, cyclin E, string, or in components of the insulin/PI(3) kinase pathway. If it is assumed that bantam acts as a negative regulator of target genes (as for hid), its targets might be negative regulators of cell growth or cell proliferation. No bantam target sites were found in the following genes: PTEN, TSC1, TSC2, salvador, warts/lats, merlin, expanded, lethal giant larvae, discs large, and discs overgrown. It seems likely that bantam may regulate an as yet unidentified negative regulator of cell proliferation. In depth analysis of additional predicted targets will be required to determine how bantam controls cell proliferation (Brennecke, 2003).

The three proapoptotic genes hid, reaper, and grim downregulate levels of the IAP proteins in Drosophila, thereby preventing caspase activation. Unlike reaper and grim, whose activity appears to be regulated primarily at the transcriptional level, hid mRNA is also detected in cells that do not undergo apoptosis. Evidence has been presented for transcriptional regulation of hid and for posttranslational regulation of Hid activity by the MAPK signaling pathway. By showing that bantam blocks the activity of Hid(Ala5), which is insensitive to MAPK regulation, an indirect effect of bantam mediated by regulation of the MAPK pathway is excluded. The hid 3'UTR confers bantam-mediated regulation on a heterologous reporter. These findings provide evidence that hid is subject to translational regulation in vivo by the bantam miRNA (Brennecke, 2003).

hid is known to play an important role in regulating apoptosis in eye development. Removing one copy of the endogenous bantam gene enhances the severity of the Hid-induced apoptosis phenotype in the eye, whereas the severity of the reaper-induced apoptosis phenotype is affected much less strongly. Similarly, overexpression of bantam suppresses both the GMR-hid and GMR-reaper phenotypes, but has a stronger effect on hid. The severity of the GMR-reaper phenotype is sensitive to the levels of hid activity. By overexpressing bantam, Hid levels are reduced, providing an explanation for the observed suppression of the GMR-reaper phenotype. Similarly, by removing one copy of bantam an increase in endogenous hid activity in the eye would be expected. By altering the level of Hid, bantam can indirectly alter the threshold for reaper-induced apoptosis. This provides an explanation for the slight increase in severity of the GMR-reaper phenotype observed. There are no bantam target sites in the reaper gene, suggesting that bantam's effect on the GMR-reaper phenotype must be indirect. Finally, no increase in apoptosis was observed in bantam mutant clones in the wing disc. Endogenous hid has not been implicated in developmental control of cell death in the wing (Brennecke, 2003).

The secreted signaling proteins that control spatial pattern during Drosophila development also control tissue growth. Hedgehog promotes cell growth and proliferation in the eye and wing discs by inducing cyclin D and cyclin E expression. The Dpp morphogen gradient has been implicated in control of proliferation and appears to provide cell survival cues as well. Wingless appears to act as both a positive and negative regulator of growth in a context-dependent manner in the wing disc. Evidence is presented that blocking Wnt signaling by overexpression of a dominant-negative form of the Fz2 receptor protein restores cell proliferation in the ZNC, at least in part by regulating bantam miRNA expression. It will be of interest to learn whether bantam homologs play a comparable role as regulators of cell proliferation during vertebrate embryogenesis. BLAST searches did not find sequences identical to the bantam miRNA in the mouse or human genomes, however the possibility exists that a functionally homologous miRNA may differ slightly in nucleotide sequence. The putative Anopheles homolog has a single nucleotide alteration. In addition, three cloned C. elegans miRNAs, mir80, mir81, and miR82, are similar to bantam. It remains to be determined if they have a similar function (Brennecke, 2003).

Is bantam an oncogene? The possibility that misexpression or misregulation of bantam homologs might be responsible for diseases of cell proliferation is intriguing. bantam can suppress apoptosis while stimulating cell proliferation in Drosophila. In Drosophila, cyclinD/CDK4 overexpression causes a proliferation phenotype similar to bantam; cyclinD/CDK4 human homologs are oncogenes. Oncogenes such as Myc and E2F promote apoptosis as well as cell proliferation. Tumor formation requires additional anti-apoptotic inputs. bantam is able to do both and so its putative vertebrate homologs may be oncogenes. In this context, it is noteworthy that Argonaute family genes, which encode components of the cellular machinery needed to produce miRNAs, have been implicated in numerous biological processes including tumorigenesis (Brennecke, 2003).

miRNAs are short noncoding sequences and so present small targets for chemical mutagenesis. Identification of bantam depended on a gain-of-function genetic strategy making use of EP elements to overexpress genes tagged by P element insertion. Several other miRNAs are located next to EP elements in the Drosophila genome, opening the possibility for analysis of their functions in vivo (Brennecke, 2003).

bantam miRNA promotes systemic growth by connecting insulin signaling and ecdysone production

During the development of multicellular organisms, body growth is controlled at the scale of the organism by the activity of long-range signaling molecules, mostly hormones. These systemic factors coordinate growth between developing tissues and act as relays to adjust body growth in response to environmental changes. In target organs, long-range signals act in concert with tissue-autonomous ones to regulate the final size of a given tissue. In Drosophila, the steroid hormone ecdysone plays a dual role: peaks of secretion promote developmental transitions and maturation, while basal production negatively controls the speed of growth. The antagonistic action of ecdysone and the conserved insulin/insulin growth factor (IGF) signaling pathway regulate systemic growth and modulate final body size. This study has unraveled an unexpected role of bantam microRNA in controlling body size in Drosophila. The data reveals that, in addition to its well-characterized function in autonomously inducing tissue growth, bantam activity in ecdysone-producing cells promotes systemic growth by repressing ecdysone release. Evidence is provided that the regulation of ecdysone production by insulin signaling relies on the repression of bantam activity. These results identify a molecular mechanism that underlies the crosstalk between these two hormones and add a new layer of complexity to the well-characterized role of bantam in growth control (Boulan, 2013).

Because ban and ecdysone affect systemic growth in an opposite manner, it is likely that ban acts in PG cells by preventing ecdysone production. The circulating levels of the active form of ecdysone (20E) were measured in in P0206>ban animals at two developmental time points: at the beginning of the wandering phase (early L3w), and just before the larva/ pupa transition (late L3w). To do so, wandering larvae were precisely staged by monitoring gut clearance of blue food. As expected, 20E levels were already high in early L3w (blue gut) control larvae and further peaked in late L3w (clear gut). In contrast, P0206>ban larvae showed lower circulating 20E levels than controls at both stages, and the amplitude of the peak was strongly reduced. Ecdysone signaling in target tissues was also reduced in late larval development upon ban overexpression in the PG (in phm>banD animals) as measured by the expression levels of E75A and Broad-Complex (BR-C), two targets of EcR. Furthermore, the phantom (phm), disembodied (dib), and shade (sad) genes, which are specifically expressed in the PG and encode enzymes required for ecdysteroid biosynthesis, showed reduced expression in phm>ban animals. Consistent with the reduced levels of 20E production and signaling in phm>ban larvae, an increase in 20E levels, produced by feeding the animals ecdysone- supplemented medium, rescued pupa formation. These findings indicate that targeted expression of ban in ecdysone-producing cells has a negative impact on 20E production (Boulan, 2013).

banΔ1 mutant larvae displayed high lethality in late larval development. Thus, ecdysone signaling was assessed in these larvae during earlier stages. Two different developmental points precisely staged with respect to the transition from second (L2) to third (L3) larval instar were selected: 2 hr and 20 hr after ecdysis to the third instar (AL3E). No changes in the expression of BR-C were detected, most probably because at that time, 20E levels had not yet reached the minimum threshold to activate this target. However, the quantification of E75A mRNA levels revealed higher expression in banΔ1 larvae when compared to controls, as did the quantification of dib, phm, and sad mRNA levels. Ni predicted ban target site was found in the 3' UTR of these genes, suggesting that this repression is not direct (Boulan, 2013).

Ecdysone signaling negatively regulates body growth. To test whether the undergrowth phenotype observed in ban mutants is a result of abnormally high ecdysone levels, whole-mount ecdysone signaling was reduced by removing one copy of EcR or impaired ecdysone synthesis by depleting the levels of Sad and Phm in the PG of banΔ1 animals. In all cases, the size of banΔ1 pupae was largely rescued. Collectively, these results suggest that ban participates in reducing ecdysone production in PG cells and corroborate the hypothesis that the systemic growth defects observed in ban mutants are caused by increased ecdysone levels. Consistent with the cell-autonomous growth-promoting role of ban, the PG was larger in P0206>ban animals (that produce less ecdysone) than in controls, whereas it was much smaller in ban mutants. Thus, the impact of ban on ecdysone production is not a consequence of changes in PG size (Boulan, 2013).

Despite displaying higher levels of ecdysone, banΔ1 mutant animals reached metamorphosis with a delay. It has been reported that a strong reduction in larval growth rates can affect developmental timing as a result of a delay in the attainment of critical size for metamorphosis. In order to address whether banΔ1 animals are delayed as a consequence of their reduced growth rates, as a simple proxy of critical size, the time at which the minimal viable size for metamorphosis was achieved in was determined banΔ1 mutant and wild-type animals. Larvae were synchronized at the second (L2) to third (L3) instar transition and then starved at fixed time points to assess survival and capacity to enter into metamorphosis. Remarkably, banΔ1 larvae reached the threshold of 50% of survival with a delay when compared to wild-type animals. These data, together with the fact that targeted expression of ban in the ring gland largely rescued growth rates and developmental delay of banΔ1 animals, support the proposal that the developmental delay is at least in part a consequence of reduced growth rates. Other activities of ban, such as reduced growth of the imaginal tissues or impaired dendrite development, might also affect the timing of metamorphosis (Boulan, 2013).

The production of ecdysone is tightly controlled during larval development. Under normal conditions, ecdysone levels are low during the growth period, thereby allowing optimal body growth rates, and peak at the end of the third-instar larval stage to induce entry into metamorphosis. To monitor whether ban activity levels are also dynamically regulated in the PG, use was made of a ban sensor that expresses GFP under control of a ubiquitously active tubulin promoter and carries two perfect ban fixation sites in its 3' UTR thus making it repressed in the presence of the miRNA. A control sensor lacking the fixation sites showed high GFP expression both in early and late larval PGs. ban sensor levels, however, were low in the PG of second- and early third-instar larvae and considerably increased in wandering third-instar larvae. This observation leads to the proposal that high ban activity in young larvae contributes to the maintenance of low ecdysone titers and the promotion of systemic growth, whereas reduced activity in late PGs contributes to the generation of the ecdysone peak, the cessation of growth, and entry into metamorphosis (Boulan, 2013).

What is the upstream signal that regulates ban activity? The conserved insulin/insulin growth factor (IGF) signaling pathway directly promotes growth in target tissues and is the main relay to couple body growth to nutritional state. In young feeding larvae, insulin signaling in the PG also promotes the basal production of ecdysone, which in turn inhibits body growth. This buffering mechanism, based on the antagonistic action of insulin and ecdysone, modulates final body size in response to nutritional changes. Interestingly, ban activity levels were strongly reduced in early PGs expressing different transgenes that activate the insulin pathway. Increased levels of circulating Dilp2 also reduced ban activity in early PGs, as monitored by increased expression of the ban sensor. Thus, insulin signaling represses ban activity in ecdysone-producing cells (Boulan, 2013).

Thanks to a nutrient-sensing mechanism in the fat body, the equivalent to the vertebrate liver, food conditions control the secretion or expression of brain-derived Dilps, which are the main systemic supply of this hormone during the growth period. Consistent with the repression caused by increased insulin signaling, young feeding larvae growing on amino acid-rich medium showed a clear decrease in ban activity in PG cells. This reduction depended on the enhanced activity of Dilps, because the inhibition of insulin signaling in the PG was sufficient to restore ban activity to normal levels (Boulan, 2013).

In order to address whether ban mediates the action of insulin in regulating ecdysone production, genetic interactions were performed in gain- and loss-of-function conditions. Remarkably, the reduced body size phenotype obtained by enhanced insulin signaling in the PG via several transgenes was completely rescued by simultaneously increasing ban levels in these cells. Given the fact that this rescue implies a much greater effect on body size than the overexpression of UAS-ban transgene alone, it was concluded that the effects of ban and insulin signaling are not additive but rather epistatic. This conclusion is further supported by the observation that modulation of insulin signaling in the PG no longer affected body size in a banΔ1 mutant background. Altogether, these results indicate that the regulation of ecdysone production by insulin signaling relies on the modulation of ban activity in PG cells (Boulan, 2013).

So far, these results unravel a novel role of ban in promoting larval body growth by reducing ecdysone production. In contrast, ban was initially identified by its capacity to induce organ growth in a cell-autonomous manner. That finding prompted an exploration of the contribution of the systemic and cell-autonomous activities of ban to organ growth. The imaginal discs of Drosophila are epithelial sacs that grow in feeding larvae to give rise after metamorphosis to the ectodermal structures of the adult flies, such as legs, wings, or eyes. In banΔ1 mutant larvae, the size of the wing imaginal discs was strongly reduced when compared to control animals. Targeted expression of ban in the PG partially rescued the wing growth defects observed in ban mutant larvae. This result supports the proposal that both the systemic and cell-autonomous activities of ban are required to promote organ growth (Boulan, 2013).

In conclusion, these results establish that ban promotes systemic growth by inhibiting the synthesis of the steroid hormone ecdysone. During the growth period, ban mediates the insulin-dependent regulation of ecdysone production and therefore acts as a buffering mechanism to adjust final body size in response to nutrient availability. Such a crosstalk between insulin and steroid hormones and its impact on the modulation of growth and developmental decisions are also observed in Caenorhabditis elegans. Depending on environmental conditions, the juvenile form of C. elegans either enters maturation to give rise to an adult worm or arrests development to form a dauer larva, a state that is specifically adapted for survival. This decision is determined by the levels of the steroid hormone dafachronic acid (DA). Mutations that enhance insulin signaling, thereby mimicking a favorable environment, increase DA levels and cause animals to become incapable of forming dauer larvae. Remarkably, the deletion of ban orthologs in C. elegans (the mir-58 family) causes severe growth defects and prevents entry into the dauer state under environmental stress (Alvarez-Saavedra, 2010). On the basis of these observations, it is proposed that the role of ban in preventing the production of steroid hormones in function of insulin and nutrient levels might be conserved in other organisms in order to regulate body growth and maturation (Boulan, 2013).

The bantam microRNA acts through Numb to exert cell growth control and feedback regulation of Notch in tumor-forming stem cells in the Drosophila brain

Notch (N) signaling is central to the self-renewal of neural stem cells (NSCs) and other tissue stem cells. Its deregulation compromises tissue homeostasis and contributes to tumorigenesis and other diseases. How N regulates stem cell behavior in health and disease is not well understood. This study shows that Notch regulates bantam (ban) microRNA to impact cell growth, a process key to NSC maintenance and particularly relied upon by tumor-forming cancer stem cells. Notch signaling directly regulates ban expression at the transcriptional level, and ban in turn feedback regulates N activity through negative regulation of the Notch inhibitor Numb. This feedback regulatory mechanism helps maintain the robustness of N signaling activity and NSC fate. Moreover, this study shows that a Numb-Myc axis mediates the effects of ban on nucleolar and cellular growth independently or downstream of N. These results highlight intricate transcriptional as well as translational control mechanisms and feedback regulation in the N signaling network, with important implications for NSC biology and cancer biology (Wu, 2017).

By revealing the involvement of the miRNA pathway, this study highlights the complexity of the N signaling network in normal NSCs and tumor-forming cancer stem cell (CSC)-like NSCs. Previous studies implicated critical roles for both canonical and non-canonical N signaling pathways in NSCs and CSC-like NSCs, and revealed particular dependence of CSC-like NB growth on non-canonical N signaling, which involves PINK1, mTORC2, and mitochondrial quality control. The current study reveals a particular requirement for ban in CSC-like NBs induced by N hyperactivation. The CSC-like NB overproliferation induced by hyperactivation of N or N pathway component Dpn can all be assumed to be of type II NB origin, since previous studies have clearly established that Notch signaling is essential for the development and/or maintenance of type II NBs, but dispensable for type I NBs, and that hyperactivation of Notch or its downstream effector Dpn induced ectopic CSC-like NB growth by altering the lineage homeostasis of the type II but not type I NBs. It would be interesting to test whether, in addition to ban's role in canonical N signaling, there exists a link between ban and non-canonical N signaling. The data indicate that the ban-Numb signaling motif regulates NSC/CSC behavior through at least two mechanisms. On one hand, it regulates cell growth and particularly nucleolar growth, through Myc, a known regulator of cellular and nucleolar growth. Consistently, negative regulation of Myc protein level by Numb was observed through E3 ubiquitin-protein ligase, Huwe1, and the UPS. c-Myc is an essential regulator of embryonic stem cell (ESC) self-renewal and cellular reprogramming, and Myc level and stability can be controlled in stem cells through targeted degradation by the UPS, suggesting conserved mechanisms. A key function of the nucleolus is the biogenesis of ribosomes, the cellular machinery for mRNA translation, and previous studies in Drosophila have supported the critical role of nucleolar growth in NSC self-renewal and maintenance. On the other hand, the ban-Numb axis feedback regulates the activity of N by a double negative regulation, with the end result being positive feedback regulation. This feedback mechanism may help transform initial not so dramatic differences in N activity between NB and its daughter cell generated by the asymmetric segregation of Numb during NB division [33] into 'all-or-none' decision of cell fates. Feed-forward regulatory loops, both coherent and incoherent, are frequently found in gene regulatory networks, and although ban miRNA is not conserved in mammals, miRNAs have been implicated in an incoherent feed-forward loop in the Numb/Notch signaling network in colon CSCs in mammals (Wu, 2017).

Given the role of ban in a positive feedback regulation of N and the potency of N hyperactivity in inducing tumorigenesis, one may wonder why ban overexpression is not sufficient to cause tumorigenesis. As in any biological systems, feedback regulation is meant to increase the robustness and maintain homeostasis of a pathway. Feedback alone, either negative or positive, should not override the main effect of the signaling pathway. Thus, in the NB system feedback regulation by ban is built on top of the available N signaling activity in a given cell and serving to maintain N activity. Because of ban's 'fine-tuning' rather than 'on/off switching' of Numb expression, its effect on N activity during feedback regulation will also be 'fine-tuning', serving to maintain N activity in NB within a certain range. Overexpression of ban in a wild type background may not be sufficient to cause tumorigenesis because N activity is not be elevated to the level sufficient to induce brain tumor as in N-v5 overexpression condition. Consistent with this, the extent of Numb inhibition by ban is also modest, not reaching the threshold level of Numb inhibition needed to cause tumorigenesis. Consistent with the notion that feedback regulation by ban is built on top of the available N signaling activity in a given cell, and that there is dosage effect of N activity in tumorigenesis, overexpression of ban in N-v5 overexpression background further enhanced N-v5 induced tumorigenesis. It is likely that ban or other miRNAs may participate in additional regulatory mechanisms in the N signaling network in Drosophila. Of particular interest, it would be interesting to test whether miRNAs may impinge on the asymmetric cell division machinery to influence the symmetric vs. asymmetric division pattern, a key mechanism employed by NSCs and transit-amplifying IPs to balance self-renewal with differentiation (Wu, 2017).

The results emphasize the critical role of translational control mechanisms in NSCs and CSC-like NSCs. Compared to the heavily studied transcriptional control, knowledge of the translational control of NSCs and CSCs is rather limited. As fundamental regulators of mRNA translation, miRNAs can interact with both positive and negative regulators of translation to influence gene expression. Thus, miRNA activity can be regulated context-dependently at both the transcriptional and translational levels, which may account for the opposite effect of N on ban activity in the fly brain and wing disc, although the ban genomic locus is bound by Su(H) in both tissues. Whether N regulates the transcription of ban or its activity as a translational repressor in the wing disc remains to be tested. With regard to the translation of numb mRNA, the conserved RNA-binding protein (RNA-BP) Musashi has been shown to critically regulate the level of Numb protein in mammalian hematopoietic SCs and leukemia SCs. Further investigation into the potential interplay between miRNAs and RNA-BPs in the translational control of Numb in NBs and CSC-like NBs promises to reveal new mechanisms and logic in stem cell homeostasis regulation, with important implications for stem cell biology and cancer biology (Wu, 2017).

REGULATION

bantam modulates polyglutamine-induced neurodegeneration

Nine human neurodegenerative diseases are due to expansion of a CAG repeat- encoding glutamine within the open reading frame of the respective genes. Polyglutamine (polyQ) expansion confers dominant toxicity, resulting in neuronal degeneration. MicroRNAs (miRNAs) have been shown to modulate programmed cell death during development. To address whether miRNA pathways play a role in neurodegeneration, whether genes critical for miRNA processing modulate toxicity induced by the spinocerebellar ataxia type 3 (SCA3) protein was tested. These studies reveal a striking enhancement of polyQ toxicity upon reduction of miRNA processing in Drosophila and human cells. In parallel genetic screens, the miRNA bantam (ban) was identified as a potent modulator of both polyQ and tau toxicity in flies. These studies suggest that ban functions downstream of toxicity of the SCA3 protein, to prevent degeneration. These findings indicate that miRNA pathways dramatically modulate polyQ- and tau-induced neurodegeneration, providing the foundation for new insight into therapeutics (Bilen, 2006).

This study reveals a striking role for miRNA-regulated pathways in modulation of polyQ toxicity in both flies and human cells. Notably, reduction of genes that affect miRNA processing, but not siRNA processing, in Drosophila modulates polyQ degeneration, underscoring the specificity to miRNA pathways. In Drosophila, one of these miRNAs is ban, which modulates cell survival upon polyQ- and tau-induced neurodegeneration. Moreover, reduction of miRNA processing in human cells also strikingly enhances polyQ toxicity, indicating that miRNAs also play a protective role in human cells. These data suggest that ban and potentially additional miRNAs are involved in mitigating polyQ- and tau-induced neurodegeneration (Bilen, 2006).

Given the role of miRNAs in modulation of developmental programmed cell death, this study tested whether miRNA pathways modulated neurodegeneration. This was addressed by reducing miRNA processing in flies and in human cells in the presence of pathogenic polyQ protein. In flies, loss of dcr-1 or R3D1 had striking effects to enhance SCA3-induced neurodegeneration. In contrast, loss of dcr-2, which is specific to siRNA pathways, had little effect in the situation tested. These studies indicate that miRNA-regulated activities, and not siRNA-regulated activities, are critical to neurodegeneration in vivo. In human cells, reduction of dicer activity also dramatically enhances cell toxicity induced by pathogenic Ataxin-3. Although, in vertebrate cells in culture, dicer activity affects both miRNA- and siRNA-regulated activities, siRNA-dependent activities like heterochromatic silencing do not become disrupted until later time periods. The effect of reducing dicer in human cells could be rescued in part by complementing the treated cells with a fraction containing miRNAs, indicating that the enhanced cell loss was likely due to reduction in one or more miRNAs. Since miRNAs could affect many cellular processes, it was confirmed that enhanced degeneration with polyQ is unlikely due to sensitizing cells to programmed cell death but rather resembles normal polyQ degeneration; identification of miRNAs and target genes will further define the pathways involved. There are hundreds of miRNAs in humans, with a subset expressed in both human brain and HeLa cells. Focus on these common miRNAs, coupled with the demonstrated ability to rescue polyQ toxicity associated with dicer deprivation, promises to reveal genes with a critical role in neuroprotection from polyQ-induced degeneration. These findings were extended with ban and genes of the miRNA pathway beyond polyQ toxicity to modulation of tau; these findings suggest a broader role for miRNA regulated pathways in neuroprotection (Bilen, 2006).

A genetic modifier screen in Drosophila revealed that one miRNA that functions to modulate Ataxin-3 degeneration is ban. ban is a critical regulator in that both loss of activity and upregulation modulated degeneration. ban mitigated not only degeneration induced by polyQ protein but also by tau, an unrelated neurodegenerative disease protein. Although ban mitigates programmed cell death through hid, these studies indicate that hid is not involved in modulation of degeneration induced by pathogenic Ataxin-3 (Bilen, 2006).

These studies suggest that ban modulates survival of cells to pathogenic polyQ protein downstream of protein accumulation, cellular stress response, and inherent protein toxicity. In the presence or absence of added ban, the pathogenic polyQ protein was present at similar levels and elicited a similar stress response. This indicates that ban regulates progression of degeneration downstream of these events. Taken together, these results suggest that ban may modulate the survival of cells. Indeed, ban may modulate cell survival in multiple situations: after initiation of programmed cell death, as well as in response to neurodegenerative disease proteins, including polyQ and tau. These studies also suggest a role for additional miRNAs, due to the stronger enhancement of polyQ degeneration upon reduction of miRNA processing compared to reduction of ban function alone. This suggests that miRNAs in addition to ban in Drosophila likely play a role in regulating neurodegeneration. Moreover, although loss of miRNA processing results in an overall enhancement, specific miRNAs may be protective, whereas others promote degeneration. It is also possible that proteins involved in miRNA processing could themselves be targets of polyQ toxicity (Bilen, 2006).

These findings expand the role of miRNA function from programmed cell death pathways, developmental processes, and cancer to suggest a striking role in protection from cellular degeneration associated with human neurodegenerative disease proteins. Further identification of the miRNAs and their targets will reveal new insight into mechanisms and therapeutics for the treatment of polyQ, tau-associated, and potentially other neurodegenerative diseases (Bilen, 2006).

Regeneration of Drosophila sensory neuron axons and dendrites is regulated by the Akt pathway involving Pten and microRNA bantam

Both cell-intrinsic and extrinsic pathways govern axon regeneration, but only a limited number of factors have been identified and it is not clear to what extent axon regeneration is evolutionarily conserved. Whether dendrites also regenerate is unknown. This study reports that, like the axons of mammalian sensory neurons, the axons of certain Drosophila dendritic arborization (da) neurons are capable of substantial regeneration in the periphery but not in the CNS, and activating the Akt pathway enhances axon regeneration in the CNS. Moreover, those da neurons capable of axon regeneration also display dendrite regeneration, which is cell type-specific, developmentally regulated, and associated with microtubule polarity reversal. Dendrite regeneration is restrained via inhibition of the Akt pathway in da neurons by the epithelial cell-derived microRNA bantam but is facilitated by cell-autonomous activation of the Akt pathway. This study begins to reveal mechanisms for dendrite regeneration, which depends on both extrinsic and intrinsic factors, including the PTEN-Akt pathway that is also important for axon regeneration. This study has thus established an important new model system -- the fly da neuron regeneration model that resembles the mammalian injury model -- with which to study and gain novel insights into the regeneration machinery (Song, 2012).

The present study shows that Drosophila sensory neuron dendrites and axons are capable of regeneration in a cell type-specific manner. While dendrites and axons share the same cell type specificity in their capacity for regeneration, they differ in their developmental regulation, with axons but not dendrites retaining the regeneration ability throughout larval development. It was further shown that the evolutionarily conserved PTEN-Akt signaling pathway is important for the regeneration of dendrites as well as axons and that both axon regeneration and dendrite regeneration are accompanied by the reversal of microtubule polarity (Song, 2012).

It is known that Drosophila larval axons undergo a scaling process in which axons substantially increase their length in accordance with the growth of the organism. Thus, it raises an important issue of whether larval axons regenerate or simply scale after axotomy. This question may be addressed with the following two considerations. First, larval axons scale while maintaining their neural connections; da neuron axons have already formed synaptic connections with neurons in the VNC, and these axon projections are not significantly altered as larvae grow in size. Therefore, this increase of axon length does not involve bona fide axon pathfinding or synaptogenesis. Thus, axon scaling differs from the developmental axon outgrowth before synapse formation and is different from axon regeneration, which involves reinitiation of the developmental program for severed axons to generate growth cones or growth cone-like structures and pathfind to reach their targets. In the larval injury model, the axon is severed and therefore develops a new growing tip, reroutes following the presumptive trajectory by active or passive cues, and may or may not eventually establish synaptic contacts with their right targets in the CNS. This process resembles the regeneration program rather than axon scaling. Second, all of the da neurons, including class I, class III, and class IV, show similar axon scaling during larval stages. However, class IV but not class I or class III da neurons displayed axon regrowth after axotomy. The fact that only class IV da neurons are capable of regrowth, although all of these different types of da neurons undergo scaling, strongly suggests that class IV da neurons possess a unique regrowth potential that allows their severed axons to reinitiate the developmental program for axon outgrowth. For these reasons, it is believed that a subset of the larval axons can regenerate after injury, although the possibility cannot be excluded that the ability of these axons to scale contributes to their regeneration potential. While this regeneration process may or may not fully recapitulate the regeneration program in adults, understanding how this process takes place in larvae will provide invaluable insights into the axon regeneration machinery (Song, 2012).

The ability of class IV but not class I or class III da neurons to readily regenerate their axons and dendrites could conceivably reflect cell type-specific features, including the transcription programs. One interesting question is whether the same program that governs the cell type morphology may also influence their regeneration capacity. For dendrite regeneration of class IV da neurons, either the regenerated dendrite or the neighboring dendritic branch continues to grow to fill the available space. Thus, they may possess a persistent growing potential that is inhibited by neighboring branches nearby so that the branches might overgrow if those inhibitory signals are removed. Therefore, with some branches removed due to injury, the remaining branches will regrow to take over the vacant space. Since class I and class III da neuron dendrites show very limited space-filling ability, these dendrites may lack the growth potential required for regeneration (Song, 2012).

In response to injury, class IV da neurons regenerate their axons substantially, while class I da neurons partially reverse the microtubule polarity of nearby dendrites and convert one of these dendrites into a pseudo-axon (Stone, 2010). Taken together with the current finding that class IV but not class I or class III da neurons are able to regenerate their dendrites, which are also associated with the reversal of microtubule polarity, these observations raise the question of whether pathways controlling neuronal polarity and/or cytoskeletal rearrangement may influence dendrite and axon regeneration (Song, 2012).

Several lines of evidence suggest that dendrite regeneration depends on a balance of influences. First, there may be competition between de novo dendrite regeneration and invasion of neighboring branches. Successful regeneration prevents invasion and vice versa. Second, there could be a balance of extrinsic inhibitory cues, as in the form of the ban miRNA in epithelial cells, and intrinsic growth-promoting signals, as conveyed by the activation of the Akt pathway (Song, 2012).

Moreover, given that activation of the Akt pathway at later stages of development is not sufficient to elevate the extent of dendrite regeneration to that during early larval development, it seems likely that either factors downstream from Akt are developmentally regulated to turn off the regeneration program at later stages or, alternatively, other pathways may contribute to this inhibition (Song, 2012).

Whereas dendrite and axon regeneration display differences with respect to developmental regulation, the PTEN-Akt pathway is important for regeneration of axons as well as dendrites. This pathway not only regulates the extent of dendrite growth of class IV da neurons during development, but also affects their dendrite regeneration and axon regeneration in the CNS. Together with previous work (Park, 2008; Liu, 2010), these results support the notion that modulating neuronal intrinsic PTEN and Akt activity is a potential therapeutic strategy for promoting axon regeneration and functional repair after CNS trauma (Song, 2012).

This work focuses on class IV da neurons, which behave very differently from class I da neurons in regeneration. In particular, unlike class I da neurons, the class IV da neuron is capable of regenerating its axon in the periphery but not inside the CNS, thereby providing the first example of this phenomenon in invertebrates. A recent study of Caenorhabditis elegans PLM neurons, a type of mechanosensory neurons that consistently regrow their axons upon laser-mediated axotomy, has identified multiple genes important for axon regeneration (Chen, 2011), illustrating the power of the genetic approach. The injury model involving Drosophila class IV da neuron axotomy in the CNS (VNC) in the current study has the additional feature that it resembles the injury model involving mammalian DRG neuron axotomy in the CNS (spinal cord) at the cellular and molecular level: Both da neuron and DRG neuron axons regenerate poorly in the CNS even though they display robust regeneration in the periphery, and in both cases, axon regeneration in the CNS is enhanced by activation of the PTEN-Akt pathway. Importantly, while the PTEN-Akt pathway has been shown to be critical for mammalian axon regeneration in the CNS, this has not been shown in invertebrate models; for example, in C. elegans, PTEN (DAF-18) has no effect on axon regrowth. The current finding underscores the usefulness of the Drosophila system that this study developed as a model to uncover evolutionarily conserved mechanisms for CNS axon regeneration. Moreover, the elaborate and stereotyped dendritic branching pattern of da neurons provides a sensitive assay system to begin studying the injury responses and regeneration of dendrites, which may yield clues to facilitate studies of mammalian neuronal dendrites and identify novel approaches to promote dendrite recovery for the treatment of nervous system trauma (Song, 2012).

Yorkie drives supercompetition by non-autonomous induction of autophagy via bantam microRNA in Drosophila

Mutations in the tumor-suppressor Hippo pathway lead to activation of the transcriptional coactivator Yorkie (Yki), which enhances cell proliferation autonomously and causes cell death non-autonomously. The mechanism by which Yki causes cell death in nearby wild-type cells, a phenomenon called supercompetition, and its role in tumorigenesis remained unknown. This study shows that Yki-induced supercompetition is essential for tumorigenesis and is driven by non-autonomous induction of autophagy. Clones of cells mutant for a Hippo pathway component fat activate Yki and cause autonomous tumorigenesis and non-autonomous cell death in Drosophila eye-antennal discs. This study found that mutations in autophagy-related genes or NF-κB genes in surrounding wild-type cells block both fat^-induced tumorigenesis and supercompetition. Mechanistically, fat mutant cells upregulate Yki-target microRNA bantam, which elevates protein synthesis levels via activation of TOR signaling. This induces elevation of autophagy in neighboring wild-type cells, which leads to downregulation of IκB Cactus and thus causes NF-κB-mediated induction of the cell death gene hid. Crucially, upregulation of bantam is sufficient to make cells to be supercompetitors and downregulation of endogenous bantam is sufficient for cells to become losers of cell competition. These data indicate that cells with elevated Yki-bantam signaling cause tumorigenesis by non-autonomous induction of autophagy that kills neighboring wild-type cells (Nagata, 2022).

The data reveal that the Hippo pathway mutant fat clones cause supercompetition by inducing autophagy-mediated cell death in surrounding wild-type cells via NF-κB-mediated induction of hid. The autophagy induction in wild-type cells depends on Yki-bantam-mediated activation of TOR signaling in neighboring fat mutant cells. This mechanism is similar to what was observed in the elimination of ribosomal protein or Hel25E mutant loser clones when surrounded by wild-type cells. This is particularly interesting in two ways: first, it suggests that different types of cell competition, namely elimination of unfit cells by wild-type cells and elimination of wild-type cells by supercompetitors, are driven by the common mechanism, and second, it indicates that induction of autophagy in loser cells is non-autonomous, as even wild-type cells elevate autophagy when juxtaposed to supercompetitors. Although the mechanism by which autophagy is induced in loser cells nearby winner cells remains unknown, observations in this study in conjunction with the previous data on the elimination of ribosomal protein or Hel25E mutant clones suggest the possibility that relative difference in protein synthesis levels between cells plays a critical role in autophagy induction (Nagata, 2022).

The mechanism by which elevated autophagy induces hid expression via NF-κB still remains to be elucidated. Elevated autophagy results in downregulation of IκB protein Cactus. IκB is known to be degraded by the ubiquitin-proteasome system. On the other hand, elevated autophagy by starvation or rapamycin treatment was shown to cause degradation of IκB and thus activate NF-κB in mouse fibroblast. Together, the data suggest the possibility that IκB is degraded by selective autophagy in losers of cell competition (Nagata, 2022).

The observations of this studsy intriguingly show that non-autonomous cell death in wild-type cells promotes fat-induced tumorigenesis. This supports the idea that cancer cells expand their territories within the tissue by cell competition during malignant progression of tumors. While the mechanism by which wild-type cell death fuels neighboring tumorigenesis is an important open question, it may involve compensatory proliferation triggered by mitogenic factors secreted from dying cells. Intriguingly, it has been reported in Drosophila eye-antennal discs that clones of malignant tumors caused by Ras activation and cell polarity defects induce autophagy in surrounding wild-type cells, which in this case do not cause cell death but provide nutrient such as amino acids to neighboring tumors to promote their growth. Clones of cells overexpressing activated form of Yki were also shown to induce autophagy in neighboring cells, but in this case non-autonomous autophagy does not have a role in promoting tumorigenesis. Thus, non-autonomous autophagy may have multiple roles and mechanisms in regulating tissue homeostasis and tumorigenesis (Nagata, 2022).

Given that the Hippo pathway is conserved throughout evolution and that YAP-mediated cell competition occurs in mammalian systems as well, autophagy-mediated cell death may play an important role in mammalian cell competition. Notably, in a mouse liver cancer model, hyperactivation of YAP in peritumoral hepatocytes triggers regression of primary liver tumors and melanoma-derived liver metastases. Thus, further studies on the mechanism of Hippo-signaling-mediated supercompetition in Drosophila may provide a novel therapeutic strategy against human cancers (Nagata, 2022).

Transcriptional Regulation

The correlation between elevated sensor (expressing GFP under control of the tubulin promoter and two copies of the bantam target in the 3'UTR) expression and the zone of nonproliferating cells adjacent to the dorsoventral boundary (ZNC) suggests that Wingless might control cell proliferation in the ZNC by reducing bantam miRNA levels. To test this, use was made of a dominant-negative form of the Wingless receptor DFz2 to locally reduce Wingless activity. Expression of DFz2-GPI under engrailed-Gal4 control reduces bantam sensor levels in the ZNC of the posterior compartment, indicating that bantam miRNA expression increases when Wingless signaling is impaired. Consequently, cells in the posterior ZNC continue to undergo DNA synthesis and are labeled by BrdU incorporation. Comparable results were obtained by overexpression of the Wingless-pathway inhibitor naked. These observations indicate that Wnt signaling contributes to bantam miRNA expression to exert developmental control over cell proliferation in the ZNC. It is noted that the entire posterior compartment is small under these conditions because Wingless is required earlier to promote overall growth of the wing pouch, in addition to its later role in specifying the ZNC. A second area of reduced bantam sensor expression was noted immediately anterior to the AP boundary, where Hedgehog signaling has been shown to induce cell proliferation. These observations provide a link between signaling proteins that serve as morphogens to control spatial pattern and bantam, a regulator of cell proliferation (Brennecke, 2003).

bantam is a target of the hippo tumor-suppressor pathway

The Hippo tumor-suppressor pathway has emerged as a key signaling pathway that controls tissue size in Drosophila. Hippo signaling restricts tissue size by promoting apoptosis and cell-cycle arrest, and animals carrying clones of cells mutant for hippo develop severely overgrown adult structures. The Hippo pathway is thought to exert its effects by modulating gene expression through the phosphorylation of the transcriptional coactivator Yorkie. However, how Yorkie regulates growth, and thus the identities of downstream target genes that mediate the effects of Hippo signaling, are largely unknown. This study reports that the bantam microRNA is a downstream target of the Hippo signaling pathway. In common with Hippo signaling, the bantam microRNA controls tissue size by regulating cell proliferation and apoptosis. hippo mutant cells had elevated levels of bantam activity; and bantam is required for Yorkie-driven overgrowth. Additionally, overexpression of bantam is sufficient to rescue growth defects of yorkie mutant cells and to suppress the cell death induced by Hippo hyperactivation. Hippo regulates bantam independently of cyclin E and diap1, two other Hippo targets, and overexpression of bantam mimics overgrowth phenotypes of hippo mutant cells. These data indicate that bantam is an essential target of the Hippo signaling pathway to regulate cell proliferation, cell death, and thus tissue size (Nolo, 2006).

To test whether the activity of the bantam miRNA is regulated by Hpo signaling, use was made of a GFP bantam sensor that reports the spatial activity of bantam. This bantam sensor expresses GFP under the control of a ubiquitously active tubulin promoter and has two perfect bantam target sites in its 3′ UTR. When present, the bantam miRNA reduces GFP expression through its RNAi effect. The expression pattern of GFP is thus a negative image of the activity pattern of the bantam miRNA. In third-instar wing imaginal discs, the bantam sensor is expressed in a complex pattern with higher levels along the presumptive wing margin, in the anterior compartment along the anteroposterior compartment boundary, and in several patches in the thorax region. Overexpression of the bantam miRNA in the developing wing eliminated the GFP expression of the bantam sensor in the corresponding region, demonstrating that the expression of GFP is indeed under the control of bantam. In developing eye discs, the bantam sensor is also broadly expressed, with higher levels in differentiating photoreceptor cells. As in wing discs, overexpression of bantam downregulated GFP expression in eye discs. The bantam sensor thus reflects the activity of the bantam miRNA in eye and wing discs (Nolo, 2006).

To address whether Hpo signaling regulates the activity of the bantam miRNA, GFP expression of the bantam sensor was monitored in imaginal discs that had defects in Hpo signaling. It was found that hpo or wts mutant cells had lower levels of bantam-sensor-driven GFP expression throughout the mutant clones. Significantly, hpo and wts mutant clones showed lower levels of GFP in multiple tissues, including the wing, antenna, and eye imaginal discs. In eye imaginal discs, wts clones affected the bantam sensor anterior to the morphogenetic furrow, where cells are still uncommitted as well as posterior to the furrow in differentiating photoreceptor cells. In all cases, the regulation of the bantam sensor was cell autonomous. In addition, wing imaginal discs that overexpressed Yki had lower levels of bantam sensor expression in the entire region of Yki overexpression. In summary, it is concluded that Hpo signaling generally regulates bantam expression in multiple imaginal discs and cell types (Nolo, 2006).

A model is postulated in which bantam is an essential target of the Hpo signaling pathway to regulate cell proliferation, cell death, and thus tissue size. This model is based on several observations. First, it was found that bantam is regulated by Hpo signaling broadly and in various tissues. This regulation is a specific downstream effect of Hpo signaling and is not simply the consequence of the cell proliferation induced in hpo mutant cells. Second, bantam is required for Yki to drive tissue overgrowth, because removal of bantam suppresses the overgrowth phenotypes caused by overexpression of Yki in the retina. Third, overexpression of bantam rescues the cell death induced by overexpressed Hpo and significantly rescues growth defects of yki mutant cells. And fourth, bantam overexpression mimics the phenotypes of hypomorphic hpo mutations. Taken together, these data support a model in which bantam is an important downstream target of the Hpo pathway (Nolo, 2006).

The finding that Hpo signaling regulates the expression of bantam raises the question of how important this effect is for Hpo signaling to control tissue size. Removal of bantam suppresses the induction of extra interommatidial cells in the retina by Yki overexpression but does not cause a general elimination of retinal cells in a wild-type background. These data indicate that the regulation of bantam is an essential downstream effect of Hpo signaling to regulate tissue size. However, loss of bantam only partially suppresses the effects of Yki overexpression, indicating that Yki regulates other targets in addition to bantam. Hpo was found to regulate bantam independently of cyclin E and diap1, two other genes known to be regulated by Hpo signaling. bantam is thus not a component of the Hpo signal transduction pathway itself, but is one of several downstream target genes. Yki must have targets in addition to bantam, cyclin E, and diap1, because overexpression of bantam, Cyclin E, and DIAP1 together did not induce the amount of overgrowth caused by Yki overexpression in wing discs. Nevertheless, overexpression of bantam alone caused phenotypes resembling hypomorphic situations for Hpo signaling, indicating that bantam is a critical mediator of Hpo function. Whether the regulation of bantam by a Yki-containing transcription factor complex is direct remains to be determined. However, the fact that Hpo regulates bantam cell autonomously and in multiple tissues is consistent with such a model (Nolo, 2006).

bantam expression is spatially modulated, and patterning signals such as Wg and Dpp also regulate the expression of bantam to generate its expression pattern. These patterning signals regulate specific aspects of the bantam expression pattern, and they have different effects on cell proliferation as well as bantam activity in different regions in various imaginal discs. In contrast, hpo mutant cells upregulate bantam activity independently of cell type and in multiple imaginal discs, indicating an intimate relationship. Hpo is thus a more general and ubiquitous regulator of bantam expression in imaginal discs. An important question that remains to be answered is how these patterning signals regulate tissue growth and bantam expression and whether they regulate bantam expression directly and independently of Hpo signaling or through the regulation of Hpo activity (Nolo, 2006).

Surprisingly, just the opposite of hpo mutant cells, TSC1 mutant cells had lower levels of bantam activity although these cells overgrow, indicating that TSC1 mutant cells induce growth independently of bantam. Neither Myc, Ras, nor Cyclin D-Cdk4 expression induced bantam, although they induce cell growth and proliferation. bantam is thus not simply a part of the cell-intrinsic machinery that executes cell growth and division but rather acts as an upstream component to instruct cells to proliferate. In summary, although Hpo is a key regulator of bantam expression, bantam is also regulated by other pathways potentially integrating the effects of several growth-regulatory and patterning pathways (Nolo, 2006).

miRNAs and their target genes often show mutually exclusive expression patterns, and miRNAs induced during differentiation tend to target messages that were abundant in the previous developmental stage. miRNAs may thus provide a rapid and effective means to suppress expression of residual, unwanted mRNAs while the transcriptional program in a cell is changing. Hpo signaling is involved in regulating cell proliferation and apoptosis in developing imaginal discs. Cell lineages and cell proliferation show significant plasticity in growing imaginal discs, which can rapidly respond to surgical ablation or genetic insults by regenerating missing (eliminated) cells or by ablating unwanted (extra) cells. This adjustment of cell proliferation and apoptosis requires a mechanism that can rapidly change the growth properties of a cell. Yki appears to regulate cell number on the one hand by inducing the expression of positive regulators of cell proliferation and cell survival and on the other hand by inducing the expression of bantam, which posttranscriptionally suppresses the expression of proteins that inhibit cell proliferation and induce apoptosis. An example of such cooperative action of Yki and bantam is the regulation of Hid: Yki suppresses the expression of hid, but also induces bantam, which then suppresses the translation of hid mRNAs that may still be present in a cell. The induction of bantam by Yki may also accelerate the repression of negative growth regulators, thereby enabling a cell to more quickly and robustly adjust its rate of cell proliferation. It will be interesting to elucidate how bantam regulates growth and how its growth targets are integrated with other targets of Hpo signaling (Nolo, 2006).

The Hippo pathway regulates the bantam microRNA to control cell proliferation and apoptosis in Drosophila

The Hippo signaling pathway acts upon the Yorkie transcriptional activator to control tissue growth in Drosophila. Activated Yorkie drives growth by stimulating cell proliferation and inhibiting apoptosis, but how it achieves this is not understood. Yorkie is known to activate Cyclin E (CycE) and the apoptosis inhibitor DIAP1. However, overexpression of these targets is not sufficient to cause tissue overgrowth. This study shows that Yorkie also activates expression of the bantam microRNA, a known regulator of both proliferation and apoptosis. bantam overexpression mimics Yorkie activation while loss of bantam function slows the rate of cell proliferation. bantam is necessary for Yorkie-induced overproliferation and bantam overexpression is sufficient to rescue survival and proliferation of yorkie mutant cells. Finally, bantam levels are shown to be regulated during both developmentally programmed proliferation arrest and apoptosis. In summary, the results show that the Hippo pathway regulates expression of bantam to control tissue growth in Drosophila (Thompson, 2006).

The Hippo pathway is unique in its direct and dedicated role in the intrinsic program of growth in proliferating tissues. The potency of the Hippo pathway in driving tissue growth appears to reside in its ability to coordinately stimulate cell proliferation and suppress apoptosis. A key goal is to understand how this coordinate control is achieved. The results show that the bantam microRNA, a known regulator of both cell proliferation and apoptosis, is a critical target of the Hippo pathway. Activated Yki is necessary and sufficient to induce bantam expression and to stimulate cell survival and proliferation. bantam appears to be a key target of Yki because loss of Yki can be rescued by overexpression of bantam. Finally, bantam clearly has an important role in both normal growth and Yki-driven overgrowth because loss of bantam strongly reduces the rate of cell proliferation in either case. Although the bantam microRNA appears not to be conserved in vertebrates, it is possible that other microRNAs play a functionally equivalent role as effectors of the Hippo pathway. Recent work has identified human microRNAs involved in this pathway (Thompson, 2006).

Two lines of evidence indicate that bantam is not the only relevant target of the Hippo pathway. Firstly, loss of bantam does not completely mimic loss of Yki in every respect, because bantam mutant cells do not undergo apoptosis. This difference is likely to reflect the contribution of the Yki target DIAP1, whose absence is known to trigger apoptosis. Secondly, Yki retains some ability to stimulate cell proliferation even in the absence of bantam. Again, this activity may reflect the role of other Yki targets, including CycE, in driving cell proliferation. Thus, the results favor the view that bantam acts in a highly cooperative way with other Yki target genes to mediate the effects of the Hippo pathway on cell proliferation and apoptosis (Thompson, 2006).

The expression of bantam during normal development shows a striking pattern of regulation; it is expressed in proliferating cells but not in quiescent cells or, as has been shown in this work, in certain cells destined for apoptosis. These findings indicate that regulation of bantam is a key feature of the normal program of tissue growth. Previous work has shown that high levels of the Wingless (Wnt) morphogen represses bantam as cells arrest proliferation at the presumptive wing margin. Since the results show that the Hippo pathway regulates bantam, the pattern of bantam expression may reflect regulation of Hippo pathway activity by positional signals. Thus, positional signals could determine the behavior of cells along the spectrum from rapid proliferation to apoptosis simply by controlling the Hippo pathway. Alternatively, positional signals and the Hippo pathway may act independently, with the bantam locus being a regulatory nexus that integrates information from a number of different signaling pathways (Thompson, 2006).

The final finding is that the Hippo pathway also influences epithelial morphogenesis. This function appears to be independent of its role in controlling cell survival and proliferation and does not involve bantam. Interestingly, expression of Yki and Hippo have reciprocal effects on the epithelium, with overexpressed Yki driving apical bulging and overexpressed Hippo causing basal outfolding. In both cases, the cells remain epithelial, indicating that the Hippo pathway controls cell shape without affecting epithelial polarity or integrity. These observations are consistent with previous reports that clones of cells with elevated pathway activity (i.e. mutant for Hippo or other negatively acting components) have a rounded appearance, indicating altered cell affinities, and that mutation of warts also causes apical bulging of epithelia that is attributed to an expanded apical membrane domain. Why cells use the same pathway to control survival, proliferation, and shape remains an intriguing question (Thompson, 2006).

A fuller understanding of the network connecting the Hippo pathway with the basic machinery controlling survival, proliferation, and morphology will be needed to understand how size regulation is connected to pattern formation during normal development. This work allows sketching of the outline of one facet of this network, with the Yki targets bantam, CycE, and DIAP1 cooperating to control survival and proliferation (Thompson, 2006).

Transcription factor choice in the Hippo signaling pathway: homothorax and yorkie regulation of the microRNA bantam in the progenitor domain of the Drosophila eye imaginal disc

The accurate control of cell proliferation and survival is critical for animal development. The Hippo tumor suppressor pathway regulates both of these parameters by controlling the nuclear availability of the transcriptional coactivator Yorkie (Yki), which regulates downstream target genes together with Scalloped (Sd), a DNA-binding protein. This study provides evidence that Yki can also regulate target genes in conjunction with Homothorax (Hth) and Teashirt (Tsh), two DNA-binding transcription factors expressed in the uncommitted progenitor cells of the Drosophila eye imaginal disc. Clonal analyses demonstrate that Hth and Tsh promote cell proliferation and protect eye progenitor cells from apoptosis. Genetic epistasis experiments suggest that Hth and Tsh execute these functions with Yki, in part by up-regulating the microRNA bantam. A physical interaction between Hth and Yki can be detected in cell culture, and this study shows that Hth and Yki are bound to a DNA sequence approximately 14 kb upstream of the bantam hairpin in eye imaginal disc cells, arguing that this regulation is direct. These data suggest that the Hippo pathway uses different DNA-binding transcription factors depending on the cellular context. In the eye disc, Hth and Tsh provide spatial information to this pathway, promoting cell proliferation and survival in the progenitor domain (Peng, 2009).

The evidence suggests that Hth and Tsh function as partners to carry out two main functions in anterior eye disc cells: They repress the expression of the later-acting retinal determination factors, and they promote cell proliferation. That these functions require hth is supported by both loss-of-function studies as well as gain-of-function studies. For example, hth^P2 clones fail to survive anterior to the MF, and Tsh's ability to induce overgrowths when ectopically expressed is abolished in the absence of hth. The involvement of Tsh is supported by gain-of-function experiments and the finding that Hth and Tsh directly interact with each other in vivo. Carrying out loss-of-function genetics for tsh is difficult because this gene is located proximal to the standard Flp recombination targets (FRTs) used to generate mitotic recombination. In addition, the highly related gene tio, which is closely linked to tsh, functions redundantly with tsh in several instances, including some aspects of eye development. Nevertheless, knocking down tsh using RNAi in a tio-null background results in poor survival in the progenitor domain. Taken together, these data provide a compelling argument for Hth + Tsh functioning together to promote cell survival in the anterior eye disc (Peng, 2009).

A functional relationship between Hth and Tsh also exists in other tissues in Drosophila, most notably in both wing and leg imaginal discs, where they are coexpressed in cells that will give rise to the proximal domains of these appendages. In both wings and legs, Tsh has the capacity to regulate hth when expressed in clones, and both tsh and hth have the ability to suppress distal appendage development when misexpressed. However, in these tissues, and unlike the eye disc, Hth + Tsh expression is not correlated with proliferation, which occurs uniformly throughout these discs. Consistently, the expression pattern exhibited by the bantam sensor does not correlate with Hth or Tsh in the leg or wing. The special relationship between proliferation and Hth + Tsh in the eye may be due in part to the Drosophila Pax6 homolog Eyeless (Ey), which is critical for eye identity. Moreover, Ey is found in a complex with Hth in vivo and participates with Hth and Tsh in the repression of retinal determination genes. Thus, it may also be the case that Ey directly participates in the regulation of bantam together with Hth and Yki (Peng, 2009).

Although hth^P2 clones fail to survive in the eye progenitor domain, the data demonstrate that hth is not absolutely required for cells in this domain to proliferate. The effects observed on the bantam sensor are consistent with the idea that hth promotes, but is not essential for, cells to proliferate in the eye progenitor domain. In hth^P2 clones, bantam sensor levels increased above those normally observed in progenitor cells, but not as high as the levels observed in differentiated photoreceptors. Thus, if the level in photoreceptors represents the complete absence of bantam, these data imply that hth only up-regulates bantam over a basal hth-independent level. Moreover, the levels of the bantam sensor in other tissues, such as the wing disc, rarely approach those observed in photoreceptors, suggesting that most cells have some bantam expression, and that bantam regulators, such as hth, only serve to modulate bantam levels (Peng, 2009).

If eye progenitor cells have the capacity to proliferate in the absence of hth, how important is the proliferation-promoting function of hth? Although normal eyes can develop in animals in which hth^P2 clones are generated, this is likely due to the ability of neighboring wild-type cells to compensate in this mosaic situation. In contrast, when wild-type and heterozygous cells are killed (using the EGUF method [ey-Gal4/UAS-flp/GMF-hidwe find that the remaining hth^P2 tissue is unable to produce normal-sized eyes. This experiment suggests that the proliferation-promoting functions of hth in the eye progenitor domain are critical for normal eye development, likely by providing a sufficient pool of progenitor cells prior to differentiation (Peng, 2009).

The Hippo pathway has emerged recently as an important regulator of cell proliferation and survival in both vertebrates and invertebrates. In Drosophila, this pathway appears to regulate proliferation in nearly all tissues. For example, wts^- clones or Yki⁺ clones have the capacity to induce overgrowths throughout the body. As Yki and its mammalian ortholog Yap are transcriptional coactivators that do not have their own DNA-binding domain, they are thought to partner with DNA-binding transcription factors to regulate gene expression. Prior to this work, the only transcription factor proposed to work directly with Yki was Sd, a member of the TEAD/TEF family of DNA-binding proteins. However, unlike other components of the Hippo pathway, the available data suggest that Sd plays a more limited role in cell proliferation and survival in Drosophila. In contrast to its essential role in the wing pouch, sd^- clones survive well in other tissues, including the region of the wing disc that will give rise to the notum of the fly. Similarly, sd-null clones grow well in the eye progenitor domain. Thus, unlike in the wing pouch, sd is not required for cell survival and proliferation in the eye progenitor domain (Peng, 2009).

In contrast to the survival of sd clones in this domain, hth^P2 clones fail to survive in the eye progenitor domain. Thus, analogous to sd in the wing pouch, hth is required for cells to survive and proliferate in the anterior eye imaginal disc. This observation suggests that hth could play an analogous role to sd in this progenitor domain, a view that is supported by the results. This evidence includes (1) Hth can interact with Yki when coexpressed in S2 cells, (2) Hth + Tsh regulate the Yki target bantam, and (3) Hth and Yki are both bound to the same region of the bantam locus in eye discs. Genetically, it was shown that the Hippo pathway is unable to induce overgrowths in the eye progenitor domain without hth, and that Hth + Tsh cannot induce overgrowths in the absence of Yki. These results suggest that Hth + Tsh comprise the DNA-binding transcription factors that function with Yki to regulate proliferation and survival genes, such as bantam. Thus, analogous to Sd in the wing pouch, Hth + Tsh are transcription factors used by the Hippo signaling pathway in eye progenitor cells (Peng, 2009).

The finding that Hth + Tsh play an analogous role in the eye progenitor domain as Sd does in the wing pouch has several implications for how the Hippo pathway is regulated in vivo. For one, the use of different DNA-binding transcription factors to regulate Hippo target genes suggests a previously unknown degree of specificity available to this pathway. Hth, a TALE family homeodomain protein, and Tsh, a Zn finger protein, are likely to bind very different target DNA sequences than Sd, a TEAD/TEF domain DNA-binding factor. Accordingly, it was found that ectopic Hth + Tsh clones in the eye disc do not consistently up-regulate diap1 or expanded, known Sd-Yki targets in the wing disc (Peng, 2009).

These results also imply that the transcriptional regulation of hth, tsh, and sd has the potential to change the output of the Hippo pathway. Because hth and tsh are transcriptionally repressed by signals coming from the MF, these factors are not available to work with the Hippo pathway posterior to the MF. However, loss of Hippo kinase activity can lead to proliferation of differentiated cells posterior to the MF. In these cells, sd is expressed, suggesting that Yki may use this transcription factor in this context. Analogously, loss of Hippo kinase activity can cause overgrowths in the notum as well as in the wing pouch. As sd^- clones grow well in the notum, but not in the wing pouch, these data suggest that the notum overgrowths may be mediated by a transcription factor other than Sd. hth clones also survive well in the notum, implying that yet another transcription factor or factors may work with Yki in this tissue. In sum, it is suggested that Yki, and thus the Hippo pathway, may be able to work with multiple transcription factors to regulate target genes. In principle, the use of several transcription factors that are themselves developmentally regulated allows the Hippo pathway to be interpreted in different ways in different contexts (Peng, 2009).

Although the data suggest that the Hippo pathway uses Hth + Tsh to up-regulate bantam, they also suggest that both Hth + Tsh and Yki have additional, independent targets. For example, the loss of Hippo kinase activity leads to the up-regulation of diap1 throughout the eye disc. Because diap1 is not affected when Hth + Tsh are coexpressed, the Hippo pathway has the capacity to regulate some genes independently of Hth + Tsh, even in the eye progenitor domain. Moreover, at least when Yki is ectopically expressed, sd appears to be required in all regions of the eye disc for diap1 activation. Thus, although it has not been shown that sd is required for endogenous diap1 expression in this tissue, these data suggest that Yki may use both Sd and Hth + Tsh to regulate gene expression in the eye disc. In fact, it has been suggested that sd is also a modifier of bantam expression in the eye disc and that sd is required for normal-sized eyes. However, these clones, which used RNAi to knock down Sd, grew well in the eye progenitor domain. In addition, the smaller eyes observed when sd was knocked down could be due to the earlier embryonic expression of the Gal4 driver used in these experiments. In contrast, when generated during larval stages, hth^- clones, but not sd^- clones, fail to survive in the eye progenitor domain, arguing that, at least post-embryonically, gene regulation by Hth + Tsh, not Sd, is critical for cell survival in this tissue. This conclusion is also supported by the finding that Hth + Tsh can induce proliferation in the absence of sd (Peng, 2009).

As shown previously, Hth + Tsh play a key role in blocking eye differentiation by repressing the retinal determination genes eya and so. The available data do not yet resolve whether this repression works independently of the Hippo pathway. In contrast, the loss of Hippo kinase activity leads to overgrowths without blocking differentiation, arguing that nuclear Yki promotes proliferation without changing cell fate. Consistently, it was found that wts^- or Yki⁺ clones do not alter Elav expression in differentiated photoreceptors. Curiously, however, ectopic expression of Hth + Tsh did not block differentiation in the absence of Yki. Although these data could be interpreted to suggest that Yki is directly required for repressing differentiation, they could alternatively suggest that repression requires cell proliferation. Consistently, Hth + Tsh were also unable to block differentiation in the absence of bantam. These observations raise the possibility that the absence of bantam or yki indirectly inhibits Hth + Tsh's ability to repress differentiation by compromising the proliferation of these cells, although other indirect affects are also possible (Peng, 2009).

Hth + Tsh are also likely to regulate genes in addition to bantam to promote proliferation and survival in the eye progenitor domain. This is most strongly supported by the observation that ectopic expression of bantam only partially rescues the survival of hth^P2 clones. In addition, it was found that the overgrowths generated by ectopic expression of Hth + Tsh are only partially suppressed by the coexpression of Hpo, whose overexpression removes Yki from the nucleus. These data suggest that some of the Hth + Tsh targets that mediate growth and survival in the eye progenitor domain are regulated independently of Yki (Peng, 2009). In summary, these results suggest that the transcriptional regulation of hth and tsh along the anterior-posterior axis of the eye disc changes the output of the Hippo pathway. In the eye progenitor domain, where Hth and Tsh are both present, the pathway uses these transcription factors to promote proliferation and cell survival, at least in part by up-regulating bantam. Once hth and tsh are repressed by signals coming from the MF, the Hippo pathway may use other transcription factors, such as Sd, to regulate a different set of target genes. Thus, together with other functions carried out by these transcription factors, their regulation across the anterior-posterior axis coordinates the complex switch from proliferation to differentiation during eye development (Peng, 2009).

Regulation of Drosophila glial cell proliferation by Merlin-Hippo signaling

Glia perform diverse and essential roles in the nervous system, but the mechanisms that regulate glial cell numbers are not well understood. This study identified and characterize a requirement for the Hippo pathway and its transcriptional co-activator Yorkie in controlling Drosophila glial proliferation. Yorkie was found to be both necessary for normal glial cell numbers and, when activated, sufficient to drive glial over-proliferation. Yorkie activity in glial cells is controlled by a Merlin-Hippo signaling pathway, whereas the upstream Hippo pathway regulators Fat, Expanded, Crumbs and Lethal giant larvae have no detectable role. Functional characterization of Merlin-Hippo signaling was extended by showing that Merlin and Hippo can be physically linked by the Salvador tumor suppressor. Yorkie promotes expression of the microRNA gene bantam in glia, and bantam promotes expression of Myc, which is required for Yorkie and bantam-induced glial proliferation. These results provide new insights into the control of glial growth, and establish glia as a model for Merlin-specific Hippo signaling. Moreover, as several of these genes have been linked to human gliomas, the results suggest that this linkage could reflect their organization into a conserved pathway for the control of glial cell proliferation (Reddy, 2011).

Merlin was first identified as the product of a human tumor suppressor gene, NF2, loss of which in peripheral glial cells results in benign tumors. Merlin has also been identified as an inhibitor of gliomas. The current observations indicate that the role of Merlin as a negative regulator of glial cell proliferation is conserved from humans to Drosophila and, thus, that Drosophila can serve as a model for understanding Merlin-dependent regulation of glial growth (Reddy, 2011).

Studies in Drosophila imaginal discs first linked Merlin to Hippo signaling, and Merlin was subsequently linked to Hippo signaling in mammalian cells, including its role in meningioma. However, the tumor suppressor activity of Merlin has also been linked to other downstream effectors in mammals, including Erb2, Src, ras, rac, TORC1 (CRTC1 -- Human Gene Nomenclature Database) and CRL4 (IL17RB -- Human Gene Nomenclature Database), creating some uncertainty regarding the general importance of the linkage of Merlin to Hippo in growth control. This study found that depletion of Merlin, depletion of other tumor suppressors in the Hippo pathway, or expression of an activated form of Yki, all result in similar glial overgrowth phenotypes. Moreover, depletion of Merlin increased nuclear localization of Yki, and depletion of Yki suppressed the overgrowth phenotype of Merlin. Together, these observations clearly establish that the glial overgrowth phenotype associated with Merlin depletion in Drosophila is mediated through the Hippo signaling pathway (Reddy, 2011).

A noteworthy feature of Hippo signaling in Drosophila glial cells is that Merlin appears to be uniquely required as an upstream regulator of Hippo signaling, as the Fat-dependent, Ex-dependent and Lgl-dependent branches have no detectable role. Glia might, thus, provide an ideal model for mechanistic investigations of the Merlin branch of Hippo signaling. Fat-Hippo signaling employs Fat as a transmembrane receptor and Dachsous as its transmembrane ligand, whereas Ex-Hippo signaling appears to employ Crumbs as a transmembrane receptor and ligand. By contrast, Drosophila transmembrane proteins that mediate extracellular signaling and interact with Merlin have not yet been identified. Distinct mechanisms might also be involved in signal transduction downstream of Merlin. Although there is evidence that Ex and Merlin can both influence Hippo activity, Ex, but not Mer, can directly associate with Hpo. Conversely, Merlin, but not Ex, can interact directly with Salvador, and Merlin, Salvador and Hippo can form a trimeric complex. Moreover, the kibra loss-of-function phenotype is weaker than expanded in imaginal discs, but comparable to Merlin, and it was found that depletion of kibra also has a significant effect on glial cell proliferation. Kibra is highly expressed in mammalian brain, and alleles of KIBRA (WWC1 -- Human Gene Nomenclature Database) have been linked to human memory performance. The role of kibra in regulating glial cell numbers in Drosophila thus raise the possibility that the influence of KIBRA on human memory might reflect a role in glial cells (Reddy, 2011).

Finally, it is noted that although Hippo signaling has been investigated in several different organs in Drosophila, including imaginal discs, ovarian follicle cells, neuroepithelial cells and intestinal cells, these all involve roles in epithelial cells, in which upstream regulators of the pathway (e.g. Fat, Ex, Mer) all have a distinctive localization near adherens junctions. The identification of a requirement for Hippo signaling in glia is the first time in Drosophila that a role for the pathway has been identified in non-epithelial cells. Indeed, in previous studies it was found that Hippo signaling influences proliferation of neuroepithelial cells, but other neuronal cell types, including neuroblasts, ganglion mother cells and neurons, are insensitive to Yki (Reddy, 2011).

Considerable attention has been paid to genes for which mutation or inappropriate activation can cause over-proliferation of glial cells, resulting in glial tumors. However, less is known about the mechanisms required for normal glial growth. Through loss-of-function studies, several genes essential for normal glial cell numbers were identified, including yki, sd, ban, mad and myc. The requirement for yki, mad and sd, together with epistasis studies, identifies a requirement for active Yki in glial growth. This in turn implies that downregulation of Hippo signaling is important for normal glial growth. Understanding how this is achieved will provide further insights into the regulation of glial cell numbers (Reddy, 2011).

A requirement for Mad, together with its upstream regulator Thickveins (Tkv), in promoting retinal glial cell proliferation was has been established in previous studies. Current studies of glial cells, together with recent work in imaginal discs, emphasize that in mediating the growth-regulating activity of Hippo signaling, Yki utilizes multiple DNA-binding partners (i.e. Mad and Sd) in the same cells at the same time to regulate distinct downstream target genes required for tissue growth (Reddy, 2011).

Although Yki activity influenced glial cell numbers throughout the nervous system, direct analysis of cell proliferation by EdU labeling revealed that retinal glia were more sensitive to Yki activation at late third instar than central brain glia, and significant induction of central brain glial cell proliferation was only observed when Yki activation was combined with Myc over-expression. Further studies will be required to define the basis for this differential sensitivity, but the implication that the proliferative response to Yki is modulated by developmental stage and/or glial cell type has important implications for diseases associated with both excess and deficits of glial cells (Reddy, 2011).

These studies in Drosophila delineate functional relationships among genes involved in the control of glial cell proliferation. Mammalian homologs of Merlin, Yki and Myc have been implicated in glioma. Although a mammalian homolog of ban has not been described, other miRNAs have also been linked to glioma. These observations imply that these genes can be placed into a pathway, in which Merlin, through Hippo signaling, regulates Yki, Yki regulates ban, and ban regulates Myc. However, as expression of Myc alone did not lead to substantial overgrowth of glia, Yki and ban must also have other downstream targets important for the promotion of glial cell proliferation. Moreover, the current observations indicate that a Yki-Sd complex is also required for glial growth. In addition to the well characterized downstream target Diap1, Yki-Sd complexes in glial cells might regulate Myc directly, as suggested by studies in imaginal discs, and might regulate cell cycle genes in conjunction with E2F1 (Reddy, 2011).

The influence of activated-Yki on a ban-GFP sensor, together with the observations that yki is not required for ban-mediated overgrowth, whereas ban is required for Yki-mediated overgrowth, position ban downstream of Yki. This is consistent with studies of Hippo signaling in imaginal discs, in which ban has also been identified as a target of Yki for growth regulation. The placement of Myc downstream of Yki and ban is supported by the observation that Myc levels can be increased by expression of ban or activated-Yki, and by genetic tests that indicate that Myc is required for Yki- and ban-promoted glial overgrowth. A mechanism by which ban can regulate Myc levels, involving downregulation of a ubiquitin ligase that negatively regulates Myc, was identified recently in imaginal discs, and might also function in glial cells. Myc has been reported to downregulate Yki expression in imaginal discs and, although this study has not investigated whether a similar negative-feedback loop exists in glial cells, the synergistic enhancement of glial cell proliferation observed when Yki and Myc were co-expressed is consistent with this possibility, as the expression of both genes under heterologous promoters could bypass negative regulation of Yki by Myc (Reddy, 2011).

The Myc proto-oncogene is de-regulated or amplified in several human cancers, including gliomas. The sensitivity of Yki/ban-induced overgrowth to reduced Myc levels parallels studies of glioma models involving other signaling pathways. For example, Myc is upregulated by EGFR, and is limiting for EGFR-PI3K-induced glial cell overgrowth in a Drosophila glioma model, and p53 and Pten-driven glioma in mouse models is also Myc dependent. Considering the evidence linking Merlin and Yap to glial growth in mammals, and the identification of Myc as a downstream target of Yap in cultured cells, it is likely that Yap could also influence glial growth in mammals, in part, through regulation of Myc (Reddy, 2011).

The full-length transcripts and promoter analysis of intergenic microRNAs in Drosophila melanogaster

MicroRNA (miRNA) transcription is poorly understood until now. To increase miRNA abundance, miRNA transcription was stimulated with CuSO(4) and Drosha enzyme was knocked down using dsRNA in Drosophila S2 cells. The full length transcripts of bantam, miR-276a and miR-277, the 5'-end of miR-8, the 3'-end of miR-2b and miR-10 were obtained. A series of miRNA promoter analyses was conducted to prove the reliability of RACE results. Luciferase-reporter assays proved that both bantam and miR-276a promoters successfully drove the expressions of downstream luciferase genes. The promoter activities were impaired by introducing one or multiple mutations at predicted transcription factor binding sites. Chromatin immunoprecipitation analysis confirmed that hypophosphorylated RNA polymerase II and transcription factor c-Myc physically bind at miRNA promoters. RNA interference of transcription factors Mad and Prd led to down-expression of bantam, miR-277 and miR-2b but not miR-276a, whereas RNAi of Dorsal had the opposite effect (Qian, 2011).

The bHLH factors Dpn and members of the E(spl) complex mediate the function of Notch signalling regulating cell proliferation during wing disc development

The Notch signalling pathway plays an essential role in the intricate control of cell proliferation and pattern formation in many organs during animal development. In addition, mutations in most members of this pathway are well characterized and frequently lead to tumour formation. The Drosophila imaginal wing discs have provided a suitable model system for the genetic and molecular analysis of the different pathway functions. During disc development, Notch signalling at the presumptive wing margin is necessary for the restricted activation of genes required for pattern formation control and disc proliferation. Interestingly, in different cellular contexts within the wing disc, Notch can either promote cell proliferation or can block the G1-S transition by negatively regulating the expression of dmyc and bantam micro RNA. The target genes of Notch signalling that are required for these functions have not been identified. This study shows that the Hes vertebrate homolog, deadpan (dpn), and the Enhancer-of-split complex (E(spl)C) genes act redundantly and cooperatively to mediate the Notch signalling function regulating cell proliferation during wing disc development (San Juan, 2012).

The sterile 20-like kinase tao controls tissue homeostasis by regulating the hippo pathway in Drosophila adult midgut

The proliferation and differentiation of adult stem cells must be tightly controlled in order to maintain resident tissue homeostasis. Dysfunction of stem cells is implicated in many human diseases, including cancer. However, the regulation of stem cell proliferation and differentiation is not fully understood. This study shows that the sterile-like 20 kinase, Tao, controls tissue homeostasis by regulating the Hippo pathway in the Drosophila adult midgut. Depletion of Tao in the progenitors leads to rapid intestinal stem cell (ISC) proliferation and midgut homeostasis loss. Meanwhile, it was find that the STAT signaling activity and cytokine production are significantly increased, resulting in stimulated ISC proliferation. Furthermore, expression of the Hippo pathway downstream targets, Diap1 and bantam, is dramatically increased in Tao knockdown intestines. Consistently, it was shown that the Yorkie (Yki) acts downstream of Tao to regulate ISC proliferation. Together, these results provide insights into understanding of the mechanisms of stem cell proliferation and tissue homeostasis control (X. Huang, 2014).

Inverse regulation of two classic Hippo pathway target genes in Drosophila by the dimerization hub protein Ctp

The LC8 family of small ~8 kD proteins are highly conserved and interact with multiple protein partners in eukaryotic cells. LC8-binding modulates target protein activity, often through induced dimerization via LC8:LC8 homodimers. Although many LC8-interactors have roles in signaling cascades, LC8's role in developing epithelia is poorly understood. Using the Drosophila wing as a developmental model, this study found that the LC8 family member Cut up (Ctp) is primarily required to promote epithelial growth, which correlates with effects on the pro-growth factor dMyc and two genes, diap1 and bantam, that are classic targets of the Hippo pathway coactivator Yorkie. Genetic tests confirm that Ctp supports Yorkie-driven tissue overgrowth and indicate that Ctp acts through Yorkie to control bantam (ban) and diap1 transcription. Quite unexpectedly however, Ctp loss has inverse effects on ban and diap1: it elevates ban expression but reduces diap1 expression. In both cases these transcriptional changes map to small segments of these promoters that recruit Yorkie. Although LC8 complexes with Yap1, a Yorkie homolog, in human cells, an orthologous interaction was not detected in Drosophila cells. Collectively these findings reveal that that Drosophila Ctp is a required regulator of Yorkie-target genes in vivo and suggest that Ctp may interact with a Hippo pathway protein(s) to exert inverse transcriptional effects on Yorkie-target genes (Barron, 2016).

The LC8 family of cytoplasmic dynein light-chains, which includes vertebrate LC8 (aka DYNLL1/DYNLL2) and Drosophila Cut-up (Ctp), are small highly conserved proteins that are ubiquitously expressed and essential for viability. The LC8 protein is 8 kilodaltons (kD) in size and was first identified as an accessory subunit in the dynein motor complex, within which an LC8 homodimer binds to and stabilizes a pair of dynein intermediate chains (DIC). However, the LC8 protein has since emerged as a general interaction hub with multiple dynein/motor-independent roles and binding partners. In fact the majority of LC8 protein in mammalian cells is not associated with either dynein or microtubules, and LC8 orthologs are encoded in the genomes of flowering plants that otherwise lack genes encoding heavy-chain dynein motors (Barron, 2016 and references therein).

Accumulating evidence has reinforced the idea that the primary role of LC8 in mammalian cells is to facilitate dimerization of its binding partners via LC8 self-association, a mechanism that has been termed 'molecular velcro'. LC8 can be found in association with over 40 proteins that function in diverse cellular processes, including intracellular transport, nuclear translocation, cell cycle progression, apoptosis, autophagy, and gene expression. LC8 is found in both the nucleus and cytoplasm and can interact with partners in either compartment. For example the mammalian kinase Pak1 binds and phosphorylates LC8 in the cytoplasm, which in turn enhances the ability of LC8 to interact with the BH3-only protein Bim and inhibit its pro-apoptotic activity. Accordingly, overexpression of LC8 or the phosphorylation of LC8 by Pak1 enhances survival and proliferation of breast cancer cells in culture. LC8 also binds estrogen receptor-α (ERα) and facilitates ERα nuclear translocation, which in turn recruits LC8 to the chromatin of ERα-target genes. In the cytoplasm, LC8 is also found in association with the kidney and brain expressed protein (KIBRA), which is an upstream regulator of the Hippo tumor suppressor pathway. KIBRA binding potentiates the effect of LC8 on nuclear translocation of ERα, suggesting crosstalk may occur between LC8-regulated pathways. The KIBRA-LC8 complex also interacts with Sorting Nexin-4 (Snx4) to promote dynein-driven traffic of cargo between the early and recycling endosomal compartments. Thus, LC8 has been linked to a variety of proteins in both the cytoplasm and nucleus that play important roles in signaling, membrane dynamics, and gene expression (Barron, 2016).

Drosophila Ctp differs from vertebrate LC8/DYNLL by only four conservative amino acid substitutions across its 89 amino acid length. Similar to mammalian LC8, phenotypes produced by Ctp loss in flies imply roles in multiple developmental mechanisms. Drosophila completely lacking Ctp die during embryogenesis due to excessive and widespread apoptosis. Partial loss of Ctp function causes thinned wing bristles and morphogenetic defects in wing development, as well as ovarian disorganization and female sterility. Within salivary gland cells, Ctp promotes autophagy during pupation, while in neuronal stem cells it localizes to centrosomes and influences mitotic spindle orientation and the symmetry of cell division. Testes mutant for ctp have motor-dependent defects in spermatagonial divisions as well as motor-independent defects in cyst cell differentiation. A recent study linked ctp mRNA expression to the zinc-finger transcription factor dASCIZ and showed that knockdown of either Ctp or dASCIZ reduces wing size. In sum, this diversity of effects produced by Ctp loss in different Drosophila cell types suggest that Ctp plays important yet context specific roles in vivo. However, knowledge of molecular pathways that require Ctp, and in turn underlie these developmental phenotypes associated with Ctp loss, remain poorly characterized (Barron, 2016).

This study used a genomic null allele of ctp and a validated ctp RNAi transgene to assess the role of the Ctp/LC8/DYNLL protein family in pathways that act within the developing Drosophila wing epithelium. Clones of ctp null cells are quite small relative to controls and RNAi depletion of Ctp shrinks the size of the corresponding segment of the adult wing without clear defects in mitotic progression or tissue patterning. The effect of Ctp depletion on adult wing size is primarily associated with a reduction in cell size, rather than cell division or cell number, implying a role for Ctp in supporting mechanisms that enable developmental growth. In assessing the effect of Ctp loss on multiple pathways that control wing growth, robust effects were detected on one-the Hippo pathway. The Hippo pathway is a conserved growth suppressor pathway that acts via its core kinase Warts to inhibit nuclear translocation of the coactivator Yorkie (Yki), which otherwise enters the nucleus, complexes with the DNA-binding factor Scalloped (Sd), and activates transcription of growth and survival genes. In parallel to the effect of Ctp loss on clone and wing size, Ctp loss alters expression of the classic Yki target genes bantam and thread(th)/diap-1 in wing pouch cells. Parallel genetic tests confirm a requirement for ctp in Yki-driven tissue growth in the wing or eye. Quite unexpectedly however, Ctp loss has opposing effects on bantam and diap1 transcription in wing pouch cells: bantam transcription is strongly elevated while diap1 expression is strongly decreased in cells lacking Ctp. In each case, these effects map to small segments of DNA in the ban and diap1 promoters that recruit Yki transcriptional complexes. Epistasis experiments confirm that Yki is required to activate the bantam promoter in Ctp-depleted cells, and that transgenic expression of Yki can overcome the block to diap1 transcription. In sum these data argue that Ctp supports physiologic Hippo signaling in wing disc epithelial cells, and that Ctp likely interacts with an as yet unidentified Hippo pathway protein(s) to exert inverse transcriptional effects on Yorkie-target genes. These types of inverse effects have not previously been described within the Hippo pathway, and imply that distinct subsets of genes within the Yorkie transcriptome can be simultaneously activated and repressed in developing tissues via a mechanism that involves Ctp (Barron, 2016).

Notch-dependent epithelial fold determines boundary formation between developmental fields in the Drosophila antenna

Compartment boundary formation plays an important role in development by separating adjacent developmental fields. Drosophila imaginal discs have proven valuable for studying the mechanisms of boundary formation. This study examined the boundary separating the proximal A1 segment and the distal segments, defined respectively by Lim1 and Dll expression in the eye-antenna disc. Sharp segregation of the Lim1 and Dll expression domains precedes activation of Notch at the Dll/Lim1 interface. By repressing bantam miRNA and elevating the actin regulator Enabled, Notch signaling then induces actomyosin-dependent apical constriction and epithelial fold. Disruption of Notch signaling or the actomyosin network reduces apical constriction and epithelial fold, so that Dll and Lim1 cells become intermingled. These results demonstrate a new mechanism of boundary formation by actomyosin-dependent tissue folding, which provides a physical barrier to prevent mixing of cells from adjacent developmental fields (Ku, 2017).

This study attempted to unravel the molecular and cellular mechanisms of boundary formation in the Drosophila head. Focus was placed on the antennal A1 fold that separates the A1 and A2-Ar segments. The results showed that the expression of the selector genes Lim1 and Dll, which are expressed in A1 and A2-Ar, respectively, was sharply segregated. This step was followed by differential expression of Dl, Ser and Fng, as well as activation of N signaling at the interface between A1 and A2. N signaling then induced apical constriction and epithelial fold, possibly through repression of bantam to allow levels of the bantam target Ena to become elevated, with this latter inducing the actomyosin network. The actomyosin-dependent epithelial fold then provided a mechanical force to prevent cell mixing. When N signaling or actomyosin was disrupted, or when bantam was overexpressed, the epithelial fold was disrupted and Dll and Lim1 cells become mixed. Thus this study describes a clear temporal and causal sequence of events leading from selector gene expression to the establishment of a lineage-restricting boundary (Ku, 2017).

Sharp segregation of Dll/Lim1 expressions began before formation of the A1 fold, suggesting that fold formation is not the driving force for segregation of Dll/Lim1 expression. Instead, the fold functions to safeguard the segregated lineages from mixing. Whether Dll/Lim1 segregated expression is due to direct or indirect antagonism between the two proteins is not known (Ku, 2017).

Actomyosin-dependent apical constriction is an important mechanism for tissue morphogenesis in diverse developmental processes, e.g., gastrulation in vertebrates, neural closure and Drosophila gastrulation, as well as dorsal closure and formation of the ventral furrow and segmental groove in embryos. This study describes a new function of actomyosin, i.e., the formation of lineage-restricting boundaries via apical constriction during development (Ku, 2017).

This actomyosin-dependent epithelial fold provides a mechanism distinctly different from other known types of boundary formation. The cells at the A1 fold still undergo mitosis, suggesting that mitotic quiescence is not involved. Perhaps epithelial fold as a lineage barrier is needed in situations in which mitotic quiescence does not happen. Mechanically and physically, epithelial folds could serve as stronger barriers than intercellular cables when mitotic activity is not suppressed. The drastic and sustained morphological changes, including reduced apical area and cell volume, may be accompanied by increased cortical tension of cells along the A1 fold, with such high interfacial tension then preventing cell intermingling and ensuring Dll and Lim1 cell segregation. Although similar to actomyosin boundaries, the epithelial fold in the A1 boundary is distinctly different from the supracellular actomyosin cable structure in fly parasegmental borders, the wing D/V border, and the interrhombomeric boundaries of vertebrates. The adherens junction protein Echinoid, which is known to promote the formation of supracellular actomyosin cables, is not involved in A1 fold formation. Although actomyosin is enriched in a ring of cells in the A1 fold, it does not exert a centripetal force to close the ring, unlike the circumferential cable described in dorsal closure and wound healing (see review. In the A1 fold, the constricting cells become smaller in both their apical and basolateral domains, thus differing from ventral furrow cells where cell volume remains constant (Ku, 2017).

A tissue fold probably provides a strong physical or mechanical barrier to prevent cell mixing. In addition, whereas in a flat tissue where the boundary involves only one to two rows of cells, the tissue fold involves more cells engaging in cell-cell communication. The close apposition of cells within the fold may allow efficient signaling within a small volume. This may be an evolutionarily conserved mechanism for boundary formation that corresponds to stable morphological constrictions such as the joints in the antennae and leg segments (Ku, 2017).

Although N signaling has been reported to be involved in many developmental processes, a role in inducing actomyosin-dependent apical constriction and epithelial fold is a novel described function for N. For the A1 boundary, N activity is possibly mediated through repression of bantam and consequent upregulation of Ena. In the wing D/V boundary, N signaling is also mediated through bantam and Ena, but the outcome is formation of actomyosin cables, i.e., without apical constriction and epithelial fold [19]. Thus, the N/bantam/Ena pathway for tissue morphological changes is apparently context-dependent (Ku, 2017).

Tissue constriction also occurs later in joint formation of the legs and antennae. N activation also occurs in the joints of the leg disc and is required for joint formation. This role is conserved from holometabolous insects like the fruitfly Drosophila melanogaster and the red flour beetle Tribolium castaneum to the hemimetabolous cricket Gryllus bimaculatus. It is possible that for segmented structures that telescope out in the P/D axis, like the antennae, legs, proboscis and genitalia, N signaling is used to demarcate the boundaries between segments, which are characterized by tissue constriction. N-dependent epithelial fold morphogenesis has also been reported in mice cilia body development without affecting cell fate, suggesting that such N-dependent regulation in morphogenesis is evolutionarily-conserved (Ku, 2017).

It is proposed that N signaling is important in all boundaries that involve stable tissue morphogenesis. For those boundaries corresponding to stable morphological constrictions, e.g., the joints in insect appendages, N acts via actomyosin-mediated epithelial fold. The wing D/V boundary represents a different type of stable tissue morphogenesis. It becomes bent into the wing margin and involves N signaling via actomyosin cables, rather than apical constriction. In contrast, actomyosin-dependent apical constrictions do not involved N signaling and are involved in transient tissue morphogenesis, such as gastrulation in vertebrates, neural closure, Drosophila gastrulation, dorsal closure, as well as formation of the ventral furrow, eye disc morphogenetic furrow, and segmental groove in embryos (Ku, 2017).

N signaling is also involved in the boundary between new bud and the parent body of Hydra, where it is required for sharpening of the gene expression boundary and tissue constriction at the base of the bud [78]. Whether the role of N in these tissue constrictions is due to actomyosin-dependent apical constriction and epithelial fold is not known (Ku, 2017).

Boundaries may be established early in development. As the tissue grows in size through cell divisions and growth, boundary maintenance become essential. This study found that N activity is maintained by actomyosin, suggesting feedback regulation to stably maintain the boundary. Mechanical tension generated by actomyosin networks has been suggested to enhance actomyosin assembly in a feedback manner. Interestingly, the N-mediated wing A/P and D/V boundaries, which form actomyosin cables rather than tissue folds, did not exhibit such positive feedback regulation. Instead, the stability of the Drosophila wing D/V boundary is maintained by a complex gene regulatory network involving N, Wg, N ligands and Cut. Perhaps this is necessary for a boundary not involving tissue morphogenesis (Ku, 2017).

The segmented appendages of arthropods (antennae, legs, mouth parts) are homologous structures of common evolutionary origin. It has been proposed that the generalized arthropod appendage is composed of a proximal segment called the coxopodite and a distal segment called the telopodite, either of which can further develop into more segments. The coxopodite is believed to be an extension of the body wall, whereas the telopodite represents the true limb, and thus represents an evolutionary addition. Dll mutants lack all distal segments except for the coxa in legs and the A1 segment in antennae. Lineage tracing studies have shown that Dll-expressing cells contributed to all parts of the legs except the coxa. These results indicate that the leg coxa and antenna A1 segment correspond to the Dll-independent coxopodite, and that Dll is the selector gene for the telopodite. Therefore, the antennal A1 fold is the boundary between the coxopodite and telopodite. It is postulated that the same N-mediated epithelial fold mechanism also operates in the coxopodite/telopodite boundary of legs and other appendages (Ku, 2017).

Growth suppressor lingerer regulates bantam microRNA to restrict organ size

The evolutionarily conserved Hippo signaling pathway plays an important role in organ size control by regulating cell proliferation and apoptosis. This study identified Lingerer (Lig) as a growth suppressor using RNAi modifying screen in Drosophila melanogaster. Loss of lig increases organ size and promotes bantam (ban) and the expression of the Hippo pathway target genes, while overexpression of lig results in diminished ban expression and organ size reduction. Lig C-terminal exhibits dominant-negative function on growth and ban expression, and thus plays an important role in organ size control and ban regulation. In addition, evidence is provided that both Yki and Mad are essential for Lig-induced ban expression. Lig was shown to regulate the expression of the Hippo pathway target genes partially via Yorkie. Moreover, Lig was found to physically interact with and requires Salvador to restrict cell growth. Taken together, this study demonstrates that Lig functions as a critical growth suppressor to control organ size via ban and Hippo signaling (Dong, 2015).

Targets of Activity

Studies on the Myc and E2F oncogenes in vertebrates have shown that strong proliferative stimuli induce apoptosis. Cell proliferation results only when apoptosis is simultaneously prevented. Overexpression of E2F with its cofactor DP causes apoptosis in the Drosophila wing disc, and net cell proliferation results only when apoptosis is blocked by coexpression of the caspase inhibitor P35. In contrast, stimulation of growth by bantam overexpression is not associated with an increase in apoptosis. This raises the possibility that bantam might stimulate cell proliferation and simultaneously suppress apoptosis. It was asked whether bantam could suppress proliferation-induced apoptosis, caused by E2F and DP. Cells expressing E2F and DP under ptc-GAL4 overproliferate, indicated by increased nuclear density in apical optical sections. In basal optical sections, elevated levels of activated caspase 3 are seen. Many of these cells drop out of the epithelial layer and have pyknotic nuclei, indicative of apoptosis. Coexpression of bantam enhances the overproliferation phenotype, indicated by the broader region of high nuclear density and reduces the levels of activated caspase. Fewer cells show pyknotic nuclei, although many cells drop out of the epithelial layer, indicating that they are not entirely healthy. Even the modest level of bantam overexpression produced by EP(3)3622 is sufficient to suppress apoptosis induced by E2F and DP overexpression (Brennecke, 2003).

To identify targets of the bantam miRNA, a computational method was developed based on the known C. elegans miRNA-target pairs and a general understanding of RNA-RNA interactions. A target search using bantam miRNA revealed three independent targets in the 3'UTR of the apoptosis-inducing gene hid. By visual inspection of the 3'UTR of hid, two additional sequences complementary to the bantam miRNA were identified. All five target sites are highly conserved in the predicted hid 3'UTR of D. pseudoobscura. The bantam precursor hairpin from D. pseudoobscura is identical to that from D. melanogaster, except for one base in the terminal loop that is not in the miRNA product. The conservation of these sequences suggests a conserved functional relationship between bantam and hid (Brennecke, 2003).

To assess the function of the predicted bantam target sites, a tubulin-EGFP sensor transgene was produced using the 3'UTR of the hid mRNA. The resulting GFP pattern was identical to that produced by the bantam sensor. In addition, the hid UTR sensor is downregulated when EP(3)3622 is overexpressed under ptc-Gal4 control, indicating that the hid UTR confers bantam-dependent regulation on the transgene. The hid UTR sensor was compared with a version from which bantam target sites one and four were deleted. The mutated sensor with the three gapped bantam sites showed a similar pattern to the complete hid UTR sensor, but was downregulated less strongly by endogenous bantam. Overexpressed bantam reduced its expression, but the difference in magnitude was less than for the intact hid UTR sensor with five sites. These observations indicate that the gapped sites are functional in mediating bantam induced repression, but show that five sites mediate stronger repression than three sites. Cooperativity among multiple sites has also been reported for siRNA-mediated translational repression (Brennecke, 2003).

Having shown that bantam can block expression of a transgene containing the hid 3'UTR, it was asked whether bantam regulates the endogenous hid gene. hid was expressed from EP(3)30060 under ptc-Gal4 control, either alone or together with EP(3)3622. Hid protein levels were reduced by coexpression with, but hid transcript levels were comparable. This indicates that Hid protein expression is repressed by bantam, most likely by blocking translation of the hid mRNA. The ability of bantam to suppress the apoptosis-inducing effects of hid was examined. hid expression induces apoptosis, visualized by caspase 3 activation. This is suppressed by coexpression of bantam. These observations show that bantam effectively suppresses hid-induced apoptosis. The ability of bantam to suppress proliferation-induced apoptosis may reflect its ability to block Hid expression, though the possibility of other indirect effects cannot be excluded (Brennecke, 2003).

Induction of cell death in postmitotic cells of the eye imaginal disc by GMR-hid, GMR-hid(Ala5) and GMR-reaper transgenes leads to a small, rough eye phenotype. Eye size is largely restored by coexpression of bantam using GMR-Gal4 to direct expression of EP(3)3622, though suppression of the GMR-hid and GMR-hid(Ala5) phenotypes is much better. Ommatidial structure is largely restored in the GMR-hid eyes, but not in the GMR-reaper eyes, suggesting a more specific suppression of hid activity. Hid(Ala5) has the 5 consensus ERK phosphorylation sites mutated to alanine and cannot be suppressed by activation of the ERK MAPK pathway. The observation that bantam coexpression blocks the activity of Hid(Ala5) excludes an indirect effect mediated by regulation of the MAPK pathway. To explore the question of specificity further, the effects of removing one copy of the endogenous bantam gene in these three backgrounds was compared. This had a minor effect on the severity of the GMR-reaper, but clearly enhanced the severity of the GMR-hid and GMR-hid(Ala5) phenotypes. This suggests that endogenous expression of the bantam gene in the developing eye imaginal disc contributes to controlling the level of hid-induced apoptosis, which is normally involved in reducing cell number in the pupal eye disc (Brennecke, 2003).

A role for microRNAs in the Drosophila circadian clock

Little is known about the contribution of translational control to circadian rhythms. To address this issue and in particular translational control by microRNAs (miRNAs), the miRNA biogenesis pathway was knocked down in Drosophila circadian tissues. In combination with an increase in circadian-mediated transcription, this severely affected Drosophila behavioral rhythms, indicating that miRNAs function in circadian timekeeping. To identify miRNA-mRNA pairs important for this regulation, immunoprecipitation of AGO1 followed by microarray analysis identified mRNAs under miRNA-mediated control. They included three core clock mRNAs: clock (clk), vrille (vri), and clockworkorange (cwo). To identify miRNAs involved in circadian timekeeping, circadian cell-specific inhibition of the miRNA biogenesis pathway was exploited followed by tiling array analysis. This approach identified miRNAs expressed in fly head circadian tissue. Behavioral and molecular experiments show that one of these miRNAs, the developmental regulator bantam, has a role in the core circadian pacemaker. S2 cell biochemical experiments indicate that bantam regulates the translation of clk through an association with three target sites located within the clk 3' untranslated region (UTR). Moreover, clk transgenes harboring mutated bantam sites in their 3' UTRs rescue rhythms of clk mutant flies much less well than wild-type CLK transgenes (Kadener, 2009).

This study demonstrates a role for miRNAs in the Drosophila central circadian clock. By performing AGO1 immunoprecipitation followed by microarray analysis, a population of mRNAs under miRNA control in fly heads. Among them was the master circadian gene clk. In addition, circadian cell-specific inhibition of the miRNA biogenesis pathway followed by tiling arrays identified several miRNAs prominently expressed in circadian tissues. In combination with bioinformatics analyses, the two approaches identified 10 candidate miRNAs involved in circadian rhythms. For one miRNA, the developmental regulator bantam, evidence is presented for a direct role in circadian timekeeping. Overexpression of bantam using a circadian cell-specific GAL4 line delays by almost 3 h the circadian clock at the molecular and behavioral levels. Moreover, this miRNA regulates clk. This regulation is achieved through three conserved bantam sites in the 3' UTR of this gene. Two are located downstream from the previously annotated clk mRNA 3' end, and other data indicate that the real clk 3' UTR includes these sites. Genetic experiments in flies demonstrate that the integrity of these three bantam sites is critical for robust circadian rhythmicity. Therefore a miRNA-mRNA pair involved in central circadian timekeeping was identified (Kadener, 2009).

This is one of the few studies to use miRNP IP to identify miRNA-regulated mRNAs, and may be the first from adult fly tissues. The data fit well with those derived from the PicTar algorithm and should allow a comparison of different miRNA target prediction algorithms (Kadener, 2009).

The second approach for studying specific miRNA expression relies on cell type-specific inhibition of miRNA synthesis pathways in vivo followed by RNA analysis on tiling arrays. Although very sensitive in identifying many circadian miRNAs, the strategy probably still fails to identify low abundance miRNAs or miRNAs present in small numbers of circadian cells. However, they should be detectable with the same approach, but after a cell purification or cell sorting step. This sensitivity issue is the reason the broad tim-gal4 driver was used rather than the more restricted pdf-gal4 driver. Tim-gal4 is expressed strongly in all circadian tissues of the fly head, including circadian neurons, eyes, fat body, and antennae. This broad expression also explains the strong effect of TIM-Dcr IR on the AGO1 IP enrichment. Consistent with data indicating that core clock components work similarly in both central (brain) and peripheral tissues, bantam overexpression slows the clock pace in both locations: in the central brain as demonstrated by behavior, and in the periphery as demonstrated by luciferase assays (Kadener, 2009).

Intersecting the Ago1 IP data with the tiling array data from Tim-DroshaIR/PashaIR flies as well as with the published fly head miRNA data led to a selection of 10 candidate circadian miRNAs. Since this analysis only used miRNAs with PicTar target predictions and therefore screened only half of the known miRNA population, 10 is likely to be an underestimate. In contrast, of the 27 miRNAs identified as expressed in circadian cells by the Tim-DroshaIR/PashaIR approach, 23 have mRNAs with PicTar predictions in the Ago IP data. This suggests that 10 is not a gross underestimate (Kadener, 2009).

Some of these 10 miRNAs are likely responsible for the decrease in locomotor activity rhythm strength due to inhibition of the miRNA pathway. It is notable that there are no prior reports of a miRNA contribution to circadian behavior in Drosophila and only a single report in mammals. This may be related to the fact that an effect was only manifest at 29°C and with the addition of the UAS-CYC-VP16 transgene. The failure to observe a phenotype in Tim-DcrIR flies at 25°C may reflect a relatively weak effect of the dicer-1 IR transgene on miRNA expression, consistent with the fact that miRNA biosynthesis is not rate-limiting for miRNA-mediated translational regulation. Nonetheless, it is likely that the lack of a circadian defect in Tim-DcrIR flies is not a consequence of inadequate inhibitory transgene expression. This is because the same strain (Tim-DcrIR) still displays normal rhythms even after increasing the temperature to 29°C. Moreover, Tim-Dcr seems to strongly down-regulate the miRNA pathway, as illustrated by the accumulation of pre-bantam and the substantial change in the AGO1 IP profile (Kadener, 2009).

It is therefore suspected that the additional requirement for UAS-CYC-VP16 reflects more than just an increase in UAS-dcr 1 IR expression. It is possible that the transcription and translation of key circadian core components are tightly connected and may buffer each other. Such a regulatory feature could explain why a major increase in transcription, like that caused by the CYC-VP16 transgene, results in only a modest increase in mRNA abundance and probably an even more modest increase in translated protein. A comparable explanation posits that inhibition of the miRNA pathway by the UAS-dcr 1 IR transgene leads to an increase in the translation of circadian repressors, which could then decrease circadian transcription. The use of UAS-CYC-VP16 as well as 29°C might be required to push the system sufficiently far from equilibrium so that pacemaker regulatory mechanisms can no longer compensate for the change in miRNA levels. This type of regulation fits recent data demonstrating that a Drosophila miRNA can function as a buffering agent against environmental perturbations during development (Li, 2009). In any case, the observed behavioral defects observed in Tim-DcrIR-CYCVP16 flies are likely a consequence of down-regulation of several circadian-relevant miRNAs (Kadener, 2009).

Behavioral, genetic, and biochemical evidence indicates that bantam contributes to clk mRNA translational regulation as well as more generally to circadian pacemaker regulation: bantam is highly expressed in circadian tissues, and overexpression with either tim-gal4 or pdf-gal4 significantly lengthens circadian period. The milder effect of the pdf driver may be due to its lower strength in pacemaker cells relative to tim-gal4 and/or to an additional contribution from non-PDF cells to period determination (Kadener, 2009).

Although the period phenotype could be misleading -- due, for example, to an effect of bantam overexpression on a circadian output pathway -- strains with a completely normal central pacemaker do not manifest altered periods, by definition. Another possibility, that bantam overexpression renders the circadian neurons sick or unhealthy, would be expected to result in weak rhythms or arrhythmicity rather than in strong rhythms with long periods. The central pacemaker is therefore the most parsimonious explanation, especially because of the good correlation between the behavioral and the molecular data; i.e., the tim-luciferase results. Unfortunately, the bantam deletion is embryonic lethal, precluding a straightforward behavioral assay of the null phenotype (Kadener, 2009).

The effect of bantam on clk mRNA translation was aided by the finding that the clk 3' UTR extends >700 bases downstream from its predicted 3' end. This error is attributed to priming by oligo (dT) within an A-rich region present near this annotated 3' end. Consistent with this interpretation, a strongly conserved cleavage and polyadenylation site is present near the end of the clk-lg isoform; no obvious site is in the vicinity of the annotated clk 3' end. In addition, RNA protection data indicate that all fly head clk transcripts extend well beyond the annotated clk 3' end. Taken together with the 3' RACE data, these results demonstrate that the clk 3' UTR is significantly longer than previously indicated. Importantly, two of the three clk 3' UTR bantam-binding sites are located downstream from the annotated 3' end (Kadener, 2009).

These clk 3' UTR bantam sites appear to be major circadian targets of bantam in flies. First, clk mRNA is strongly associated with RISC. Second, bantam is strongly expressed in the circadian cells, as demonstrated by the accumulation of precursors of this miRNA when Dicer-1, drosha, or pasha was down-regulated in fly circadian tissues. Third, the effect of bantam (lengthening of the circadian period) resembles the period effect observed in flies carrying fewer genomic copies of clk, and it is opposite to the period effect observed in flies with additional clk copies. Fourth, the three evolutionarily conserved bantam sites are necessary for circadian rhythmicity. Nonetheless, the period effect due to bantam overexpression may be due to effects on other mRNAs in addition to clk (Kadener, 2009).

It is concluded that miRNAs have a role in the central pacemaker and, more specifically, that bantam regulates the central clock component clk. Whereas previous studies have identified miRNAs relevant to circadian rhythms, this one identifies a mRNA-miRNA pair involved in the core timekeeping process. Given the in vivo methods used to study miRNA function (including principally in neuronal tissue), it is suspected that they will have a broad impact on the study of miRNAs and their roles in regulating diverse aspects of Drosophila behavior (Kadener, 2009).

Notch-mediated repression of bantam miRNA contributes to boundary formation in the Drosophila wing

Subdivision of proliferating tissues into adjacent compartments that do not mix plays a key role in animal development. The Actin cytoskeleton has recently been shown to mediate cell sorting at compartment boundaries, and reduced cell proliferation in boundary cells has been proposed as a way of stabilizing compartment boundaries. Cell interactions mediated by the receptor Notch have been implicated in the specification of compartment boundaries in vertebrates and in Drosophila, but the molecular effectors remain largely unidentified. This study presents evidence that Notch mediates boundary formation in the Drosophila wing in part through repression of bantam miRNA. bantam induces cell proliferation and the Actin regulator Enabled was identified as a new target of bantam. Increased levels of Enabled and reduced proliferation rates contribute to the maintenance of the dorsal-ventral affinity boundary. The activity of Notch also defines, through the homeobox-containing gene cut, a distinct population of boundary cells at the dorsal-ventral (DV) interface that helps to segregate boundary from non-boundary cells and contributes to the maintenance of the DV affinity boundary (Becam, 2011).

Cell divisions lead to cell rearrangements that may challenge straight and sharp compartment boundaries. The DV boundary of mid- and late third instar wing primordia is characterized by a reduced rate of cell proliferation which defines the zone of non-proliferating cells (ZNC). The contribution of the ZNC to the maintenance of the DV affinity boundary was proposed many years ago but this notion was subsequently questioned. This study provides evidence that the ZNC does indeed play a role in boundary formation. bantam miRNA positively modulates the activity of the E2F transcription factor and drives G1-S transition in Drosophila tissues. Notch-mediated downregulation of bantam miRNA defines the ZNC and contributes to maintain a stable DV affinity boundary. Induction of proliferation in boundary cells by the ectopic expression of bantam, the cell cycle regulators Cyclin E and String, or the proto-oncogene dMyc, which is known to drive G1-S transition, compromises the formation of a smooth DV affinity boundary. A similar reduction in proliferation rates is observed at the rhombomere boundaries in the developing hindbrain, suggesting that reduced rate of cell proliferation might often be used in compartment boundary formation (Becam, 2011).

Notch-mediated downregulation of bantam activity is not only required to define the ZNC but also to establish the actomyosin cables observed at the interface between boundary and non-boundary cells. Ena, a regulator of Actin elongation, was identified as a direct target of bantam that is involved in DV boundary formation. The multiple roles of bantam in promoting G1-S transition and tissue growth, blocking apoptosis and regulating Actin dynamics unveil a new molecular connection between these three processes that might have relevance in growth control and tumorigenesis (Becam, 2011).

Intriguingly, bantam miRNA has no major role in the maintenance of the anterior-posterior compartment boundary of the developing wing and this boundary is not affected upon depletion of Ena protein levels. Thus, different regulators of actin elongation might be at work to regulate the actomyosin cytoskeleton and direct cell sorting in diverse developmental contexts. Whether reduced levels of bantam miRNA and increased levels of Ena protein are required to maintain differential cell sorting in the embryonic ectoderm or other imaginal tissues remains to be elucidated (Becam, 2011).

Cut is a late target of Notch that is expressed in boundary cells and is required to induce a stable Notch signaling center. This study demonstrate that Cut activity has also a specific function in reducing Ena mRNA and protein levels in boundary cells. Although depletion of Cut compromises the formation of the actomyosin cables at the interface of boundary and non-boundary cells and the maintenance of a stable DV affinity boundary, cell lineage and clonal analysis of wild-type and cut mutant cells reveal that Cut plays a major role in sorting boundary from non-boundary cells. The finding that the Notch signaling pathway defines, through Cut, a distinct population of boundary cells at the DV interface reinforces the mechanistic similarities in the maintenance of compartment boundaries within the vertebrate hindbrain and the Drosophila wing. In both developmental contexts, Notch defines a distinct population of boundary cells and contributes to segregating boundary from non-boundary cells. Although Cut mediates the role of Notch in the Drosophila wing, the molecular effectors mediating the role of vertebrate Notch in boundary formation remain uncharacterized. The data indicate that the later subdivision into boundary and non-boundary cells contributes to the maintenance of a stable DV affinity barrier in the mature wing primordium (Becam, 2011).

A miR-130a-YAP positive feedback loop promotes organ size and tumorigenesis

Organ size determination is one of the most intriguing unsolved mysteries in biology. Aberrant activation of the major effector and transcription co-activator YAP (see Drosophila Yorkie) in the Hippo pathway causes drastic organ enlargement in development. This study reports that the YAP signaling is sustained through a novel microRNA-dependent positive feedback loop. miR-130a, which is directly induced by YAP, could effectively repress VGLL4 (a vestigial homolog), an inhibitor of YAP activity, thereby amplifying the YAP signals. Inhibition of miR-130a reversed liver size enlargement induced by Hippo pathway inactivation and blocked YAP-induced tumorigenesis. Furthermore, the Drosophila Hippo pathway target bantam functionally mimics miR-130a by repressing the VGLL4 homolog Tondu-domain-containing Growth Inhibitor (SdBP/Tgi). These findings reveal an evolutionarily conserved positive feedback mechanism underlying robustness of the Hippo pathway in size control and tumorigenesis (Shen, 2015).

The equilibrium between antagonistic signaling pathways determines the number of synapses in Drosophila

Using the Drosophila larval neuromuscular junction, this study shows a PI3K-dependent pathway for synaptogenesis (a pro-syaptogenesis pathway) which is functionally connected with other previously known elements including the Wit receptor, its ligand Gbb, and the MAPkinases cascade. Based on epistasis assays, the functional hierarchy within the pathway was determined. Wit seems to trigger signaling through PI3K, and Ras85D also contributes to the initiation of synaptogenesis. However, contrary to other signaling pathways, PI3K does not require Ras85D binding in the context of synaptogenesis. In addition to the MAPK cascade, Bsk/JNK undergoes regulation by Puc and Ras85D which results in a narrow range of activity of this kinase to determine normalcy of synapse number. The transcriptional readout of the synaptogenesis pathway involves the Fos/Jun complex and the repressor Cic. In addition, an antagonistic pathway (an anti-synaptogenesis pathway) was identified that uses the transcription factors Mad and Medea and the microRNA bantam to down-regulate key elements of the pro-synaptogenesis pathway. Like its counterpart, the anti-synaptogenesis signaling uses small GTPases and MAPKs including Ras64B, Ras-like-a, p38a and Licorne. Bantam downregulates the pro-synaptogenesis factors PI3K, Hiw, Ras85D and Bsk, but not AKT. AKT, however, can suppress Mad which, in conjunction with the reported suppression of Mad by Hiw, closes the mutual regulation between both pathways. Thus, the number of synapses seems to result from the balanced output from these two pathways (Jordan-Alvarez, 2017).

The epistasis assays have determined the in vivo functional links between PI3K and other previously known pro-synaptogenesis factors. Epistasis assays are based on the combined expression of two or more UAS constructs. Several double combinations in this study have produced a phenotype in spite of the apparent ineffectiveness of the single constructs. This type of results underscores the necessity to use epistasis assays in order to reveal functional interactions in vivo, hence, biologically relevant. In addition to the pro-synaptogenesis signaling, the study has revealed an anti-synaptogenesis pathway that composes a signaling equilibrium to determine the actual number of synapses. The magnitude of the synapse number changes elicited by the factors tested here are mostly within the range of 20%-50%. Are these values significant to cause behavioral changes? Reductions in the order of 30% of excitatory or inhibitory synapses in adult Drosophila local olfactory interneurons transform perception of certain odorants from attraction to repulsion and vice versa. In schizophrenia patients, a 16% loss of inhibitory synapses in the brain cortex has been reported. In Rhesus monkeys, the pyramidal neurons in layer III of area 46 in dorsolateral prefrontal cortex show a 33% spine loss, and a significant reduction in learning task performance during normal aging. Thus, it seems that behavior is rather sensitive to small changes in synapse number irrespective of the total brain mass (Jordan-Alvarez, 2017).

The signaling interactions analyzed here were chosen because they were reported in other cellular systems and species previously. Some of these interactions have been confirmed (e.g., Gbb/Wit), while others have proven ineffective in the context of synaptogenesis (e.g., Ras85D/PI3K binding). Likely, the two signaling pathways, pro- and anti-synaptogenesis, are not the only ones relevant for synapse formation. For example, in spite of the null condition of the gbb and wit mutant alleles used here, the resulting synaptic phenotypes are far less extreme than expected if these two factors would be the only source of signaling for synaptogenesis. Although it could be argued that the incomplete absence of synapses in the mutant phenotypes could result from maternal perdurance, Wit is not part of the oocyte endowment while Gbb is. Three alternative possibilities may be considered, additional ligands for Wit, additional receptors for Gbb, and a combination of the previous two. Beyond the identity of these putative additional ligands and receptors, the stoichiometry between ligands and receptors may certainly be relevant. Actually, Gbb levels are titrated by Crimpy. An equivalent quantitative regulation could operate on Wit. The reported data on Wit illustrate already the diversity of the functional repertoire of this receptor. Wit can form heteromeric complexes with Thick veins (Tkv) or Saxophone (Sax) receptors to receive Dpp/BMP4 or Gbb/BMP7 as ligands. However, the same study also showed that Wit could dimerize with another receptor, Baboon, upon binding of Myoglianin to activate a different and antagonistic signaling pathway, TGFβ/activin-like (Jordan-Alvarez, 2017).

The Gbb/Wit/PI3K signaling analyzed in this study is likely not the only pro-synaptogenesis pathway in flies and vertebrates. The ligand Wingless (Wg), member of the Wnt family, and the receptors Frizzled have been widely documented as relevant in neuromuscular junction development, albeit data on synapse number are scant. Interestingly, however, the downstream intermediaries can be as diverse as those mentioned above for Wit. Although generally depicted as linear pathways, a more realistic image would be a network of cross-interacting signaling events whose in situ regulation and cellular compartmentalization remains fully unexplored (Jordan-Alvarez, 2017).

The quantitative regulation of receptors is most relevant to understand their biological effects. In that context, is worth noting that Tkv levels are distinctly regulated from those of Wit and Sax through ubiquitination in the context of neurite growth. On the other hand, although the receptor Wit is considered a RSTK type, the functional link with PI3K is a feature usually associated to the RTK type instead. The link of Wit with a kinase has a precedent with LIMK1 that binds to, and is functionally downstream from, Wit in the context of synapse stabilization. Thus, Wit should be considered a wide spectrum receptor in terms of its ligands, co-receptor partners and, consequently, signaling pathways elicited. Actually, the Wit amino acid sequence shows both, Tyr and Ser/Thr motifs justifying its initial classification as a 'dual' type of receptor. In this report this study did not determine if Wit heterodimerizes with other receptors, as canonical RSTKs do, or if it forms homodimers, as canonical RTKs do. However, the lack of synaptogenesis effects by the putative co-receptors, Tkv and Sax, and the phenotypic similarity with the manipulation of the standard RTK signaling effector Cic, leaves open the possibility that Wit could play RTK-like functions, at least in the context of synaptogenesis (Jordan-Alvarez, 2017).

Consistent with the proposal of a dual mechanism for Wit, its activation seems to be a requirement to elicit two independent signaling steps, PI3K and Ras85D, that could reflect RTK and RSTK mechanisms, respectively. Both steps are independent because the mutated form of PI3K unable to bind Ras85D, PI3KΔRBD, is as effective as the normal PI3K to elicit synaptogenesis. PI3K and Ras85D signaling, however, seem to converge on Bsk revealing a novel feature of this crossroad point. The activity level of Bsk is known to be critical in many signaling processes. The peculiarities of Bsk/JNK activity include its coordinated regulation by p38a and Slpr in the context of stress heat response without interference on the developmental context. Another modulator, Puc, was described as a negative feed-back loop in the context of oxidative stress. The Puc mediated loop is operative also for synaptogenesis, while that of p38a/Slpr is relevant for p38a only, as shown here. Further, Ras85D represents an additional regulator in the neural scenario. The triple regulation of Bsk/JNK by Ras85D, Puc and the MAPKs seems to stablish a narrow range of activity thresholds within which normal number of synapses is determined (Jordan-Alvarez, 2017).

The concept of signaling thresholds is also unveiled in this study by the identification of another signaling pathway that opposes synapse formation. The pro- and anti-synaptogenesis pathways have similar constituents, including small GTPases, MAPKs and transcriptional effectors, Mad/Smad, which are canonical for RSTK receptors. The RSTK type II receptor Put, which can mediate diverse signaling pathways according to the co-receptor bound can be discarded in either the pro- or the anti-synaptogenesis pathways. Thus, the main receptor for the anti-synaptogenesis pathway remains to be identified (Jordan-Alvarez, 2017).

Concerning small GTPases, the pro-synaptogenesis pathway uses Ras85D while its counterpart uses the poorly studied Ras64B. The anti-synaptogenesis pathway includes an additional member of this family of enzymes, Rala. This small GTPase plays a role in the exocyst-mediated growth of the muscle membrane specialization that surrounds the synaptic bouton as a consequence of synapse activity. That is, Rala can influence synapse physiology acting from the postsynaptic side. The experimental expression of a constitutively active form of Rala in the neuron does not seem to affect the overall synaptic terminal branching. However, the null ral mutant shows reduced synapse branching and its vertebrate homolog is expressed in the central nervous system. This study found that Rala under-expression in neurons yields an elevated number of synapses. Thus, it is likely that this small GTPase acts as a break to synaptogenesis, hence its inclusion in the antagonistic pathway (Jordan-Alvarez, 2017).

Synaptogenesis and neuritogenesis are distinct processes since each one can be differentially affected by the same mutant (e.g.: Hiw). Both features, however, share some signals (e.g., Wnd, Hep). This signaling overlap is akin to the case of axon specification versus spine formation for constituents of the apico/basal polarity complex Par3-6/aPKC [127]. These and other examples illustrated in this study underscore the need to discriminate between synapses and boutons. This study is focused on the cell autonomous signaling that takes place in the neuron. Non-cell autonomous signals (e.g., originated in the glia or hemolymph circulating) have not been considered. The active role of glia in axon pruning and bouton number has been the subject of other studies. Considering the reported role of Hiw through the midline glia in the remodeling of the giant fiber interneuron it is not unlikely that the glia-to-neuron signaling may share components with the neuron autonomous signaling addressed here (Jordan-Alvarez, 2017).

The summary scheme (see Summary diagram of antagonistic signaling pathways for synaptogenesis and their interactions) describes the scenario where two signaling pathways mutually regulate each other. Epistasis assays are the only experimental approach for in vivo studies of more than one signaling component, albeit this type of assay is only feasible in Drosophila Thus, it is plausible that vertebrate synaptogenesis will be regulated by a similar antagonistic signaling (Jordan-Alvarez, 2017).

The regulatory equilibrium as a mechanism to determine a biological parameter is the most relevant feature in this scenario for several reasons. First, because this type of mechanism can respond very fast to changes in the physiological status of the cell, and, second because it provides remarkable precision to the trait to be regulated, synapse number in this case. Although bi-stable regulatory mechanisms are known in other contexts, the case of synapse number may seem unexpected because the highly dynamic nature of synapse number has been recognized only recently. Consequently, a molecular signaling mechanism endowed with proper precision and time resolution must sustain this dynamic process. The balanced equilibrium uncovered in this study, although most likely still incomplete in terms of its components, offers such a mechanism (Jordan-Alvarez, 2017).

Dicer-1 regulates proliferative potential of Drosophila larval neural stem cells through bantam miRNA based down-regulation of the G1/S inhibitor Dacapo

This study elucidates the role of miRNA in cell cycle regulation during brain development in Drosophila. It was found that lineage specific depletion of dicer-1, a classically acknowledged miRNA biogenesis protein in neuroblasts leads to a reduction in their numbers and size in the third instar larval central brain. These brains also show lower number of mitotically active cells and when homozygous mitotic clones were generated in an otherwise heterozygous dicer-1 mutant background via MARCM technique, they show reduced number of progeny cells in individual clones, substantiating the adverse effect of the loss of dicer-1 on the proliferative potential of neuroblasts. bantam miRNA, which has been classically reported to be involved in tissue growth was found to be expressed in neuroblasts and undergo reduced expression in Dicer-1 depleted background in the third instar larval brain. Reduction in the number and proliferative potential of neuroblasts in bantam mutant background implies a pivotal role played by bantam miRNA in maintenance of neuroblast number. Since in both Dicer-1 and bantam depleted genetic backgrounds, Dacapo, an inhibitor of cyclin E-Cdk complex, was found to have elevated expression, the study postulates a molecular mechanism involving bantam-Dacapo-Cyclin E/Cdk complex that regulates the G1-S phase transition of Drosophila neuroblasts (Banerjee, 2017).

Processing of Pre-miR-bantam

microRNAs (miRNAs) are a large family of 21- to 22-nucleotide non-coding RNAs that interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. miRNAs are excised in a stepwise process from primary miRNA (pri-miRNA) transcripts. The Drosha-Pasha/DGCR8 complex in the nucleus cleaves pri-miRNAs to release hairpin-shaped precursor miRNAs (pre-miRNAs). These pre-miRNAs are then exported to the cytoplasm and further processed by Dicer to mature miRNAs. Drosophila Dicer-1 interacts with Loquacious, a double-stranded RNA-binding domain protein. Depletion of Loquacious results in pre-miRNA accumulation in Drosophila S2 cells, as is the case for depletion of Dicer-1. Immuno-affinity purification experiments revealed that along with Dicer-1, Loquacious resides in a functional pre-miRNA processing complex, and stimulates and directs the specific pre-miRNA processing activity. Efficient miRNA-directed silencing of a reporter transgene, complete repression of white by a dsRNA trigger, and silencing of the endogenous Stellate locus by Suppressor of Stellate, all require Loqs. In loqs^f00791 mutant ovaries, germ-line stem cells are not appropriately maintained. Loqs associates with Dcr-1, the Drosophila RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known Drosophila RNase-III endonuclease is paired with a dsRBD protein that facilitates its function in small RNA biogenesis. These results support a model in which Loquacious mediates miRNA biogenesis and, thereby, the expression of genes regulated by miRNAs (Forstemann, 2005; Saito, 2005).

Pre-miR-bantam was used as a substrate for pre-miRNA processing assays. It has been shown that S2 cell extracts contain primary-miRNA processing activity that cleaves pri-miRNA into an approximately 60- to 70-bp pre-miRNA precursor. This processing is known to occur in the nucleus; thus pre-miR-ban was prepared by in vitro processing of pri-miR-ban incubated with S2 nuclear extracts. Uniformly labeled pre-miR-ban was then gel-purified and used as a substrate for analysis of pre-miRNA processing. Incubation of the pre-miRNA with S2 cytoplasmic extracts results in the appearance of a mature 21-nucleotide miR-ban. Then the requirement of Dicer-1 and Loqs in pre-miR-ban processing was examined. Incubation of pre-miRNA with Dicer-1- and Loqs-depleted S2 cytoplasmic extracts results in a marked reduction in mature miRNA levels. In contrast, depletion of Dicer-2 or R2D2 shows no measurable reduction of mature miRNA levels. Then the pre-miRNA processing activity of the purified complexes (both Flag-Dicer-1 and Flag-Loqs complexes) was assayed. That the Flag-Loqs complex contains Dicer-1 was confirmed by immunoblotting. Both Dicer-1 and Loqs complexes are capable of generating maturemiR-ban from pre-miR-ban. Several steps in the RNAi and miRNA pathways are known to require a divalent metal ion. In addition, it is well known that RNase III-type enzymes require divalent metals for cleavage. Flag-Dicer-1 complex was employed and the processing was performed in the presence of magnesium ions or EDTA in a buffer. No pre-miRNA processing activity is detected at 10 mM EDTA. These results demonstrate that the Dicer-1-Loqs complex converts pre-miRNAs into mature miRNAs in a divalent metal ion-dependent manner (Saito, 2005).

To confirm the function of loqs in pre-miRNA processing, cultured Drosophila S2 cells were depleted of loqs mRNA by RNAi. Eight days after incubating S2 cells with dsRNA corresponding to the first 300 nucleotides of the loqs coding sequence, the steady-state levels of pre-miRNA and mature miRNA were determined for miR-277 and bantam. Relative to an unrelated dsRNA control, dsRNA corresponding to dcr-1 caused an approximately 9-fold and approximately 23-fold increase in steady-state pre-miR-277 and bantam levels, respectively, and dsRNA corresponding to loqs caused an approximately 2-fold and approximately 6-fold increase in steady-state pre-miR-277 and bantam levels, respectively. In these experiments, RNAi of dcr-1 more completely depleted Dcr-1 protein than RNAi of loqs reduced Loqs protein. RNAi of dcr-2,r2d2, or drosha did not alter pre-miRNA levels for either miR-277 or bantam, nor did it alter Dcr-1 or Loqs levels. The Drosha/Pasha protein complex functions before pre-miRNA processing, converting primary miRNA (pri-miRNA) to pre-miRNA. Consistent with the idea that Loqs functions with Dcr-1 to convert pre-miRNA to mature miRNA, RNAi of drosha together with loqs alleviates the high pre-miRNA levels observed for RNAi of loqs alone, demonstrating that Loqs acts after Drosha (Forstemann, 2005).

Mutual repression by bantam miRNA and Capicua links the EGFR/MAPK and Hippo pathways in growth control

The epidermal growth factor receptor (EGFR) and Hippo signaling pathways control cell proliferation and apoptosis to promote tissue growth during development. Misregulation of these pathways is implicated in cancer. Understanding of the mechanisms that integrate the activity of these pathways remains fragmentary. This study identifies bantam microRNA as a common target of these pathways and suggests a mechanistic link between them. The EGFR pathway acts through bantam to control tissue growth. bantam expression is regulated by the EGFR pathway, acting via repression of the transcriptional repressor Capicua. Thus EGFR signaling induces bantam expression by alleviating the effects of a repressor. bantam in turn acts in a negative feedback loop to limit Capicua expression. bantam appears to be a transcriptional target of both the EGFR and Hippo growth control pathways. Feedback regulation by bantam on Capicua provides a means to link signal propagation by the EGFR pathway to activity of the Hippo pathway and may play an important role in integration of these two pathways in growth control (Herranz, 2012).

The ability of the EGFR pathway to drive tissue growth resides in its ability to coordinately stimulate cell proliferation and suppress apoptosis. Understanding how coordinated control is achieved depends on identification of the effector mechanisms that mediate these outputs along with the connections to other growth regulatory pathways. The results show that the bantam miRNA is a critical target of the EGFR pathway. Further, a mechanism is outlined by which bantam serves as a link between the EGFR and Hippo pathways (Herranz, 2012).

In Drosophila, EGFR pathway effectors include the transcription factors Pointed, Tramtrack, and Yan, and the HMG-box repressor Capicua. Capicua has an important role in early embryonic patterning and as a negative growth regulator. Although several Capicua targets involved in embryonic patterning have been identified, how Capicua regulates tissue growth was unknown. These results identify bantam as an important target of Capicua required to mediate EGFR-dependent tissue growth (Herranz, 2012).

A key finding of this study is the regulatory feedback relationship between bantam and Capicua. Each represses the activity of the other. Viewed from the perspective of the EGFR/MAPK pathway alone, the outcome of this relationship would be signal amplification, with downregulation of Capicua levels by bantam reinforcing direct MAPK-induced turnover of Capicua protein. This adds a new mechanism to the repertoire of positive and negative feedback loops affecting EGFR pathway activity. These feedback mechanisms are thought to be important in disease, and their regulation is complex. Relatively little is known about Capicua in cancer, although one recent study reports mutants in the human Cic protein in oligodendroglioma (Bettegowda, 2012; Herranz, 2012 and references therein).

An alternative logic for the relationship between bantam and Capicua may be seen in the fact that it links the output of the EGFR pathway to the output of the Hippo pathway, mediated through transcriptional regulation of bantam by Yorkie. EGFR signaling via MAPK and bantam cooperate to downregulate Capicua protein levels. Thus the transcriptional output of the Hippo pathway via Yorkie can be seen as potentiating EGFR signaling by 'lowering' the effective threshold of MAPK activity needed to reduce Capicua to a given level. Alternatively, the lack of sufficient Yorkie activity would lower bantam activity and thereby raise the threshold of EGFR activity required to reach an effective level of Capicua downregulation. This provides a mechanism to ensure coordination of the growth regulatory pathways. Signaling via the Hippo pathway has also been shown to induce the EGFR ligand amphiregulin to promote tissue growth in a nonautonomous manner. Thus, there appear to be multiple levels of crosstalk between these pathways (Herranz, 2012).

Considerable evidence is emerging linking miRNAs to robustness of regulatory feedback networks. It is intriguing that miRNAs are now implicated in regulation of all three of the known transcriptional effectors of EGFR signaling. miR-7 acts in two feed-forward loops downstream of EGFR to control photoreceptor specification and differentiation in the Drosophila eye. EGFR acts via the transcription factors Yan and Pointed. Yan is a direct target of miR-7. Yan also represses miR-7 transcription directly as well as indirectly. In the same cells, the ETS-1 factor Pointed-P1 activates miR-7 to repress Yan as well as acting directly to repress Yan. Use of interlinked motifs is thought to provide stability to the cell differentiation program controlled by EGFR. The current findings link bantam to regulation of a third EGFR transcriptional effector, Capicua, in addition to its regulation by the Hippo pathway. Coordination of diverse growth control inputs by miRNAs might contribute to robustness (Herranz, 2012).

HDAC inhibitor misprocesses bantam oncomiRNA, but stimulates hid induced apoptotic pathway

Apoptosis or programmed cell death is critical for embryogenesis and tissue homeostasis. Uncontrolled apoptosis leads to different human disorders including immunodeficiency, autoimmune disorder and cancer. Several small molecules that control apoptosis have been identified. This study has shown the functional role of triazole derivative (DCPTN-PT) that acts as a potent HDAC inhibitor (reducing the level of Rpd3/HDAC1 in Drosophila) and leads to the misexpression of proto onco microRNA (miRNA) bantam. To further understanding the mechanism of action of the molecule in the apoptotic pathway, a series of experiments were also performed in Drosophila, a well known model organism in which the nature of human apoptosis is very analogous. DCPTN-PT mis processes bantam microRNA and alters its down regulatory target hid function and cleavage of Caspase-3 which in turn influence components of the mitochondrial apoptotic pathway in Drosophila. However regulatory microRNAs in other pro-apoptotic genes are not altered. Simultaneously, treatment of same molecule also affects the mitochondrial regulatory pathway in human tumour cell lines suggesting its conservative nature between fly and human. It is reasonable to propose that triazole derivative (DCPTN-PT) controls bantam oncomiRNA and increases hid induced apoptosis and is also able to influence mitochondrial apoptotic pathway (Bhadra, 2015).

DEVELOPMENTAL BIOLOGY

bantam miRNA is expressed at all developmental stages, though at varying levels. To ask whether bantam expression is spatially regulated during development, an assay was developed based on the ability of miRNAs to inactivate genes by RNAi. A transgene was prepared expressing GFP under control of the tubulin promoter and two copies of a perfect bantam target were placed in the 3'UTR. A comparable construct without the bantam target sequences in the 3'UTR was used as a control. Where present, bantam miRNA should reduce expression of the transgene containing the target sequences by RNAi, providing an in vivo sensor for bantam activity. When expression of the two transgenes was compared using the same settings on the confocal microscope, it was apparent that the control transgene was expressed at much higher levels overall. The bantam sensor transgene showed a complex pattern of spatial modulation in the third instar wing disc, being higher in cells near the anteroposterior and dorsoventral boundaries and in patches in the dorsal thorax. The control transgene showed limited spatial modulation (Brennecke, 2003).

The difference in the overall levels of control and bantam sensor transgenes suggested that bantam miRNA is expressed broadly in the wing disc, lowering the expression of the specific sensor transgene. Indeed, removing bantam miRNA in clones of cells homozygous for the bantam^Δ1 deletion increased expression of the bantam sensor. The level of sensor expression in the clones was considerably higher than the maximal endogenous level at the DV boundary, indicating that the miRNA is present throughout the disc, though at varying levels. The P element EP(3)3622 is inserted 2.7 kb from the hairpin and has been identified as a hypomorphic allele of bantam (Hipfner, 2002). Clones of cells homozygous mutant for EP(3)3622 also show upregulation of the bantam sensor, demonstrating that this insertion reduces bantam miRNA levels. In this case, the maximal level of sensor expression is similar to the level at the DV boundary. The level of sensor expression is lower in the twin spots, which express two copies of the endogenous bantam gene than in the surrounding cells, which have one wild-type and one mutant copy of the gene. This suggests that elevated bantam activity would reduce sensor expression. Indeed, clones overexpressing bantam do reduce sensor levels. The control sensor was not affected by overexpression of bantam. Taken together, these observations indicate that the sensor is capable of reflecting even subtle increases and decreases in bantam miRNA levels in vivo. In all cases, the effects on the sensor were cell autonomous. The sensor transgene method may provide a generally useful tool to visualize miRNA activity in vivo (Brennecke, 2003).

Bantam is essential for Drosophila intestinal stem cell proliferation in response to Hippo signaling

The Drosophila midgut has emerged as an attractive model system to study stem cell biology. Extensive studies have been carried out to investigate the mechanisms of how the signaling pathways integrate to regulate intestinal stem cells (ISCs), yet, whether the microRNAs are involved in ISC self-renewal and maintenance is unknown. This study demonstrates that the bantam microRNA is expressed specifically at high levels in Drosophila midgut precursor cells (including ISCs and enteroblasts) and secretory enteroendocrine cells while at extremely low levels in enterocytes. Furthermore, overexpression of bantam microRNA results in increase of the division of the midgut precursor cells, whereas loss of bantam microRNA decreases their proliferation. The mechanical studies show that bantam microRNA is essential for the Hpo pathway induced cell-autonomous ISC self-renewal, while it is disposable for EGFR and Notch pathways mediated ISC proliferation. More interestingly, bantam microRNA was found to not be required for the Hpo pathway mediated non-cell-autonomous ISC proliferation, revealing a novel mechanism by which the Hpo signaling pathway specifies its transcriptional targets in specific tissue to exhibit its biological functions (H. Huang, 2014).

Dying cells protect survivors from radiation-induced cell death in Drosophila

Induction of cell death by a variety of means in wing imaginal discs of Drosophila larvae resulted in the activation of an anti-apoptotic microRNA, bantam. Cells in the vicinity of dying cells also become harder to kill by ionizing radiation (IR)-induced apoptosis. Both ban activation and increased protection from IR required receptor tyrosine kinase Tie, which was identified in a genetic screen for modifiers of ban. tie mutants are hypersensitive to radiation, and radiation sensitivity of tie mutants was rescued by increased ban gene dosage. It is proposed that dying cells activate ban in surviving cells through Tie to make the latter cells harder to kill, thereby preserving tissues and ensuring organism survival. The protective effect reported in this study differs from classical radiation bystander effect in which neighbors of irradiated cells become more prone to death. The protective effect also differs from the previously described effect of dying cells that results in proliferation of nearby cells in Drosophila larval discs. If conserved in mammals, a phenomenon in which dying cells make the rest harder to kill by IR could have implications for treatments that involve the sequential use of cytotoxic agents and radiation therapy (Bilak, 2014).

In metazoa where cells exist in the context of other cells, the behavior of one affects the others. The consequences of such interactions include not just cell fate choices but also life and death decisions. In wing imaginal discs of Drosophila melanogaster larvae, dying cells release mitogenic signals. Signaling from dying cells, or dying cells kept alive by the caspase inhibitor p35 (the so-called 'undead' cells), in wing discs operate through activation of Wingless (Drosophila Wnt) and JNK, and through repression of the tumor suppressor Salvador/Warts/Hippo pathway. A crosstalk between JNK and Hpo has also been reported. The consequences on the neighbors include increased number of cells in S phase and activation of targets of Yki, a transcription factor that is normally repressed by Hpo signaling. Mitogenic signals from dying cells results in increased proliferation of neighbors, which is proposed to compensate for cell loss and help regenerate the disc (Bilak, 2014).

A target of Yki is bantam microRNA, but ban was not examined in above-described studies. ban was first uncovered in a genetic screen for promoters of tissue growth when overexpressed in Drosophila. Further study found a role for ban in both preventing apoptosis and promoting proliferation. A key target of ban in apoptosis is hid, a Drosophila ortholog of mammalian SMAC/Diablo proteins. These proteins antagonize DIAP1 to liberate active caspases and allow apoptosis. Hid is pro-apoptotic; repression of Hid by ban via binding sites in hid 3′UTR curbs apoptosis (Bilak, 2014).

Since the initial characterization of ban, the role of this miRNA has expanded to include coordinating differentiation and proliferation in neural and glial lineages, cell fate decisions in germ line stem cells, in circadian rhythm, and in ecdyson hormone production. In these and other contexts, ban is regulated by a number of transcriptional factors and signaling pathways including, Hpo/Yki, Wg, Myc, Mad, Notch and Htx. The regulatory region of ban gene is likely to be complex and substantial; p-element insertions more than 10 kb away from ban sequences produce ban phenotypes (Bilak, 2014).

The experimental evidence in Drosophila that dying cells promote proliferation presaged by several years the experimental evidence for a similar but mechanistically different phenomenon in mammals. A response called 'Phoenix Rising' occurs in mice after cell killing by ionizing radiation. Here, the activity of Caspase 3 and 7 is required in dying cells and mediates the release of prostaglandin E₂, a stimulator of cell proliferation. These signals act non-autonomously to stimulate proliferation and tissue regeneration. A follow-up study in mice found a requirement for Caspase 3 in tumor regeneration after radiation treatment. Not all consequences on neighboring cells are protective or mitogenic. In the classical 'radiation bystander effect', seen in cell culture and in mice, the effect of irradiated cells on the neighbors is destructive, making the latter more prone to death. There is evidence for a soluble signal; media from irradiated cells can induce the bystander effect on naïve cells. Inhibitors of the bystander effect include antioxidants, suggesting that oxidative stress and energy metabolism may be involved in radiation bystander effect (Bilak, 2014).

It has been shown previously that ban activity increased after exposure to ionizing radiation (IR) in wing imaginal discs of Drosophila larvae (Jaklevic, 2008). IR-induced increase in ban activity required caspase activity: expression of a viral caspase inhibitor, p35, or mutations in p53 that reduced and delayed the onset of caspase activation attenuated ban activation. It is noted that while IR-induced cell death is scattered throughout the disc, ban activation is homogeneous. This suggested a non-cell-autonomous component in activation of ban. The current study came out of efforts to understand how ban is activated in response to IR. Drosophila tie, which encodes a receptor tyrosine kinase of VGFR/PDGFR family, was identified as an important mediator of IR-induced changes in ban. Previous knowledge of Tie function in Drosophila was limited to long range signaling for border cell migration during oogenesis (Wang, 2006). This study reports that Tie was needed to activate ban in response to cell death. One consequence of ban activation was that remaining cells were harder to kill by IR (Bilak, 2014).

This study has documented a previously unknown phenomenon in wing imaginal discs of Drosophila larvae; dying cells protected nearby cells from death. Killing cells by any one of three methods -- ptc-GAL4-driven expression of dE2F1^RNAi or pro-apoptotic genes hid and rpr, exposure to ionizing radiation (IR) and clonal induction of Hid/Rpr -- activated an anti-apoptotic microRNA, bantam. Death by ptc-GAL4 or clonal expression of Hid/Rpr also made surviving cells more resistant to killing by IR. The protective effect was sensitive to ban gene dosage. This phenomenon was named 'Mahakali effect', after the Hindu goddess of death who protects her followers. Mahakali effect differs from classical radiation 'bystander effect' in which byproducts from cell corpses make surviving cells more prone to death. The Mahakali effect appears to operate in a non-cell-autonomous fashion. Disc-wide protection by ptc4>Rpr and Hid/Rpr that included even cells in the P compartment that did not express ptc, provides the strongest evidence for non-autonomy. This idea is supported by the finding that IR-induced caspase activation was reduced in cells outside Hid/Rpr flip-out clones (Bilak, 2014).

A recent paper describes a non-autonomous induction of apoptosis by apoptotic cells. These results do not necessarily contradict what is reported in this study. Most of the experiments in the published work used undead cells kept alive by p35; Mahakali effect is seen without p35. Non-autonomous apoptosis was assayed at, typically, 3-4 days after induction of undead cells; this study detected Mahakali effect 6 hr after cell death induction using similar death-inducing stimuli (Hid/Rpr). It would be interesting to see how long Mahakali effect persists and whether non-autonomous apoptosis, occurring at longer time points, also produces Mahakali effects of its own. Another recent paper describes tissue regeneration after massive cell ablation in wing discs. It would also be interesting to see if the Mahakali effect operates among regenerating cells (Bilak, 2014).

The data shown in this study suggest that the basic components of the Mahakali effect are caspase activity in dying cells (because expression in dying cells of p35, an inhibitor of effector caspases, blocked ban activation), ban (because ban activation resulted from cell death and the protective effect was sensitive to ban gene dosage), and tie (because tie was required to activate ban and the protective effect was sensitive to tie gene dosage). A model is proposed in which caspase activity in dying cells acts through Tie to cause non-autonomous activation of ban and the Mahakali effect. A validated target of ban in apoptosis inhibition is hid, whose 3'UTR includes 4 potential ban binding sites. Previous work has shown that a GFP sensor with hid 3'UTR is reduced after IR (Jaklevic, 2008), reflecting repression of hid by ban. Deletion of two potential ban-binding sites in the hid 3'UTR abolished the IR-induced changes in GFP (Bilak, 2014).

The Mahakali effect differs in two ways from previously described effects of dead/dying cells in wing discs. First, the Mahakali effect extended further than previously reported signaling from dead/dying cells. In the extreme case of ptc4>Hid/Rpr, the protection reached as far as the edge of the disc. This distance, on of order of 100 or more mm is comparable to the distance of border cell migration, in which Tie is known to function. In contrast, the mitogenic effect that occurs through JNK/Wingless in response to undead cells in the wing disc is seen up to 5 cells away. Activation of proliferation through the Hpo/Yki axis also spans 3-5 cells away. This can be seen as activation of Yki targets such as DIAP1. This result could be reproduced: ptc4>dE2f1^RNAi activated a Yki target, DIAP1, but only within or close to the ptc domain. Yki^B5 allele, which disrupts cell death-induced proliferation, did not alter the Mahakali effect, further supporting the idea that the two effects are different. Second, ban activation in response to cell death was sensitive to the caspase inhibitor p35. In contrast, the mitogenic effect of dying cells in wing imaginal discs is not sensitive to p35. It is noted that the mitogenic effect of dying cells is inhibited by p35 in the differentiating posterior region of eye imaginal discs, which is similar to what was seen for ban activation in the wing discs (Bilak, 2014).

This study found that tie was required for IR-induced activation of ban and for larval survival after irradiation. There were similarities as well as differences in the role of ban and tie. tie mutants were IR-sensitive, as are viable alleles of ban (Jaklevic, 2008). Tissue-specific overexpression of ban results in abnormal growth; this study found that 6 independent UAS-tie transgenic lines were lethal when driven by actin-GAL4. Thus, too much ban or tie has consequences. On the other hand, reducing tie or ban gene dosage by half attenuated the Mahakali effect. Thus, too little ban or tie also has consequences. In fact, UAS-ban or UAS-tie without a GAL4-driver was sufficient to rescue ban and tie mutant phenotypes. Thus, intermediate levels of expression may be important for the function of these genes (Bilak, 2014).

The biggest difference between ban and tie, of course, was that while tie homozygous larvae were viable (this study), ban homozygous larvae are lethal. tie became necessary only after radiation exposure. This suggests that tie was needed to regulate ban not during normal development but after radiation exposure. How is IR and cell death linked to Tie? mRNA for Pvf1, a ligand for Tie in border cell migration, was found to be induced by IR and this induction appeared to be dependent on cell death (abolished in p53 mutants). Pvf1^EP1624 mutants that are mRNA and protein null, also showed reduced Mahakali effect. The degree of reduction was significant but not back to the level seen in control discs without ptc4>dE2f1^RNAi, suggesting the involvement of additional ligands or mechanisms for Tie activation. In agreement, no ban activation or the Mahakali effect was seen after overproduction of Pvf1. Pvf1 was necessary but insufficient to produce these effects without cell death (Bilak, 2014).

Tie activated ban, at least in part by increasing ban levels. How IR and caspase activity promotes Pvf1 expression and how Tie activity increases ban levels will be key questions to address in the future. Testing the role of known apoptosis regulators, such as Diap1, and signaling molecules, such as Wg, may help address these questions. The genetic screen that identified Tie will be completed in future studies; it has the potential to identify additional components of the Mahakali effect (Bilak, 2014).

Pvr, a PDGF/VEGF receptor homolog that function redundantly with Tie in border cell migration, also plays an anti-apoptotic role in embryonic hemocytes. A recent study in wing discs found that Pvr is activated in neighbors of dying cells in a JNK-dependent manner, to result in cytoskeletal changes that allow the engulfment of the dead cell by the neighbor. It is interesting that two PDGF/VEGF receptor homologs that function redundantly in cell migration during oogenesis may also play non-redundant roles in non-autonomous responses to cell death in wing discs (Bilak, 2014).

Cancer therapy routinely comprises the application of two or more cytotoxic agents (taxol and radiation, for example) to cancer cells. A phenomenon in which cell killing by one agent influence resistance to the second agent is, therefore, of potential clinical significance. The bulk of the current analysis focused on protection from IR-induced cell death. But preliminary evidence indicates that the Mahakali effect can also protect against cell death induced by maytansinol, a microtubule depolymerizing agent with relevance to cancer therapy that we found before to induce cell death in Drosophila wing discs. An important question is whether a phenomenon like Mahakali effect exists in mammals and acts as a survival mechanism in response to cell death. Ang-1, a ligand for mammalian Tie-2, is a pro-survival factor for endothelial cells during serum deprivation and after irradiation in cell culture models (Holash, 1999; Kwak, 2000; Papapetropoulos 2000). Interestingly, Ang1 is produced not by endothelial cells but by neighbors, at least in cell culture. Based on these data, it is possible that radiation exposure results in Ang1 production by dead/dying cells that promote the survival of endothelial cells via Tie-2. Consistent, an Ang-1 derivative that is a potent activator of Tie-2 has been shown to protect endothelial cells from radiation-induced apoptosis (Bilak, 2014 and references therein).

Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster

Many types of normal and cancer stem cells are resistant to killing by genotoxins, but the mechanism for this resistance is poorly understood. This study shows that adult stem cells in Drosophila melanogaster germline and midgut are resistant to ionizing radiation (IR) or chemically induced apoptosis; the mechanism for this protection was dissected. Upon IR the receptor tyrosine kinase Tie/Tie-2 is activated, leading to the upregulation of microRNA bantam that represses FOXO-mediated transcription of pro-apoptotic Smac/DIABLO orthologue, Hid in germline stem cells. Knockdown of the IR-induced putative Tie ligand, PDGF- and VEGF-related factor 1 (Pvf1), a functional homologue of human Angiopoietin, in differentiating daughter cells renders germline stem cells sensitive to IR, suggesting that the dying daughters send a survival signal to protect their stem cells for future repopulation of the tissue. If conserved in cancer stem cells, this mechanism may provide therapeutic options for the eradication of cancer (Xing, 2015).

A form of programmed cell death, apoptosis, is characterized as controlled, caspase-induced degradation of cellular compartments to terminate the activity of the cell. Apoptosis plays a vital role in various processes including normal cell turnover, proper development and function of the immune system and embryonic development. Apoptosis is also induced by upstream signals, such as DNA double-strand breaks (DSB), to destruct severely damaged cells. DSB activate ATM checkpoint kinase and Chk2 kinase-dependent p53 phosphorylation and induction of repair genes. However, if DSB are irreparable, p53 activation will result in pro-apoptotic gene expression and cell death. However, aggressive cancers contain cells that show inability to undergo apoptosis in response to stimuli that trigger apoptosis in sensitive cells. This feature is responsible for the resistance to anticancer therapies, as well as the relapse of tumours after treatment, yet the molecular mechanism of this resistance is poorly understood (Xing, 2015).

As the cell type that constantly regenerates and gives rise to differentiated cell types in a tissue, stem cells share high similarities with cancer stem cells, including unlimited regenerative capacity and resistance to genotoxic agents. Adult stem cells in model organisms such as Drosophila melanogaster, have been utilized to study stem cell biology and for conducting drug screens, thanks to their intrinsic niche, which provides authentic in vivo microenvironment. This study shows that Drosophila adult stem cells are resistant to radiation/chemical-induced apoptosis, and the mechanism for this protection was dissected. A previously reported cell survival gene with a human homologue, pineapple eye (pie) , acts in both stem cells and in differentiating cells to repress the transcription factor FOXO. Elevated FOXO levels in pie mutants lead to apoptosis in differentiating cells, but not in stem cells, indicating the presence of an additional anti-apoptotic mechanism(s) in the latter. We show that this mechanism requires Tie, encoding a homologue of human receptor tyrosine kinase Tie-2, and its target, bantam, encoding a microRNA. The downstream effector of FOXO, Tie and ban, is show to be Hid, encoding a Smac/DIABLO orthologue. Knocking down the ligand Pvf1/PDGF/VEGF/Ang in differentiating daughter cells made stem cells more sensitive to radiation-induced apoptosis, suggesting that Pvf1 from the apoptotic differentiating daughter cells protects stem cells (Xing, 2015).

This study shows that an anti-apoptotic gene, pie, is required for stem cell self-renewal but not for resistance to apoptosis, indicating a compensatory anti-apoptotic mechanism in stem cells. The cell cycle marker profile of pie GSCs resembles that of InR deficient GSCs, leading to the finding that pie controls GSC, as well as ISC self-renewal/division through FOXO protein levels. Surprisingly, pie targets FOXO as well in differentiating cells, failing to explain why the loss of pie does not induce apoptosis in stem cells. However, while the upregulation of FOXO leads to the upregulation of its apoptotic target Hid in differentiating cells, in adult stem cells Hid is not upregulated. Hence additional regulatory pathway is in place to repress Hid and thereby apoptosis in stem cells. This study identified Tie-receptor as the key gatekeeper for the process in the GSCs. The signal (Pvf1) from the dying daughter cells activates Tie in GSCs to upregulate bantam microRNA that represses Hid, thereby protecting the stem cells. Bantam is known to repress apoptosis and activate the cell cycle. However, while protected from apoptosis in this manner, the stem cells do not activate the cell cycle but rather stay in protective quiescence through FOXO activity. When the challenge is passed, stem cells repopulate the tissue (Xing, 2015).

The mammalian pie homologue, G2E3 was reported to be an ubiquitin ligase with amino terminal catalytic PHD/RING domains. G2E3 is essential for early embryonic development (Brooks, 2008). Importantly, microarray data show significant enrichment of G2E3 expression levels in human embryonic stem (ES) cell lines. These observations suggest a critical role of G2E3 in embryonic development, potentially in maintaining the pluripotent capacity. Since FOXO is shown to be an important ESC regulator, it will be interesting to test whether defects in G2E3 result in changes in FOXO levels. Furthermore, future studies are required to test whether human ES cells also are protected from apoptosis due to external signals from dying neighbouring cells (Xing, 2015).

The cell cycle defects of pie mutant stem cells, such as abnormal cell cycle marker profile, can be a consequence of elevated FOXO levels, since FOXO is a transcription factor with wide array of target genes, many of which are involved with cell cycle progress, such as the cyclin-dependent kinase inhibitor p21/p27 (Dacapo in Drosophila). This may be critical when bantam function is considered in the stem cells. Bantam is known to function as anti-apoptotic and cell cycle inducing microRNA. While in GSC bantam is critical through its anti-apoptotic function as a Hid repressor, it has no capacity to induce GSC cell cycle after irradiation. In a challenging situation, such as irradiation, an additional protection mechanism for the tissue is to keep the stem cell in a quiescent state during challenge. bantam's pro-cell division activity may be dampened by FOXO's capacity to upregulate p21/Dacapo (Xing, 2015).

The FOXO family is involved in diverse cellular processes such as tumor suppression, stress response and metabolism. The FOXO group of human Forkhead proteins contains four members: FOXO1, FOXO3a, FOXO4, and FOXO6. Studies to elucidate their function in various stem cell types in vivo using knockout mice have shown some potential redundancy of FOXO proteins. Recent publications have demonstrated a requirement for some of the FOXO family members in mouse hematopoietic stem cell proliferation, mouse neural stem cells, leukaemia stem cells and human and mouse ES cells in vitro. However, FOXO is shown to be dispensable in the early embryonic development in mouse. Drosophila genome has only one FOXO, allowing a definitive study of FOXO's function in stem cells. This study now demonstrates that tight regulation of FOXO protein levels is essential for in vivo GSC and ISC self-renewal in Drosophila. While the loss of FOXO function generates supernumerary stem cells, inappropriately high level of FOXO results in stem cell loss. Under challenge, such as exposure to irradiation, stem cells depleted of FOXO fail to stay quiescent and become more sensitive to the damage, leading to the loss of GSC population. These data demonstrate the importance of the balanced FOXO expression level for stem cell fate (Xing, 2015).

Previous studies have shown that multiple adult stem cell types manage to avoid cell death in response to severe DNA damage. This work has studied the mechanisms that stem cells utilize to avoid apoptosis in absence of pie and revealed that apoptosis is protected through a receptor, Tie and its target miRNA bantam that can repress the pro-apoptotic gene Hid. The ligand for Tie is likely secreted from the dying neighbours since Tie is essential in GSC only after irradiation challenge, IR induces Tie's potential ligand Pvf1 expression in cystoblasts and knockdown of Pvf1 in cystoblasts eliminates stem cells' protection against apoptosis. Further studies will reveal whether the same protective pathway is utilized in other stem cells. Community phenomenon have been described previously around dying cells: compensatory proliferation, Phoenix rising, bystander effect and Mahakali. While Bystander effect describes dying cells inducing death in the neighbours, compensatory proliferation, Phoenix rising and Mahakali describe positive effects in cells neighbouring the dying cells. The present work shows that adult stem cell can survive but show no immediate induction of proliferation when neighboured by dying cells. However, since adult stem cells can repopulate the tissue when death signals have passed, it is proposed that in adult stem cells these phenomenon merge. First, the GSCs survive by bantam repressing the apoptotic inducer, Hid, and later repopulate the tissue by activating cell cycle. Recent findings have suggested that p53 might play an important role in re-entry to cell cycle in stem cells51. The results from the current studies shed light on the general understanding of stem cell behaviour in response to surrounding tissue to ensure the normal tissue homeostasis. It is also plausible that cancer stem cells hijack these normal capacities of stem cells (Xing, 2015).

Control of Drosophila type I and type II central brain neuroblast proliferation by bantam microRNA

Post-transcriptional regulation of stem cell self-renewal by microRNAs is emerging as an important mechanism controlling tissue homeostasis. This report providse evidence that the bantam microRNA controls neuroblast number and proliferation in the Drosophila central brain. bantam also supports proliferation of the transit-amplifying intermediate neural progenitor cells in type II neuroblast lineages. The stem cell factors brat, prospero are identified as bantam targets acting on different aspects of these processes. Thus bantam appears to act in multiple regulatory steps in the maintenance and proliferation of neuroblasts and their progeny to regulate growth of the central brain (Weng, 2015).