Morphogen control of wing growth through the fat signaling pathway

Organ growth is influenced by organ patterning, but the molecular mechanisms that link patterning to growth have remained unclear. The Dpp morphogen gradient in the Drosophila wing influences growth by modulating the activity of the Fat signaling pathway. Dpp signaling regulates the expression and localization of Fat pathway components, and Fat signaling through Dachs is required for the effect of the Dpp gradient on cell proliferation. Juxtaposition of cells that express different levels of the Fat pathway regulators four-jointed and dachsous stimulates expression of Fat/Hippo pathway target genes and cell proliferation, consistent with the hypothesis that the graded expression of these genes contributes to wing growth. Moreover, uniform expression of four-jointed and dachsous in the wing inhibits cell proliferation. These observations identify Fat as a signaling pathway that links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth (Rogulja, 2008).

Studies of regeneration first led to models that proposed that growth could be influenced by gradients of positional values, with steep gradients promoting growth and shallow gradients suppressing growth. Experimental manipulations of Dpp pathway activity in the Drosophila wing supported this concept, but have left unanswered the question of how differences in the levels of Dpp pathway activity perceived by neighboring cells are actually linked to growth. This study has established that the Fat signaling pathway provides this link. Dpp signaling influences the Fat pathway; the expression of upstream Fat pathway regulators, the subcellular localization of Fat pathway components, and downstream transcriptional outputs of Fat signaling are all affected by Dpp signaling. The effects that Tkv and Brk expression have on the expression of Fat target genes parallels their effects on BrdU labeling and depend genetically on Fat signaling (Rogulja, 2008).

Dpp signaling impinges on Fat signaling upstream of Fat, as the expression of both of its known regulators, Fj and Ds, is regulated by Dpp signaling. Although the Fat signaling pathway was only recently discovered, and understanding of Fat signaling and its regulation remains incomplete, the inference that Fat signaling is normally influenced by the Dpp morphogen gradient is supported by the polarized localization of Dachs in wild-type wing discs. Near the D-V compartment boundary, the vector of Dachs polarization parallels the vector of the Dpp morphogen gradient, and the consequences of altered Dpp pathway activity confirm that the correlation between them is reflective of a functional link. The expression of Fj and Ds and the localization of Dachs are also polarized along the D-V axis. The implication that signaling downstream of the D-V compartment boundary thus also impinges on Fat signaling, and indeed may also influence growth through this pathway, is consistent with the observation that normal wing growth requires both A-P and D-V compartment boundary signals, and is further supported here by the observation that Notch activation affects both fj expression and Dachs localization (Rogulja, 2008).

The results argue that Fat signaling is influenced by the graded expression of its regulators: uniform expression of Fj and Ds can activate Fat signaling and thereby inhibit growth, whereas juxtaposition of cells expressing different levels of either Fj or Ds can inhibit Fat signaling and thereby promote growth. Here, a model is proposed to explain how Fat signaling can be modulated by Fj and Ds gradients. Although aspects of the model remain speculative, it provides an explanation for a number of observations that would otherwise appear puzzling, and serves as a useful framework for future studies (Rogulja, 2008).

Central to the model is the inference that the interaction between Ds and Fat activates Fat. This inference is well supported by the observations that mutation or downregulation of ds results in overgrowth and upregulation of Diap1, whereas uniform overexpression of Ds inhibits growth and Diap1 expression. A second key aspect of the model is that once activated by Ds, Fat locally transmits a signal to a complex at the membrane. An important corollary to this is that if Fat and Ds are not engaged around the entire circumference of a cell, then there could be a region where Fat is locally inactive. This is hypothetical, but the Fat-dependent polarization of Dachs implies that there can be regional differences in Fat activity within a cell. Local Fat signaling is then proposed to locally promote Warts stability and activity, and thereby locally antagonize Yki activity. Conversely, a local absence of Fat signaling could result in a local failure to phosphorylate Yki, which could then transit to the nucleus, where it would promote the expression of downstream target genes. Formally, this model treats Fat signaling like a contact inhibition pathway: if Fat is engaged by Ds around the entire circumference of a cell, then Fat is active everywhere and downstream gene expression is off; however, if Fat is not active on even one side of a cell, then Yki-dependent gene expression can be turned on and growth can be promoted (Rogulja, 2008).

In this model, graded expression of Fat regulators, like Fj and Ds, could modulate Fat signaling by polarizing Fat activity within a cell. In theoretical models of PCP, even shallow gradients of polarizing activity can be converted to strong polarity responses through positive-feedback mechanisms. How this might be achieved in Fat signaling is not yet clear, but the polarized localization of Dachs implies that, at some level, Fat activity is normally polarized in wild-type animals, even where the Fj and Ds expression gradients appear relatively shallow. Importantly, this polarization hypothesis provides a solution to the puzzle of how Ds could act as a ligand to activate Fat, yet inhibit Fat along the edges of Ds-expressing clones. In this model, Ds overexpression in clones polarizes Fat activity, possibly through its ability to relocalize Fat. This would allow a strong derepression of Yki on the side of the cell opposite to where Ds and Fat are actually bound, resulting in the induction of Yki:Scalloped target gene expression and promotion of cell proliferation. Propagation of this polarization, e.g., through the influence of Fat-Ds binding on Fat and Ds localization, might explain the spread of effects beyond immediately neighboring cells. Conversely, uniform expression of Ds would generate cells presenting a ligand that activates Fat and dampens the relative difference in expression levels between neighboring cells. Yki would thus remain sequestered around the entire cell circumference, consistent with the reduced growth and Diap1 expression observed. A dampening of gradients could also explain why the induction of Fat/Hippo target gene expression or BrdU labeling associated with clones expressing Ds, Fj, or TkvQ-D is biased toward cells outside of clones (Rogulja, 2008).

The hypothesis of Fat polarization and local signal transduction also suggests a solution to another puzzle. In terms of their effects on tissue polarity and Dachs localization, Fj and Ds always behave as though they have opposite effects on Fat. Conversely, in terms of their effects on cell proliferation and downstream gene expression, Fj and Ds behave as though they have identical effects on Fat. To explain this, it is proposed that Fj acts oppositely to Ds, by, for example, antagonizing Ds-Fat binding. The influence of Ds and Fj on polarity would be a function of the direction in which they polarize Fat activity, which, based on their effects on epitope-tagged protein Dachs:V5, is opposite. In contrast, their influence on downstream gene expression and growth would be a function of the degree to which they polarize Fat activity, which could be the same. In other words, their influence on polarity would be a function of the vector of their expression gradients, and their influence on growth would be a function of the slope. However, since Dachs:V5 generally appears to be strongly polarized, the actual interpretation of Fj and Ds gradients may involve feedback amplification and threshold responses rather than providing a continuous response proportional to the gradient slope (Rogulja, 2008).

The results have provided a molecular understanding of a how a gradient of positional values, established by the morphogen Dpp and reflected, at least in part, in the graded expression of Fj and Ds, can influence growth. However, it is clear that other mechanisms must also contribute to the regulation of wing growth. The relative contribution of Fat gradients to wing growth can be estimated by considering the size of the wing in dachs mutants, or when Fj and Ds are expressed ubiquitously, as, in either case, it would be expected that the derepression of Yki associated with normal Fat signaling gradients was abolished. In both cases, the wing is less than half its normal size. Fat signaling could thus be considered a major, but by no means the sole, mechanism for regulating wing growth. The determination that not all wing growth depends on the regulation of Fat activity fits with the observation that Dpp signaling promotes growth in at least two distinct ways, one dependent upon its gradient, and the other dependent upon its levels. Other models for wing growth, including a Vestigial-dependent recruitment of new cells into the wing, and an inhibition of Dpp-promoted wing growth by mechanical strain, have also been proposed. It is emphasized that these models are not incompatible with the conclusion that a Fat gradient influences growth. Rather, it is plausible, and even likely, that multiple mechanisms contribute to the appropriate regulation of wing growth. Indeed, it is expected that a critical challenge for the future will be to define not only the respective contributions of these or other mechanisms to growth control, but also to understand feedback and crosstalk processes that influence how these different mechanisms interact with each other (Rogulja, 2008).

A feed-forward circuit linking wingless, fat-dachsous signaling, and the warts-hippo pathway to Drosophila wing growth

During development, the Drosophila wing primordium undergoes a dramatic increase in cell number and mass under the control of the long-range morphogens Wingless (Wg, a Wnt) and Decapentaplegic (Dpp, a BMP). This process depends in part on the capacity of wing cells to recruit neighboring, non-wing cells into the wing primordium. Wing cells are defined by activity of the selector gene vestigial (vg) and recruitment entails the production of a vg-dependent 'feed-forward signal' that acts together with morphogen to induce vg expression in neighboring non-wing cells. This study identifies the protocadherins Fat (Ft) and Dachsous (Ds), the Warts-Hippo tumor suppressor pathway, and the transcriptional co-activator Yorkie (Yki, a YES associated protein, or YAP) as components of the feed-forward signaling mechanism; this mechanism promotes wing growth in response to Wg. vg generates the feed-forward signal by creating a steep differential in Ft-Ds signaling between wing and non-wing cells. This differential down-regulates Warts-Hippo pathway activity in non-wing cells, leading to a burst of Yki activity and the induction of vg in response to Wg. It is posited that Wg propels wing growth at least in part by fueling a wave front of Ft-Ds signaling that propagates vg expression from one cell to the next (Zecca, 2010).

During larval life, the Drosophila wing primordium undergoes a dramatic ~200-fold increase in cell number and mass driven by the morphogens Wg and Dpp. Focusing on Wg, it has been established that this increase depends at least in part on a reiterative process of recruitment in which wing cells send a feed-forward (FF) signal that induces neighboring cells to join the primordium in response to morphogen. The present results identify Ft-Ds signaling, the Wts-Hpo tumor suppressor pathway, and the transcriptional co-activator Yki as essential components of the FF process and define the circuitry by which it propagates from one cell to the next. This discussion considers, in turn, the nature of the circuit, the parallels between FF signaling and PCP, and the implications for the control of organ growth by morphogen (Zecca, 2010).

Several lines of evidence are presented that expression of the wing selector gene vg drives production of the FF signal by promoting a non-autonomous signaling activity of Ft. First, it was shown that vg acts both to up-regulate fj and down-regulate ds, two outputs known to elevate an outgoing, signaling activity of Ft in PCP. Second, it was demonstrated that experimental manipulations that elevate Ft signaling -- specifically, over-expression of Ft or removal of Ds -- generate ectopic FF signal. Third, and most incisively, it was shown that ft is normally essential in wing cells to send FF signal (Zecca, 2010).

Ft and Ds are both required in non-wing cells to receive the FF signal, functioning in this capacity to prevent the activation of vg unless countermanded by FF input. Notably, the removal of either Ft or Ds from non-wing cells constitutively activates the FF signal transduction pathway, mimicking receipt of the FF signal. However, the pathway is only weakly activated in this condition and the cells are refractory to any further elevation in pathway activity (Zecca, 2010).

Previous studies have defined a transduction pathway that links Ft-Ds signaling via the atypical myosin myosin Dachs (D) to suppression of the Wts kinase and enhanced nuclear import of Yki. Likewise, Ft and Ds operate through the same pathway to transduce the FF signal. Specifically, it was shown that manipulations of the pathway that increase nuclear activity of Yki (over-expression of D or Yki, or loss of Wts or Ex) cause non-wing cells to adopt the wing state. Conversely, removal of D, an intervention that precludes down-regulation of Wts by Ft-Ds signaling, prevents non-wing cells from being recruited into the wing primordium (Zecca, 2010).

To induce non-wing cells to become wing cells, transduction of the FF signal has to activate vg transcription. Activation is mediated by the vg QE and depends on binding sites for Scalloped (Sd), a member of the TEAD/TEF family of DNA binding proteins that can combine with either Yki or Vg to form a transcriptional activator Hence, it is posited that Yki transduces the FF signal by entering the nucleus and combining with Sd to activate vg. In addition, it is posited that once sufficient Vg produced under Yki-Sd control accumulates, it can substitute for Yki to generate a stable auto-regulatory loop in which Vg, operating in complex with Sd, sustains its own expression. Accordingly, recruitment is viewed as a ratchet mechanism. Once the auto-regulatory loop is established, neither FF signaling nor the resulting elevation in Yki activity would be required to sustain vg expression and maintain the wing state (Zecca, 2010).

Both the activation of the QE by Yki as well as the maintenance of its activity by Vg depend on Wg and Dpp input and hence define distinct circuits of vg auto-regulation fueled by morphogen. For activation, the circuit is inter-cellular, depending on Ft-Ds signaling for vg activity to propagate from one cell to the next. For maintenance, the circuit is intra-cellular, depending on Vg to sustain its own expression. Accordingly, it is posited that growth of the wing primordium is propelled by the progressive expansion in the range of morphogen, which acts both to recruit and to retain cells in the primordium (Zecca, 2010).

To date, Ft-Ds signaling has been studied in two contexts: the control of Yki target genes in tissue growth and the orientation of cell structures in PCP. Most work on tissue growth has focused on Yki target genes that control basic cell parameters, such as survival, mass increase, and proliferation (e.g., diap, bantam, and cyclinE). In this context, Ds and Ft are thought to function as a ligand-receptor pair, with tissue-wide gradients of Ds signal serving to activate Ft to appropriate levels within each cell. In contrast, Ft and Ds behave as dual ligands and receptors in PCP, each protein having intrinsic and opposite signaling activity and both proteins being required to receive and orient cells in response to each signal (Zecca, 2010).

This study has analyzed a different, Yki-dependent aspect of growth, namely the control of organ size by the regulation of a selector gene, vg. In this case, Ft appears to correspond to a ligand, the FF signal, and Ds to a receptor required to receive the ligand -- the opposite of the Ds-Ft ligand-receptor relationship inferred to regulate other Yki target genes. Moreover, as in PCP, evidence was found that Ft and Ds operate as bidirectional ligands and receptors: like Ds, Ft is also required for receipt of the FF signal, possibly in response to an opposing signal conferred by Ds (Zecca, 2010).

Studies of Ft-Ds interactions, both in vivo and in cell culture, have established that Ft and Ds interact in trans to form hetero-dimeric bridges between neighboring cells, the ratio of Ft to Ds presented on the surface of any given cell influencing the engagement of Ds and Ft on the abutting surfaces of its neighbors. These interactions are thought, in turn, to polarize the sub-cellular accumulation and activity of D. Accordingly, it is posited that vg activity generates the FF signal by driving steep and opposing differentials of Ft and Ds signaling activity between wing (vgON) and non-wing (vgOFF) cells. Further, it is posited that these differentials are transduced in cells undergoing recruitment by the resulting polarization of D activity, acting through the Wts-Hpo pathway and Yki to activate vg (Zecca, 2010).

Thus, it is proposeed that FF propagation and PCP depend on a common mechanism in which opposing Ft and Ds signals polarize D activity, both proteins acting as dual ligands and receptors for each other. However, the two processes differ in the downstream consequences of D polarization. For FF propagation, the degree of polarization governs a transcriptional response, via regulation of the Wts-Hpo pathway and Yki. For PCP, the direction of polarization controls an asymmetry in cell behavior, through a presently unknown molecular pathway (Zecca, 2010).

FF propagation and PCP may also differ in their threshold responses to D polarization. vg expression is graded, albeit weakly, within the wing primordium, due to the response of the QE to graded Wg and Dpp inputs. Hence, a shallow differential of Ft-Ds signaling reflecting that of Vg may be sufficient to orient cells in most of the prospective wing territories, but only cells in the vicinity of the recruitment interface may experience a steep enough differential to induce Yki to enter the nucleus and activate vg (Zecca, 2010).

Finally, FF propagation and PCP differ in at least one other respect, namely, that they exhibit different dependent relationships between Ft and Ds signaling. In PCP, clonal removal of either Ft or Ds generates ectopic polarizing activity, apparently by creating an abrupt disparity in the balance of Ft-to-Ds signaling activity presented by mutant cells relative to that of their wild type neighbors. By contrast, in FF propagation, only the removal of Ds, and not that of Ft, generates ectopic FF signal. This difference is attributed to the underlying dependence of Ft and Ds signaling activity on vg. In dso cells, Ft signaling activity is promoted both by the absence of Ds and by the Vg-dependent up-regulation of fj. However, in fto cells, Ft is absent and Vg down-regulates ds, rendering the cells equivalent to dso fto cells (which are devoid of signaling activity in PCP). Thus, for FF propagation, the underlying circuitry creates a context in which only the loss of Ds, but not that of Ft, generates a strong, ectopic signal. For PCP, no such circuit bias applies (Zecca, 2010).

Morphogens organize gene expression and cell pattern by dictating distinct transcriptional responses at different threshold concentrations, a process that is understood conceptually, if not in molecular detail. At the same time, they also govern the rate at which developing tissues gain mass and proliferate, a process that continues to defy explanation (Zecca, 2010).

One long-standing proposal, the 'steepness hypothesis,' is that the slope of a morphogen gradient can be perceived locally as a difference in morphogen concentration across the diameter of each cell, providing a scalar value that dictates the rate of growth. Indeed, in the context of the Drosophila wing, it has been proposed that the Dpp gradient directs opposing, tissue-wide gradients of fj and ds transcription, with the local differential of Ft-Ds signaling across every cell acting via D, the Wts-Hpo pathway, and Yki to control the rate of cell growth and proliferation. The steepness hypothesis has been challenged, however, by experiments in which uniform distributions of morphogen, or uniform activation of their receptor systems, appear to cause extra, rather than reduced, organ growth (Zecca, 2010).

The current results provide an alternative interpretation (see The vestigial feed-forward circuit, and the control of wing growth by morphogen). It is posited that 'steepness,' as conferred by the local differential of Ft-Ds signaling across each cell, is not a direct reflection of morphogen slope but rather an indirect response governed by vg activity. Moreover, it is proposed that it promotes wing growth not by functioning as a relatively constant parameter to set a given level of Wts-Hpo pathway activity in all cells but rather by acting as a local, inductive cue to suppress Wts-Hpo pathway activity and recruit non-wing cells into the wing primordium (Zecca, 2010).

How important is such local Ft-Ds signaling and FF propagation to the control of wing growth by morphogen? In the absence of D, cells are severely compromised for the capacity to transduce the FF signal, and the wing primordium gives rise to an adult appendage that is around a third the normal size, albeit normally patterned and proportioned. A similar reduction in size is also observed when QE-dependent vg expression is obviated by other means. Both findings indicate that FF signaling makes a significant contribution to the expansion of the wing primordium driven by Wg and Dpp. Nevertheless, wings formed in the absence of D are still larger than wings formed when either Wg or Dpp signaling is compromised. Hence, both morphogens must operate through additional mechanisms to promote wing growth (Zecca, 2010).

At least three other outputs of signaling by Wg (and likely Dpp) have been identified that work in conjunction with FF propagation. First, as discussed above, Wg is required to maintain vg expression in wing cells once they are recruited by FF signaling, and hence to retain them within the wing primordium. Second, it functions to provide a tonic signal necessary for wing cells to survive, gain mass, and proliferate at a characteristic rate. And third, it acts indirectly, via the capacity of wing cells, to stimulate the growth and proliferation of neighboring non-wing cells, the source population from which new wing cells will be recruited. All of these outputs, as well as FF propagation, depend on, and are fueled by, the outward spread of Wg and Dpp from D-V and A-P border cells. Accordingly, it is thought that wing growth is governed by the progressive expansion in the range of Wg and Dpp signaling (Zecca, 2010).

Identification of Ft-Ds signaling, the Wts-Hpo pathway, and Yki as key components of the FF recruitment process provides a striking parallel with the recently discovered involvement of the Wts-Hpo pathway and Yki/YAP in regulating primordial cell populations in vertebrates, notably the segregation of trophectoderm and inner cell mass in early mammalian embryos and that of neural and endodermal progenitor cells into spinal cord neurons and gut. As in the Drosophila wing, Wts-Hpo activity and YAP appear to function in these contexts in a manner that is distinct from their generic roles in the regulation of cell survival, growth, and proliferation, namely as part of an intercellular signaling mechanism that specifies cell type. It is suggested that this novel employment of the pathway constitutes a new, and potentially general, mechanism for regulating tissue and organ size (Zecca, 2010).

Notch inhibits yorkie activity in Drosophila wing discs

During development, tissues and organs must coordinate growth and patterning so they reach the right size and shape. During larval stages, a dramatic increase in size and cell number of Drosophila wing imaginal discs is controlled by the action of several signaling pathways. Complex cross-talk between these pathways also pattern these discs to specify different regions with different fates and growth potentials. This study shows that the Notch signaling pathway is both required and sufficient to inhibit the activity of Yorkie (Yki), the Salvador/Warts/Hippo (SWH) pathway terminal transcription activator, but only in the central regions of the wing disc, where the TEAD factor and Yki partner Scalloped (Sd) is expressed. This cross-talk between the Notch and SWH pathways is shown to be mediated, at least in part, by the Notch target and Sd partner Vestigial (Vg). It is proposed that, by altering the ratios between Yki, Sd and Vg, Notch pathway activation restricts the effects of Yki mediated transcription, therefore contributing to define a zone of low proliferation in the central wing discs (Djiane, 2014).

In order to investigate the possibility of cross talk between the Notch and Sav/Warts/Hippo (SWH) pathways, the expression pattern of ex-lacZ, a reporter of Yki activity, which reveals the places where SWH activity is lowest was compared with NRE-GFP, which gives a direct read out of Notch activity. In the wing pouch these reporters direct expression in patterns that are complementary. Thus, ex-lacZ expression is completely absent from the dorso-ventral boundary where Notch activity, reported by NRE-GFP, is at its highest. Conversely, in late stage discs, ex-lacZ expression is higher in pro-vein regions where Notch activity (NRE-GFP) is low. Because ex-lacZ gives a mirror image of SWH activity, these results suggest that both Notch and SWH pathways are active together in the D/V boundary and are largely inactive in the pro-veins (Djiane, 2014).

The consequences of modulating Notch activity on the expression of ex-lacZ was tested as an indicator of its effects on SWH pathway. Expression of Nicd, the constitutively active form of the Notch receptor, promoted a strong down-regulation of ex-lacZ in the wing pouch. This effect was stronger in the region surrounding the D/V boundary and weaker towards the periphery. Little down-regulation occurred outside the pouch. Conversely, when Notch activity was impaired, through RNAi mediated knock-down in randomly generated overexpression clones, ex-lacZ levels were up-regulated. This effect was also only evident within the wing-pouch. Notch activity is therefore necessary and sufficient for the inhibition of ex-lacZ in the wing pouch, suggesting that it contributes to the normal down-regulation of ex-lacZ at the D/V boundary (Djiane, 2014).

In the wing pouch, ex-lacZ expression requires Yki. Therefore, to mediate the observed inhibition of ex-lacZ expression, Notch could either exert its actions upstream of Yki, by activating the SWH pathway, or downstream of Yki, by inhibiting Yki's transcriptional activity. To determine which of these alternatives is correct, the consequences of Notch activity on ectopic Yki expression were assessed. When over-expressed in a stripe of cells along the A/P boundary, Yki was able to promote strong expression of ex-lacZ at the periphery of the wing pouch. Strikingly, the high levels of Yki were not able to force ex-lacZ expression at the D/V boundary where Notch activity is highest. These results suggest that the actions of Notch, ie ex-lacZ down-regulation, are epistatic to Yki. This was further verified when high levels of Yki were expressed together with high levels of Nicd. In this case, Nicd suppressed the ex-lacZ expression, demonstrating that it wins out over Yki in the wing pouch. However, at the periphery of the discs, Nicd was unable to modify the effects of Yki over-expression on ex-lacZ levels. Taken together these results suggest that Notch-mediated down-regulation of ex-lacZ occurs at the level or downstream of Yki (Djiane, 2014).

Since a major output of Notch pathway activity is the up-regulation of gene expression, whether any of the directly regulated Notch target-genes could be responsible for antagonizing Yki was assessed. Amongst the direct Notch targets identified in wing discs, several are predicted to encode transcriptional repressors. These include members of the HES family, E(spl)mβ, E(spl)m5, E(spl)m7, E(spl)m8 and Deadpan (dpn), as well as the homeodomain protein Cut. All of these proteins are normally expressed at high levels along the D/V boundary, in response to Notch activity, and hence are candidates to mediate the repression of ex-lacZ (Djiane, 2014).

Over-expression of E(spl)mβ, E(spl)m5 or E(spl)m7 repressors had no effect on ex-lacZ expression. In contrast, over-expression of either E(spl)m8 or of dpn resulted in a robust down-regulation of ex-lacZ. The effect differed slightly from that from Nicd expression, in that ex-lacZ expression was not completely abolished and low levels persisted throughout the wing pouch domain. These results indicate that a subset of the HES bHLH proteins have the capability to repress ex-lacZ, and hence are candidates to antagonize Yki. Previous experiments have demonstrated that the E(spl)bHLH genes and dpn have overlapping functions, especially at the D/V boundary. Therefore to determine whether these factors normally contribute to the repression of ex-lacZ, it was necessary to eliminate all of the E(spl)bHLH genes in combination with dpn. To achieve this a potent RNAi directed against dpn was expressed in MARCM clones that were homozygous mutant for a deficiency removing the entire E(spl) complex. No derepression of ex-lacZ was detectable in such clones, suggesting that none of the E(spl)bHLH/dpn genes can account for the repression of ex-lacZ at the D/V boundary or in the wing pouch. Therefore even though E(spl)m8 and dpn expression is sufficient for ex-lacZ repression, they do not appear to be essential in the context of the wing pouch (Djiane, 2014).

An alternative candidate was Cut, which encodes a transcriptional repressor and is expressed at the D/V boundary in response to Notch signaling. Similar to some of the HES genes, over-expression of Cut promoted a down-regulation of ex-lacZ. This was most clearly evident at early developmental stages because Cut induced a strong epithelial delamination at later stages, confounding the interpretation. However no up-regulation of ex-lacZ was detectable when Cut function was ablated, using RNAi, even though Cut levels where efficiently reduced. Thus, as with the HES genes, Cut is capable of inhibiting ex-lacZ expression but does not appear to be essential for the regulation of ex under normal conditions in the wing pouch (Djiane, 2014).

Recent studies have demonstrated that, in the absence of Yki, several SWH target genes are kept repressed by Sd, the DNA-binding partner of Yki. This so-called 'default repression' requires Tondu-domain-containing growth inhibitor (Tgi), an evolutionarily conserved tondu domain containing protein, which acts as a potent co-repressor with Sd. There is no evidence that Drosophila tgiM is a target of Notch in the wing disc, making it an unlikely candidate to mediate the inhibitory effects on Yki-mediated ex-lacZ expression. However, vg, which encodes another Sd binding-partner with a tondu domain, is directly regulated by Notch in the wing pouch. It was therefore hypothesized that Vg could mediate the effects of Notch on Yki function and ex-lacZ down-regulation (Djiane, 2014).

In agreement with the hypothesis, when Vg was over-expressed it strongly inhibited ex-lacZ expression in the pouch and promoted a modest overgrowth of the tissue. This overgrowth is somewhat puzzling since it appears that Yki activity, as monitored by ex-lacZ, is lowered in the presence of excess Vg. How over-expressed Vg triggers overgrowth remains poorly understood, but has been proposed to involve a cross-talk with the wg pathway. More recently, it has been shown that the expansion of the pouch region is achieved by Vg activating transiently and non-autonomously Yki in cells not expressing Vg. These cells are then recruited to become wing pouch cells and turn on vg expression. This model predicts a wave of Yki activation around Vg positive cells. Therefore, the overgrowth seen when Vg is over-expressed, could be due to a non-autonomous effect where more cells are recruited as pouch cells at the expense of more peripheral cells. Alternatively, Vg could promote proliferation of the pouch cells by an as yet unidentified mechanism, independent of Yki (Djiane, 2014).

Conversely to over-expressed Vg inhibiting ex-lacZ expression, lowering the levels of vg using RNA interference in the whole posterior compartment resulted in a significant up-regulation of ex-lacZ. Vg knock down has proven difficult to achieve in small populations of cells, due to their elimination from the wing pouch, probably by cell competition. Thus, unlike the other factors tested, Vg is required for the repression of ex-lacZ in the wing pouch. It was further shown that, co-expressing with NICD a vg RNAi transgene in the patched domain, suppresses the NICD mediated ex-lacZ repression in the wing pouch. Taken together, these results suggest that Vg mediates the repressive effects of Notch on expanded expression (Djiane, 2014).

If the involvement of Vg downstream of Notch is a general mechanism for cross-talk between Notch and Yki, other targets of the Sd-Yki complex should be inhibited by Notch in a similar manner to ex-lacZ. However, apart from expanded, all other known Yki targets in the wing pouch, such as thread/DIAP1, diminutive/myc, and Cyclin E are also direct Notch targets. Their final expression patterns are therefore a reflection of the balance between different transcriptional inputs, in particular Notch and Yki. A model predicts that Notch could have a dual effect on the expression of genes: a positive direct effect through the NICD/Su(H) complex when bound in their promoters, but also a negative effect through the induction of Vg, which prevents the positive effect of Yki on Sd bound promoters (Djiane, 2014).

In agreement with this model, thread/DIAP1 and diminutive/myc, two well established Yki targets in wing discs, which are normally refractory to Notch mediated activation in the centre of the pouch, become susceptible to Nicd when Vg or Sd levels are lowered through RNAi (Djiane, 2014).

Focusing on DIAP1, it was decided to separate the Notch and Yki direct inputs on transcription by isolating the Hippo pathway Responsive Elements (HREs) from any potential Notch Responsive Elements (NREs). IAP2B2C-lacZ is a previously described DIAP1-HRE driving lacZ reporter expression that does not contain any NRE, at least based on Su(H) ChIP data and bio-informatics prediction of Su(H) binding sites. The model predicts that this IAP2B2C-lacZ reporter should be inhibited by Vg. In control wing discs, it was confirmed that IAP2B2C-lacZ is expressed at uniform low levels with a slight increase at the periphery of the pouch, where Vg protein levels have been shown to fade. The D/V boundary expression of DIAP1 is not reported by IAP2B2C-lacZ confirming that the NRE is absent in this reporter (Djiane, 2014).

Vg levels were lowered using moderate RNAi knocked down in the whole posterior compartment using the hh-Gal4 driver. In this experimental set-up, the posterior compartment is smaller than normal, and vg knock-down induced a 12% up-regulation of IAP2B2C-lacZ expression when compared to IAP2B2C-lacZ levels in the anterior control compartment, demonstrating that Vg has a negative effect on this reporter activity (there was no difference in IAP2B2C-lacZ expression between the anterior and posterior compartment in the pouch region of control discs). It is noted that IAP2B2C-lacZ expression was up-regulated in a small stripe of cells in the anterior compartment just at the boundary with vg depleted cell. This region was excluded from the quantifications, but suggests that the IAP2B2C-lacZ reporter fragment could be sensitive to a non-autonomous input acting around the boundary of cells with different Vg levels (Djiane, 2014).

It appears therefore, that at least for the two Yki targets ex-lacZ and IAP2B2C-lacZ, Vg inhibits their expression in the wing pouch. Previous studies reported independent roles of Vg and Yki on the activation of their targets, and could appear to contradict this newly described inhibitory role of Vg on Yki targets. However, in these previous studies, it was demonstrated that Vg and Yki do not require each other to promote wing pouch cell survival and to activate their respective targets, which does not rule out any negative cross regulation, as shown in this report (Djiane, 2014).

This analysis brings therefore new evidence of the central role of Vg in the complex network regulating wing disc growth, adding a new level of complexity in its interaction with the SWH pathway effector Yki. Thus, Notch induced expression of Vg could give rise to an Sd-Vg repressive complex that prevents expression of Yki targets. In situations where SWH signaling is lowest, Yki levels may be sufficiently high to overcome this repression. This suggests that in the wing pouch, Notch and SWH would act co-operatively rather than antagonistically (Djiane, 2014).

Outside of the pouch, at the wing disc periphery, sd and vg expressions are not promoted by Notch activity. Furthermore, other binding partners for Yki, such as Homothorax are expressed there and might substitute for Sd to control the expression of Yki targets in a way similar to what has been described in the Drosophila eye. The differential expression of these transcription factors in the disc could explain why Notch only has an inhibitory effect on Yki targets in the wing pouch. Furthermore, it is also worth noting that Notch has very different effect outside of the pouch, where it promotes Yki stabilization non-autonomously via its regulation of ligands for the Jak/Stat pathway (Djiane, 2014).

In summary, the evidence demonstrates that Notch activity can inhibit Yki under circumstances where Yki acts together with Sd. It does so by promoting the expression of Vg, a co-factor for Sd, counteracting the effects of Yki. This cross talk potentially extends to mammalian systems as the active form of NOTCH1, NICD1 promotes the up-regulation of VGLL3 (a human homologue of vg) in MCF-10A breast cancer derived cells. Thus, similar mechanisms may also be important in mediating interactions between the NOTCH and SWH pathways in human diseases (Djiane, 2014).

Because the end-point of SWH pathway activity is to prevent Yki function, the inhibitory effects of Notch on Yki could provide an explanation for those cellular contexts where the two pathways act co-operatively, as at the D/V boundary in the wing discs. Similar co-operative effects have been noted in the Drosophila follicle cells. However, in this case it is the SWH activity that is involved in promoting the expression of Notch targets. In other contexts, such as the mouse intestine, accumulation of Yap1, the mouse Yki homolog, and therefore inhibition of the SWH promotes Notch activity. These examples demonstrate that the interactions between Notch and the SWH are highly dependent on cellular context. The results suggest that some of these differences may be explained by the nature of the target genes that are regulated and by which Yki co-operating transcription factors are present in the receiving cells (Djiane, 2014).

Critical role for Fat/Hippo and IIS/Akt pathways downstream of Ultrabithorax during haltere specification in Drosophila

In Drosophila, differential development of wing and haltere, which differ in cell size, number and morphology, is dependent on the function of Hox gene Ultrabithorax (Ubx). This paper reports studies on Ubx-mediated regulation of the Fat/Hippo and IIS/dAkt pathways, which control cell number and cell size during development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded, caused considerable increase in haltere size, mainly due to increase in cell number. These phenotypes were also associated with the activation of Akt pathways in developing haltere. Although activation of Akt alone did not affect the cell size or the organ size, dramatic increase was observed in haltere size when Akt was activated in the background where expanded is down regulated. This was associated with the increase in both cell size and cell number. The organ appeared flatter than wildtype haltere and the trichome morphology and spacing resembled that of wing suggesting homeotic transformations. Thus, these results suggest a link between cellular growth and pattern formation and the final differentiated state of the organ (Singh, 2015).

Wing and haltere are the dorsal appendages of second and third thoracic segments, respectively, of adult Drosophila. They are homologous structures, although differ greatly in their morphology. The homeotic gene Ultrabithorax (Ubx), which is required and sufficient to confer haltere fate to epithelial cells, is known to regulate many wing patterning genes to specify haltere, but the mechanism is still poorly understood (Singh, 2015).

There are a number of differences between wing and haltere at the cellular and organ levels. Wing is a large, flat and thin structure, while haltere is a small globular structure, although both are made up of 2-layered sheet of epithelial cells. Space between the two layers of cells in haltere is filled with haemocytes. Cuticle area of each wing cell is 8 fold more than a haltere cell. Haltere has smaller and fewer cells than the wing. Trichomes of wing cells are long and thin, while haltere trichomes are short and stout in morphology. The ratio of anterior to posterior compartment size in the haltere (~2.5:1) is much different from that in the wing (~1.2:1). Haltere also lacks wing-type vein and sensory bristles. Haltere cells are more cuboidal compared to flatter wing cells (Roch, 2000). Thus, cell number, size and shape all add to the differences in the size and shape of the two organs (Singh, 2015).

However, cells of the third instar larval wing and haltere discs are similar in size and shape (Makhijani, 2007). The difference between cell size and shape becomes evident at late pupal stages (Roch, 2000). Wing cells become much larger, compared to haltere cells. At pupal stages, they also exhibit differences in the organization of actin cytoskeleton elements viz. F-actin levels are much higher in haltere cells compared to wing cells (Roch, 2000) (Singh, 2015).

In the context of final shape of wings and halteres, one needs to understand the mechanism by which Ubx influences cell size, shape and arrangement. It is possible that Ubx regulates overall shape of the haltere by regulating either cell size and/or shape. The current understanding of mechanisms by which wing and haltere differ at cellular, tissue and organ level is ambiguous (Sanchez-Herrero, 2013). For example, while removal of Ubx from the entire haltere, or at least from one entire compartment, leads to haltere to wing transformation with increased growth of Ubx minus tissues, mitotic clones of Ubx (using the null allele Ubx6.28) show similar sized twin spot in small clones (Crickmore, 2006, De Navas, 2006; Makhijani, 2007). Only when very large clones of Ubx6.28Ubx6.28 are generated, one can see increased growth compared to their twin spots (Crickmore, 2006). This suggests that unless a certain threshold level of growth factors is de-repressed, the haltere does not show any overgrowth phenotype (Singh, 2015).

There have been several efforts to identify functional and molecular mechanisms by which Ubx regulates genes/pathways to provide haltere its distinct morphology. Various approaches have been used to identify targets of Ubx that are expected to differentially express between wing and haltere, e.g., loss-of-function genetics, deficiency screens, enhancer-trap screening and genome wide approaches such as microarray analysis and chromatin immunoprecipitation (ChIP). Targets include genes involved in diverse cellular functions like components of the cuticle and extracellular matrix, genes involved in cell specification, cell proliferation, cell survival, cell adhesion, or cell differentiation, structural components of actin and microtubule filaments, and accessory proteins controlling filament dynamics (reviewed in Sanchez-Herrero, 2013; Singh, 2015).

Decapentaplegic (Dpp), Wingless (Wg), and Epidermal growth factor receptor (EGFR) are some of the major growth and pattern regulating pathways that are repressed by Ubx in the haltere (Weatherbee, 1998, Shashidhara, 1999; Prasad, 2003; Mohit, 2006; Crickmore, 2006, Pallavi, 2006; De Navas, 2006; Makhijani, 2007). However, over-expression of Dpp, Wg, Vestigial (Vg) or Vein (Vn) provides only marginal growth advantage to haltere compared to the wildtype. In this context, additional growth regulating pathways amongst the targets of Ubx were examined. Genome wide studies have identified many components of Fat/Hippo and Insulin-insulin like/dAkt signalling (IIS/dAkt) pathways as potential targets of Ubx. The Fat/Hippo pathway is a crucial determinant of organ size in both Drosophila and mammals. It regulates cell proliferation, cell death, and cell fate decisions and coordinates these events to specify organ size. In contrast, the IIS/dAkt pathway is known to regulate cell size (Singh, 2015).

Recent studies have revealed that the Fat/Hippo pathway networks with other signalling pathways. For example, during wing development, Fat/Hippo pathway activities are dependent on Four-jointed (Fj) and Dachous (Ds) gradients, which are influenced by Dpp, Notch, Wg and Vg. Glypicans, which play a prominent role in morphogen signalling, are regulated by Fat/Hippo signalling (Baena-Lopez, 2008). EGFR activates Yorkie (Yki; effector of Fat/Hippo pathway) through its EGFR-RAS-MAPK signalling by promoting the phosphorylation of Ajuba family protein WTIP (Reddy, 2013). However, EGFR negatively regulates events downstream of Yki (Herranz, 2012). The Fat/Hippo pathway is also known to inhibit EGFR signalling, which makes the interaction between the two pathways very complex and context-dependent. IIS/dAkt pathway is also known to activate Yki signalling and vice-versa. Thus, Fat/Hippo pathway may specify organ size by regulating both cell number (directly) and cell size (via regulating IIS/dAkt pathway) (Singh, 2015).

This study reports studies on the functional implication of regulation of Fat/Hippo and IIS/dAkt pathways by Ubx in specifying haltere development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded (ex), induced considerable increase in haltere size, mainly due to increase in cell number. Although activation of dAkt alone did not affect the organ size or the cell size, activation of Yki or down regulation of ex in the background of over-expressed dAkt caused dramatic increase in haltere size, much severe than Yki or ex alone. In this background, increase was observed in both cell size and cell number. The resulted haltere appeared flatter than wildtype haltere and the morphology of trichomes and their spacing resembled that of wing suggesting homeotic transformations. Thus, these results suggest a link between cellular growth and pattern formation and the final differentiated state of the organ (Singh, 2015).

The findings suggest that, downstream of Ubx, the Fat/Hippo pathway is critical for haltere specification. It is required for Ubx-mediated specification of organ size, sensory bristle repression, trichome morphology and arrangement. The Fat/Hippo pathway cooperates with the IIS/dAkt pathway, which is also a target of Ubx, in specifying cell size and compartment size in developing haltere. The fact that over-expression of Yki or downregulation of ex show haltere-to-wing transformations at the levels of organ size and shape, and trichome morphology and arrangement, suggest that regulation of the Fat/Hippo pathway by Ubx is central to the modification of wing identity to that of the haltere (Singh, 2015).

The observations made in this study pose new questions and suggest various interesting possibilities to study the Fat/Hippo pathway with a new perspective.

(1) It was observed that while Yki is nuclear in haltere discs, it appears to be non-functional. Yki is a transcriptional co-activator protein, which requires other DNA-binding partners for its activity. In this context, understanding the precise relationship between Yki and Ubx may provide an insight into mechanism of haltere specification (Singh, 2015).

(2) The Fat/Hippo pathway (along with the IIS/dAkt pathway) may be involved in the specification of cell size, trichome morphology and their arrangement, all of which are important parameters in determining organ morphology. Recent studies indicate that the Fat/Hippo pathway regulates cellular architecture and the mechanical properties of cells in response to the environment. It would be interesting to study the role of the Fat/Hippo pathway in regulating the cytoskeleton of epithelial cells during development. Haltere cells at pupal stages exhibit higher levels of F-actin than wing cells. One possible mechanism that is currently being investigated is lowering of F-actin levels in transformed haltere cells due to over-expression of Yki or down regulation of ex. This may cause flattening of cells during morphogenesis leading to larger organ size (Singh, 2015).

(3) Reversing cell size and number was sufficient to induce homeotic transformations at the level of haltere morphology. This suggests the importance of negative regulation of genetic mechanisms that determine cell size and number, in specifying an organ size and shape. As a corollary, Ubx-mediated regulation of Fat/Hippo and IIS/dAkt pathways provides an opportunity to study cooperative repression of cell number and cell size during organ specification (Singh, 2015).

(4) Certain genetic backgrounds investigated in this study showed severe effect on cell proliferation in haltere discs than in wing discs. This could be due to the fact that, the wing disc has already attained a specific size by the third instar larval stage (the developmental stage examined in this study), which is controlled by several pathways. Any change to this size may need more drastic alteration to the controlling mechanisms. As Ubx specifies haltere by modulating various wing-patterning events, there may still exist a certain degree of plasticity in mechanisms that determine the size of the haltere. However, in absolute terms, the haltere is also resistant to changes in growth control due to regulation by Ubx at multiple levels. Thus, differential development of wing and haltere provides a very good assay system to study not only growth control, but also to dissect out function of important growth regulators (tumour suppressor pathways) such as the Fat/Hippo pathway using various genome-wide approaches (Singh, 2015).

Warts and Yorkie mediate intestinal regeneration by influencing stem cell proliferation

Homeostasis in the Drosophila midgut is maintained by stem cells. The intestinal epithelium contains two types of differentiated cells that are lost and replenished: enteroendocrine (EE) cells and enterocytes (ECs). Intestinal stem cells (ISCs) are the only cells in the adult midgut that proliferate, and ISC divisions give rise to an ISC and an enteroblast (EB), which differentiates into an EC or an EE cell. If the midgut epithelium is damaged, then ISC proliferation increases. Damaged ECs express secreted ligands (Unpaired proteins) that activate Jak-Stat signaling in ISCs and EBs to promote their proliferation and differentiation]. This study shows that the Hippo pathway components Warts and Yorkie mediate a transition from low- to high-level ISC proliferation to facilitate regeneration. The Hippo pathway regulates growth in diverse organisms and has been linked to cancer. Yorkie is activated in ECs in response to tissue damage or activation of the damage-sensing Jnk pathway. Activation of Yorkie promotes expression of unpaired genes and triggers a nonautonomous increase in ISC proliferation. These observations uncover a role for Hippo pathway components in regulating stem cell proliferation and intestinal regeneration (Staley, 2010).

Hippo signaling can have both autonomous and nonautonomous effects on growth, and this study reports that in the adult Drosophila midgut, Yki has profound nonautonomous effects on growth via the Jak-Stat pathway. Jak-Stat signaling is important for proliferation control and stem cell biology, not only in the Drosophila intestine, but also in other tissues, both in Drosophila and in vertebrates. Members of the interleukin (IL) family of cytokines are homologous to Upd ligands, and a microarray study in cultured mammalian cells found that the Yki homolog Yap could regulate IL cytokines, which raises the possibility that a regulatory connection between Hippo signaling and Jak-Stat signaling might be conserved. Increased levels and nuclear localization of Yap have been reported in colon cancer patient samples, and ubiquitous Yap1 overexpression causes overproliferation of progenitor cells in the murine intestine. These observations suggest that future considerations of the potential contributions of Hippo signaling to colon cancer should include evaluations both of its possible regulation by Jnk signaling and of possible nonautonomous effects mediated by cytokines (Staley, 2010).

Influence of fat-hippo and notch signaling on the proliferation and differentiation of Drosophila optic neuroepithelia

The Drosophila optic lobe develops from neuroepithelial cells, which function as symmetrically dividing neural progenitors. This study describes a role for the Fat-Hippo pathway in controlling the growth and differentiation of Drosophila optic neuroepithelia. Mutation of tumor suppressor genes within the pathway, or expression of activated Yorkie, promotes overgrowth of neuroepithelial cells and delays or blocks their differentiation; mutation of yorkie inhibits growth and accelerates differentiation. Neuroblasts and other neural cells, by contrast, appear unaffected by Yorkie activation. Neuroepithelial cells undergo a cell cycle arrest before converting to neuroblasts; this cell cycle arrest is regulated by Fat-Hippo signaling. Combinations of cell cycle regulators, including E2f1 and CyclinD, delay neuroepithelial differentiation, and Fat-Hippo signaling delays differentiation in part through E2f1. Roles for Jak-Stat and Notch signaling were also characterized. These studies establish that the progression of neuroepithelial cells to neuroblasts is regulated by Notch signaling, and suggest a model in which Fat-Hippo and Jak-Stat signaling influence differentiation by their acceleration of cell cycle progression and consequent impairment of Delta accumulation, thereby modulating Notch signaling. This characterization of Fat-Hippo signaling in neuroepithelial growth and differentiation also provides insights into the potential roles of Yes-associated protein in vertebrate neural development and medullablastoma (Reddy, 2010).

Both normal development and homeostasis require that cells transition from proliferating undifferentiated cells to quiescent differentiated cells. Failure to undergo this transition results in tumor formation, whereas premature differentiation results in hypotrophy. Some tissues balance proliferation and differentiation by employing stem cells that divide asymmetrically to yield both a stem cell and a progenitor cell, which will then give rise to differentiated cells. Most of the Drosophila central nervous system develops in this way: individual cells within the embryonic ectoderm become specified as neural stem cells called neuroblasts (NBs), which divide asymmetrically to yield a neuroblast and a progenitor cell called a ganglion mother cell (GMC). By contrast, much of the vertebrate central nervous system initially develops from neuroepithelia (NE), sheets of epithelial neural progenitor cells that function as symmetrically dividing neural stem cells. This provides for rapid expansion of neural tissue, and then, as development proceeds, asymmetrically dividing progenitor cells arise, although the mechanisms that govern their appearance are not well understood. The optic lobe of Drosophila is unlike the rest of the Drosophila nervous system in that, akin to the vertebrate nervous system, it develops from NE. The optic lobe may thus serve as a model in which the powerful experimental approaches available in Drosophila can be used to investigate mechanisms that control the growth and differentiation of NE (Reddy, 2010).

At the end of larval development, the optic lobes comprise the lateral half of each of the two brain hemispheres, and are organized into lamina, medulla and lobula layers. The optic lobes originate from clusters of epithelial cells that invaginate from a small region on the surface of the embryo (the optic placode). During larval development, these cells separate into an inner optic anlagen (IOA), which will give rise to the lobula and inner part of the medulla, and an outer optic anlagen (OOA), which will give rise to the outer part of the medulla and the lamina. Initially, the IOA and OOA are composed entirely of NE cells, but during the third larval instar they begin to differentiate. Along the lateral margin of the OOA, NE cells undergo cell cycle arrest in G1, and then are recruited to differentiate into lamina neurons by signals from the arriving retinal axons. Along the medial margin of the OOA, a wave of differentiation sweeps across the NE from medial to lateral, converting NE cells into medulla NBs. These NBs divide perpendicularly to the plane of the neuroepithelium, and appear to follow a NB developmental program, giving rise to additional self-renewing NBs, and to GMCs, which ultimately give rise to neurons (Reddy, 2010).

The Fat-Hippo signaling pathway encompasses distinct downstream branches that regulate planar cell polarity and gene expression. Transcriptional targets of the pathway include genes that influence cell proliferation and cell survival, and consequently Fat-Hippo signaling is an important regulator of growth from Drosophila to vertebrates. The influence of Fat-Hippo signaling on transcription is mediated by a co-activator protein, called Yorkie (Yki) in Drosophila and Yes-associated protein (YAP) in vertebrates. Warts (Wts)-mediated phosphorylation and binding to cytoplasmic proteins negatively regulate Yki by promoting its retention in the cytoplasm. Wts is regulated in at least two ways: Wts kinase activity is promoted by Hippo; and Wts protein levels are influenced by Dachs. Upstream regulators of the pathway include the large cadherin Fat, and the FERM-domain proteins Merlin (Mer) and Expanded (Ex). Fat acts as a transmembrane receptor, regulated by the cadherin Dachsous (Ds), and the cadherin-domain kinase Four-jointed (Fj). The mechanisms that regulate Ex and Mer are not completely understood, but Ex localization can be influenced by Fat, and, in mammalian cells, Mer mediates an influence of contact inhibition on Hippo signaling. Genetic studies in Drosophila have also revealed that the relative contributions of pathway components can vary among different tissues (Reddy, 2010).

Optic NE cells proliferate during larval development, but aside from a requirement for the transcription factor DVSX1 (Erclik, 2008), how this proliferation is regulated is not understood. The progression of NE cells to medulla NBs in the OOA is antagonized by Jak-Stat signaling (Yasugi, 2008), but, aside from this, the regulation of this differentiation wave is not understood. This study demonstrates that Fat-Hippo signaling regulates the proliferation and differentiation of NE cells in the optic lobe. By contrast, Fat-Hippo signaling does not detectably influence the proliferation or differentiation of NBs or their progeny. A role is identified for Notch signaling in controlling the progression of NE cells to medulla NBs, and relationships are characterized between the Fat-Hippo, Jak-Stat and Notch signaling pathways. The results indicate that a transient pause in the cell cycle is needed for cells to transition from NE cells to NBs, and suggest a model in which a cell cycle arrest modulates Notch signaling by contributing to accumulation of Delta expression. The insights these results provide into the role of Fat-Hippo signaling in NE growth and differentiation in Drosophila are likely to be relevant to recently described roles of YAP in vertebrate neural development and medulloblastoma (Reddy, 2010).

The Fat-Hippo pathway has emerged as an important regulator of growth, but has not previously been implicated in neural development in Drosophila. The observation that expression of an activated form of Yki, or mutation of tumor suppressors in the pathway (i.e. fat, ex or wts), promotes growth, whereas mutation of yki impairs growth, identify a crucial role for Fat-Hippo signaling in regulating the proliferation of optic neural progenitor cells (i.e. NE). Indeed, expression of activated Yki can result in massive overgrowths that are taken up in folded sheets of NE, which push into the central brain, forming tumors of undifferentiated NE cells. Although the influence of Fat-Hippo signaling on NE growth parallels its influence on imaginal discs, the influence of Fat-Hippo signaling on NE differentiation does not, as clones of cells mutant for tumor suppressors in the pathway can differentiate cuticle in the head, thorax and abdomen (Reddy, 2010).

In contrast to the extensive overgrowth and suppressed differentiation of NE, NBs and their more differentiated progeny appear refractory to Fat-Hippo signaling. Developing tissues that are unaffected by Fat-Hippo signaling have not been well characterized. The restriction of Fat-Hippo signaling to the NE is matched by the preferential expression of several pathway components, but even when a constitutively activated form of Yki was expressed outside of the NE, neural development in the central brain was not obviously perturbed. Given the emerging importance of Hippo signaling in cancer, determination of what makes different cell types sensitive or resistant to activated Yki is an important direction for future studies (Reddy, 2010).

The progressive nature of NE to NB differentiation in the optic lobe, with different stages displayed in a spatial pattern, make it a sensitive system for investigating differentiation. The extent of delay associated with Fat-Hippo pathway tumor suppressors varied depending on strength of the mutations, which suggests that progression of NE to NB involves a balance of positive and negative influences. The silencing of Yki expression as cells differentiate further suggests that there is negative feedback of differentiation signals onto Yki, which might normally help to ensure a sharp transition between NE and NBs. When Yki activity is further elevated, by overexpression of activated Yki, a complete block in differentiation could be achieved. The observation that a complete block in differentiation could also be achieved by combining overexpression of wild-type Yki with a mutation that influences Yki phosphorylation (wts) is intriguing in light of observations that several human cancers are associated with an increase in levels of Yki expression, rather than a simple change in its localization or phosphorylation. Thus, it is suggested that the two-hit scenario observed in the optic lobe, in which both Yki activity and Yki levels need to be affected in order to transform cells permanently, could also be relevant to human tumors (Reddy, 2010).

This analysis of optic lobe development and the influence of Fat-Hippo signaling implies that a transient pause in the cell cycle is required for cells to transition from NE to medulla NBs, and that Fat-Hippo signaling influences differentiation via an effect on the cell cycle. This model is supported by several observations: there is normally a cell cycle pause along the edge of the outer optic anlagen NE; inhibition of Fat-Hippo signaling, or activation of Yki, impairs both this cell cycle pause and differentiation; and direct manipulation of multiple cell cycle regulators can delay NE differentiation. Although multiple cell cycle regulators appear to be involved in this cell cycle pause, this analysis implicates E2f1 as a key player. PCNA-GFP is downregulated at the edge of the NE, which indicates that E2f1 activity is low there. As E2f1 activity is negatively regulated by association with Rb, and Rb is negatively regulated by phosphorylation by Cdks, expression of CycD+Cdk4 is expected to increase E2f1 activity. Thus, the significant delay in differentiation observed when CycD+Cdk4 were co-expressed with E2F1+DP could all be due to increased E2f1 activity. Importantly, E2f1 is normally regulated by Fat-Hippo signaling in the optic NE, and E2f1 is functionally important for the influence of Fat-Hippo signaling on NE differentiation, because mutation of E2f1 suppressed the wts-mediated differentiation delay. A cell cycle pause also occurs in conjunction with a wave of differentiation that sweeps across the developing eye imaginal disc; however, direct manipulation of cell cycle progression did not affect the differentiation wave in the eye disc, nor does mutation of wts, hpo or sav affect differentiation of photoreceptor cells, even though it does prevent the normal cell cycle pause in the eye disc (Reddy, 2010).

The transition from NE to NB is regulated by Notch signaling, and the results of this study suggest a model in which high level expression of Dl at the edge of the NE autonomously inhibits Notch activation, resulting in upregulation of L(1)sc, which promotes NB fate. This model is supported by the observations that activation of Notch or mutation of Dl can inhibit NE differentiation. At the same time, high-level expression of Dl should enhance Notch activation in neighboring cells, which, as Dl is upregulated by Notch activation, would contribute to the progressive spread of elevated Dl expression across the NE. This simple model allows for the input of other pathways into NE to NB progression via effects on Dl expression, and indeed this appears to be the point at which Fat-Hippo and Jak-Stat signaling intersect with Notch. As a unifying model, it is proposed that a cell cycle pause facilitates the accumulation of the high levels of Dl expression needed to autonomously block Notch signaling, and thereby to upregulate the expression of proneural genes like L(1)sc. A possible mechanism for this hypothesized effect on Delta is suggested by the recent observation in vertebrate NE that Delta1 transcripts are unstable during S-phase. The hypothesis that the influence of Fat-Hippo signaling on differentiation is due to its effect on Dl expression also provides an explanation for the specificity of this phenotype, as Dl is not generally required for the differentiation of imaginal disc cells (Reddy, 2010).

Studies of homologues of Yki, Sd, Hpo and Wts in the chick neural tube identified influences on proliferation and differentiation (Cao, 2008). These studies identified effects on Sox2-expressing neural progenitor cells, but could not distinguish between effects on NE cells versus other neural progenitor cells. A recent study has also implicated YAP in Hedgehog-associated medulloblastoma. Vertebrate NE cells give rise to progenitor cells (e.g. radial glial cells and basal progenitors) that share with neuroblasts the ability to divide asymmetrically to give rise to both another progenitor cell and a more differentiated cell. Since this analysis of the Drosophila optic lobe indicates that Fat-Hippo signaling functions specifically to regulate the proliferation and differentiation of NE, it is suggested that YAP might also function specifically within NE cells in vertebrates. Notably, the observation that depending on the level of expression, Yki can delay rather than block differentiation, provides for the possibility that YAP-dependent tumors could nonetheless contain a mixture of NE cells and more differentiated cells. In Drosophila, each of the three upstream branches of the pathway (i.e. Fat-dependent, Ex-dependent and Mer-dependent, contribute to Yki regulation in NE. Studies in vertebrates have not addressed how the pathway is normally regulated, but Fat-, Ds- and Fj-related genes are all normally expressed in vertebrate NE, consistent with the possibility that they function there (Reddy, 2010).

Artificially slowing the cell cycle can promote precocious differentiation in the cortex, although in this context increasing cell cycle length was associated with a transition from proliferative to differentiative divisions of basal progenitors, which appear functionally similar to NBs rather than to NE cells. The differentiation of optic lobe NE cells into medulla NBs also differs from the general model of increasing cell cycle length causing differentiation, because NBs proliferate even more rapidly than NE cells, and thus this step is not associated with a general lengthening of the cell cycle, but rather a transient pause. Nonetheless, it is intriguing that, in the spinal cord, overexpression of CyclinD did not block differentiation, but did appear to transiently delay it, reminiscent of the delay in NE to NB progression that this study identified in the optic lobe. Moreover, CyclinD expression is regulated by Hippo signaling in the chick neural tube, and overexpression of CyclinD inhibits differentiation there. Although further studies are required to identify the CyclinD-sensitive mechanism in the vertebrate nervous system, the reported instability of Delta1 transcripts during S phase, together with the role of Notch signaling in maintaining NE progenitors in vertebrates and the analysis of NE differentiation and Dl expression in the Drosophila optic lobe, suggest that the possibility of a general influence of cell cycle progression on Notch signaling warrants further investigation as a contributor to the link between cell cycle progression and differentiation in the nervous system across different phyla (Reddy, 2010).

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; see Drosophila CRTC) 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).

Polyploidization and cell fusion contribute to wound healing in the adult Drosophila epithelium

Reestablishing epithelial integrity and biosynthetic capacity is critically important following tissue damage. The adult Drosophila abdominal epithelium provides an attractive new system to address how postmitotic diploid cells contribute to repair. Puncture wounds to the adult Drosophila epidermis close initially by forming a melanized scab. Epithelial cells near the wound site fuse to form a giant syncytium, which sends lamellae under the scab to re-epithelialize the damaged site. Other large cells arise more peripherally by initiating endocycles and becoming polyploid, or by cell fusion. Rac GTPase activity is needed for syncytium formation, while the Hippo signaling effector Yorkie modulates both polyploidization and cell fusion. Large cell formation is functionally important because when both polyploidization and fusion are blocked, wounds do not re-epithelialize. These observations indicate that cell mass lost upon wounding can be replaced by polyploidization instead of mitotic proliferation. It is proposed that large cells generated by polyploidization or cell fusion are essential because they are better able than diploid cells to mechanically stabilize wounds, especially those containing permanent acellular structures, such as scar tissue (Losick, 2013).

Puncture wounds of the adult epithelium such as those in this study present multiple challenges. Biosynthetic capacity has been reduced by cell loss, the epithelial barrier has been breached, and regional mechanical stability has been compromised by irreversible muscle damage. It was found that the epithelium employs two novel processes, polyploidization and cell fusion rather than cell proliferation, to respond to these challenges (Losick, 2013).

One function of polyploidization is to restore the tissue mass destroyed during wounding. The number of nuclei induced to leave quiescence in the adult abdomen and reenter S phase correlates closely with the wound size. Furthermore, the levels of epithelial polyploidy observed are sufficient to generate approximately the same number of new genomes as were initially lost. More severe cell losses in the abdomen caused by repeated wounding induce higher levels of endoreplication. This correspondence even holds in the case of the severe damage to the hindgut pyloric region. A region of 300 cells (600 genomes) was reduced to 100 cells following damage, but their ploidy increased sufficiently to restore a total of 550 genomes, approximately the starting number. Thus, previously quiescent diploid cells can sense the severity of tissue damage, reenter the cell cycle, and endoreplicate to levels that replace the lost cells (Losick, 2013).

During development, animals and their component organs are able to precisely control their size. Following injury, as after liver resection, cells proliferate until the normal organ size is again attained. In many tissues, certain cell types complete differentiation while undergoing endocycles; hence, mechanisms to modulate endocycling as well as cell proliferation in response to tissue size must exist. Indeed, during development, the ploidy level of cells can increase beyond that normally attained to compensate for overall reductions in cell number caused by mutation or damage, both in Drosophila embryos, ovarian follicles, and rectum and in the mammalian liver. This study has extended the known versatility of endocycle regulation by showing that polyploidization can also be induced in previously differentiated, quiescent diploid cells and then terminated at an appropriate level as a repair response (Losick, 2013).

Another striking response to adult epidermal wounding is the generation of a large syncytium by cell fusion. Barrier function is transiently restored by scab formation, after which a continuous epithelium must be regenerated. These studies suggest that syncytium formation at the site of the scab accelerates this process. When cell fusion was blocked by expressing RacN17, wound re-epithelialization was significantly slowed. Previous studies described cell fusion and syncytium formation at the site of larval epidermal wounds. Larval epidermal cells facilitate wound closure by sending lamellae under the scab using actin treadmilling and myosin II-dependent crawling. The giant syncytia that were studied contain five to ten times as many cells as in these larval epidermal wounds but may utilize many of the same processes to speed re-epithelialization. The giant syncytium may provide several additional advantages. Concentrating most nuclei at the periphery of the scar may allow thin cytoplasmic lamellae to rapidly move under the scab to seal the wound while the cell itself remains firmly fixed at the wound periphery. Individual diploid cells would have to migrate under the scab, a zone that is probably not conducive to organized cellular movement due to the absence of a basement membrane, polarity signals, and supporting muscle. Even after the epithelium is restored, the large syncytial cell may continue to function by stabilizing the scar, a large rigid structure susceptible to motions that could damage the tissue and reopen the wound (Losick, 2013).

How does the injured abdomen induce polyploidization and cell fusion at the appropriate levels and locations? Both JNK and Hippo signaling are upregulated at the wound site, suggesting that these pathways regulate the wound response. Hippo signaling, in conjunction with the TOR and insulin/IGF pathways, plays an important role in organ size control in both mammals and insects. Yki, the major effector of Hippo signaling, is required for the polyploidization response. Hippo signaling in response to wound damage may activate cycE, a gene known to be regulated by Yki in other tissues to stimulate S phase reentry. The modest 2- to 3-fold increase in the Yki-regulated genes expanded and dIAP may reflect the relative mild nature of these wounds, which only caused a 25% reduction in cell number (233 of 913) within the wound region. Thus, Hippo signaling via Yki may specify how much polyploidization ensues in response to the magnitude of the wound as well as its spatial location (Losick, 2013).

In order to fulfill this role, Hippo signaling would have to be activated locally within the tissue in proportion to the magnitude of the damage. Changes in cell polarity, actin cytoskeletal dynamics, and cell density can all induce the expression of Yki-regulated genes. In addition, other signals may play Hippo-independent roles in controlling abdominal wound repair. Hemocytes are known to be recruited to the wound site to help clear debris and microorganisms. Hemocytes are also recruited to adult abdominal wounds and are present at the 24 hr time point, when cells are both undergoing cell fusion and reentering S phase. This suggests that blood cells could liberate factors that facilitate either of these wound-healing processes. Another possibility is DNA damage from genotoxic stress, such as that produced from reactive oxygen species (ROS). ROS are known to be released after injury to many tissues, and these products can also induce the endocycle in plant cells. The syncytial and polyploid cells induced by wounding might themselves send both local and long-range signals that participate in sensing when organ size and stability have been restored (Losick, 2013).

These experiments provided insight into the distinct and overlapping roles of polyploidy and cell fusion in the healing process. Polyploidization appears to be solely responsible for replacing the lost cells and restoring the tissue back to its initial mass. However, the 25% reduction in synthetic capacity generated by the wounds made in this study is probably too small to register in assays when polyploidization alone is blocked. A 25% reduction in the time required for re-epithelialization or in the thickness of the lamella under the scar would not have been detected. Despite this, a distinct function was detected for polyploidization by analyzing its role in conjunction with that of cell fusion (Losick, 2013).

Blocking cell fusion clearly perturbs wound healing, as an extra day is now required to complete wound closure but healing is not prevented. In the absence of fusion, polyploidization still takes place. Indeed, the level of ploidy is slightly increased under these conditions. However, when polyploid cells cannot form and cell fusion is also blocked, wounds usually fail to heal. Thus, polyploid cells, which are located near the edge of the scab and extend several cell diameters away, contribute something critically important to wound repair in addition to restoring cell mass, but this function is redundant when cell fusion can operate (Losick, 2013).

It is proposed that large cells, whether syncytial or polyploid in origin, provide a unique mechanical function that helps organize and control the healing process, and that cannot be provided by surviving diploid cells. The large size of either type of cell allows more robust cytoskeletal structures to form and function than is possible in diploid cells. This is likely the reason that muscle cells fuse into large syncytia prior to organizing myofibrils. However, other large cytoplasmic mechanical structures are present and function in many other types of polyploid cells that are less familiar. For example, megakaryocytes, which extrude segments of their cytoplasm as platelets, contain long branching β1-tubulin-based processes that are required for platelet release. Polyploid jump reflex neurons in Drosophila produce exceptionally long and thick axons that allow signals to be transmitted with great speed. Trophoblast giant cells accumulate stress fibers and specialized podosomes, which may structurally support placenta development (Losick, 2013).

Mechanical tension is already known to play a critical role in mammalian epithelial wound healing. Cells tend to migrate toward regions of higher ECM rigidity ('durotaxis'). Large cells may be necessary at the wound site to generate an appropriate mechanical environment for migrating cells to complete their movements under the scar and close the wound. In the abdomen, the transverse muscle bands that normally span the abdomen did not undergo repair. Large cells may also be particularly advantageous for dealing with mechanical stability issues that require balancing forces over a substantial area due to the size of the damaged region and the presence of altered structures such as scar tissue. The central syncytium may be advantageous not only in rapidly closing the wound but also because a large cell can better stabilize the scar and prevents it from breaking loose. The enlarged peripheral cells, whether polyploid or syncytial, may more easily generate stabilizing forces to protect the wounded region during the normal flexure and stress on the abdomen (Losick, 2013).

These same considerations would apply equally to mammalian as well as Drosophila tissue. In many damaged mammalian tissues, extracellular matrix deposits of fibrin and collagen initially form a fibrin clot/scab to hold edges of the wound together, but collagen protein deposits can persist in a lasting mark at the injury site in the form of a scar. In tissues where cell proliferation is limited, such as the heart, scar formation is necessary to maintain tissue integrity but also leads to stiffness and reduced heart function. Polyploidization may frequently take place in response to mammalian tissue damage that repairs imperfectly and leaves scar tissue, such as in the heart, but this has received little attention. Cardiomyocytes reenter the cell cycle after injury, leading to a low level of cell division as well as polyploidy and multinucleation at the scar periphery. Consequently, the establishment of a model system for studying the control of polyploidization and syncytium formation in response to wounding is likely to provide insight to questions of wide significance (Losick, 2013).

The Hippo pathway controls border cell migration through distinct mechanisms in outer border cells and polar cells of the Drosophila ovary

The Hippo pathway is a key signaling cascade in controlling organ size. The core components of this pathway are two kinases, Hippo (Hpo) and Warts (Wts), and a transcriptional coactivator Yorkie (Yki). YAP (a Yki homolog in mammals) promotes epithelial-mesenchymal transition and cell migration in vitro. This study used border cells in the Drosophila ovary as a model to study Hippo pathway functions in cell migration in vivo. During oogenesis, polar cells secrete Unpaired (Upd), which activates JAK/STAT signaling of neighboring cells and specifies them into outer border cells. The outer border cells form a cluster with polar cells and undergo migration. This study found that hpo and wts are required for migration of the border cell cluster. In outer border cells, over-expression of hpo disrupts polarization of the actin cytoskeleton and attenuates migration. In polar cells, knockdown of hpo, wts, or over-expression of yki impairs border cell induction and disrupts migration. These manipulations in polar cells reduce JAK/STAT activity in outer border cells. Expression of upd-lacZ is increased and decreased in yki and hpo mutant polar cells, respectively. Furthermore, forced-expression of upd in polar cells rescues defects of border cell induction and migration caused by wts knockdown. These results suggest that Yki negatively regulates border cell induction by inhibiting JAK/STAT signaling. Together, these data elucidate two distinct mechanisms of the Hippo pathway in controlling border cell migration: 1) in outer border cells, it regulates polarized distribution of the actin cytoskeleton; 2) in polar cells, it regulates upd expression to control border cell induction and migration (T. Lin, 2014).

Systemic organ wasting induced by localized expression of the secreted Insulin/IGF antagonist ImpL2

Organ wasting, related to changes in nutrition and metabolic activity of cells and tissues, is observed under conditions of starvation and in the context of diseases, including cancers. A model for organ wasting in adult Drosophila is described, whereby overproliferation induced by activation of Yorkie, the Yap1 oncogene ortholog, in intestinal stem cells leads to wasting of the ovary, fat body, and muscle. These organ-wasting phenotypes are associated with a reduction in systemic insulin/IGF signaling due to increased expression of the secreted insulin/IGF antagonist ImpL2 from the overproliferating gut. Strikingly, expression of rate-limiting glycolytic enzymes and central components of the insulin/IGF pathway is upregulated with activation of Yorkie in the gut, which may provide a mechanism for this overproliferating tissue to evade the effect of ImpL2. Altogether, this study provides insights into the mechanisms underlying organ-wasting phenotypes in Drosophila and how overproliferating tissues adapt to global changes in metabolism (Kwon, 2015).

This study describes the unexpected observation that the overproliferating midgut due to aberrant Yki activity in ISCs induces the bloating syndrome and systemic organ wasting. Additionally, the overproliferating midgut perturbs organismal metabolism, resulting in an increase of hemolymph trehalose and depletion of glycogen and triglyceride storage. Strikingly, it was shown that the accumulation of hemolymph trehalose and organ-wasting processes are dependent on the antagonist of insulin/IGF signaling, ImpL2, which is specifically upregulated in the proliferating midgut. This study provides strong genetic evidence supporting that systemic organ wasting associated with the aberrant activation of Yki in ISCs cannot be explained solely by the perturbation of general gut function. Based on these findings, it is proposed that ImpL2 is a critical factor involved in systemic organ wasting in Drosophila (Kwon, 2015).

An accompanying paper (Figueroa-Clarevega, 2015) shows that transplantation of scrib1/RasV12 disc tumors into wild-type flies induces the bloating syndrome phenotype and systemic organ wasting, affecting ovaries, fat bodies, and muscles. That study also identified ImpL2 as a tumor-driven factor that plays a critical role in the organ-wasting process. These results are consistent with earlier findings and indicate that the bloating syndrome and organ-wasting phenotypes are not associated specifically with perturbation of gut function. Interestingly, Figueroa-Clarevega and Bilder observe that disc tumors derived by the expression of ykiS/A (an active form of yki that is less potent than ykiact used in this study) did not cause organ wasting, which can be explained by the low level of ImpL2 induction in the ykiS/A tumors as compared to scrib1/RasV12 tumors (Kwon, 2015).

The current results do not rule out the existence of an additional factor(s) contributing to the bloating syndrome and organ-wasting phenotypes. Indeed, the partial rescue of the bloating syndrome and organ-wasting phenotypes by depletion of ImpL2 in esgts>ykiact midguts suggests the existence of an additional factor(s). Moreover, this study observed that ectopic expression of ImpL2 in ECs was not sufficient to reduce whole-body triglyceride and glycogen levels, although it caused hyperglycemia, reduction of Akt1 phosphorylation, and increase of hemolymph volume. Thus, given the involvement of diverse factors in the wasting process in mammals, it is likely that in addition to ImpL2, another factor(s) contributes to systemic organ wasting in Drosophila (Kwon, 2015).

This study shows that the bloating syndrome caused by esgts>ykiact is associated with ImpL2, as depletion of ImpL2 from esgts>ykiact midguts significantly rescues the bloating phenotype. Given the observation that elevated expression of ImpL2 from esgts>ykiact midgut induces hyperglycemia, it is speculated that the accumulation of trehalose in hemolymph is a factor involved in bloating, because a high concentration of trehalose can cause water influx to adjust hemolymph osmolarity to physiological levels. Interestingly, recent findings have shown that disruption of l(2)gl in discs activates yki, suggesting that the bloating syndrome observed in flies with transplanted l(2)gl mutant discs may be due to aberrant yki activity (Kwon, 2015).

The current findings are reminiscent of a previous study showing that in Drosophila, humoral infection with the bacterial pathogen Mycobacterium marinum (closely related to Mycobacterium tuberculosis) causes a progressive loss of energy stores in the form of fat and glycogen—a wasting-like phenotype. Similar to the current observation, the previous study found that infection with M. marinum caused a downregulation of Akt1 phosphorylation. Given the observation that ImpL2 produced from esgts>ykiact affects systemic insulin/IGF signaling, it will be of interest to test whether ImpL2 expression is increased upon infection with M. marinum and mediates the effect on the loss of fat and glycogen storage (Kwon, 2015).

yki plays critical roles in tissue growth, repair, and regeneration by inducing cell proliferation, a process requiring additional nutrients to support rapid synthesis of macromolecules including lipids, proteins, and nucleotides. In particular, increased aerobic glycolysis metabolizing glucose into lactate is a characteristic feature of many cancerous and normal proliferating cells. Interestingly, the aberrant activation of yki in ISCs caused a disparity in the gene expression of glycolytic enzymes and the activity of insulin/IGF signaling between the proliferating midgut and other tissues, such as muscle and ovaries. Thus, it is speculated that this disparity favors Yki-induced cell proliferation by increasing the availability of trehalose/glucose to the proliferating midgut, which presumably requires high levels of trehalose/glucose. Additionally, it will be of interest to test whether activation of Yki during tissue growth, repair, and regeneration alters systemic metabolism in a similar manner (Kwon, 2015).

Niche appropriation by Drosophila intestinal stem cell tumours

Mutations that inhibit differentiation in stem cell lineages are a common early step in cancer development, but precisely how a loss of differentiation initiates tumorigenesis is unclear. This study investigated Drosophila intestinal stem cell (ISC) tumours generated by suppressing Notch(N) signalling, which blocks differentiation. Notch-defective ISCs require stress-induced divisions for tumour initiation and an autocrine EGFR ligand, Spitz, during early tumour growth. On achieving a critical mass these tumours displace surrounding enterocytes, competing with them for basement membrane space and causing their detachment, extrusion and apoptosis. This loss of epithelial integrity induces JNK and Yki/YAP activity in enterocytes and, consequently, their expression of stress-dependent cytokines (Upd2, Upd3). These paracrine signals, normally used within the stem cell niche to trigger regeneration, propel tumour growth without the need for secondary mutations in growth signalling pathways. The appropriation of niche signalling by differentiation-defective stem cells may be a common mechanism of early tumorigenesis (Patel, 2015).

This paper described a step-wise series of events during the earliest stage of tumour development in a stem cell niche. First, the combination of environmentally triggered mitogenic signalling and a mutation that compromises differentiation generates small clusters of differentiation-defective stem-like cells. Autocrine (Spi/EGFR) signalling between these cells then promotes their expansion into clusters, which quickly reach a size capable of physically disrupting the surrounding epithelium and driving the detachment and apical extrusion of surrounding epithelial cells (that is, ECs). This loss of normal cells seems to involve tumour cell/epithelial cell competition through integrin-mediated adhesion. Subsequently, the loss of epithelial integrity (specifically, EC detachment) triggers stress signalling (JNK, Yki/YAP) in the surrounding epithelium and underlying VM, and these stressed tissues respond by producing cytokines (Upd2,3) and growth factors (Vn, Pvf, Wg, dILP3). These signals are normally used within the niche to activate stem cells for epithelial repair, but in this context they further stimulate tumour growth in a positive feedback loop. It is noteworthy that in this example a single mutation that blocks differentiation is sufficient to drive early tumour development, even without secondary mutations in growth signalling pathways that might make the tumour-initiating cells growth factor- and niche-independent (for example, Ras, PTEN). Thus, tumour cell-niche interactions can be sufficient to allow tumour-initiating cells to rapidly expand, increasing their chance to acquire secondary mutations that might enhance their growth or allow them to survive outside their normal niche. This study highlights the importance of investigating the factors that control paracrine stem cell mitogens and survival signals in the niche environment. Tumour-niche interactions may be important to acquire a sizable tumour mass before the recruitment of a tumour-specific microenvironment that supports further tumour progression. A careful analysis of similar interactions in other epithelia, such as in the lung, skin or intestine could yield insights relevant to the early detection, treatment and prevention of cancers in such tissues (Patel, 2015).

Effects of Mutation or Overexpression

yki is required for tissue growth and normal diap1 transcription. To further explore the role of Yki in Hpo signaling, a loss-of-function mutation of yki was generated by homologous recombination. The targeting construct was designed in such a way that all of the coding sequence of yki was replaced by the w+ marker, thus resulting in a null allele. yki null mutants are homozygous lethal and die as late embryos and early first instar larvae. A full-length yki cDNA driven by the ubiquitous α-tubulin promoter completely rescues yki null animals to viable and phenotypically normal adult flies (Huang, 2005).

eyeless-FLP was used to selectively remove yki function in over 90% of the eye disc cells. Eyes composed predominantly of yki mutant cells are markedly reduced in size when compared to control animals, thus revealing an essential function for yki in tissue growth. To follow yki mutant cells during development, FLP/FRT was used to examine genetically marked clones of yki mutant cells. yki mutant clones generated at 40 hr AED were hardly observed in third instar wing discs , with rare clones recovered containing only a few cells. yki mutant clones generated at a similar stage were more frequently recovered in the eye discs but contained much fewer cells than the wild-type twin spots. Despite the severe growth defects, loss of yki does not perturb early retina differentiation, as shown by the normal expression of the neuronal marker Elav. Taken together, these results reveal a specific requirement for yki in tissue growth (Huang, 2005).

To further probe the requirement of Yki in the Hpo pathway, diap1 transcription was examined in yki mutant clones using the thj5c8 diap1-lacZ reporter. Consistent with the overexpression results, diap1-lacZ expression is reduced in yki null cells in a cell-autonomous manner. Similar results were seen in the wing discs. DIAP1 protein level was also reduced in a cell-autonomous manner in yki mutant clones. Thus, yki is required for the normal level of diap1 transcription in Drosophila (Huang, 2005).

The Hippo tumor-suppressor pathway regulates apical-domain size in parallel to tissue growth

The Hippo tumor-suppressor pathway controls tissue growth in Drosophila and mammals by regulating cell proliferation and apoptosis. The Hippo pathway includes the Fat cadherin, a transmembrane protein, which acts upstream of several other components that form a kinase cascade that culminates in the regulation of gene expression through the transcriptional coactivator Yorkie (Yki). Work in Drosophila has indicated indicated that Merlin (Mer) and Expanded (Ex) are members of the Hippo pathway and act upstream of the Hippo kinase. In contrast to this model, it was suggested that Mer and Ex primarily regulate membrane dynamics and receptor trafficking, thereby affecting Hippo pathway activity only indirectly. This study examined the effects of Mer, Ex and the Hippo pathway on the size of the apical membrane and on apical-basal polarity complexes. It was found that mer;ex double mutant imaginal disc cells have significantly increased levels of apical membrane determinants, such as Crb, aPKC and Patj. These phenotypes were shared with mutations in other Hippo pathway components and required Yki, indicating that Mer and Ex signal through the Hippo pathway. Interestingly, however, whereas Crb was required for the accumulation of other apical proteins and for the expansion of the apical domain observed in Hippo pathway mutants, its elimination did not significantly reverse the overgrowth phenotype of warts mutant cells. Therefore, Hippo signaling regulates cell polarity complexes in addition to and independently of its growth control function in imaginal disc cells (Hamaratoglu, 2009).

The results show that the Hippo pathway regulates the amount of apical protein complexes and thereby the size of the apical domain and that this effect is independent of its growth control function. Importantly, the regulation of apical complexes is a specific effect of the Hippo pathway, since other growth control pathways do not regulate apical complexes. In addition, this effect of the Hippo pathway is a general effect, since upregulation of apical complexes was observed in multiple tissues and cell types. Although overexpression of Crb and aPKC are sufficient to drive extra growth, the results show that the upregulation of apical complexes is not required for the overgrowth phenotype and for the induction of Hippo target genes in wts mutant cells. It is thus concluded that the Hippo pathway regulates the amount of apical complexes in Drosophila imaginal disc cells in addition to and independently of its growth control function (Hamaratoglu, 2009).

It has been suggested that Mer and Ex regulate the levels of membrane receptors independently of the Hippo pathway. However, the current results show that the upregulation of DER, Ft and apical complexes was similar in hpo and wts mutant cells and mer;ex double mutant cells, and that this effect requires Yki. These results thus indicate that Mer and Ex act through the Hippo pathway to exert their effect and that they are bona fide members of the Hippo pathway. Similar conclusions have been drawn based on observations that overexpression of wts suppresses the lethality and overgrowth phenotypes of ex mutants (Hamaratoglu, 2009 and references therein).

How does the Hippo pathway regulate the size of the apical domain and the amount of the apical complexes? The observation that Yki is required and sufficient for the effect on the apical domain indicates that this effect of the Hippo pathway is mediated by transcriptional regulation. However, although the upregulation of Crb is necessary and sufficient for the expansion of the apical domain and for the accumulation of the other apical polarity complex proteins, it is not required for the upregulation of DER and Ft, which still accumulate in wts,crb double mutant cells. Thus a model is favored in which the Hippo pathway regulates the turnover of several apical membrane components, for example through regulation of endocytosis. Notably mer;ex mutant cells in wing imaginal discs have defects in Notch (N) endocytosis, which leads to accumulation of N. Moreover, the endosomal protein Hrs accumulates in hpo mutant follicle cells in Drosophila ovaries, and this study observed a similar accumulation of Hrs in wts mutant clones in imaginal discs. These observations thus support the hypothesis that Hippo signaling regulates the amount of endocytosis and membrane turnover, thereby affecting the amount of apical membrane proteins. The target of Yki that mediates these effects, however, is currently not known (Hamaratoglu, 2009).

Several other studies also demonstrated roles for the Hippo pathway beyond its function in growth control. For example, the Hippo pathway is required for the proper selection of photoreceptor subtypes in the Drosophila eye, and it is required in follicle cells to generate a signal that polarizes the underlying oocyte. For both of these functions, Hippo signals through Yki, but Yki may regulate different sets of target genes, since the phenotypic effects are different. In addition, the Hippo pathway regulates cellular behavior through pathways that may not require Yki and thus may not involve the regulation of gene expression. For example, Yki-independent functions of the Hippo pathway may regulate dendritic tiling of larval neurons and the death of salivary gland cells during metamorphosis. The finding that Hippo regulates apical polarity complexes in addition to and independently of its growth control function in imaginal discs cells thus further reveals the complex function of this pathway in the regulation of cellular behavior (Hamaratoglu, 2009).

Differential requirement of Salvador-Warts-Hippo pathway members for organ size control in Drosophila melanogaster

The Salvador-Warts-Hippo (SWH) pathway contains multiple growth-inhibitory proteins that control organ size during development by limiting activity of the Yorkie oncoprotein. Increasing evidence indicates that these growth inhibitors act in a complex network upstream of Yorkie. This complexity is emphasised by the distinct phenotypes of tissue lacking different SWH pathway genes. For example, eye tissue lacking the core SWH pathway components salvador, warts or hippo is highly overgrown and resistant to developmental apoptosis, whereas tissue lacking fat or expanded is not. This study explores the relative contribution of SWH pathway proteins to organ size control by determining their temporal activity profile throughout Drosophila eye development. Eye tissue lacking fat, expanded or discs overgrown displays elevated Yorkie activity during the larval growth phase of development, but not in the pupal eye when apoptosis ensues. Fat and Expanded do possess Yorkie-repressive activity in the pupal eye, but loss of fat or expanded at this stage of development can be compensated for by Merlin. Fat appears to repress Yorkie independently of Dachs in the pupal eye, which would contrast with the mode of action of Fat during larval development. Fat is more likely to restrict Yorkie activity in the pupal eye together with Expanded, given that pupal eye tissue lacking both these genes resembles that of tissue lacking either gene. This study highlights the complexity employed by different SWH pathway proteins to control organ size at different stages of development (Milton, 2010).

The SWH pathway controls Drosophila eye size by limiting growth during the larval stage of development and by restricting proliferation and promoting apoptosis during pupal development. Eyes lacking core SWH pathway components (e.g. sav, wts or hpo) are significantly larger than eyes lacking the non-core components ft, ex, dco or Mer. Owing to this disparity, it has been hypothesized that ft and ex only partially affect SWH pathway activity, whereas sav, wts and hpo have stronger effects, or, alternatively, that non-core components affect pathway activity in a temporally restricted fashion. Analysis of tissue recessive for ft, ex or dco3 revealed that Yki activity was elevated during larval eye development when tissues are actively growing and proliferating, but not during pupal development when apoptosis ensues, supporting the idea that Ft, Ex and Dco influence SWH pathway activity in a temporally restricted fashion. However, when tissue lacking both Mer and ft, or Mer and ex, was analysed, Yki activity was found to be elevated during both larval and pupal development, similar to the Yki activity profile observed in tissue lacking core SWH pathway proteins. This is consistent with previous reports showing that Mer acts in parallel to both Ft and Ex, and that these proteins can compensate for each other to control SWH pathway activity. Therefore, Ft and Ex do contribute to SWH pathway regulation in the pupal eye to ensure appropriate exit from the cell cycle and developmental apoptosis, but these functions can be executed by Mer in their absence, suggesting a degree of plasticity in the regulation of Yki activity by non-core SWH pathway proteins. The ability of Mer to compensate for Ft or Ex cannot simply be explained by compensatory increases in Mer protein in pupal eye tissues lacking ft or ex, since Mer expression levels were found to be unaltered in these tissues (Milton, 2010).

Previous analyses of tissue lacking both ft and ex showed that these proteins function, at least in part, in parallel to control growth of larval imaginal discs. The current analysis of ft,ex double-mutant tissue suggests that these proteins are likely to function together to control Yki activity in the pupal eye. Yki activity was not elevated in tissue lacking ft, ex or both genes, showing that these genes cannot compensate for each other in the pupal eye. This is consistent with the notion that Ft influences the activity of downstream SWH pathway proteins by multiple mechanisms, an idea that is supported by THE analysis of the requirement of the atypical myosin, Dachs, for Ft signalling in the pupal eye. During larval imaginal disc development, Ft can influence Yki activity by repressing Dachs activity, which in turn can repress the core SWH pathway protein Wts. Analysis of pupal eye tissue that lacks both Mer and ft, or Mer, ft and dachs, showed that Yki activity was elevated in each scenario. This shows that in the pupal eye, the ability of Ft to compensate for Mer is not reliant on Dachs, and implies that Ft can employ different modes of signal transduction throughout eye development. However, because Ft and Mer can compensate for each other it is not possible to formally conclude that normal signal transduction by Ft in the pupal eye occurs independently of Dachs (Milton, 2010).

Expression of Ex is tightly controlled in response to alterations in SWH pathway activity at both the transcriptional and post-transcriptional levels. Interestingly, it was also found that Ex expression is controlled in a temporal fashion throughout eye development; Ex is expressed at relatively high levels in the larval eye, but at very low levels in the pupal eye. Despite the fact that Ex expression is very low in the pupal eye, it clearly retains function at this stage of development because it can compensate for loss of Mer to restrict Yki activity. The dynamic expression profile of Ex suggests that factors that influence its expression play an important role in defining overall eye size in Drosophila. At present, only two transcriptional regulatory proteins have been shown to influence the expression of ex: Yki and Sd. There are conflicting reports on whether Yki and Sd control basal expression of ex in larval imaginal discs. It is clear, however, that Yki and Sd collaborate to drive ex expression when the activity of the SWH pathway is suppressed, presumably as part of a negative-feedback loop. Despite the fact that basal ex expression is low in the pupal eye, the ex promoter is still responsive to Yki, as Ex expression is substantially elevated in pupal eye clones lacking hpo or Mer and ex. Future investigation of the ex promoter will help to clarify understanding of the complex fashion by which expression of the ex gene is controlled, and should aid understanding of eye size specification in Drosophila (Milton, 2010).

This study emphasises the complexity of the means by which the activity of core SWH pathway proteins is regulated by non-core proteins such as Ft, Ex, Mer and Dco. The signalling mechanisms employed by non-core proteins appear to differ at discrete stages of development in order to achieve appropriate organ size during the larval growth period of eye development, and to subsequently sculpt the eye by regulating apoptosis during pupal development (Milton, 2010).

Yki/YAP, Sd/TEAD and Hth/MEIS control tissue specification in the Drosophila eye disc epithelium

During animal development, accurate control of tissue specification and growth are critical to generate organisms of reproducible shape and size. The eye-antennal disc epithelium of Drosophila is a powerful model system to identify the signaling pathway and transcription factors that mediate and coordinate these processes. This study shows that the Yorkie (Yki) pathway plays a major role in tissue specification within the developing fly eye disc, in squamous peripodial portion of the epithelium (PE), at a time when organ primordia and regional identity domains are specified. RNAi-mediated inactivation of Yki, or its partner Scalloped (Sd), or increased activity of the upstream negative regulators of Yki cause a dramatic reorganization of the eye disc fate map leading to specification of the entire disc epithelium into retina. On the contrary, constitutive expression of Yki suppresses eye formation in a Sd-dependent fashion. It was also showm that knockdown of the transcription factor Homothorax (Hth), known to partner Yki in some developmental contexts, also induces an ectopic retina domain, that Yki and Scalloped regulate Hth expression, and that the gain-of-function activity of Yki is partially dependent on Hth. These results support a critical role for Yki - and its partners Sd and Hth - in shaping the fate map of the eye epithelium independently of its universal role as a regulator of proliferation and survival (Zhang, 2011).

The process of regional specification in the eye-antennal disc of Drosophila involves a number of signaling pathways that regulate the expression of identity-defining transcriptional regulators, often referred to as selector factors. This study shows that the Hippo signaling pathway and the transcription factors Yki, Sd, and Hth play critical roles in the regional specification of this disc. Specifically, all three factors are required for the establishment of the peripodial cell layer of the eye disc. Yki function is required in the PE to maintain tissue identity and appears to works in concert with Sd and Hth. Moreover, negative regulation of Yki through the Hippo/Warts genetic pathway is required in the disc proper (DP) to modulate Yki activity such that it promotes proliferation and survival of retina progenitor cells without interfering with their specification. The specification role of Yki and its partners in the eye epithelium is central to proper disc development, occurs in the early stages of regional specification within this disc, and appears to be distinct from its more general role in proliferation and survival (Zhang, 2011).

Yki and Sd have been shown to form a complex and regulate the anti-apoptotic gene Diap1 in the wing and eye discs of Drosophila. In the eye, Yki is also strongly required for proliferation and survival, whereas Sd makes a lesser contribution to these processes (Zhang, 2011).

Does Yki function in a complex with Sd in the context of regional specification within the eye disc and how? The evidence presented in this study shows that (1) the RNAi-induced knock-down of either gene induces essentially identical PE-to-DP transformations and (2) sd-RNAi can suppress the gain-of-function, anti-retina effect of Yki over-expression. These findings, together with the biochemical evidence of protein-protein interactions, suggest that Yki and Sd may indeed control transcription together in a complex during PE specification (Zhang, 2011).

Surprisingly, a comparison of loss-of-function analyses in mosaic discs versus RNAi-mediated disc-wide knock-downs has uncovered a discrepancy in the induced phenotypes. Specifically, loss-of-function clones of either yki or sd do not show signs of cell fate transformation. This is the case for traditional flip-FRT loss-of-function clones generated using mutant alleles as well as for knock-down MARCM clones induced by RNAi expression. Differences in reagents cannot explain this discrepancy, because the same yki-RNAi or sd-RNAi lines were used in disc-wide versus clonal analyses. Hence, this discrepancy must depend on the two different loss-of-function approaches employed. The two approaches differ significantly in two ways: (1) the amount of mutant tissue induced, (2) and the timing of loss-of-gene function (Zhang, 2011).

Thus far, two major functions of Hth have been uncovered in the DP cell layer: 1) Hth, together with Tsh, suppresses the expression of the late retinal determination (RD) genes eya and so, thus slowing down the conversion of early eye progenitors (Ey, Tsh and Hth positive) into more mature (Ey, Tsh, Eya and So positive) eye precursor cells, and 2) Hth together with Tsh and Yki enhances proliferation by inducing expression of the proliferation-promoting microRNA Bantam. The combined effect of these two activities results in the generation of an abundant pool of eye progenitor cells. Protein-protein interactions among these factors have been documented in vitro and/or in vivo, leading to the proposal that two complexes (inclusive of Hth/Tsh and Hth/Yki/Tsh) may perform these major functions (Zhang, 2011).

The data presented in this study uncover another critical function of Hth in the eye disc. In the PE cell layer, fated to give rise to portions of the head cuticle, Hth prevents conversion of this tissue into retina. As in the eye progenitors within the DP, this anti-retina activity of Hth involves suppression of late-RD factors (Eya, So, Dac). However, unlike its role in eye progenitors, it also entails down-regulation of the early factors Ey and Tsh. Thus, Hth suppresses late but not early RD factors in early eye disc progenitors in conjunction with Tsh, whereas it suppresses retina formation at the level if the early factors in the PE through a process that appears to involve Yki and Sd, but not Tsh (whose expression is normally absent from the PE). In agreement with this context dependent role of Hth, hth-RNAi reverses the anti-retina effect of Yki over-expression, at least in part, by relieving Yki suppression of the early retinal determination factor Ey, and expression of exogenous Tsh within the PE cell layer leads to the formation of an ectopic retina (Zhang, 2011).

Lastly, this study has shown that Hth expression is under the genetic, positive control of Yki and Sd, and that neither yki-RNAi nor sd-RNAi can suppress the gain-of-function activity of Hth. These findings, together with the reported association of the Hth and Yki proteins in S2 cells and on the regulatory region of Bantam, suggest a potentially more complex relationship between Hth and Yki in the development of the PE. The observation that Yki and Sd control Hth expression does not preclude the possibility that the Hth protein also functions in a complex with these factors. Indeed, a related example is offered by the retinal determination factor Eya, which first induces and then partners with the Sine oculis to regulate retina development. It is proposed therefore that Hth is one of the direct or indirect targets of a Yki/Sd complex in the PE. Thereafter, association with Hth may modify Yki/Sd activity eventually resulting in the transcriptional silencing of critical retinal determination genes, such as Tsh (Zhang, 2011).

The Hippo/Yki signaling pathway is a general regulator of cell proliferation and survival in metazoans. In a handful of cases, the Hippo pathway has been shown to regulate processes other than proliferation or cell death, such as the maintenance of the undifferentiated state of progenitor cell types in the neural tube and in the gut and, in two cases, aspects of neuronal differentiation. In one, the pathway controls the choice of opsin gene expressed in the R8 cells of the adult fly eye; in the other, it is required for maintenance of the dendritic harbors of body-wall sensory neurons in the Drosophila larva. While transcriptional regulation by Yki is thought to play a role in these non-proliferation-related processes, it is difficult to draw parallels between these cases and what is seen in the eye disc (Zhang, 2011).

Two more recent reports implicate Yki in processes related to tissue specification. In one case, a transient, expanding burst of Yki activity within rows of cells at the border of an already established wing field results in a marginal expansion of the wing primordium, ultimately ensuring proper wing size. In a second example, the YAP/Yki and TEAD4/Sd proteins specify the trophoectoderm as distinct from the inner cell mass (ICM) in mammalian embryos. It is thought that the circumferential cell-cell contacts experienced by ICM cells triggers Hpo activity and cytoplasmic retention of YAP, whereas the less extensive cell-cell contacts experienced by outer cells do not, thus allowing nuclear accumulation of YAP (Zhang, 2011).

Based on similarities with the latter example, it is tempting to hypothesize that less extensive cell-cell contacts in the squamous cell layer, than in the columnar one, would promote a more efficient nuclear localization of Yki in the PE, than in the DP cells. At moderate activity levels in the DP, Yki would then promote proliferation and survival without interfering with retina specification, whereas at higher activity levels in the PE, Yki would be able to not only promote cell proliferation and survival but also PE-identity by suppressing retina formation. This scenario would be consistent with the observed need for down-regulation of Yki activity by Warts in the DP and the stronger expression of Diap1-2-lacZ in the PE. However, the observation that the two cell layers differ significantly in the availability of factors found in Yki-based complexes suggests that 'Yki-complex composition' plays a critical role in the PE versus DP/retina distinction in the developing fly eye (Zhang, 2011).

Yki association with specific co-factors would, therefore, modify the output of the Hpo-Yki pathway to bring about different outcomes in distinct regions of the eye epithelium. A Yki/Hth/Tsh complex would ensure maintenance and expansion of the retina progenitors pool, whereas a Yki/Sd, and possibly Hth, complex would contribute to regional specification within the eye disc by ensuring formation of PE-derived head structures. That Yki/YAP-based complexes can include a variety of different co-factors is supported by other recent examples, including the above mentioned role of Yki, Hth and Tsh in promoting eye progenitors' proliferation in the fly, and its interaction with IRS1 to promote proliferation of neural precursors or with Smad1 to enhance BMP-mediated suppression of neuronal differentiation of embryonic stem cells in the mouse (Zhang, 2011).

In the L2 eye disc, the Sd protein is believed to be available throughout, but does not appear to contribute greatly to Yki-induced proliferation in either cell layer. On the contrary, in the PE, it behaves as a critical co-factor in the control of tissue identity. Unlike Sd, Tsh expression has been shown to be restricted specifically to the DP cell layer. Thus, Yki must perform its PE-promoting tasks in transcriptional complexes that do not include Tsh. Interestingly, misexpression of Tsh within the PE has been shown to induce retina development in this tissue. Whether Tsh does so, at least in part, by diverting Yki activity away from 'anti-retina-fate' functions remains to be determined. Hth, also present broadly in both cells layer at the L2 stage, would play a more general role contributing to Yki's function in both proliferation and tissue specification (Zhang, 2011).

One possible scenario is that the tissue specification functions of Yki in the PE are critically dependent on its association with Sd, and that the availability of Tsh specifically in the DP interferes with the ability of Sd and Yki to associate or work together on fate-determining tasks, thereby preventing any interference with retina formation (Zhang, 2011).

As shown by the paucity of examples, this analysis is still in the early days of deciphering how Yki and its partners regulate cell fate. Nonetheless, this role is apparently separate from its contribution to cell proliferation and survival, and likely involves a number of distinct molecular mechanisms including co-factor specificity and differing levels of Yki activity (Zhang, 2011).

A modifier screen in Drosophila melanogaster implicates cytoskeletal regulators, Jun N-terminal kinase, and Yorkie in Draper signaling

The Drosophila melanogaster homolog of the ced-1 gene from Caenorhabditis elegans is draper, which encodes a cell surface receptor required for the recognition and engulfment of apoptotic cells, glial clearance of axon fragments and dendritic pruning, and salivary gland autophagy. To further elucidate mechanisms of Draper signaling, a genetic screen of chromosomal deficiencies was performed to identify loci that dominantly modify the phenotype of over-expression of Draper isoform II, which suppresses differentiation of the posterior crossvein in the wing. The existence of 43 genetic modifiers of Draper II was deduced. 24 of the 37 suppressor loci and 3 of the 6 enhancer loci have been identified. A further 5 suppressors and 2 enhancers were identified from mutations in functionally related genes. These studies indicated positive contributions to Drpr signaling for the Jun N-terminal Kinase pathway, supported by genetic interactions with hemipterous, basket, jun, and puckered, and for cytoskeleton regulation as indicated by genetic interactions with rac1, rac2, RhoA, myoblast city, Wiskcott-Aldrich syndrome protein, and the formin CG32138, and for yorkie and expanded. These findings indicate that Jun N-terminal Kinase activation and cytoskeletal remodeling collaborate in the engulfment process downstream of Draper activation. The relationships between Draper signaling and Decapentaplegic signaling, insulin signaling, Salvador-Warts-Hippo signaling, apical-basal cell polarity, and cellular responses to mechanical forces are further investigated and discussed (Fullard, 2014).


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yorkie: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 20 February 2017

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