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).
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).
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).
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).
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 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).
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).
Reference names in red indicate recommended papers.
Aqeilan, R. I., et al. (2005). WW domain-containing proteins, WWOX and YAP, compete for interaction with ErbB-4 and modulate its transcriptional function. Cancer Res. 65(15): 6764-72. 16061658
Alarcon, C., et al.. (2009). Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-beta pathways. Cell 139(4): 757-69. PubMed Citation: 19914168
Badouel, C., et al. (2009). The FERM-domain protein Expanded regulates Hippo pathway activity via direct interactions with the transcriptional activator Yorkie. Dev. Cell 16(3): 411-20. PubMed Citation: 19289086
Basu, S., et al. (2003). Akt phosphorylates the Yes-associated protein, YAP, to induce interaction with 14-3-3 and attenuation of p73-mediated apoptosis. Mol. Cell 11: 11-23. 12535517
Bothos, J., et al. (2005). Human LATS1 is a mitotic exit network kinase. Cancer Res 65: 6568-6575. PubMed Citation: 16061636
Cao X., Pfaff S. L. and Gage F. H. (2008). YAP regulates neural progenitor cell number via the TEA domain transcription factor. Genes Dev. 22: 3320-3334. PubMed Citation: 19015275
Chan, E. H., et al. (2005). The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1. Oncogene 24(12): 2076-86. 15688006
Chen, L., et al. (2010). Structural basis of YAP recognition by TEAD4 in the hippo pathway. Genes Dev. 24(3): 290-300. PubMed Citation: 20123908
Colombani, J., Polesello, C., Josue, F. and Tapon, N. (2006). Dmp53 activates the Hippo pathway to promote cell death in response to DNA damage. Curr. Biol. 16(14): 1453-8. 16860746
Cooper, J. A. and Sept, D. (2008). New insights into mechanism and regulation of actin capping protein. Int. Rev. Cell Mol. Biol. 267: 183-206. PubMed Citation: 18544499
Corbit, K. C., et al. (2008). Kif3a constrains β-catenin-dependent Wnt signalling through dual ciliary and non-ciliary mechanisms. Nat. Cell Biol. 10: 70-76. PubMed Citation: 18084282
Delalle I., et al. (2005). Mutations in the Drosophila orthologs of the F-actin capping protein alpha- and beta-subunits cause actin accumulation and subsequent retinal degeneration. Genetics 171: 1757-1765. PubMed Citation: 16143599
Densham R. M., et al. (2009). MST kinases monitor actin cytoskeletal integrity and signal via c-Jun N-terminal kinase stress-activated kinase to regulate p21Waf1/Cip1 stability. Mol. Cell. Biol. 29: 6380-6390. PubMed Citation: 19822666
Dong, J., et al. (2007). Elucidation of a universal size-control mechanism in Drosophila and mammals. Cell 130(6): 1120-33. PubMed citation: 17889654
Emoto, K, et al. (2004). Control of dendritic branching and tiling by the Tricornered-kinase/Furry signaling pathway in Drosophila sensory neurons. Cell 119: 245-256. PubMed Citation: 16061636
Erclik, T., Hartenstein V., Lipshitz H. D. and McInnes R. R. (2008). Conserved role of the Vsx genes supports a monophyletic origin for bilaterian visual systems. Curr. Biol. 18: 1278-1287. PubMed Citation: 18723351
Espanel, X. and Sudol, M. (2001). Yes-associated protein and p53-binding protein-2 interact through their WW and SH3 domains. J. Biol. Chem. 276(17): 14514-23 11278422
Fernandez-L, A., et al. (2009). YAP1 is amplified and up-regulated in hedgehog-associated medulloblastomas and mediates Sonic hedgehog-driven neural precursor proliferation. Genes Dev. 23(23): 2729-41. PubMed Citation: 19952108
Fernández, B. G., et al. (2011). Actin-Capping Protein and the Hippo pathway regulate F-actin and tissue growth in Drosophila. Development 138(11): 2337-46. PubMed Citation: 21525075
Ferrigno, O., et al. (2002). Yes-associated protein (YAP65) interacts with Smad7 and potentiates its inhibitory activity against TGF-beta/Smad signaling. Oncogene 21(32): 4879-8412118366
Goulev, Y., et al. (2008). SCALLOPED interacts with YORKIE, the nuclear effector of the hippo tumor-suppressor pathway in Drosophila. Curr. Biol. 18(6): 435-41. PubMed Citation: 18313299
Grzeschik, N. A., Parsons, L. M., Allott, M. L., Harvey, K. F. and Richardson, H. E. (2010). Lgl, aPKC, and Crumbs regulate the Salvador/Warts/Hippo pathway through two distinct mechanisms. Curr. Biol. 20(7): 573-581. PubMed Citation: 20362447
Hamaratoglu, F., et al. (2009). The Hippo tumor-suppressor pathway regulates apical-domain size in parallel to tissue growth. J. Cell Sci. 122(Pt 14): 2351-9. PubMed Citation: 19531584
Hariharan, I. K. (2006). Growth regulation: a beginning for the hippo pathway. Curr. Biol. 16: R1037-1039. PubMed Citation: 17174912
Hergovich, A., Bichsel, S. J. and Hemmings, B. A. (2005). Human NDR kinases are rapidly activated by MOB proteins through recruitment to the plasma membrane and phosphorylation. Mol. Cell. Biol. 25: 8259-8272. PubMed Citation: 16135814
Hergovich, A., Schmitz, D. and Hemmings, B. A. (2006a). The human tumour suppressor LATS1 is activated by human MOB1 at the membrane. Biochem. Biophys. Res. Commun. 345: 50-58. PubMed Citation: 16674920
Hergovich, A., Stegert, M. R., Schmitz, D. and Hemmings, B. A. (2006b). NDR kinases regulate essential cell processes from yeast to humans. Nat. Rev. Mol. Cell Biol. 7: 253-264. PubMed Citation: 16607288
Ho L. L., Wei X., Shimizu T. and Lai Z. C. (2010). Mob as tumor suppressor is activated at the cell membrane to control tissue growth and organ size in Drosophila. Dev. Biol. 337: 274-283. PubMed Citation: 19913529
Hou, M. C., Salek, J. and McCollum, D. (2000). Mob1p interacts with the Sid2p kinase and is required for cytokinesis in fission yeast. Curr. Biol. 10: 619-622. PubMed Citation: 10837231
Howell, M., Borchers, C. and Milgram, S. L. (2004). Heterogeneous nuclear ribonuclear protein U associates with YAP and regulates its co-activation of Bax transcription. J. Biol. Chem. 279(25): 26300-6. 15096513
Huang, J., Wu, S., Barrera, J., Matthews, K. and Pan, D. (2005). The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila homolog of YAP. Cell 122: 421-434. 16096061
Janody, F. and Treisman, J. E. (2006). Actin capping protein {alpha} maintains vestigial-expressing cells within the Drosophila wing disc epithelium. Development 133: 3349-3357. PubMed Citation: 16887822
Johnson R. I., Seppa M. J. and Cagan R. L. (2008). The Drosophila CD2AP/CIN85 orthologue Cindr regulates junctions and cytoskeleton dynamics during tissue patterning. J. Cell Biol. 180: 1191-1204. PubMed Citation: 18362180
Oh, H., Reddy B. V. and Irvine K. D. (2009). Phosphorylation-independent repression of Yorkie in Fat-Hippo signaling. Dev. Biol. 335: 188-197. PubMed Citation: 19733165
Reddy B. V. and Irvine K. D. (2008). The Fat and Warts signaling pathways: new insights into their regulation, mechanism and conservation. Development 135: 2827-2838. PubMed Citation: 18697904
Johnson R. I., Seppa M. J. and Cagan R. L. (2008). The Drosophila CD2AP/CIN85 orthologue Cindr regulates junctions and cytoskeleton dynamics during tissue patterning. J. Cell Biol. 180: 1191-1204. PubMed Citation: 18362180
Komuro, A., Nagai, M., Navin, N. E. and Sudol, M. (2003). WW domain-containing protein YAP associates with ErbB-4 and acts as a co-transcriptional activator for the carboxyl-terminal fragment of ErbB-4 that translocates to the nucleus. J. Biol. Chem. 278(35): 33334-41. 12807903
Lai, Z. C., et al. (2005). Control of cell proliferation and apoptosis by mob as tumor suppressor Mats. Cell 120: 675-685. 15766530
Lal, M., et al. (2008). Polycystin-1 C-terminal tail associates with β-catenin and inhibits canonical Wnt signaling. Hum. Mol. Genet. 17: 3105-3117. PubMed Citation: 18632682
Lee, S. E., et al. (2001). Order of function of the budding-yeast mitotic exit-network proteins Tem1, Cdc15, Mob1, Dbf2, and Cdc5. Curr. Biol. 11: 784-788. PubMed Citation: 11378390
Lei, Q. Y., et al. (2008). TAZ promotes cell proliferation and epithelial-mesenchymal transition and is inhibited by the hippo pathway. Mol. Cell Biol. 28(7): 2426-36. PubMed Citation: 18227151
Li, Z., et al. (2010). Structural insights into the YAP and TEAD complex. Genes Dev. 24(3): 235-40. PubMed Citation: 20123905
Lian, I., et al. (2010). The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation. Genes Dev. 24(11): 1106-18. PubMed Citation: 20516196
Lowe, S. W., Cepero, E. and Evan, G. (2004). Intrinsic tumour suppression. Nature 432: 307-315. 15549092
Milton, C. C., Zhang, X., Albanese, N. O. and Harvey, K. F. (2010). Differential requirement of Salvador-Warts-Hippo pathway members for organ size control in Drosophila melanogaster. Development 137(5): 735-43. PubMed Citation: 20110315
Moreno, C. S., Lane, W. S., Pallas, D. C. (2001). A mammalian homolog of yeast MOB1 is both a member and a putative substrate of striatin family-protein phosphatase 2A complexes. J. Biol. Chem. 276: 24253-24260. PubMed Citation: 11319234
Nicolay, B. N., et al. (2011). Cooperation between dE2F1 and Yki/Sd defines a distinct transcriptional program necessary to bypass cell cycle exit. Genes Dev. 25(4): 323-35. PubMed Citation: 21325133
Nishioka, N., et al. (2009). The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass. Dev. Cell 16(3): 398-410. PubMed Citation: 19289085
Nolo, R., Morrison, C. M., Tao, C., Zhang, X. and Halder, G. (2006). The bantam microRNA is a target of the hippo tumor-suppressor pathway. Curr. Biol. 16(19): 1895-904. Medline abstract: 16949821
Oh, H., Reddy B. V. and Irvine K. D. (2009). Phosphorylation-independent repression of Yorkie in Fat-Hippo signaling. Dev. Biol. 335: 188-197. PubMed Citation: 19733165
Omerovic, J., et al. (2004). Ligand-regulated association of ErbB-4 to the transcriptional co-activator YAP65 controls transcription at the nuclear level. Exp. Cell Res. 294(2): 469-7915023535
Ota, M. and Sasaki, H. (2009). Mammalian Tead proteins regulate cell proliferation and contact inhibition as transcriptional mediators of Hippo signaling. Development 135: 4059-4069. PubMed Citation: 19004856
Overholtzer, M., et al. (2006). Transforming properties of YAP, a candidate oncogene on the chromosome 11q22 amplicon. Proc. Natl. Acad. Sci. 103(33): 12405-10. Medline abstract: 16894141
Park, T. J., et al. (2008). Dishevelled controls apical docking and planar polarization of basal bodies in ciliated epithelial cells. Nat. Genet. 40: 871-879. PubMed Citation: 18552847
Peng, H. W., Slattery, M. and Mann, R. S. (2009). Transcription factor choice in the Hippo signaling pathway: homothorax and yorkie regulation of the microRNA bantam in the progenitor domain of the Drosophila eye imaginal disc. Genes Dev. 23(19): 2307-19. PubMed Citation: 19762509
Reddy B. V. and Irvine K. D. (2008). The Fat and Warts signaling pathways: new insights into their regulation, mechanism and conservation. Development 135: 2827-2838. PubMed Citation: 18697904
Reddy, B. V., Rauskolb, C. and Irvine, K. D. (2010)
Ribeiro, P. S., et al. (2010). Combined functional genomic and proteomic approaches identify a PP2A complex as a negative regulator of Hippo signaling. Mol. Cell 39: 521-534. PubMed Citation: 20797625
Rogulja, D., Rauskolb, C. and Irvine, K. D. (2008). Morphogen control of wing growth through the fat signaling pathway. Dev. Cell 15: 309-321. PubMed Citation: 18694569
Staley, B. K. and Irvine, K. D. (2010). Warts and Yorkie mediate intestinal regeneration by influencing stem cell proliferation. Curr. Biol. 20(17): 1580-7. PubMed Citation: 20727758
Strano, S., et al. (2001). Physical interaction with Yes-associated protein enhances p73 transcriptional activity, J. Biol. Chem. 276: 15164-15173. 11278685
Strano S., et al. (2005). The transcriptional coactivator Yes-associated protein drives p73 gene-target specificity in response to DNA damage. Mol. Cell 18(4): 447-59. 15893728
Sudol, M. (1994). Yes-associated protein (YAP65) is a proline-rich phosphoprotein that binds to the SH3 domain of the Yes proto-oncogene product, Oncogene 9: 2145-2152. 8035999
Sun, G. and Irvine, K. D. (2011). Regulation of Hippo signaling by Jun kinase signaling during compensatory cell proliferation and regeneration, and in neoplastic tumors. Dev. Biol. 350(1): 139-51. PubMed Citation: 21145886
Tamaskovic, S. J., et al. (2003). NDR family of AGC kinases-essential regulators of the cell cycle and morphogenesis, FEBS Lett. 546: 73-80. 12829239
Thompson, B. J. and Cohen, S. M. (2006). The Hippo pathway regulates the bantam microRNA to control cell proliferation and apoptosis in Drosophila. Cell 126(4): 767-74. Medline abstract: 16923395
Van Hateren, N. J., et al. (2011). FatJ acts via the Hippo mediator Yap1 to restrict the size of neural progenitor cell pools. Development 138(10): 1893-902. PubMed Citation: 21521736
Varelas, X., et al. (2010). The Hippo pathway regulates Wnt/beta-catenin signaling. Dev. Cell 18(4): 579-91. PubMed Citation: 20412773
Vassilev, A., et al. (2001). TEAD/TEF transcription factors utilize the activation domain of YAP65, a Src/Yes-associated protein localized in the cytoplasm, Genes Dev. 15: 1229-1241. 11358867
Wada, K., et al. (2011). Hippo pathway regulation by cell morphology and stress fibers. Development 138(18): 3907-14. PubMed Citation: 21831922
Wei, X., Shimizu, T. and Lai, Z. C. (2007). Mob as tumor suppressor is activated by Hippo kinase for growth inhibition in Drosophila. EMBO J. 26(7): 1772-81. PubMed Citation: 17347649
Wu, S., Huang, J., Dong, J. and Pan, D. (2003). hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts. Cell 114: 445-456. 12941273
Wu, S., Liu, Y., Zheng, Y., Dong, J. and Pan, D. (2008). The TEAD/TEF family protein Scalloped mediates transcriptional output of the Hippo growth-regulatory pathway. Dev. Cell 14(3): 388-98. PubMed Citation: 18258486
Yagi, R., et al. (1999). A WW domain-containing yes-associated protein (YAP) is a novel transcriptional co-activator. EMBO J. 18: 2551-2562. 10228168
Yang, X., et al. (2004). LATS1 tumour suppressor affects cytokinesis by inhibiting LIMK1. Nat. Cell Biol. 6: 609-617. PubMed Citation: 15220930
Yasugi T., Umetsu D., Murakami S., Sato M. and Tabata T. (2008). Drosophila optic lobe neuroblasts triggered by a wave of proneural gene expression that is negatively regulated by JAK/STAT. Development 135: 1471-1480. PubMed Citation: 18339672
Zaidi, S. K., et al. (2004). Tyrosine phosphorylation controls Runx2-mediated subnuclear targeting of YAP to repress transcription. EMBO J. 23(4): 790-9. 14765127
Zecca, M. and Struhl, G. (2010). A feed-forward circuit linking wingless, fat-dachsous signaling, and the warts-hippo pathway to Drosophila wing growth. PLoS Biol. 8(6): e1000386. PubMed Citation: 20532238
Zhang, J., Smolen, G. A. and Haber, D. A. (2009). Negative regulation of YAP by LATS1 underscores evolutionary conservation of the Drosophila Hippo pathway. Cancer Res. 68: 2789-2794. PubMed Citation: 18413746
Zhang, L., et al. (2008). The TEAD/TEF family of transcription factor Scalloped mediates Hippo signaling in organ size control. Dev. Cell 14: 377-387. PubMed Citation: 18258485
Zhang, N., et al. (2010). The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals. Dev. Cell 19(1): 27-38. PubMed Citation: 20643348
Zhao, B., et al. (2008). TEAD mediates YAP-dependent gene induction and growth control. Genes Dev. 22: 1962-1971. PubMed Citation: 18579750
Zhao, B., Li, L., Tumaneng, K., Wang, C. Y. and Guan, K. L. (2010). A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCFβ-TRCP. Genes Dev. 24(1): 72-85. PubMed Citation: 20048001
Zhu, M., et al. (2010). Activation of JNK signaling links lgl mutations to disruption of the cell polarity and epithelial organization in Drosophila imaginal discs. Cell Res. 20: 242-245. PubMed Citation: 20066009
date revised: 15 December 2011
Home page: The Interactive Fly © 2003 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.