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).
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).
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
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
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
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
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
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
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
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
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
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
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
Lowe, S. W., Cepero, E. and Evan, G. (2004). Intrinsic tumour suppression. Nature 432: 307-315. 15549092
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
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
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
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
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
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
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
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
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
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
Zhao, B., et al. (2008). TEAD mediates YAP-dependent gene induction and growth control. Genes Dev. 22: 1962-1971. PubMed Citation: 18579750
date revised: 25 August 2009
Home page: The Interactive Fly © 2003 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.