NEWS & VIEWS
doi:10.1038/nature12848
FORUM: Developmental biology
Tethered wings Wnt signalling molecules are thought to direct the development of an organism by spreading through tissues. But flies grow with almost normal appendages even when their main Wnt protein cannot move. Two scientists discuss the implications of this finding for our understanding of development.
THE PAPER IN BRIEF ●●The Drosophila (fruitfly) protein Wingless
(Wg) is the prototype member of the Wnt family of proteins, which regulate tissue patterning and growth during development. ●●Wg is thought to act as a morphogen — a protein that forms concentration gradients as it spreads from its site of synthesis and that regulates gene expression as a
Non-essential spread G I N É S M O R ATA
M
orphogen regulation of target genes depends on the physical distance from the morphogen-secreting cell population, such that the levels of this molecule provide a genetic reading of position, a key issue in morphogenesis. The best examples of morphogens come from Drosophila: the secreted molecules Hedgehog, Decapentaplegic (Dpp) and Wingless (Wg) have been identified as morphogens2, and for Dpp and Wg there is compelling evidence that they act at long range3,4. It follows from the very definition of a morphogen that the spread of the molecule is an essential component of its function. But Alexandre and colleagues’ results suggest that this idea needs to be reconsidered. A clear demonstration of long-range action by Wg came from the finding4 that the protein activates target genes, including vestigial (vg) and Distal-less (Dll), in cells distant from Wg-secreting cells. By contrast, a Wg variant protein (Nrt–Wg) that is functional but anchored to the cell membrane, through the addition of part of the transmembrane protein Neurotactin, was found to activate these target genes only in neighbouring cells. These original experiments were performed by artificial overexpression of Nrt–Wg; Alexandre et al. have now used sophisticated genomeediting technology to generate flies in which the wg gene encodes the Nrt–Wg protein. The
function of its concentration. ●●In a paper published on Nature’s website today1, Alexandre et al. describe wing formation in flies expressing a form of Wg that is tethered to the cell membrane, in place of the secreted protein. ●●They observe normal wing morphology, although development is delayed and the final wings are smaller than those of normal flies.
edited gene contains all the normal regulatory sequences and is therefore expressed normally, and the method seems to work with high efficiency, opening up the possibility of performing similar manipulations in other Drosophila genes of interest5. Considering the many functions of Wg during embryogenesis and during larval and adult life, and the essential role assigned to the protein’s spread, any expert would have confidently predicted that a fly with only tethered Wg would not develop. But Alexandre and colleagues’ flies survive and are normal in appearance, although they grow more slowly than normal flies and their wings are smaller (Fig. 1). The authors examined the situation only in the wing disc, but the fact that the flies survive indicates that other Wg functions are more or less normal. The implication of their findings is that, at least for Drosophila, the long-range diffusion of Wg may be of minor significance — bringing into question the functional value of its role as a morphogen. How should these results be interpreted in light of the compelling evidence for longrange Wg action? Alexandre et al. confirm that Nrt–Wg can induce Dll and vg gene expression only in adjoining cells, so the almost-normal expression of these target genes in their mutant flies is hard to explain. The authors propose a ‘cellular memory’ model, in which cells initially expressing target genes continue to express them even when they no longer receive the expression-inducing signal. That implies that Dll and vg expression is perpetuated through cell divisions, but this is not supported by published evidence6. An alternative explanation is that, although the Nrt–Wg protein is considered
to be functionally equivalent to Wg (except for its diffusion), there might be undetected differences in its expression levels or stability. Despite the need for clarifying some aspects of these findings, the survival of flies that have only membrane-tethered Wg is telling, and the authors’ results call for a reassessment of how we think about Wg function, and perhaps about that of other morphogens. Wg and Dpp are evolutionarily conserved in all animals, so their mode of function is likely to be conserved as well. The two proteins have acted as model morphogens, and understanding how they work is of major importance to biology. Wnt signalling is also of biomedical interest, because its misregulation is associated with human cancers and other diseases7. Although there is no question that these molecules have a crucial role in development and disease, reexamining how they work might change our picture of these processes. Ginés Morata is in the Centro de Biología Molecular, CSIC-UAM, Universidad Autónoma de Madrid, Madrid 28049, Spain. e-mail: [email protected]
Long-range thinking GARY STRUHL
T
here is compelling evidence3,6,8 that Wg can, and normally does, act over many cell diameters to control gene expression and growth of the Drosophila wing. So the remarkable discovery that a membrane-tethered form of Wg can substitute for the normal protein poses the question: must morphogens move to organize development? When considering this challenge to how we think of morphogens, the devil is in the details. The main phase of Drosophila wing development begins with the induction of Wg expression in all cells of the nascent wing and lasts for around two days, during which time the wing increases by about 50 times in size and | NAT U R E | 1
© 2013 Macmillan Publishers Limited. All rights reserved
RESEARCH NEWS & VIEWS
a
b Nrt–Wgexpressing cell
Wg-secreting cell
Figure 1 | Wing development and Wingless spread. a, The Wingless protein (Wg) is thought to regulate the development of Drosophila wings by diffusing from Wg-secreting cells, thereby activating Wg target genes in distant cells as the wing grows. b, Alexandre et al.1 show that, in flies expressing the Wg variant Nrt–Wg, which is tethered to the cell membrane and cannot diffuse, wings with normal morphology develop, although development is delayed and the wings are smaller than normal. Previous work3,6 has shown that Nrt–Wg can activate Wg target genes in nearby cells but not in distant cells (green arrow), so it remains unclear how the long-distance Wg signalling thought to be required for wing development is exerted in these flies.
cell number. On the first day, Wg is broadly expressed, but its expression fades progressively in the more dorsally and ventrally positioned cells, generating Wg gradients. These gradients might well suffice to initially direct normal gene expression and growth without requiring Wg to spread, and Alexandre and colleagues’ results support this idea. During the second day, Wg production becomes restricted to a central stripe of cells, but the protein continues to control gene expression and growth in cells up to 15–20 cell diameters away. The conventional view is that this is because they continue to receive Wg secreted by the central cells. Alexandre and colleagues propose instead that, once prospective wing cells receive Wg, they acquire a long-term cellular memory of Wg exposure that controls the behaviour of their descendants thereafter. According to this model, the descendants of cells exposed to Nrt–Wg should grow and express Wg target genes even when they are many cell diameters away from
Nrt–Wg-expressing cells. But previous work3,6 shows that this is not so; rather, only those cells that remain close to Nrt–Wg-expressing cells continue to express Wg target genes and grow. Without invoking a memory model, how might tethered Wg mimic the long-range action of the normal protein? One possible answer comes from the observation5 that Nrt–Wg accumulates in secreting cells to several times higher levels than normal Wg, indicating either that it is significantly more stable and/or that it provides a more potent signal to neighbouring cells because it is not attenuated by release and dispersion. Accordingly, Nrt–Wg that is expressed during the first day might persist and function adventitiously during the second day, providing a signal that would otherwise require spread of the protein. Another possibility is that Wg moves to some extent via cellular projections9,10; membrane-tethered Wg might also be able to do this, allowing it to influence cells at least a few cell diameters away. A third option is
2 | NAT U R E |
© 2013 Macmillan Publishers Limited. All rights reserved
that the downstream effects of Wg signalling (including the function of proteins encoded by target genes) might persist for several hours, even after cells cease to receive Wg. All of these factors, some normal and others artefacts of the Nrt–Wg system, would extend the range over which Nrt–Wg can influence cell behaviour during the second day of wing development. Notably, Alexandre et al. find that the tethered protein fails to sustain normal patterns of gene expression or support normal growth after its expression becomes restricted to the central stripe. This results in delayed wing development, and wings that never reach full size even when they have an extra day or longer to catch up. Thus, the new results do not falsify the interpretation of Wg as a classic morphogen in the Drosophila wing. Instead, they highlight that Wg acts at short range during early wing development but must act at long range at later times, as its production becomes restricted and the population of cells requiring Wg input expands. The Drosophila wing will continue to serve as a model for understanding how morphogens act to organize the development of larger tissues (such as butterfly wings and vertebrate limbs), and further studies using the methods introduced by Alexandre et al. will contribute to this understanding. ■ Gary Struhl is in the Department of Genetics and Development, Columbia University, New York, New York 10032, USA. e-mail: [email protected] 1. Alexandre, C., Baena-Lopez, A. & Vincent, J.-P. Nature http://dx.doi.org/10.1038/nature12879 (2013). 2. Lawrence, P. A. & Struhl, G. Cell 85, 951–961 (1996). 3. Zecca, M., Basler, K. & Struhl, G. Cell 87, 833–844 (1996). 4. Nellen, D., Burke, R., Struhl, G. & Basler, K. Cell 85, 357–368 (1996). 5. Baena-Lopez, L. A., Alexandre, C., Mitchell, A., Pasakarnis, L. & Vincent, J.-P. Development 140, 4818–4825 (2013). 6. Zecca, M. & Struhl, G. Development 134, 3001–3010 (2007). 7. MacDonald, B. T., Tamai, K. & He, X. Dev. Cell 17, 9–26 (2009). 8. Neumann, C. J. & Cohen, S. M. Development 124, 871–880 (1997). 9. Locke, M. & Huie, P. Tissue Cell 13, 787–803 (1981). 10. Gradilla, A.-C. & Guerrero, I. Curr. Opin. Genet. Dev. 23, 363–373 (2013).
doi:10.1038/nature12848
FORUM: Developmental biology
Tethered wings Wnt signalling molecules are thought to direct the development of an organism by spreading through tissues. But flies grow with almost normal appendages even when their main Wnt protein cannot move. Two scientists discuss the implications of this finding for our understanding of development.
THE PAPER IN BRIEF ●●The Drosophila (fruitfly) protein Wingless
(Wg) is the prototype member of the Wnt family of proteins, which regulate tissue patterning and growth during development. ●●Wg is thought to act as a morphogen — a protein that forms concentration gradients as it spreads from its site of synthesis and that regulates gene expression as a
Non-essential spread G I N É S M O R ATA
M
orphogen regulation of target genes depends on the physical distance from the morphogen-secreting cell population, such that the levels of this molecule provide a genetic reading of position, a key issue in morphogenesis. The best examples of morphogens come from Drosophila: the secreted molecules Hedgehog, Decapentaplegic (Dpp) and Wingless (Wg) have been identified as morphogens2, and for Dpp and Wg there is compelling evidence that they act at long range3,4. It follows from the very definition of a morphogen that the spread of the molecule is an essential component of its function. But Alexandre and colleagues’ results suggest that this idea needs to be reconsidered. A clear demonstration of long-range action by Wg came from the finding4 that the protein activates target genes, including vestigial (vg) and Distal-less (Dll), in cells distant from Wg-secreting cells. By contrast, a Wg variant protein (Nrt–Wg) that is functional but anchored to the cell membrane, through the addition of part of the transmembrane protein Neurotactin, was found to activate these target genes only in neighbouring cells. These original experiments were performed by artificial overexpression of Nrt–Wg; Alexandre et al. have now used sophisticated genomeediting technology to generate flies in which the wg gene encodes the Nrt–Wg protein. The
function of its concentration. ●●In a paper published on Nature’s website today1, Alexandre et al. describe wing formation in flies expressing a form of Wg that is tethered to the cell membrane, in place of the secreted protein. ●●They observe normal wing morphology, although development is delayed and the final wings are smaller than those of normal flies.
edited gene contains all the normal regulatory sequences and is therefore expressed normally, and the method seems to work with high efficiency, opening up the possibility of performing similar manipulations in other Drosophila genes of interest5. Considering the many functions of Wg during embryogenesis and during larval and adult life, and the essential role assigned to the protein’s spread, any expert would have confidently predicted that a fly with only tethered Wg would not develop. But Alexandre and colleagues’ flies survive and are normal in appearance, although they grow more slowly than normal flies and their wings are smaller (Fig. 1). The authors examined the situation only in the wing disc, but the fact that the flies survive indicates that other Wg functions are more or less normal. The implication of their findings is that, at least for Drosophila, the long-range diffusion of Wg may be of minor significance — bringing into question the functional value of its role as a morphogen. How should these results be interpreted in light of the compelling evidence for longrange Wg action? Alexandre et al. confirm that Nrt–Wg can induce Dll and vg gene expression only in adjoining cells, so the almost-normal expression of these target genes in their mutant flies is hard to explain. The authors propose a ‘cellular memory’ model, in which cells initially expressing target genes continue to express them even when they no longer receive the expression-inducing signal. That implies that Dll and vg expression is perpetuated through cell divisions, but this is not supported by published evidence6. An alternative explanation is that, although the Nrt–Wg protein is considered
to be functionally equivalent to Wg (except for its diffusion), there might be undetected differences in its expression levels or stability. Despite the need for clarifying some aspects of these findings, the survival of flies that have only membrane-tethered Wg is telling, and the authors’ results call for a reassessment of how we think about Wg function, and perhaps about that of other morphogens. Wg and Dpp are evolutionarily conserved in all animals, so their mode of function is likely to be conserved as well. The two proteins have acted as model morphogens, and understanding how they work is of major importance to biology. Wnt signalling is also of biomedical interest, because its misregulation is associated with human cancers and other diseases7. Although there is no question that these molecules have a crucial role in development and disease, reexamining how they work might change our picture of these processes. Ginés Morata is in the Centro de Biología Molecular, CSIC-UAM, Universidad Autónoma de Madrid, Madrid 28049, Spain. e-mail: [email protected]
Long-range thinking GARY STRUHL
T
here is compelling evidence3,6,8 that Wg can, and normally does, act over many cell diameters to control gene expression and growth of the Drosophila wing. So the remarkable discovery that a membrane-tethered form of Wg can substitute for the normal protein poses the question: must morphogens move to organize development? When considering this challenge to how we think of morphogens, the devil is in the details. The main phase of Drosophila wing development begins with the induction of Wg expression in all cells of the nascent wing and lasts for around two days, during which time the wing increases by about 50 times in size and | NAT U R E | 1
© 2013 Macmillan Publishers Limited. All rights reserved
RESEARCH NEWS & VIEWS
a
b Nrt–Wgexpressing cell
Wg-secreting cell
Figure 1 | Wing development and Wingless spread. a, The Wingless protein (Wg) is thought to regulate the development of Drosophila wings by diffusing from Wg-secreting cells, thereby activating Wg target genes in distant cells as the wing grows. b, Alexandre et al.1 show that, in flies expressing the Wg variant Nrt–Wg, which is tethered to the cell membrane and cannot diffuse, wings with normal morphology develop, although development is delayed and the wings are smaller than normal. Previous work3,6 has shown that Nrt–Wg can activate Wg target genes in nearby cells but not in distant cells (green arrow), so it remains unclear how the long-distance Wg signalling thought to be required for wing development is exerted in these flies.
cell number. On the first day, Wg is broadly expressed, but its expression fades progressively in the more dorsally and ventrally positioned cells, generating Wg gradients. These gradients might well suffice to initially direct normal gene expression and growth without requiring Wg to spread, and Alexandre and colleagues’ results support this idea. During the second day, Wg production becomes restricted to a central stripe of cells, but the protein continues to control gene expression and growth in cells up to 15–20 cell diameters away. The conventional view is that this is because they continue to receive Wg secreted by the central cells. Alexandre and colleagues propose instead that, once prospective wing cells receive Wg, they acquire a long-term cellular memory of Wg exposure that controls the behaviour of their descendants thereafter. According to this model, the descendants of cells exposed to Nrt–Wg should grow and express Wg target genes even when they are many cell diameters away from
Nrt–Wg-expressing cells. But previous work3,6 shows that this is not so; rather, only those cells that remain close to Nrt–Wg-expressing cells continue to express Wg target genes and grow. Without invoking a memory model, how might tethered Wg mimic the long-range action of the normal protein? One possible answer comes from the observation5 that Nrt–Wg accumulates in secreting cells to several times higher levels than normal Wg, indicating either that it is significantly more stable and/or that it provides a more potent signal to neighbouring cells because it is not attenuated by release and dispersion. Accordingly, Nrt–Wg that is expressed during the first day might persist and function adventitiously during the second day, providing a signal that would otherwise require spread of the protein. Another possibility is that Wg moves to some extent via cellular projections9,10; membrane-tethered Wg might also be able to do this, allowing it to influence cells at least a few cell diameters away. A third option is
2 | NAT U R E |
© 2013 Macmillan Publishers Limited. All rights reserved
that the downstream effects of Wg signalling (including the function of proteins encoded by target genes) might persist for several hours, even after cells cease to receive Wg. All of these factors, some normal and others artefacts of the Nrt–Wg system, would extend the range over which Nrt–Wg can influence cell behaviour during the second day of wing development. Notably, Alexandre et al. find that the tethered protein fails to sustain normal patterns of gene expression or support normal growth after its expression becomes restricted to the central stripe. This results in delayed wing development, and wings that never reach full size even when they have an extra day or longer to catch up. Thus, the new results do not falsify the interpretation of Wg as a classic morphogen in the Drosophila wing. Instead, they highlight that Wg acts at short range during early wing development but must act at long range at later times, as its production becomes restricted and the population of cells requiring Wg input expands. The Drosophila wing will continue to serve as a model for understanding how morphogens act to organize the development of larger tissues (such as butterfly wings and vertebrate limbs), and further studies using the methods introduced by Alexandre et al. will contribute to this understanding. ■ Gary Struhl is in the Department of Genetics and Development, Columbia University, New York, New York 10032, USA. e-mail: [email protected] 1. Alexandre, C., Baena-Lopez, A. & Vincent, J.-P. Nature http://dx.doi.org/10.1038/nature12879 (2013). 2. Lawrence, P. A. & Struhl, G. Cell 85, 951–961 (1996). 3. Zecca, M., Basler, K. & Struhl, G. Cell 87, 833–844 (1996). 4. Nellen, D., Burke, R., Struhl, G. & Basler, K. Cell 85, 357–368 (1996). 5. Baena-Lopez, L. A., Alexandre, C., Mitchell, A., Pasakarnis, L. & Vincent, J.-P. Development 140, 4818–4825 (2013). 6. Zecca, M. & Struhl, G. Development 134, 3001–3010 (2007). 7. MacDonald, B. T., Tamai, K. & He, X. Dev. Cell 17, 9–26 (2009). 8. Neumann, C. J. & Cohen, S. M. Development 124, 871–880 (1997). 9. Locke, M. & Huie, P. Tissue Cell 13, 787–803 (1981). 10. Gradilla, A.-C. & Guerrero, I. Curr. Opin. Genet. Dev. 23, 363–373 (2013).
