- Email: [email protected]
Sticking Around: Short-Range Activity of Wnt Ligands
Developmental Cell
Previews K.A., et al. (2016). Cell Metab. http://dx.doi.org/10. 1016/j.cmet.2016.01.007. DeBerardinis, R.J., Mancuso, A., Daikhin, E., Nissim, I., Yudkoff, M., Wehrli, S., and Thompson, C.B. (2007). Proc. Natl. Acad. Sci. USA 104, 19345–19350. Deberardinis, R.J., Sayed, N., Ditsworth, D., and Thompson, C.B. (2008). Curr. Opin. Genet. Dev. 18, 54–61.
Fan, J., Ye, J., Kamphorst, J.J., Shlomi, T., Thompson, C.B., and Rabinowitz, J.D. (2014). Nature 510, 298–302.
Owen, O.E., Kalhan, S.C., and Hanson, R.W. (2002). J. Biol. Chem. 277, 30409–30412.
Hosios, A.M., Hecht, V.C., Danai, L.V., Johnson, M.O., Rathmell, J.C., Steinhauser, M.L., Manalis, S.R., and Vander Heiden, M.G. (2016). Dev. Cell 36, this issue, 540–549.
Vander Heiden, M.G., Cantley, L.C., and Thompson, C.B. (2009). Science 324, 1029–1033.
Locasale, J.W. (2013). Nat. Rev. Cancer 13, 572–583.
Yuneva, M., Zamboni, N., Oefner, P., Sachidanandam, R., and Lazebnik, Y. (2007). J. Cell Biol. 178, 93–105.
Sticking Around: Short-Range Activity of Wnt Ligands Michael Boutros1,* and Christof Niehrs2,3 1Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Department of Cell and Molecular Biology, Faculty of Medicine Mannheim, Heidelberg University, 69120 Heidelberg, Germany 2Division of Molecular Embryology, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany 3Institute of Molecular Biology (IMB), 55128 Mainz, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2016.02.018
Wnt ligands are secreted lipid-modified proteins that are essential for development and homeostasis. Published recently in Nature, Farin et al. (2016) report that Wnt3 acts at a short distance during growth of mouse intestinal organoids, suggesting that Wnt proteins remain membrane-bound to activate signaling at a short range from their source. The question of how a uniform tissue gives rise to patterned structures has fascinated developmental biologists for many decades. First discussed in detail by Gierer and Meinhardt in the 1970s, morphogen gradients have since been implicated in many developmental processes, ranging from pattern formation in hydra to patterning decisions of wings in flies and limbs in mouse (reviewed in Rogers and Schier, 2011). Morphogen gradients are thought to be generated by secreted molecules that elicit distinct cellular responses at different concentrations or activities, thereby leading to cell fate changes and patterning of tissues. However, while the existence of activity gradients is generally accepted, different mechanisms have been proposed for how ligands travel from their source to their target sites. This has been a particularly ‘‘sticky’’ issue for Wnt ligands, which are secreted lipid-modified glycoproteins and are unlikely to diffuse freely in the extracellular space without carriers. How Wnt
morphogen gradients—both short- and long-range—are formed and how Wnt proteins are transported have remained major open questions. Proposed molecular mechanisms included diffusion, protein carriers, different forms of exovesicles and cytonemes, and filopodia that transport Wnt proteins from the site of production (reviewed in Stanganello and Scholpp, 2016). In the intestinal crypts, Wnt signaling plays an important role for stem cell growth and differentiation (Clevers, 2013). Multiple Wnt proteins are expressed in the intestine, which can act redundantly (Farin et al., 2012). Knockout experiments have shown that Wnt3, the main Wnt expressed in the epithelium, is not essential for maintenance of intestinal stem cells but can be compensated for by other Wnt proteins or non-epithelial sources of Wnts. In contrast, in organoids, Wnt3 is essential for growth and differentiation (Farin et al., 2012). Wnt activity in crypts is mainly found at their base, where Paneth cells and pro-
genitor cells are found. Studies using various methods for detecting signaling activity demonstrated a Wnt activity gradient along the colonic crypt axis (Davies et al., 2008). In vivo gain-of function experiments demonstrated that high levels of Wnt signaling activity lead to growth of Lgr5-positive stem cells and de novo crypt formation, whereas low Wnt activity induces proliferation of epithelial progenitors (Hirata et al., 2013). However, these studies are only suggestive for the existence of a relevant Wnt activity gradient in the colonic crypt axis. Not only is the topological dimension of the proposed gradient field ill-defined, but also, more importantly, definitive proof of a physiologically relevant Wnt gradient awaits the demonstration of a requirement for graded Wnt activity in the colonic crypt axis. Accepting the gradient hypothesis, Farin et al. (2016) have now asked how a Wnt3 gradient could be formed by intestinal cells. To address this issue, they generated a functional epitope knockin
Developmental Cell 36, March 7, 2016 ª2016 Elsevier Inc. 485
Developmental Cell
Previews allele of Wnt3 to visualize the Wnt protein gradient in the intestine. They demonstrated that homozygous Wnt3HA/HA mice are viable and fertile, indicating that the function of Wnt3 is not impaired by addition of the epitope tag. They then made use of the organoid model, which the Clevers lab pioneered to analyze the distribution of tagged Wnt3 protein in fixed cells. To test whether Wnt3 is secreted from Paneth cells, Farin et al. (2016) co-cultured Wnt3 knockout cells with wild-type organoids and showed that co-culturing could not rescue organoid growth, suggesting that Wnt3 might act in a rather autocrine manner. In contrast, using elegant cell dispersal and reaggregation experiments, they showed that wild-type Paneth cells could restore organoid growth of Wnt3 knockout cells, where Wnt3 remains close to the producing Paneth cells. These findings are interesting in the light of a recent report in Drosophila in which the existence of a ‘‘diffusible’’ long-range gradient has been challenged by the ability of membrane-tethered Wg to rescue Wg loss-of-function animals to adulthood (Alexandre et al., 2014). This study also supported a model, similar to Farin et al., that Wnt proteins remain membrane-bound to exert their function over a distance. Assuming that Wnt3 generates a colonic activity gradient, Farin et al. (2016) asked how the protein could func-
tion over a distance if it acts only short range. To test whether cell division may play a role in propagation of surfacebound Wnt3, they stalled Wnt cell-surface release with a Porcupine inhibitor, which they then washed out to generate a wave of surface-released Wnt3. Interestingly, under these conditions cell-cycle inhibitors lead to an accumulation of surface-bound Wnt3 protein, suggesting that cell division normally propagates and dilutes the signal. The authors propose a model in which the establishment of a short-range gradient would involve two steps: first, the Wnt ligand is transferred to the neighboring Lgr5+ cell, where it remains bound to the Frizzled receptor. This cell would then distribute the ligand through cell division, thereby further diluting the ligand. Many open questions remain. Assuming a direct action of membrane-bound Wnt, what is the mechanism by which Wnt proteins are transferred from the source cell to the nearest neighbor? What keeps the Wnt protein on the surface of the receiving cell (and not internalized by endocytosis) to be distributed to daughter cells and thereby establishing a gradient? Would this require ‘‘attenuating’’ the ability of receiving cells to endocytose Wnt/Fz receptor complexes and forming signalosomes? Importantly, the results of Farin et al. (2016) are also compatible with filopodia and/or exovesicle transporting membrane-bound Wnt protein to distant
486 Developmental Cell 36, March 7, 2016 ª2016 Elsevier Inc.
sites, for which there is now good evidence from a variety of model systems (Gross et al., 2012; Huang and Kornberg, 2015; Stanganello and Scholpp, 2016). These would go undetected in the microscopy of fixed samples used in their study. In the future, live-cell imaging of fluorescently labeled Wnt may yield deeper insights into the mechanism of Wnt signaling. REFERENCES Alexandre, C., Baena-Lopez, A., and Vincent, J.-P. (2014). Nature 505, 180–185. Clevers, H. (2013). Cell 154, 274–284. Davies, P.S., Dismuke, A.D., Powell, A.E., Carroll, K.H., and Wong, M.H. (2008). BMC Gastroenterol. 8, 57. Farin, H.F., Van Es, J.H., and Clevers, H. (2012). Gastroenterology 143, 1518–1529.e7. Farin, H.F., Jordens, I., Mosa, M.H., Basak, O., Korving, J., Tauriello, D.V., de Punder, K., Angers, S., Peters, P.J., Maurice, M.M., and Clevers, H. (2016). Nature 530, 340–343. Gross, J.C., Chaudhary, V., Bartscherer, K., and Boutros, M. (2012). Nat. Cell Biol. 14, 1036–1045. Hirata, A., Utikal, J., Yamashita, S., Aoki, H., Watanabe, A., Yamamoto, T., Okano, H., Bardeesy, N., Kunisada, T., Ushijima, T., et al. (2013). Development 140, 66–75. Huang, H., and Kornberg, T.B. (2015). Elife 4, e06114. Rogers, K.W., and Schier, A.F. (2011). Annu. Rev. Cell Dev. Biol. 27, 377–407. Stanganello, E., and Scholpp, S. (2016). J. Cell Sci., pii:jcs.182469.
Previews K.A., et al. (2016). Cell Metab. http://dx.doi.org/10. 1016/j.cmet.2016.01.007. DeBerardinis, R.J., Mancuso, A., Daikhin, E., Nissim, I., Yudkoff, M., Wehrli, S., and Thompson, C.B. (2007). Proc. Natl. Acad. Sci. USA 104, 19345–19350. Deberardinis, R.J., Sayed, N., Ditsworth, D., and Thompson, C.B. (2008). Curr. Opin. Genet. Dev. 18, 54–61.
Fan, J., Ye, J., Kamphorst, J.J., Shlomi, T., Thompson, C.B., and Rabinowitz, J.D. (2014). Nature 510, 298–302.
Owen, O.E., Kalhan, S.C., and Hanson, R.W. (2002). J. Biol. Chem. 277, 30409–30412.
Hosios, A.M., Hecht, V.C., Danai, L.V., Johnson, M.O., Rathmell, J.C., Steinhauser, M.L., Manalis, S.R., and Vander Heiden, M.G. (2016). Dev. Cell 36, this issue, 540–549.
Vander Heiden, M.G., Cantley, L.C., and Thompson, C.B. (2009). Science 324, 1029–1033.
Locasale, J.W. (2013). Nat. Rev. Cancer 13, 572–583.
Yuneva, M., Zamboni, N., Oefner, P., Sachidanandam, R., and Lazebnik, Y. (2007). J. Cell Biol. 178, 93–105.
Sticking Around: Short-Range Activity of Wnt Ligands Michael Boutros1,* and Christof Niehrs2,3 1Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Department of Cell and Molecular Biology, Faculty of Medicine Mannheim, Heidelberg University, 69120 Heidelberg, Germany 2Division of Molecular Embryology, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany 3Institute of Molecular Biology (IMB), 55128 Mainz, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2016.02.018
Wnt ligands are secreted lipid-modified proteins that are essential for development and homeostasis. Published recently in Nature, Farin et al. (2016) report that Wnt3 acts at a short distance during growth of mouse intestinal organoids, suggesting that Wnt proteins remain membrane-bound to activate signaling at a short range from their source. The question of how a uniform tissue gives rise to patterned structures has fascinated developmental biologists for many decades. First discussed in detail by Gierer and Meinhardt in the 1970s, morphogen gradients have since been implicated in many developmental processes, ranging from pattern formation in hydra to patterning decisions of wings in flies and limbs in mouse (reviewed in Rogers and Schier, 2011). Morphogen gradients are thought to be generated by secreted molecules that elicit distinct cellular responses at different concentrations or activities, thereby leading to cell fate changes and patterning of tissues. However, while the existence of activity gradients is generally accepted, different mechanisms have been proposed for how ligands travel from their source to their target sites. This has been a particularly ‘‘sticky’’ issue for Wnt ligands, which are secreted lipid-modified glycoproteins and are unlikely to diffuse freely in the extracellular space without carriers. How Wnt
morphogen gradients—both short- and long-range—are formed and how Wnt proteins are transported have remained major open questions. Proposed molecular mechanisms included diffusion, protein carriers, different forms of exovesicles and cytonemes, and filopodia that transport Wnt proteins from the site of production (reviewed in Stanganello and Scholpp, 2016). In the intestinal crypts, Wnt signaling plays an important role for stem cell growth and differentiation (Clevers, 2013). Multiple Wnt proteins are expressed in the intestine, which can act redundantly (Farin et al., 2012). Knockout experiments have shown that Wnt3, the main Wnt expressed in the epithelium, is not essential for maintenance of intestinal stem cells but can be compensated for by other Wnt proteins or non-epithelial sources of Wnts. In contrast, in organoids, Wnt3 is essential for growth and differentiation (Farin et al., 2012). Wnt activity in crypts is mainly found at their base, where Paneth cells and pro-
genitor cells are found. Studies using various methods for detecting signaling activity demonstrated a Wnt activity gradient along the colonic crypt axis (Davies et al., 2008). In vivo gain-of function experiments demonstrated that high levels of Wnt signaling activity lead to growth of Lgr5-positive stem cells and de novo crypt formation, whereas low Wnt activity induces proliferation of epithelial progenitors (Hirata et al., 2013). However, these studies are only suggestive for the existence of a relevant Wnt activity gradient in the colonic crypt axis. Not only is the topological dimension of the proposed gradient field ill-defined, but also, more importantly, definitive proof of a physiologically relevant Wnt gradient awaits the demonstration of a requirement for graded Wnt activity in the colonic crypt axis. Accepting the gradient hypothesis, Farin et al. (2016) have now asked how a Wnt3 gradient could be formed by intestinal cells. To address this issue, they generated a functional epitope knockin
Developmental Cell 36, March 7, 2016 ª2016 Elsevier Inc. 485
Developmental Cell
Previews allele of Wnt3 to visualize the Wnt protein gradient in the intestine. They demonstrated that homozygous Wnt3HA/HA mice are viable and fertile, indicating that the function of Wnt3 is not impaired by addition of the epitope tag. They then made use of the organoid model, which the Clevers lab pioneered to analyze the distribution of tagged Wnt3 protein in fixed cells. To test whether Wnt3 is secreted from Paneth cells, Farin et al. (2016) co-cultured Wnt3 knockout cells with wild-type organoids and showed that co-culturing could not rescue organoid growth, suggesting that Wnt3 might act in a rather autocrine manner. In contrast, using elegant cell dispersal and reaggregation experiments, they showed that wild-type Paneth cells could restore organoid growth of Wnt3 knockout cells, where Wnt3 remains close to the producing Paneth cells. These findings are interesting in the light of a recent report in Drosophila in which the existence of a ‘‘diffusible’’ long-range gradient has been challenged by the ability of membrane-tethered Wg to rescue Wg loss-of-function animals to adulthood (Alexandre et al., 2014). This study also supported a model, similar to Farin et al., that Wnt proteins remain membrane-bound to exert their function over a distance. Assuming that Wnt3 generates a colonic activity gradient, Farin et al. (2016) asked how the protein could func-
tion over a distance if it acts only short range. To test whether cell division may play a role in propagation of surfacebound Wnt3, they stalled Wnt cell-surface release with a Porcupine inhibitor, which they then washed out to generate a wave of surface-released Wnt3. Interestingly, under these conditions cell-cycle inhibitors lead to an accumulation of surface-bound Wnt3 protein, suggesting that cell division normally propagates and dilutes the signal. The authors propose a model in which the establishment of a short-range gradient would involve two steps: first, the Wnt ligand is transferred to the neighboring Lgr5+ cell, where it remains bound to the Frizzled receptor. This cell would then distribute the ligand through cell division, thereby further diluting the ligand. Many open questions remain. Assuming a direct action of membrane-bound Wnt, what is the mechanism by which Wnt proteins are transferred from the source cell to the nearest neighbor? What keeps the Wnt protein on the surface of the receiving cell (and not internalized by endocytosis) to be distributed to daughter cells and thereby establishing a gradient? Would this require ‘‘attenuating’’ the ability of receiving cells to endocytose Wnt/Fz receptor complexes and forming signalosomes? Importantly, the results of Farin et al. (2016) are also compatible with filopodia and/or exovesicle transporting membrane-bound Wnt protein to distant
486 Developmental Cell 36, March 7, 2016 ª2016 Elsevier Inc.
sites, for which there is now good evidence from a variety of model systems (Gross et al., 2012; Huang and Kornberg, 2015; Stanganello and Scholpp, 2016). These would go undetected in the microscopy of fixed samples used in their study. In the future, live-cell imaging of fluorescently labeled Wnt may yield deeper insights into the mechanism of Wnt signaling. REFERENCES Alexandre, C., Baena-Lopez, A., and Vincent, J.-P. (2014). Nature 505, 180–185. Clevers, H. (2013). Cell 154, 274–284. Davies, P.S., Dismuke, A.D., Powell, A.E., Carroll, K.H., and Wong, M.H. (2008). BMC Gastroenterol. 8, 57. Farin, H.F., Van Es, J.H., and Clevers, H. (2012). Gastroenterology 143, 1518–1529.e7. Farin, H.F., Jordens, I., Mosa, M.H., Basak, O., Korving, J., Tauriello, D.V., de Punder, K., Angers, S., Peters, P.J., Maurice, M.M., and Clevers, H. (2016). Nature 530, 340–343. Gross, J.C., Chaudhary, V., Bartscherer, K., and Boutros, M. (2012). Nat. Cell Biol. 14, 1036–1045. Hirata, A., Utikal, J., Yamashita, S., Aoki, H., Watanabe, A., Yamamoto, T., Okano, H., Bardeesy, N., Kunisada, T., Ushijima, T., et al. (2013). Development 140, 66–75. Huang, H., and Kornberg, T.B. (2015). Elife 4, e06114. Rogers, K.W., and Schier, A.F. (2011). Annu. Rev. Cell Dev. Biol. 27, 377–407. Stanganello, E., and Scholpp, S. (2016). J. Cell Sci., pii:jcs.182469.