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EGF Receptor Signaling: Putting a New Spin on Eye Development
Current Biology, Vol. 13, R813–R814, October 14, 2003, ©2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/j.cub.2003.09.053
EGF Receptor Signaling: Putting a New Spin on Eye Development Tanya Wolff
The coordinated polarization of cells within an epithelium is required for the development and function of some tissues. Recent work has shown that the EGF receptor signaling pathway plays a key role in establishing epithelial polarity in the compound eye of Drosophila.
The 800-fold reiteration of facets that tile the Drosophila compound eye is exquisite not only in its flawlessness and aesthetic appeal, but also, from a molecular and cell biological vantage point, in its elaborate execution of multiple signaling pathways and cellular processes. For example, the epidermal growth factor (EGF) receptor signaling pathway plays a myriad roles in eye development, including the regulation of cell proliferation and recruitment, ommatidial spacing, and apoptosis [1]. Recent studies [2–4] have now identified a role for this pathway in a specialized type of cellular movement that occurs during eye development. One of the early events in patterning the fly eye is recruitment of the future photoreceptor cells into symmetric ommatidial precursors [5] (Figure 1A). These cells then adopt unique photoreceptor fates with respect to their proximity to the dorsoventral midline, thereby breaking the symmetry and giving rise to two chiral forms of ommatidia. These chiral forms lie on opposite sides of the dorsoventral midline (Figure 1B). Next, these clusters undergo a striking morphogenetic movement, known as ommatidial rotation, which both polarizes the epithelium and organizes it to optimize visual acuity. Ommatidial rotation is an elegant and unique type of cellular movement in which groups of cells rotate within an epithelium as individual units, independent of their undifferentiated neighbors. Ommatidial precursors rotate precisely 90° counterclockwise in the dorsal half of the eye and 90° clockwise in the ventral half of the eye (Figure 1A). This specialized movement requires the coordinated maintenance of cell–cell contacts between photoreceptor cells within a cluster and the simultaneous abolition of contacts between cells within a cluster and their undifferentiated neighbors. Subsequently, the cytoskeleton must drive the physical movement of rotating clusters. Little is known about the control of rotation at a mechanistic level. Only three genes in which mutatons specifically affect the degree of rotation, and not the direction of rotation or chirality, have been identified: nemo, scabrous and roulette. Mutations in nemo cause ommatidia to arrest rotation at 45° [6], whereas mutations in scabrous [7,8] cause ommatidia to rotate more Washington University School of Medicine, Department of Genetics, Box 8232, St. Louis, Missouri 63110, USA.
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than 90° [9]; roulette mutant eyes contain a mixture of over-rotated and under-rotated ommatidia [2–4,6]. The molecular identities of the nemo and scabrous gene products have been known for some time, but the roulette gene product has only just been characterized. Three groups [2–4] have now reported evidence that roulette is an allele of argos — henceforth referred to as argosrlt — which encodes a secreted, inhibitory ligand of the EGF receptor [10,11]. Mapping and sequence data for argosrlt [3,4], as well as information on the argos mutant phenotype [2,4], indicate that the argosrlt allele carries a regulatory mutation of argos. The three groups all found that altering levels of EGF receptor activity, either positively or negatively, results in defects in ommatidial rotation. The authors propose three distinct interpretations for a mechanism by which EGF receptor signaling regulates ommatidial rotation: that it plays a role in cementing rotated clusters in position to prevent them from getting jostled around during later morphogenetic events; that it is generally required for the physical process of rotation; or that, as a consequence of extra cells being recruited into ommatidial clusters when the EGF receptor signaling level is abnormal, localization of rotational cues is disrupted. Brown and Freeman [2] explored the effects of increasing EGF receptor signaling and found that it resulted in defects in ommatidial orientation in adult eyes. Surprisingly, ommatidial precursors rotate normally, suggesting the EGF receptor pathway is dispensable for the process of rotation per se. By early pupal life, though, ommatidia are out of register with the 90° axis. Given that ommatidia initiate and stop rotation normally, these authors conclude that EGF receptor signaling functions to anchor rotated ommatidial precursors in place, thereby insulating them from physical stresses imposed on the retina during subsequent morphogenetic events. Gaengel and Mlodzik [3] also examined the effect of genetically increasing EGF receptor activity, but they found contrasting results: in an argosrlt mutant background, both over-rotated and under-rotated ommatidial precursors were observed, suggesting that normal argos expression is required for proper ommatidial rotation. These discrepant results might point towards an additional signaling pathway, mediated by Argos, which controls rotation. As reported recently in Current Biology, Strutt and Strutt [4] base their model on the discerning observation that ommatidial clusters have extra photoreceptor cells, leading them to suggest yet a different role for EGF receptor signaling. As the photoreceptors play an essential role in directing rotation, Strutt and Strutt [4] speculate that the presence of too many photoreceptors might interfere with rotational cues that are propagated by these cells. One such cue is the subcellular localization of Frizzled, a Wingless receptor protein known to be involved in the
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affected by EGF receptor signaling, given their roles in ommatidial rotation. The papers show that DE-Cadherin/shotgun interacts genetically with the EGF receptor pathway [2,3], and that Flamingo, a tissue polarity protein that co-localizes with Frizzled, is mislocalized in argosrlt eye discs [3,4]. While this work has shown that Argos, and more broadly, EGF receptor signaling, regulates ommatidial rotation, further work is necessary to better define the mechanism.
B
References
D/V
Current Biology
Figure 1. Ommatidial rotation in development of the Drosophila compound eye. (A) Pattern formation in the Drosophila eye begins in the third larval instar. Photoreceptors (colored cells) are recruited into ommatidial precursors. These future ommatidia then rotate 90° counterclockwise in the dorsal half of the eye and 90° clockwise in the ventral half of the eye. Upon the completion of rotation, ommatidia in the dorsal and ventral halves of the eye are mirror-symmetrical to one another, about the dorsoventral (D/V) midline. Arrows indicate direction of rotation. (B) Two chiral forms of ommatidia (red in dorsal half, blue in ventral half) lie on opposite sides of the dorsoventral midline.
establishment of tissue polarity. Their finding that Frizzled localization was drastically disrupted in argosrlt mutant clusters at the time the clusters are beginning to rotate supports their hypothesis. Signaling pathways are used repeatedly throughout development to direct diverse events. One means of achieving this versatility is through the use of different transducers, which exert their effects at multiple levels. In Drosophila, the canonical Ras–Raf–MAP kinase pathway serves as the main effector pathway for EGF receptor signaling (reviewed in [12]), and the transcription factor Pointed is frequently part of this cascade. The three groups [2–4] sought to determine if the Ras–Raf–MAP kinase pathway is involved in the control of ommatidial rotation by the EGF receptor, and also to identify additional effectors in this event. Together, they demonstrated links between the EGF receptor pathway and transcription factors, cytoskeletal elements and cadherins. The link to the nucleus and transcriptional regulation was made by showing that Ras [3,4] and Pointed [2,3] act downstream of EGF receptor signaling to mediate rotation. Given that rotation involves remodeling of the cytoskeleton and that Ras, by acting through a subset of its effectors, can mediate such events [13,14], Gaengel and Mlodzik [3] tested several candidate genes for their ability to genetically modify the Ras rotation phenotype. This work, in addition to loss-offunction studies, provides evidence that Canoe — a component of adherens junctions [15] and an effector of Ras — is the link between EGF receptor signaling and the cytoskeleton. Finally, cadherins could also be
1. Dominguez, M., Wasserman, J.D. and Freeman, M. (1998). Multiple functions of the EGF receptor in Drosophila eye development. Curr. Biol. 8, 1039-1048. 2. Brown, K.E. and Freeman, M. (2003). EGF receptor signalling defines a protective function for ommatidial orientation in the Drosophila eye. Development, in press. 3. Gaengel, K. and Mlodzik, M. (2003). EGF receptor signaling regulates ommatidial rotation and cell motility in the Drosophila eye via MAPK/Pnt signaling and the Ras effector Canoe/AF6. Development, in press. 4. Strutt, H. and Strutt, D. (2003). EGF Signaling and Ommatidial Rotation in the Drosophila Eye. Curr. Biol. 13, 1451-1457. 5. Wolff, T. and Ready, D.F. (1993). The Development of Drosophila melanogaster. Plainview, N.Y.: Cold Spring Harbor Laboratory Press. 2 v. 6. Choi, K.W., and Benzer, S. (1994). Rotation of photoreceptor clusters in the developing Drosophila eye requires the nemo gene. Cell 78, 1251-1236. 7. Baker, N.E., Mlodzik, M., and Rubin, G.M. (1990). Spacing differentiation in the developing Drosophila eye: a fibrinogen-related lateral inhibitor encoded by scabrous. Science 250, 1370-1377. 8. Mlodzik, M., Baker, N.E., and Rubin, G.M. (1990). Isolation and expression of scabrous, a gene regulating neurogenesis in Drosophila. Genes Dev. 4, 1848-1861. 9. Chou, Y.H. and Chien, C.T. (2002). Scabrous controls ommatidial rotation in the Drosophila compound eye. Dev, Cell 3, 839-850. 10. Freeman, M., Klambt, C., Goodman, C.S. and Rubin, G.M. (1992). The argos gene encodes a diffusible factor that regulates cell fate decisions in the Drosophila eye. Cell 69, 963-975. 11. Schweitzer, R., Howes, R., Smith, R., Shilo, B.Z. and Freeman, M. (1995). Inhibition of Drosophila EGF receptor activation by the secreted protein Argos. Nature 376, 699-702. 12. Shilo, B.Z. (2003). Signaling by the Drosophila epidermal growth factor receptor pathway during development. Exp. Cell Res. 284, 140-149. 13. Joneson, T., White, M.A., Wigler, M.H. and Bar-Sagi, D. (1996). Stimulation of membrane ruffling and MAP kinase activation by distinct effectors of RAS. Science 271, 810-812. 14. Rodriguez-Viciana ,P., Warne, P.H., Khwaja, A., Marte, B.M., Pappin, D., Das, P., Waterfield, M.D., Ridley, A. and Downward, J. (1997). Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell 89, 457-467. 15. Matsuo, T., Takahashi, K., Suzuki, E. and Yamamoto, D. (1999). The Canoe protein is necessary in adherens junctions for development of ommatidial architecture in the Drosophila compound eye. Cell Tissue Res. 298, 397-404.
EGF Receptor Signaling: Putting a New Spin on Eye Development Tanya Wolff
The coordinated polarization of cells within an epithelium is required for the development and function of some tissues. Recent work has shown that the EGF receptor signaling pathway plays a key role in establishing epithelial polarity in the compound eye of Drosophila.
The 800-fold reiteration of facets that tile the Drosophila compound eye is exquisite not only in its flawlessness and aesthetic appeal, but also, from a molecular and cell biological vantage point, in its elaborate execution of multiple signaling pathways and cellular processes. For example, the epidermal growth factor (EGF) receptor signaling pathway plays a myriad roles in eye development, including the regulation of cell proliferation and recruitment, ommatidial spacing, and apoptosis [1]. Recent studies [2–4] have now identified a role for this pathway in a specialized type of cellular movement that occurs during eye development. One of the early events in patterning the fly eye is recruitment of the future photoreceptor cells into symmetric ommatidial precursors [5] (Figure 1A). These cells then adopt unique photoreceptor fates with respect to their proximity to the dorsoventral midline, thereby breaking the symmetry and giving rise to two chiral forms of ommatidia. These chiral forms lie on opposite sides of the dorsoventral midline (Figure 1B). Next, these clusters undergo a striking morphogenetic movement, known as ommatidial rotation, which both polarizes the epithelium and organizes it to optimize visual acuity. Ommatidial rotation is an elegant and unique type of cellular movement in which groups of cells rotate within an epithelium as individual units, independent of their undifferentiated neighbors. Ommatidial precursors rotate precisely 90° counterclockwise in the dorsal half of the eye and 90° clockwise in the ventral half of the eye (Figure 1A). This specialized movement requires the coordinated maintenance of cell–cell contacts between photoreceptor cells within a cluster and the simultaneous abolition of contacts between cells within a cluster and their undifferentiated neighbors. Subsequently, the cytoskeleton must drive the physical movement of rotating clusters. Little is known about the control of rotation at a mechanistic level. Only three genes in which mutatons specifically affect the degree of rotation, and not the direction of rotation or chirality, have been identified: nemo, scabrous and roulette. Mutations in nemo cause ommatidia to arrest rotation at 45° [6], whereas mutations in scabrous [7,8] cause ommatidia to rotate more Washington University School of Medicine, Department of Genetics, Box 8232, St. Louis, Missouri 63110, USA.
Dispatch
than 90° [9]; roulette mutant eyes contain a mixture of over-rotated and under-rotated ommatidia [2–4,6]. The molecular identities of the nemo and scabrous gene products have been known for some time, but the roulette gene product has only just been characterized. Three groups [2–4] have now reported evidence that roulette is an allele of argos — henceforth referred to as argosrlt — which encodes a secreted, inhibitory ligand of the EGF receptor [10,11]. Mapping and sequence data for argosrlt [3,4], as well as information on the argos mutant phenotype [2,4], indicate that the argosrlt allele carries a regulatory mutation of argos. The three groups all found that altering levels of EGF receptor activity, either positively or negatively, results in defects in ommatidial rotation. The authors propose three distinct interpretations for a mechanism by which EGF receptor signaling regulates ommatidial rotation: that it plays a role in cementing rotated clusters in position to prevent them from getting jostled around during later morphogenetic events; that it is generally required for the physical process of rotation; or that, as a consequence of extra cells being recruited into ommatidial clusters when the EGF receptor signaling level is abnormal, localization of rotational cues is disrupted. Brown and Freeman [2] explored the effects of increasing EGF receptor signaling and found that it resulted in defects in ommatidial orientation in adult eyes. Surprisingly, ommatidial precursors rotate normally, suggesting the EGF receptor pathway is dispensable for the process of rotation per se. By early pupal life, though, ommatidia are out of register with the 90° axis. Given that ommatidia initiate and stop rotation normally, these authors conclude that EGF receptor signaling functions to anchor rotated ommatidial precursors in place, thereby insulating them from physical stresses imposed on the retina during subsequent morphogenetic events. Gaengel and Mlodzik [3] also examined the effect of genetically increasing EGF receptor activity, but they found contrasting results: in an argosrlt mutant background, both over-rotated and under-rotated ommatidial precursors were observed, suggesting that normal argos expression is required for proper ommatidial rotation. These discrepant results might point towards an additional signaling pathway, mediated by Argos, which controls rotation. As reported recently in Current Biology, Strutt and Strutt [4] base their model on the discerning observation that ommatidial clusters have extra photoreceptor cells, leading them to suggest yet a different role for EGF receptor signaling. As the photoreceptors play an essential role in directing rotation, Strutt and Strutt [4] speculate that the presence of too many photoreceptors might interfere with rotational cues that are propagated by these cells. One such cue is the subcellular localization of Frizzled, a Wingless receptor protein known to be involved in the
Dispatch R814
A
affected by EGF receptor signaling, given their roles in ommatidial rotation. The papers show that DE-Cadherin/shotgun interacts genetically with the EGF receptor pathway [2,3], and that Flamingo, a tissue polarity protein that co-localizes with Frizzled, is mislocalized in argosrlt eye discs [3,4]. While this work has shown that Argos, and more broadly, EGF receptor signaling, regulates ommatidial rotation, further work is necessary to better define the mechanism.
B
References
D/V
Current Biology
Figure 1. Ommatidial rotation in development of the Drosophila compound eye. (A) Pattern formation in the Drosophila eye begins in the third larval instar. Photoreceptors (colored cells) are recruited into ommatidial precursors. These future ommatidia then rotate 90° counterclockwise in the dorsal half of the eye and 90° clockwise in the ventral half of the eye. Upon the completion of rotation, ommatidia in the dorsal and ventral halves of the eye are mirror-symmetrical to one another, about the dorsoventral (D/V) midline. Arrows indicate direction of rotation. (B) Two chiral forms of ommatidia (red in dorsal half, blue in ventral half) lie on opposite sides of the dorsoventral midline.
establishment of tissue polarity. Their finding that Frizzled localization was drastically disrupted in argosrlt mutant clusters at the time the clusters are beginning to rotate supports their hypothesis. Signaling pathways are used repeatedly throughout development to direct diverse events. One means of achieving this versatility is through the use of different transducers, which exert their effects at multiple levels. In Drosophila, the canonical Ras–Raf–MAP kinase pathway serves as the main effector pathway for EGF receptor signaling (reviewed in [12]), and the transcription factor Pointed is frequently part of this cascade. The three groups [2–4] sought to determine if the Ras–Raf–MAP kinase pathway is involved in the control of ommatidial rotation by the EGF receptor, and also to identify additional effectors in this event. Together, they demonstrated links between the EGF receptor pathway and transcription factors, cytoskeletal elements and cadherins. The link to the nucleus and transcriptional regulation was made by showing that Ras [3,4] and Pointed [2,3] act downstream of EGF receptor signaling to mediate rotation. Given that rotation involves remodeling of the cytoskeleton and that Ras, by acting through a subset of its effectors, can mediate such events [13,14], Gaengel and Mlodzik [3] tested several candidate genes for their ability to genetically modify the Ras rotation phenotype. This work, in addition to loss-offunction studies, provides evidence that Canoe — a component of adherens junctions [15] and an effector of Ras — is the link between EGF receptor signaling and the cytoskeleton. Finally, cadherins could also be
1. Dominguez, M., Wasserman, J.D. and Freeman, M. (1998). Multiple functions of the EGF receptor in Drosophila eye development. Curr. Biol. 8, 1039-1048. 2. Brown, K.E. and Freeman, M. (2003). EGF receptor signalling defines a protective function for ommatidial orientation in the Drosophila eye. Development, in press. 3. Gaengel, K. and Mlodzik, M. (2003). EGF receptor signaling regulates ommatidial rotation and cell motility in the Drosophila eye via MAPK/Pnt signaling and the Ras effector Canoe/AF6. Development, in press. 4. Strutt, H. and Strutt, D. (2003). EGF Signaling and Ommatidial Rotation in the Drosophila Eye. Curr. Biol. 13, 1451-1457. 5. Wolff, T. and Ready, D.F. (1993). The Development of Drosophila melanogaster. Plainview, N.Y.: Cold Spring Harbor Laboratory Press. 2 v. 6. Choi, K.W., and Benzer, S. (1994). Rotation of photoreceptor clusters in the developing Drosophila eye requires the nemo gene. Cell 78, 1251-1236. 7. Baker, N.E., Mlodzik, M., and Rubin, G.M. (1990). Spacing differentiation in the developing Drosophila eye: a fibrinogen-related lateral inhibitor encoded by scabrous. Science 250, 1370-1377. 8. Mlodzik, M., Baker, N.E., and Rubin, G.M. (1990). Isolation and expression of scabrous, a gene regulating neurogenesis in Drosophila. Genes Dev. 4, 1848-1861. 9. Chou, Y.H. and Chien, C.T. (2002). Scabrous controls ommatidial rotation in the Drosophila compound eye. Dev, Cell 3, 839-850. 10. Freeman, M., Klambt, C., Goodman, C.S. and Rubin, G.M. (1992). The argos gene encodes a diffusible factor that regulates cell fate decisions in the Drosophila eye. Cell 69, 963-975. 11. Schweitzer, R., Howes, R., Smith, R., Shilo, B.Z. and Freeman, M. (1995). Inhibition of Drosophila EGF receptor activation by the secreted protein Argos. Nature 376, 699-702. 12. Shilo, B.Z. (2003). Signaling by the Drosophila epidermal growth factor receptor pathway during development. Exp. Cell Res. 284, 140-149. 13. Joneson, T., White, M.A., Wigler, M.H. and Bar-Sagi, D. (1996). Stimulation of membrane ruffling and MAP kinase activation by distinct effectors of RAS. Science 271, 810-812. 14. Rodriguez-Viciana ,P., Warne, P.H., Khwaja, A., Marte, B.M., Pappin, D., Das, P., Waterfield, M.D., Ridley, A. and Downward, J. (1997). Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell 89, 457-467. 15. Matsuo, T., Takahashi, K., Suzuki, E. and Yamamoto, D. (1999). The Canoe protein is necessary in adherens junctions for development of ommatidial architecture in the Drosophila compound eye. Cell Tissue Res. 298, 397-404.