The International Journal of Biochemistry & Cell Biology 64 (2015) 11–14
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Molecules in focus
Celsr3 and Fzd3 in axon guidance Guoliang Chai, Andre M. Goffinet ∗ , Fadel Tissir ∗∗ Institute of Neuroscience, Université catholique de Louvain, 73 Avenue Mounier, B1.73.16, Brussels 1200, Belgium
a r t i c l e
i n f o
Article history: Received 26 January 2015 Received in revised form 10 March 2015 Accepted 16 March 2015 Available online 23 March 2015 Keywords: Cytoskeleton Growth cone Wnt signaling Linx E3 ligase Disheveled
a b s t r a c t The assembly of functional neuronal circuits depends on the correct wiring of axons and dendrites. To reach their targets, axons are guided by a variety of extracellular guidance cues, including Netrins, Ephrins, Semaphorins and Slits. Corresponding receptors in the growth cone, the dynamic structure at the tip of the growing axon, sense and integrate these positional signals, and activate downstream effectors to regulate cytoskeletal organization. In addition to the four canonical families of axon guidance cues mentioned above, some proteins that regulate planar cell polarity were recently found to be critical for axon guidance. The seven-transmembrane domain receptors Celsr3 and Fzd3, in particular, control the development of most longitudinal tracts in the central nervous system, and axon navigation in the peripheral, sympathetic and enteric nervous systems. Despite their unequivocally important role, however, underlying molecular mechanisms remain elusive. We do not know which extracellular ligands they recognize, whether they have co-receptors in the growth cone, and what their downstream effectors are. Here, we review some recent advances and discuss future trends in this emerging field. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Axon guidance is a critical step in the wiring of the nervous system. The “active” component that guides axons to their target is the growth cone which senses signals in the microenvironment and translates them into appropriate motility behavior. Extracellular signals are attractive or repulsive molecules that are diffusible, or anchored in the extracellular matrix and/or at the surface of intermediate target (also called “guidepost”) cells. Growth cone sensors are membrane proteins that recognize a variety of extracellular cues. They regulate growth cone adhesion and transduce information via intracellular adaptors to the actin and microtubule cytoskeleton (Fig. 1). Like other axon guidance systems, the function of Celsr3/Fzd3 should be considered in this framework. Important findings, published since the role of Celsr3 and Fzd3 in axon guidance was reported, point to candidate mechanisms that will be the focus of this mini review. Celsr3 and Fzd3 belong to the so called “Planar cell polarity” (PCP) genes that regulate organization of cells in a sheet. PCP genes, identified in Drosophila, encode the transmembrane
∗ Corresponding author. Tel.: +32 2764 7386. ∗∗ Corresponding author. E-mail addresses: andre.goffi[email protected] (A.M. Goffinet), [email protected] (F. Tissir). http://dx.doi.org/10.1016/j.biocel.2015.03.013 1357-2725/© 2015 Elsevier Ltd. All rights reserved.
proteins Frizzled (Fz), Flamingo/Starry night (Fmi/Stan) and Van Gogh (Vang), and the adaptors Dishevelled (Dsh), Prickle (Pk) and Diego (Dgo). They participate in a signaling cascade that controls the alignment of actin hairs in the wing, the rotation of ommatidia in the eye, the asymmetric cell division of sensory organ precursors and other events (Goodrich and Strutt, 2011). Vertebrate PCP genes are Celsr1–3 (orthologues of Fmi/Stan); Fzd3 (Frizzled3) and Fzd6; Vangl1 (Vang like protein 1) and Vangl2; Dvl1–3 (Dishevelled1–3); Prickle1–4 and Ankrd6 (the orthologue of Dgo). In mice, PCP proteins mediate classical “epithelial” PCP including patterning of skin hair follicles, the organization of the inner ear epithelium, convergent extension during gastrulation, neural tube closure, as well as directional neuronal migration (Wang and Nathans, 2007). PCP proteins also play a critical role in the organization of cilia in ependymal and airway cells (Boutin et al., 2014; Tissir and Goffinet, 2010; Vladar et al., 2012). Unexpectedly, PCP genes Celsr3 and Fzd3 were found to regulate axon guidance, an event that takes place outside of epithelia. Their inactivation leads to severe defects in major axon tracts such as the anterior commissure, internal capsule, and corticospinal tract (Tissir and Goffinet, 2013). Both genes are also required in the guidance of monoaminergic axons along the anterior–posterior axis, in the anterior turning of commissural axons in the spinal cord (Fenstermaker et al., 2010; Lyuksyutova et al., 2003), as well as in axon pathfinding in the peripheral, sympathetic and enteric nervous systems (Armstrong et al., 2011; Chai et al., 2014; Hua et al., 2013; Sasselli et al., 2013).
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G. Chai et al. / The International Journal of Biochemistry & Cell Biology 64 (2015) 11–14
3. Potential partners of Celsr3/Fzd3 in axon guidance 3.1. Upstream ligands of Celsr3/Fzd3
Fig. 1. Schematic representation of a growth cone. There is a clear segregation of actin and microtubules. Stable bundled microtubules enter the growth cone from the axon shaft to the central (C) domain, while dynamic microtubules project through the transition (T) zone to the peripheral (P) domain. P domain contains meshworks of filamentous (F) actin called lamellipodia and finger-like structures filled with bundled F-actin, called filopodia. Surface receptors sense and integrate attractive and repulsive cues, including diffusible factors that form gradients in the environment, ligands at the surface of intermediate target cells, and molecules in the extracellular matrix (ECM). Signals are transduced to instruct the growth cone to turn, extend, or retract. The movement of the growth cone relies on its intrinsic motile property and its capability to reorganize the actin and microtubule cytoskeleton in response to the environment.
2. Is there a role of Vangl2 in axon guidance? Van Gogh is a key regulator of PCP in the fly, and in vertebrate epithelia such as the neuroepithelium, inner ear, epidermis, or ependyma (Goodrich and Strutt, 2011). Some axon guidance defects described in Fzd3 and Celsr3 mutant mice, such as the abnormal trajectory of spinal commissural axons (Shafer et al., 2011) and brainstem monoaminergic axons (Fenstermaker et al., 2010), are also observed in Vangl2Lp/Lp (Lp: Looptail) mice which have a S464N substitution in Vangl2, suggesting that the latter may be a partner of Celsr3 and Fzd3 in axon guidance. However, studies using a null Vangl1 and a conditional Vangl2 allele challenge this view. In contrast to phenotypes in Celsr3 and Fzd3 mutant mice (Hua et al., 2014; Qu et al., 2014; Zhou et al., 2008), the anterior commissure, thalamocortical, corticothalamic, and corticospinal tracts develop normally in the absence of both Vangl1 and Vangl2. Moreover, whereas inactivation of Celsr3 or Fzd3 in motor neurons leads to defective innervation of the dorsal hindlimb (Chai et al., 2014; Hua et al., 2013), this is unaffected in Vangl2 mutants (Chai et al., 2014). In the same line, the superior cervical ganglion is atrophic, and orthosympatic nerves are poorly fasciculated in Fzd3 but not in Vangl2Lp mutant mice (Armstrong et al., 2011). This indicates that, contrary to epithelial polarity, axon guidance does not require Vangl genes, at least not for the formation of major axonal bundles and peripheral nerve trunks. How can we reconcile those findings? The Vangl2Lp mutant protein is unable to exit the endoplasmic reticulum (ER) and reach the plasma membrane, and has strong dominant negative activity (Yin et al., 2012). Both Vangl1 and Vangl2 interact physically and recruit Dvl1 to the membrane. In yeast two hybrid assays, Vangl2Lp mutant protein does not bind to Dvl1, but still binds to Dvl2 and Dvl3 (Torban et al., 2004). It could compete with Fzd3 and sequester Dvl in the ER, thereby affecting the targeting of Dvl and Fzd3 at the membrane. In epithelial cells, Vangl and Celsr accumulate and could interact at the proximal side of the cell. Thus, Vangl2Lp might hamper the targeting of Celsr3 and affect Celsr3–Fzd3 signaling. Axon guidance defects in Vangl2LP mutants could therefore reflect the indirect perturbation of Celsr3–Fzd3 signaling, rather than a direct function of Vangl2.
Studies of conditional Celsr3 mutant mice argue against simple homophilic interactions and suggest that Celsr3/Fzd3 binding proteins are present in growth cones or guidepost cells, perhaps as a complex. Graded expression of ligands and/or surface receptors is classically evoked to explain growth cone steering (Zou, 2004). A key question is which ligands bind to the complex and activate signals to steer growth cones. Wnt proteins, ligands of Fzd receptors, are obvious candidates (Zou, 2004). Spinal commissural axons turn rostrally after crossing the midline, and this is defective in Fzd3 and in Celsr3 mutant mice. Wnt4 and Wnt7b form an anterior high–posterior low gradient in the floor plate. Wnt4 is able to attract postcrossing commissural axons, and inactivation of Wnts by sFRP impairs rostral turning of axons explants. When Wnt4 expressing cells are placed posteriorly, some axons are rerouted towards them (Lyuksyutova et al., 2003). Wnt4 may therefore act as an attractant through Celsr3 and Fzd3 receptors. However, thus far no ligand receptor-binding interaction was identified, and alterations of commissural axons were not demonstrated in Wnt4 mutant mice. Another evidence for the role of Wnts is during development of monoaminergic axons, which harbor guidance defects in Fzd3 and Celsr3 mutants. In cultured explants, Wnt5a attracts serotoninergic but repels mdDA axons, and Wnt7b attracts mdDA axons. Fzd3 and Celsr3 are expressed in serotoninergic and mdDA neurons, and Fzd3 mutant mdDA axons cannot respond to Wnt5a and Wnt7b. Therefore, Wnt ligands may act as diffusible guidance cues by activating Celsr3–Fzd3 signaling (Fenstermaker et al., 2010) (Fig. 2). The debate remains open, and issues are complicated by the existence of 19 Wnts (in mammals) that can bind to 10 different Fzd receptors, while some Wnts also bind to other receptors
Fig. 2. A working model for Celsr3/Fzd3 signal in axon guidance. Wnt ligands can bind to Fzd3 and may act as diffusible guidance cues. EphrinAs, Ret and Linx may interact with Celsr3/Fzds and form a complex to integrate steering signals in the growth cone. Activated Fzd3 recruits Dvl to the membrane and activates Formins, RhoA and ROCK, ultimately leading to the rearrangement of the cytoskeleton. E3 ligases interact with Dvl and control the endocytosis of the Frizzled-Dvl3 complex which may be required for axon guidance.
G. Chai et al. / The International Journal of Biochemistry & Cell Biology 64 (2015) 11–14
such as the tyrosine kinases Ror1,2 (Minami et al., 2010) or Ryk (“receptor related to tyrosine kinase”) (Yoshikawa et al., 2003). 3.2. Co-receptors of Celsr3/Fzd3 Given that growth cones encounter and integrate multiple guidance cues, do Celsr3 and Fzd3 act in collaboration with, or in parallel to other systems? Recent studies on hindlimb innervation indicate that Celsr3–Fzd3 interacts with EphA-ephrinA reverse signaling (Chai et al., 2014). In the developing hindlimb buds, the sciatic nerve divides into dorsal/peroneal and ventral/tibial nerves, as a result of at least three signals. ephrinAs in ventral limb mesenchyme repels dorsal peroneal axons by binding to EphA receptors in their growth cones. In parallel, EphA receptors and GDNF expressed by mesenchymal cells in the dorsal hindlimb bind respectively to ephrinA2/A5 and the GDNF receptor (Ret and GFR␣1) in growth cones, synergistically promoting growth of dorsal axons (Bonanomi et al., 2012). Celsr3 and Fzd3 mutant motor axons of the deep peroneal nerve stall after the sciatic nerve division, and fail to extend distally in the dorsal hindlimb. Their ability to respond to attractive GDNF or repellent ephrinA, presumably expressed as gradients in the limb,is preserved. But, intriguingly, Celsr3 or Fzd3 mutant axons cannot respond to attractive ephrinA reverse signaling (EphAs in the limb acting on ephrinAs in the axon). Moreover, Celsr3 and Fzd3 coimmunoprecipitate with ephrinA2 ephrinA5 and Ret in transfected cells. EphrinAs, which are GPI-anchored proteins residing in lipid microdomains (“rafts”), could help assemble signaling platforms containing Celsr3/Fzd3, Ret/GFRA1 and probably other partners, enabling growth cones to integrate multiple steering signals (Fig. 2). Linx, a LIG family transmembrane protein, mediates the extension and guidance of corticofugal, thalamocortical, sensory and spinal motor axons (Mandai et al., 2009, 2014). It interacts with receptor tyrosine kinases such as TrkA, Ret, and Trk-associated protein p75NTR . Peripheral nerve projection defects in Linx mutants are reminiscent of those in Ngf, TrkA and Ret mutants. In neurons, Linx is required for GDNF–GFR␣1/Ret induced motor axon extension, and NGF–TrkA mediated sensory axon extension (Mandai et al., 2009). Thus, Linx can modulate multiple growth factor signals to control axonal growth. Intriguingly, Linx mutants share phenotypic defects with Celsr3 or Fzd3 deficient mice. Like Celsr3 and Fzd3, Linx is required in guidepost cells in the basal forebrain for internal capsule formation, in neocortex for the development of corticospinal tracts, and in motoneurons for hindlimb innervation. In transfected cells, Linx and Celsr3–Fzd3 co-immunoprecipitate with Ret (Mandai et al., 2009). Altogether, these data suggest that Linx may interact with Celsr3–Fzd3 (Fig. 2). 3.3. Downstream effectors of Celsr3/Fzd3 signaling What could be the transduction molecules that mediate the signal sensed by Celsr3–Fzd3? Dvl is a key cytoplasmic protein adapter of Fzd receptors and PCP signaling. Three Dvl orthologs (Dvl1–3) are widely expressed in the mouse nervous system. Single Dvl mutants exhibit specific phenotypes, Dvl1 and Dvl2 double mutants show neural tube closure defects, and Dvl1–3 triple mutants cannot undergo gastrulation. This indicates that Dvl1–3 are crucial intracellular effectors and display redundancy. In PCP signaling, Fzd3 can recruit and activate Dvl, which further signals to JNK, RhoA and Rac to regulate the cytoskeleton (Wallingford and Habas, 2005). Given the importance of Dvl in PCP, an obvious question is whether Dvl1–3 act downstream of Celsr3–Fzd3 signaling in axon guidance. Unfortunately, there is thus far no genetic evidence for the participation of Dvl in axon guidance, due to lack of conditional Dvl alleles. The CRISPR/Cas system, which allows simultaneous targeting of multiple genes (Wang et al., 2013; Yang et al., 2013), could generate such in vivo evidence. In contrast, several in vitro
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experiments support such a role. Overexpression of Dvl in cortical or commissural neurons enhances axon outgrowth. In dissociated commissural neurons, Fzd3 is able to recruit Dvl from intracellular vesicles to the membrane, and this depends on the Dvl DEP domain required in PCP (Shafer et al., 2011). This suggests that Dvl is a downstream effector of Fzd3 in axon guidance, sharing mechanism with PCP signaling (Fig. 2). Formin proteins, key regulators of actin polymerization and bundling, and microtubule dynamics, are strong candidate PCP regulators. Formin Daam1 (“Disheveled associated activator of morphogenesis 1”) interacts physically and functionally with Dvl and Rho (Habas et al., 2001). Daam1 switches between open active and closed inactive conformations, through the interaction between its amino-terminal GTPase binding, and C-terminal diaphanous autoregulatory domain. Binding to Dvl disrupts that auto-inhibition and activates Daam1, which further activates Rho to mediate cytoskeletal reorganization. Daam1 cooperates with Dvl to regulate directed cell migration during convergent extension and gastrulation in Xenopus (Liu et al., 2008). In Drosophila, dDaam regulates axon morphogenesis; dDaam mutants exhibit disorganized nerve cords in CNS, and filopodia are reduced in cultured mutant neurons (Matusek et al., 2008). mDia1, a member of the diaphanous group of formins, is necessary and sufficient for SDF-1␣ (stromal cell derived factor-1␣) dependent axon elongation in cultured cerebellar granule cells (Arakawa et al., 2003). Formins are therefore strong candidates to remodel the actin skeleton in growth cones, downstream of Fzd3/Celsr3. However, in vivo evidence for the role of formins in axon growth is still lacking. Lastly, E3 ubiquitin ligases interact with Dvl and control PCP. Mice lacking both Smurf1 and Smurf2 exhibit PCP defects in cochlea, and convergent extension defects with an open neural tube. Smurf1 and Smurf2 interact with phosphorylated Dvl2 and form a complex with Par6. The complex binds Prickle 1, a key PCP regulator, and leads to its ubiquitination and downregulation (Narimatsu et al., 2009). Another E3 ligase, PDZRN3, ubiquitinates Dvl3 to enhance endocytosis of the Frizzled-Dvl3 complex which is essential for the transduction of PCP signals (Sewduth et al., 2014). Phr1 is another E3 ubiquitin ligase expressed in mouse nervous system. Like Celsr3 and Fzd3 mutants, Phr1 knockout mice die at birth and exhibit axonal defects: corticofugal and thalamocortical projections are lost, the anterior commissure is absent, the corpus callosum is reduced, and peripheral nerves are reduced (Bloom et al., 2007). Phr1 inhibits the dual leucine kinase Map3k12 and forms a complex with the F-box protein Fbxo45 (Saiga et al., 2009) that regulates growth cone pausing at guidepost cells through microtubule disassembly (Hendricks and Jesuthasan, 2009). Rnf165 (Ark2C), another E3 ligase expressed in neural tissue, controls the extension of spinal motor axons in the dorsal hindlimb. Rnf165 mutant mice have tiny peroneal nerves and hindlimb paresis, which is attributed to interference with BMP-Smad signaling (Kelly et al., 2013). The phenotype is very reminiscent of that in Celsr3 and Fzd3 mutants, raising the possibility of yet another plausible interaction. 4. Conclusion Since the demonstration about ten years ago that Fzd3 and Celsr3 are two key regulators in the formation of axonal tracts, significant progress has been made but we still lack an integrated view of molecular mechanisms. We need to understand better how Fzd3 and Celsr3 organize a multicomponent complex, which ligand(s) they recognize in guidepost cells and in the extracellular matrix, how they remodel cytoskeletal organization in growth cones, and how they interact with other guidance signals. Answers to those questions are required not solely to grasp the basic biology, but also to assess the potential of these molecules in regeneration, and as candidate pharmacological targets.
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Acknowledgements This work was supported by the following grants: Actions de Recherches Concertées (ARC-10/15-026), FRSM 3.4550.11, FNRS T0002.13, Interuniversity Poles of Attraction (SSTC, PAI p6/20 and PAI7/20), Welbio CR-2012A-07, Fondation JED Belgique, and Fondation médicale Reine Elisabeth all from Belgium. G.C. and F.T. are respectively research fellow and senior research associate of the Belgian Fund for Scientific Research (FNRS). Authors have no conflict of interest. References Arakawa Y, Bito H, Furuyashiki T, Tsuji T, Takemoto-Kimura S, Kimura K, et al. Control of axon elongation via an SDF-1alpha/Rho/mDia pathway in cultured cerebellar granule neurons. J Cell Biol 2003;161:381–91. Armstrong A, Ryu YK, Chieco D, Kuruvilla R. Frizzled3 is required for neurogenesis and target innervation during sympathetic nervous system development. J Neurosci 2011;31:2371–81. Bloom AJ, Miller BR, Sanes JR, DiAntonio A. The requirement for Phr1 in CNS axon tract formation reveals the corticostriatal boundary as a choice point for cortical axons. Genes Dev 2007;21:2593–606. Bonanomi D, Chivatakarn O, Bai G, Abdesselem H, Lettieri K, Marquardt T, et al. Ret is a multifunctional coreceptor that integrates diffusible- and contact-axon guidance signals. Cell 2012;148:568–82. Boutin C, Labedan P, Dimidschstein J, Richard F, Cremer H, Andre P, et al. A dual role for planar cell polarity genes in ciliated cells. Proc Natl Acad Sci U S A 2014;111:E3129–38. Chai G, Zhou L, Manto M, Helmbacher F, Clotman F, Goffinet AM, et al. Celsr3 is required in motor neurons to steer their axons in the hindlimb. Nat Neurosci 2014;17:1171–9. Fenstermaker AG, Prasad AA, Bechara A, Adolfs Y, Tissir F, Goffinet A, et al. Wnt/planar cell polarity signaling controls the anterior-posterior organization of monoaminergic axons in the brainstem. J Neurosci 2010;30:16053–64. Goodrich LV, Strutt D. Principles of planar polarity in animal development. Development 2011;138:1877–92. Habas R, Kato Y, He X. Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel Formin homology protein Daam1. Cell 2001;107:843–54. Hendricks M, Jesuthasan S. PHR regulates growth cone pausing at intermediate targets through microtubule disassembly. J Neurosci 2009;29:6593–8. Hua ZL, Smallwood PM, Nathans J. Frizzled3 controls axonal development in distinct populations of cranial and spinal motor neurons. eLife 2013;2:e01482. Hua ZL, Jeon S, Caterina MJ, Nathans J. Frizzled3 is required for the development of multiple axon tracts in the mouse central nervous system. Proc Natl Acad Sci U S A 2014;111:E3005–14. Kelly CE, Thymiakou E, Dixon JE, Tanaka S, Godwin J, Episkopou V. Rnf165/Ark2C enhances BMP-Smad signaling to mediate motor axon extension. PLoS Biol 2013;11:e1001538. Liu W, Sato A, Khadka D, Bharti R, Diaz H, Runnels LW, et al. Mechanism of activation of the Formin protein Daam1. Proc Natl Acad Sci U S A 2008;105:210–5. Lyuksyutova AI, Lu CC, Milanesio N, King LA, Guo N, Wang Y, et al. Anterior–posterior guidance of commissural axons by Wnt-frizzled signaling. Science 2003;302:1984–8.
Mandai K, Guo T, St Hillaire C, Meabon JS, Kanning KC, Bothwell M, et al. LIG family receptor tyrosine kinase-associated proteins modulate growth factor signals during neural development. Neuron 2009;63:614–27. Mandai K, Reimert DV, Ginty DD. Linx mediates interaxonal interactions and formation of the internal capsule. Neuron 2014;83:93–103. Matusek T, Gombos R, Szecsenyi A, Sanchez-Soriano N, Czibula A, Pataki C, et al. Formin proteins of the DAAM subfamily play a role during axon growth. J Neurosci 2008;28:13310–9. Minami Y, Oishi I, Endo M, Nishita M. Ror-family receptor tyrosine kinases in noncanonical Wnt signaling: their implications in developmental morphogenesis and human diseases. Dev Dyn 2010;239:1–15. Narimatsu M, Bose R, Pye M, Zhang L, Miller B, Ching P, et al. Regulation of planar cell polarity by Smurf ubiquitin ligases. Cell 2009;137:295–307. Qu Y, Huang Y, Feng J, Alvarez-Bolado G, Grove EA, Yang Y, et al. Genetic evidence that Celsr3 and Celsr2, together with Fzd3, regulate forebrain wiring in a Vanglindependent manner. Proc Natl Acad Sci U S A 2014;111:E2996–3004. Saiga T, Fukuda T, Matsumoto M, Tada H, Okano HJ, Okano H, et al. Fbxo45 forms a novel ubiquitin ligase complex and is required for neuronal development. Mol Cell Biol 2009;29:3529–43. Sasselli V, Boesmans W, Vanden Berghe P, Tissir F, Goffinet AM, Pachnis V. Planar cell polarity genes control the connectivity of enteric neurons. J Clin Invest 2013;123:1763–72. Sewduth RN, Jaspard-Vinassa B, Peghaire C, Guillabert A, Franzl N, Larrieu-Lahargue F, et al. The ubiquitin ligase PDZRN3 is required for vascular morphogenesis through Wnt/planar cell polarity signalling. Nat Commun 2014;5:4832. Shafer B, Onishi K, Lo C, Colakoglu G, Zou Y. Vangl2 promotes Wnt/planar cell polarity-like signaling by antagonizing Dvl1-mediated feedback inhibition in growth cone guidance. Dev Cell 2011;20:177–91. Tissir F, Goffinet AM. Planar cell polarity signaling in neural development. Curr Opin Neurobiol 2010;20:572–7. Tissir F, Goffinet AM. Shaping the nervous system: role of the core planar cell polarity genes. Nat Rev Neurosci 2013;14:525–35. Torban E, Wang HJ, Groulx N, Gros P. Independent mutations in mouse Vangl2 that cause neural tube defects in looptail mice impair interaction with members of the Dishevelled family. J Biol Chem 2004;279:52703–13. Vladar EK, Bayly RD, Sangoram AM, Scott MP, Axelrod JD. Microtubules enable the planar cell polarity of airway cilia. Curr Biol 2012;22:2203–12. Wallingford JB, Habas R. The developmental biology of Dishevelled: an enigmatic protein governing cell fate and cell polarity. Development 2005;132: 4421–36. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 2013;153:910–8. Wang Y, Nathans J. Tissue/planar cell polarity in vertebrates: new insights and new questions. Development 2007;134:647–58. Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 2013;154:1370–9. Yin H, Copley CO, Goodrich LV, Deans MR. Comparison of phenotypes between different vangl2 mutants demonstrates dominant effects of the Looptail mutation during hair cell development. PloS One 2012;7:e31988. Yoshikawa S, McKinnon RD, Kokel M, Thomas JB. Wnt-mediated axon guidance via the Drosophila Derailed receptor. Nature 2003;422:583–8. Zhou L, Bar I, Achouri Y, Campbell K, De Backer O, Hebert JM, et al. Early forebrain wiring: genetic dissection using conditional Celsr3 mutant mice. Science 2008;320:946–9. Zou Y. Wnt signaling in axon guidance. Trends Neurosci 2004;27:528–32.
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Molecules in focus
Celsr3 and Fzd3 in axon guidance Guoliang Chai, Andre M. Goffinet ∗ , Fadel Tissir ∗∗ Institute of Neuroscience, Université catholique de Louvain, 73 Avenue Mounier, B1.73.16, Brussels 1200, Belgium
a r t i c l e
i n f o
Article history: Received 26 January 2015 Received in revised form 10 March 2015 Accepted 16 March 2015 Available online 23 March 2015 Keywords: Cytoskeleton Growth cone Wnt signaling Linx E3 ligase Disheveled
a b s t r a c t The assembly of functional neuronal circuits depends on the correct wiring of axons and dendrites. To reach their targets, axons are guided by a variety of extracellular guidance cues, including Netrins, Ephrins, Semaphorins and Slits. Corresponding receptors in the growth cone, the dynamic structure at the tip of the growing axon, sense and integrate these positional signals, and activate downstream effectors to regulate cytoskeletal organization. In addition to the four canonical families of axon guidance cues mentioned above, some proteins that regulate planar cell polarity were recently found to be critical for axon guidance. The seven-transmembrane domain receptors Celsr3 and Fzd3, in particular, control the development of most longitudinal tracts in the central nervous system, and axon navigation in the peripheral, sympathetic and enteric nervous systems. Despite their unequivocally important role, however, underlying molecular mechanisms remain elusive. We do not know which extracellular ligands they recognize, whether they have co-receptors in the growth cone, and what their downstream effectors are. Here, we review some recent advances and discuss future trends in this emerging field. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Axon guidance is a critical step in the wiring of the nervous system. The “active” component that guides axons to their target is the growth cone which senses signals in the microenvironment and translates them into appropriate motility behavior. Extracellular signals are attractive or repulsive molecules that are diffusible, or anchored in the extracellular matrix and/or at the surface of intermediate target (also called “guidepost”) cells. Growth cone sensors are membrane proteins that recognize a variety of extracellular cues. They regulate growth cone adhesion and transduce information via intracellular adaptors to the actin and microtubule cytoskeleton (Fig. 1). Like other axon guidance systems, the function of Celsr3/Fzd3 should be considered in this framework. Important findings, published since the role of Celsr3 and Fzd3 in axon guidance was reported, point to candidate mechanisms that will be the focus of this mini review. Celsr3 and Fzd3 belong to the so called “Planar cell polarity” (PCP) genes that regulate organization of cells in a sheet. PCP genes, identified in Drosophila, encode the transmembrane
∗ Corresponding author. Tel.: +32 2764 7386. ∗∗ Corresponding author. E-mail addresses: andre.goffi[email protected] (A.M. Goffinet), [email protected] (F. Tissir). http://dx.doi.org/10.1016/j.biocel.2015.03.013 1357-2725/© 2015 Elsevier Ltd. All rights reserved.
proteins Frizzled (Fz), Flamingo/Starry night (Fmi/Stan) and Van Gogh (Vang), and the adaptors Dishevelled (Dsh), Prickle (Pk) and Diego (Dgo). They participate in a signaling cascade that controls the alignment of actin hairs in the wing, the rotation of ommatidia in the eye, the asymmetric cell division of sensory organ precursors and other events (Goodrich and Strutt, 2011). Vertebrate PCP genes are Celsr1–3 (orthologues of Fmi/Stan); Fzd3 (Frizzled3) and Fzd6; Vangl1 (Vang like protein 1) and Vangl2; Dvl1–3 (Dishevelled1–3); Prickle1–4 and Ankrd6 (the orthologue of Dgo). In mice, PCP proteins mediate classical “epithelial” PCP including patterning of skin hair follicles, the organization of the inner ear epithelium, convergent extension during gastrulation, neural tube closure, as well as directional neuronal migration (Wang and Nathans, 2007). PCP proteins also play a critical role in the organization of cilia in ependymal and airway cells (Boutin et al., 2014; Tissir and Goffinet, 2010; Vladar et al., 2012). Unexpectedly, PCP genes Celsr3 and Fzd3 were found to regulate axon guidance, an event that takes place outside of epithelia. Their inactivation leads to severe defects in major axon tracts such as the anterior commissure, internal capsule, and corticospinal tract (Tissir and Goffinet, 2013). Both genes are also required in the guidance of monoaminergic axons along the anterior–posterior axis, in the anterior turning of commissural axons in the spinal cord (Fenstermaker et al., 2010; Lyuksyutova et al., 2003), as well as in axon pathfinding in the peripheral, sympathetic and enteric nervous systems (Armstrong et al., 2011; Chai et al., 2014; Hua et al., 2013; Sasselli et al., 2013).
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G. Chai et al. / The International Journal of Biochemistry & Cell Biology 64 (2015) 11–14
3. Potential partners of Celsr3/Fzd3 in axon guidance 3.1. Upstream ligands of Celsr3/Fzd3
Fig. 1. Schematic representation of a growth cone. There is a clear segregation of actin and microtubules. Stable bundled microtubules enter the growth cone from the axon shaft to the central (C) domain, while dynamic microtubules project through the transition (T) zone to the peripheral (P) domain. P domain contains meshworks of filamentous (F) actin called lamellipodia and finger-like structures filled with bundled F-actin, called filopodia. Surface receptors sense and integrate attractive and repulsive cues, including diffusible factors that form gradients in the environment, ligands at the surface of intermediate target cells, and molecules in the extracellular matrix (ECM). Signals are transduced to instruct the growth cone to turn, extend, or retract. The movement of the growth cone relies on its intrinsic motile property and its capability to reorganize the actin and microtubule cytoskeleton in response to the environment.
2. Is there a role of Vangl2 in axon guidance? Van Gogh is a key regulator of PCP in the fly, and in vertebrate epithelia such as the neuroepithelium, inner ear, epidermis, or ependyma (Goodrich and Strutt, 2011). Some axon guidance defects described in Fzd3 and Celsr3 mutant mice, such as the abnormal trajectory of spinal commissural axons (Shafer et al., 2011) and brainstem monoaminergic axons (Fenstermaker et al., 2010), are also observed in Vangl2Lp/Lp (Lp: Looptail) mice which have a S464N substitution in Vangl2, suggesting that the latter may be a partner of Celsr3 and Fzd3 in axon guidance. However, studies using a null Vangl1 and a conditional Vangl2 allele challenge this view. In contrast to phenotypes in Celsr3 and Fzd3 mutant mice (Hua et al., 2014; Qu et al., 2014; Zhou et al., 2008), the anterior commissure, thalamocortical, corticothalamic, and corticospinal tracts develop normally in the absence of both Vangl1 and Vangl2. Moreover, whereas inactivation of Celsr3 or Fzd3 in motor neurons leads to defective innervation of the dorsal hindlimb (Chai et al., 2014; Hua et al., 2013), this is unaffected in Vangl2 mutants (Chai et al., 2014). In the same line, the superior cervical ganglion is atrophic, and orthosympatic nerves are poorly fasciculated in Fzd3 but not in Vangl2Lp mutant mice (Armstrong et al., 2011). This indicates that, contrary to epithelial polarity, axon guidance does not require Vangl genes, at least not for the formation of major axonal bundles and peripheral nerve trunks. How can we reconcile those findings? The Vangl2Lp mutant protein is unable to exit the endoplasmic reticulum (ER) and reach the plasma membrane, and has strong dominant negative activity (Yin et al., 2012). Both Vangl1 and Vangl2 interact physically and recruit Dvl1 to the membrane. In yeast two hybrid assays, Vangl2Lp mutant protein does not bind to Dvl1, but still binds to Dvl2 and Dvl3 (Torban et al., 2004). It could compete with Fzd3 and sequester Dvl in the ER, thereby affecting the targeting of Dvl and Fzd3 at the membrane. In epithelial cells, Vangl and Celsr accumulate and could interact at the proximal side of the cell. Thus, Vangl2Lp might hamper the targeting of Celsr3 and affect Celsr3–Fzd3 signaling. Axon guidance defects in Vangl2LP mutants could therefore reflect the indirect perturbation of Celsr3–Fzd3 signaling, rather than a direct function of Vangl2.
Studies of conditional Celsr3 mutant mice argue against simple homophilic interactions and suggest that Celsr3/Fzd3 binding proteins are present in growth cones or guidepost cells, perhaps as a complex. Graded expression of ligands and/or surface receptors is classically evoked to explain growth cone steering (Zou, 2004). A key question is which ligands bind to the complex and activate signals to steer growth cones. Wnt proteins, ligands of Fzd receptors, are obvious candidates (Zou, 2004). Spinal commissural axons turn rostrally after crossing the midline, and this is defective in Fzd3 and in Celsr3 mutant mice. Wnt4 and Wnt7b form an anterior high–posterior low gradient in the floor plate. Wnt4 is able to attract postcrossing commissural axons, and inactivation of Wnts by sFRP impairs rostral turning of axons explants. When Wnt4 expressing cells are placed posteriorly, some axons are rerouted towards them (Lyuksyutova et al., 2003). Wnt4 may therefore act as an attractant through Celsr3 and Fzd3 receptors. However, thus far no ligand receptor-binding interaction was identified, and alterations of commissural axons were not demonstrated in Wnt4 mutant mice. Another evidence for the role of Wnts is during development of monoaminergic axons, which harbor guidance defects in Fzd3 and Celsr3 mutants. In cultured explants, Wnt5a attracts serotoninergic but repels mdDA axons, and Wnt7b attracts mdDA axons. Fzd3 and Celsr3 are expressed in serotoninergic and mdDA neurons, and Fzd3 mutant mdDA axons cannot respond to Wnt5a and Wnt7b. Therefore, Wnt ligands may act as diffusible guidance cues by activating Celsr3–Fzd3 signaling (Fenstermaker et al., 2010) (Fig. 2). The debate remains open, and issues are complicated by the existence of 19 Wnts (in mammals) that can bind to 10 different Fzd receptors, while some Wnts also bind to other receptors
Fig. 2. A working model for Celsr3/Fzd3 signal in axon guidance. Wnt ligands can bind to Fzd3 and may act as diffusible guidance cues. EphrinAs, Ret and Linx may interact with Celsr3/Fzds and form a complex to integrate steering signals in the growth cone. Activated Fzd3 recruits Dvl to the membrane and activates Formins, RhoA and ROCK, ultimately leading to the rearrangement of the cytoskeleton. E3 ligases interact with Dvl and control the endocytosis of the Frizzled-Dvl3 complex which may be required for axon guidance.
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such as the tyrosine kinases Ror1,2 (Minami et al., 2010) or Ryk (“receptor related to tyrosine kinase”) (Yoshikawa et al., 2003). 3.2. Co-receptors of Celsr3/Fzd3 Given that growth cones encounter and integrate multiple guidance cues, do Celsr3 and Fzd3 act in collaboration with, or in parallel to other systems? Recent studies on hindlimb innervation indicate that Celsr3–Fzd3 interacts with EphA-ephrinA reverse signaling (Chai et al., 2014). In the developing hindlimb buds, the sciatic nerve divides into dorsal/peroneal and ventral/tibial nerves, as a result of at least three signals. ephrinAs in ventral limb mesenchyme repels dorsal peroneal axons by binding to EphA receptors in their growth cones. In parallel, EphA receptors and GDNF expressed by mesenchymal cells in the dorsal hindlimb bind respectively to ephrinA2/A5 and the GDNF receptor (Ret and GFR␣1) in growth cones, synergistically promoting growth of dorsal axons (Bonanomi et al., 2012). Celsr3 and Fzd3 mutant motor axons of the deep peroneal nerve stall after the sciatic nerve division, and fail to extend distally in the dorsal hindlimb. Their ability to respond to attractive GDNF or repellent ephrinA, presumably expressed as gradients in the limb,is preserved. But, intriguingly, Celsr3 or Fzd3 mutant axons cannot respond to attractive ephrinA reverse signaling (EphAs in the limb acting on ephrinAs in the axon). Moreover, Celsr3 and Fzd3 coimmunoprecipitate with ephrinA2 ephrinA5 and Ret in transfected cells. EphrinAs, which are GPI-anchored proteins residing in lipid microdomains (“rafts”), could help assemble signaling platforms containing Celsr3/Fzd3, Ret/GFRA1 and probably other partners, enabling growth cones to integrate multiple steering signals (Fig. 2). Linx, a LIG family transmembrane protein, mediates the extension and guidance of corticofugal, thalamocortical, sensory and spinal motor axons (Mandai et al., 2009, 2014). It interacts with receptor tyrosine kinases such as TrkA, Ret, and Trk-associated protein p75NTR . Peripheral nerve projection defects in Linx mutants are reminiscent of those in Ngf, TrkA and Ret mutants. In neurons, Linx is required for GDNF–GFR␣1/Ret induced motor axon extension, and NGF–TrkA mediated sensory axon extension (Mandai et al., 2009). Thus, Linx can modulate multiple growth factor signals to control axonal growth. Intriguingly, Linx mutants share phenotypic defects with Celsr3 or Fzd3 deficient mice. Like Celsr3 and Fzd3, Linx is required in guidepost cells in the basal forebrain for internal capsule formation, in neocortex for the development of corticospinal tracts, and in motoneurons for hindlimb innervation. In transfected cells, Linx and Celsr3–Fzd3 co-immunoprecipitate with Ret (Mandai et al., 2009). Altogether, these data suggest that Linx may interact with Celsr3–Fzd3 (Fig. 2). 3.3. Downstream effectors of Celsr3/Fzd3 signaling What could be the transduction molecules that mediate the signal sensed by Celsr3–Fzd3? Dvl is a key cytoplasmic protein adapter of Fzd receptors and PCP signaling. Three Dvl orthologs (Dvl1–3) are widely expressed in the mouse nervous system. Single Dvl mutants exhibit specific phenotypes, Dvl1 and Dvl2 double mutants show neural tube closure defects, and Dvl1–3 triple mutants cannot undergo gastrulation. This indicates that Dvl1–3 are crucial intracellular effectors and display redundancy. In PCP signaling, Fzd3 can recruit and activate Dvl, which further signals to JNK, RhoA and Rac to regulate the cytoskeleton (Wallingford and Habas, 2005). Given the importance of Dvl in PCP, an obvious question is whether Dvl1–3 act downstream of Celsr3–Fzd3 signaling in axon guidance. Unfortunately, there is thus far no genetic evidence for the participation of Dvl in axon guidance, due to lack of conditional Dvl alleles. The CRISPR/Cas system, which allows simultaneous targeting of multiple genes (Wang et al., 2013; Yang et al., 2013), could generate such in vivo evidence. In contrast, several in vitro
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experiments support such a role. Overexpression of Dvl in cortical or commissural neurons enhances axon outgrowth. In dissociated commissural neurons, Fzd3 is able to recruit Dvl from intracellular vesicles to the membrane, and this depends on the Dvl DEP domain required in PCP (Shafer et al., 2011). This suggests that Dvl is a downstream effector of Fzd3 in axon guidance, sharing mechanism with PCP signaling (Fig. 2). Formin proteins, key regulators of actin polymerization and bundling, and microtubule dynamics, are strong candidate PCP regulators. Formin Daam1 (“Disheveled associated activator of morphogenesis 1”) interacts physically and functionally with Dvl and Rho (Habas et al., 2001). Daam1 switches between open active and closed inactive conformations, through the interaction between its amino-terminal GTPase binding, and C-terminal diaphanous autoregulatory domain. Binding to Dvl disrupts that auto-inhibition and activates Daam1, which further activates Rho to mediate cytoskeletal reorganization. Daam1 cooperates with Dvl to regulate directed cell migration during convergent extension and gastrulation in Xenopus (Liu et al., 2008). In Drosophila, dDaam regulates axon morphogenesis; dDaam mutants exhibit disorganized nerve cords in CNS, and filopodia are reduced in cultured mutant neurons (Matusek et al., 2008). mDia1, a member of the diaphanous group of formins, is necessary and sufficient for SDF-1␣ (stromal cell derived factor-1␣) dependent axon elongation in cultured cerebellar granule cells (Arakawa et al., 2003). Formins are therefore strong candidates to remodel the actin skeleton in growth cones, downstream of Fzd3/Celsr3. However, in vivo evidence for the role of formins in axon growth is still lacking. Lastly, E3 ubiquitin ligases interact with Dvl and control PCP. Mice lacking both Smurf1 and Smurf2 exhibit PCP defects in cochlea, and convergent extension defects with an open neural tube. Smurf1 and Smurf2 interact with phosphorylated Dvl2 and form a complex with Par6. The complex binds Prickle 1, a key PCP regulator, and leads to its ubiquitination and downregulation (Narimatsu et al., 2009). Another E3 ligase, PDZRN3, ubiquitinates Dvl3 to enhance endocytosis of the Frizzled-Dvl3 complex which is essential for the transduction of PCP signals (Sewduth et al., 2014). Phr1 is another E3 ubiquitin ligase expressed in mouse nervous system. Like Celsr3 and Fzd3 mutants, Phr1 knockout mice die at birth and exhibit axonal defects: corticofugal and thalamocortical projections are lost, the anterior commissure is absent, the corpus callosum is reduced, and peripheral nerves are reduced (Bloom et al., 2007). Phr1 inhibits the dual leucine kinase Map3k12 and forms a complex with the F-box protein Fbxo45 (Saiga et al., 2009) that regulates growth cone pausing at guidepost cells through microtubule disassembly (Hendricks and Jesuthasan, 2009). Rnf165 (Ark2C), another E3 ligase expressed in neural tissue, controls the extension of spinal motor axons in the dorsal hindlimb. Rnf165 mutant mice have tiny peroneal nerves and hindlimb paresis, which is attributed to interference with BMP-Smad signaling (Kelly et al., 2013). The phenotype is very reminiscent of that in Celsr3 and Fzd3 mutants, raising the possibility of yet another plausible interaction. 4. Conclusion Since the demonstration about ten years ago that Fzd3 and Celsr3 are two key regulators in the formation of axonal tracts, significant progress has been made but we still lack an integrated view of molecular mechanisms. We need to understand better how Fzd3 and Celsr3 organize a multicomponent complex, which ligand(s) they recognize in guidepost cells and in the extracellular matrix, how they remodel cytoskeletal organization in growth cones, and how they interact with other guidance signals. Answers to those questions are required not solely to grasp the basic biology, but also to assess the potential of these molecules in regeneration, and as candidate pharmacological targets.
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Acknowledgements This work was supported by the following grants: Actions de Recherches Concertées (ARC-10/15-026), FRSM 3.4550.11, FNRS T0002.13, Interuniversity Poles of Attraction (SSTC, PAI p6/20 and PAI7/20), Welbio CR-2012A-07, Fondation JED Belgique, and Fondation médicale Reine Elisabeth all from Belgium. G.C. and F.T. are respectively research fellow and senior research associate of the Belgian Fund for Scientific Research (FNRS). Authors have no conflict of interest. References Arakawa Y, Bito H, Furuyashiki T, Tsuji T, Takemoto-Kimura S, Kimura K, et al. Control of axon elongation via an SDF-1alpha/Rho/mDia pathway in cultured cerebellar granule neurons. J Cell Biol 2003;161:381–91. Armstrong A, Ryu YK, Chieco D, Kuruvilla R. Frizzled3 is required for neurogenesis and target innervation during sympathetic nervous system development. J Neurosci 2011;31:2371–81. Bloom AJ, Miller BR, Sanes JR, DiAntonio A. The requirement for Phr1 in CNS axon tract formation reveals the corticostriatal boundary as a choice point for cortical axons. Genes Dev 2007;21:2593–606. Bonanomi D, Chivatakarn O, Bai G, Abdesselem H, Lettieri K, Marquardt T, et al. Ret is a multifunctional coreceptor that integrates diffusible- and contact-axon guidance signals. Cell 2012;148:568–82. Boutin C, Labedan P, Dimidschstein J, Richard F, Cremer H, Andre P, et al. A dual role for planar cell polarity genes in ciliated cells. Proc Natl Acad Sci U S A 2014;111:E3129–38. Chai G, Zhou L, Manto M, Helmbacher F, Clotman F, Goffinet AM, et al. Celsr3 is required in motor neurons to steer their axons in the hindlimb. Nat Neurosci 2014;17:1171–9. Fenstermaker AG, Prasad AA, Bechara A, Adolfs Y, Tissir F, Goffinet A, et al. Wnt/planar cell polarity signaling controls the anterior-posterior organization of monoaminergic axons in the brainstem. J Neurosci 2010;30:16053–64. Goodrich LV, Strutt D. Principles of planar polarity in animal development. Development 2011;138:1877–92. Habas R, Kato Y, He X. Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel Formin homology protein Daam1. Cell 2001;107:843–54. Hendricks M, Jesuthasan S. PHR regulates growth cone pausing at intermediate targets through microtubule disassembly. J Neurosci 2009;29:6593–8. Hua ZL, Smallwood PM, Nathans J. Frizzled3 controls axonal development in distinct populations of cranial and spinal motor neurons. eLife 2013;2:e01482. Hua ZL, Jeon S, Caterina MJ, Nathans J. Frizzled3 is required for the development of multiple axon tracts in the mouse central nervous system. Proc Natl Acad Sci U S A 2014;111:E3005–14. Kelly CE, Thymiakou E, Dixon JE, Tanaka S, Godwin J, Episkopou V. Rnf165/Ark2C enhances BMP-Smad signaling to mediate motor axon extension. PLoS Biol 2013;11:e1001538. Liu W, Sato A, Khadka D, Bharti R, Diaz H, Runnels LW, et al. Mechanism of activation of the Formin protein Daam1. Proc Natl Acad Sci U S A 2008;105:210–5. Lyuksyutova AI, Lu CC, Milanesio N, King LA, Guo N, Wang Y, et al. Anterior–posterior guidance of commissural axons by Wnt-frizzled signaling. Science 2003;302:1984–8.
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