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Branch management: mechanisms of axon branching in the developing vertebrate CNS

Key Points

  • Axon branching connects single neurons with multiple targets, which, along with the formation of highly branched terminal arbors, underlies the complex circuitry of the vertebrate CNS.

  • Axon collateral branches extend interstitially from the axon shaft as dynamic filopodia that develop into branches at appropriate targets regions to form functional maps. Extrinsic guidance cues, growth factors and morphogens regulate axon branching and shape terminal arbors that develop from axon branches.

  • Growth and guidance of axon branches in response to extracellular cues require dynamic reorganization of the actin and microtubule cytoskeleton. Cycles of cytoskeletal polymerization and depolymerization are highly regulated by actin- and microtubule-associated proteins during branch formation.

  • Complex signalling pathways that are activated by extracellular cues through their receptors regulate axon branching. The ultimate target of signal transduction pathways is the cytoskeleton, which can reorganize by changes in dynamics to promote or suppress axon branching.

  • Neuronal activity, which is often stimulated by extracellular cues, can regulate axon branching by transient fluctuations in the levels of intracellular calcium, which acts as a second messenger to activate downstream cytoskeletal effectors. Effects of neural activity can involve competition among neighbouring axon arbors, such as in the retinotectal system, where competitive activity-dependent mechanisms regulate arbor size and complexity.

  • Future directions in the study of axon branch formation will involve the use of preparations of the vertebrate CNS that recapitulate the complexity of the in vivo environment. Improvements in labelling techniques and high-resolution time-lapse microscopy should facilitate such studies.

Abstract

The remarkable ability of a single axon to extend multiple branches and form terminal arbors enables vertebrate neurons to integrate information from divergent regions of the nervous system. Axons select appropriate pathways during development, but it is the branches that extend interstitially from the axon shaft and arborize at specific targets that are responsible for virtually all of the synaptic connectivity in the vertebrate CNS. How do axons form branches at specific target regions? Recent studies have identified molecular cues that activate intracellular signalling pathways in axons and mediate dynamic reorganization of the cytoskeleton to promote the formation of axon branches.

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Figure 1: Stages of axon branching in developing CNS pathways.
Figure 2: Cytoskeletal reorganization at different stages of axon branching.
Figure 3: Signalling pathways that promote axon branching.
Figure 4: Competition shapes the morphology of terminal arbors.

References

  1. Acebes, A. & Ferrus, A. Cellular and molecular features of axon collaterals and dendrites. Trends Neurosci. 23, 557–565 (2000).

    CAS  PubMed  Google Scholar 

  2. Bilimoria, P. M. & Bonni, A. Molecular control of axon branching. Neuroscientist 19, 16–24 (2013).

    CAS  PubMed  Google Scholar 

  3. Gallo, G. The cytoskeletal and signaling mechanisms of axon collateral branching. Dev. Neurobiol. 71, 201–220 (2011).

    PubMed  Google Scholar 

  4. Gibson, D. A. & Ma, L. Developmental regulation of axon branching in the vertebrate nervous system. Development 138, 183–195 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Kornack, D. R. & Giger, R. J. Probing microtubule +TIPs: regulation of axon branching. Curr. Opin. Neurobiol. 15, 58–66 (2005).

    CAS  PubMed  Google Scholar 

  6. Schmidt, H. & Rathjen, F. G. Signalling mechanisms regulating axonal branching in vivo. Bioessays 32, 977–985 (2010).

    CAS  PubMed  Google Scholar 

  7. Feldheim, D. A. & O'Leary, D. D. Visual map development: bidirectional signaling, bifunctional guidance molecules, and competition. Cold Spring Harb. Perspect. Biol. 2, a001768 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Stanfield, B. B. The development of the corticospinal projection. Prog. Neurobiol. 38, 169–202 (1992).

    CAS  PubMed  Google Scholar 

  9. O'Leary, D. D. & Terashima, T. Cortical axons branch to multiple subcortical targets by interstitial axon budding: implications for target recognition and “waiting periods”. Neuron 1, 901–910 (1988).

    CAS  PubMed  Google Scholar 

  10. Lu, S. M. & Lin, R. C. Thalamic afferents of the rat barrel cortex: a light- and electron-microscopic study using Phaseolus vulgaris leucoagglutinin as an anterograde tracer. Somatosens. Mot. Res. 10, 1–16 (1993).

    CAS  PubMed  Google Scholar 

  11. Fame, R. M., MacDonald, J. L. & Macklis, J. D. Development, specification, and diversity of callosal projection neurons. Trends Neurosci. 34, 41–50 (2011).

    CAS  PubMed  Google Scholar 

  12. Cajal, S. R. Histology of the Nervous System of Man and Vertebrates (Oxford Univ. Press, 1995).

    Google Scholar 

  13. Dickson, B. J. Molecular mechanisms of axon guidance. Science 298, 1959–1964 (2002).

    CAS  PubMed  Google Scholar 

  14. Ma, L. & Tessier-Lavigne, M. Dual branch-promoting and branch-repelling actions of Slit/Robo signaling on peripheral and central branches of developing sensory axons. J. Neurosci. 27, 6843–6851 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Schmidt, H. et al. The receptor guanylyl cyclase Npr2 is essential for sensory axon bifurcation within the spinal cord. J. Cell Biol. 179, 331–340 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. O'Leary, D. D. et al. Target selection by cortical axons: alternative mechanisms to establish axonal connections in the developing brain. Cold Spring Harb. Symp. Quant. Biol. 55, 453–468 (1990).

    CAS  PubMed  Google Scholar 

  17. Bastmeyer, M. & O'Leary, D. D. Dynamics of target recognition by interstitial axon branching along developing cortical axons. J. Neurosci. 16, 1450–1459 (1996).

    CAS  PubMed  Google Scholar 

  18. Kuang, R. Z. & Kalil, K. Development of specificity in corticospinal connections by axon collaterals branching selectively into appropriate spinal targets. J. Comp. Neurol. 344, 270–282 (1994).

    CAS  PubMed  Google Scholar 

  19. Luo, L. & O'Leary, D. D. Axon retraction and degeneration in development and disease. Annu. Rev. Neurosci. 28, 127–156 (2005).

    CAS  PubMed  Google Scholar 

  20. Norris, C. R. & Kalil, K. Guidance of callosal axons by radial glia in the developing cerebral cortex. J. Neurosci. 11, 3481–3492 (1991).

    CAS  PubMed  Google Scholar 

  21. Halloran, M. C. & Kalil, K. Dynamic behaviors of growth cones extending in the corpus callosum of living cortical brain slices observed with video microscopy. J. Neurosci. 14, 2161–2177 (1994).

    CAS  PubMed  Google Scholar 

  22. Szebenyi, G., Callaway, J. L., Dent, E. W. & Kalil, K. Interstitial branches develop from active regions of the axon demarcated by the primary growth cone during pausing behaviors. J. Neurosci. 18, 7930–7940 (1998).

    CAS  PubMed  Google Scholar 

  23. Agmon, A., Yang, L. T., O'Dowd, D. K. & Jones, E. G. Organized growth of thalamocortical axons from the deep tier of terminations into layer IV of developing mouse barrel cortex. J. Neurosci. 13, 5365–5382 (1993).

    CAS  PubMed  Google Scholar 

  24. Portera-Cailliau, C., Weimer, R. M., De Paola, V., Caroni, P. & Svoboda, K. Diverse modes of axon elaboration in the developing neocortex. PLoS Biol. 3, e272 (2005). An elegant live-cell in vivo imaging study using two-photon time-lapse microscopy to follow the postnatal development of thalamocortical and Cajal–Retzius axons and their collaterals in the mouse cortex over timescales from minutes to days during the first 3 weeks of postnatal development. Although these axons have different morphologies and dynamics, branches of both types of axons develop interstitially and not by growth cone splitting, providing direct evidence that in vivo vertebrate CNS axons primarily form branches interstitially.

    PubMed  PubMed Central  Google Scholar 

  25. Yates, P. A., Roskies, A. L., McLaughlin, T. & O'Leary, D. D. Topographic-specific axon branching controlled by ephrin-As is the critical event in retinotectal map development. J. Neurosci. 21, 8548–8563 (2001).

    CAS  PubMed  Google Scholar 

  26. Simon, D. K. & O'Leary, D. D. Limited topographic specificity in the targeting and branching of mammalian retinal axons. Dev. Biol. 137, 125–134 (1990).

    CAS  PubMed  Google Scholar 

  27. McLaughlin, T. & O'Leary, D. D. Molecular gradients and development of retinotopic maps. Annu. Rev. Neurosci. 28, 327–355 (2005).

    CAS  PubMed  Google Scholar 

  28. Kaethner, R. J. & Stuermer, C. A. Dynamics of terminal arbor formation and target approach of retinotectal axons in living zebrafish embryos: a time-lapse study of single axons. J. Neurosci. 12, 3257–3271 (1992).

    CAS  PubMed  Google Scholar 

  29. O'Rourke, N. A., Cline, H. T. & Fraser, S. E. Rapid remodeling of retinal arbors in the tectum with and without blockade of synaptic transmission. Neuron 12, 921–934 (1994).

    CAS  PubMed  Google Scholar 

  30. Metin, C., Deleglise, D., Serafini, T., Kennedy, T. E. & Tessier-Lavigne, M. A role for netrin-1 in the guidance of cortical efferents. Development 124, 5063–5074 (1997).

    CAS  PubMed  Google Scholar 

  31. Richards, L. J., Koester, S. E., Tuttle, R. & O'Leary, D. D. Directed growth of early cortical axons is influenced by a chemoattractant released from an intermediate target. J. Neurosci. 17, 2445–2458 (1997).

    CAS  PubMed  Google Scholar 

  32. Serafini, T. et al. Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system. Cell 87, 1001–1014 (1996).

    CAS  PubMed  Google Scholar 

  33. Lai Wing Sun, K., Correia, J. P. & Kennedy, T. E. Netrins: versatile extracellular cues with diverse functions. Development 138, 2153–2169 (2011).

    PubMed  Google Scholar 

  34. Dent, E. W., Barnes, A. M., Tang, F. & Kalil, K. Netrin-1 and semaphorin 3A promote or inhibit cortical axon branching, respectively, by reorganization of the cytoskeleton. J. Neurosci. 24, 3002–3012 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Lebrand, C. et al. Critical role of Ena/VASP proteins for filopodia formation in neurons and in function downstream of netrin-1. Neuron 42, 37–49 (2004).

    CAS  PubMed  Google Scholar 

  36. Tang, F. & Kalil, K. Netrin-1 induces axon branching in developing cortical neurons by frequency-dependent calcium signaling pathways. J. Neurosci. 25, 6702–6715 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Manitt, C., Nikolakopoulou, A. M., Almario, D. R., Nguyen, S. A. & Cohen-Cory, S. Netrin participates in the development of retinotectal synaptic connectivity by modulating axon arborization and synapse formation in the developing brain. J. Neurosci. 29, 11065–11077 (2009). An in vivo imaging study of netrin 1-mediated development of retinal axon arbors in the frog optic tectum, which shows that netrin 1induces dynamic axon branching, increasing branch addition and retraction to ultimately increase total branch number. Importantly, comparison between netrin 1-induced retinotectal axon branching in this study and BDNF-induced retinal axon branching from previous work shows that, although both cues increase arbor complexity, they do so through different dynamic strategies. Thus, different molecular cues can use different strategies that lead to a similar end point in shaping terminal arbors.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Palmer, A. & Klein, R. Multiple roles of ephrins in morphogenesis, neuronal networking, and brain function. Genes Dev. 17, 1429–1450 (2003).

    CAS  PubMed  Google Scholar 

  39. Wilkinson, D. G. Multiple roles of EPH receptors and ephrins in neural development. Nature Rev. Neurosci. 2, 155–164 (2001).

    CAS  Google Scholar 

  40. Castellani, V., Yue, Y., Gao, P. P., Zhou, R. & Bolz, J. Dual action of a ligand for Eph receptor tyrosine kinases on specific populations of axons during the development of cortical circuits. J. Neurosci. 18, 4663–4672 (1998).

    CAS  PubMed  Google Scholar 

  41. Mann, F., Peuckert, C., Dehner, F., Zhou, R. & Bolz, J. Ephrins regulate the formation of terminal axonal arbors during the development of thalamocortical projections. Development 129, 3945–3955 (2002).

    CAS  PubMed  Google Scholar 

  42. Klein, R. Bidirectional modulation of synaptic functions by Eph/ephrin signaling. Nature Neurosci. 12, 15–20 (2009).

    CAS  PubMed  Google Scholar 

  43. Marler, K. J. et al. A TrkB/EphrinA interaction controls retinal axon branching and synaptogenesis. J. Neurosci. 28, 12700–12712 (2008). In vitro ephrin A5 stripe assays with chick retinal ganglion cell axons reveal a novel interaction between ephrin A5 and the BDNF receptor TRKB in retinal ganglion cell axons. This interaction was shown to promote axon branching by augmenting the PI3K pathway, leading to a model in which BDNF acts as a global branch-promoting factor in the tectum and ephrin As–EPHAs locally suppress this activity, resulting in topographically specific retinotectal axon branching.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Mann, F., Ray, S., Harris, W. & Holt, C. Topographic mapping in dorsoventral axis of the Xenopus retinotectal system depends on signaling through ephrin-B ligands. Neuron 35, 461–473 (2002).

    CAS  PubMed  Google Scholar 

  45. Pasterkamp, R. J. Getting neural circuits into shape with semaphorins. Nature Rev. Neurosci. 13, 605–618 (2012).

    CAS  Google Scholar 

  46. Polleux, F., Giger, R. J., Ginty, D. D., Kolodkin, A. L. & Ghosh, A. Patterning of cortical efferent projections by semaphorin-neuropilin interactions. Science 282, 1904–1906 (1998).

    CAS  PubMed  Google Scholar 

  47. Bagnard, D., Lohrum, M., Uziel, D., Puschel, A. W. & Bolz, J. Semaphorins act as attractive and repulsive guidance signals during the development of cortical projections. Development 125, 5043–5053 (1998).

    CAS  PubMed  Google Scholar 

  48. Liu, Y. & Halloran, M. C. Central and peripheral axon branches from one neuron are guided differentially by Semaphorin3D and transient axonal glycoprotein-1. J. Neurosci. 25, 10556–10563 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Bagri, A., Cheng, H. J., Yaron, A., Pleasure, S. J. & Tessier-Lavigne, M. Stereotyped pruning of long hippocampal axon branches triggered by retraction inducers of the semaphorin family. Cell 113, 285–299 (2003).

    CAS  PubMed  Google Scholar 

  50. Cioni, J. M. et al. SEMA3A signaling controls layer-specific interneuron branching in the cerebellum. Curr. Biol. 23, 850–861 (2013). This study shows that SEMA3A, acting through its neuropilin 1 receptor, induces the branching of basket cell axons onto Purkinje cells in vivo and in vitro . Importantly, localized signalling mechanisms involving the SRC kinase FYN in basket cell axons promote layer-specific branching without affecting overall axon organization, demonstrating the importance of localized signalling in the specificity of axon branching.

    CAS  PubMed  Google Scholar 

  51. Lopez-Bendito, G. et al. Robo1 and Robo2 cooperate to control the guidance of major axonal tracts in the mammalian forebrain. J. Neurosci. 27, 3395–3407 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Wang, K. H. et al. Biochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching. Cell 96, 771–784 (1999).

    CAS  PubMed  Google Scholar 

  53. Yeo, S. Y. et al. Involvement of Islet-2 in the Slit signaling for axonal branching and defasciculation of the sensory neurons in embryonic zebrafish. Mech. Dev. 121, 315–324 (2004).

    CAS  PubMed  Google Scholar 

  54. Campbell, D. S. et al. Slit1a inhibits retinal ganglion cell arborization and synaptogenesis via Robo2-dependent and -independent pathways. Neuron 55, 231–245 (2007). An in vivo time-lapse imaging study in the zebrafish retinotectal system, showing that Slit1a inhibits arborization of retinal ganglion cell axons in the zebrafish optic tectum, thereby preventing the premature maturation of terminal arbors. These results are in contrast with the growth-promoting effects of SLITs on peripheral axon arbors, demonstrating that one guidance cue can have different effects on axon branching in different cell types.

    CAS  PubMed  Google Scholar 

  55. Gallo, G. & Letourneau, P. C. Localized sources of neurotrophins initiate axon collateral sprouting. J. Neurosci. 18, 5403–5414 (1998).

    CAS  PubMed  Google Scholar 

  56. Gallo, G. & Letourneau, P. C. Neurotrophins and the dynamic regulation of the neuronal cytoskeleton. J. Neurobiol. 44, 159–173 (2000).

    CAS  PubMed  Google Scholar 

  57. Gibney, J. & Zheng, J. Q. Cytoskeletal dynamics underlying collateral membrane protrusions induced by neurotrophins in cultured Xenopus embryonic neurons. J. Neurobiol. 54, 393–405 (2003).

    CAS  PubMed  Google Scholar 

  58. Jeanneteau, F., Deinhardt, K., Miyoshi, G., Bennett, A. M. & Chao, M. V. The MAP kinase phosphatase MKP-1 regulates BDNF-induced axon branching. Nature Neurosci. 13, 1373–1379 (2010). An important study in identifying the novel signalling mechanisms by which the extracellular cue BDNF induces cortical axon branching in vivo and in vitro . Transient induction of the MAPK phosphatase MKP1, which inactivates JNK, leads to activation of STMN1 and destabilization of microtubules, which facilitates BDNF-induced axon branching. This study therefore links extracellular cues to signalling pathways that influence cytoskeletal reorganization involved in axon branching.

    CAS  PubMed  Google Scholar 

  59. Szebenyi, G. et al. Fibroblast growth factor-2 promotes axon branching of cortical neurons by influencing morphology and behavior of the primary growth cone. J. Neurosci. 21, 3932–3941 (2001).

    CAS  PubMed  Google Scholar 

  60. Danzer, S. C., Crooks, K. R., Lo, D. C. & McNamara, J. O. Increased expression of brain-derived neurotrophic factor induces formation of basal dendrites and axonal branching in dentate granule cells in hippocampal explant cultures. J. Neurosci. 22, 9754–9763 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Marshak, S., Nikolakopoulou, A. M., Dirks, R., Martens, G. J. & Cohen-Cory, S. Cell-autonomous TrkB signaling in presynaptic retinal ganglion cells mediates axon arbor growth and synapse maturation during the establishment of retinotectal synaptic connectivity. J. Neurosci. 27, 2444–2456 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Cohen-Cory, S., Kidane, A. H., Shirkey, N. J. & Marshak, S. Brain-derived neurotrophic factor and the development of structural neuronal connectivity. Dev. Neurobiol. 70, 271–288 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Alsina, B., Vu, T. & Cohen-Cory, S. Visualizing synapse formation in arborizing optic axons in vivo: dynamics and modulation by BDNF. Nature Neurosci. 4, 1093–1101 (2001).

    CAS  PubMed  Google Scholar 

  64. Ciani, L. & Salinas, P. C. WNTs in the vertebrate nervous system: from patterning to neuronal connectivity. Nature Rev. Neurosci. 6, 351–362 (2005).

    CAS  Google Scholar 

  65. Salinas, P. C. Modulation of the microtubule cytoskeleton: a role for a divergent canonical Wnt pathway. Trends Cell Biol. 17, 333–342 (2007).

    CAS  PubMed  Google Scholar 

  66. Hall, A. C., Lucas, F. R. & Salinas, P. C. Axonal remodeling and synaptic differentiation in the cerebellum is regulated by WNT-7a signaling. Cell 100, 525–535 (2000).

    CAS  PubMed  Google Scholar 

  67. Krylova, O. et al. WNT-3, expressed by motoneurons, regulates terminal arborization of neurotrophin-3-responsive spinal sensory neurons. Neuron 35, 1043–1056 (2002).

    CAS  PubMed  Google Scholar 

  68. Purro, S. A. et al. Wnt regulates axon behavior through changes in microtubule growth directionality: a new role for adenomatous polyposis coli. J. Neurosci. 28, 8644–8654 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Keeble, T. R. et al. The Wnt receptor Ryk is required for Wnt5a-mediated axon guidance on the contralateral side of the corpus callosum. J. Neurosci. 26, 5840–5848 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Liu, Y. et al. Ryk-mediated Wnt repulsion regulates posterior-directed growth of corticospinal tract. Nature Neurosci. 8, 1151–1159 (2005).

    CAS  PubMed  Google Scholar 

  71. Zou, Y. & Lyuksyutova, A. I. Morphogens as conserved axon guidance cues. Curr. Opin. Neurobiol. 17, 22–28 (2007).

    CAS  PubMed  Google Scholar 

  72. Hutchins, B. I., Li, L. & Kalil, K. Wnt/calcium signaling mediates axon growth and guidance in the developing corpus callosum. Dev. Neurobiol. 71, 269–283 (2011).

    CAS  PubMed  Google Scholar 

  73. Li, L., Hutchins, B. I. & Kalil, K. Wnt5a induces simultaneous cortical axon outgrowth and repulsive axon guidance through distinct signaling mechanisms. J. Neurosci. 29, 5873–5883 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Bodmer, D., Levine-Wilkinson, S., Richmond, A., Hirsh, S. & Kuruvilla, R. Wnt5a mediates nerve growth factor-dependent axonal branching and growth in developing sympathetic neurons. J. Neurosci. 29, 7569–7581 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Dent, E. W. & Gertler, F. B. Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron 40, 209–227 (2003).

    CAS  PubMed  Google Scholar 

  76. Kalil, K., Szebenyi, G. & Dent, E. W. Common mechanisms underlying growth cone guidance and axon branching. J. Neurobiol. 44, 145–158 (2000).

    CAS  PubMed  Google Scholar 

  77. Dent, E. W., Gupton, S. L. & Gertler, F. B. The growth cone cytoskeleton in axon outgrowth and guidance. Cold Spring Harb. Perspect. Biol. 3, a001800 (2011).

    PubMed  PubMed Central  Google Scholar 

  78. Lowery, L. A. & Van Vactor, D. The trip of the tip: understanding the growth cone machinery. Nature Rev. Mol. Cell Biol. 10, 332–343 (2009).

    CAS  Google Scholar 

  79. Vitriol, E. A. & Zheng, J. Q. Growth cone travel in space and time: the cellular ensemble of cytoskeleton, adhesion, and membrane. Neuron 73, 1068–1081 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Dent, E. W. & Kalil, K. Axon branching requires interactions between dynamic microtubules and actin filaments. J. Neurosci. 21, 9757–9769 (2001).

    CAS  PubMed  Google Scholar 

  81. Ketschek, A. & Gallo, G. Nerve growth factor induces axonal filopodia through localized microdomains of phosphoinositide 3-kinase activity that drive the formation of cytoskeletal precursors to filopodia. J. Neurosci. 30, 12185–12197 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Spillane, M. et al. Nerve growth factor-induced formation of axonal filopodia and collateral branches involves the intra-axonal synthesis of regulators of the actin-nucleating Arp2/3 complex. J. Neurosci. 32, 17671–17689 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Spillane, M. et al. The actin nucleating Arp2/3 complex contributes to the formation of axonal filopodia and branches through the regulation of actin patch precursors to filopodia. Dev. Neurobiol. 71, 747–758 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Medeiros, N. A., Burnette, D. T. & Forscher, P. Myosin II functions in actin-bundle turnover in neuronal growth cones. Nature Cell Biol. 8, 215–226 (2006).

    CAS  PubMed  Google Scholar 

  85. Bear, J. E. & Gertler, F. B. Ena/VASP: towards resolving a pointed controversy at the barbed end. J. Cell Sci. 122, 1947–1953 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Dwivedy, A., Gertler, F. B., Miller, J., Holt, C. E. & Lebrand, C. Ena/VASP function in retinal axons is required for terminal arborization but not pathway navigation. Development 134, 2137–2146 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. Dent, E. W. et al. Filopodia are required for cortical neurite initiation. Nature Cell Biol. 9, 1347–1359 (2007).

    CAS  PubMed  Google Scholar 

  88. Ahuja, R. et al. Cordon-bleu is an actin nucleation factor and controls neuronal morphology. Cell 131, 337–350 (2007). This paper identifies a novel actin nucleator, cordon-bleu, which gives rise to long non-bundled unbranched filaments that elongate by barbed-end growth; by contrast, ARP2/3 gives rise to branched filaments. When overexpressed in hippocampal neurons in vitro , cordon-bleu increases axon branching without affecting axon length, but interestingly it also increases the number of dendrites and their branching.

    CAS  PubMed  PubMed Central  Google Scholar 

  89. Korobova, F. & Svitkina, T. Arp2/3 complex is important for filopodia formation, growth cone motility, and neuritogenesis in neuronal cells. Mol. Biol. Cell 19, 1561–1574 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. Strasser, G. A., Rahim, N. A., VanderWaal, K. E., Gertler, F. B. & Lanier, L. M. Arp2/3 is a negative regulator of growth cone translocation. Neuron 43, 81–94 (2004).

    CAS  PubMed  Google Scholar 

  91. Lu, M., Witke, W., Kwiatkowski, D. J. & Kosik, K. S. Delayed retraction of filopodia in gelsolin null mice. J. Cell Biol. 138, 1279–1287 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. Chen, T. J., Gehler, S., Shaw, A. E., Bamburg, J. R. & Letourneau, P. C. Cdc42 participates in the regulation of ADF/cofilin and retinal growth cone filopodia by brain derived neurotrophic factor. J. Neurobiol. 66, 103–114 (2006).

    CAS  PubMed  Google Scholar 

  93. Dent, E. W., Callaway, J. L., Szebenyi, G., Baas, P. W. & Kalil, K. Reorganization and movement of microtubules in axonal growth cones and developing interstitial branches. J. Neurosci. 19, 8894–8908 (1999).

    CAS  PubMed  Google Scholar 

  94. Conde, C. & Caceres, A. Microtubule assembly, organization and dynamics in axons and dendrites. Nature Rev. Neurosci. 10, 319–332 (2009).

    CAS  Google Scholar 

  95. Mitchison, T. & Kirschner, M. Cytoskeletal dynamics and nerve growth. Neuron 1, 761–772 (1988).

    CAS  PubMed  Google Scholar 

  96. Gallo, G. & Letourneau, P. C. Different contributions of microtubule dynamics and transport to the growth of axons and collateral sprouts. J. Neurosci. 19, 3860–3873 (1999).

    CAS  PubMed  Google Scholar 

  97. Yu, W., Ahmad, F. J. & Baas, P. W. Microtubule fragmentation and partitioning in the axon during collateral branch formation. J. Neurosci. 14, 5872–5884 (1994).

    CAS  PubMed  Google Scholar 

  98. Yu, W. et al. The microtubule-severing proteins spastin and katanin participate differently in the formation of axonal branches. Mol. Biol. Cell 19, 1485–1498 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  99. Qiang, L., Yu, W., Andreadis, A., Luo, M. & Baas, P. W. Tau protects microtubules in the axon from severing by katanin. J. Neurosci. 26, 3120–3129 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  100. Zhou, F. Q., Zhou, J., Dedhar, S., Wu, Y. H. & Snider, W. D. NGF-induced axon growth is mediated by localized inactivation of GSK-3β and functions of the microtubule plus end binding protein APC. Neuron 42, 897–912 (2004).

    CAS  PubMed  Google Scholar 

  101. Yokota, Y. et al. The adenomatous polyposis coli protein is an essential regulator of radial glial polarity and construction of the cerebral cortex. Neuron 61, 42–56 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  102. Chen, Y., Tian, X., Kim, W. Y. & Snider, W. D. Adenomatous polyposis coli regulates axon arborization and cytoskeleton organization via its N-terminus. PLoS ONE 6, e24335 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  103. Koizumi, H., Tanaka, T. & Gleeson, J. G. Doublecortin-like kinase functions with doublecortin to mediate fiber tract decussation and neuronal migration. Neuron 49, 55–66 (2006).

    CAS  PubMed  Google Scholar 

  104. Homma, N. et al. Kinesin superfamily protein 2A (KIF2A) functions in suppression of collateral branch extension. Cell 114, 229–239 (2003).

    CAS  PubMed  Google Scholar 

  105. Rodriguez, O. C. et al. Conserved microtubule-actin interactions in cell movement and morphogenesis. Nature Cell Biol. 5, 599–609 (2003).

    CAS  PubMed  Google Scholar 

  106. Schaefer, A. W., Kabir, N. & Forscher, P. Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones. J. Cell Biol. 158, 139–152 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  107. Zhou, F. Q., Waterman-Storer, C. M. & Cohan, C. S. Focal loss of actin bundles causes microtubule redistribution and growth cone turning. J. Cell Biol. 157, 839–849 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  108. Geraldo, S., Khanzada, U. K., Parsons, M., Chilton, J. K. & Gordon-Weeks, P. R. Targeting of the F-actin-binding protein drebrin by the microtubule plus-tip protein EB3 is required for neuritogenesis. Nature Cell Biol. 10, 1181–1189 (2008).

    CAS  PubMed  Google Scholar 

  109. Flynn, K. C. et al. ADF/cofilin-mediated actin retrograde flow directs neurite formation in the developing brain. Neuron 76, 1091–1107 (2012).

    CAS  PubMed  Google Scholar 

  110. Hu, J. et al. Septin-driven coordination of actin and microtubule remodeling regulates the collateral branching of axons. Curr. Biol. 22, 1109–1115 (2012). This study demonstrates that sensory axon branching involves septin proteins that interact with actin and microtubules. SEPT6 triggers the formation of filopodia by recruiting cortactin, whereas SEPT7 promotes the entry of microtubules into filopodia, suggesting that although each septin performs distinct functions, together they coordinate the cytoskeletal reorganization that is required for axon branching

    CAS  PubMed  PubMed Central  Google Scholar 

  111. Govek, E. E., Newey, S. E. & Van Aelst, L. The role of the Rho GTPases in neuronal development. Genes Dev. 19, 1–49 (2005).

    CAS  PubMed  Google Scholar 

  112. Huber, A. B., Kolodkin, A. L., Ginty, D. D. & Cloutier, J. F. Signaling at the growth cone: ligand-receptor complexes and the control of axon growth and guidance. Annu. Rev. Neurosci. 26, 509–563 (2003).

    CAS  PubMed  Google Scholar 

  113. Hall, A. & Lalli, G. Rho and Ras GTPases in axon growth, guidance, and branching. Cold Spring Harb. Perspect. Biol. 2, a001818 (2010).

    PubMed  PubMed Central  Google Scholar 

  114. Doble, B. W. & Woodgett, J. R. GSK-3: tricks of the trade for a multi-tasking kinase. J. Cell Sci. 116, 1175–1186 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  115. Etienne-Manneville, S. From signaling pathways to microtubule dynamics: the key players. Curr. Opin. Cell Biol. 22, 104–111 (2010).

    CAS  PubMed  Google Scholar 

  116. Goold, R. G. & Gordon-Weeks, P. R. Glycogen synthase kinase 3β and the regulation of axon growth. Biochem. Soc. Trans. 32, 809–811 (2004).

    CAS  PubMed  Google Scholar 

  117. Kim, W.-Y. et al. Essential roles of GSK-3s and GSK-3-primed substrates in neurotrophin-induced and hippocampal axon growth. Neuron 52, 981–996 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  118. Ren, G., Crampton, M. S. & Yap, A. S. Cortactin: coordinating adhesion and the actin cytoskeleton at cellular protrusions. Cell. Motil. Cytoskeleton 66, 865–873 (2009).

    CAS  PubMed  Google Scholar 

  119. Mingorance-Le Meur, A. & O'Connor, T. P. Neurite consolidation is an active process requiring constant repression of protrusive activity. EMBO J. 28, 248–260 (2009). This is one of the few studies to demonstrate that axon branching is actively repressed. Calpain, through proteolysis of cortactin, represses actin polymerization and maintains axon consolidation. In hippocampal neurons, local calpain inhibition by branching factors allows branching to occur.

    CAS  PubMed  Google Scholar 

  120. Lucas, F. R., Goold, R. G., Gordon-Weeks, P. R. & Salinas, P. C. Inhibition of GSK-3β leading to the loss of phosphorylated MAP-1B is an early event in axonal remodelling induced by WNT-7a or lithium. J. Cell Sci. 111, 1351–1361 (1998).

    CAS  PubMed  Google Scholar 

  121. Goold, R. G., Owen, R. & Gordon-Weeks, P. R. Glycogen synthase kinase 3β phosphorylation of microtubule-associated protein 1B regulates the stability of microtubules in growth cones. J. Cell Sci. 112, 3373–3384 (1999).

    CAS  PubMed  Google Scholar 

  122. Bilimoria, P. M. et al. A JIP3-regulated GSK3β/DCX signaling pathway restricts axon branching. J. Neurosci. 30, 16766–16776 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  123. Drinjakovic, J. et al. E3 ligase Nedd4 promotes axon branching by downregulating PTEN. Neuron 65, 341–357 (2010). This in vivo study identifies a novel signalling pathway in frog retinal ganglion neurons in which the ubiquitin ligase E3 NEDD4 ubiquitylates the phosphatase PTEN, thereby targeting it for degradation and promoting the PI3K pathway, which leads to retinal axon branching. Importantly, this signalling pathway regulates terminal branching of retinal axons in the optic tectum without affecting long-range axon pathfinding, providing a mechanism for independent regulation of axon guidance and branching.

    CAS  PubMed  PubMed Central  Google Scholar 

  124. Spitzer, N. C. Electrical activity in early neuronal development. Nature 444, 707–712 (2006).

    CAS  PubMed  Google Scholar 

  125. Gomez, T. M. & Zheng, J. Q. The molecular basis for calcium-dependent axon pathfinding. Nature Rev. Neurosci. 7, 115–125 (2006).

    CAS  Google Scholar 

  126. Ohnami, S. et al. Role of RhoA in activity-dependent cortical axon branching. J. Neurosci. 37, 9117–9121 (2008).

    Google Scholar 

  127. Singh, K. K. & Miller, F. D. Activity regulates positive and negative neurotrophin-derived signals to determine axon competition. Neuron 45, 837–845 (2005).

    CAS  PubMed  Google Scholar 

  128. Hutchins, B. I. & Kalil, K. Differential outgrowth of axons and their branches is regulated by localized calcium transients. J. Neurosci. 28, 143–153 (2008). By induction of localized calcium transients with photolysis of caged calcium in axons and branches of the same cortical neuron in vitro , this study shows that a process with higher-frequency calcium transients grows more rapidly than a process with lower-frequency calcium transients, which stalls or retracts. This paper suggests a novel activity-dependent mechanism for regulating competitive growth of an axon and its branches that might occur in vivo.

    CAS  PubMed  PubMed Central  Google Scholar 

  129. Katz, L. C. & Shatz, C. J. Synaptic activity and the construction of cortical circuits. Science 274, 1133–1138 (1996).

    CAS  PubMed  Google Scholar 

  130. Uesaka, N., Ruthazer, E. S. & Yamamoto, N. The role of neural activity in cortical axon branching. Neuroscientist 12, 102–106 (2006).

    PubMed  Google Scholar 

  131. Ruthazer, E. S., Akerman, C. J. & Cline, H. T. Control of axon branch dynamics by correlated activity in vivo. Science 301, 66–70 (2003).

    CAS  PubMed  Google Scholar 

  132. Mizuno, H., Hirano, T. & Tagawa, Y. Evidence for activity-dependent cortical wiring: formation of interhemispheric connections in neonatal mouse visual cortex requires projection neuron activity. J. Neurosci. 27, 6760–6770 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  133. Yamada, A. et al. Role of pre- and postsynaptic activity in thalamocortical axon branching. Proc. Natl Acad. Sci. USA 107, 7562–7567 (2010).

    CAS  PubMed  Google Scholar 

  134. Uesaka, N., Hayano, Y., Yamada, A. & Yamamoto, N. Interplay between laminar specificity and activity-dependent mechanisms of thalamocortical axon branching. J. Neurosci. 27, 5215–5223 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  135. Hua, J. Y., Smear, M. C., Baier, H. & Smith, S. J. Regulation of axon growth in vivo by activity-based competition. Nature 434, 1022–1026 (2005).

    CAS  PubMed  Google Scholar 

  136. Ben Fredj, N. et al. Synaptic activity and activity-dependent competition regulates axon arbor maturation, growth arrest, and territory in the retinotectal projection. J. Neurosci. 30, 10939–10951 (2010).

    CAS  PubMed  Google Scholar 

  137. Gosse, N. J., Nevin, L. M. & Baier, H. Retinotopic order in the absence of axon competition. Nature 452, 892–895 (2008). Using a clever strategy to create zebrafish chimeras that have eyes with only a single retinal ganglion cell, this study showed that the axon arbors of these neurons terminate at correct tectal positions, revealing that competitionis not involved in innervation of the posterior tectum. However, the single arbors were larger and more complex than normal, showing that axon–axon interaction restricts arbor size and shape.

    CAS  PubMed  PubMed Central  Google Scholar 

  138. Courchet, J. et al. Terminal axon branching is regulated by the LKB1–NUAK1 kinase pathway via presynaptic mitochondrial capture. Cell 153, 1510–1525 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  139. Kner, P., Chhun, B. B., Griffis, E. R., Winoto, L. & Gustafsson, M. G. Super-resolution video microscopy of live cells by structured illumination. Nature Methods 6, 339–342 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  140. Chmyrov, A. et al. Nanoscopy with more than 100,000 'doughnuts'. Nature Methods 10, 737–740 (2013).

    CAS  PubMed  Google Scholar 

  141. Sengupta, P., Van Engelenburg, S. & Lippincott-Schwartz, J. Visualizing cell structure and function with point-localization superresolution imaging. Dev. Cell 23, 1092–1102 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  142. Fenno, L., Yizhar, O. & Deisseroth, K. The development and application of optogenetics. Annu. Rev. Neurosci. 34, 389–412 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  143. Hama, H. et al. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nature Neurosci. 14, 1481–1488 (2011).

    CAS  PubMed  Google Scholar 

  144. Chung, K. et al. Structural and molecular interrogation of intact biological systems. Nature 497, 332–337 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  145. Ke, M. T., Fujimoto, S. & Imai, T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nature Neurosci. 16, 1154–1161 (2013).

    CAS  PubMed  Google Scholar 

  146. Kuwajima, T. et al. ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue. Development 140, 1364–1368 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We apologize that, owing to space constraints, we could not cite many excellent studies. We thank members of our laboratories for reviewing the manuscript and the helpful comments of the anonymous reviewers. Work from our laboratories is supported by US National Institutes of Health grants NS014428, NS064014 and NS080928.

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Correspondence to Katherine Kalil.

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PowerPoint slides

Glossary

Terminal arbors

Highly branched tree-like structures that are found at the ends of axons that innervate target regions.

Collateral branches

Branches that extend from the sides of an axon, often interstitially, and innervate a target by re-branching to form a terminal arbor.

Cytoskeletal dynamics

Cycles of polymerization and depolymerization that result in growth and shrinkage of microtubules and actin filaments, which enable their reorganization.

Filopodia

Finger-like membrane protrusions that contain bundled actin filaments. Filopodia extend transiently from the growth cone, the axon shaft and axon branches.

Neurotrophic factor

A type of molecule, such as brain-derived neurotrophic factor or nerve growth factor, that regulates neuronal growth and survival.

Lamellipodia

Thin sheet-like veils of cytoplasm at the growth cone periphery that are comprised of actin filament networks.

Plus-end-tracking proteins

Plus-end-tracking proteins, such as end-binding protein 1 (EB1) and EB3, associate with the growing plus ends of dynamic microtubules.

RHO GTPases

A family of molecules that regulate cytoskeletal dynamics downstream of guidance cue receptors.

Calcium transients

Transient increases in the level of intracellular calcium that can occur repetitively at different frequencies.

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Kalil, K., Dent, E. Branch management: mechanisms of axon branching in the developing vertebrate CNS. Nat Rev Neurosci 15, 7–18 (2014). https://doi.org/10.1038/nrn3650

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