Spontaneous activity regulates Robo1 transcription to mediate a switch in thalamocortical axon growth

Journal name:
Nature Neuroscience
Volume:
15,
Pages:
1134–1143
Year published:
DOI:
doi:10.1038/nn.3160
Received
Accepted
Published online

Abstract

Developing axons must control their growth rate to follow the appropriate pathways and establish specific connections. However, the regulatory mechanisms involved remain elusive. By combining live imaging with transplantation studies in mice, we found that spontaneous calcium activity in the thalamocortical system and the growth rate of thalamocortical axons were developmentally and intrinsically regulated. Indeed, the spontaneous activity of thalamic neurons governed axon growth and extension through the cortex in vivo. This activity-dependent modulation of growth was mediated by transcriptional regulation of Robo1 through an NF-κB binding site. Disruption of either the Robo1 or Slit1 genes accelerated the progression of thalamocortical axons in vivo, and interfering with Robo1 signaling restored normal axon growth in electrically silent neurons. Thus, modifications to spontaneous calcium activity encode a switch in the axon outgrowth program that allows the establishment of specific neuronal connections through the transcriptional regulation of Slit1 and Robo1 signaling.

At a glance

Figures

  1. The speed of growth of TCAs is developmentally regulated.
    Figure 1: The speed of growth of TCAs is developmentally regulated.

    (a) Schematic representation of the experimental procedure used for time-lapse imaging of in-growing TCAs ex vivo. (b) Bright-field image showing an oblique thalamocortical slice use for the recordings. Ncx, neocortex; Th, thalamus. (c) Gfp-electroporated axons traversing several forebrain structures. (dg) Examples of TCAs recorded at the level of the vTel (d), angle (e), entering the cortex (f) and extending through the neocortex (g) from E13.5 (df) and E15.5 (g) slices. (hk) Track of axons in dg measured after 4 h imaging. (l) Quantification of the data shown in hk. **P < 0.01, ***P < 0.001, one-way ANOVA test with Tukey's post hoc analysis. (m) Composite images of axons growing from E11.5 (blue), E13.5 (green) and E15.5 (red) thalamic explants from a 1-h time lapse showing axonal extension at 0, 30 and 60 min. For comparison, axonal growth cones were oriented in parallel and aligned at 0 min. Both E11.5 and E13.5 axons extended faster than E15.5. (n) The mean axon growth rate was calculated as the total extension over 1 h of the growth cone between the first and last frame. #P < 0.01, Kruskal-Wallis test with Dunn's post hoc analysis. (o) Proportion of the time spent by axons advancing, pausing or retracting at each stage studied. (p) The instantaneous velocity was defined as the average advancing velocity. *P < 0.05, Kruskal-Wallis test with Dunn's post hoc analysis. The data are presented as mean ± s.e.m. Scale bars represent 500 μm (b,c), 200 μm (dg) and 20 μm (m).

  2. Spontaneous thalamic activity is developmentally regulated.
    Figure 2: Spontaneous thalamic activity is developmentally regulated.

    (a) Schematic representation of the experimental procedure used for Ca2+ imaging. (b) Bright-field image showing the level of the thalamus from which recordings were obtained. (c) Pseudocolored image showing the Ca2+ levels in numerous Oregon Green BAPTA-AM (OG)-loaded cells. Red to blue indicates high to low Ca2+ levels. (d) Ca2+ activity in thalamic neurons at different ages. Typically, Ca2+ transients (asterisks) displayed a rise time of 3 s and they lasted for <12 s, with an amplitude of twice the s.d. of the noise (dotted lines). E12.5 and E14.5 neurons exhibited twice as many spikes as E16.5 and E17.5 neurons. (e) Mean incidence (left) and frequency (right) of Ca2+ spiking at early (E12.5 and E14.5) and late (E16.5 and E17.5) developmental stages. *P = 0.04, **P = 0.003, two-tailed Student's t test. (f) Mean spike amplitude (left) and mean spike area (right) of Ca2+ activity at early (E12.5 and E14.5) and late (E16.5 and E17.5) developmental stages. Example of mean Ca2+ spike traces at early and late developmental stages. #P = 0.002, two-tailed Student's t test. (g) Resting membrane potential recorded by whole-cell patch-clamp in thalamic neurons. GluK, potassium gluconate. ***P < 0.001, two-tailed Student's t test. Data are presented as mean ± s.e.m. Scale bars represent 300 μm (b) and 50 μm (c).

  3. Manipulation of spontaneous thalamic activity alters the axon growth rate.
    Figure 3: Manipulation of spontaneous thalamic activity alters the axon growth rate.

    (a) Experimental procedure used for Ca2+ imaging in the presence of 10 μM nifedipine or 10 mM KCl. Blocking L-type VOCCs with nifedipine decreased spontaneous Ca2+ activity to levels similar to those seen at later stages. KCl increased the frequency of Ca2+ activity in E14.5 neurons (49.5 ± 5.3 h−1, n = 53 cells). *P < 0.05, ***P < 0.001, Kruskal-Wallis test with Dunn's post hoc analysis. (b,c) Tuj1 revealed that blocking L-type VOCC with 10 μM nifedipine decreased TCA length. (d) Quantification of the data shown in b and c (control, 173 ± 15 μm; nifedipine, 101 ± 7 μm). #P < 0.001, Mann-Whitney U-test. (e,f) Tuj1 revealed increased TCA growth following chronic depolarization. (g) Quantification of the data shown in e and f. When [KCl]o was elevated to 7.5–10 mM, axon growth increased significantly when compared with control conditions (control, 116 ± 10 μm; 7.5–10 mM, 154 ± 6 μm; 12.5–15 mM, 141 ± 8 μm). (h) Experimental procedure used to assess the effect of Kir2.1 on the resting membrane potential at two developmental stages (E13.5 and E17.5). ##P < 0.001, two-tailed Student's t test. (i,j) GFP immunostaining revealed a decrease in axon length after forced hyperpolarization. (k) Quantification of the data shown in i and j and data not shown. **P < 0.01, Kruskal-Wallis test with Dunn's post hoc analysis. Data are presented as mean ± s.e.m. Scale bar represents 300 μm.

  4. Silencing spontaneous thalamic activity attenuates TCA elongation in vivo.
    Figure 4: Silencing spontaneous thalamic activity attenuates TCA elongation in vivo.

    (a) Schematic representation of the experimental procedure used to test the effect of silencing thalamic activity in vivo. The mutant Kir2.1 or Kir2.1 plasmids were co-electroporated with Gfp into the thalamus in utero at E12.5 and the brains were analyzed at E15.5, at the peak of TCA cortical extension. (b,c) GFP expression in the thalamus reflecting the extent of electroporation in both conditions. (d) Schematic representation of the method to quantify and calculate the cortical extension along the rostro-caudal axis of the cerebral wall. The maximum distance reached by GFP-positive axons (x, green line) was measured at two rostro-caudal levels from a horizontal line passing tangential to the ventricular edge of the pallial-subpallial boundary (red line) and normalized to the total length (y, blue line) of the cortical area defined by the lateral and medial intersections of this line in the cortical wall. (eh) Coronal sections from rostral (e,g) and intermediate (f,h) levels of electroporated brains revealed the delay in cortical invasion by GFP-positive axons (arrowheads) in Kir2.1 and mutant Kir2.1 electroporated neurons. (i) Quantification of the data shown in eh. ***P < 0.001, two-tailed Student's t test. The data are presented as mean ± s.e.m. Scale bar represents 300 μm and applies to all panels.

  5. Silencing thalamic activity upregulates Robo1 transcription.
    Figure 5: Silencing thalamic activity upregulates Robo1 transcription.

    (a) Semi-quantitative PCR comparing transcript levels in active and silenced thalamic cells using primers for Gapdh (control), Kir2.1, Robo2 and two sets of primers for Robo1 (generating 155-bp and 232-bp products). Total thalamic cDNA was used as a positive control (c+). M, molecular marker. c–, negative control. (b) Real-time PCR quantification of the transcripts for Kir2.1, Robo1 and Robo2 in Gfp and Kir2.1-electroporated cells. *P = 0.03, **P = 0.003, two-tailed Student's t test. (c) Real-time PCR quantification of the transcripts for Robo1 and Robo2 after decreasing spontaneous Ca2+ activity by nifedipine treatment. #P = 0.006, two-tailed Student's t test. (dg) Immunohistochemistry for Robo1 in control (d,e) and Kir2.1-expressing axons (f,g). (h) Semiquantitative PCR for Kir2.1, Robo1 and Gapdh. Expression of Kir2.1 and Robo1 were normalized to that of Gapdh and to the E12.5 value. Full-length gels are presented in Supplementary Figure 9. ##P = 0.02, ***P < 0.001, two-tailed Student's t test. (ik) Immunohistochemistry for Robo1 in E13.5 (GFP-positive) and E15.5 isolated thalamic growth cones. Thalamic explants from E13.5 GFP-expressing mice were co-cultured together with explants from wild-type E15.5 embryos on poly-L-lysine/laminin coverslips. (l) Quantification of the data shown in j. ***P < 0.001, two-tailed Student's t test. The data are presented as mean ± s.e.m. Scale bars represent 15 μm (dg), 50 μm (i) and 15 μm (j,k).

  6. Robo1 expression is regulated at the transcriptional level through a NF-[kappa]B binding site.
    Figure 6: Robo1 expression is regulated at the transcriptional level through a NF-κB binding site.

    (ac) CHO-A1 cells transfected with the Kir2.1 construct and loaded with Fura2 calcium indicator. Following tetracycline administration, TRPA1 channels were upregulated and a strong spontaneous Ca2+ activity was generated (black traces in b and c), which could be blocked by Kir2.1 electroporation (green traces). WT, wild type. (d) Silencing spontaneous activity induced a 1.5-fold increase in expression of the luciferase reporter. ***P < 0.001, two-tailed Student's t test. (e) Schematic representations of the wild-type and mutated forms of the 2,446-bp cloned region of the Robo1 promoter. (f) There was strong reduction in the basal transcription of Robo1 when the NF-κB site 1 was mutated, but no significant reduction when the AP-1 or the NF-κB sites 2 were mutated (n ≥ 3 replicates). **P < 0.001, Kruskal-Wallis test with Dunn's post hoc analysis. (g) Luciferase assays to evaluate the contribution of AP-1 and NF-κB sites to the Robo1 induction by Kir2.1. Only mutating the NF-κB site 1 significantly reduced the induction of Robo1 transcription after silencing spontaneous activity (n ≥ 3 replicates). *P < 0.05, Mann-Whitney U-test. Data are presented as mean ± s.e.m. Scale bar represents 50 μm.

  7. Loss of Robo1 increases the intrinsic capacity for thalamic outgrowth.
    Figure 7: Loss of Robo1 increases the intrinsic capacity for thalamic outgrowth.

    (a,b) Tuj1 immunostaining revealed that thalamic axon growth increased in the absence of Robo1. (c) Quantification of the data shown in a and b. ***P < 0.001, Student's t test. (d,e) Tuj1 immunostaining revealed that the absence of the Robo2 receptor did not affect thalamic axon growth in vitro. (f) Quantification of the data shown in d and e, and data not shown. #P < 0.001, two-tailed Student's t test. (g,i) Wild-type thalamic axons from a GFP-expressing mouse grew significantly more in Slit1−/− slices than in control slices. Islet1 labeled the corridor at the subpallial region. (h) Experimental procedure used to analyze the involvement of Slit1 in thalamic growth. (j) Quantification of the data shown in g and i. Normalized fluorescence was measured from the border of the explant. *P = 0.03 and **P = 0.007, two-tailed Student's t test. (k,l) Progression of TCAs labeled by DiI in the E14.5 thalamus of Slit1+/+ and Slit1−/− brains. (m,n) Progression of TCAs labeled by DiI in the E14.5 thalamus in Robo1+/+ and Robo1−/− brains. (oq) GFP-immunoreactive axons in coronal sections at intermediate cortical levels. Overexpression of Robo1 (p) led to a significant delay in the cortical extension of TCAs when compared with controls. Electroporation of a truncated Robo1 lacking the CC2 and CC3 domains (q) failed to significantly alter TCA cortical extension when compared with controls. (r) Quantification of the data shown in oq. ##P < 0.05, one-way ANOVA test with Tukey's post hoc analysis. Data are presented as mean ± s.e.m. Scale bars represent 300 μm.

  8. Robo1 controls axon extension in vivo downstream of spontaneous activity.
    Figure 8: Robo1 controls axon extension in vivo downstream of spontaneous activity.

    (a,b) GFP (green) and Hoechst (blue) staining in control (Gfp, a) and Kir2.1-tranfected (b) dissociated thalamic cells after treatment with 5 nM Slit1. Addition of recombinant Slit1 significantly increased thalamic growth cone collapse in Kir2.1-transfected cells compared with controls. (c) Quantification of the data shown in a and b. ***P < 0.001, one-way ANOVA test with Tukey's post hoc analysis. (d,e) Tuj1 staining in E13.5 Robo1−/− thalamic explants after treatment with 10 μM nifedipine. Addition of nifedipine did not significantly change the increased thalamic growth observed in the Robo1 mutants. (f) Quantification of the data shown in d and e (Robo1+/+ control, 288 ± 9.8 μm; Robo1+/+ + nifedipine, 201 ± 8.8 μm; Robo1−/− control, 437 ± 24 μm; Robo1−/− + nifedipine, 372 ± 21 μm). *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA test with Tukey's post hoc analysis. (gi) Coronal sections showing GFP-labeled axons at intermediate cortical levels following electroporation with mutant Kir2.1, Kir2.1 and Kir2.1 + Robo1ΔC. Co-electroporation of Robo1ΔC with Kir2.1 rescued the defective cortical progression of TCAs following electroporation with Kir2.1 alone. (j) Quantification of the data shown in gi. *P < 0.05, ***P < 0.001, one-way ANOVA test with Tukey's post hoc analysis. Data are presented as mean ± s.e.m. Scale bars represent 50 μm (a,b) and 300 μm (d,e and gi).

References

  1. López-Bendito, G. et al. Tangential neuronal migration controls axon guidance: a role for neuregulin-1 in thalamocortical axon navigation. Cell 125, 127142 (2006).
  2. Métin, C., Deleglise, D., Serafini, T., Kennedy, T.E. & Tessier-Lavigne, M. A role for netrin-1 in the guidance of cortical efferents. Development 124, 50635074 (1997).
  3. Garel, S. & Rubenstein, J.L.R. Intermediate targets in formation of topographic projections: inputs from the thalamocortical system. Trends Neurosci. 27, 533539 (2004).
  4. Braisted, J.E. et al. Netrin-1 promotes thalamic axon growth and is required for proper development of the thalamocortical projection. J. Neurosci. 20, 57925801 (2000).
  5. Tucker, K.L., Meyer, M. & Barde, Y. Neurotrophins are required for nerve growth during development. Nat. Neurosci. 4, 2937 (2001).
  6. Blackmore, M. & Letourneau, P.C. Changes within maturing neurons limit axonal regeneration in the developing spinal cord. J. Neurobiol. 66, 348360 (2006).
  7. Filbin, M.T. Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS. Nat. Rev. Neurosci. 4, 703713 (2003).
  8. Spitzer, N.C. Electrical activity in early neuronal development. Nature 444, 707712 (2006).
  9. Ming, G., Henley, J., Tessier-Lavigne, M., Song, H. & Poo, M. Electrical activity modulates growth cone guidance by diffusible factors. Neuron 29, 441452 (2001).
  10. Goldberg, J.L. et al. Retinal ganglion cells do not extend axons by default: promotion by neurotrophic signaling and electrical activity. Neuron 33, 689702 (2002).
  11. Fields, R.D., Neale, E. & Nelson, P. Effects of patterned electrical activity on neurite outgrowth from mouse sensory neurons. J. Neurosci. 10, 29502964 (1990).
  12. Gomez, T.M. & Zheng, J.Q. The molecular basis for calcium-dependent axon pathfinding. Nat. Rev. Neurosci. 7, 115125 (2006).
  13. Tang, F., Dent, E.W. & Kalil, K. Spontaneous calcium transients in developing cortical neurons regulate axon outgrowth. J. Neurosci. 23, 927936 (2003).
  14. Cohan, C.S. & Kater, S. Suppression of neurite elongation and growth cone motility by electrical activity. Science 232, 16381640 (1986).
  15. Borodinsky, L.N. et al. Activity-dependent homeostatic specification of transmitter expression in embryonic neurons. Nature 429, 523530 (2004).
  16. Hanson, M.G. & Landmesser, L.T. Normal patterns of spontaneous activity are required for correct motor axon guidance and the expression of specific guidance molecules. Neuron 43, 687701 (2004).
  17. Marek, K.W., Kurtz, L. & Spitzer, N. cJun integrates calcium activity and tlx3 expression to regulate neurotransmitter specification. Nat. Neurosci. 13, 944950 (2010).
  18. López-Bendito, G. & Molnár, Z. Thalamocortical development: how are we going to get there? Nat. Rev. Neurosci. 4, 276289 (2003).
  19. Skaliora, I., Adams, R. & Blakemore, C. Morphology and growth patterns of developing thalamocortical axons. J. Neurosci. 20, 36503662 (2000).
  20. Gomez, T.M. & Spitzer, N.C. In vivo regulation of axon extension and pathfinding by growth-cone calcium transients. Nature 397, 350 (1999).
  21. Burrone, J., O'Byrne, M. & Murthy, V.N. Multiple forms of synaptic plasticity triggered by selective suppression of activity in individual neurons. Nature 420, 414418 (2002).
  22. Demarque, M. & Spitzer, N.C. Activity-dependent expression of Lmx1b regulates specification of serotonergic neurons modulating swimming behavior. Neuron 67, 321334 (2010).
  23. 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, 67606770 (2007).
  24. Itoh, K., Ozaki, M., Stevens, B. & Fields, R. Activity-dependent regulation of N-cadherin in DRG neurons: differential regulation of N-cadherin, NCAM, and L1 by distinct patterns of action potentials. J. Neurobiol. 33, 735748 (1997).
  25. Dolmetsch, R.E., Lewis, R.S., Goodnow, C.C. & Healy, J.I. Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature 386, 855858 (1997).
  26. Fajardo, O., Meseguer, V., Belmonte, C. & Viana, F. TRPA1 channels: novel targets of 1,4-dihydropyridines. Channels (Austin) 2, 429438 (2008).
  27. Sugiyama, C. et al. Activator protein-1 responsive to the group II metabotropic glutamate receptor subtype in association with intracellular calcium in cultured rat cortical neurons. Neurochem. Int. 51, 467475 (2007).
  28. Xiang, G. et al. Identification of activity-dependent gene expression profiles reveals specific subsets of genes induced by different routes of Ca2+ entry in cultured rat cortical neurons. J. Cell. Physiol. 212, 126136 (2007).
  29. Bagri, A. et al. Slit proteins prevent midline crossing and determine the dorsoventral position of major axonal pathways in the mammalian forebrain. Neuron 33, 233248 (2002).
  30. Stein, E. & Tessier-Lavigne, M. Hierarchical organization of guidance receptors: silencing of netrin attraction by slit through a Robo/DCC receptor complex. Science 291, 19281938 (2001).
  31. Sann, S.B., Xu, L., Nishimune, H., Sanes, J.R. & Spitzer, N.C. Neurite outgrowth and in vivo sensory innervation mediated by a Ca(V)2.2-laminin beta 2 stop signal. J. Neurosci. 28, 23662374 (2008).
  32. Porter, B.E., Weis, J. & Sanes, J.R. A motoneuron-selective stop signal in the synaptic protein S-laminin. Neuron 14, 549559 (1995).
  33. Butler, A.K., Dantzker, J., Shah, R. & Callaway, E. Development of visual cortical axons: layer-specific effects of extrinsic influences and activity blockade. J. Comp. Neurol. 430, 321331 (2001).
  34. Uesaka, N., Hayano, Y., Yamada, A. & Yamamoto, N. Interplay between laminar specificity and activity-dependent mechanisms of thalamocortical axon branching. J. Neurosci. 27, 52155223 (2007).
  35. Nishimaru, H., Iizuka, M., Ozaki, S. & Kudo, N. Spontaneous motoneuronal activity mediated by glycine and GABA in the spinal cord of rat fetuses in vitro. J. Physiol. (Lond.) 497, 131143 (1996).
  36. Scain, A.L. et al. Glycine release from radial cells modulates the spontaneous activity and its propagation during early spinal cord development. J. Neurosci. 30, 390403 (2010).
  37. Picken Bahrey, H.L. & Moody, W.J. Early development of voltage-gated ion currents and firing properties in neurons of the mouse cerebral cortex. J. Neurophysiol. 89, 17611773 (2003).
  38. Xiao, Q., Xu, L. & Spitzer, N.C. Target-dependent regulation of neurotransmitter specification and embryonic neuronal calcium spike activity. J. Neurosci. 30, 57925801 (2010).
  39. Ibarretxe, G., Perrais, D., Jaskolski, F., Vimeney, A. & Mulle, C. Fast regulation of axonal growth cone motility by electrical activity. J. Neurosci. 27, 76847695 (2007).
  40. Gutierrez, H. & Davies, A.M. Regulation of neural process growth, elaboration and structural plasticity by NF-kappaB. Trends Neurosci. 34, 316325 (2011).
  41. Gutierrez, H., O'Keeffe, G.W., Gavalda, N., Gallagher, D. & Davies, A.M. Nuclear factor kappa B signaling either stimulates or inhibits neurite growth depending on the phosphorylation status of p65/RelA. J. Neurosci. 28, 82468256 (2008).
  42. Riquelme, D. et al. High-frequency field stimulation of primary neurons enhances ryanodine receptor–mediated Ca2+ release and generates hydrogen peroxide, which jointly stimulate NF-kappaB activity. Antioxid. Redox Signal. 14, 12451259 (2011).
  43. Tcherkezian, J., Brittis, P.A., Thomas, F., Roux, P.P. & Flanagan, J.G. Transmembrane receptor DCC associates with protein synthesis machinery and regulates translation. Cell 141, 632644 (2010).
  44. Jung, H., Yoon, B.C. & Holt, C.E. Axonal mRNA localization and local protein synthesis in nervous system assembly, maintenance and repair. Nat. Rev. Neurosci. 13, 308324 (2012).
  45. Andrews, W. et al. Robo1 regulates the development of major axon tracts and interneuron migration in the forebrain. Development 133, 22432252 (2006).
  46. López-Bendito, G. et al. Robo1 and Robo2 cooperate to control the guidance of major axonal tracts in the mammalian forebrain. J. Neurosci. 27, 33953407 (2007).
  47. Zallen, J.A., Yi, B.A. & Bargmann, C.I. The conserved immunoglobulin superfamily member SAX-3/Robo directs multiple aspects of axon guidance in C. elegans. Cell 92, 217227 (1998).
  48. Hadjantonakis, A.K., Gertsenstein, M., Ikawa, M., Okabe, M. & Nagy, A. Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech. Dev. 76, 7990 (1998).
  49. Andrews, W. et al. The role of Slit-Robo signaling in the generation, migration and morphological differentiation of cortical interneurons. Dev. Biol. 313, 648658 (2008).
  50. Long, H. et al. Conserved roles for Slit and Robo proteins in midline commissural axon guidance. Neuron 42, 213223 (2004).
  51. Agmon, A. & Connors, B.W. Thalamocortical responses of mouse somatosensory (barrel) cortex in vitro. Neuroscience 41, 365379 (1991).

Download references

Author information

  1. These authors contributed equally to this work.

    • Erik Mire &
    • Cecilia Mezzera

Affiliations

  1. Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-Consejo Superior de Investigaciones Científicas (UMH-CSIC), San't Joan d'Alacant, Spain.

    • Erik Mire,
    • Cecilia Mezzera,
    • Eduardo Leyva-Díaz,
    • Ana V Paternain,
    • Lisa Bluy,
    • Mar Castillo-Paterna,
    • María José López,
    • Sandra Peregrín,
    • Joan Galcerán,
    • Juan Lerma &
    • Guillermina López-Bendito
  2. Ecole Normale Supérieure, Institut de Biologie de l'ENS, Paris, France.

    • Paola Squarzoni &
    • Sonia Garel
  3. INSERM, U1024, Avenir Team, Paris, France.

    • Paola Squarzoni &
    • Sonia Garel
  4. CNRS, UMR 8197, Paris, France.

    • Paola Squarzoni &
    • Sonia Garel
  5. Genentech, South San Francisco, California, USA.

    • Marc Tessier-Lavigne
  6. The Rockefeller University, New York, New York, USA.

    • Marc Tessier-Lavigne

Contributions

G.L.-B. conceived the idea. E.M. and G.L.-B. designed the study. E.M., C.M., E.L.-D. and G.L.-B. performed the in vitro, ex vivo and in utero electroporation experiments. E.L.-D. and L.B. performed the luciferase assays. A.V.P. performed the electrophysiological recordings. P.S. and S.G. performed the experiments on Slit1 mutant mice. M.C.-P. performed the semiquantitative PCR and the collapse assay. M.J.L. performed the calcium recordings in CHO-A1 cells. S.P. subcloned the Kir2.1 plasmid. M.T.-L. produced the Robo1, Robo2 and Robo1; Robo2 mutant mice. J.G. supervised the luciferase assays. J.L. supervised the electrophysiological experiments. E.M., C.M. and G.L.-B. conducted the data analysis and wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (17M)

    Supplementary Figures 1–9

Movies

  1. Supplementary Video 1 (4M)

    Speed of growth of TCA travelling at the vTel.

  2. Supplementary Video 2 (7M)

    Speed of growth of TCA extending at the neocortex.

  3. Supplementary Video 3 (4M)

    Speed of growth of TCA travelling at the angle at the PSPB.

  4. Supplementary Video 4 (1M)

    Speed of growth of TCA travelling at the entrance of the neocortex.

  5. Supplementary Video 5 (6M)

    Spontaneous activity in the thalamus of E12.5 embryo.

  6. Supplementary Video 6 (7M)

    Spontaneous activity in the thalamus of E16.5 embryo.

  7. Supplementary Video 7 (4M)

    Spontaneous activity in early thalamocortical growth cones.

  8. Supplementary Video 8 (4M)

    Spontaneous activity in late thalamocortical growth cones.

Additional data