(. Robo3 and G. , TAG-1 (D-F) immunostaining and 3DISCO clearing in E16 Slit and Robo mutant embryos, Robo1 À/À

D. , In a Robo3 À/À mutant, the FR reaches the IPN, where they cross the midline and extend caudally within the midline. (G-I)

J. Shh,

À. Slit1, In both cases, FR axons reach the IPN and then form a dense meshwork at the floor plate level from which two axon bundles emerge and extend along the floor plate rostrally (1) and caudally

M. Shh,

À. Slit1, FR axons from two large commissures: a rostral one (1) and a caudal one (2). A small tract leaves the rostral commissure to grow along the floor plate (arrowheads in M and N). In the second case (O), three commissures are observed, Slit2 lox/lox ;Slit3 À/À embryos (#1 and #2)

, Moreover, two small fascicles extend caudally on both sides (arrowheads)

A. , B. , D. , E. , G. et al., 50 mm (C and I), 100 mm (O), 150 mm (K), and 200 mm, Scale bars represent 40 mm (F)

R. J. Ferland, T. J. Cherry, P. O. Preware, E. E. Morrisey, W. et al., Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain, J. Comp. Neurol, vol.460, pp.266-279, 2003.

M. C. Figdor and C. D. Stern, Segmental organization of embryonic diencephalon, Nature, vol.363, pp.630-634, 1993.

C. D. Fowler, Q. Lu, P. M. Johnson, M. J. Marks, K. et al., , 2011.

, Habenular a5 nicotinic receptor subunit signalling controls nicotine intake, Nature, vol.471, pp.597-601

H. Fujita and I. Sugihara, FoxP2 expression in the cerebellum and inferior olive: development of the transverse stripe-shaped expression pattern in the mouse cerebellar cortex, J. Comp. Neurol, vol.520, pp.656-677, 2012.

H. Funato, Y. Saito-nakazato, and H. Takahashi, Axonal growth from the habenular nucleus along the neuromere boundary region of the diencephalon is regulated by semaphorin 3F and netrin-1, Mol. Cell. Neurosci, vol.16, pp.206-220, 2000.

D. A. Gibson, S. Tymanskyj, R. C. Yuan, H. C. Leung, J. L. Lefebvre et al., Dendrite self-avoidance requires cell-autonomous slit/robo signaling in cerebellar purkinje cells, Neuron, vol.81, pp.1040-1056, 2014.

U. Grieshammer, L. Ma, A. S. Plump, F. Wang, M. Tessier-lavigne et al., SLIT2-mediated ROBO2 signaling restricts kidney induction to a single site, Dev. Cell, vol.6, pp.709-717, 2004.

H. Hama, H. Kurokawa, H. Kawano, R. Ando, T. Shimogori et al., Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain, Nat. Neurosci, vol.14, pp.1481-1488, 2011.

B. D. Harfe, P. J. Scherz, S. Nissim, H. Tian, A. P. Mcmahon et al., Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities, Cell, vol.118, pp.517-528, 2004.

M. Helmstaedter, K. L. Briggman, S. C. Turaga, V. Jain, H. S. Seung et al., Connectomic reconstruction of the inner plexiform layer in the mouse retina, Nature, vol.500, pp.168-174, 2013.

M. Herkenham and W. J. Nauta, Efferent connections of the habenular nuclei in the rat, J. Comp. Neurol, vol.187, pp.19-47, 1979.

O. Hikosaka, The habenula: from stress evasion to value-based decision-making, Nat. Rev. Neurosci, vol.11, pp.503-513, 2010.

S. Hippenmeyer, E. Vrieseling, M. Sigrist, T. Portmann, C. Laengle et al., A developmental switch in the response of DRG neurons to ETS transcription factor signaling, PLoS Biol, vol.3, p.159, 2005.

N. Iwahori, K. Nakamura, and S. Kameda, Terminal patterns of the fasciculus retroflexus in the interpeduncular nucleus of the mouse: a Golgi study, Anat. Embryol. (Berl.), vol.187, pp.523-528, 1993.

A. Jaworski, H. Long, and M. Tessier-lavigne, Collaborative and specialized functions of Robo1 and Robo2 in spinal commissural axon guidance, J. Neurosci, vol.30, pp.9445-9453, 2010.

M. T. Ke, S. Fujimoto, and T. Imai, SeeDB: a simple and morphologypreserving optical clearing agent for neuronal circuit reconstruction, Nat. Neurosci, vol.16, pp.1154-1161, 2013.
DOI : 10.1038/nn.3447

A. L. Kolodkin, D. J. Matthes, and C. S. Goodman, The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules, Cell, vol.75, pp.1389-1399, 1993.

D. Kopinke, M. Brailsford, F. C. Pan, M. A. Magnuson, C. V. Wright et al., Ongoing Notch signaling maintains phenotypic fidelity in the adult exocrine pancreas, Dev. Biol, vol.362, pp.57-64, 2012.
DOI : 10.1016/j.ydbio.2011.11.010
URL : https://doi.org/10.1016/j.ydbio.2011.11.010

M. J. Kuhar, R. N. Dehaven, H. I. Yamamura, H. Rommel-spacher, and J. R. Simon, Further evidence for cholinergic habenulo-interpeduncular neurons: pharmacologic and functional characteristics, Brain Res, vol.97, pp.265-275, 1975.
DOI : 10.1016/0006-8993(75)90449-7

J. W. Lewcock, N. Genoud, K. Lettieri, and S. L. Pfaff, The ubiquitin ligase Phr1 regulates axon outgrowth through modulation of microtubule dynamics, Neuron, vol.56, pp.604-620, 2007.
DOI : 10.1016/j.neuron.2007.09.009
URL : https://doi.org/10.1016/j.neuron.2007.09.009

H. Long, C. Sabatier, L. Ma, A. Plump, W. Yuan et al., Conserved roles for Slit and Robo proteins in midline commissural axon guidance, Neuron, vol.42, pp.213-223, 2004.
DOI : 10.1016/s0896-6273(04)00179-5
URL : https://doi.org/10.1016/s0896-6273(04)00179-5

L. Madisen, T. A. Zwingman, S. M. Sunkin, S. W. Oh, H. A. Zariwala et al., A robust and high-throughput Cre reporting and characterization system for the whole mouse brain, Nat. Neurosci, vol.13, pp.133-140, 2010.
DOI : 10.1038/nn.2467
URL : http://europepmc.org/articles/pmc2840225?pdf=render

V. Marillat, O. Cases, K. T. Nguyen-ba-charvet, M. Tessier-lavigne, C. Sotelo et al., Spatiotemporal expression patterns of slit and robo genes in the rat brain, J. Comp. Neurol, vol.442, pp.130-155, 2002.

V. Marillat, C. Sabatier, V. Failli, E. Matsunaga, C. Sotelo et al., The slit receptor Rig-1/Robo3 controls midline crossing by hindbrain precerebellar neurons and axons, Neuron, vol.43, pp.69-79, 2004.
DOI : 10.1016/j.neuron.2004.06.018
URL : https://hal.archives-ouvertes.fr/hal-00080701

J. Merte, Q. Wang, C. W. Vander-kooi, S. Sarsfield, D. J. Leahy et al., A forward genetic screen in mice identifies Sema3A(K108N), which binds to neuropilin-1 but cannot signal, J. Neurosci, vol.30, pp.5767-5775, 2010.
DOI : 10.1523/jneurosci.5061-09.2010
URL : http://www.jneurosci.org/content/30/16/5767.full.pdf

A. S. Plump, L. Erskine, C. Sabatier, K. Brose, C. J. Epstein et al., Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system, Neuron, vol.33, pp.219-232, 2002.

L. A. Quina, S. Wang, L. Ng, and E. E. Turner, Brn3a and Nurr1 mediate a gene regulatory pathway for habenula development, J. Neurosci, vol.29, pp.14309-14322, 2009.
DOI : 10.1523/jneurosci.2430-09.2009
URL : http://www.jneurosci.org/content/jneuro/29/45/14309.full.pdf

S. Ramon-y-cajal, Histologie Systè me Nerveux de L'homme et des Verté bré s, Maloine), vol.2, 1911.

C. Sabatier, A. S. Plump, L. Ma, K. Brose, A. Tamada et al., The divergent Robo family protein rig, 2004.

, Robo3 is a negative regulator of slit responsiveness required for midline crossing by commissural axons, Cell, vol.117, pp.157-169

A. Sahay, M. E. Molliver, D. D. Ginty, and A. L. Kolodkin, Semaphorin 3F is critical for development of limbic system circuitry and is required in neurons for selective CNS axon guidance events, J. Neurosci, vol.23, pp.6671-6680, 2003.

J. Schindelin, I. Arganda-carreras, E. Frise, V. Kaynig, M. Longair et al., Fiji: an open-source platform for biological-image analysis, Nat. Methods, vol.9, pp.676-682, 2012.

E. R. Schmidt, S. Brignani, Y. Adolfs, S. Lemstra, J. Demmers et al., Subdomain-mediated axon-axon signaling and chemoattraction cooperate to regulate afferent innervation of the lateral habenula, Neuron, vol.83, pp.372-387, 2014.

M. Seeger, G. Tear, D. Ferres-marco, and C. S. Goodman, Mutations affecting growth cone guidance in Drosophila: genes necessary for guidance toward or away from the midline, Neuron, vol.10, pp.409-426, 1993.

T. Serafini, S. A. Colamarino, E. D. Leonardo, H. Wang, R. Beddington et al., , 1996.

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara et al., Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis, Cell, vol.157, pp.726-739, 2014.

R. Tomer, L. Ye, B. Hsueh, K. Deisseroth, D. M. Van-den-heuvel et al., Spatiotemporal expression of repulsive guidance molecules (RGMs) and their receptor neogenin in the mouse brain, Nat. Protoc, vol.9, p.55828, 2013.

D. P. Wolfer, A. Henehan-beatty, E. T. Stoeckli, P. Sonderegger, and H. P. Lipp, Distribution of TAG-1/axonin-1 in fibre tracts and migratory streams of the developing mouse nervous system, J. Comp. Neurol, vol.345, pp.1-32, 1994.

T. Yamaguchi, T. Danjo, I. Pastan, T. Hikida, and S. Nakanishi, Distinct roles of segregated transmission of the septo-habenular pathway in anxiety and fear, Neuron, vol.78, pp.537-544, 2013.

M. Yamamoto, A. M. Boyer, J. E. Crandall, M. Edwards, and H. Tanaka, Distribution of stage-specific neurite-associated proteins in the developing murine nervous system recognized by a monoclonal antibody, 1986.

, J. Neurosci, vol.6, pp.3576-3594

B. Yang, J. B. Treweek, R. P. Kulkarni, B. E. Deverman, C. K. Chen et al., Single-cell phenotyping within transparent intact tissue through whole-body clearing, Cell, vol.158, pp.945-958, 2014.

W. Yuan, Y. Rao, R. P. Babiuk, J. J. Greer, J. Y. Wu et al., , 2003.

, A genetic model for a central (septum transversum) congenital diaphragmatic hernia in mice lacking Slit3, Proc. Natl. Acad. Sci. USA, vol.100, pp.5217-5222

J. A. Zallen, B. A. Yi, and C. I. Bargmann, The conserved immunoglobulin superfamily member SAX-3/Robo directs multiple aspects of axon guidance in C. elegans, Cell, vol.92, pp.217-227, 1998.