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Developmental guidance of the retroflex tract at its bending point involves Robo1-Slit2-mediated floor plate repulsion

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Abstract

The retroflex tract contains medial habenula efferents that target the hindbrain interpeduncular complex and surrounding areas. This tract displays a singular course. Initially, habenular axons extend ventralwards in front of the pretectum until they reach the basal plate. Next, they avoid crossing the local floor plate, sharply changing course caudalwards (the retroflexion alluded by the tract name) and navigate strictly antero-posteriorly across basal pretectum, midbrain and isthmus. Once they reach rhombomere 1, the habenular axons criss-cross the floor plate several times within the interpeduncular nuclear complex as they innervate it. Here we described the timing and details of growth phenomena as these axons navigate to their target. The first dorsoventral course apparently obeys Ntn1 attraction. We checked the role of local floor plate signaling in the decision to avoid the thalamic floor plate and bend caudalwards. Analyzing the altered floor and basal plates of Gli2 knockout mice, we found a contralateral projection of most habenular axons, plus ulterior bizarre navigation rostralwards. This crossing phenotype was due to a reduced expression of Slit repulsive cues, suggesting involvement of the floor-derived Robo-Slit system in the normal guidance of this tract. Using Slit and Robo mutant mice, open neural tube and co-culture assays, we determined that Robo1-Slit2 interaction is specifically required for impeding that medial habenular axons cross the thalamic floor plate. This pathfinding mechanism is essential to establish the functionally important habenulo-interpeduncular connection.

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Acknowledgments

Work supported by ‘‘Ministerio de Economía y Competitividad’’ BFU2010-16548 and BFU2013-48230-P (FEDER Fonds) to E. Puelles, BFU2012-34298 to G. Lopez-Bendito; Consolider Grant (CSD2007-00023) and European commission (EUCOMMTOOLS, contract 261492) to S.M. J.A. Moreno-Bravo was supported by the Predoctoral Program of the ‘‘Consejo Superior de Investigaciones Científicas-Junta de Ampliación de Estudios’’, co-financed by the European Social Fund. The Instituto de Neurociencias is a “Centre of Excellence Severo Ochoa”. Funding was also provided to G.S. Mastick by NIH grants R21NS077169, with core facility support by NIH COBREs 1 P20 RR024210 and 1 P20 GM103650, and the Nevada INBRE 8 P20 GM103440-11. We thank E. Turner for kindly providing the Pou4f1-TauLacZ transgenic line and J. Pasterkamp for kindly providing the Slit2 mutant embryos. We are also grateful to O. Marín for providing the Robo1, Robo2, Slit1, Slit2, Slit3 probes; O. Reiner for providing the Netrin1 probe; M. Frohman for proving Gbx2, A. McMahon for Shh and J.L Rubenstein for Dlx2 probe. M. Tessier-Lavigne for providing the Slit1 functional construct and A. Nieto for providing the PCX-GFP plasmid. We thank E. Leyva-Diaz, R. Susin and C. Merino from Lopez-Bendito lab for helping us in the generation and genotyping of the Robo transgenic embryos. We are also grateful with E. Domiguez and A. Sempere for statistical advice, and L. Puelles and the staff from the S. Martínez lab, especially D. Echevarría, for helpful discussions and comments.

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Authors and Affiliations

  1. Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-CSIC, Sant Joan d’Alacant, 03550, Alicante, Spain

    Juan A. Moreno-Bravo, Jesus E. Martinez-Lopez, M. Pilar Madrigal, Guillermina Lopez-Bendito, Salvador Martinez & Eduardo Puelles

  2. Department of Biology, University of Nevada, Reno, NV, 89557, USA

    Minkyung Kim & Grant S. Mastick

  3. Instituto Murciano de Investigación Biomédica IMIB-Arrixaca, Murcia, Spain

    Salvador Martinez

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  1. Juan A. Moreno-Bravo
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  2. Jesus E. Martinez-Lopez
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  3. M. Pilar Madrigal
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  4. Minkyung Kim
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  5. Grant S. Mastick
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  6. Guillermina Lopez-Bendito
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  8. Eduardo Puelles
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Correspondence to Eduardo Puelles.

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ESM1: Video of an OPT scanned E15.5 whole wild-type brain with DiI in right and left Habenula. (MP4 334 kb)

429_2014_932_MOESM2_ESM.jpg

ESM2: Open neural tube technic (ONT). (a, b) Schematic design of the ONTs, we dissected the neural tube and cut the rostral forebrain and caudal hindbrain out. The remaining neural tube was opened like a book through the roof plate where we maintained the adjacent territories involved in the guidance of the rft. (c) X-gal stained-E12.5 ONT in which the Pou4f1 positive neurons and their axons are labeled, habenula and the rft among them. (d, e) In situ hybridization for Gbx2 (d) and Shh (e) displaying the location of the thalamus and the basal and floor plate, respectively. We demonstrated that the ONTs allow us to study the course of the rtf due to the maintenance of the positional relations of the territories involved. Abbreviations: ap, alar plate; bp, basal plate; fp, floor plate; Hb, Habenula; Ist, Isthmus; Mb, Midbrain; p1, prosomere 1; p2, prosomere 2; p3, prosomere 3; pc, posterior commissure; r1, rhombomere 1; rft, retroflex tract; rp, roof plate; Th, Thalamus; zli, zona limitans intrathalamica. Scale bar: 200 μm. (JPEG 1154 kb)

429_2014_932_MOESM3_ESM.jpg

ESM3: Phenotype of the rft and neural territories in Gli2 mutant. Medial (a, c, e) and lateral (b, d, f) sagittal paraffin sections from an E18.5 Gli2 mutant brain. (a-b) Immunostaining against DCC reveals the rostral navigation of the rft in the midline (see arrow in a). (b) In a lateral section, the dorsoventral trajectory is showed. (c-f) In situ hybridization for Gbx2 (c, d) and Dlx2 (e, f) displaying the thalamus, and the hypothalamus–prethalamus, respectively. Abbreviations: Cb, Cerebellum; Hb, habenula; Hyp, Hypothalamus; Mb, Midbrain; pc, posterior commissure; PTh, Prethalamus; rft, retroflex tract; Th, Thalamus. Scale bar: 200 μm (JPEG 1884 kb)

ESM4: Video of an OPT scanned E18.5 whole Gli2 –/– brain with DiI in right and DiA in left Habenula. (MP4 602 kb)

429_2014_932_MOESM5_ESM.jpg

ESM5: Robo3 and Robo4 expression pattern in the habenula. (a-b) In situ hybridization for Robo3 (a, a’) and Robo4 (b, b’) in Hb paraffin coronal sections of an E12.5 (a, b) and E18.5 (a’, b’) embryo. Robo3 is strongly expressed in the mHb and Robo4 is not expressed in this region. The arrow in b shows the Robo4 expression in the cortex. The dotted line indicates the location of the habenular complex. Abbreviations: Hb, habenula; lHb, lateral habenula; mHb, medial habenula. Scale bar: 200 μm (JPEG 514 kb)

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Moreno-Bravo, J.A., Martinez-Lopez, J.E., Madrigal, M.P. et al. Developmental guidance of the retroflex tract at its bending point involves Robo1-Slit2-mediated floor plate repulsion. Brain Struct Funct 221, 665–678 (2016). https://doi.org/10.1007/s00429-014-0932-4

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