231363912013013120260128
1477-9129139242012DecDevelopment (Cambridge, England)DevelopmentGbx2 regulates thalamocortical axon guidance by modifying the LIM and Robo codes.463346434633-4310.1242/dev.086991Combinatorial expression of transcription factors forms transcriptional codes to confer neuronal identities and connectivity. However, how these intrinsic factors orchestrate the spatiotemporal expression of guidance molecules to dictate the responsiveness of axons to guidance cues is less understood. Thalamocortical axons (TCAs) represent the major input to the neocortex and modulate cognitive functions, consciousness and alertness. TCAs travel a long distance and make multiple target choices en route to the cortex. The homeodomain transcription factor Gbx2 is essential for TCA development, as loss of Gbx2 abolishes TCAs in mice. Using a novel TCA-specific reporter, we have discovered that thalamic axons are mostly misrouted to the ventral midbrain and dorsal midline of the diencephalon in Gbx2-deficient mice. Furthermore, conditionally deleting Gbx2 at different embryonic stages has revealed a sustained role of Gbx2 in regulating TCA navigation and targeting. Using explant culture and mosaic analyses, we demonstrate that Gbx2 controls the intrinsic responsiveness of TCAs to guidance cues. The guidance defects of Gbx2-deficient TCAs are associated with abnormal expression of guidance receptors Robo1 and Robo2. Finally, we demonstrate that Gbx2 controls Robo expression by regulating LIM-domain transcription factors through three different mechanisms: Gbx2 and Lhx2 compete for binding to the Lmo3 promoter and exert opposing effects on its transcription; repressing Lmo3 by Gbx2 is essential for Lhx2 activity to induce Robo2; and Gbx2 represses Lhx9 transcription, which in turn induces Robo1. Our findings illustrate the transcriptional control of differential expression of Robo1 and Robo2, which may play an important role in establishing the topography of TCAs.ChatterjeeMallikaMDepartment of Genetics and Developmental Biology, University of Connecticut Health Center, 400 Farmington Avenue, Farmington, CT 06030-6403, USA.LiKairongKChenLiLMaisanoXuXGuoQiuxiaQGanLinLLiJames Y HJYengR01 MH094914MHNIMH NIH HHSUnited StatesR01MH094914MHNIMH NIH HHSUnited StatesJournal ArticleResearch Support, N.I.H., Extramural20121107
EnglandDevelopment87017440950-19910Homeodomain Proteins0LIM-Homeodomain Proteins0Nerve Tissue Proteins0Receptors, Immunologic0Transcription Factors0Roundabout Proteins0Gbx2 protein, mouse0Robo2 protein, mouseIMAnimalsAxonsmetabolismphysiologyCells, CulturedCerebral CortexembryologymetabolismphysiologyEmbryo, MammalianFemaleGene Expression Regulation, DevelopmentalHomeodomain ProteinsgeneticsmetabolismphysiologyLIM-Homeodomain ProteinsgeneticsmetabolismMiceMice, TransgenicNerve Tissue ProteinsgeneticsmetabolismNeurogenesisgeneticsphysiologyPregnancyReceptors, ImmunologicgeneticsmetabolismThalamusembryologymetabolismphysiologyTranscription FactorsgeneticsmetabolismRoundabout Proteins2012119602012119602013216020131215ppublish23136391PMC350972510.1242/dev.086991dev.086991 Andrews W., Liapi A., Plachez C., Camurri L., Zhang J., Mori S., Murakami F., Parnavelas J. G., Sundaresan V., Richards L. J. (2006). Robo1 regulates the development of major axon tracts and interneuron migration in the forebrain. Development 133, 2243–2252 16690755 Bach I. (2000). The LIM domain: regulation by association. Mech. Dev. 91, 5–17 10704826 Bagri A., Marín O., Plump A. S., Mak J., Pleasure S. J., Rubenstein J. L., Tessier-Lavigne M. (2002). Slit proteins prevent midline crossing and determine the dorsoventral position of major axonal pathways in the mammalian forebrain. Neuron 33, 233–248 11804571 Berger M. F., Badis G., Gehrke A. R., Talukder S., Philippakis A. A., Peña-Castillo L., Alleyne T. M., Mnaimneh S., Botvinnik O. B., Chan E. T., et al. (2008). Variation in homeodomain DNA binding revealed by high-resolution analysis of sequence preferences. Cell 133, 1266–1276 PMC253116118585359 Bielle F., Marcos-Mondejar P., Keita M., Mailhes C., Verney C., Nguyen Ba-Charvet K., Tessier-Lavigne M., Lopez-Bendito G., Garel S. (2011a). Slit2 activity in the migration of guidepost neurons shapes thalamic projections during development and evolution. Neuron 69, 1085–1098 21435555 Bielle F., Marcos-Mondéjar P., Leyva-Diaz E., Lokmane L., Mire E., Mailhes C., Keita M., Garcia N., Tessier-Lavigne M., Garel S., et al. (2011b). Emergent growth cone responses to combinations of Slit1 and Netrin 1 in thalamocortical axon topography. Curr. Biol. 21, 1748–1755 22000108 Bonnin A., Torii M., Wang L., Rakic P., Levitt P. (2007). Serotonin modulates the response of embryonic thalamocortical axons to netrin-1. Nat. Neurosci. 10, 588–597 17450135 Braisted J. E., Ringstedt T., O’Leary D. D. (2009). Slits are chemorepellents endogenous to hypothalamus and steer thalamocortical axons into ventral telencephalon. Cereb. Cortex 19, i144–i151 PMC269353419435711 Brose K., Bland K. S., Wang K. H., Arnott D., Henzel W., Goodman C. S., Tessier-Lavigne M., Kidd T. (1999). Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96, 795–806 10102268 Bulfone A., Puelles L., Porteus M. H., Frohman M. A., Martin G. R., Rubenstein J. L. (1993). Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries. J. Neurosci. 13, 3155–3172 PMC65766887687285 Buratowski S., Chodosh L. A. (2001). Mobility shift DNA-binding assay using gel electrophoresis. Curr. Protoc. Mol. Biol. 36, 12.2.1–12.2.11 18265085 Chen L., Guo Q., Li J. Y. (2009). Transcription factor Gbx2 acts cell-nonautonomously to regulate the formation of lineage-restriction boundaries of the thalamus. Development 136, 1317–1326 PMC268746319279136 Chen L., Chatterjee M., Li J. Y. (2010). The mouse homeobox gene Gbx2 is required for the development of cholinergic interneurons in the striatum. J. Neurosci. 30, 14824–14834 PMC307164621048141 Dwyer N. D., Manning D. K., Moran J. L., Mudbhary R., Fleming M. S., Favero C. B., Vock V. M., O’Leary D. D., Walsh C. A., Beier D. R. (2011). A forward genetic screen with a thalamocortical axon reporter mouse yields novel neurodevelopment mutants and a distinct emx2 mutant phenotype. Neural Dev. 6, 3 PMC302492221214893 Garel S., Rubenstein J. L. (2004). Intermediate targets in formation of topographic projections: inputs from the thalamocortical system. Trends Neurosci. 27, 533–539 15331235 Hevner R. F., Miyashita-Lin E., Rubenstein J. L. (2002). Cortical and thalamic axon pathfinding defects in Tbr1, Gbx2, and Pax6 mutant mice: evidence that cortical and thalamic axons interact and guide each other. J. Comp. Neurol. 447, 8–17 11967891 Jones E. G. (2007). The Thalamus. New York, NY: Cambridge University Press; Jones E. G., Rubenstein J. L. (2004). Expression of regulatory genes during differentiation of thalamic nuclei in mouse and monkey. J. Comp. Neurol. 477, 55–80 15281080 Kain S. R., Ganguly S. (2001). Overview of Genetic Reporter Systems. Curr. Protoc. Mol. Biol. 68, 9.6.1–9.6.12 18265284 Lakhina V., Falnikar A., Bhatnagar L., Tole S. (2007). Early thalamocortical tract guidance and topographic sorting of thalamic projections requires LIM-homeodomain gene Lhx2. Dev. Biol. 306, 703–713 17493606 Lee S., Lee B., Joshi K., Pfaff S. L., Lee J. W., Lee S. K. (2008). A regulatory network to segregate the identity of neuronal subtypes. Dev. Cell 14, 877–889 PMC307174318539116 Li J. Y., Lao Z., Joyner A. L. (2002). Changing requirements for Gbx2 in development of the cerebellum and maintenance of the mid/hindbrain organizer. Neuron 36, 31–43 12367504 Loots G. G., Ovcharenko I., Pachter L., Dubchak I., Rubin E. M. (2002). rVista for comparative sequence-based discovery of functional transcription factor binding sites. Genome Res. 12, 832–839 PMC18658011997350 López-Bendito G., Molnár Z. (2003). Thalamocortical development: how are we going to get there? Nat. Rev. Neurosci. 4, 276–289 12671644 López-Bendito G., Flames N., Ma L., Fouquet C., Di Meglio T., Chedotal A., Tessier-Lavigne M., Marín O. (2007). Robo1 and Robo2 cooperate to control the guidance of major axonal tracts in the mammalian forebrain. J. Neurosci. 27, 3395–3407 PMC667212817392456 Lund R. D., Mustari M. J. (1977). Development of the geniculocortical pathway in rats. J. Comp. Neurol. 173, 289–305 856885 Madisen L., Zwingman T. A., Sunkin S. M., Oh S. W., Zariwala H. A., Gu H., Ng L. L., Palmiter R. D., Hawrylycz M. J., Jones A. R., et al. (2010). A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 PMC284022520023653 Marcos-Mondéjar P., Peregrin S., Li J. Y., Carlsson L., Tole S., López-Bendito G. (2012). The lhx2 transcription factor controls thalamocortical axonal guidance by specific regulation of robo1 and robo2 receptors. J. Neurosci. 32, 4372–4385 PMC662204722457488 Métin C., Godement P. (1996). The ganglionic eminence may be an intermediate target for corticofugal and thalamocortical axons. J. Neurosci. 16, 3219–3235 PMC65791428627360 Milán M., Diaz-Benjumea F. J., Cohen S. M. (1998). Beadex encodes an LMO protein that regulates Apterous LIM-homeodomain activity in Drosophila wing development: a model for LMO oncogene function. Genes Dev. 12, 2912–2920 PMC3171639744867 Miyashita-Lin E. M., Hevner R., Wassarman K. M., Martinez S., Rubenstein J. L. (1999). Early neocortical regionalization in the absence of thalamic innervation. Science 285, 906–909 10436162 Molnar Z., Adams R., Blakemore C. (1998). Mechanisms underlying the early establishment of thalamocortical connections in the rat. J. Neurosci. 18, 5723–5745 PMC67930539671663 Nagy A., Gertsenstein M., Vintersten K., Behringer R. (2003). Manipulating the Mouse Embryo. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; Nakagawa Y., O’Leary D. D. (2001). Combinatorial expression patterns of LIM-homeodomain and other regulatory genes parcellate developing thalamus. J. Neurosci. 21, 2711–2725 PMC676251811306624 O’Donnell M., Chance R. K., Bashaw G. J. (2009). Axon growth and guidance: receptor regulation and signal transduction. Annu. Rev. Neurosci. 32, 383–412 PMC476543319400716 O’Leary D. D., Koester S. E. (1993). Development of projection neuron types, axon pathways, and patterned connections of the mammalian cortex. Neuron 10, 991–1006 8318235 Porter F. D., Drago J., Xu Y., Cheema S. S., Wassif C., Huang S. P., Lee E., Grinberg A., Massalas J. S., Bodine D., et al. (1997). Lhx2, a LIM homeobox gene, is required for eye, forebrain, and definitive erythrocyte development. Development 124, 2935–2944 9247336 Powell A. W., Sassa T., Wu Y., Tessier-Lavigne M., Polleux F. (2008). Topography of thalamic projections requires attractive and repulsive functions of Netrin-1 in the ventral telencephalon. PLoS Biol. 6, e116 PMC258457218479186 Quina L. A., Wang S., Ng L., Turner E. E. (2009). Brn3a and Nurr1 mediate a gene regulatory pathway for habenula development. J. Neurosci. 29, 14309–14322 PMC280283219906978 Rajagopalan S., Vivancos V., Nicolas E., Dickson B. J. (2000). Selecting a longitudinal pathway: Robo receptors specify the lateral position of axons in the Drosophila CNS. Cell 103, 1033–1045 11163180 Rakic P. (1977). Prenatal development of the visual system in rhesus monkey. Philos. Trans. R. Soc. Lond. B Biol. Sci. 278, 245–260 19781 Rubenstein J. L., Martinez S., Shimamura K., Puelles L. (1994). The embryonic vertebrate forebrain: the prosomeric model. Science 266, 578–580 7939711 Seibt J., Schuurmans C., Gradwhol G., Dehay C., Vanderhaeghen P., Guillemot F., Polleux F. (2003). Neurogenin2 specifies the connectivity of thalamic neurons by controlling axon responsiveness to intermediate target cues. Neuron 39, 439–452 12895419 Simpson J. H., Kidd T., Bland K. S., Goodman C. S. (2000). Short-range and long-range guidance by slit and its Robo receptors. Robo and Robo2 play distinct roles in midline guidance. Neuron 28, 753–766 11163264 Sunmonu N. A., Li K., Guo Q., Li J. Y. (2011). Gbx2 and Fgf8 are sequentially required for formation of the midbrain-hindbrain compartment boundary. Development 138, 725–734 PMC302641621266408 Thaler J. P., Lee S. K., Jurata L. W., Gill G. N., Pfaff S. L. (2002). LIM factor Lhx3 contributes to the specification of motor neuron and interneuron identity through cell-type-specific protein-protein interactions. Cell 110, 237–249 12150931 Tse E., Smith A. J., Hunt S., Lavenir I., Forster A., Warren A. J., Grutz G., Foroni L., Carlton M. B., Colledge W. H., et al. (2004). Null mutation of the Lmo4 gene or a combined null mutation of the Lmo1/Lmo3 genes causes perinatal lethality, and Lmo4 controls neural tube development in mice. Mol. Cell. Biol. 24, 2063–2073 PMC35056214966285 Wassarman K. M., Lewandoski M., Campbell K., Joyner A. L., Rubenstein J. L., Martinez S., Martin G. R. (1997). Specification of the anterior hindbrain and establishment of a normal mid/hindbrain organizer is dependent on Gbx2 gene function. Development 124, 2923–2934 9247335