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2167-835912013PeerJPeerJThe homeodomain factor Gbx1 is required for locomotion and cell specification in the dorsal spinal cord.e142e142e14210.7717/peerj.142Dorsal horn neurons in the spinal cord integrate and relay sensory information to higher brain centers. These neurons are organized in specific laminae and different transcription factors are involved in their specification. The murine homeodomain Gbx1 protein is expressed in the mantle zone of the spinal cord at E12.5-13.5, correlating with the appearance of a discernable dorsal horn around E14 and eventually defining a narrow layer in the dorsal horn around perinatal stages. At postnatal stages, Gbx1 identifies a specific subpopulation of GABAergic neurons in the dorsal spinal cord. We have generated a loss of function mutation for Gbx1 and analyzed its consequences during spinal cord development. Gbx1 (-/-) mice are viable and can reproduce as homozygous null mutants. However, the adult mutant mice display an altered gait during forward movement that specifically affects the hindlimbs. This abnormal gait was evaluated by a series of behavioral tests, indicating that locomotion is impaired, but not muscle strength or motor coordination. Molecular analysis showed that the development of the dorsal horn is not profoundly affected in Gbx1 (-/-) mutant mice. However, analysis of terminal neuronal differentiation revealed that the proportion of GABAergic inhibitory interneurons in the superficial dorsal horn is diminished. Our study unveiled a role for Gbx1 in specifying a subset of GABAergic neurons in the dorsal horn of the spinal cord involved in the control of posterior limb movement.MezianeHamidHMouse Clinical Institute / Institut Clinique de la Souris, PHENOMIN, GIE CERBM, Illkirch Cedex, France.FraulobValérieVInstitut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université de Strasbourg, Illkirch Cedex, France.RietFabriceFMouse Clinical Institute / Institut Clinique de la Souris, PHENOMIN, GIE CERBM, Illkirch Cedex, France.KrezelWojciechWInstitut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université de Strasbourg, Illkirch Cedex, France.SelloumMohammedMMouse Clinical Institute / Institut Clinique de la Souris, PHENOMIN, GIE CERBM, Illkirch Cedex, France.GeffarthMichaelaMDFG-Center for Regenerative Therapies / Cluster of Excellence, and Biotechnology Center, Technische Universität Dresden, Dresden, Germany.AcamporaDarioDInstitute of Genetics and Biophysics "Adriano Buzzati-Traverso", Naples, Italy.IRCCS Neuromed, Pozzilli, Italy.HéraultYannYMouse Clinical Institute / Institut Clinique de la Souris, PHENOMIN, GIE CERBM, Illkirch Cedex, France.SimeoneAntonioAInstitute of Genetics and Biophysics "Adriano Buzzati-Traverso", Naples, Italy.IRCCS Neuromed, Pozzilli, Italy.BrandMichaelMDFG-Center for Regenerative Therapies / Cluster of Excellence, and Biotechnology Center, Technische Universität Dresden, Dresden, Germany.DolléPascalPInstitut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université de Strasbourg, Illkirch Cedex, France.RhinnMurielMInstitut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université de Strasbourg, Illkirch Cedex, France.engJournal Article20130829
United StatesPeerJ1016034252167-8359GABAergic neuronsGbx genesLocomotionMouse mutantSpinal cord201211272013842013976020139760201397612013829epublish24010020PMC375746510.7717/peerj.142142Bellocchio EE, Reimer RJ, Fremeau RT, Edwards RH. Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter. Science. 2000;289:957–960. doi: 10.1126/science.289.5481.957.10.1126/science.289.5481.95710938000Ben-Ari Y, Gaiarsa JL, Tyzio R, Khazipov R. GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations. Physiological Review. 2007;87:1215–1284. doi: 10.1152/physrev.00017.2006.10.1152/physrev.00017.200617928584Bermingham NA, Hassan BA, Wang VY, Fernandez M, Banfi S, Bellen HJ, Fritzsch B, Zoghbi HY. Proprioceptor pathway development is dependent on Math1. Neuron. 2001;30:411–422. doi: 10.1016/S0896-6273(01)00305-1.10.1016/S0896-6273(01)00305-111395003Bouillet P, Chazaud C, Oulad-Abdelghani M, Dollé P, Chambon P. Sequence and expression pattern of the Stra7 (Gbx-2) homeobox-containing gene induced by retinoic acid in P19 embryonal carcinoma cells. Developmental Dynamics. 1995;204:372–382. doi: 10.1002/aja.1002040404.10.1002/aja.10020404048601031Briscoe J, Ericson J. The specification of neuronal identity by graded sonic hedgehog signalling. Seminars in Cell & Developmental Biology. 1999;10:353–362. doi: 10.1006/scdb.1999.0295.10.1006/scdb.1999.029510441550Brown A. Organisation in the spinal cord. New York: Springer; 1981.Brown SDM, Chambon P, Hrabé de Angelis M. EMPReSS: standardized phenotype screens for functional annotation of the mouse genome. Nature Genetics. 2005;37:1155. doi: 10.1038/ng1105-1155.10.1038/ng1105-115516254554Buckley DM, Burroughs-Garcia J, Lewandoski M, Waters ST. Characterization of the Gbx1(−/−) mouse mutant: a requirement for Gbx1 in normal locomotion and sensorimotor circuit development. PLoS ONE. 2013;8(2):e56214. doi: 10.1371/journal.pone.0056214.10.1371/journal.pone.0056214PMC357202723418536Burroughs-Garcia J, Sittaramane V, Chandrasekhar A, Waters ST. Evolutionarily conserved function of Gbx2 in anterior hindbrain development. Developmental Dynamics. 2011;240:828–838. doi: 10.1002/dvdy.22589.10.1002/dvdy.2258921360792Caspary T, Anderson KV. Patterning cell types in the dorsal spinal cord: what the mouse mutants say. Nature Reviews Neuroscience. 2003;4:289–289. doi: 10.1038/nrn1073.10.1038/nrn107312671645Chen ZF, Rebelo S, White F, Malmberg AB, Baba H, Lima D, Woolf CJ, Basbaum AI, Anderson DJ. The paired homeodomain protein DRG11 is required for the projection of cutaneous sensory afferent fibers to the dorsal spinal cord. Neuron. 2001;31:59–73. doi: 10.1016/S0896-6273(01)00341-5.10.1016/S0896-6273(01)00341-511498051Cheng L, Arata A, Mizuguchi R, Qian Y, Karunaratne A, Gray PA, Arata S, Shirasawa S, Bouchard M, Luo P, Chen CL, Busslinger M, Goulding M, Onimaru H, Ma Q. Tlx3 and Tlx1 are post-mitotic selector genes determining glutamatergic over GABAergic cell fates. Nature Neuroscience. 2004;7:510–517. doi: 10.1038/nn1221.10.1038/nn122115064766Chiang C, Young KE, Beachy PA. Control of Drosophila tracheal branching by the novel homeodomain gene unplugged, a regulatory target for genes of the bithorax complex. Development. 1995;121:3901–3912.8582298Chotteau-Lelièvre A, Dollé P, Gofflot F. Expression analysis of murine genes using in situ hybridization with radioactive and nonradioactively labeled RNA probes. In: Darby IA, Hewitson TD, editors. In situ hybridization protocols. Totowa: Humana Press; 2006. pp. 61–87.10.1385/1-59745-007-3:6116780194Cui X, Doe CQ. The role of the cell cycle and cytokinesis in regulating neuroblast sublineage gene expression in the Drosophila CNS. Development. 1995;121:3233–3243.7588058Dickenson AH. Gate control theory of pain stands the test of time. The British Journal of Anaesthesia. 2002;88:755–757. doi: 10.1093/bja/88.6.755.10.1093/bja/88.6.75512173188Escurat M, Djabali K, Gumpel M, Gros F, Portier MM. Differential expression of two neuronal intermediate-filament proteins, peripherin and the low-molecular-mass neurofilament protein (NF-L), during the development of the rat. Journal of Neuroscience. 1990;10:764–784.PMC65701382108230Fremeau RT, Troyer MD, Pahner I, Nygaard GO, Tran CH, Reimer RJ, Bellocchio EE, Fortin D, Storm-Mathisen J, Edwards RH. The expression of vesicular glutamate transporters defines two classes of excitatory synapse. Neuron. 2001;31:247–260. doi: 10.1016/S0896-6273(01)00344-0.10.1016/S0896-6273(01)00344-011502256Ganguly K, Schinder AF, Wong ST, Poo M. GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition. Cell. 2001;105:521–532. doi: 10.1016/S0092-8674(01)00341-5.10.1016/S0092-8674(01)00341-511371348Glasgow SM, Henke RM, MacDonald RJ, Wright CVE, Johnson JE. Ptf1a determines GABAergic over glutamatergic neuronal cell fate in the spinal cord dorsal horn. Development. 2005;132:5461–5469. doi: 10.1242/dev.02167.10.1242/dev.0216716291784Goulding M, Lanuza G, Sapir T, Narayan S. The formation of sensorimotor circuits. Current Opinion Neurobiology. 2002;12:508–515. doi: 10.1016/S0959-4388(02)00371-9.10.1016/S0959-4388(02)00371-912367629Gowan K, Helms AW, Hunsaker TL, Collisson T, Ebert PJ, Odom R, Johnson JE. Crossinhibitory activities of Ngn1 and Math1 allow specification of distinct dorsal interneurons. Neuron. 2001;31:219–232. doi: 10.1016/S0896-6273(01)00367-1.10.1016/S0896-6273(01)00367-111502254Graham BA, Brichta AM, Callister RJ. Moving from an averaged to specific view of spinal cord pain processing circuits. Journal of Neurophysiology. 2007;98:1057–1063. doi: 10.1152/jn.00581.2007.10.1152/jn.00581.200717567772Gross MK, Dottori M, Goulding M. Lbx1 specifies somatosensory association interneurons in the dorsal spinal cord. Neuron. 2002;16:535–549. doi: 10.1016/S0896-6273(02)00690-6.10.1016/S0896-6273(02)00690-612062038Guo Z, Zhao C, Huang M, Huang T, Fan M, Xie Z, Chen Y, Zhao X, Xia G, Geng J, Cheng L. Tlx1/3 and Ptf1a control the expression of distinct sets of transmitter and peptide receptor genes in the developing dorsal spinal cord. Journal of Neuroscience. 2012;32:8509–8520. doi: 10.1523/JNEUROSCI.6301-11.2012.10.1523/JNEUROSCI.6301-11.2012PMC662099322723691Hatcher JP, Jones DNC, Rogers DC, Hatcher PD, Reavill PD, Hagan JJ, Hunter AJ. Development of SHIRPA to characterize the phenotype of gene-targeted mice. Behaviour Brain Research. 2001;125:43–47. doi: 10.1016/S0166-4328(01)00275-3.10.1016/S0166-4328(01)00275-311682092Helms AW, Johnson JE. Specification of dorsal spinal cord interneurons. Current Opinion Neurobiology. 2003;13:42–49. doi: 10.1016/S0959-4388(03)00010-2.10.1016/S0959-4388(03)00010-212593981Honda CN. Differential distribution of calbindin-D28k and parvalbumin in somatic and visceral sensory neurons. Neuroscience. 1995;68:883–892. doi: 10.1016/0306-4522(95)00180-Q.10.1016/0306-4522(95)00180-Q8577381Irwin S. Comprehensive observational assessment: Ia. A systematic, quantitative procedure for assessing the behavioral and physiologic state of the mouse. Psychopharmacologia. 1968;13:222–257. doi: 10.1007/BF00401402.10.1007/BF004014025679627John A, Wildner H, Britsch S. The homeodomain transcription factor Gbx1 identifies a subpopulation of late-born GABAergic interneurons in the developing dorsal spinal cord. Developmental Dynamics. 2005;234:767–771. doi: 10.1002/dvdy.20568.10.1002/dvdy.2056816193514Kaneko T, Fujiyama F. Complementary distribution of vesicular glutamate transporters in the central nervous system. Neuroscience Research. 2002;42:243–250. doi: 10.1016/S0168-0102(02)00009-3.10.1016/S0168-0102(02)00009-311985876Larsson M, Broman J. Synaptic plasticity and pain: role of ionotropic glutamate receptors. Neuroscientist. 2011;17:256–273. doi: 10.1177/1073858409349913.10.1177/107385840934991320360599Lee KJ, Jessell TM. The specification of dorsal cell fates in the vertebrate central nervous system. Annual Review of Neuroscience. 1999;22:261–294. doi: 10.1146/annurev.neuro.22.1.261.10.1146/annurev.neuro.22.1.26110202540Leonardo ED, Hinck L, Masu M, Keino-Masu K, Fazeli A, Stoeckli ET, Ackerman SL, Weinberg RA, Tessier-Lavigne M. Guidance of developing axons by netrin-1 and its receptors. Cold Spring Harbor Symposia on Quantitative Biology. 1997;62:467–478. doi: 10.1101/SQB.1997.062.01.054.10.1101/SQB.1997.062.01.0549598381Le Mouellic H, Lallemand Y, Brûlet P. Targeted replacement of the homeobox gene Hox-3.1 by the Escherichia coli lacZ in mouse chimeric embryos. Proceedings of the National Academy of Sciences of the United States of America. 1990;87:4712–4716. doi: 10.1073/pnas.87.12.4712.10.1073/pnas.87.12.4712PMC541871972279Lewis KE. Do genes regulate simple behaviours? Understanding how different neurons in the vertebrate spinal cord are genetically specified. Philosophical Transactions of the Royal Society B: Biological Sciences. 2006;361:45–66. doi: 10.1098/rstb.2005.1778.10.1098/rstb.2005.1778PMC162654516553308Lin X, Swaroop A, Vaccarino FM, Murtha MT, Haas M, Ji X, Ruddle FH, Leckman JF. Characterization and sequence analysis of the human homeobox-containing gene GBX2. Genomics. 1996;31:335–342. doi: 10.1006/geno.1996.0056.10.1006/geno.1996.00568838315Malcangio M, Bowery NG. GABA and its receptors in the spinal cord. Trends in Pharmacological Sciences. 1996;17:457–462. doi: 10.1016/S0165-6147(96)01013-9.10.1016/S0165-6147(96)01013-99014500Magin TM, McWhir J, Melton DW. A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency. Nucleic Acids Research. 1992;20:3795–3796. doi: 10.1093/nar/20.14.3795.10.1093/nar/20.14.3795PMC3340451641353Melzack R, Wall PD. Pain mechanisms: a new theory. Science. 1965;150:971–979. doi: 10.1126/science.150.3699.971.10.1126/science.150.3699.9715320816Müller T, Brohmann H, Pierani A, Heppenstall PA, Lewin GR, Jessell TM, Birchmeier C. The homeodomain factor lbx1 distinguishes two major programs of neuronal differentiation in the dorsal spinal cord. Neuron. 2002;34:551–562. doi: 10.1016/S0896-6273(02)00689-X.10.1016/S0896-6273(02)00689-X12062039Nagy A, Gertsenstein M, Vintersten K, Behringer R. Manipulating the mouse embryo: a laboratory manual. New York: Cold Spring Harbor Laboratory Press; 2003.Niss K, Leutz A. Expression of the homeobox gene GBX2 during chicken development. Mechanisms of Development. 1998;76:151–155. doi: 10.1016/S0925-4773(98)00103-8.10.1016/S0925-4773(98)00103-89767154Ozaki S, Snider WD. Initial trajectories of sensory axons toward laminar targets in the developing mouse spinal cord. Journal of Comparative Neurology. 1997;380:215–229. doi: 10.1002/(SICI)1096-9861(19970407)380:2<215::AID-CNE5>3.0.CO;2-6.10.1002/(SICI)1096-9861(19970407)380:2<215::AID-CNE5>3.0.CO;2-69100133Rexed B. The cytoarchitectonic organization of the spinal cord in the cat. Journal of Comparative Neurology. 1952;96:414–495. doi: 10.1002/cne.900960303.10.1002/cne.90096030314946260Ramírez-Solis R, Davis AC, Bradley A. [51] Gene targeting in embryonic stem cells. Methods in Enzymology. 1993;225:855–878. doi: 10.1016/0076-6879(93)25054-6.10.1016/0076-6879(93)25054-68231891Rebelo S, Reguenga C, Lopes C, Lima D. Prrxl1 is required for the generation of a subset of nociceptive glutamatergic superficial spinal dorsal horn neurons. Developmental Dynamics. 2010;239:1684–1694. doi: 10.1002/dvdy.22305.10.1002/dvdy.2230520503365Rhinn M, Brand M. The midbrain-hindbrain boundary organizer. Current Opinion in Neurobiology. 2001;11:34–42. doi: 10.1016/S0959-4388(00)00171-9.10.1016/S0959-4388(00)00171-911179870Rhinn M, Lun K, Amores A, Yan YL, Postlethwait JH, Brand M. Cloning, expression and relationship of zebrafish gbx1 and gbx2 genes to Fgf signaling. Mechanisms of Development. 2003;120:919–936. doi: 10.1016/S0925-4773(03)00135-7.10.1016/S0925-4773(03)00135-712963112Rhinn M, Lun K, Werner M, Simeone A, Brand M. Isolation and expression of the homeobox gene Gbx1 during mouse development. Developmental Dynamics. 2003;229:334–339. doi: 10.1002/dvdy.10435.10.1002/dvdy.1043514745958Rhinn M, Lun K, Ahrendt R, Geffarth M, Brand M. Zebrafish gbx1 refines the midbrain-hindbrain boundary border and mediates the Wnt8 posteriorization signal. Neural Development. 2009;4:12. doi: 10.1186/1749-8104-4-12.10.1186/1749-8104-4-12PMC267443919341460Robertson EJ. Embryo-derived stem cell lines. In: Robertson EJ, editor. Teratocarcinomas and embryonic stem cells: a practical approach. Oxford: IRL Press; 1987. pp. 71–112.Shamim H, Mason I. Expression of Gbx-2 during early development of the chick embryo. Mechanisms of Development. 1998;76:157–159. doi: 10.1016/S0925-4773(98)00102-6.10.1016/S0925-4773(98)00102-69767156Sibilla S, Ballerini L. GABAergic and glycinergic interneuron expression during spinal cord development: dynamic interplay between inhibition and excitation in the control of ventral network outputs. Progress in Neurobiology. 2009;9:46–60. doi: 10.1016/j.pneurobio.2009.06.001.10.1016/j.pneurobio.2009.06.00119539686Simeone A. Positioning the isthmic organizer where Otx2 and Gbx2 meet. Trends in Genetics. 2000;16:237–240. doi: 10.1016/S0168-9525(00)02000-X.10.1016/S0168-9525(00)02000-X10827447Somogyi R, Wen X, Ma W, Barker JL. Developmental kinetics of GAD family mRNAs parallel neurogenesis in the rat spinal cord. Journal of Neuroscience. 1995;15:2575–2591.PMC65777537722616Stein V, Hermans-Borgmeyer I, Jentsch TJ, Hübner CA. Expression of the KCl cotransporter KCC2 parallels neuronal maturation and the emergence of low intracellular chloride. Journal of Comparative Neurology. 2004;468:57–64. doi: 10.1002/cne.10983.10.1002/cne.1098314648690Todd AJ. Neuronal circuitry for pain processing in the dorsal horn. Nature Reviews Neuroscience. 2010;11:823–836. doi: 10.1038/nrn2947.10.1038/nrn2947PMC327794121068766Wassarman KM, Lewandoski M, Campbell K, Joyner AL, Rubenstein JL, Martinez S, Martin GR. Specification of the anterior hindbrain and establishment of a normal mid/hindbrain organizer is dependent on Gbx2 gene function. Development. 1997;124:2923–2923.9247335Waters ST, Wilson CP, Lewandoski M. Cloning and embryonic expression analysis of the mouse Gbx1 gene. Gene Expression Patterns. 2003;3:313–317. doi: 10.1016/S1567-133X(03)00041-3.10.1016/S1567-133X(03)00041-312799077Waters ST, Lewandoski M. A threshold requirement for Gbx2 levels in hindbrain development. Development. 2006;133:1991–2000. doi: 10.1242/dev.02364.10.1242/dev.0236416651541White FA, Keller-Peck CR, Knudson CM, Korsmeyer SJ, Snider WD. Widespread elimination of naturally occurring neuronal death in Bax-deficient mice. Journal of Neuroscience. 1998;18:1428–1439.PMC67927259454852Willis W, Coggeshall RE. Sensory mechanisms of the spinal cord. NY: Plenum Press; 1991.