PATTERNS & PHENOTYPES

The Homeodomain Transcription Factor Gbx1Identifies a Subpopulation of Late-BornGABAergic Interneurons in the DevelopingDorsal Spinal CordAnita John, Hendrik Wildner, and Stefan Britsch*

The dorsal spinal cord processes somatosensory information and relays it to higher brain centers and tomotoneurons in the ventral spinal horn. These functions reside in a large number of distinct sensoryinterneurons that are organized in specific laminae within the dorsal spinal horn. Homeodomain and bHLHtranscription factors can control the development of neuronal cell types in the dorsal horn. Here, wedemonstrate that the murine homeodomain transcription factor Gbx1 is expressed specifically in a subsetof Lbx1� (class B) neurons in the dorsal horn. Expression of Gbx1 in the dorsal spinal cord depends on Lbx1function. Immunohistological analyses revealed that Gbx1 identifies a distinct population of late-born,Lhx1/5�, Pax2� neurons. In the perinatal period as well as in the adult spinal cord, Gbx1 marks asubpopulation of GABAergic neurons. The expression of Gbx1 suggests that it controls development of aspecific subset of GABAergic neurons in the dorsal horn of the spinal cord. Developmental Dynamics 234:767–771, 2005. © 2005 Wiley-Liss, Inc.

Key words: Gbx1; Gbx2; Lbx1; homeodomain transcription factor; GABA; spinal cord; dorsal horn; mouse

Received 27 April 2005; Revised 4 July 2005; Accepted 3 August 2005

INTRODUCTION

The dorsal spinal cord processes so-matosensory information and relays itto motor neurons located in the ven-tral horn and to higher brain centers,like the brainstem, thalamus, and cer-ebellum. These functions reside in alarge number of distinct interneurontypes that are arranged in a highlyorganized laminar structure, the dor-sal horn (Rexed, 1952; Brown, 1981).Such interneurons participate in spi-nal reflexes and are major targets ofdescending neuronal systems that canmodulate incoming somatosensory in-formation, for example pain stimuli

(Gillespie and Walker, 2001; Juliusand Basbaum, 2001). The functionalarchitecture of the mature dorsal hornis the result of developmental pro-cesses that involve cell-type specifica-tion and differentiation as well as mi-gration of neurons that weregenerated in the dorsal neural tube(Lee and Jessell, 1999; Caspary andAnderson, 2003).

Recent studies demonstrate thathomeodomain transcription factorsplay a central role during develop-ment of neurons in the dorsal horn (forrecent reviews see Goulding et al.,2002; Helms and Johnson, 2003). Sen-

sory interneurons of the superficialdorsal horn are generated from neu-rons that are born during a late phaseof neurogenesis (E11.5–E14.0 in mice)in the alar plate. At the time of theirbirth, these neurons are characterizedby the expression of the homeodomaintranscription factor Lbx1 and are des-ignated as class B neurons. Correctspecification of late-born class B neu-rons depends on Lbx1 function. Inmice that carry a null mutation of theLbx1 gene, the neurons that arise inthe dorsal spinal cord assume aber-rant molecular characteristics and aresubsequently eliminated by apoptosis,

Max Delbruck Center for Molecular Medicine (MDC), Berlin-Buch, GermanyGrant sponsor: DFG; Grant number: SFB 665-A1.*Correspondence to: Stefan Britsch, Max Delbruck Center for Molecular Medicine (MDC), Robert-Rossle-Str. 10, 13125Berlin-Buch, Germany. E-mail: sbritsch@mdc-berlin.de

DOI 10.1002/dvdy.20568Published online 28 September 2005 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 234:767–771, 2005

© 2005 Wiley-Liss, Inc.

Fig. 1.

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768 JOHN ET AL.

resulting in a loss of the superficialdorsal horn (Gross et al., 2002; Mulleret al., 2002). Late-born class B neu-rons comprise two neuron types (dILAand dILB neurons). The two neuronstypes can be distinguished by the ex-pression of different sets of homeodo-main transcription factors: dILB neu-rons express the homeodomainfactors, Lmx1b and Tlx3 (Rnx),whereas dILA neurons are identifiedby the expression of Lhx1/5 and Pax2.These homeodomain factors controldistinct steps during the further dif-ferentiation of these neuron types.Tlx3 and the closely related Tlx1 de-termine a glutamatergic cell fate inpostmitotic dorsal neurons and in theabsence of these genes the neuronstake on an aberrant GABAergic fate(Cheng et al., 2004). Lmx1b and a fur-ther paired homeodomain factor,Drg11, are both essential for the dif-ferentiation of these neurons in thesuperficial dorsal horn. In addition,the innervation of the spinal cord bysensory afferents does not occur cor-rectly in Tlx1/3, Lmx1b, and Drg11mutant mice (Chen et al., 2001; Dinget al., 2004). Pax2 is required forGABAergic differentiation of dILAcells (Cheng et al., 2004). The molec-ular mechanisms that control subtypediversification and terminal differen-tiation of dorsal spinal interneuronsare incompletely understood, and mo-lecular markers that allow distin-guishing the diverse neuronal sub-types in the dorsal horn are stilllimited.

Here, we show that the murine ho-meodomain factor Gbx1 is expressedspecifically in late-born neurons thatsubsequently populate the superficialdorsal horn. Gbx1� spinal neurons co-express Lbx1, Lhx1/5, and Pax2.

Gbx1� cells are not generated in Lbx1mutant mice. Gbx1 expression identi-fies a specific subpopulation ofGABAergic neurons in the dorsal spi-nal cord. The distinct expression ofGbx1 suggests a function in the mat-uration of a particular GABAergicneuronal subtype of the dorsal horn.

RESULTS AND DISCUSSION

Identification and Isolationof Gbx1 cDNA

In order to identify genes differen-tially expressed in the dorsal spinalcord at E13.5, a microarray-based ge-nome wide expression analysis wasperformed (Britsch and Birchmeier,unpublished data). Among the genesspecifically expressed in the develop-ing dorsal neural tube, we identifiedan EST sequence (Affymetrix-ID116752_at, corresponding to GenBankAI464517). Computational analysiswith CELERA DISCOVERY SYSTEMindicated that the EST mapped to the3�-UTR of the murine Gbx1 gene. Toisolate the complete Gbx1 cDNA, wescreened a cDNA library generatedfrom E13.5 mouse spinal cord with aprobe derived from the EST. We iso-lated two independent cDNA clones.One contained the entire ORF of themurine Gbx1 gene including 5� and 3�untranslated sequences.

Expression Analysis of Gbx1and Gbx2 During SpinalCord Development

We determined the expression of theGbx1 gene in the developing mousespinal cord by in situ hybridization.Gbx1 transcripts were first detected atE11.5 in the ventricular zone of the

spinal cord (Fig. 1A). At E12.5–13.5,Gbx1 was broadly expressed in themantle zone of the dorsal spinal cord(Fig. 1B,C). With the appearance of adiscernable dorsal horn around E14,Gbx1 expression became more re-stricted (Fig. 1D). Perinatally, Gbx1expression was observed in a narrowlayer in the superficial dorsal horn(Fig. 1F). Gbx1 expression was stilldetectable in a small number of neu-rons in the superficial dorsal horn inthe adult (not shown).

Gbx2 is closely related to Gbx1 andhas been reported to be expressed inthe neural tube (Rhinn et al., 2004).Functional analyses have demon-strated an essential role of Gbx2 dur-ing development of the mid-hindbrainboundary (Wassarman et al., 1997; forreview, see Joyner et al., 2000). As yet,a detailed analysis of the expression ofGbx2 in the spinal cord has not beenreported. We compared expression ofGbx1 and Gbx2 during spinal cord de-velopment. At E11.5, Gbx2 expressionwas detected in the dorsal ventricularzone and in the mantle zone withinthe ventral half of the neural tube(Fig. 1G). Strong expression of Gbx2was observed at E12.5 in the ventric-ular zone and the mantle zone of theentire dorsal spinal cord. At this de-velopmental stage, the expression do-mains of Gbx1 and Gbx2 overlapped(Fig. 1B and H). After E12.5, Gbx2expression was rapidly downregu-lated (Fig. 1I,J). Gbx1 and Gbx2 ap-pear thus to be transiently co-ex-pressed in neurons of the dorsal spinalcord. In addition to its dorsal expres-sion, Gbx2 is also expressed in theventral spinal cord. In this region, noGbx1 co-expression was detectable.Ventral expression of Gbx2 was ob-served between E11.5 and E14.5. In

Fig. 1. Expression analysis of Gbx1 and Gbx2 in the developing spinal cord. Vibratome sections from mouse cervical spinal cords at E11.5–E19.5 (A–J).Spinal cord tissues were hybridized with probes specific for Gbx1 (A–F) or Gbx2 (G–J). Note that both genes are transiently expressed in overlappingregions within the alar plate (B and H). ap, alar plate; vz, ventricular zone; mz, mantle zone.Fig. 2. Lbx1 controls expression of Gbx1 in the dorsal spinal cord. Immunofluorescence analysis of the developing cervical spinal cord from wildtype(A, B) and Lbx1�/� (C, D) embryos at E12.5 using antibodies directed against Gbx1 (A–D), Lbx1 (A, C), and Lhx1/5 (B, D). Within the alar plate of controlembryos, Gbx1� cells co-express Lbx1 and Lhx1/5 (A, B). In homozygous mutants, expression of either Lbx1 or Gbx1 is no longer detectable,demonstrating that Gbx1 expression in dorsal spinal neurons depends on Lbx1 function (C, D). Lhx1/5 expressing cells are still present on E12.5 inhomozygous mutants, indicating that the loss of Gbx1 expression is not caused by a depletion of the corresponding cells.Fig. 3. Characterization of Gbx1 expressing dorsal spinal neurons. Co-expression analysis of Gbx1� dorsal spinal neurons at E12.5 (A, B), E14.5 (C,D), E16.5 (E, F), and P28 (G, H). At E12.5–E14.5, Gbx1� cells co-express Pax2 (A, C) but exclude expression of Lmx1b and Tlx3 (B, D) indicating thatGbx1 expressing dorsal neurons share molecular characteristics of dILA cells. Note that only part of Pax2� cells co-express Gbx1. At E16.5, Gbx1�

neurons are detectable in the superficial dorsal horn and only few of them co-express Lbx1 (E). With the onset of GABA expression, both GABA andGbx1 are co-expressed in the superficial dorsal horn (F). At postnatal day 28 (P28), Gbx1-expressing cells are distributed throughout the entiresuperficial dorsal horn (G), and most of the Gbx1� cells co-express GABA (G, H).

Gbx1 EXPRESSION IN THE MOUSE SPINAL CORD 769

this part of the spinal cord, Gbx2 mayidentify a distinct population of ven-trally located spinal interneurons.

Characterization of Gbx1�

Neurons in the DorsalSpinal Cord

Dorsal interneurons are generatedduring two distinct waves of neuro-genesis in the spinal cord (Gross et al.,2002; Muller et al., 2002, 2005). Dur-ing the second phase of neurogenesis(E11.5–14.0), the majority of neuronsgenerated from dorsal progenitors areclass B neurons that express the ho-meodomain transcription factor Lbx1.During further development, theselate-born neurons colonize the super-ficial dorsal horn (Gross et al., 2002;Muller et al., 2002). In situ hybridiza-tion indicated that Gbx1 is expressedin the neurons generated during thesecond phase of neurogenesis. To de-fine this neuronal population, anti-Gbx1 antibodies were generated. Im-munohistological analyses of thespinal cord at E12.5 demonstratedthat Gbx1 protein was detected incells located in the alar plate, but notin the ventricular zone or in the ven-tral spinal cord. In addition, whenGbx1 in situ hybridization and Gbx1immunohistology were carried out onidentical sections, both signals werefound to co-localize. Together, this in-dicates that the antibody specificallyrecognizes Gbx1 protein (compare Fig.1B,H and Fig. 2A,B; and not shown).Further analysis revealed that Gbx1�

cells co-express Lbx1 at E12.5. Gbx1�

cells correspond thus to late-bornclass B neurons (Fig. 2A). Lbx1 is akey determinant for the specificationof class B neurons (Gross et al., 2002;Muller et al., 2002). We, therefore,tested if Gbx1 expression is detectablein the dorsal spinal cord of Lbx1 mu-tant mice, and found that Gbx1 wasabsent at E12.5 (Fig. 2C). In Lbx1 mu-tants, miss-specified neurons thatarise in the dorsal spinal cord areeliminated by apoptosis during fur-ther development (Gross et al., 2002;Muller et al., 2002). At E12.5 Gbx1�

wildtype spinal neurons co-expressthe homeodomain factor Lhx1/5 (Fig.2B). In Lbx1 mutants, Lhx1/5� neu-rons are still detectable in similarnumbers in the dorsal spinal cord(Fig. 2D), indicating that the lack of

Gbx1 expression is not caused by theapoptotic loss of dorsal neurons inLbx1 mutants. We conclude that Gbx1expression depends on Lbx1 functionand that the Gbx1� neuronal cell pop-ulation is not generated in the dorsalhorn of Lbx1 mutant animals.

Late-born class B neurons com-prise initially two neuron popula-tions, dILA and dILB, which areborn in an apparent salt and pepperpattern in the dorsal spinal cord.dILA neurons express Lbx1, Pax2,and Lhx1/5, whereas dILB cells ex-press Lbx1, Lmx1b, and Tlx3 (Mulleret al., 2002). At E12.5, only a sub-population of the Lbx1� cells co-ex-pressed Gbx1 (Fig. 2A). We, there-fore, characterized the Gbx1�

neuronal population further. AtE12.5 and 14.5, Gbx1� neurons co-express the transcription factorsLhx1/5 and Pax2, but are negativefor Lmx1b and Tlx3. This indicatesthat Gbx1� neurons correspond tothe dILA neuronal subtype (Figs. 2B,3A–D, and not shown). We observedat no developmental stage that allPax2� or Lhx1/5� cells expressedGbx1, and many Pax2� or Lhx1/5�

cells that do not express Gbx1 weredetectable. Thus, Gbx1 expressiondistinguishes a subpopulation ofdILA cells (Figs. 2B, 3A,C, and notshown). We analyzed Gbx1-express-ing cells also during the maturationof the dorsal spinal cord. At E16.5,most Gbx1� cells were located in thesuperficial dorsal horn (Fig. 3E). Atthis stage, only few Gbx1� neuronsco-expressed Lbx1; however, most ofthe Gbx1� cells co-expressed Pax2 orLhx1/5 (Fig. 3E, and not shown). Ithas been shown previously thatdILA neurons undergo GABAergicdifferentiation (Cheng et al., 2004).We have, therefore, tested if mark-ers for GABAergic differentiationare expressed in Gbx1� cells. WhenGABA� or Gad67� neurons are firstdetectable in the dorsal spinal cord,the GABA� or Gad67� neurons co-express Gbx1. Gbx1� cells can thusdifferentiate into GABAergic neu-rons (Fig. 3F, and not shown). Re-cent studies demonstrated that Pax2is essential for GABAergic differen-tiation in the dorsal spinal cord,since GABAergic differentiationdoes not occur correctly in Pax2 mu-tant mice (Cheng et al., 2004). Gain-

of-function studies demonstratedthat Pax2, when over-expressed inthe chick spinal cord, is however notsufficient to induce GABAergic dif-ferentiation (Cheng et al., 2004).This suggests that additional factorsmight be involved in this process.

In order to further determine thefate of Gbx1� cells, we have analyzedGbx1 expression in the early postna-tal (P7) and in the adult spinal cord(Fig. 3G and H). Here, Gbx1 identifiesa small population of interneuronsthat are scattered throughout the en-tire superficial dorsal horn. Very fewGbx1� cells are located in the deeperlayers of dorsal horn, close to the cen-tral canal (Fig. 3G and not shown).GABA expression was found to beabundant in the adult superficial dor-sal horn. Within this region, the greatmajority of Gbx1� neurons co-expressGABA, further indicating that Gbx1comprises a distinct subpopulation ofGABAergic dorsal spinal neurons(Fig. 3G and H).

We have analyzed here the expres-sion pattern of the homeodomain tran-scription factor Gbx1 during develop-ment of the mouse spinal cord. Weshow that Gbx1 expression is re-stricted to the dorsal spinal cord andthat it coincides with the generation ofa late neurogenic cell population. Im-munohistological analyses identifiedGbx1� neurons as a subpopulation ofdILA neurons that require the home-odomain transcription factor Lbx1 fordevelopment. As development pro-ceeds, Gbx1� interneurons differenti-ate into a subpopulation of GABAergicneurons and colonize the superficiallayers of the dorsal horn. Physiologi-cal experiments have indicated thatGABAergic neurons are not homoge-neous, but GABAergic subpopulationsare ill defined on a molecular level(Heinke et al., 2004). Further work isrequired to define the functional prop-erties of the Gbx1� population ofGABAergic neurons, and to determinethe role of Gbx1 in the development ofthis neuronal lineage.

EXPERIMENTALPROCEDURES

In Situ Hybridization

Whole mount in situ hybridizationwas performed as previously de-

770 JOHN ET AL.

scribed (Britsch et al., 1998). In brief,spinal cords were micro-dissectedfrom mouse embryos at E12.5–19.5and fixed overnight with 4% PFA. Af-ter whole mount in situ hybridization,cervical spinal cords that werematched for their axial levels wereembedded in 20% gelatin and post-fixed for several days. Thirty �m vi-bratome sections were examined on aZeiss Axiophot microscope. For gener-ation of Gbx1 specific riboprobes aSalI fragment of a plasmid that con-tained the entire Gbx1 cds was used. Aplasmid for the generation of Gbx2specific probes was obtained from A.Joyner (Skirball Institute, NY).

Generation of Anti-Gbx1Antisera

A 741-bp fragment from the murineGbx1 cDNA, corresponding to aa 61–308 was amplified by PCR with thefollowing primers: Gbx1upper 5�-CA-TATGCAGAGAGCGGCAGGCGGCG-GC and Gbx1lower 5�-GGATCCCC-TGTGGCCACTGGTGTCCCCTCCTC.The PCR fragment was cloned into thebacterial expression vector pET-14b(Novagen), which provided coding se-quences for a His6-tag. His6-Gbx1was propagated in BL21(DE3)pLysScells, affinity purified on TALONmetal resin (BD Biosciences), and in-jected into rabbits and guinea pigs(Sequence Laboratories, Gottingen,Germany).

Immunohistology

For immunofluorescence staining, em-bryonic mouse tissue was fixed with4% PFA in 0.1 M sodium phosphatebuffer (pH 7.4). After cryoprotectiontissue was embedded into OCT com-pound (Sakura) and 12 �m cryosec-tions were made from matched cervi-cal parts (C5/6) of spinal cords on aMicrom cryostat. Stained sectionswere examined on a confocal micro-scope (Pascal, Zeiss). The followingantibodies were used: rabbit andguinea pig anti-Gbx1, rabbit andguinea pig anti-Lbx1 (Muller et al.,2002), rabbit anti-Tlx3 (Muller et al.,2005), guinea pig anti-Lmx1b (giftfrom T. Jessel, New York), rabbit anti-

Pax2 (Chemicon, Temecula, CA), rab-bit anti-GABA (Sigma, St. Louis, MO),rabbit anti-Gad67 (Sigma), mousemonoclonal anti-Lhx1/5 (Developmen-tal Studies Hybridoma Bank, Univer-sity of Iowa), and fluorophore-conju-gated secondary antibodies (Dianova).

Animals

Heterozygous Lbx1 mutant mice wereintercrossed to obtain homozygousmutant embryos. Genotyping was per-formed as previously described (Broh-mann et al., 2000).

ACKNOWLEDGMENTSThe authors thank Verena Sasse andKarin Gottschling for excellent techni-cal assistance, Carmen Birchmeier,Thomas Muller, and Martin Sieber forcritical reading of the manuscript andfor helpful discussions. We gratefullyacknowledge the following scientistsfor gifts of antibodies and plasmids:Alexandra Joyner (New York),Thomas Jessell (New York), andThomas Muller (Berlin). This workhas been supported by a grant fromthe DFG (SFB 665-A1) to S.B.

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