Molecular and Cellular Neuroscience 21, 410–420 (2002)

CNS Expression Pattern of Lmx1b andCoexpression with Ptx Genes Suggest FunctionalCooperativity in the Development of ForebrainMotor Control Systems

Ceriel H. J. Asbreuk, Christina F. Vogelaar, Anita Hellemons,Marten P. Smidt, and J. Peter H. BurbachRudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy,University Medical Center, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands

In the central nervous system, acquisition of regionalspecification is an important developmental process. Theregional specification is reflected by restricted and over-lapping expression of homeobox genes, which are regu-lators of this event. Here, we detail the expression patternof Lmx1b during late embryonic brain development andshow that this gene is expressed in multiple regions anddiverse sets of neurons. Noteworthy, the Lmx1b expres-sion domain is shared by Ptx2 in posterior hypothalamicregions and by Ptx3 in the dopaminergic neurons of theventral midbrain. In addition, the mutual cofactor Ldb1 isexpressed in these regions. The expression of these genesets is maintained in the adult brain. The subthalamicnucleus, where Lmx1b is coexpressed with Ptx2, and thesubstantia nigra/ventral tegmental area, where Lmx1b iscoexpressed with Ptx3, are both ancillary nuclei of themotor control circuitry, but use different neurotransmit-ters. These data point to a combinatorial gene networkthat allows Lmx1b to diversify its regulatory actions bycooperation with specific Ptx genes.

INTRODUCTION

The nervous system acquires cell diversity throughdevelopmentally regulated cell fate restriction. Early indevelopment, after closure of the neural tube, brainpatterning takes place. Regions are specified by restrict-ing multipotent precursor cells to appropriate fate de-cisions. Different regions acquire specific responsive-ness to cues that instruct cell fate decisions and cell-intrinsic properties have generated characteristic

lineage trees. In brain patterning, transcription factorsof the homeobox gene family play a significant role

410

(Lumsden and Krumlauf, 1996; Puelles and Rubenstein,1993). In the ventral neural tube, graded activity of themorphogen sonic hedgehog (Shh) results in patterningof five main progenitor cell domains. This patterning isgenerated by homeobox genes that are either repressedor activated by specific Shh concentrations. The combi-natorial action of these homeobox genes is crucial, andcross-repressive interactions between the genes defineboundaries between ventral regions (Jessell, 2000). Sim-ilar cross-repressive interactions between homeoboxgenes define other boundaries, such as the formation ofthe midbrain–hindbrain boundary that involves Gbx2and Otx2 (Broccoli et al., 1999; Millet et al., 1999; Sime-one, 2000). Transcription factor interactions determinenot only boundaries between regions but also the dif-ferentiation of cell lineages, as highlighted by the gen-eration of diverse endocrine cells in the pituitary gland(Dasen et al., 1999; Dasen and Rosenfeld, 1999). Singleor combinatorial expression of homeobox genes marksregions of the central nervous system that are anatom-ically coherent (Bishop et al., 2000; Puelles et al., 2000;Smidt et al., 1997; Sussel et al., 1999), share cellularproperties such as neurotransmitter phenotype (Arberet al., 1999; Asbreuk et al., 2002; Pattyn et al., 1999; Smidtet al., 1997; Thaler et al., 1999), or are engaged in thesame physiological circuitry (Lee et al., 2001; Liu et al.,1994; van Schaick et al., 1997). In the developing spinalcord, specific expression of homeobox genes, particu-larly of the LIM class, has aided in discrimination ofseveral neuronal subtypes (Sharma and Peng, 2001;Tsuchida et al., 1994). Albeit the full transcriptional code

MCN

doi:10.1006/mcne.2002.1182

for specification of neuronal subtypes requires addi-tional genes of different classes (Marquardt and Pfaff,

1044-7431/02 $35.00© 2002 Elsevier Science (USA)

All rights reserved.

2001), LIM-HD genes play such a prominent role in theacquisition of regionalization, axonal projection pat-terns, and neurotransmitter phenotypes that a LIM-HDcombinatorial code was suggested (Jessell, 2000; Thor etal., 1999). Similarly, in the brain a LIM-HD combinato-rial code may be applicable (Bachy et al., 2001; Retaux etal., 1999).

Examples of interactions between LIM-HD and othertranscription factors are found in the pituitary gland(Dasen and Rosenfeld, 1999), where LIM domains, inaddition to mediating direct transcription factor inter-actions, were shown to bind to cofactors that do notbind DNA. These Ldb cofactors Ldb1/CLIM2/NLI/Chip and Ldb2/CLIM1 can homodimerize and formmultiprotein complexes consisting of LIM-HD, LMO,Ptx, bHLH, and GATA transcription factors (Bach,2000). Cooperation of Ldb cofactors with LIM-HD pro-teins and other factors is suggested to support remoteenhancer–promoter interactions (Morcillo et al., 1997).

Ptx genes belong to the subfamily of K50 paired-likehomeodomain genes (Galliot et al., 1999). Ptx1 (Pitx1,P-otx, Hindfoot) was isolated first, on the basis of inter-actions with the Pit1 protein (Szeto et al., 1996) and withregulatory sequences of the POMC gene (Lamonerie etal., 1996), and the gene product was shown to be ex-pressed in the developing pituitary gland, firstbranchial arch, duodenum, and hindlimb. And indeed,Ptx1 is necessary for development of the hindlimb,mandible, and some pituitary cell lines (Lanctot et al.,1999; Szeto et al., 1999). Ptx2 (Pitx2, Rieg, Brx, Otlx2, Arp)expression overlaps with Ptx1 in the pituitary and par-tially in craniofacial mesenchyme. In the developingbrain, Ptx2 expression follows the prosomeric model(Rubenstein et al., 1994) and is confined to the basalplate of the caudal prosencephalon and rostral mesen-cephalon (Muccielli et al., 1996). A second domain isfound at the alar plate of the rostral mesencephalon.Later expression is still detected in most of these areas,in the mammillary complex, including the subthalamicnucleus, the zona incerta, the pretectum, and the supe-rior colliculus (Muccielli et al., 1996).

In adult brain, Ptx2 expression was identified in thesubthalamic nucleus, posterior hypothalamus, medialmammillary nucleus, supramammillary nucleus, thered nucleus, and the superior colliculus deep gray layer(Smidt et al., 2000b). Mutations in the Ptx2 gene havebeen associated with Rieger syndrome (Semina et al.,1996), and Ptx2 is involved in the Nodal/Sonic hedge-hog pathway, which determines left/right polarity ofmesoderm-derived organs such as heart, gut, and stom-ach (Logan et al., 1998; Lu et al., 1999; Piedra et al., 1998;Ryan et al., 1998; Yoshioka et al., 1998). The third Ptx

gene, Ptx3 (Pitx3), has a highly restricted expressionpattern in mesencephalic dopaminergic neurons. Dur-ing development, these encompass the ventral tegmen-tum, and in adult brain the substantia nigra and ventraltegmental area are marked by Ptx3 (Smidt et al., 1997).

A striking synergistic action of Ptx1 and Lhx3 to-gether with Ldb2 was shown in the activation of the�GSU promoter (Bach et al., 1997), suggesting that Ptx1and Lhx3 cooperate in the activation and regulation ofglycohormone genes in the pituitary gland. In thatstudy, it was noted that related genes show overlappingexpression patterns and functions, such as Otx2 andLhx1 and Ldb2. We suggest more such possible func-tional relationships between Ptx, Lhx, and Ldb genes inthe development of the ventral forebrain. We have pro-posed Lmx1b and Ptx3 to be active in a developmentalcascade in mesencephalic dopaminergic (mesDA) neu-rons (Smidt et al., 2000a), suggesting that cooperativeactivation of target genes in dopaminergic midbrainneurons may well be possible. Unlike Ptx3, brain ex-pression of Lmx1b can be detected outside the mesDAsystem. Closer examination of the Lmx1b expressionpattern in the central nervous system, with special at-tention for overlapping patterns with Ptx factors couldprove worthwhile.

Lmx1b expression in the central nervous system hasnot been described in detail. In the periphery, Lmx1b isknown to be responsible for dorsoventral patterning oflimbs, by specifying dorsal fate under control of Wnt7a(Riddle et al., 1995; Vogel et al., 1995). Loss of Lmx1bresults in nail patella syndrome, a disorder character-ized by limb-patterning defects and kidney malfunc-tions (Chen et al., 1998; Dreyer et al., 1998). We previ-ously identified Lmx1b as a homeobox gene expressedin the ventral forebrain (Smidt et al., 2000a). Lmx1bexpression has been reported in the developing spinalcord: in the roofplate, floorplate, and D4 interneurons(Gowan et al., 2001; Matise and Joyner, 1997; Pierani etal., 2001; Smidt et al., 2000a; Yuan and Schoenwolf,1999). In addition to expression at the dorsal and ven-tral midline, Lmx1b was detected in chick caudal dien-cephalon and in a ring just rostral to the isthmus (Ad-ams et al., 2000), where its expression is maintained byFGF8, and Lmx1b in turn maintains expression of Wnt1(Adams et al., 2000).

These preliminary expression data suggest thatLmx1b may play an important regulatory role in differ-ent parts of the central nervous system, and that itsmultiple involvements require a regulatory network ofcooperation with other transcription factors. In thisstudy we detail the neural systems that express Lmx1band focus on Ptx genes as potential cooperative part-

411Coexpression of Lmx1b and Ptx Genes in the CNS

ners. The results reveal a notable overlapping expres-sion in systems belonging to the same physiologicalcircuitry.

RESULTS

In order to start understanding the role of Lmx1b indifferentiating neurons, its expression in mouse brainwas studied during late embryonic development. Fig-ure 1 shows the expression pattern at E14.5 in sagittalsections. The most rostral area where Lmx1b was de-tected comprised brain regions that will give rise to thesubthalamic nucleus, the posterior hypothalamus, andpart of the mammillary bodies (Figs. 1C and 1D). Al-though discrete regions were discriminated, the rostralexpression field of Lmx1b formed a continuity (Fig. 1D).More caudally, Lmx1b was detected in the tegmentum,especially at the ventral midline (Figs. 1B–1F). Thisdomain will give rise to the substantia nigra and theventral tegmental area. Dorsally, Lmx1b was expressedin the neuroepithelium of the pretectum and the tectum(Figs. 1D and 1E). Directly caudal to the tegmentum,Lmx1b was detected in the isthmic neuroepithelium(Fig. 1F). In the pons, ventral neuroepithelium wasLmx1b-positive and differentiating fields in the anteriorand posterior pons (Figs. 1A, 1B, 1F, and 1G). Many ofthese pontine areas will develop into Raphe nuclei.Additionally, Lmx1b was detected in the differentiatingfield of the principle sensible trigeminal nucleus (Fig.1G). Dorsally, a small patch of cells in the cerebellarneuroepithelium was Lmx1b-positive. Finally, in thespinal cord, Lmx1b expression was most prominent dor-sally, and more faint at the ventral side (Figs. 1D, 1E,and 1H).

To monitor spatiotemporal aspects of Lmx1b expres-sion during brain development, the rostral Lmx1b-pos-itive area was compared at different stages from E12.5to E16.5 (Fig. 2). The area comprising subthalamic nu-cleus, posterior hypothalamus, and mammillary nucleiconsistently remained Lmx1b-positive over time. Ap-parently, surrounding areas of thalamus and hypothal-amus expand at these late developmental stages, whilethe ventral area expressing Lmx1b remained constricted.

The tegmental area acquired an Lmx1b expressionpattern that became more restricted to substantia nigraand ventral tegmentum area as development pro-gressed. At late developmental stages (E16.5), Lmx1bwas no longer detected in neuroepithelia of pretectum,tectum, and cerebellum.

Thus, Lmx1b expression in E12.5 differentiating fieldspersisted until at least E16.5 in subthalamic nucleus,

posterior hypothalamus, mammillary nuclei, and sub-stantia nigra and ventral tegmental area, while it dis-appeared from undifferentiated neuroepithelia of tec-tum and cerebellum.

Expression that is persistent at late developmentalstages often resembles adult expression. Therefore, theexpression of Lmx1b was determined in coronal sectionsfrom adult mouse brain by in situ hybridization (Fig. 3).It should be noted that the hippocampus and cerebel-

FIG. 1. Lmx1b expression at E14.5. Saggital section of the embryonichead showing Lmx1b expression in the mammillary region/subtha-lamic nucleus, ventral tegmentum, anterior, and posterior pontineregions and dorsal spinal cord. PH, posterior hypothalamus; MM,mammillary region; AP, anterior pons; PP, posterior pons; TG, teg-mentum; SpC, spinal cord; STN, subthalamic nucleus; PT, pretectum;T, tectum; I, isthmic neuroepithelium; n5p, principle sensible trigem-inal nucleus; C, cerebellar neuroepithelium.

412 Asbreuk et al.

lum display background staining due to their denselypopulated granule cell layers, sense and antisenseprobes alike. The most rostral Lmx1b expression wasdetected in the subthalamic nucleus (Fig. 3A). Also, theposterior hypothalamus, premammillary nucleus, andsupramammillary nucleus were found to containLmx1b-expressing cells (Figs. 3B–3F). Lmx1b expressionwas not detected in the medial mammillary nuclei.

Directly caudal to the STN, Lmx1b was detected in thesubstantia nigra and in the adjacent ventral tegmentalarea (Figs. 3E–3G). This expression occurred at the samerostro-caudal level as the most caudal mammillary ex-pression in the lateral mammillary nucleus and thesupramammillary nucleus. Caudal to the latter, the in-terpeduncular nucleus contained Lmx1b-expressingcells (Figs. 3G and 3H).

In the pons, Lmx1b was detected in several of theRaphe nuclei: the dorsal nucleus Raphe, the caudallinear nucleus Raphe, and the superior central nucleusRaphe (medial Raphe nucleus). In addition, Lmx1b wasfound in the parabrachial nucleus and the pontine re-ticular nucleus (Figs. 3H–3M). Ventrolaterally, Lmx1bwas expressed in the principle sensible trigeminal nu-cleus (Figs. 3K–3M).

In adult rat spinal cord Lmx1b was detected all overthe gray matter. Expression levels appeared highest inthe dorsal horn (Figs. 3N and 3O).

In conclusion, Lmx1b was detected in all brain areaswhere late embryonic expression persisted, in restrictedneuronal populations, and was not found in those areaswhere early embryonic neuroepithelium expressedLmx1b, such as the tectum and cerebellum.

The expression pattern of Lmx1b in the adult brainsuggested overlapping patterns with other homeoboxgenes. In particular, a possible overlap with Ptx ho-

meobox genes was striking. Therefore, we comparedthe expression patterns of Lmx1b and the brain-ex-pressed Ptx genes Ptx2 and Ptx3 by radioactive in situhybridization on coronal sections of adult rat brain.Sections at similar positions were compared. The ex-pression pattern of Ptx2 was more restricted than that ofLmx1b. Clearly, Lmx1b and Ptx2 expression patternswere overlapping in the STN and posterior hypothala-mus (Figs 4A, 4F, 4B, and 4G). Also in some, but not all,mammillary nuclei, Lmx1b and Ptx2 expression over-lapped (not shown).

Interestingly, in several of the Lmx1b-positive regionsin which Ptx2 was not expressed, specifically in thesubstantia nigra and ventral tegmental area, Lmx1b ex-pression was found overlapping with Ptx3 (Figs. 4C–4Eand 4H–4J).

Notably, Lmx1b is expressed in a restricted fashion insome ventral brain nuclei. The rostral part of the Lmx1b-positive area, comprising STN, PH, and mammillarynuclei, is coexpressing Ptx2. A more caudal domain,comprising the mesencephalic dopaminergic neuronsof the substantia nigra and VTA, is coexpressing Ptx3.

To show that Ldb, a cofactor able to bridge Lhx andPtx proteins (Bach et al., 1997), is expressed in overlap-ing regions with Lmx1b, Ptx2, and Ptx3, in situ hybrid-ization of Ldb1/NLI was performed on coronal sectionsfrom adult mouse brain. Ldb gene expression has beenshown to be quite ubiquitous (Bach et al., 1997; Bach,2000; Jurata et al., 1996). In Fig. 5, Ldb1 expression in theSTN and SNc is confirmed (Figs. 5B and 5D). Ldb1 wasbroadly expressed in the brain (B, D, data not shown),while the sense control showed no labeling (Figs. 5Aand 5C). So, the areas where Lmx1b is coexpressed withPtx2 or Ptx3 express the cofactor Ldb1 as well.

FIG. 2. Lmx1b expression during development. Midline expression of Lmx1b is compared in saggital sections from embryonic head at E12.5,E14.5, and E16.5. Note that dorsal expression in the neuroepithelium of the tectum is transient, while that in ventral tegmentum and posteriorhypothalamus is persistent. Scale bar � 250 �m. PH, posterior hypothalamus; MM, mammillary region; STN, subthalamic nucleus; SN/VTA,substantia nigra/ventral tegmental area.

413Coexpression of Lmx1b and Ptx Genes in the CNS

DISCUSSION

The key question in this study was whether Lmx1bcan display functional diversity in different regions

where it is expressed. In this respect, a striking featureemanating from the expression data obtained in thisstudy, was the complementary expression of homeoboxgenes Ptx2 and Ptx3, overlapping with Lmx1b. The ex-

FIG. 3. Expression pattern of Lmx1b in the adult CNS. Coronal sections of adult mouse brain showing Lmx1b prominently in the subthalamicnucleus, some mammillary nuclei, substantia nigra/VTA, medial Raphe nuclei, prabrachial nucleus, pontine reticular nucleus, principle sensiblenucleus of the trigeminal nerve, and throughout the gray matter of the adult rat spinal cord. STN, subthalamic nucleus; PH, posteriorhypothalamus; PM, premammillary nucleus; SUM, supramammillary nucleus; LM, lateral mammillary nucleus; SN, substantia nigra (parscompacta); VTA vantral tegmental area; IPN, interpeduncular nucleus; DR, dorsal nucleus Raphe; CLI, central linear nucleus Raphe; CS, superiorcentral nucleus Raphe; PRN, pontine reticular nucleus; PB, parabrachial nucleus; PSV, prinsiple sensible trigeminal nucleus; DH, dorsal horn;c, central canal; VH, ventral horn.

414 Asbreuk et al.

pression of Lmx1b in a ventral midline domain, andPtx2 and Ptx3 in rostral and caudal subdomains, respec-tively, is summarized in Fig. 6. This observation isparticularly interesting because nuclei in which thiscoexpression occurs—STN for Ptx2/Lmx1b andVTA/SN for Ptx3/Lmx1b—are strongly linked in an in-

tegrated neuronal network that serves a physiologicalsystem. Both the STN and the SN are ancillary nuclei ofthe indirect pathway of the basal ganglia. Besides manyconnections to the basal ganglia they have been shownto be reciprocally interconnected (Nambu et al., 1996;Parent et al., 2000; Smith and Kieval, 2000). To clarifythe position of the SN and STN in the network, a briefdescription of this system and afferent and efferentprojections is given here and schematically depicted inFig. 7. The major target, by far, of midbrain dopaminer-gic neurons is the striatum. However, the globus palli-dus, ventral pallidum, and subthalamic nucleus receivesignificant dopaminergic input from SNc and VTA neu-rons (Smith and Kieval, 2000). Nigrostriatal projectionsseem to obey a simple rule, which is that the degree ofarborization of any given SNc neuron is inversely pro-portional to its degree of branching in extrastriatalstructures (Parent et al., 2000). The subthalamic nucleusis classically known to be reciprocally linked to theglobus pallidus (Parent and Hazrati, 1995). However, itis now known that subthalamic projection neurons in-teract with neurons in the globus pallidus, entopedun-cular nucleus, the substantia nigra pars reticulata, and,sparsely, in the striatum (Parent et al., 2000). In addition,some of the fibers projecting to the SNr ascend alongdopaminergic cell columns of the SNc. Also limbic do-paminergic neurons of the VTA are targeted by STNprojection neurons (Parent and Hazrati, 1995). The STNreceives massive input from the GP and the cerebralcortex and also from the centromedial/parafascicularthalamic complex, the pedunculopontine nucleus, dor-sal Raphe, and, importantly, the SNc (Parent and Haz-rati, 1995). An important notion is the fact that neuronsof the GP do not project to the STN territory that con-tains most of the neurons projecting to the basal gangliaoutput nuclei, entopeduncular nucleus, and SNr, but toa territory that primarily projects back to the GP (Parentand Hazrati, 1995).

The STN and SN share several characteristics. Botharise from a contiguous region of the ventral midbrain.They are intricately involved in the same circuitry, andwe found overlapping expression of a set of homeoboxgenes (Fig. 7). Both nuclei receive input from cortex,thalamus, and pedunculopontine nucleus, and bothproject to the striatum, pallidum, and each other. How-ever, SNc axons project predominantly to the striatum,with far fewer collaterals connecting to the pallidum.The STN, on the other hand, projects massively to thepallidum with only a few axons running to the stria-tum. The STN and SN have the possibility to innervatethe same targets, but they do not necessarily do so.Furthermore, they use a different neurotransmitter. The

FIG. 4. Complementary expression of Ptx2 and Ptx3 within theLmx1b expression domain. Anterior Lmx1b-positive nuclei in the pos-terior hypothalamus coexpress Ptx2, whereas the more dorsal Lmx1b-positive nuclei in the ventral midbrain coexpress Ptx3. STN, subtha-lamic nucleus; PH, posterior hypothalamus; SNc, substantia nigrapars compacta; VTA, ventral tegmental area.

415Coexpression of Lmx1b and Ptx Genes in the CNS

STN is composed of glutamatergic neurons, and istherefore excitatory, while the SNc consists of modula-tory dopaminergic neurons.

Recently, we have shown that Lmx1b and Ptx3 are notinvolved in specification of the neurotransmitter phe-notype of the mesDA neurons (Smidt et al., 2000a). Ptx2,which is expressed in the glutamatergic STN, has evenbeen implicated in GABAergic development (West-moreland et al., 2001). These data rather suggest that notthe neurotransmitter identity but other characteristics,like axon trajectory, are regulated by Ptx genes. Analy-ses in Caenorhabditis elegans have also shown that Ptxgenes of different species are functionally homologous.Taken together, a combinatorial cooperation of Lmx1bwith Ptx genes that differ markedly in spatiotemporalexpression can result in diversification of the regulatoryroles of Lmx1b. In this respect, the complementary ex-pression of Ptx2 and Ptx3 within a larger domain ofLmx1b expression is intriguing. These expression datapoint out differences between STN and SN, such asexpression of Ptx2 versus Ptx3, but they share the ex-pression of Lmx1b in both the STN and the SN. Presum-ably, the germinal sources of STN and SN do sharesome characteristics. Using tritiated thymidine, it hasbeen shown that the germinative zone lying caudally

along the dorsal aspect of the mammillary recess givesrise to subthalamic neurons and also to the supramam-millary and submammillothalamic nuclei (Marchand,1987). Differentiating neurons migrate laterally andthen dorsally and tangentially to reach their adult po-sition. This, together with additional studies indicatedthat the functionally interrelated interpeduncular nu-cleus, substantia nigra and subthalamic nucleus are notderived from the same germinal source but from threedistinct neuroepithelial domains (Marchand, 1987). Thedevelopmental expression pattern of Lmx1b, as shownhere, combined with that of Ptx2 (Muccielli et al., 1996)allows for distinction of future STN and supramammil-lary nuclei during development, while Ptx3 marks themesDA system (Smidt et al., 1997, 2000a).

The data suggest a functional relationship betweenLmx1b and Ptx genes, but the nature of cooperationbetween Lmx1b and the Ptx genes is not exactly known.However, parallels may be drawn between neuronsand endocrine cells of the pituitary gland or pancreaticislets, where examples of interactions of Lhx and Ptxtranscription factors are found (Dasen and Rosenfeld,1999; German et al., 1992; Johnson et al., 1997). Lhx3interactions with Pit1 promote synergistic activationevents of prolactin ( prf ), thyroid-stimulating hormone beta

FIG. 5. Expression of Ldb1/NLI in the STN and SN/VTA. Ldb1 is expressed throughout the adult brain, and was clearly detected in the STNand SN (B, D). The sense control shows no labeling (A, C). Scale bar � 250 �m. STN, subthalamic nucleus; v3, third ventricle; SNc, substantianigra, pars compacta; VTA, ventral tegmental area.

416 Asbreuk et al.

(TSH�), and Pit1 gene regulatory elements (Bach et al.,1995), while Ptx1 activates promoters of the �GSU gene,POMC, and growth hormone and synergizes with Pit1 onthe prolactin distal enhancer (Szeto et al., 1996). Lmx1aand Lmx1b can interact with the bHLH gene E47/Pan-1and synergize with it to activate the insulin promoter(German et al., 1992; Johnson et al., 1997). A strikingsynergistic action of Ptx1 and Lhx3 together with Ldb2was shown in the activation of the �GSU promoter(Bach et al., 1997). In that study, it was noted that related

genes show overlapping expression patterns and func-tions, such as Otx2 and Lhx1 and Ldb2. The coexpressionof Lmx1b with Ptx2 or Ptx3, as demonstrated in thisstudy, points at such a possible functional relationship.Since Ldb genes are expressed in the STN and SN/VTA(Fig. 5), in addition to other areas (Bach et al., 1997;Bach, 2000; Jurata et al., 1996), higher-order complexesare likely. It raises the issue of a Lmx1b–Ptx2 interactionconferring a different gene-controling activity thanLmx1b–Ptx3. From genetic experiments in C. elegans it

FIG. 6. Schematic overview showing expression paterns of Lmx1b, Ptx2, and Ptx3 at E12.5, in adult mouse posterior hypothalamus and in adultmouse anterior midbrain. Lmx1b (gray) is expressed along the ventral midline at E12.5. The most anterior domain is in prosomere 4, forming the futuremammilary region and subthalamic nucleus, and this domain is essentially continuous through the hindbrain. In the spinal cord, almost a Lmx1bexpression is detected in a dorsal domain. Additional dorsal domains are found in the tectum/pretectum, isthmus region, anterior pons, and posteriorpons. Ptx2 (vertical stripes) expression coincides with Lmx1b in its rostral domain, but shows some additional dorsal expression, i.e., in the ZLI.Remarkably, Ptx3 (horizontal stripes) also coincides with Lmx1b, in a ventral mesencephalic area just caudal to the Ptx2 domain. Adapted fromMucchielli (Muccielli et al., 1996). Os, optic stalk; p1–p6, prosomeres 1–6 (Rubenstein et al., 1994); ZLI, zona limitans intrathalamica; MES, mesenceph-alon; i, isthmus; r1–r8, rhombomeres 1–8 (Rubenstein et al., 1994); SpC, spinal cord. Lmx1b (gray) is expressed in the adult STN, PH, PM, and SUM (B),and in the SNc and VTA. This expression overlaps with Ptx2 (vertical stripes), or with Ptx3 (horizontal stripes). Additional Ptx2 expression is foundin the MM and TM. Adapted from Swanson (Swanson, 1992). V3, third ventricle; MM, medial mammillary nucleus; PM, premammillary nucleus; TM,tuberomammilary nucleus; SUM, supramammillary nucleus; STN, subthalamic nucleus; pm, principle mammillary tract; cpd, central peduncle; VL,lateral ventricle; SNc, substantia nigra pars compacta; VTA vantral tegmental area; AQ, cerebral aquaduct.

417Coexpression of Lmx1b and Ptx Genes in the CNS

has been concluded that the endogenous Ptx-like genein C. elegans, unc-30, can be substituted by mammalianPtx2, which rescues the neuronal unc-30 null phenotype(Westmoreland et al., 2001). If Ptx2 and Ptx3 have sim-ilar properties, they still differ in temporal expression:Ptx2 is already expressed in the developing mouse em-bryo at E9.5 (Muccielli et al., 1996), while Ptx3 is onlyinduced around E12. This would allow to provide in-dividual specificity to the spatiotemporal expression ofPtx2 versus Ptx3.

Interestingly, the C. elegans ortholog of Lmx1b, lim-6,and the Ptx ortholog unc-30 are both expressed inGABAergic neurons, but in complementary, nonover-lapping sets (Hobert et al., 1999). Lim-6 and unc-30 eachact in a subsets of GABAergic neurons to determinetheir correct axonal trajectory and regulate the expres-sion of glutamic acid decarboxylase, the rate-limitingenzyme of GABA synthesis in these neurons (Hobert etal., 1999). In mammalians, Lmx1b, Ptx2, and Ptx3 mayinfluence similar processes in the neurons where theyare expressed.

EXPERIMENTAL METHODS

CBAxC57BL6 mice were mated, and the morning whena vaginal plug was detected was considered E0.5. Preg-nant mice were killed by cervical dislocation, and em-bryos were dissected, and directly frozen in powdereddry-ice. Digoxigenin labeled sense and antisense RNAprobes were generated according to the manufacturer’sinstructions (Roche Molecular Biochemicals). In situ hy-bridization with DIG-probes was performed essentiallyaccording to Jessel (http://cpmcnet.columbia.edu/dept/neurobeh/jessell/insitu.html) (Schaeren-Wiemers and

Gerfin-Moser, 1993). Briefly, hybridization was carried outat 72°C, in 50% formamide and 5x SSC. The Digoxigeninwas detected with an alkaline phosphatase-labeled anti-body (Roche Molecular Biochemicals) using NBT/BCIP asa substrate.

Sections from rat and mouse brain were preparedand subjected to radioactive in situ hybridization essen-tially as described (Lopes da Silva et al., 1995). Sectionswere exposed on Biomax MR films (Kodak) or Betamaxfilms (Amersham Life Science).

The following probes were used: for radioactive insitu hybridization a rat Ptx2EcoRI fragment (332 bp), arat Ptx3 EcoRI/PstI fragment (285 bp), and a PCR frag-ment from rat the Lmx1b homeobox region (115 bp;AF390073) were labeled with 35S, and for nonradioac-tive in situ hybridization the mouse Lmx1b cDNA (1262bp; provided by R. L. Johnson) and mouse Ldb1/NLICDS (1128 bp; provided by G. N. Gill) were labeledwith Digoxigenin (Roche Molecular Biochemicals).Sense probes were used as control and did not showany labeling.

ACKNOWLEDGMENTS

We are grateful to G. Gill and R. Johnson for supplying Ldb1 (NLI)and Lmx1b probes and for discussing data. This work was supported bygrants of the Netherlands Organization for Scientific Research NWO-SLW (805-33.211-P) for C.A. and an NWO fellowship (903-42-7S) for M.S.

REFERENCES

Adams, K. A., Maida, J. M., Golden, J. A., and Riddle, R. D. (2000). Thetranscription factor Lmx1b maintains Wnt1 expression within theisthmic organizer. Development 127: 1857–1867.

FIG. 7. Schematic view of brain areas participating in motor control, with connections and expression of Lmx1b, Ptx2, and Ptx3. Lmx1b isexpressed in ancillary nuclei of the motor control circuitry. Remarkable coexpression occurs with Ptx2 in the STN and Ptx3 in the SNc. The STNand SNc receive from many the same nuclei and both send efferents to each other an the GP. Gpe, globus pallidus externa; Gpi, globus pallidusinterna; EP, entopeduncular area; STN, subthalamic nucleus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; PPN,pedunculopontine nucleus; DR, dorsal Raphe.

418 Asbreuk et al.

Arber, S., Han, B., Mendelsohn, M., Smith, M., Jessell, T. M., andSockanathan, S. (1999). Requirement for the homeobox gene Hb9 inthe consolidation of motor neuron identity [see comments]. Neuron23: 659–674.

Asbreuk, C. H. J., van Schaick, H. S. A., Cox, J. J., Kromkamp, M.,Smidt, M. P., and Burbach, J. P. H. (2002). The homeobox genesLhx7 and Gbx1 are expressed in the basal forebrain cholinergicsystem. Neuroscience 109: 287–298.

Bach, I. (2000). The LIM domain: Regulation by association. Mech.Dev. 91: 5–17.

Bach, I., Carriere, C., Ostendorff, H. P., Andersen, B., and Rosenfeld,M. G. (1997). A family of LIM domain-associated cofactors confertranscriptional synergism between LIM and Otx homeodomainproteins. Genes Dev. 11: 1370–1380.

Bach, I., Rhodes, S. J., Pearse, R. V., Heinzel, T., Gloss, B., Scully, K. M.,Sawchenko, P. E., and Rosenfeld, M. G. (1995). P-Lim, a LIMhomeodomain factor, is expressed during pituitary organ and cellcommitment and synergizes with Pit-1. Proc. Natl. Acad. Sci. USA92: 2720–2724.

Bachy, I., Vernier, P., and Retaux, S. (2001). The LIM-homeodomaingene family in the developing Xenopus brain: Conservation anddivergences with the mouse related to the evolution of the fore-brain. J. Neurosci. 21: 7620–7629.

Bishop, K. M., Goudreau, G., and O’Leary, D. D. (2000). Regulation ofarea identity in the mammalian neocortex by Emx2 and Pax6.Science 288: 344–349.

Broccoli, V., Boncinelli, E., and Wurst, W. (1999). The caudal limit ofOtx2 expression positions the isthmic organizer. Nature 401: 164–168.

Chen, H., Lun, Y., Ovchinnikov, D., Kokubo, H., Oberg, K. C., Pepi-celli, C. V., Gan, L., Lee, B., and Johnson, R. L. (1998). Limb andkidney defects in Lmx1b mutant mice suggest an involvement ofLMX1B in human nail patella syndrome. Nat. Genet. 19: 51–55.

Dasen, J. S., O’Connell, S. M., Flynn, S. E., Treier, M., Gleiberman,A. S., Szeto, D. P., Hooshmand, F., Aggarwal, A. K., and Rosenfeld,M. G. (1999). Reciprocal interactions of Pit1 and GATA2 mediatesignaling gradient-induced determination of pituitary cell types.Cell 97: 587–598.

Dasen, J. S., and Rosenfeld, M. G. (1999). Combinatorial codes insignaling and synergy: lessons from pituitary development. Curr.Opin. Genet. Dev. 9: 566–574.

Dreyer, S. D., Zhou, G., Baldini, A., Winterpacht, A., Zabel, B., Cole,W., Johnson, R. L., and Lee, B. (1998). Mutations in LMX1B causeabnormal skeletal patterning and renal dysplasia in nail patellasyndrome. Nat. Genet. 19: 47–50.

Galliot, B., de Vargas, C., and Miller, D. (1999). Evolution of ho-meobox genes: Q50 Paired-like genes founded the Paired class. Dev.Genes Evol. 209: 186–197.

German, M. S., Wang, J., Chadwick, R. B., and Rutter, W. J. (1992).Synergistic activation of the insulin gene by a LIM-homeo domainprotein and a basic helix–loop–helix protein: Building a functionalinsulin minienhancer complex. Genes Dev. 6: 2165–2176.

Gowan, K., Helms, A. W., Hunsaker, T. L., Collisson, T., Ebert, P. J.,Odom, R., and Johnson, J. E. (2001). Crossinhibitory activities ofNgn1 and Math1 allow specification of distinct dorsal interneurons.Neuron 31: 219–232.

Hobert, O., Tessmar, K., and Ruvkun, G. (1999). The Caenorhabditiselegans lim-6 LIM homeobox gene regulates neurite outgrowth andfunction of particular GABAergic neurons. Development 126: 1547–1562.

Jessell, T. M. (2000). Neuronal specification in the spinal cord: induc-tive signals and transcriptional codes. Nat. Rev. Genet. 1: 20–29.

Johnson, J. D., Zhang, W., Rudnick, A., Rutter, W. J., and German,M. S. (1997). Transcriptional synergy between LIM-homeodomainproteins and basic helix–loop–helix proteins: The LIM2 domaindetermines specificity. Mol. Cell Biol. 17: 3488–3496.

Jurata, L. W., Kenny, D. A., and Gill, G. N. (1996). Nuclear LIMinteractor, a rhombotin and LIM homeodomain interacting protein,is expressed early in neuronal development. Proc. Natl. Acad. Sci.USA 93: 11,693–11,698.

Lamonerie, T., Tremblay, J. J., Lanctot, C., Therrien, M., Gauthier, Y.,and Drouin, J. (1996). Ptx1, a bicoid-related homeo box transcrip-tion factor involved in transcription of the pro-opiomelanocortingene. Genes Dev. 10: 1284–1295.

Lanctot, C., Moreau, A., Chamberland, M., Tremblay, M. L., andDrouin, J. (1999). Hindlimb patterning and mandible developmentrequire the Ptx1 gene. Development 126: 1805–1810.

Lee, B. J., Cho, G. J., Norgren, R. B., Jr., Junier, M. P., Hill, D. F., Tapia,V., Costa, M. E., and Ojeda, S. R. (2001). TTF-1, a homeodomaingene required for diencephalic morphogenesis, is postnatally ex-pressed in the neuroendocrine brain in a developmentally regu-lated and cell-specific fashion. Mol. Cell Neurosci. 17: 107–126.

Liu, I. S., Chen, J. D., Ploder, L., Vidgen, D., van der, K. D., Kalnins,V. I., and Mcinnes, R. R. (1994). Developmental expression of anovel murine homeobox gene (Chx10): Evidence for roles in deter-mination of the neuroretina and inner nuclear layer. Neuron 13:377–393.

Logan, M., Pagan-Westphal, S. M., Smith, D. M., Paganessi, L., andTabin, C. J. (1998). The transcription factor Pitx2 mediates situs-specific morphogenesis in response to left–right asymmetric sig-nals. Cell 94: 307–317.

Lopes da Silva, S., Van Horssen, A. M., Chang, C., and Burbach, J. P.(1995). Expression of nuclear hormone receptors in the rat supraop-tic nucleus. Endocrinology 136: 2276–2283.

Lu, M. F., Pressman, C., Dyer, R., Johnson, R. L., and Martin, J. F.(1999). Function of Rieger syndrome gene in left-right asymmetryand craniofacial development. Nature 401: 276–278.

Lumsden, A., and Krumlauf, R. (1996). Patterning the vertebrateneuraxis. Science 274: 1109–1115.

Marchand, R. (1987). Histogenesis of the subthalamic nucleus. Neu-roscience 21: 183–195.

Marquardt, T., and Pfaff, S. L. (2001). Cracking the transcriptionalcode for cell specification in the neural tube. Cell 106: 651–654.

Matise, M. P., and Joyner, A. L. (1997). Expression patterns of devel-opmental control genes in normal and Engrailed-1 mutant mousespinal cord reveal early diversity in developing interneurons.J. Neurosci. 17: 7805–7816.

Millet, S., Campbell, K., Epstein, D. J., Losos, K., Harris, E., andJoyner, A. L. (1999). A role for Gbx2 in repression of Otx2 andpositioning the mid/hindbrain organizer. Nature 401: 161–164.

Morcillo, P., Rosen, C., Baylies, M. K., and Dorsett, D. (1997). Chip, awidely expressed chromosomal protein required for segmentationand activity of a remote wing margin enhancer in Drosophila. GenesDev. 11: 2729–2740.

Muccielli, M. L., Martinez, S., Pattyn, A., Goridis, C., and Brunet, J. F.(1996). Otlx2, an Otx-related homeobox gene expressed in the pi-tuitary gland and in a restricted pattern in the forebrain. Mol. CellNeurosci. 8: 258–271.

Nambu, A., Takada, M., Inase, M., and Tokuno, H. (1996). Dualsomatotopical representations in the primate subthalamic nucleus:Evidence for ordered but reversed body-map transformations fromthe primary motor cortex and the supplementary motor area.J. Neurosci. 16: 2671–2683.

Parent, A., and Hazrati, L. N. (1995). Functional anatomy of the basal

419Coexpression of Lmx1b and Ptx Genes in the CNS

ganglia. II. The place of subthalamic nucleus and external pallidumin basal ganglia circuitry. Brain Res. Brain Res. Rev. 20: 128–154.

Parent, A., Sato, F., Wu, Y., Gauthier, J., Levesque, M., and Parent, M.(2000). Organization of the basal ganglia: The importance of axonalcollateralization. Trends Neurosci. 23: S20–S27.

Pattyn, A., Morin, X., Cremer, H., Goridis, C., and Brunet, J. F. (1999).The homeobox gene Phox2b is essential for the development ofautonomic neural crest derivatives. Nature 399: 366–370.

Piedra, M. E., Icardo, J. M., Albajar, M., Rodriguez-Rey, J. C., and Ros,M. A. (1998). Pitx2 participates in the late phase of the pathwaycontrolling left–right asymmetry. Cell 94: 319–324.

Pierani, A., Moran-Rivard, L., Sunshine, M. J., Littman, D. R., Goul-ding, M., and Jessell, T. M. (2001). Control of interneuron fate in thedeveloping spinal cord by the progenitor homeodomain proteinDbx1. Neuron 29: 367–384.

Puelles, L., Kuwana, E., Puelles, E., Bulfone, A., Shimamura, K.,Keleher, J., Smiga, S., and Rubenstein, J. L. (2000). Pallial andsubpallial derivatives in the embryonic chick and mouse telenceph-alon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1,Pax-6, and Tbr-1. J. Comp. Neurol. 424: 409–438.

Puelles, L., and Rubenstein, J. L. (1993). Expression patterns of ho-meobox and other putative regulatory genes in the embryonicmouse forebrain suggest a neuromeric organization. Trends Neuro-sci. 16: 472–479.

Retaux, S., Rogard, M., Bach, I., Failli, V., and Besson, M. J. (1999).Lhx9: A novel LIM-homeodomain gene expressed in the develop-ing forebrain. J. Neurosci. 19: 783–793.

Riddle, R. D., Ensini, M., Nelson, C., Tsuchida, T., Jessell, T. M., andTabin, C. (1995). Induction of the LIM homeobox gene Lmx1 byWNT7a establishes dorsoventral pattern in the vertebrate limb. Cell83: 631–640.

Rubenstein, J. L., Martinez, S., Shimamura, K., and Puelles, L. (1994).The embryonic vertebrate forebrain: The prosomeric model. Science266: 578–580.

Ryan, A. K., Blumberg, B., Rodriguez-Esteban, C., Yonei-Tamura, S.,Tamura, K., Tsukui, T., de la, P. J., Sabbagh, W., Greenwald, J.,Choe, S., Norris, D. P., Robertson, E. J., Evans, R. M., Rosenfeld,M. G., and Izpisua Belmonte, J. C. (1998). Pitx2 determines left–right asymmetry of internal organs in vertebrates. Nature 394: 545–551.

Schaeren-Wiemers, N., and Gerfin-Moser, A. (1993). A single protocolto detect transcripts of various types and expression levels in neuraltissue and cultured cells: In situ hybridization using digoxigenin-labelled cRNA probes. Histochemistry 100: 431–440.

Semina, E. V., Reiter, R., Leysens, N. J., Alward, W. L., Small, K. W.,Datson, N. A., Siegel-Bartelt, J., Bierke-Nelson, D., Bitoun, P., Zabel,B. U., Carey, J. C., and Murray, J. C. (1996). Cloning and character-ization of a novel bicoid-related homeobox transcription factorgene, RIEG, involved in Rieger syndrome. Nat. Genet. 14: 392–399.

Sharma, K., and Peng, C. Y. (2001). Spinal motor circuits: Mergingdevelopment and function. Neuron 29: 321–324.

Simeone, A. (2000). Positioning the isthmic organizer where Otx2 andGbx2meet. Trends Genet. 16: 237–240.

Smidt, M. P., Asbreuk, C. H., Cox, J. J., Chen, H., Johnson, R. L., andBurbach, J. P. (2000a). A second independent pathway for devel-opment of mesencephalic dopaminergic neurons requires Lmx1b.Nat. Neurosci. 3: 337–341.

Smidt, M. P., Cox, J. J., van Schaick, H. S., Coolen, M., Schepers, J., van

der Kleij, A. M., and Burbach, J. P. (2000b). Analysis of three Ptx2splice variants on transcriptional activity and differential expres-sion pattern in the brain. J. Neurochem. 75: 1818–1825.

Smidt, M. P., van Schaick, H. S., Lanctot, C., Tremblay, J. J., Cox, J. J.,van der Kleij, A. A., Wolterink, G., Drouin, J., and Burbach, J. P.(1997). A homeodomain gene Ptx3 has highly restricted brain ex-pression in mesencephalic dopaminergic neurons. Proc. Natl. Acad.Sci. USA 94: 13,305–13,310.

Smith, Y., and Kieval, J. Z. (2000). Anatomy of the dopamine systemin the basal ganglia. Trends Neurosci. 23: S28–S33.

Sussel, L., Marin, O., Kimura, S., and Rubenstein, J. L. (1999). Loss ofNkx2.1 homeobox gene function results in a ventral to dorsalmolecular respecification within the basal telencephalon: Evidencefor a transformation of the pallidum into the striatum. Development126: 3359–3370.

Swanson, L. W. (1992). Brain Maps: Structure of the Rat Brain. Elsevier,Amsterdam.

Szeto, D. P., Rodriguez-Esteban, C., Ryan, A. K., O’Connell, S. M., Liu,F., Kioussi, C., Gleiberman, A. S., Izpisua-Belmonte, J. C., andRosenfeld, M. G. (1999). Role of the Bicoid-related homeodomainfactor Pitx1 in specifying hindlimb morphogenesis and pituitarydevelopment. Genes Dev. 13: 484–494.

Szeto, D. P., Ryan, A. K., O’Connell, S. M., and Rosenfeld, M. G.(1996). P-OTX: A PIT-1-interacting homeodomain factor expressedduring anterior pituitary gland development. Proc. Natl. Acad. Sci.USA 93: 7706–7710.

Thaler, J., Harrison, K., Sharma, K., Lettieri, K., Kehrl, J., and Pfaff,S. L. (1999). Active suppression of interneuron programs withindeveloping motor neurons revealed by analysis of homeodomainfactor HB9. Neuron 23: 675–687. [See comments]

Thor, S., Andersson, S. G., Tomlinson, A., and Thomas, J. B. (1999). ALIM-homeodomain combinatorial code for motor-neuron pathwayselection. Nature 397: 76–80.

Tsuchida, T., Ensini, M., Morton, S. B., Baldassare, M., Edlund, T.,Jessell, T. M., and Pfaff, S. L. (1994). Topographic organization ofembryonic motor neurons defined by expression of LIM homeoboxgenes. Cell 79: 957–970 [See comments].

van Schaick, H. S., Smidt, M. P., Rovescalli, A. C., Luijten, M., van derKleij, A. A., Asoh, S., Kozak, C. A., Nirenberg, M., and Burbach, J. P.(1997). Homeobox gene Prx3 expression in rodent brain and extra-neural tissues. Proc. Natl. Acad. Sci. USA 94: 12,993–12,998.

Vogel, A., Rodriguez, C., Wamrnken, W., and Izpisua Belmonte, J. C.(1995). Dorsal cell fate specified by chick Lmx1 during vertebratelimb development [published erratum appears in Nature 1996 Feb29;379(6568):848]. Nature 378: 716–720.

Westmoreland, J. J., McEwen, J., Moore, B. A., Jin, Y., and Condie,B. G. (2001). Conserved function of Caenorhabditis elegans UNC-30and mouse Pitx2 in controlling GABAergic neuron differentiation.J. Neurosci. 21: 6810–6819.

Yoshioka, H., Meno, C., Koshiba, K., Sugihara, M., Itoh, H., Ishimaru,Y., Inoue, T., Ohuchi, H., Semina, E. V., Murray, J. C., Hamada, H.,and Noji, S. (1998). Pitx2, a bicoid-type homeobox gene, is involvedin a lefty-signaling pathway in determination of left-right asymme-try. Cell 94: 299–305.

Yuan, S., and Schoenwolf, G. C. (1999). The spatial and temporalpattern of C-Lmx1 expression in the neuroectoderm during chickneurulation. Mech. Dev. 88: 243–247.

Received May 20, 2002Accepted June 18, 2002

420 Asbreuk et al.