PATTERNS & PHENOTYPES

Selective Expression of Bhlhb5 in Subsets ofEarly-Born Interneurons and Late-BornAssociation Neurons in the Spinal CordBen Liu,1,2 Zijing Liu,2 Tao Chen,1 Hong Li,2 Boqin Qiang,1 Jiangang Yuan,1 Xiaozhong Peng1 andMengsheng Qiu2*

Accumulating evidence has suggested that the basic helix-loop-helix transcription factors play importantroles in controlling neuronal fate specification and differentiation in the developing central nervoussystem. In this study, we report a detailed immunological study on the expression of Bhlhb5 in embryonicmouse spinal cord with a newly developed antibody. At the early stage of neural development, Bhlhb5 isspecifically expressed in dI6 dorsal interneurons and in V1 and V2 ventral interneurons. At late stages ofdevelopment, Bhlhb5 expression is detected in a subset of late-born dorsal association interneurons thatmigrate into the uppermost layer of the dorsal horn. Developmental Dynamics 236:829–835, 2007.© 2007 Wiley-Liss, Inc.

Key words: Bhlh; transcription factor; immunostaining; expression pattern; neural development

Accepted 11 December 2006

INTRODUCTION

Spinal cord has served as an excellentmodel for studying molecular and ge-netic control of cell fate specificationand differentiation. At an early stageof spinal cord development, the homo-geneous neuroepithelial cells undergodorsal–ventral patterning by signal-ing molecules produced from both thedorsal and ventral midline structures.It has been proposed that Sonic hedge-hog produced from the notochord andfloor plate functions as a morphogento selectively induce or repress expres-sion of homeodomain transcriptionfactors (TFs) in a concentration-de-pendent manner, creating a nestedpattern of TF expression in the ven-

tral neural progenitor cells (Roelink etal., 1995; Ericson et al., 1997). Thecombinatory expression of these TFsconstitutes a homeodomain code thatdivides the ventral neuroepitheliuminto five distinct domains (pMN, p0–p3), with each domain expressing aunique combination of TFs and gener-ating a specific neuronal subtype (mo-tor neurons or MNs, V0–V3 ventralinterneurons; Briscoe et al., 2000).Different subtypes of ventral neuronscan be readily identified by their ex-pression of specific postmitotic home-odomain TFs, such as HB9 (MNs),Evx1 (V0), En1 (V1), and Chx10 (V2).Recent studies have suggested thatthese homeodomain proteins function

primarily as postmitotic determinantsof neuronal subtype identities. For in-stance, HB9 and Evx1 are known to berequired for the consolidation of motorneuron fate and V0 interneuron fate,respectively (Arber et al., 1999; Thaleret al., 1999; Moran-Rivard et al.,2001).

In the dorsal spinal cord, severalmembers of the bone morphogeneticprotein (BMP) family are produced inthe dorsal midline structures (ecto-derm and roof plate; Lee and Jessell,1999). There is both in vitro and invivo evidence for a gradient of BMP-dependent activity in controlling neu-ronal subtype identities (Barth et al.,1999; Timmer et al., 2002). The gradi-

1The National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences andPeking Union Medical College, Beijing, China2Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, KentuckyGrant sponsor: NIH; Grant number: R01 NS37703; Grant sponsor: National Sciences Foundation of China; Grant number: 30421003.*Correspondence to: Mengsheng Qiu, Department of Anatomical Sciences and Neurobiology, University of Louisville Schoolof Medicine, Louisville, KY 40202. E-mail: m0qiu001@louisville.edu

DOI 10.1002/dvdy.21061Published online 10 January 2007 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 236:829–835, 2007

© 2007 Wiley-Liss, Inc.

ent BMP signaling, together withother signal molecules (e.g., Wnt),could be responsible for the differen-tial expression of many TFs in the dor-sal neural progenitor cells. Intrigu-ingly, many of the TFs that areinvolved in the dorsal neural pattern-ing are members of the basic helix-loop-helix (bHLH) family, known asproneural bHLH factors, which in-clude Math1, Ngn1, Ngn2, and Mash1(Gowan et al., 2001). Based on thecombinatorial expression of certainproneural bHLH factors and other ho-meodomain factors, the dorsal neuro-epithelium is divided into six progen-itor domains (dp1–6), from whicharise six early-born (E9.5–E12.0) dor-sal interneuron populations calleddI1–6 neurons (Gross et al., 2002;Muller et al., 2002). Like their ventralcounterparts, different subclasses ofdorsal interneurons express distinctcombinations of postmitotic homeodo-main TFs, which are similarly in-volved in cell fate consolidation (forreviews, see Matise, 2002; Casparyand Anderson, 2003; Helms and John-son, 2003). For instance, Lbx1 is spe-cifically expressed in dI4–6 neuronsand is required for their ultimate fatedetermination (Gross et al., 2002;Muller et al., 2002).

Interestingly, two later-born (E12–E14) neuronal populations, termeddILA and dILB, are generated from theMash1�/Gsh1/2� dp3–5 domains ofdorsal neuroepithelium in a salt andpepper manner (Mizuguchi et al.,2006; Wildner et al., 2006). Whileearly neurons are born at embryonicday (E) 9.5–E12, dILA and dILB neu-rons are mostly generated betweenE12 and E14. Soon after their birth,both dILA and dILB neurons migrateinto the dorsal superficial layerswhere they intermingle and differen-tiate into association interneurons.Although the dILA and dILB neuronsappear to be generated from the sameneural progenitor cells and both ex-press Lbx1, they can be readily distin-guished by their expression of distinctcombination of other homeodomainTFs. While dILA neurons expressPax2 and Lim1/2, the dILB neuronsexpress Brn3a, Lmx1b, and Tlx3(Gross et al., 2002; Muller et al.,2002). However, at later stages, thepostmitotic TFs seem to be differen-tially down-regulated in dILB neurons

so that Brn3a� and Lmx1b�/Tlx3�neurons are found in distinct laminaeof the dorsal horn (Gross et al., 2002;Muller et al., 2002).

Despite that many bHLH factorsare involved in the fate specification ofneural progenitor cells, there is grow-ing evidence that bHLH factors alsoparticipate in the differentiation ofneuronal subtypes. For instance,Bhlh4 is transcribed in bipolar neu-rons in the retina (Bramblett et al.,2002), and its expression is requiredfor rod bipolar cell maturation (Bram-blett et al., 2004). Recently, Bhlhb5, aclosely related member of Bhlhb4 inthe Olig family (Xu et al., 2002; Ber-trand et al., 2002), has also been re-ported to be expressed in embryoniccentral nervous system (CNS) and inother tissues as well (Brunelli et al.,2003). However, its expression in thedeveloping nervous system has notbeen characterized at the cellularlevel, and its function in neural devel-opment remains unknown. As a firststep to elucidate its role in neuronaldifferentiation, we developed a spe-cific polyclonal antibody against theBhlhb5 protein and performed de-tailed studies on its expression in thedeveloping spinal cord. Here we reportthat Bhlhb5 is selectively expressed insubsets of spinal interneurons, specif-ically the early-born dI6, V1 and V2neurons, and a subpopulation of late-born (E12.5 and later) dILA and dILB

dorsal interneurons that settle downin the superficial laminae relativelylate.

RESULTS

It was previously reported thatBhlhb5 mRNA is transcribed in sev-eral embryonic tissues, including theneural tube (Brunelli et al., 2003).However, its spatial and temporalpatterns of expression in the CNShave not been analyzed in detail. Tofully characterize the expression andregulation of Bhlhb5 in the developingspinal cord, we set out to develop poly-clonal antibodies against the N�-ter-minal sequence of the Bhlhb5 protein.Western immunoblotting with the af-finity-purified antibody showed that itrecognized a single protein band of theexpected size (�39 kDa) in HEK293cells transfected with pcDNA4-BHLHB5 expression vector, but not

with the control vector (Fig. 1A). Theantibody specificity was further sub-stantiated by the virtually identicalstaining patterns of Bhlhb5 in situRNA hybridization and the anti-Bhlhb5 immunofluorescence on adja-cent sections prepared from E10.5 andE17.5 mouse spinal cord tissues (Fig.1B–E). At E10.5, Bhlhb5� cells werelocated in the gray matter adjacent tothe ventricular zone mostly in theventral spinal cord (Fig. 1B,C). AtE17.5, Bhlhb5� cells remained con-fined to the gray matter but werefound in both the ventral and dorsalregions (Fig. 1D,E). The expression ofBhlhb5 in the gray matter persisted inyoung postnatal spinal cord (Fig.1F–H) but was eventually down-regu-lated in the adult (data not shown).The restriction of Bhlhb5 expressionto the gray matter strongly suggestedthat Bhlhb5 is likely to be expressedin neurons. Consistent with this idea,many Bhlhb5� neurons graduallygained the expression of the neuronalmarker NeuN (Mullen et al., 1992) asthey migrated away from the ventric-ular zone (Fig. 2), indicating theBhlhb5 is initially expressed in youngimmature neurons.

To define the specific neuronal sub-types that express Bhlhb5 at the earlystage of spinal development, we first in-vestigated whether Bhlhb5� neuronscan be generated from dorsal neuralprogenitor cells by examining the ex-pression of Bhlhb5 in relation to that ofPax7 and Mash1 in E10.5 spinal cord.At this stage, Pax7 is expressed in theentire dorsal neuroepithelium (dp1–6),whereas Mash1 specifically marks thedp3–5 domains of dorsal neuroepithe-lial cells (Fig. 3B; Matise, 2002; Helmsand Johnson, 2003; Caspary andAnderson, 2003). Double-labeling ex-periments revealed that, in the dorsalregion, Bhlhb5 was expressed in a smallnumber of neurons flanking the lowestregion of the Pax7� dorsal neuroepithe-lial cells (Fig. 3A) but below theMash1� neural progenitor cells (Fig.3B), suggesting that Bhlhb5 is selec-tively expressed in dI6 interneurons de-rived from the Pax7�/Mash1� neuralprogenitor cells. Consistently, thisgroup of Bhlhb5� neurons also coex-pressed the dI4–dI6 marker Lbx1 (Fig.3C), but not Brn3a (Fig. 3D), whichmarks dI1–3 and dI5 neurons or thedI5-specific marker Lmx1b (Fig. 3E).

830 LIU ET AL.

The dI6 identity of Bhlhb5� neurons inthe dorsal spinal cord was further sub-stantiated by their coexpression of Pax2and Lim1/2 (Fig. 3F,G), two homeodo-main TFs that are expressed in dI6 andother interneurons as well (Fig. 3H).

At E10.5, Bhlhb5� neurons were

also observed in the ventral spinalcord, and an apparent gap was de-tected between the dorsal and ventralBhlhb5� neurons at approximatelythe position of V0 interneuron. Dou-ble-labeling experiments revealedthat, in the ventral part, Bhlhb5 neu-

rons were generated immediately ad-jacent to the Pax6� neuroepithelialcells (Fig. 4A) above the Olig2� pMNdomain (Fig. 4B). Thus Bhlhb5 islikely to be expressed in the V1 and V2ventral interneurons interposed be-tween V0 and MNs. Consistent withthis idea, Bhlhb5� neurons coex-pressed the V1 marker EN1 (Fig. 4D)and V2 marker Chx10 (Fig. 4E), butnot the V0 marker Evx1 (Fig. 4C) northe MN marker HB9 (Fig. 4F). In fur-ther support, Bhlhb5� neurons in theV1 position also coexpressed Lim1/2and Pax2, two postmitotic TFs ex-pressed in V0 and V1 interneurons inthe ventral spinal cord (Fig. 3F,G).

Of interest, at later stages of embry-ogenesis (E17.5), Bhlhb5 expressionwas also detected in the dorsal spinalcord, primarily in the superficial lay-ers (laminae 1–3) of the dorsal horn(Fig. 1D,E). Recent studies demon-strated that neurons that populatethis region are predominantly thelate-born (after E11) dILA and dILB

dorsal interneurons that originatefrom the Mash1�/Gsh� dp3–dp5 do-

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 1. Characterization of Bhlhb5 antibodyspecificity and Bhlhb5 expression in the spinalcord. A: Detection of Bhlhb5 protein by poly-clonal anti-Bhlhb5 antibody in Western immu-noblotting of HEK293 cells transfected withpcDNA4 (lane 1) or pcDNA4-BHLHB5 (lane 2).B–E: Comparison of Bhlhb5 in situ RNA hybrid-ization (B,D) and anti-Bhlhb5 immunofluores-cent staining (C,E) on adjacent spinal cord sec-tions from embryonic day (E) 10.5 (B,C) andE17.5 (D,E) embryos. D: Spinal cord. Anti-Bhlhb5 immunofluorescent staining of mousespinal cord from E12.5 (B), E13.5 (C), and E14.5(D) embryos. F–H: Anti-Bhlhb5 immunostainingin postnatal day 0 (F), day 7 (G), and day 15 (H)spinal cord.

Fig. 2. Coexpression of Bhlhb5 and NeuN inneurons. Embryonic day (E) 12.5 spinal cordsections were subject to double-immunofluo-rescent staining with Bhlhb5 (red) and anti-NeuN (green). Bhlhb5� cells gradually acquiredNeuN expression after they migrated away fromthe ventricular zone. Representative double-positive neurons are indicated by arrows.

Fig. 3. Expression of Bhlhb5 in relation to vari-ous dorsal markers at embryonic day (E) 10.5.A–G: Spinal cord sections from E10.5 embryoswere subjected to double-immunofluorescentstaining with anti-Bhlhb5 and anti-Pax7 (A), an-ti-Mash1(B), anti-Lbx1(C), anti-Brn3a (D), anti-Lmx1b (E), anti-Lim1/2 (F), or anti-Pax2(G). H: Asummary of the expression of Bhlhb5 in relationto other transcription factor markers in the dor-sal spinal cord at E10.5.

BHLHB5 EXPRESSION IN THE SPINAL CORD 831

mains and subsequently migrate dor-sally into the superficial laminae(Muller et al., 2002; Gross et al.,2002). To test the possibility thatBhlhb5 is transcribed in dILA and/ordILB neurons, we examined the dorsalexpression of Bhlhb5 in relation to thedILA marker Pax2 and the dILB mark-ers Brn3a, Lmx1b, or Tlx3. At E14.5, asmall number of Bhlhb5� cells weredetected in the uppermost layers pop-ulated by Pax2� dILA neurons (Fig.5A), and Lmx1b�/Tlx3� dILB neu-rons (Fig. 5B,C). In this region, a fewBhlhb5� cells coexpressed Pax2, butnot Lmx1b or Tlx3. In contrast, themajority of Bhlhb5� cells in the dor-sal horn were situated in the moreventral layers rich in Bran3a� neu-rons, with some of Bhlhb5� cells co-expressing Brn3a (Fig. 5D). Later atE17.5, most Bhlhb5� neurons werefound in the more dorsal layers, coex-

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 4. Expression of Bhlhb5 in relation to vari-ous ventral markers at embryonic day (E) 10.5.A–F: Spinal cord sections from E10.5 were sub-jected to double-immunofluorescent stainingwith anti-Bhlhb5 and anti-Pax6 (A), anti-Olig2(B), anti-Evx1(C), anti-En1 (D), anti-Chx10 (E), oranti-HB9 (F). G: A summary of the expression ofBhlhb5 in relation to various transcription factormarkers in the ventral spinal cord at E10.5.

Fig. 5. Dynamic expression of Bhlhb5 in thedorsal interneurons. A–H: Spinal cord sectionsfrom embryonic day (E) 14.5 (A–D) and E17.5(E–H) embryos were simultaneously immuno-stained with anti-Bhlhb5 and anti-Pax2 (A,E),Lmx1b (B,F), Tlx3 (C,G), and Brn3a (D,H). Rep-resentative double-positive neurons are indi-cated by arrows. From E14.5 to E17.5, theBlhb5� neurons progressively migrated dor-sally into the superficial laminae.

Fig. 6. Birthdating of Bhlhb5� neurons in thedorsal spinal cord. A–F: Pregnant mice wereinjected with bromodeoxyuridine (BrdU) at em-bryonic day (E) 11.5 (A,D), E12.5 (B,E), or E13.5(D,F) and killed at E18.5. Spinal cord tissueswere simultaneously immunostained with anti-BrdU (green) and anti-Bhlhb5 (red). Double-la-beled neurons in superficial layers were pre-dominantly found in embryos that were treatedwith BrdU at E12.5 and E13.5. D,E,F: Highermagnifications of the dorsal horn regions of A,B, and C, respectively. Representative double-positive cells in E and F are indicated by arrows.G–L: Double labeling of Bhlhb5 and Mash1 inE12.5 and E13.5 spinal cord. At E12.5, a weakexpression of Bhlhb5 adjacent to the Mash1�neuroepithelial cells is indicated by arrows, andthe staining in the ventricular zone ventral to theMash1 expression was nonspecific due to anoverexposure to show the weakly stainedBhlhb5� neurons.

pressing either the dILA marker Pax2,or the dILB markers Lmx1b, Tlx3, orBrn3a (Fig. 5E–H). However, Bhlhb5expression was only detected in a frac-tion of dIL neurons in the dorsal lay-ers at this stage. Together, these re-sults suggest that Bhlhb5� neuronsprogressively migrate into the super-ficial laminae of the dorsal horn be-tween E14.5 and E17.5.

The late arrival of Bhlhb5� neu-rons in the superficial layers as com-pared with other Pax2� and Lmx1b�dIL neurons has raised the possibilitythat they represent a late-born popu-lation of dorsal association interneu-rons. To examine this possibility, webirthdated the dorsal Bhlhb5 neuronsby administrating a single injection ofbromodeoxyuridine (BrdU) into preg-nant mice at E11.5, E12.5, or E13.5.Spinal cord tissues were collected atE18.5 and subject to double immuno-staining with anti-BrdU and anti-Bhlhb5. In embryos that receivedBrdU injection at E11.5, numerousBrdU� cells were observed in the dor-sal horn, but few Bhlhb5� neurons inthis region were BrdU� (Fig. 6A,D).However, in E12.5-labeled embryos, alarge number of Bhlhb5� neurons inthe dorsal horn were strongly immu-noreactive to BrdU (Fig. 6B,E). InE13.5-injected embryos, despite amuch reduced number of BrdU� cellsin the dorsal horn, Bhlhb5�/BrdU�double-positive neurons were still de-tected in the superficial layers (Fig.6C,F). Double immunostaining fur-ther confirmed that a small number ofBhlhb5� neurons started to be pro-duced at E12.5 from the Mash1� neu-roepithelial cells (Fig. 6G–I), and thenumber was dramatically increased atE13.5 (Fig. 6J–L). Taken together,these BrdU birthdating and double-immunostaining experiments sug-gested that Bhlhb5� dIL neuronswere predominantly generated fromthe dorsal neural progenitor cells atE12.5 and later.

DISCUSSION

In this study, we report the detailedanalyses of Bhlhb5 expression in thedeveloping spinal cord by immuno-staining with a newly generated anti-Bhlhb5 polyclonal antibody. The affin-ity-purified antibody appears to bespecific to the Bhlhb5 protein based on

the detection of a single protein bandof the expected size by Western immu-noblotting in cells transfected withBhlhb5 expression vector and theidentical patterns of Bhlhb5 in situRNA hybridization and immunostain-ing signals (Fig. 1). The Bhlhb5 anti-body did not seem to cross-react withother related proteins, as its immuno-staining pattern did not overlap withthose of other members of the Oligfamily, which includes Olig1, Olig2,Olig3, Bhlhb4, and Bhlh5 itself (Ber-trand et al., 2002). For instance, inE10.5 spinal cord, Olig1 and Olig2 areexpressed in the pMN domain of theventral neuroepithelium (Fig. 3B;Takebayashi et al., 2000; Lu et al.,2000; Zhou et al., 2000), whereasOlig3 is expressed in the dI1–3 do-mains of the dorsal neuroepithelium(Takebayashi et al., 2002; Muller etal., 2005). Bhlhb4 is specifically ex-pressed in the diencephalon–mesen-cephalon boundary, but not in the spi-nal cord (Bramblett et al., 2002).

Comparison of Bhlhb5 expressionwith other well-characterized mark-ers for neural progenitor cells andneuronal subtypes revealed a dynamicpattern of Bhlhb5 expression in em-bryonic mouse spinal cord. At earlystage of spinal cord development(E10.5), Bhlhb5 is expressed in post-mitotic immature neurons derivedfrom the dp6, p1 and p2 domains ofneural progenitor cells (Figs. 3, 4). In-deed, Bhlhb5� neurons did not incor-porate BrdU in a 2-hr short pulsechase and did not coexpress the ma-ture neuronal marker NeuN (data notshown). However, at E12.5, coexpres-sion of Bhlhb5 and NeuN can beclearly detected in the ventral spinalcord as Bhlhb5 neurons migrate intothe mantle zone (Fig. 2).

At later stages, Bhlhb5 expressionis also up-regulated in a small numberof late-born dIL dorsal interneurons inthe superficial layers, coexpressingdIL markers Pax2, Brn3a, Lmx1b, orTlx3 (Fig. 5). Pervious studies showedthat the majority of dIL neurons wereborn between E11 and E13 (Gross etal., 2002; Muller et al., 2002). OurBrdU birthdating studies revealedthat the Bhlhb5� dorsal associationneurons are mostly generated at andafter E12.5, with many being pro-duced as late as E13.5 (Fig. 6). Indeed,at E12.5, a small number of Bhlhb5�

neurons started to emerge from theMash1� dorsal neural progenitorcells (arrows in Fig. 6G–I). However,this number was dramatically in-creased at E13.5 (Fig. 6J–L), suggest-ing that, like other dIL neurons,Bhlhb5� dorsal association neuronsare also generated from the dp3–5neural progenitor cells. In keepingwith the idea that Bhlhb5 labels alate-born population of dIL neurons,they migrate into the dorsal superfi-cial laminae later than most of otherdIL neurons. At E14.5, Bhlhb5� neu-rons in the dorsal spinal cord werelargely localized in the Brn3a� layerventral to the dorsal layers populatedby Pax2� and Lmx1b� dIL neurons(Fig. 5A–D). By E17.5, a majority ofBhlhb5� neurons were found in themore superficial layers interminglingwith other dILA and dILB neurons(Fig. 5E–H), indicating that Bhlhb5�interneurons migrate into the upper-most layers between E14.5 and E17.5.

The function of Bhlhb5 in neuronaldevelopment in the spinal cord is notknown at this stage. Previous workshowed that mouse Bhlhb5 proteincan repress the human Pax6 promoter(Xu et al., 2002). Consistent with thisobservation, Pax6 expression is down-regulated in Bhlhb5� neurons as theyleave the ventricular zone. Given thatBhlhb5 is initially expressed in NeuN-negative immature neurons beforeterminal differentiation, it is quitepossible that Bhlhb5 may participatein neuronal fate specification and dif-ferentiation. Recent studies have indi-cated that expression of bHLH TFsduring the differentiation process cancontribute to the specification of dis-tinct neuronal identities (Lee andPfaff, 2003; Helms et al., 2005). Amore direct support for the possiblerole of Bhlhb5 in the control of neuralspecification and differentiation camefrom the most recent observation thatBhlhb5 function is required for thecorrect specification of �-aminobutyricacid (GABA)ergic amacrine and Type2 OFF-cone bipolar subtypes in thedeveloping retina (Feng et al., 2006).

Recent studies have shown thatmany postmitotic transcription fac-tors are involved in the control or reg-ulation of the synthesis of particularneurotransmitters. For instance,Pft1a and Pax2 specify GABAergicneurons (Glasgow et al., 2005),

BHLHB5 EXPRESSION IN THE SPINAL CORD 833

whereas Tlx1/Tlx3 homeodomaintranscription factors produce gluta-matergic neurotransmitter phenotype(Cheng et al., 2005). All early-bornBhlhb5� neurons and many late-bornBhlhb5� dorsal association neuronscoexpressed Pax2 and, thus, are likelyto be GABAergic neurons (Fig. 5;Cheng et al., 2004). However, a smallnumber of Bhlhb5� dorsal interneu-rons appear to belong to the glutama-tergic dILB neurons as they coex-pressed Lmx1b and Tlx3 (Fig. 5;Cheng et al., 2004). Thus, Bhlhb5 ex-pression is not directly related to theswitch of GABAergic versus glutama-tergic neurotransmitter phenotypes.However, it cannot be ruled out thatBhlhb5 may regulate other neuro-transmitter phenotypes (e.g., sero-toninergic, cholinergic, or peptider-gic). In addition, it is also conceivablethat Bhlhb5 may be involved in otherdevelopmental processes such as neu-ronal migration, axonal pathfinding,or circuitry formation. Further func-tional studies are required to eluci-date the role of Bhlhb5 in neuronaldevelopment in the developing spinalcord.

EXPERIMENTALPROCEDURES

Generation of Anti-Bhlhb5Antibody

The 5�-terminus sequence (residues2–213) and 3�-terminus sequence(277–356) of Bhlh5 were subclonedseparately into the pET-32a vector(Novagen). The fused Trx-Bhlhb5 pro-teins were expressed in Escherichiacoli and purified by the NTA-Ni2� col-umn according to the manufacturer’sinstructions. Fusion proteins (1 mg)was then injected into rabbits orguinea pigs (PRF&L Inc.), and the an-tisera were then collected 2 monthslater and purified by antigen-conju-gated affinity columns. However, onlythe antiserum against the 5�-terminusfusion protein produced specific im-munostaining signal. Western and im-munofluorescence data were gener-ated with antisera from guinea pig.

Immunofluorescent Staining

Embryonic and postnatal spinal cordtissues were dissected out and sub-

merged in 4% paraformaldehyde at4°C overnight. After fixation, tissueswere transferred to 20% sucrose inphosphate buffered saline overnight,embedded in OCT medium, and thensectioned (15 �m thickness) on a cry-ostat. Immunofluorescent procedureswith guinea pig anti-Bhlhb5 were pre-viously described by Xu et al. (2000).Anti-Nkx2.2, anti-Evx1, anti-En1, an-ti-HB9, anti-Pax7, and anti-BrdUwere obtained from the Developmen-tal Studies Hybridoma Bank. Anti-Brn3a (Chemicon, Inc.), anti-Pax2(Zymed, Inc.), and anti-Lmx1b (Ab-cam Inc.) were purchased from com-mercial sources. Anti-Chx10, anti-Tlx3, and anti-Lbx1 were generouslyprovided by Drs. Sam Pfaff and Car-men Birchmeier.

BrdU Birthdating Analysisof Bhlhb5� Cells

Specifically, BrdU (Sigma, 15 mg/ml in7 mM NaOH) was injected intraperito-neally into pregnant mice (0.12 mg/g ofbody weight) at E11.5, E12.5, or E13.5.Embryos were harvested at E18.5 andanalyzed for incorporation of BrdU inBhlhb5� neurons by double immuno-fluorescence with anti-BrdU (Develop-mental Studies Hybridoma Bank, Iowacity, IA) and anti-Bhlhb5 antibody aspreviously described in our previousstudies (Xu et al., 2000). The percentageof BrdU� cells in Bhlhb5� cells wascalculated from three separate sections.

ACKNOWLEDGMENTSWe thank Dr. Sam Pfaff for gener-ously providing the anti-Chx10 anti-body and Dr. Carmen Birchmeier forgenerously providing the anti-Lbx1and anti-Tlx3 antibody. M.Q. wasfunded by the NIH, and X.P. wasfunded by the National SciencesFoundation of China.

REFERENCES

Arber S, Han B, Mendelsohn M, Smith M,Jessell TM, Sockanathan S. 1999. Re-quirement for the homeobox gene Hb9 inthe consolidation of motor neuron iden-tity. Neuron 23:659–674.

Barth K, Kishimoto Y, Rohr K, Seydler C,Schulte-Merker S, Wilson S. 1999. BMPactivity establishes a gradient of posi-tional information throughout the entireneural plate. Development 126:4977–4987.

Bertrand N, Castro DS, Guillemot F. 2002.Proneural genes and the specification ofneural cell types. Nat Rev Neurosci 3:517–530.

Bramblett DE, Copeland NG, Jenkins NA,Tsai MJ. 2002. BHLHB4 is a bHLH tran-scriptional regulator in pancreas andbrain that marks the dimesencephalicboundary. Genomics 79:402–412.

Bramblett DE, Pennesi ME, Wu SM, TsaiMJ. 2004. The transcription factorBhlhb4 is required for rod bipolar cellmaturation. Neuron 43:779–793.

Briscoe J, Pierani A, Jessell TM, Ericson J.2000. A homeodomain protein code spec-ifies progenitor cell identity and neuro-nal fate in the ventral neural tube. Cell101:435–445.

Brunelli S, Innocenzi A, Cossu G. 2003.Bhlhb5 is expressed in the CNS and sen-sory organs during mouse embryonic de-velopment. Gene Expr Patterns 3:755–759.

Caspary T, Anderson KV. 2003. Patterningcell types in the dorsal spinal cord: whatthe mouse mutants say. Nat Rev Neuro-sci 4:289–297.

Cheng L, Arata A, Mizuguchi R, Qian Y,Karunaratne A, Gray PA, Arata S,Shirasawa S, Bouchard M, Luo P, ChenCL, Busslinger M, Goulding M, OnimaruH, Ma Q. 2004. Tlx3 and Tlx1 are post-mitotic selector genes determining gluta-matergic over GABAergic cell fates. NatNeurosci 7:510–517.

Cheng L, Samad OA, Xu Y, Mizuguchi R,Luo P, Shirasawa S, Goulding M, Ma Q.2005. Lbx1 and Tlx3 are opposingswitches in determining GABAergic ver-sus glutamatergic transmitter pheno-types. Nat Neurosci 8:1510–1515.

Ericson J, Rashbass P, Schedl A, Brenner-Morton S, Kawakami A, van HeyningenV, Jessell TM, Briscoe J. 1997. Pax6 con-trols progenitor cell identity and neuro-nal fate in response to graded Shh sig-naling. Cell 90:169–180.

Feng L, Xie X, Joshi PS, Yang Z, ShibasakiK, Chow RL, Gan L. 2006. Requirementfor Bhlhb5 in the specification of ama-crine and cone bipolar subtypes in mouseretina. Development 133:4815–4825.

Glasgow SM, Henke RM, MacDonald RJ,Wright CVE, Johnson JE. 2005. Ptf1adetermines GABAergic over glutamater-gic neuronal cell fate in the spinal corddorsal horn. Development 132:5461–5469.

Gowan K, Helms AW, Hunsaker TL, Col-lisson T, Ebert PJ, Odom R, Johnson JE.2001. Crossinhibitory activities of Ngn1and Math1 allow specification of distinctdorsal interneurons. Neuron 31:219–232.

Gross MK, Dottori M, Goulding M. 2002.Lbx1 specifies somatosensory associa-tion interneurons in the dorsal spinalcord. Neuron 34:535–549.

Helms AW, Johnson JE. 2003. Specifica-tion of dorsal spinal cord interneurons.Curr Opin Neurobiol 13:42–49.

Helms AW, Battiste J, Henke RM, NakadaY, Simplicio N, Guillemot F, Johnson JE.2005. Sequential roles for Mash1 and

834 LIU ET AL.

Ngn2 in the generation of dorsal spinalcord interneurons. Development 132:2709–2719.

Lee KJ, Jessell TM. 1999. The specificationof dorsal cell fates in the vertebrate cen-tral nervous system. Annu Rev Neurosci22:261–294.

Lee SK, Pfaff S. 2003. Synchronization ofneurogenesis and motor neuron specifi-cation by direct coupling of bHLH andhomeodomain transcription factors.Neuron 38:731–745.

Lu QR, Yuk D, Alberta JA, Zhu Z,Pawlitzky I, Chan J, McMahon AP,Stiles CD, Rowitch DH. 2000. Sonichedgehog—regulated oligodendrocytelineage genes encoding bHLH proteins inthe mammalian central nervous system.Neuron 25:317–329.

Matise M. 2002. A dorsal elaboration in thespinal cord. Neuron 34:491–493.

Mizuguchi R, Kriks S, Cordes R, Gossler A,Ma Q, Goulding M. 2006. Ascl1 andGsh1/2 control inhibitory and excitatorycell fate in spinal sensory interneurons.Nat Neurosci 9:770–778.

Moran-Rivard L, Kagawa T, Saueressig H,Gross MK, Burrill J, Goulding M. 2001.Evx1 is a postmitotic determinant of v0interneuron identity in the spinal cord.Neuron 29:385–399.

Mullen RJ, Buck CR, Smith AM. 1992.NeuN, a neuronal specific nuclear pro-tein in vertebrates. Development 116:201–211.

Muller T, Brohmann H, Pierani A, Hep-penstall PA, Lewin GR, Jessell TM,Birchmeier C. 2002. The homeodomainfactor lbx1 distinguishes two major pro-grams of neuronal differentiation in thedorsal spinal cord. Neuron 34:551–562.

Muller T, Anlag K, Wildner H, Britsch S,Treier M, Birchmeier C. 2005. ThebHLH factor Olig3 coordinates the spec-ification of dorsal neurons in the spinalcord. Genes Dev 19:733–743.

Roelink H, Porter JA, Chiang C, Tanabe Y,Chang DT, Beachy PA, Jessell TM. 1995.Floor plate and motor neuron inductionby different concentrations of the amino-terminal cleavage product of sonichedgehog autoproteolysis. Cell 81:445–455.

Takebayashi H, Yoshida S, Sugimori M,Kosako H, Kominami R, Nakafuku M,Nabeshima Y. 2000. Dynamic expressionof basic helix-loop-helix Olig familymembers: implication of Olig2 in neuronand oligodendrocyte differentiation andidentification of a new member, Olig3.Mech Dev 99:143–148.

Takebayashi H, Ohtsuki T, Uchida T,Kawamoto S, Okubo K, Ikenaka K,Takeichi M, Chisaka O, Nabeshima Y.2002. Non-overlapping expression ofOlig3 and Olig2 in the embryonic neuraltube. Mech Dev 113:169–174.

Thaler J, Harrison K, Sharma K, LettieriK, Kehrl J, Pfaff SL. 1999. Active sup-pression of interneuron programs within

developing motor neurons revealed byanalysis of homeodomain factor HB9.Neuron 23:675–687.

Timmer J, Wang C, Niswander L. 2002.BMP signaling patterns the dorsal andintermediate neural tube via regulationof homeobox and helix-loop-helix tran-scriptionfactors.Development129:2459–2472.

Wildner H, Muller T, Cho SH, Brohl D,Cepko CL, Guillemot F, Birchmeier C.2006. dILA neurons in the dorsal spinalcord are the product of terminal and non-terminal asymmetric progenitor cell di-visions, and require Mash1 for their de-velopment. Development 133:2105–2113.

Xu X, Cai J, Fu H, Wu R, Qi Y, ModdermanG, Liu R, Qiu M. 2000. Selective expres-sion of Nkx-2.2 transcription factor inchicken oligodendrocyte progenitors andimplications for the embryonic origin ofoligodendrocytes. Mol Cell Neurosci 16:740–753.

Xu ZP, Dutra A, Stellrecht CM, Wu C, Pi-atigorsky J, Saunders GF. 2002. Func-tional and structural characterization ofthe human gene BHLHB5, encoding abasic helix-loop-helix transcription fac-tor. Genomics 80:311–318.

Zhou Q, Wang S, Anderson DJ. 2000. Iden-tification of a novel family of oligoden-drocyte lineage-specific basic helix-loop-helix transcription factors. Neuron 25:331–343.

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