a retinoic acid-hox hierarchy controls both anterior/posterior patterning and neuronal specification...

96 (2006) 190–202www.elsevier.com/locate/ydbio

Developmental Biology 2

A retinoic acid-Hox hierarchy controls both anterior/posterior patterning andneuronal specification in the developing central nervous system of the

cephalochordate amphioxus

Michael Schubert a,b, Nicholas D. Holland c, Vincent Laudet a, Linda Z. Holland c,⁎

a Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5161/INRA UMR1237/ENS Lyon, IFR128 BioSciences Lyon-Gerland,Ecole Normale Supérieure de Lyon, 46 allée d’Italie, 69364 Lyon Cedex 07, France

b Institut des Sciences de l’Evolution-Montpellier, CNRS-UMR5554, Université de Montpellier II, CC 065, 34095 Montpellier Cedex 05, Francec Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, 8750 Biological Grade,

La Jolla, CA, 92093-0202, USA

Received for publication 1 February 2006; revised 9 April 2006; accepted 19 April 2006Available online 30 April 2006

Abstract

Retinoic acid (RA) mediates both anterior/posterior patterning and neuronal specification in the vertebrate central nervous system (CNS).However, the molecular mechanisms downstream of RA are not well understood. To investigate these mechanisms, we used the invertebratechordate amphioxus, in which the CNS, although containing only about 20,000 neurons in adults, like the vertebrate CNS, has a forebrain,midbrain, hindbrain, and spinal cord and is regionalized by RA-signaling. Here we show, first, that domains of genes with expression normallylimited to diencephalon and midbrain are generally not affected by altered RA-signaling, second, that contrary to previous reports, not only Hox1,3, and 4, but also Hox2 and Hox6 are collinearly expressed in the amphioxus CNS, and third, that collinear expression of all these Hox genes iscontrolled by RA-signaling. Finally, we show that Hox1 is involved in mediating both the role of RA-signaling in regionalization of the hindbrainand in specification of hindbrain motor neurons. Thus, morpholino knock-down of the single amphioxus Hox1 mimics the effects of treatmentswith an RA-antagonist. This analysis establishes RA-dependent regulation of collinear Hox expression as a feature common to the chordate CNSand indicates that the RA-Hox hierarchy functions both in proper anterior/posterior patterning of the developing CNS and in specification ofneuronal identity.© 2006 Elsevier Inc. All rights reserved.

Keywords: Branchiostoma; Chordate evolution; Hindbrain regionalization; Hox collinearity; Morpholino; Motor neuron; Neurogenesis; Retinoic acid-antagonist

Introduction

In chordates, retinoic acid (RA) is a potent, vitamin A-derived teratogen that in excess can cause rostral truncations ofthe head. In vertebrates, RA has two main roles in patterning thecentral nervous system (CNS): (1) determination of anterior/posterior position within the hindbrain and (2) neuronalspecification (Maden, 2002; Wilson et al., 2003). Thus, forexample, excess RA posteriorizes the CNS and increases the

⁎ Corresponding author. Fax: +1 858 534 7313.E-mail address: [email protected] (L.Z. Holland).

0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.ydbio.2006.04.457

number of primary neurons, while decreased RA-signalinglevels anteriorize it resulting in hindbrain expansion anddiminution of primary neurons. RA functions via binding toretinoic acid receptors (RARs), which form heterodimers withretinoid X receptors (RXRs) before binding to retinoic acidresponse elements (RAREs) in the regulatory regions of targetgenes. Although a number of targets of RA-signaling have beenidentified (Arima et al., 2005; Ishibashi et al., 2005), themolecular mechanisms downstream of RA are not wellunderstood.

To address the question of how RA patterns the chordateCNS, we are using the invertebrate chordate amphioxus as asimple model. Amphioxus has a genome that is vertebrate-like,

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191M. Schubert et al. / Developmental Biology 296 (2006) 190–202

but lacks the extensive gene duplications of vertebrates. Thus, ithas a single Hox cluster (Minguillon et al., 2005) and singleRAR and RXR genes (Escriva et al., 2002). In addition,amphioxus has a prototypical chordate body plan with anotochord, pharyngeal gill slits, muscular somites and dorsalhollow CNS, but not paired eyes, ears or a cartilaginous or bonyendoskeleton. The amphioxus CNS is divided into two mainregions, a small, anterior, expanded portion termed the cerebralvesicle or brain and a long, narrow posterior region, often calledthe spinal cord. Based on microanatomy and patterns of geneexpression, the cerebral vesicle corresponds to the vertebratediencephalon plus a small midbrain, while the remainder of thenerve cord corresponds to the vertebrate hindbrain plus spinalcord (Holland and Holland, 1999; Holland and Chen, 2001;Wicht and Lacalli, 2005). It has been estimated that only about20,000 neurons are present in the CNS of adult amphioxus andeven fewer in the larval nerve cord (Nicol and Meinertzhagen,1991). Therefore, at least for the brain of larval amphioxus, ithas been possible to map each cell and its connections withserial transmission electron microscopy (Lacalli et al., 1994;Lacalli, 1996, 2002, 2003; Lacalli and Kelly, 1999, 2003a,b)and, in the absence of extensive cell migration, to localizeexpression of neural marker genes to specific nerve cells(Lacalli, 2000; Bardet et al., 2005).

The cerebral vesicle is first distinguishable from thehindbrain/spinal cord at the early neurula by the expression ofOtx (Williams and Holland, 1996, 1998). By the mid-neurula,the cerebral vesicle is visibly expanded, and the hindbrain isregionalized as shown by collinear expression of the singleHox1,Hox3 andHox4 genes (Holland and Holland, 1996; Wadaet al., 1999). At this stage, several other genes have expressionlimited to either the cerebral vesicle (e.g., Wnt5, Pax6 andNkx2.2) or to the hindbrain and spinal cord (e.g., Pax2/5/8,Wnt3 and Wnt6) (Glardon et al., 1998; Holland et al., 1998;Kozmik et al., 1999; Schubert et al., 2001). Moreover, by thelate neurula, the cellular retinoic acid binding protein (CRABP),which modulates RA-signaling (Dong et al., 1999), is stronglyexpressed in a stripe at the posterior end of the cerebral vesicle(Jackman et al., 2004) suggesting that differences in levels ofRA-signaling may be important in maintaining the distinctionbetween the cerebral vesicle and the hindbrain/spinal cord inamphioxus.

When neurons initially become detectable in the amphioxushindbrain at the mid-neurula, among the first to develop arethose associated with the primary photoreceptor and the sixpairs of dorsal compartment (DC) motor neurons. Theseneurons, which are located in the posterior midbrain andanterior hindbrain from slightly anterior to the rostral limit ofHox1 to about the rostral limit of Hox4, express the pan-neuralmarkers Neurogenin and Coe, as well as those that are morespecific for motor neurons, such as Islet and the estrogen-receptor related gene ERR (Holland et al., 2000; Jackman et al.,2000; Mazet et al., 2004; Bardet et al., 2005). ERR is aparticularly good marker, since unlike the other three genes, itsexpression is almost exclusively limited to the DC motorneurons through at least the two gill slit stage (Bardet et al.,2005). The DC motor neurons are serially arranged and

innervate the paraxial muscles used in slow undulatoryswimming (Lacalli and Kelly, 1999, 2003b; Bardet et al.,2005). It is not surprising that these motor neurons developearly, since slow swimming begins during the late neurula stage,about 18–20 h after fertilization (Stokes, 1997).

Neither elevated nor reduced levels of RA-signaling affectthe size or shape of the cerebral vesicle or the domain of thecompetitive RAR-inhibitor, the orphan receptor TR2/4, within it(Holland and Holland, 1996; Escriva et al., 2002). However,excess RA shifts the expression of the retinoic acid receptor(RAR) and of Hox1 to the anterior tip of the CNS, while an RA-antagonist downregulates expression of RAR (Holland andHolland, 1996; Escriva et al., 2002). The effects of altered RA-signaling on expression of other Hox genes in the amphioxusCNS have not been studied.

Hox genes are also expressed in mesoderm, endoderm andgeneral ectoderm in amphioxus (Schubert et al., 2004, 2005).Although often thought of as non-neural, the general ectodermalso includes some sensory neurons, which appear to bespecified by a combinatorial gene code that includes pan-neuronal markers, such as Coe, as well as particular combina-tions of Hox genes, Islet and ERR (Schubert et al., 2004). Theeffect of excess RA on expression of Hox1 in the generalectoderm and endoderm is similar to that on its expression in theCNS (Holland and Holland, 1996; Schubert et al., 2004, 2005).However, the downstream effectors of RA-signaling in theamphioxus CNS and general ectoderm are not known, althoughin the endoderm Hox1 has been shown to mediate the effect ofRA-signaling in establishing the posterior limit of the pharynx(Schubert et al., 2005). This previous work allowed us topropose a model in which RA-signaling directly activates Hox1in the midgut and Hox1 in turn suppresses expression ofpharyngeal specifiers (Schubert et al., 2005). Whether a similarmodel applies to the CNS is uncertain especially in light ofreports that Hox2 and Hox6 are not expressed collinearly in theCNS as they are in the general ectoderm (Wada et al., 1999;Cohn, 2002; Schubert et al., 2004). Therefore, in the presentstudy, we have investigated the role of RA-signaling and Hoxgenes in global patterning and specification of neuronal identityin the amphioxus CNS.

The present results show that expression domains of genesnormally transcribed only in the cerebral vesicle (includingCRABP) are generally not affected by altered RA-signaling. Incontrast, elevated RA-signaling levels tend to expand expres-sion of genes normally transcribed in the hindbrain and spinalcord into the cerebral vesicle. Furthermore, we show that Hox2and Hox6 are also collinearly expressed in the amphioxus CNS,and exogenous RA and an RA-antagonist shift their rostralexpression limits anteriorly and posteriorly, respectively, as theydo those of Hox1, Hox3 and Hox4. These results establish theRA-dependent regulation of collinear Hox expression as afeature common to the chordate CNS. Moreover, we show thatknock-down of Hox1 function with an antisense morpholino-oligonucleotide mimics the effects of RA-antagonist treatmentsboth during global regionalization of the CNS and inspecification of DC motor neurons. Taken together, our resultssuggest that genes expressed in the anterior CNS (such as

192 M. Schubert et al. / Developmental Biology 296 (2006) 190–202

CRABP and TR2/4) normally act to reduce levels of RA-signaling and thereby inhibit expression of hindbrain/spinalcord markers in the cerebral vesicle. In addition, we show thatRA-signaling acts via Hox1 not only during broad regionaliza-tion of the hindbrain and spinal cord but also in neurogenesis toinfluence the specification of DC motor neurons. Thus, bothroles – in anterior/posterior patterning of the CNS and in

Fig. 1. RA-signaling has differential effects on gene expression in the amphioxus cerPax2/5/8 is expressed throughout the hindbrain and spinal cord and more weakly in prneither RA (A) nor the RA-antagonist BMS009 (C) has an obvious effect. By the earthe cerebral vesicle (D), but there is no obvious effect of BMS009 on Pax2/5/8 exthroughout the hindbrain and spinal cord. RA (G) expands expression to the anterior(J) does not affect expression, nor does BMS009 (L). (M–R) Wnt5 expression. In novesicle. Expression is unaffected by RA and BMS009 at both 16 h (M, O) and 30 h (Pposterior border of the cerebral vesicle. Treatments with RA (S) and BMS009 (U) havleft. Arrowheads in (A–I) = Anterior limits of continuous gene expression in the CNScale bars = 50 μm.

neuronal specification – arose relatively early during chordateevolution before the large-scale gene duplications and thesignificant increase in complexity of the CNS occurred in thevertebrate lineage. These results underscore the utility ofamphioxus as a relatively simple model for understanding thecomplex mechanisms whereby RA-signaling patterns thechordate CNS.

ebral vesicle and hindbrain. (A–F) Pax2/5/8 expression. In normal larvae (B, E),ecursors of pigment cells associated with the frontal eye. In 18-h neurulae (A–C),ly larval stage (30 h) (D–F), RA expands the domain of Pax2/5/8 anteriorly intopression (F). (G–I) Wnt3 expression. In normal larvae (H), Wnt3 is expressedtip of the nerve cord, but BMS009 (I) has no effect. (J–L) Notch expression. RArmal embryos (N, Q), Wnt5 is conspicuously expressed throughout the cerebral, R). (S–U) CRABP expression. In normal larvae (T), CRABP is expressed at thee no effect on CRABP expression. Whole mounts in side view with anterior to theS. Arrowheads in (M–U) = Domains of gene expression in the cerebral vesicle.


Materials and methods

Embryonic culture and treatment with RA and RA-antagonist(BMS009)

Ripe amphioxus adults (Branchiostoma floridae) were collected in OldTampa Bay Florida, USA, during the summer. Spawning of the collectedanimals was induced by electric stimulation (Holland and Yu, 2004). Afterfertilization, embryos were reared in filtered seawater at 25°C. Treatments withRA (in DMSO), the RA-antagonist BMS009 (in DMSO) or DMSO were carriedout at the late blastula stage at a final concentration of 1 × 10−6 M (Holland andHolland, 1996; Escriva et al., 2002). At the early neurula stage, embryos weretransferred to untreated filtered seawater. The DMSO treatments did not affectamphioxus development. Embryos and larvae were fixed for in situhybridization at frequent intervals. Fixation was at 4°C overnight with 4%paraformaldehyde in a buffer containing 0.1 M MOPS, 0.5 M NaCl, 2 mMMgSO4, 1 mM EGTA, pH 7.4 (Holland et al., 1996).

Microinjection of unfertilized amphioxus eggs

Unfertilized amphioxus eggs were injected as described by Holland and Yu(2004) with either a control antisense morpholino-oligonucleotide (5′-CCTCTTACCTCAGTTACAATTTATA-3′) or an antisense morpholino-oligo-

Fig. 2. RA-signaling controls expression of five Hox genes in the CNS of the amphioIn normal embryos (B, E, H, K, N), the five Hox genes are expressed collinearly wexpression of all five genes anteriorly in the CNS, while BMS009 (C, F, I, L, O) shiftsin side view, anterior to the left. Arrowheads = Anterior limit of Hox expression in

nucleotide targeting B. floridae AmphiHox1 (5′-ATTCTTGCCGTGTC-CATTTGCTCCA-3′) (Gene Tools, LLC, Philomath, OR, USA). Prior to usein microinjection experiments, the specificity of the AmphiHox1morpholino forAmphiHox1 mRNAwas verified by in vitro translation assays (Schubert et al.,2005). The total volume injected into amphioxus eggs was approximately 2 pl.Morpholinos were injected at a final concentration of 500 μM in 15% glycerolwith 5 mg/ml Texas Red dextran (Molecular Probes, Inc., Eugene, OR, USA).Just prior to use, the morpholino solutions were heated to 65°C for 5 min toinsure an unfolded configuration. After injection, eggs were fertilized andallowed to develop either for 26–30 h or for 40–48 h before fixation. Followingfixation, only embryos and larvae showing conspicuous fluorescence of TexasRed dextran were analyzed by in situ hybridization (Holland et al., 1996). Allanimals successfully injected with the control morpholino developed normally.

In situ hybridization, microscopy and photography

In situ hybridizations were carried out as previously described (Holland etal., 1996). Gene markers used in this analysis were: AmphiHox1 (GenBankaccession number: AB050886), AmphiHox2 (AB028207), AmphiHox3(X68045), AmphiHox4 (AB028208) and AmphiHox6 (Z35146) (all fiveprovided by P. W. H. Holland), AmphiPax2/5/8 (AF053762) (Kozmik et al.,1999), AmphiWnt3 (AF361013) and AmphiWnt5 (AF361014) (Schubert et al.,2001), AmphiNotch (Y12539) (Holland et al., 2001), AmphiCRABP

xus neurula. (A–C) Hox1. (D–F) Hox2. (G–I) Hox3. (J–L) Hox4. (M–O) Hox6.ith anterior limits (arrowheads) in the hindbrain. RA (A, D, G, J, M) expandsthe rostral limits of expression of all five posteriorly in the CNS. Whole mountsthe CNS. Scale bars = 50 μm.


(AY365445) (provided byW. R. Jackman), AmphiOtx (AF043740) (provided byP. W. H. Holland) and AmphiERR (AY738655) (Bardet et al., 2005). In addition,AmphiIslet (AF226616) (provided by W. R. Jackman), AmphiNeurogenin(AF271788) (Holland et al., 2000), AmphiCoe (AJ580840) and AmphiKrox(AJ278149) (the latter two markers were provided by S. M. Shimeld) were alsoused as marker genes. After in situ hybridization, embryos were mounted onglass slides in 80% glycerol in NaPBS, analyzed under the microscope andsubsequently photographed as whole mounts. In order to determine the exactanatomical structures labeled by the marker genes, the animals were counter-stained (with Ponceau S), dehydrated in ethanol, embedded in plastic (Spurr'sresin) and prepared as serial sections (3–4 μm thickness) for light microscopy(Holland et al., 1996).

Cell counts and statistical analyses

Cells labeled with AmphiERR in the amphioxus nerve cord were counted indorsal views of amphioxus whole mount embryos and larvae. The weakAmphiERR signal in the anterior cerebral vesicle was not included in theanalysis, since this expression domain is neither affected by RA- or BMS009-treatments nor by AmphiHox1 morpholino injections. For control as well as forRA- or BMS009-treated animals, the specimens analyzed were between 30 and48 h old, whereas for control morpholino- or AmphiHox1 morpholino-injectedanimals, the specimens were between 40 and 48 h old. The total number ofanimals assayed was: 14 control animals, 10 RA-treated amphioxus, 10BMS009-treated specimens, 8 animals injected with control morpholino and 9

Fig. 3. Altered RA-signaling disrupts collinear expression of five Hox genes in the CN(M–O)Hox6. In normal 30-h larvae (B, E, H, K, N), nested expression of the fiveHoxthe anterior limits of all five genes to the rostral end of the CNS (A, D, G, J, M), whWhole mounts in side view, anterior at left. Arrowheads = Anterior limit of gene ex

larvae injected with the AmphiHox1 morpholino. Statistical analyses to assessthe significance of the obtained data were carried out using the softwareEnvironment R (version 2.0.1).

Results

RA-signaling affects gene expression differently in the cerebralvesicle and hindbrain/spinal cord

To investigate the role of RA-signaling on regionalization ofthe CNS, we selected five marker genes expressed at differentanteroposterior levels along the amphioxus CNS (namely:Pax2/5/8, Wnt3, Notch, Wnt5 and CRABP). In normal larvae,expression of Pax2/5/8 and Wnt3 is restricted to the hindbrain/spinal cord except for a small domain of Pax2/5/8 at the anteriortip of the CNS in pigment cell precursors of the frontal eye(Figs. 1B, E, H) (Kozmik et al., 1999; Schubert et al., 2001).Treatment with RA has no apparent effect on Pax2/5/8 expression in the CNS in the 18-h neurula (Fig. 1A), but by30 h, expression is expanded throughout the cerebral vesicle(Fig. 1D). However, the RA-antagonist BMS009 has no

S of the amphioxus larva. (A–C) Hox1. (D–F) Hox2. (G–I) Hox3. (J–L) Hox4.genes is maintained with anterior limits (arrowheads) in the hindbrain. RA shiftsereas BMS009 (C, F, I, L, O) pushes the anterior expression limits posteriorly.pression in the CNS. Scale bars = 50 μm.


apparent effect on Pax2/5/8 expression at either 18 or 30 h(Figs. 1C, F). Similarly, exogenous RA expands expression ofWnt3 anteriorly throughout the cerebral vesicle (Fig. 1G), butthere is no clear effect of BMS009 (Fig. 1I). In contrast, neitherRA nor BMS009 affects the Notch domain, which extends theentire length of the nerve cord (Figs. 1J–L) (Holland et al.,2001). Altered RA-signaling has also no apparent effect onexpression of Wnt5 in the cerebral vesicle (Figs. 1M–R)(Schubert et al., 2001). Expression of Wnt5 in patches in thehindbrain/spinal cord may be a little stronger in early neurulaetreated with RA, but the domain is unchanged (Fig. 1M).Moreover, expression of CRABP (cellular retinoic acid bindingprotein) at the posterior end of the cerebral vesicle is unaffectedby the treatments (Figs. 1S–U) (Jackman et al., 2004). Takentogether, these results suggest that in general, RA-signalingregulates, perhaps indirectly, expression of genes such as Pax2/5/8 and Wnt3 that are normally expressed throughout thehindbrain/spinal cord with an anterior limit at the boundarybetween the cerebral vesicle and hindbrain, while it typicallydoes not control expression of genes with domains normallyrestricted to the cerebral vesicle (e.g., Notch, Wnt5 andCRABP). A minor exception is Otx, which is normally broadlytranscribed in the cerebral vesicle, but which is also expressed in

Fig. 4. RA-signaling regulates motor neuron specification and Otx expression in theleft. Expression (brackets) in the CNS of normal animals (B, E) is limited to the ceredomain and BMS009 (C, F) expands expression posteriorly into the hindbrain. (G–larvae. Anterior to left. Arrows indicate the first photoreceptor. (J–L) show the larvaethe six pairs of DC motor neurons, in the two neurons of the first photoreceptor (inse(excepting the most anterior pair), which extend the full anterior/posterior length ofreduces their numbers. The first photoreceptor may be normal (inset in J), but is mposteriorly and increases their numbers and often disrupts formation of the first pho

the anteriormost hindbrain with a distinct boundary between the1st and 2nd somites (Figs. 4B, E) (Williams and Holland, 1996,1998). Although the Otx domain in the anterior 80% of thecerebral vesicle is not strongly affected by altered RA-signaling,the domain in the posterior cerebral vesicle and anteriorhindbrain is eliminated by elevated RA (Figs. 4A, D) andexpanded posteriorly by BMS009 (Figs. 4C, F). These effectsare already detectable in 16-h neurulae (Figs. 4A, C), butbecome more pronounced in 30-h larvae (Figs. 4D, F). Thus,even though RA-signaling probably does not normally regulateexpression of genes in the cerebral vesicle, levels of RA-signaling are evidently kept low there to prevent misexpressionof RA target genes.

RA-signaling controls expression of five Hox genes in theamphioxus CNS

To determine if RA-signaling affects the anterior limits of theCNS domains of amphioxus Hox genes other than Hox1(Holland and Holland, 1996), we also examined Hox2, Hox3,Hox4 andHox6. Surprisingly, in spite of reports thatHox2 is notexpressed in the amphioxus CNS (Wada et al., 1999) and thatHox6 is expressed anterior to Hox1 (Cohn, 2002), we found that

amphioxus CNS. (A–F) Otx expression. Whole mounts in side view, anterior tobral vesicle and to the anterior portion of the hindbrain. RA (A, D) reduces theL) ERR expression. Optical frontal sections through CNS and somites of earlyin (G–I) at higher magnification. In normal embryos (H, K), ERR is expressed int in K) and in a few cells in the anterior cerebral vesicle as well as in the somitesthe larva. RA (G, J) shifts ERR-positive cells anteriorly in the nerve cord andore often disrupted. BMS009 (I, L) expands the zone of ERR-expressing cellstoreceptor (inset in L). Scale bars = 50 μm.


during normal development, all five Hox genes display spatialcollinearity in the amphioxus CNS. Collinear expression ofthese genes is already apparent at the mid-neurula stage (Figs.2B, E, H, K, N) and becomes more pronounced at later stages(Figs. 3B, E, H, K, N). Although the amphioxus hindbrain lacksrhombomeres, the somites extend to the anterior extremity ofthe larva and are thus fiducial marks for anterior/posteriorposition (Figs. 4G–L). Thus, in the 30-h larva, the anteriorlimits of expression of these five genes and the correspondingsomite positions are as follows (Fig. 7): Hox1/somite 2 (Fig.3B), Hox2/somite 3 (Fig. 3E), Hox3/somite 4 (Fig. 3H), Hox4/somite 6 (Fig. 3K) and Hox6/between somite 6 and 7 (Fig. 3N).

Treatment with RA expands expression of all five Hox genesanteriorly (Figs. 2A, D, G, J, M). By the early larval stage, theyare all expressed to the most anterior tip of the CNS (Figs. 3A,D, G, J, M). Conversely, treatment with the RA-antagonistBMS009 shifts the anterior limits of expression of all five genesposteriorly in the CNS (Figs. 2C, F, I, L, O and 3C, F, I, L, O).

Fig. 5. Injection of Hox1-specific morpholino mimics the effects of RA-antagonist treCNS. (A–D) Otx expression. Whole mounts in side view, anterior at left. Injection wHox1-specific morpholino (B, D) expands theOtx domain posteriorly in the CNS. Arrthe Hox1 morpholino may be abnormal (arrow in B) or normal (arrow in D). Brackexpression. Side (E, F) and dorsal (G, H) views with anterior to the left. Arrows indmorpholino (E, G), ERR is expressed in motor neurons anterior to the first photorecemorpholino (F, H), ERR-expressing cells extend posterior to the first photoreceptor,

At the early larval stage, BMS009 shifts the rostral expressionlimits to the following anteroposterior levels (Fig. 7): Hox1 tobetween the 3rd and 4th somite (Fig. 3C), Hox2 to close to the6th somite (Fig. 3F), Hox3 to between the 6th and 7th somites(Fig. 3I), Hox4 to the 10th somite (Fig. 3L) and Hox6 tobetween the 12th and the 13th somites (Fig. 3O). Takentogether, these results show, first, that all the amphioxus Hoxgenes studied so far are collinearly expressed in the amphioxushindbrain/spinal cord (paralleling their collinear arrangement inthe Hox cluster) and, second, that expression of all of them isregulated by RA-signaling.

RA-signaling in the hindbrain activates Hox1, which in turnrestricts expression of Otx to the cerebral vesicle and regulatesmotor neuron identity

To determine if Hox1 is a downstream mediator of RA-signaling in patterning the amphioxus hindbrain, we compared

atments on regional patterning and motor neuron specification in the amphioxusith the control morpholino (A, C) does not alter expression of Otx. Injection of aows indicate pigment cell of the first photoreceptor, which in larvae injected withets = Bars anterior/posterior extent of Otx expression in the CNS. (E–H) ERRicate pigment cell of the first photoreceptor. In larvae injected with the controlptor and in the photoreceptor cells themselves. In larvae injected with the Hox1which is abnormal (arrow in H). Scale bars = 50 μm.

Fig. 6. Both RA-signaling and Hox1 regulate the total number of cellsexpressing ERR in the amphioxus CNS. (A) Number of ERR-positive cells in theCNS of normal, RA- and BMS009-treated amphioxus larvae, not includingthose in the anterior cerebral vesicle. The number of ERR-expressing cells isdecreased by RA and increased by BMS009. Differences are statisticallysignificant as assessed by a Kruskal–Wallis rank sum test (P = 3.8 × 10−7).N = 14 for control; N = 10 for RA-treated and BMS009-treated embryos. (B)Number of ERR-positive cells in the nerve cord of amphioxus larvae injectedwith either the control or Hox1 morpholino, not including those in the anteriorcerebral vesicle. The number of ERR-expressing cells is increased by the Hox1morpholino. This increase is statistically significant as assessed by a one-sidedWilcoxon rank sum test (P = 2.1 × 10−3). N = 8 for control; N = 9 for theAmphiHox1 morpholino. Error bars are +/− one standard deviation.


the effects of gene knock-down to our results from alteringlevels of RA-signaling. We used two marker genes: Otx, which,as noted above, is normally strongly expressed in the cerebralvesicle and more weakly in the anteriormost part of thehindbrain with a boundary between the 1st and 2nd somite(Figs. 4B, E) (Williams and Holland, 1996, 1998) and ERR,which is an excellent marker for the six pairs of DC motorneurons in the hindbrain as well as for the somites (Bardet et al.,2005). The only other cells in the amphioxus CNS expressingERR are a few cells in the anterior cerebral vesicle, whichcontribute to the median frontal eye, and the neurons of the firstphotoreceptor (the organ of Hesse), which forms at the level ofsomite 5 and consists of a pigment cell with two concavities,each occupied, respectively, by the perikaryon of a photore-ceptive neuron (Figs. 4H, K) (Lacalli, 2002; Bardet et al., 2005).In normal embryos, ERR is never expressed more posteriorly inthe CNS (Fig. 4H) (Bardet et al., 2005). Other motor neuronmarkers were less useful for this analysis, since they are eithertransiently transcribed in several different cell populations ofthe amphioxus hindbrain (e.g., Islet and Krox) (data not shown)(Jackman et al., 2000; Knight et al., 2000; Jackman andKimmel, 2002; Bardet et al., 2005) or they are expressed innumerous neurons in both the amphioxus hindbrain and spinalcord (e.g., Neurogenin and Coe) (data not shown) (Holland etal., 2000; Mazet et al., 2004).

Treatment with RA reduces the Otx domain (Figs. 4A, D),while BMS009-treatment expands the domain posteriorly intothe hindbrain (Figs. 4C, F). These effects are already detectableat the neurula stage (Figs. 4A, C), but become even moreconspicuous in early larvae (Figs. 4D, F). The effect of blockingHox1 function by injection of an antisense morpholino-oligonucleotide specific for Hox1 into amphioxus eggs wasindistinguishable from that of BMS009. Both the Hox1morpholino and the RA-antagonist BMS009 expanded Otxexpression posteriorly (compare Figs. 4C, F, 5B, D). This effectof the Hox1 morpholino is already detectable in 28-h embryos(Figs. 5A, B), but becomes much more conspicuous in 44-h larvae (Figs. 5C, D). We conclude that Hox1 probablymediates the effect of RA-signaling on the posterior limit of Otxexpression in the amphioxus CNS.

Altered RA-signaling not only changes the anterior expres-sion limits of the Hox genes, it also influences the number andposition of the DC motor neurons as well as the morphology ofthe first photoreceptor (Figs. 4G–L; data not shown). In controllarvae, the six pairs of DC motor neurons and the two neuronsof the first photoreceptor are marked by expression of ERR(Figs. 4H, K). Abnormal (doubled, reduced or absent) pigmentcells occurred in 55% and 70% of larvae treated with RA- orBMS009, respectively, compared to 25% of controls withabnormal pigment cells (N = 20 in each group). Moreover, RA-or BMS009-treatment, respectively, shifts the DC motorneurons and first photoreceptor neurons anteriorly or posteriorlyin the CNS (Figs. 4G–L, 7). Thus, BMS009 has the effect ofadding several ERR-expressing cells posterior to the firstphotoreceptor (Figs. 4I, L, 7). For scoring these effects ofaltered RA-signaling on cell abundance, it was convenient toadd together the number of DC motor neurons plus the neurons

of the first photoreceptor. This total number of ERR-expressingcells, which was 14 in the controls, decreased to a mean of 10after RA-treatment and increased to a mean of 17 afterBMS009-treatment (Fig. 6A). These were highly significantchanges when tested statistically (details in caption to Fig. 6).Importantly, injection of the Hox1morpholino gave an identicalresult to treatment with BMS009. In Hox1 morpholino-injectedanimals, the number of cells expressing ERR is increased to anaverage of 17 and they extend posterior to the first pigment spot(Figs. 5E–H and 6B). Moreover, in 60% of the larvae injectedwith Hox1 morpholino, compared to 15% of larvae injected


with the control morpholino, the first photoreceptor is disrupted(N = 20 in each group) (Figs. 5A–H; data not shown). Takentogether, these results show that Hox1 mediates, at least in part,the roles of RA both in regionalization of the amphioxushindbrain and in specification of motor neuron identity as wellas in formation of the first photoreceptor.

Discussion

The present study shows that RA-signaling specifies bothpositional and neuronal identity in the amphioxus hindbrain andspinal cord, but does not regulate expression of several geneswithin the cerebral vesicle, the equivalent of the diencephalonand midbrain. Morpholino knock-down of Hox1 suggests thatboth these roles of RA-signaling are mediated by Hox1. Inaddition, we show that the single Hox1, Hox2, Hox3, Hox4 andHox6 genes are collinearly expressed in the amphioxus CNS,correcting previous reports that Hox2 is not expressed in theamphioxus CNS (Wada et al., 1999) and that Hox6 expressionin the CNS breaks collinearity (Cohn, 2002).

Collinearity of Hox genes in the CNS is fundamental tochordates

Although the amphioxus hindbrain lacks obvious segments,nested expression of five Hox genes (Hox1, Hox2, Hox3, Hox4and Hox6) shows that it is, in effect, divided into rhombomereequivalents (Holland et al., 1992; Holland and Holland, 1996;Wada et al., 1999). In light of these results, it is surprising thatprevious work found no expression of Hox2 in the CNS (Wadaet al., 1999) and the anterior limit of Hox6 to be rostral to that ofHox1 (Cohn, 2002). We used the same Hox clones to synthesizeriboprobes as in these studies and sequenced the clones toconfirm their identity. The previous failure to detect Hox2expression in the amphioxus CNS could be because of acomparatively low level of Hox2 expression. However, wefound Hox6 expressed at a high level, but only in tail bud,extreme posterior somites and posterior endoderm in addition tothe posterior 40% of the nerve cord. We could not detect anyHox6 expression in the anterior third of the larva as reported byCohn (2002). Since the signals he reported are widespread andrather weak, it seems likely that they represent non-specificsticking of the probe.

Our results suggest that collinear expression of Hox genes inthe CNS is fundamental to chordates. Amphioxus has a singleHox cluster of 14 genes (Ferrier, 2004; Minguillon et al., 2005)and phylogenetic analyses indicate that the chordate ancestorprobably had a Hox cluster of at least 13 (Ikuta and Saiga,2005). Tunicates, which recent phylogenetic analyses place asthe sister group of vertebrates (Blair and Hedges, 2005; Philippeet al., 2005), have lost several Hox genes and both clusterorganization and spatio/temporal collinearity have brokendown. For example, the ascidian Ciona has lost Hox7, Hox8,Hox9 and Hox11 (Ikuta et al., 2004), while the appendicularianOikopleura has lost Hox3, Hox6, Hox7 and Hox8 (Seo et al.,2004). Only about half of the remaining Hox genes areexpressed in the CNS and they display only approximate

collinearity. For example, in Ciona, the anterior limits of Hox1and Hox3 are the same, that of Hox5 is just posterior to that ofHox3, that of Hox10 is just posterior to that of Hox5 and that ofHox12 is far posterior (Ikuta et al., 2004). In Oikopleura, therostral limit ofHox1 in the CNS is anterior to that ofHox4, thoseof Hox10 and Hox13 are posterior to that of Hox4, and Hox11breaks collinearity (Seo et al., 2004).

Although the Hox cluster has been duplicated in vertebrates,expression of Hox genes in the gnathostome CNS is collinearexcept for Hox2, which has a rostral limit of expression anteriorto that ofHox1 (McGinnis and Krumlauf, 1992; Deschamps andvan Nes, 2005). Our results suggest that this anterior expansionof Hox2 in the vertebrate CNS may be vertebrate-specific.However, it is not known whether Hox2 also breaks collinearityin agnathans. The rostral limit of lamprey Hox2 is anterior tothat of Hox3 (Takio et al., 2004), but expression ofHox1 has notbeen reported.

RA-signaling and Hox genes establish positional identity in thehindbrain/spinal cord of the amphioxus CNS

RA expands the expression domains of Hox1, Hox2, Hox3,Hox4 and Hox6 in the amphioxus CNS to the rostral limit of theCNS, while the RA-antagonist BMS009 shifts them posteriorly(Fig. 7). It is likely that at least Hox1 and Hox3 are direct targetsof RA-signaling, since DNA binding sites for the retinoic acidreceptor (RAR) are present in their regulatory regions(Manzanares et al., 2000; Wada et al., 2006). Moreover, theexpression domain of RAR, which is autoregulated, isapproximately congruent with that of Hox1 and is similarlyaffected by altered RA-signaling (Escriva et al., 2002). Inaddition, blocking Hox1 function mimics the effect of the RA-antagonist on Otx and ERR expression. It may be general thatRA regulates genes such as Wnt3 and Pax2/5/8 that are broadlyexpressed in the hindbrain/spinal cord with an anterior limit ator posterior to the cerebral vesicle/hindbrain boundary but doesnot affect expression of genes that are normally transcribedthroughout the length of the nerve cord (e.g., Notch) orexpressed only in the cerebral vesicle (e.g., Wnt5 and CRABP).

Since expression of Otx is largely limited to the cerebralvesicle (Williams and Holland, 1996, 1998), it has beensuggested that the posterior limit of Otx corresponds approx-imately to the vertebrate midbrain/hindbrain boundary (MHB).This idea is reinforced by expression of Gbx in the hindbrainwith an anterior limit abutting the posterior limit of Otx (Castroet al., in press). The Otx/Gbx expression boundary appears to bea major dividing line in the CNS with genes often expressedeither anterior (e.g., Pax6 and Nkx2.2) or posterior (Wnt6) to it(Glardon et al., 1998; Holland et al., 1998; Schubert et al.,2001). A key feature of this boundary appears to be an abruptchange in the level of RA-signaling from very low in thecerebral vesicle to very high in the hindbrain/spinal cord. Thus,as noted above, RAR is only expressed in the hindbrain andspinal cord, while the orphan nuclear receptor TR2/4, acompetitive inhibitor of RAR, is expressed strongly in thecerebral vesicle (Escriva et al., 2002) and may represstranscription of RAR there as well as that of RAR target

Fig. 7. Summary of the effects of RA and RA-antagonist on patterning andspecification of motor neurons in the amphioxus CNS. Expression of Hox1(yellow), Hox2 (turquoise), Hox3 (orange), Hox4 (red), Hox6 (green) and ERR(blue) in normal (center), RA-treated (left) and RA-antagonist-treated (right) 30-h larvae is shown relative to the position of somites (numbered at left).Expression of ERR in the anterior cerebral vesicle is not shown. The pigment cellof the first photoreceptor is depicted as a black circle. In control larvae,expression of Hox1, Hox2, Hox3, Hox4 and Hox6 in the CNS respects spatialcollinearity. RA shifts expression of all five Hox genes to the anterior tip of theCNS and shortens and shifts anteriorly the ERR expression domain. The totalnumber of neural cells labeled with ERR is decreased. In BMS009-treatedlarvae, the expression domains of the five Hox genes are shifted posteriorly, theERR domain is expanded posteriorly, and the number of cells expressing ERR isincreased. The position of the first photoreceptor is only slightly affected by thetreatments. In controls, the photoreceptor is at the level of the center of the 5thsomite. RA and RA-antagonist shift it, respectively, to the anterior and posteriorend of the 5th somite. Somite numbers were determined using as landmark theexpression of ERR in all somites, but the most anterior pair (Bardet et al., 2005).


genes. Similarly, in vertebrates, TR2 and TR4 are transcrip-tional repressors that bind to retinoic acid response elements(Lee et al., 1999, 2004), although they are broadly expressed inthe developing CNS (Wirtanen et al., 1998; van Schaick et al.,2000). Moreover, CRABP is expressed in the posterior portionof the amphioxus cerebral vesicle (Jackman et al., 2004) in apattern resembling that of CRABPIa in the zebrafish CNS (Liuet al., 2005). Evidence to date indicates that CRABPI, unlikeCRABPII, does not interact directly with RAR but inhibitstranscriptional activity of RA either by sequestering it or byenhancing its breakdown (Dong et al., 1999). Such a role seemslikely for amphioxus CRABP. Although treatment with RAshifts expression of several genes into the cerebral vesicle, itdoes not affect either the TR2/4 or the CRABP domains. Thissuggests that even if RA-levels are altered, CRABP and TR2/4may still provide some protection against the influence of RA-signaling in the cerebral vesicle. This may explain, at least inpart, why the gross morphology of the amphioxus forebrain andexpression of some genes (e.g., Otx and ERR) in the cerebral

vesicle is not substantially affected by altered RA-signaling,even though their domains at more posterior levels of the CNSare changed.

RA-signaling and Hox genes have a role in specification ofmotor neuron identity

RA-signaling, in addition to its role in regional patterning ofthe amphioxus CNS, controls the number of DC motor neurons,which, like their proposed homologs, the vertebrate branchio-motor neurons (Lacalli and Kelly, 1999), express Islet, ERR andNeurogenin and are located in the posterior midbrain andanterior half of the hindbrain (Lacalli and Kelly, 1999, 2003b;Bardet et al., 2005). However, unlike the vertebrate branchio-motor neurons, the amphioxus motor neurons do not appear tomigrate, but differentiate in place. Treatment with RA not onlyshifts the DC motor neurons anteriorly, it also reduces theirnumbers, evidently altering the fate of these neurons. It is notknown what this fate may be, since markers specific for othertypes of amphioxus neurons have not been identified.Conversely, both the RA-antagonist and the Hox1 morpholinoexpand the domains of the DC motor neurons posteriorly andincrease their numbers, suggesting that Hox1 mediates theeffect of RA in motor neuron specification as well as in anteriorposterior patterning in the amphioxus CNS (Fig. 7).

This simple amphioxus model appears to reflect thefundamental molecular basis of hindbrain patterning andmotor neuron specification in chordates. In tunicates, there areonly six motor neurons, which are arrayed in pairs as inamphioxus (Katsuyama et al., 2005). The effects of RA onmotor neuron specification have not been studied in tunicates,although RA treatment does expand the Hox1 domain in theCNS (Nagatomo and Fujiwara, 2003). In vertebrates, ascompared to amphioxus, the CNS has far more neurons and agreater diversity of motor neuron subtypes. In addition to thebranchiomotor (BM) neurons, which innervate muscles withinthe branchial arches, there are visceral motor (VM) neurons,which innervate parasympathetic ganglia, and somatic motor(SM) neurons, which innervate the extraocular and tonguemuscles (Lumsden and Keynes, 1989; Guidato et al., 2003a).Application of RA increases the number of primary neurons andspreads them anteriorly, while reduced RA levels decrease thenumber of primary neurons (Sharpe and Goldstone, 2000;Maden, 2002). In addition, RA treatment reduces the number ofhindbrain BM and VM neurons by respecifying them as SMneurons (Guidato et al., 2003a). Inhibition of the RA-degradingenzyme Cyp26 similarly promotes the differentiation of SMneurons in rostral rhombomeres of hindbrain explants as doesgrafting of RA-soaked beads beneath the rostral hindbrain(Guidato et al., 2003a,b).

The effects of altered RA- and Hox1-signaling on thedevelopment of facial BM neurons in vertebrates appear to becomparable to those on DC motor neurons in amphioxus, whichare located in a region that extends from the anterior half of theHox1 domain rostrally into the small midbrain. In amphioxus,either Hox1 knock-down or treatment with RA-antagonistincreases the numbers of DC motor neurons, suggesting that


Hox1 mediates the effect of RA-signaling in controlling theirfate. Similarly, in vertebrates, the facial BM motor neuronsdevelop in the Hox1-expressing region (rhombomere 4), whilethe trigeminal BM neurons develop more anteriorly in thehindbrain. In the mouse, knock-out of Hoxa1 and Hoxb1extends the domain of BM neurons along the entire hindbrain(Rossel and Capecchi, 1999). However, these BM neurons donot appear to be properly specified. The VIIth through XIthcranial nerves are defective (Gavalas et al., 1998) and even inmice mutant only for Hoxb1, the facial nerve is missing(Goddard et al., 1996). Simultaneous knock-down of all threeHox1 genes in Xenopus results in more severe overall patterningdefects in rhombomeres 3–5 than knock-down of only one ortwo Hox1 genes (McNulty et al., 2005). In addition, interplaybetween RA and Hox has been shown to be required for theproper specification of subtypes of spinal motor neurons(Vermot et al., 2005). Thus, given the similarities in motorneuron patterning and specification between amphioxus andvertebrates, it seems very likely that, as in amphioxus, at leastsome of the functions of Hoxa1 and Hoxb1 in BM neurondevelopment of vertebrates are controlled by RA-signaling.

Our results showing that RA-signaling acts via Hox1 both inregional patterning of the CNS and in motor neuronspecification raise the question of whether these two roles areindependent or whether the number of DC motor neurons issimply due to hindbrain size, which is reduced by excess RA(hence fewer motor neurons) and expanded either by the RA-antagonist or knock-down of Hox1 (hence more motorneurons). In vertebrates, as in amphioxus, Hox genes are directtargets of RA-signaling and RA and Hox have each been shownto affect both anterior/posterior patterning and motor neuronspecification in the hindbrain (Guidato et al., 2003b; Oosterveenet al., 2003). However, opinion is divided as to whether the rolesof RA-signaling in anterior/posterior patterning and branchio-motor neuron specification are independent (Begemann et al.,2004; Linville et al., 2004) or not (Gavalas et al., 2003). Inlampreys, excess RA shifts the domains of both Hox3 andbranchiomotor nuclei, but does not affect the morphology of thehindbrain rhombomeres (Murakami et al., 2004). Thus, theeffects of RA on rhombomere morphology probably evolvedlater than those on Hox expression and branchiomotor neurons.Our data show that the roles of both RA and Hox genes inanterior/posterior patterning of the hindbrain and in specifica-tion of motor neuron identity preceded the evolution ofrhombomeres, being present before vertebrates arose from anancestral protochordate. Subsequently, early during vertebratediversification, the hindbrain became organized into rhombo-meres but, as suggested by Murakami et al. (2004), only afterthe divergence of the lamprey lineage did RA and Hox controlof hindbrain patterning become linked to rhombomere identity.

In amphioxus, similar mechanisms are involved in anterior/posterior patterning of the general ectoderm and hindbrain

The general ectoderm of amphioxus embryos includesseveral different types of sensory neurons (Stokes and Holland,1995; Holland and Yu, 2002; Holland, 2005). As in the CNS,

Hox1, Hox3, Hox4 and Hox6 are collinearly expressed in thegeneral ectoderm, while different subsets of the ectodermalsensory neurons express particular combinations of Hox genestogether with specific combinations of the proneural markersCoe, Islet and ERR (Schubert et al., 2004). Moreover, treatmentswith RA or RA-antagonist have similar effects on the collinearexpression of Hox genes, on the combinatorial code of neuronalmarkers as well as on the numbers and distribution of neurons inthe general ectoderm as they do in the CNS. The RA-antagonistthus increases the number of sensory neurons in the generalectoderm and skews them posteriorly, while RA skews themanteriorly. Although there are some differences in patterning theCNS and general ectoderm (for example, the RA-antagonistincreases the number of ERR-expressing cells in the nerve cord,but completely downregulates the gene in the generalectoderm), the mechanisms for patterning general ectodermand CNS of amphioxus appear to be closely related.

A role for Hox genes in anterior/posterior patterning of theectoderm is probably an ancestral characteristic of alldeuterostomes. In hemichordates, which lack a CNS andinstead have a diffuse ectodermal nerve net, Hox1, Hox3,Hox4, Hox7/8 and Hox11/13 are collinearly expressed in theectoderm (Lowe et al., 2003). Whether RA-signaling patternshemichordate embryos is not known, but RA apparently doesnot appear to affect axial patterning in echinoderms, the sistergroup of hemichordates (Sciarrino and Matranga, 1995).However, echinoderms have lost bilateral symmetry and Hoxgenes are not expressed in the general ectoderm or in either thelarval or the developing adult nervous system (Arenas-Mena etal., 2000). Thus, if RA-signaling were found to regulate Hoxexpression in the hemichordate ectoderm, it would suggest thatthe linkage between RA-signaling and Hox genes originated atthe base of the deuterostomes and was lost in the echinodermlineage.

In summary, our results underscore the value of amphioxusas a model system for chordate neural development. Theamphioxus CNS has a diencephalon, midbrain, hindbrain andspinal cord (Holland and Holland, 1999; Holland and Chen,2001; Wicht and Lacalli, 2005), but there are only about 20,000neurons in the adult (Nicol and Meinertzhagen, 1991; Lacalliand Kelly, 1999; Wicht and Lacalli, 2005). The single cluster ofHox genes is collinearly expressed in the CNS, and the larvalmotor neurons, of which there are only two types, express thetypical complement of gene markers (Holland et al., 2000;Jackman and Kimmel, 2002; Jackman et al., 2000; Bardet et al.,2005; Langeland et al., 2006). RA-signaling regulates Hoxexpression, global regionalization and motor neuron identity inthe amphioxus hindbrain. Moreover, knock-down of Hox1indicates that the effects of RA-signaling are probably mediatedby Hox1. Taken together, they show that amphioxus isparticularly well suited for studying the roles of the RA-signaling cascade in the developing chordate CNS.

Acknowledgments

The authors thank J. M. Lawrence and the University ofSouth Florida, Tampa, FL, USA, for providing laboratory space


and H. Gronemeyer for generously supplying the BMS009compound. We are also grateful to P. W. H. Holland, S. M.Shimeld and W. R. Jackman for providing clones of amphioxusgenes used as markers in this analysis. The authors arefurthermore indebted to A. Chaumot and M. A. van Zuilenfor help with statistical analyses and to H. Escriva for fruitfuldiscussions. This research was supported by NSF grants IBN00-78599 and IOB-0416292 (to L.Z.H. and N.D.H.), grantNAG2-1585 from NASA (to L.Z.H.), by grant 1-FY05-108from the March of Dimes (to L.Z.H.), by funds from MENRTand CNRS (to V.L. and M.S.) and by grants from ARC and theRégion Rhône-Alpes (to V.L.). M.S. was also supported by aEuropean Community Marie Curie Individual Fellowship.

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a retinoic acid-hox hierarchy controls both anterior/posterior patterning and neuronal specification...

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