SPECIAL ISSUE RESEARCH ARTICLE

Xenopus hairy2 Functions in Neural CrestFormation by Maintaining Cells in a Mitoticand Undifferentiated StateKan-Ichiro Nagatomo and Chikara Hashimoto*

The neural crest is a population of mitotically active, multipotent progenitor cells that arise at the neuralplate border. Neural crest progenitors must be maintained in a multipotent state until after neural tubeclosure. However, the molecular underpinnings of this process have yet to be fully elucidated. Here we showthat the basic helix-loop-helix (bHLH) transcriptional repressor gene, Xenopus hairy2 (Xhairy2), is anessential early regulator of neural crest formation in Xenopus. During gastrulation, Xhairy2 is localized atthe presumptive neural crest prior to the expression of such neural crest markers as Slug and FoxD3.Morpholino-mediated knockdown of Xhairy2 results in the repression of neural crest marker geneexpression while inducing the ectopic expression of the cell cycle inhibitor p27xic1 in the presumptiveneural crest. We also found that ectopic p27xic1 disturbs neural crest formation. Furthermore, the depletionof Xhairy2 leads to the apoptosis of mitotic cells. Our results suggest that Xhairy2 functions in neural crestspecification by maintaining cells in the mitotic and undifferentiated state. Developmental Dynamics 236:1475–1483, 2007. © 2007 Wiley-Liss, Inc.

Key words: neural crest; Xhairy2; Foxd3; Slug; p27xic1; X-Delta-1

Accepted 8 March 2007

INTRODUCTION

The neural crest is a multipotent andtransient population of ectoderm de-rivatives that arise from the borderbetween the neural plate and epider-mis in vertebrate embryos. As theneural tube closes, neural crest cellsdelaminate and migrate to diverse lo-cations. They differentiate into di-verse cell types that include neuronsand glia of the peripheral nervous sys-tem, smooth muscle cells, craniofacialcartilage, pigment cells, bone, and fin(LaBonne and Bronner-Fraser, 1999;Christiansen et al., 2000).

Many studies have focused on theinduction of neural crest precursors.Some extracellular signaling mole-cules such as BMP, Wnt, FGF, and

Notch are believed to up-regulate spe-cific target transcription factors thathave been shown to be required forneural crest formation (LaBonne andBronner-Fraser, 1998; Huang andSaint-Jeannet, 2004; Steventon et al.,2005; Cornell and Eisen, 2005). Thecombinatorial action of signaling mol-ecules and these transcription factorsdefines the bona fide neural crest re-gion (reviewed by Sauka-Spengler andBronner-Fraser, 2006), although littleis known about how these signalingmolecules and transcription factorsregulate neural crest induction andmaintenance. Concomitant with theinduction of neural crest precursors,the neural crest precursors must bemultipotent progenitors. It has been

suggested that there should be someprotective mechanism that preventsneural crest precursors from adoptingthese alternate fates and immaturedifferentiation, so that the neuralcrest precursors can be generated ad-jacent to signals that influence bothneural plate and prospective epider-mal cell fates. For example, the proto-oncogenic protein c-Myc and its directtarget gene ID3 were suggested toplay an essential role in maintainingneural crest progenitors in the multi-potent state (Bellmeyer et al., 2003;Light et al., 2005; Kee and Bronner-Fraser, 2005), yet little is knownabout the molecular underpinnings ofthis process.

Hairy and enhancer of split (hes)-

Department of Biology, Graduate School of Science, Osaka University, and JT Biohistory Research Hall, Osaka, Japan*Correspondence to: Chikara Hashimoto, JT Biohistory Research Hall, 1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, Japan.E-mail: hashimoto@brh.co.jp

DOI 10.1002/dvdy.21152Published online 13 April 2007 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 236:1475–1483, 2007

© 2007 Wiley-Liss, Inc.

related genes are known to function inthe establishment of various tissueidentities in both vertebrates and in-vertebrates (Fisher and Caudy, 1998;Davis and Turner, 2001). In particu-lar, hes-related genes have a promi-nent role in inhibiting neurogenesis.Recent studies have revealed that thetiming of neural stem cell differentia-tion is critically controlled by multiplehes-related genes (reviewed byKageyama et al., 2005). Additionally,in general, differentiation is closelyrelated to the cell cycle, and hes-re-lated genes also act to control cell cy-cle progression. Hes1 directly contrib-utes to the promotion of progenitorcell proliferation through transcrip-tional repression of a cyclin-depen-dent kinase inhibitor, p27Kip1, in em-bryonic carcinoma cells (Murata et al.,2005). Zebrafish p27xic1 expression isnegatively regulated by Her5 in themidbrain-hindbrain boundary (Gelinget al., 2003). These findings suggestedthat hes-related genes might be in-volved in maintaining neural creststem cells in the mitotic and undiffer-entiated state.

In Xenopus, a member of the hes-related gene, Xhairy2, is expressed inthe entire prospective ectodermal re-gion prior to the gastrula stage. Asgastrulation progresses, the ectoder-mal expression of Xhairy2 is localizedas a narrow stripe that curves andforms a border between the neuralplate and the epidermis during gas-trulation (Tsuji et al., 2003). AlthoughXhairy2 has been suggested to be thetranscriptional effector of Notch sig-naling and to act as a repressor ofXbmp4 transcription during cranialneural crest induction (Glavic et al.,2004), its role in neural crest develop-ment is largely unknown. Here, wereport that the morpholino-mediatedknockdown of Xhairy2 results in therepression of neural crest markergene expression and neural crest de-rivatives. Our results also suggestthat the depletion of Xhairy2 affectsneural crest cell proliferation and sur-vival, and the neural crest derivatives,in turn, are abolished. Furthermore,our data indicate that Xhairy2 is es-sential for the repression of cdk inhib-itor p27xic1 expression in the pre-sumptive neural crest region from thegastrula stage, and this repression islikely to be responsible for neural

crest specification. We propose thatXhairy2 functions in early neuralcrest specification by maintainingcells in the mitotic and undifferenti-ated state.

RESULTS

Xhairy2 Is Required forNeural Crest Development

Xhairy2 expression is localized at theneural plate border that contributes tothe neural crest at the early neurulastage (Tsuji et al., 2003; see also Fig.1A), and the overexpression ofXhairy2 results in an increase of theneural crest population (Glavic et al.,2004). We, therefore, examined theroles of Xhairy2 in the normal devel-opment of the presumptive neuralcrest region by performing loss-of-function studies using morpholino an-tisense oligonucleotides (MOs). Injec-tion of Xhairy2-MO led to repressionof the expression of such neural crestmarkers as Slug and FoxD3 at theearly neurula stage (Slug, 86%, n �72; FoxD3, 90%, n � 82; Fig. 1C,F),whereas injection of Control-MO didnot affect neural crest marker expres-sion (Slug, 100% normal, n � 31;FoxD3, 100% normal, n � 42; Fig.1B,E). This disappearance of the neu-ral crest markers was rescued by co-injection of Xhairy2 mRNA that doesnot contain the morpholino annealingsite (Slug, 29% inhibition, n � 76;FoxD3, 30% inhibition, n � 64; Fig.1D,G). These results suggest thatXhairy2 is required for the early de-velopment of the neural crest.

The loss of early neural crest markerexpression in Xhairy2-depleted em-bryos suggests that Xhairy2 may playimportant role(s) in neural crest devel-opment. To this end, we next attemptedto examine what happens to neuralcrest derivatives in Xhairy2-depletedembryos at later stages. Embryos in-jected with Xhairy2-MO showed repres-sion of both Slug and FoxD3 expres-sions at mid neurula stage (Slug, 72%,n � 25; FoxD3, 66%, n � 29; Fig. 2C,D),whereas injection of Control-MO did notaffect neural crest marker expression(Slug, 100% normal, n � 24; FoxD3,100% normal, n � 25; Fig. 2A,B).Embryos injected with Xhairy2-MOshowed reduced number of migratoryneural crest cells as depicted by Xtwist

expression at stage 24 (68%, n � 28;Fig. 2F). In Xhairy2-depleted embryosat stage 25, we detected no n-tubulin-positive cranial nerves, which areknown to be the derivatives of cranialneural crest (64%, n � 22; Fig. 2H). Atstage 45, the branchial arch cartilageand other derivative tissues of the cra-nial neural crest almost completely dis-appeared (50%, n � 6; Fig. 2K) on theinjected side. The mesenchymes of dor-sal fin and melanocytes originating inthe trunk neural crest were not formedin embryos injected with Xhairy2-MO(56%, n � 16; Fig. 2M). Therefore, theloss of neural crest derivatives inXhairy2-depleted embryos appears tobe a consequence of early hypoplasia ofthe neural crest region.

Xhairy2 Affects Neural CrestDevelopment in a Cell-Autonomous Manner

As shown above, Xhairy2 seems to beinvolved in the development of theneural crest from an early stage. Sohow does Xhairy2 function in this pro-cess? It has been surmised that aninteraction between the neural plateand the epidermis is involved in theinduction of the neural crest (Mouryand Jacobson, 1990; Selleck andBronner-Fraser, 1995; Mancilla andMayor, 1996). Thus, it may be possiblethat Xhairy2 affects the formation ofeither the neural plate or the epider-mis, and the development of the neu-ral crest, in turn, is affected indirectly.However, the expression patterns ofboth neural plate markers (Xsox2 andXsox3) and the epidermal marker(Keratin) were observed in Xhairy2-depleted embryos (Xsox2, 100%, n �56; Keratin, 100%, n � 53; Fig. 1H,I,and data not shown), suggesting thatthe loss of early neural crest markerexpression by Xhairy2-MO may havea direct effect on the neural crest.

Another possibility is that Xhairy2may regulate the expression ofXbmp4, since it was reported thatNotch signaling might participatein neural crest specification by down-regulating Xbmp4 transcriptionthrough the activation of Xhairy2A(Glavic et al., 2004). However, the de-pletion of Xhairy2 did not lead to anychanges in the expression patterns ofXbmp4 from early gastrula to earlyneurula stage (100%, n � 46; Fig. 1J,K

1476 NAGATOMO AND HASHIMOTO

and data not shown). We also foundthat the overexpression of X-Delta-1stu, which inhibits Notch signaling(Chitnis et al., 1995), up-regulates

Xbmp4 expression (data not shown),in agreement with a previous study(Kuriyama et al., 2006), suggestingthat the up-regulation of Xbmp4 ex-

pression by Notch signaling inhibitionis mediated by not only Xhairy2 butalso other Notch signaling target mol-ecules.

Taken together, we conclude thatXhairy2 cell-autonomously regulatesthe expression of early neural crestmarkers, completely independent ofthe formation of other tissues.

Depletion of Xhairy2 DoesNot Lead to PrematureNeuronal Differentiation,But Induces Ectopic p27xic1

and X-Delta-1 Expression inPresumptive Neural Crest

Because previous studies have indi-cated that Xhairy2 is an antineuro-genic gene (Dawson et al., 1995;Stancheva et al., 2003) and Notch sig-naling promotes formation of neuralcrest by repressing neurogenin1 func-tion (Cornell and Eisen, 2002), the de-pletion of Xhairy2 may lead to prema-ture neuronal differentiation thatcould cause the loss of early neuralcrest markers. To examine this possi-bility, we analyzed the expression ofthe neural differentiation marker n-tubulin (Chitnis et al., 1995) and theneuronal determination gene X-ngnr-1(Ma et al., 1996) in Xhairy2-depletedembryos. Xhairy2-MO injection led toa slight increase of n-tubulin and X-ngnr-1 expression around trigeminalganglia (n-tubulin, 39%, n � 31; X-ngnr-1, 38%, n � 26; Fig. 3A,B), but

Fig. 1. Xhairy2 is required for neural crestmarker gene expression. A: Double in situ hy-bridization with Xhairy2 (magenta) and Slug(blue) at stage 13. B,E: Control-MO injectionhas no effect on any of the neural crest markersexamined at stage 13: Slug (B) and FoxD3 (E).C,F: In embryos injected with Xhairy2-MO, theexpression of Slug (C) and FoxD3 (F) is inhibitedon the injected side (red arrowhead) at stage13. D,G: Loss of neural crest marker expressionin embryos injected wit Xhairy2-MO is rescuedby injecting 20 pg of Xhairy2 mRNA (yellowarrowhead): Slug (D) and FoxD3 (G). H,I: In em-bryos injected with Xhairy2-MO, the expressionof Xsox2 (H) and Keratin (I) is not apparentlyaffected at stage 13. White brackets indicatethe width of the neural plate. J,K: In embryosinjected with Xhairy2-MO, Xbmp4 expression isnot affected at stages 11.5 (J) and 12.5 (K). A,lateral views with anterior to the left. B–K, dorsalviews with anterior to the bottom. Injected areais the right side. Turquoise staining is lineagetracer �-gal.

XHAIRY2 IN NEURAL CREST SPECIFICATION 1477

not in the neural crest. These resultsindicate that the early repression ofneural crest marker expression byXhairy2-MO may not depend upon thepremature neuronal differentiation.

We also examined the expression ofthe neurogenic gene X-Delta-1 (Chit-nis et al., 1995) and the cell cycle in-hibitor p27xic1 that is required for pri-mary neurogenesis (Vernon et al.,2003) in the absence of Xhairy2. Wefound that the expression patterns ofboth p27xic1 and X-Delta-1 were af-fected, and that those ectopic expres-sions were repressed by co-injectingXhairy2 mRNA, suggesting that thoseeffects are specific to Xhairy2. As re-gards the expression pattern ofp27xic1, broad ectopic expression wasobserved in the ectoderm includingthe presumptive neural crest (96%,n � 84; Fig. 3D), and this ectopic ex-pression was completely repressed byco-injecting Xhairy2 mRNA (91% n �46; Fig. 3E). This up-regulation wasobserved from the early gastrula stage(data not shown). In good agreementwith this result, Xhairy2 expression islocated in the anterior region andp27xic1 expression is in the posteriorregion, appearing to complement eachother from the early gastrula stage(Fig. 3I,J and data not shown). In ad-dition, we noted the weak ectopic ex-pression of X-Delta-1 in the cranialneural crest region (75%, n � 40; Fig.3G), and this ectopic expression wascompletely repressed by co-injectingXhairy2 mRNA (100% n � 33; Fig.3H). A previous study showed thatSlug expression is surrounded by X-Delta-1 expression and they neveroverlap in early neurula embryo(Glavic et al., 2004). Together, thesedata indicate that p27xic1 and/or X-Delta-1 may hinder cells from takingthe neural crest fate, and Xhairy2 re-presses the expression of these genesto establish neural crest identity.

Repression of p27xic1

Expression by Xhairy2 IsInvolved in Early NeuralCrest Specification

During gastrulation, Xhairy2 re-presses p27xic1 expression in the ecto-derm that includes the presumptiveneural crest prior to the expression ofearly neural crest markers. To exam-ine whether the repression of p27xic1

expression by Xhairy2 is required forthe expression of early neural crestmarkers, we first overexpressedp27xic1 mRNA. The overexpression ofp27xic1 inhibited Slug and FoxD3 ex-pression (Slug, 83%, n � 36; FoxD3,73%, n � 40; Fig. 4A,B). These effectsare similar to that of Xhairy2-MO. X-Delta-1 expression was slightly in-creased by p27xic1 overexpression im-mediately outside the cranial neuralcrest region (52%, n � 44; Fig. 4C).

Therefore, the repression of Slug andFoxD3 expression by Xhairy2-MOcould be realized, at least in part, bythe ectopic expression of p27xic1.

To further evaluate the effect ofp27xic1 repression by Xhairy2 on earlyneural crest development, we next ex-amined whether early changes of neu-ral crest marker and X-Delta-1 expres-sion by Xhairy2-MO are rescued byp27xic1-MO. The injection of p27xic1-MOresulted in a slightly increased number

Fig. 2. Depletion of Xhairy2 leads to loss of neural crest derivatives. A,B: Control-MO injection hasno effect on any of the neural crest markers examined at stage 17: Slug (A) and FoxD3 (B). C,D: Inembryos injected with Xhairy2-MO, the expression of Slug (C) and FoxD3 (D) in inhibited on theinjected side (red arrowhead) at stage 17. E,F: Compared with the control side (E), the Xhairy2-MOinjected side shows reduced migratory neural crest cells (F) as depicted by Xtwist expression (redarrowhead) at stage 24. G,H: Compared with the control side (G), the Xhairy2-depleted embryoshows no cranial sensory ganglia on the injected side (H) as depicted by n-tubulin expression (redarrowhead) at stage 25. I: Schematic diagram depicting ventral cranial cartilages of a stage 45embryo. J: Cartilage staining of Control-MO injected embryo shows normal structures at stage 45.K: Cartilage staining of Xhairy2-MO injected embryo shows partial loss of cartilage on the injectedside (red arrowhead) at stage 45. L,M: Phenotypes of stage 35 embryos whose bilateral trunkneural crest was injected with Control-MO or Xhairy2-MO. Melanocytes of Xhairy2-MO injectedembryo are reduced (M; yellow arrowhead) compared with Control-MO injected embryo (L). L: Thedorsal fin of Control-MO injected embryo shows normal structures M: The dorsal fin of Xhairy2-MOinjected embryo shows partial loss (red arrowhead). A–D, dorsal views with anterior to the bottom.E,G, lateral views with anterior to the right. F, H, L, M, lateral views with anterior to the left. J, K,ventral views with anterior to the top. A–D,J,K, injected area is the right side. Turquoise staining islineage tracer �-gal.

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of apoptotic cells, but did not signifi-cantly affect neural crest marker or X-Delta-1 expression (Slug, 100% normal,n � 24; FoxD3, 100% normal, n � 28;

X-Delta-1, 100% normal, n � 20; Fig.4D–F and data not shown). The repres-sion of early neural crest marker ex-pression by Xhairy2-MO was partially

rescued by co-injecting p27xic1-MO withXhairy2-MO (Slug, 48% inhibition, n �69; FoxD3, 51% inhibition, n � 67; Fig.4J,K), but not by co-injecting Con-trol-MO (Slug, 75% inhibition, n � 56;FoxD3, 83% inhibition, n � 64; Fig.4G,H). Similar results were observedfor X-Delta-1 expression in the cranialneural crest region (Control-MO �Xhairy2-MO, 84% ectopic expression,n � 61; p27xic1-MO � Xhairy2-MO, 54%ectopic expression, n � 72; Fig. 4I,L).The finding that the percentage of res-cued early neural crest marker and X-Delta-1 expression by p27xic1-MO islower than that by Xhairy2 mRNA, in-dicates that the effect of Xhairy2-MO onearly neural crest marker and X-Del-ta-1 expression is due not only to theectopic p27xic1 expression. This is sup-ported by the fact that the overexpres-sion of p27xic1 did not induce the ectopicexpression of X-Delta-1 in the cranialneural crest region.

Taken together, these results sug-gest that the repression of p27xic1 ex-pression by Xhairy2 plays a role inearly neural crest marker expressionand the repression of X-Delta-1 ex-pression in the presumptive neuralcrest region.

Xhairy2 Is Required for CellProliferation and Survival inNeural Crest

As shown above, Xhairy2 repressesthe expression of the cell cycle inhibi-tor p27xic1. Thus, Xhairy2 seems to berequired for controlling the cell cyclein the neural crest. To check this pos-sibility, we performed both immuno-staining with anti-phosphohistone H3and TUNEL staining of Xhairy2-de-pleted embryos, and found a decreasein the number of proliferating cells(42%, n � 12; Fig. 5D) as well as sig-nificantly increased apoptosis in midneurula stage embryos injected withXhairy2-MO (92%, n � 25; Fig. 5H),whereas injection of Control-MO didnot affect cell cycle regulation (100%,normal number of proliferating cells,n � 15; Fig. 5C; 100% normal numberof apoptotic cells, n � 20; Fig. 5G).Interestingly, these effects on cell cy-cle regulation were not observed fromearly gastrula through the early neu-rula stage (100%, normal number ofproliferating cells, n � 31; Fig. 5B anddata not shown; 100% normal number

Fig. 3. Depletion of Xhairy2 induces ectopic p27xic1 and X-Delta-1 expression in presumptiveneural crest. A,B: Xhairy2-MO induces slight increases of n-tubulin expression at stage 15 (A) andX-ngnr-1 expression at stage 13 (B) in trigeminal ganglia. C: Control-MO injection has no effect onp27xic1 expression at stage 13. D: In embryos injected with Xhairy2-MO, ectopic expression ofp27xic1 is induced on the injected side (red arrowhead) at stage 13. E: The ectopic expression ofp27xic1 is repressed by injecting 20 pg of Xhairy2 mRNA (yellow arrowhead). F: Control-MO injectionhas no effect on X-Delta-1 expression at stage 13. G: Xhairy2-MO induces a modest increase ofX-Delta-1 expression in the cranial neural crest region (red arrowhead) at stage 13. H: The modestincrease of X-Delta-1 expression is repressed by injecting 20 pg of Xhairy2 mRNA (yellow arrow-head). I: Double in situ hybridization with Xhairy2 (blue) and p27xic1 (magenta) at stage 12. J: Doublein situ hybridization with Xhairy2 (blue) and p27xic1 (magenta) at stage 13. Dorsal views with anteriorto the bottom. Injected area is the right side. Turquoise staining is lineage tracer �-gal.

XHAIRY2 IN NEURAL CREST SPECIFICATION 1479

of apoptotic cells, n � 53; Fig. 5F anddata not shown). How can these differ-ences between early and mid neurulastages be explained? There are atleast two interpretations of these re-sults. One is that p27xic1 protein levelsmight not be sufficient for cell cyclecontrol at the early neurula stage. Theother interpretation is that decreasedcell proliferation and increased apo-ptosis might be a direct consequenceof not only ectopic p27xic1 expressionbut also combined effects, such as therepression of some neural crest mark-ers that are known to control cell pro-liferation and apoptosis.

Taken together, the results suggestthat Xhairy2 is essential for neuralcrest cell proliferation and survival.

DISCUSSION

In this report, we show that the spec-ification of neural crest cells is depen-dent in part upon the function ofXhairy2, indicating the existence ofnovel mechanism(s) for neural crestspecification closely associated withXhairy2 function.

It is known that a high BMP con-centration induces the epidermis, anda significantly low BMP concentrationinduces the neural plate (reviewed bySasai and De Robertis, 1997). SinceBMP is a secreted molecule, an inter-mediate concentration should be auto-matically generated at the border be-tween neural and epidermal tissues.It is known that an intermediate con-centration of BMP is needed for neu-

Fig. 4.

Fig. 5.

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ral crest specification (Marchant etal., 1998). Although Xhairy2 in theprospective neural crest is known todown-regulate Xbmp4 transcription tothe suitable concentration for neuralcrest induction as a Notch signalingtarget gene (Glavic et al., 2004), ourresults showed that the ablation ofXhairy2 had no effect on the expres-sion of Xbmp4 (Fig. 1J,K). Instead, theectopic expression of a dominant-neg-ative form of XBMPRII (Frisch andWright, 1998) or BMP3b, which par-tially blocks BMP signaling (Hino etal., 2003), up-regulates Xhairy2 ex-pression ectopically (data not shown).In agreement with a previous study byKuriyama et al. (2006), our results in-dicate that an intermediate concen-tration of BMP is necessary for theinduction of Xhairy2 expression at theneural border.

The primary induction of ectoderminto epidermis, neural crest, or neuralplate occurs at the early gastrulastage. Xhairy2 is expressed initially inthe entire ectoderm prior to gastrula-tion, and then its expression is re-stricted to a narrow band at the bor-der between the neural plate and theepidermis (Tsuji et al., 2003). Thismeans that Xhairy2 is down-regu-lated in both neural and epidermalcells, or the gene expression continuesto be maintained only in prospectiveneural crest cells. It is known thatepidermal cells are specified at high

BMP concentration and that the neu-ral plate is formed in the absence ofBMP. Thus, only cells receiving inter-mediate BMP signals would incom-pletely differentiate into either tissue,and only those cells would retainXhairy2 expression.

Neural crest cells are pluripotent(reviewed by LaBonne and Bronner-Fraser, 1999). Considering this prop-erty, prospective neural crest cellsshould keep their proliferative abilityuntil the time of differentiation. Cy-clin-dependent kinases (CDKs) posi-tively regulate the cell cycle, andp27xic1 inhibits CDKs involved in theG1/S transition (Sherr and Roberts,1999). This cell cycle regulation isthought to be important for the tran-sition between differentiation andproliferation. Indeed, p27xic1 is highlyexpressed in cells destined to becomeprimary neurons, and it is required forprimary neurogenesis (Vernon et al.,2003). Previous studies have shownthat the timing of neural stem celldifferentiation is critically controlledby multiple hes-related genes (re-viewed by Kageyama et al., 2005).Generally, differentiation is closely re-lated to the cell cycle and hes-relatedgene products control cell cycle pro-gression. For example, Hes1 directlycontributes to the promotion of pro-genitor cell proliferation throughtranscriptional repression of the cdkinhibitor p27kip1 in embryonic carci-

noma cells (Murata et al., 2005). Thus,it is suggested that once p27xic1 is ex-pressed, the cells are going to assumecertain cell fates by escaping from theproliferation phase. As shown above,the ablation of Xhairy2 leads to theinhibition of neural crest formation(Figs. 1, 2), and this inhibition is par-tially dependent upon the ectopic ex-pression of p27xic1 (Figs. 3, 4). Thus,Xhairy2 may function to maintainprospective neural crest cells in themitotic and undifferentiated state.Supporting this idea, the ablation ofXhairy2 induced the apoptosis of mi-totic cells (Fig. 5), and p27xic1-MO re-duced the increased population of ap-optotic cells in the Xhairy2 ablatedembryos (data not shown). p27xic1-MO, however, did not rescue the re-duction of the number of mitotic cellscaused by Xhairy2-MO (data notshown). As shown above, p27xic1-MOdoes not completely rescue the pheno-type of Xhairy2 knockdown. There-fore, it is possible to think thatXhairy2 may have another func-tion(s), which positively progressesthe cell cycle in addition to the repres-sion of the expression of the cell cycleinhibitor, p27xic1.

We showed that the ablation ofXhairy2 leads to the ectopic expres-sion of p27xic1, and the ectopically ex-pressed p27xic1, in turn, suppressesneural crest formation. However,p27xic1 does not seem to directly regu-late the gene expression by itself. Inaddition, the ectopic expression ofXhairy2 does not cause the ectopic in-duction of gene expressions specific forthe neural crest (Glavic et al., 2004;data not shown), indicating that thegene expression should be directlyregulated by other mechanism(s). Sev-eral recent reports have indicated thatWnts are neural crest inducers (re-viewed by Raible, 2006). It was re-ported that the cooperative function ofPax3 and Zic1, in concert with Wntsignaling, is essential for neural crestspecification (Monsoro-Burq et al.,2005; Sato et al., 2005). The transcrip-tion factor Xsnail, which is expressedas early as Xhairy2 in the prospectiveneural crest, is known to have theability to induce neural crest markergenes (Aybar et al., 2003). Therefore,the gene regulatory network that in-cludes these genes may be involved inthe direct induction of the neural

Fig. 4. Repression of p27xic1 expression by Xhairy2 is involved in early neural crest specification.A,B: Embryo injected with 10 pg of p27xic1 mRNA shows repression of Slug (A) and Foxd3 (B)expression (red arrowhead). C: The injection of 10 pg of p27xic1 mRNA produces a slightly increaseof X-Delta-1 expression on the lateral side of the neural plate (red arrowhead). D–F: p27xic1-MOinjection has no effect on X-Delta-1 expression (F) or any of the neural crest markers examined:Slug (D) and Foxd3 (E). G,H,J,K: The repression of early neural crest marker expression byXhairy2-MO seems to be rescued by p27xic1-MO injection (yellow arrowhead): Slug (J) and Foxd3(K), but not by Control-MO injection (red arrowhead): Slug (G) and Foxd3 (H). I,L: The mild increaseof X-Delta-1 expression by Xhairy2-MO seems to be rescued by p27xic1-MO injection (L) but not byControl-MO injection (I). Dorsal views with anterior to the bottom at stage 13. Injected area is theright side. Turquoise staining is lineage tracer �-gal.

Fig. 5. Xhairy2 is required for cell proliferation and survival in neural crest. A–D: Phosphohistone H3immunohistochemistry of Xhairy2-MO or Control-MO injected embryos. A,C: There is no detectabledifference in the number of actively cycling cells between the side injected with Control-MO and theuninjected side at stages 13 (A) and 18 (C). B: There is no detectable difference in the number ofactively cycling cells between the side injected with Xhairy2-MO and the uninjected side at stage13. D: The number of actively cycling cells is decreased in the neural crest region injected withXhairy2-MO (red arrowhead), compared with the uninjected side at stage 18. E–H: TUNEL staining ofXhairy2-MO or Control-MO injected embryos. E,G: There is no detectable difference in the number ofapoptotic cells between the side injected with Control-MO and the uninjected side at stages 13 (E) and18 (G). F: There is no detectable difference in the number of apoptotic cells between the side injectedwith Xhairy2-MO and the uninjected side at stage 13. H: A significant increase in the number ofapoptotic cells is observed on the side injected with Xhairy2-MO (red arrowhead), compared with theuninjected side at stage 18. Dorsal views with anterior to the bottom. Injected area is the right side.

XHAIRY2 IN NEURAL CREST SPECIFICATION 1481

crest. In contrast, several recent stud-ies support the idea that BMPs set upa competency zone for the neural crest(reviewed by Raible, 2006). Therefore,it is interesting to speculate thatXhairy2 may function to confer “compe-tence” for neural crest induction down-stream of the BMP signaling pathway,and the gene regulatory network canwork only in cells expressing Xhairy2.

As only the molecular mechanism(s)for the direct induction of the neuralcrest have been examined so far, it isimportant to study the molecular na-ture of this competence for neuralcrest induction.

EXPERIMENTALPROCEDURES

Plasmid Construction

For overexpression studies, we usedXhairy2b and p27xic1. pCS2AT-Xhairy2bwas constructed as described previously(Yamaguti et al., 2005). For the con-struction of pCS2AT-p27xic1, oligonucle-otides were designed (5�CCCCGAAT-TCCACCATGGCTGCTTTCCACATC-GCCCTG3� and 5�CCCCGGCGCGC-CTCATCGAATCTTTTTCCTGGGG-GT3�). The annealed oligonucleotideswere subcloned into the EcoRI/Asc1 siteof pCS2AT� that was constructed byinserting annealed oligonucleotides(5�TCGAGGGCGCGCCGATATCTCT-AGACGCCCTATAGTGAGTCGTATT-AC3� and 5�GTAATACGACTCACTAT-AGGGCGTCTAGAGATATCGGCGC-GCCC3�) into XhoI-SnaBI-digestedpCS2�. This strategy creates new Asc1EcoRV sites in the polylinker 1 region.The expression plasmid for X-Delta-1stu

was described previously (Chitnis et al.,1995).

Embryo Manipulation andInjection

Xenopus embryos were in vitro fer-tilized and cultured as described(Hawley et al., 1995). Embryos werestaged according to Nieuwkoopand Faber (1967), and fixed inMEMFA (Harland, 1991). For micro-injection studies, synthetic cappedmRNAs (produced using mMESSAGEmMACHINE Kit; Ambion) and Mor-pholino oligonucleotides (MOs; gener-ated by Gene Tools) were injected intothe dorsal animal blastomere of eight-

cell-stage embryos. For synthesis ofmRNAs, each construct was linear-ized by Not1 and transcribed with SP6RNA polymerase (Ambion). MOs wereresuspended in sterile water to a con-centration of 0.1 mM each, and 4 or 8nl of these solutions was injected.Xhairy2a-MO, Xhairy2b-MO, five-mis-match Xhairy2a-MO, five-mismatchXhairy2b-MO, and p27xic1-MO havebeen previously published (Yamaguti etal., 2005; Murato et al., submitted; Ver-non et al., 2003). Xhairy2a andXhairy2b are likely to represent alter-native copies of the same gene (Tsuji etal., 2003). The percentage of repressedearly neural crest marker expression byXhairy2b-MO is higher than that byXhairy2a-MO (Murato et al., submit-ted). In this study, Xhairy2-MO is a 1:1mixture of Xhairy2a-MO and Xhairy2b-MO, and Control-MO is a 1:1 mixture offive-mismatch Xhairy2a-MO and five-mismatch Xhairy2b-MO. The standardcontrol morpholino oligonucleotide 5�-CCTCTTACCTCAGTTACAATTTATA-3� (Gene Tools) was used as negativecontrol of p27xic1-MO. To confirm theinjected region, �-galactosidase (�-gal)and yellow fluorescent protein (YFP)mRNA were co-injected. �-gal activityof nuclear lacZ was visualized with5-bromo-4-chloro-3-indolyl-�-D-galacto-pyranoside (Wako).

Whole-Mount In SituHybridization and ProbePreparation

Whole-mount in situ hybridizationwas performed essentially as de-scribed (Harland, 1991). Double insitu hybridization was performed asdescribed (Takada et al., 2005). Anti-sense RNA probes were synthesized byin vitro transcription in the presence ofdigoxigenin-UTP (Roche) or fluorescein-UTP (Roche). Alkaline phosphatasesubstrates used were nitroblue tetrazo-lium/5-bromo-4-chloro-3-indoxyl phos-phate (NBT/BCIP, Roche), BCIP, or5-bromo-6-chloro-3-indoxyl phosphate(BCIP RED, Biotium).

Antisense RNA probes for in situhybridization were prepared fromtemplates encoding Xhairy2b (Tsuji etal., 2003), p27xic1 (Su et al., 1995), n-tubulin (Chitnis et al., 1995), Sox2(Mizuseki et al., 1998), X-Delta-1(Chitnis et al., 1995), Keratin (Jonaset al., 1985), X-ngnr-1 (Ma et al.,

1996), FoxD3 (Sasai et al., 2001), Slug(Mayor et al., 1995), Xtwist (Hopwoodet al., 1989), and Xbmp4 (Hemmati-Brivanlou and Thomsen, 1995).

Proliferation and TUNELAssay

Phosphohistone H3immunohistochemistry.

For phosphohistone H3 detection, em-bryos were fixed in MEMFA, washedwith PBS containing 0.1% TritonX-100 (PBT), and blocked in PBT con-taining 10% heat-treated goat serum.Anti-phosphohistone H3 antibody(Upstate Biotechnology) was used at aconcentration of 5 �g/ml. Anti-rabbitIgG conjugated with alkaline phos-phatase (Chemicon, 1:1,000) was usedas a secondary antibody, and detectedwith NBT/BCIP.

TUNEL staining was performed aspreviously described (Hensey andGautier 1998). In brief, fixed embryoswere rehydrated in PBS containing0.1% Tween 20, and washed with ter-minal deoxynucleotidyl transferase(TdT) buffer (Gibco) for 30 min. Endlabeling was carried out at room tem-perature overnight in TdT buffer con-taining 2 �M digoxigenin-11-dUTP(Roche) and 150 U/ml TdT (Gibco).Embryos were washed at 65°C withPBS/1 mM EDTA for 1 h, and detectedwith NBT/BCIP.

Cartilage Staining

For cartilage staining, embryos werefixed in MEMFA at stage 45, washedwith PBS, and stained overnight with0.2% alcian blue/30% acetic acid inethanol. Embryos were washed with80% glycerol/2% KOH.

ACKNOWLEDGMENTSPlasmids used to generate mRNA fortBR2 were a kind gift from K. Ume-sono. We are grateful to the membersof Hashimoto laboratory for helpfulcomments and a critical reading of themanuscript.

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