MOLECULAR REPRODUCTION AND DEVELOPMENT 60:331±337 (2001)

Distinct Roles for Distal-Less Genes Dlx3 and Dlx5in Regulating Ectodermal Development in XenopusTING LUO, MAMI MATSUO-TAKASAKI, AND THOMAS D. SARGENT*

Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes ofHealth, Bethesda, Maryland

ABSTRACT In vertebrates, there are six ormore copies of genes related to the Drosophila patternformation homeodomain gene Distal-less. Among thisfamily, Dlx3 and Dlx5 share extensive sequencehomology and have similar, but distinctive, expressionpatterns, suggesting that these two factors may havesubstantially redundant developmental functions. Herewe show that at the earliest phases of embryogenesisin Xenopus, there are significant differences betweenDlx3 and Dlx5 expression and that this correlates withdifferent functions in the restriction of neural crest andneural plate boundaries, respectively. Mol. Reprod.Dev. 60: 331±337, 2001. ß 2001 Wiley-Liss, Inc.

Key Words: Dlx3; Dlx5; neural crest; neural plate;ectoderm

INTRODUCTION

In mouse and human, the six Dlx genes are organizedinto three pairs, each linked to one of the four Hoxclusters (Stock et al., 1996). Dlx3 and Dlx4 (alsodesignated Dlx7 or Dlx8; Beanan and Sargent, 2000)are linked to HoxB, and Dlx5 and Dlx6 to HoxA.Individual Dlx genes are expressed in a complex andpartially overlapping pattern during organogenesis(Robinson and Mahon, 1994; Ellies et al., 1997). Inthe gastrulating Xenopus embryo, Dlx3 (originallynamed ``X-dll2; Papalopulu and Kintner, 1993) isexpressed only in ventral ectoderm (Feledy et al.,1999a). Dlx5 (originally named X-dll3; Papalopuluand Kintner, 1993) is initially expressed in the anteriorneural fold in mouse, chick and Xenopus, de®ning therostral limit of the neural plate in these species(Papalopulu and Kintner, 1993; Yang et al., 1998; Peraet al., 1999). Ectopic expression studies support a rolefor Dlx3 in patterning the anterior neural plate inXenopus (Feledy et al., 1999a) and in the control ofepidermal cell differentiation in mouse (Morasso et al.,1996). Less is known about the function of Dlx5 at earlystages, but gene targeting experiments suggest thatthis factor is important in the development of cranialneural crest derivatives (Acampora et al., 1999; Depewet al., 1999).

In this paper we report the results of experimentscomparing the expression and function of Dlx3 andDlx5 in early Xenopus development, using whole mount

in situ hybridization that is optimized for detection oftranscripts in super®cial ectoderm, and in animal capoverexpression experiments. We ®nd that Dlx3 inhibitscranial neural crest induction while Dlx5 has littleeffect on this tissue but instead inhibits expression ofan early neural plate marker gene. These resultscorrespond with differences in the expression domainsof these two genes, and support a model in which Dlxfactors have distinct roles in the de®nition of theseectodermally derived tissues.

MATERIALS AND METHODS

Dlx cDNA Clones

The Dlx5 open reading frame was subcloned usingPCR into pCTS, a derivative of pCS2� (Turner andWeintraub, 1994) containing a dual T7 and SP6promoter (Feledy et al., 1999a), with an optimizedtranslational initiation site (Kozak, 1987). The primersused were AAA AGA ATT CGC CAT GAC AGG AGTCTA TGA at the 50 end and AAA ACT CGA GTT AGTAGA GAG TCC CTG ATG C at the 30 end. Theseprimers generated a fragment of 870 bp, which wascloned following digestion with EcoR1 and Xho1, andwas checked for absence of errors by DNA sequencing.Experiments with Dlx3 were carried out using a similarplasmid clone described previously (Feledy et al.,1999a).

Embryo Manipulation

Embryos were obtained from adult .Xenopus laevis byhormone-induced egg laying and arti®cial fertilizationusing standard methods, and staged according toNieuwkoop and Faber (1967). Following microinjection,embryos were cultured in 3% Ficoll-400/1�MMR untilsibling embryos reached stage 7. For animal explants,�2/3 of the pigmented ectoderm was dissected fromstage 7±8 embryos and cultured in 0.3�MMR untilsibling embryos reached the desired stage.

For the FGF induction experiment, animal caps weredissected from stage 7±8 blastula embryos in calcium±magnesium-free medium (CMFM; Sargent et al., 1986)

ß 2001 WILEY-LISS, INC.

*Correspondence to: Thomas D. Sargent, Bldg 6B, Rm 412, NIH MSC2790, 6 Center Drive, Bethesda, MD 20892-2790.E-mail: tsargent@nih.gov

Received 27 February 2001; Accepted 22 May 2001

containing 1 mg/ml bovine serum albumin (BSA;Sigma, St. Louis, MO). Isolated caps were transferredto fresh CMFM in agarose-coated dishes and gentlypipetted until inner layer cells had completely disag-gregated, and outer layer cells partly disaggregated.Half of this material was treated for 30 min at roomtemperature with 300 ng/ml recombinant human basicFGF (Upstate Biotechnology, Lake Placid, NY) inCMFM, 1 mg/ml BSA, and the other half left in CMFM,1 mg/ml BSA. After this incubation period, Ca and Mgwere both added to a ®nal concentration of 1 mM, thecells were allowed to reaggregate, and were culturedovernight at 148C, along with sibling embryos.

In Situ Hybridization

Antisense probes labeled with digoxigenin or ¯uor-escein were synthesized using an in vitro transcriptionkit (Boehringer-Mannheim, Indianapolis, IN) accord-ing to the manufacturer's instructions. The Dlx3 andDlx5 probes were made by subcloning open readingframes for these cDNAs in pBluescript 2KS� (Strata-gene), linearizing with EcoR1 (Dlx3) or XbaI (Dlx5) andtranscribing with T7 (Dlx3) or T3 (Dlx5) polymerase.The chordin probe was made by linearizing pBSSK-chdF2 (a gift from Dr. I. Dawid) with EcoR1 andtranscribing with T7 polymerase. The Xenopus slugprobe was made by linearizing the plasmid pMX392 (agift from Dr. M. Sargent), with Bgl2 and transcribingwith SP6 polymerase. The Sox2 probe was made bylinearizing pBSSK� xSox-2 (a gift from Dr. R. Grain-ger) with Xba1 and transcribing with T7 polymerase.Whole mount in situ hybridizations were carried outaccording to Harland (1991), except that ®xed embryoswere incubated in 10 mg/ml proteinase K for 5 min,rinsed twice for 5 min in 0.1 M triethanolamine, pH7.5,then treated for 10 min in 0.1 M triethanolamine pH7.5, 0.25% acetic anhydride (Sigma). Embryos werestained with BCIP (5-bromo-4-chloro-3-indolyl-phos-phate 4-toluidine salt; Roche Biochemicals, Inc., India-napolis, IN), BM-purple (Roche Biochemicals, Inc.,Indianpolis, IN) or magenta-phos (5-bromo-6-chloro-3-indolyl-phosphate 4-toluidine salt; Biosynth Interna-tional, Inc., Naperville, IL) as indicated in ®gurelegends, according to manufacturer's instructions.Frozen sections (Fig. 2) of hybridized and stainedembryos were carried out as described by Matsuo-Takasaki et al. (1999). Tissue identi®cations were madeaccording to Hausen and Reibesell (1991).

Microinjection and RNA Analysis

Full-length capped transcripts encoding Dlx3, Dlx5,chordin, constitutively active type I activin receptor(ALK4-CA), and Wnt3a were generated using an SP6 mMessage Machine kits (Ambion, Inc., Austin, TX)according to the manufacturer's instructions. Thetemplates were SP64S/Xdll2 and pCTS/Dlx5 for Dlx3and Dlx5, respectively, which contain the open readingframes with an optimized initiation context, linearizedwith Xba1 and Not1, respectively, XE1 for Wnt3a(Wolda et al., 1993), linearized with EcoR1, ALK4-CA

for the activated activin receptor (Armes and Smith,1997) linearized with Not1, and pCS2�Chd for chordin(a gift from E. DeRobertis) linearized with Not1. RNAswere checked for concentration, integrity and size bymethylmercury gel electrophoresis, and quanti®ed byabsorbence at 260 nm. Translation ef®ciency of DlxRNAs was tested in vitro using a reticulocyte lysatesystem (Promega, Inc.) and shown to result in compar-able protein production from equivalent amounts ofRNA (data not shown). For animal cap experiments,synthetic RNA was injected into two sites in the animalhemisphere at the one-cell stage in a total of 10 nl. RNAextraction, methylmercury hydroxide gel electrophor-esis and Northern blot analysis for expression of Dlx3and Dlx5 (Feledy et al., 1999a, and this paper), ZicR1(Mizuseki et al., 1998), Xag1 (Sive et al., 1989), Xtwi(Hopwood et al., 1989), Xslu (Mayor et al., 1995),muscle actin (Sargent et al., 1986), and Edd (Sasai et al.,1996) were carried out as previously described (Sargentet al., 1986). All animal cap preparations were testedand found to be free of contaminating mesoderm byhybridization with appropriate cDNA probes (data notshown). For blastomere targeting experiments, pig-mented embryos were oriented on the dorsoventral axisby visual inspection at the 4-cell stage, and theninjected into prospective dorso-anterior animal poleblastomeres at the 16±32 cell stage. Properly targetedembryos were identi®ed by inspection after staining forthe lineage tracer, b-galactosidase.

RESULTS

Dlx3 and Dlx5 Are Coordinately Activated inGastrula Ectoderm

Of the six mammalian Dlx genes, expression of fourhomologs, Dlx2, 3, 5, and 6, has been reported inXenopus (Asano et al., 1992; Dirksen et al., 1993, 1994;Papalopulu and Kintner, 1993). Developmental North-ern blot data have been published for Dlx3 (Dirksenet al., 1994) and Dlx2 (X-DLL1; Dirksen et al., 1993),but not for the other two genes. While whole mount insitu hybridizations for Dlx5 have been reported (X-dll3;Papalopulu and Kintner, 1993), these earlier experi-ments did not detect transcripts in the epidermis. Tocompare the spatiotemporal expression patterns ofDlx3 and Dlx5 in gastrula and early neurula stages,we carried out developmental Northerns and wholemount in situ hybridizations with probes for thesegenes, using conditions adjusted to ef®ciently detectRNAs in super®cial cell layers. As shown in Fig. 1,mature transcripts from both genes ®rst becomedetectable at about the same time, at the beginning ofgastrulation. Dlx5 RNA is detectable for about 1 hrprior to this point as a shortened transcript, but thesigni®cance of this transient RNA molecule isunknown. Evidence supporting the conclusion thatexpression of both Dlx genes at early stages isprimarily, if not exclusively, con®ned to the ectodermand comes from in situ hybridization and animal capexplant experiments. As shown in Fig. 2A and B, thin

332 T. LUO ET AL.

sections of embryos hybridized in situ with Dlx3 or Dlx5probes show no detectable signal in any nonectodermaltissue. The ectodermal speci®city of these genes isfurther indicated by the observation that Dlx3 and Dlx5RNAs are almost completely eliminated in animalcaps exposed to basic ®broblast growth factor (bFGF;Fig. 2C), a treatment which has been shown to down-regulate epidermal gene expression and to induceexpression of ventral and lateral mesodermal deriva-tives (Green et al., 1992). Therefore, Dlx3 and Dlx5 areexpressed in epidermal but not mesodermal cell types.Similarly, injection of 1 ng RNA encoding a constitu-tively active type I activin receptor (ALK4-CA; Armesand Smith, 1997) converts ectoderm into endoderm, asshown by expression of the endodermal markerendodermin (Sasai et al., 1996). This treatment alsorepresses Dlx3 and Dlx5 (Fig. 2D), suggesting that Dlx3and Dlx5 are absent in this tissue as well.

Dlx3 and Dlx5 Demarcate Neural Crest andNeural Plate Boundaries, Respectively

Double whole mount in situ hybridization with Dlx3,Dlx5 and the neural plate marker gene Sox2 to lategastrula/early neurula embryos show that while simi-lar, the domains of Dlx3 and Dlx5 expression differnoticeably in the lateral exclusion from the neural plate(Fig. 3A,B). This difference is especially evident in theanterior region where a gap exists between the neuralplate and the domain of Dlx3 expression, while Dlx5transcripts are found adjacent to (or possibly slightlyoverlapping with) the neural plate. The areas in thelateral/anterior region where both Dlx3 and Sox2 areexcluded is reminiscent of the cranial neural crest(CNC) domain at this stage. This identi®cation wascon®rmed by double in situ hybridization with Dlx3 andthe CNC marker Xslu (Mayor et al., 1995). As shown inFig. 3 (C,D), the anterior expression domain of Xslucorresponds to the negative region between the Dlx3and Sox2 patterns.

Dlx3 and Dlx5 Regulate Different Targets

Previous work from this laboratory (Feledy et al.,1999a) suggested a model in which Dlx3 functions torepress genes expressed in the neural plate, caudal tothe cement gland. We have also found that in the veryearly embryo, Dlx3 overexpression antagonizes theneuralization that can be triggered by activating theWnt/bcatenin signaling pathway (Beanan et al., 2000).The spatial expression pattern of Dlx3 in the cranialregion at the early neurula stage suggests thatthis factor might also inhibit CNC gene activity. Onthe other hand, Dlx5 might be expected to have lesseffect on CNC genes, but possibly to antagonize theexpression of genes transcribed in the neural plateproper.

Animal cap overexpression experiments support bothof these hypotheses. As shown in Fig. 4A, co-injection ofRNAs encoding Dlx5 and chordin results in therepression of the neural plate marker gene ZicR1,which is strongly induced by chordin RNA alone(Mizuseki et al., 1998). In contrast, expression ofXag1, a marker for the cement gland (Sive et al.,1989), an anterior nonneural ectodermal derivativethat is also induced by chordin, is signi®cantlyenhanced. Fig. 4B shows the results of similar experi-ments testing the effects of Dlx3 and Dlx5 on CNC geneexpression. In this case, CNC, marked by Xslu and Xtwi(Hopwood et al., 1989) expression, is induced in whatwould otherwise be epidermal ectoderm by co-injectionof chordin and Wnt3a RNAs (Saint-Jeannet et al.,1997). Addition of Dlx3 RNA to the mixture stronglyrepresses both CNC markers while Dlx5 overexpres-sion has a much less inhibitory effect. These data alsoshow that injection of Wnt3a results in furtherrepression of the Dlx3 gene, which is similar to theresult seen in early gastrula embryos overexpressingb-catenin (Beanan et al., 2000). Dlx5 does not exhibitthis negative response to Wnt3a. Note that at the doseof chordin RNA used in Fig. 4B, expression of theendogenous Dlx3 gene is reduced while the Dlx5 RNAlevel is essentially unaffected. This is a reproduciblephenomenon, although it usually occurs at lowerchordin doses, and re¯ects differences in the responseof Dlx genes to BMP antagonism: Dlx3 is more sensitivethan the Dlx5 gene, the expression of which is actuallyincreased by partial blockage of BMP signaling (Luoet al., 2001). The inhibitory effect of Dlx3 on neuralcrest can also be seen in the intact embryo, as shown inFig. 5: Targeted overexpression of Dlx3 in prospectiveanterior ectoderm, by injection into appropriate blas-tomeres at the 16±32 cell stage, resulted in inhibition ofXSlu expression on the injected side.

DISCUSSION

The Dlx homeobox proteins, along with the closelyrelated Msx factors, are expressed in a pattern ofclosely apposed and overlapping domains in a numberof sites in vertebrate embryos. These families arenotably active in regions of epithelial-mesenchymal

Fig. 1. Developmental Northern analysis of Dlx3 and Dlx5. TotalRNA from Xenopus laevis embryos (0.6 mg/lane) hybridized withradiolabeled probes corresponding to the open reading frames of Dlx3or Dlx5. The lanes are labeled by stage according to Nieuwkoop andFaber (1967), and gastrula stages are indicated by the black bar. Notethe presence at stage 8 and 9 of a smaller Dlx5 RNA (arrow), which isreplaced by a larger RNA by the early gastrula stage (10.5). Below thehybridization is an image of ethidium bromide staining, demonstrat-ing equal loading of lanes.

ECTODERMAL REGULATION BY Dlx GENES 333

Fig. 3.

Fig. 2.

Fig. 5. In vivo inhibition of Xslu expression by targeted Dlx3overexpression. Embryos were injected into a single dorsal blastomereat the 16/32 cell stage with 60±100 pg Dlx3 RNA mixed with 200 pg-galactosidase RNA as a lineage tracer. Embryos were ®xed at stage 14,stained with X-gal (blue color), and properly targeted specimenshybridized in situ with a probe for Xslu (purple; stain is BM purple). Inthe embryo shown, Xslu expression (white arrowheads) is onlydetectable on the noninjected side.

Fig. 4.

334 T. LUO ET AL.

interactions, such as in the developing limb bud, toothgerms, hair follicles, mammary tissue, and epidermis.Heterodimer and homodimers have been demonstratedin vitro and in overexpression studies in vivo, and it hasbeen proposed that the positive transcriptional effectsof Dlx factors might be modulated in vivo by therepressive functions of Msx proteins (Zhang et al.,1997). Gene targeting experiments in the mouse haverevealed functions for several of these genes inorganogenesis, particularly in the craniofacial deriva-tives of branchial arch mesenchyme (Qiu et al., 1997;Thomas et al., 1997; Price et al., 1998; Acampora et al.,1999; Depew et al., 1999). Less is known about earlierdevelopmental functions, but studies with Xenopushave pointed to Msx1 as a major factor in the initialspeci®cation of epidermis (Suzuki et al., 1997) and Dlx3in the patterning of the anterior neural plate/epidermalboundary (Feledy et al., 1999a). In this paper wepresent evidence supporting complementary roles forDlx3 and Dlx5 in regulating the initial differentiation ofXenopus ectoderm into epidermal, neural plate, andneural crest tissue.

Transcription of both Dlx3 and Dlx5 begins very earlyin Xenopus development. The small, transient RNAcorresponding to Dlx5 can be detected approximately atthe midblastula transition, which is the generaltranscriptional activation point in the frog embryo(Newport and Kirschner, 1982). The function of thissmall RNA in Xenopus is not known, but it isinteresting that a similar truncated Dlx5 has beendescribed in developing mouse (Liu et al., 1997; Yanget al., 1998). These transcripts are due to alternateRNA processing and some encode Dlx5 derivatives thatlack homeodomain sequences and thus might possiblyfunction in vivo as antagonists to full-length Dlx5(Yang et al., 1998). However, the smaller RNA weobserve is a minor component, and disappears by earlygastrula, making it less likely to play an importantregulatory role.

Results of in situ hybridization assays (Feledy et al.,1999a; Fig. 3, this paper, and Luo et al., 2001) andinduction experiments (Fig. 2) indicate that Dlx3 andDlx5 are not expressed in nonectodermal tissuethrough neurula stages. In the case of Dlx5, thisconclusion is relevant to the interpretation of experi-ments in which RNA encoding Dlx5 was injected intothe dorsal marginal region of 4-cell Xenopus embryos(Miyama et al., 1999), resulting in extensive ventrali-zation of the resulting embryos. Since the major sourceof axis-inducing signals in Xenopus come from theNieuwkoop center and the Spemann organizer(reviewed by Heasman, 1997), which are endodermaland mesodermal, respectively, Dlx5 probably does notplay a signi®cant role in establishing embryonicdorsoventral polarity in vivo. The ventralizationobserved in the previous work cited above could bethe result of inappropriate disruption or alteration ofregulatory mechanisms due to overexpression of ectopicDlx5. One possibility is that injected Dlx5 is mimickingsome function of Msx1, which is expressed in ventral

Fig. 2. Dlx3 and Dlx5 are ectoderm-speci®c at gastrula stages.(A,B) Transverse frozen sections of whole-mount in situ hybridiza-tions at late gastrula stage (St. 12.5) to Dlx3 (A) and or Dlx5 (B) probes(purple; stain is BM-purple) combined with chordin probe (aqua;stain is BCIP) to de®ne the notochord. Expression of both Dlx genesis detectable only in ectoderm. Tissues are indicated on panel A:(nt, notochord; arch, archenteron; pm, paraxial mesoderm; endo,endoderm; lm, lateral mesoderm; vm, ventral mesoderm; ec, ecto-derm). (C) Mesoderm induction: Dissociated ectodermal cells weretreated with bovine basic ®broblast growth factor as described inMaterials and Methods. Northern blot analysis with Dlx3 and Dlx5probes showed greater than ten-fold reduction in expression levels forboth genes, as estimated by ®lm densitometry. Induction of ventro-lateral mesoderm was con®rmed by hybridization with muscle-speci®cactin (m-actin). W12, whole stage 12 RNA; Con, control/untreatedectoderm RNA; FGF, RNA from FGF-treated cells. (D) Endoderminduction: Fertilized eggs were injected with 1 ng of RNA encoding aconstitutively active type I activin receptor (ALK4-CA), animal capsremoved at stage 7±8, cultured until sibling embryos were at stage20 and processed for RNA and Northern blot analysis. Dlx3 andDlx5 are also repressed by this treatment, concomitant with theinduction of the endodermal marker endodermin (Edd). Ethidiumbromide staining of the 18S ribosomal RNA band is shown as a controlfor equal loading of lanes. W20, whole stage 20 RNA; UI, RNA fromun-injected embryo ectoderm; ALK4-CA, RNA from injected embryoectoderm.

Fig. 3. Different lateral boundaries of Dlx3 and Dlx5 expression.(A,B). Neurula stage embryos were hybridized with probes for thegeneral neural plate marker gene Sox2 (aqua; stain is BCIP) andDlx3 (purple; A), or Dlx5 (purple; B-stain is BM-purple). There isa clear negative region between Dlx3 and Sox2 expression, par-ticularly evident in the anterior (arrows). In contrast, Dlx5 expressionlies adjacent to, or possibly slightly overlaps with, the presumptiveneural plate. (C,D). Dlx3 expression is excluded from the presumptivecranial neural crest. Neurula stage embryos were hybridized withprobes for Dlx3 (dark reddish-blue; stain is magenta-phos) andXSlu (arrows; purple-stain is BM purple), showing that the Dlx3-negative region in the anterior ectoderm corresponds to the pre-migratory cranial neural crest region. (C), dorsal view; (D), lateralview.

Fig. 4. (A) Region-speci®c regulation of neural plate markers byDlx5. Injection of 50 pg of chordin RNA resulted in inhibition of Dlx5expression and induction of the cement gland marker Xag1 and theneural prepattern gene ZicR1 in isolated animal cap ectoderm (stage14). Co-injection of 100 pg of Dlx5 RNA inhibited ZicR1 but enhancedexpression of Xag1. Note that the Dlx5 probe was not hybridized toRNA from Dlx5-injected embryos to prevent overexposure of the ®lm.(B) Antagonism of neural crest induction by Dlx3 in animal caps.Embryos were injected at the 1-cell stage with a mixture of 250 pgof chordin (Chd) RNA and 300 pg Wnt3a RNA to induce neuralcrest gene expression in the ectoderm (Saint-Jeannet et al., 1997),with and without 100 pg Dlx3 or Dlx5 RNA to assess the effects of Dlxgene expression on this induction. Animal caps were explanted atstage 7±8 and cultured until sibling embryos reached stage 19, thenRNA was isolated and analyzed by Northern blot. Addition of Dlx3RNA resulted in signi®cant inhibition of expression of neural crestmarkers Xenopus Slug (Xslu) and Twist (Xtwi), while Dlx5 over-expression had a much less severe inhibitory effect. Also, Wnt3ainhibited expression of Dlx3 but not that of Dlx5. Note that in theseexperiments, 50 pg chordin RNA injection resulted in an inhibition ofDlx5 expression (panel A), whereas 250 pg (panel B) had little if anyaffect on Dlx5. The higher dose usually is inhibitory for both Dlx3 andDlx5, but in this experiment the response to chordin was attenuated,possibly due to partial degradation of the synthetic mRNA. Also, notethat Dlx3 and Dlx5 probes were not hybridized to RNA from embryoswith the corresponding injected RNAs to prevent overexposure of the®lm.

ECTODERMAL REGULATION BY Dlx GENES 335

mesoderm and strongly ventralizes Xenopus embryonictissues when overexpressed in dorsal cells (Suzuki etal., 1997). Dlx and Msx factors can interact via protein±protein contacts within the homeodomains, and havevery similar DNA binding elements (Catron et al., 1993;Feledy et al., 1999b). Thus it is possible that high levelsof Dlx5 might interact with target genes, or withregulatory factors via protein±protein interactions,that normally respond to the homeoprotein Msx1.

Both Dlx genes are regulated by BMP signaling,consistent with the ventral expression patterns. Thereare differences, however, in the responsiveness of Dlx5vs. Dlx3 to such signaling; this can be seen in theresults of Fig. 4; in one experiment (panel A) Dlx5 isinhibited by chordin, presumably due to antagonism ofBMP signaling, while in the other experiment (panel B)Dlx5 expression is unchanged, while Dlx3 is inhibited.This seemingly contradictory behavior is probably dueto two factors: ®rst, low levels of BMP antagonismstimulate expression of Dlx5 relative to what takesplace in undisturbed ectoderm (Luo et al., 2001), andsecond, in the experiment shown in panel B theresponse to chordin was less than usually observed,possibly due to batchwise variation in the syntheticchordin mRNA preparations.

The relatively strong inhibition of neural crest (i.e.,Xslu, Xtwi) gene induction by Dlx3 compared to Dlx5(Fig. 4), is consistent with a model in which Dlx3functions to de®ne a lateral boundary for the cranialneural crest, possibly by antagonizing the inductiveactivity of Wnt3a (Saint-Jeannet et al., 1997; LaBonneand Bronner-Fraser, 1998). This is also consistent withprevious ®ndings suggesting an antagonism in the pre-gastrula Xenopus embryo between Dlx3 function and b-catenin signaling (Beanan et al., 2000) that results inspatial dorsoventral bias within the ectoderm. Theapparent absence of a similar antagonism between Wntsignaling and Dlx5 expression is an important distinc-tion between Dlx5 and Dlx3, reinforcing the conclusionthat these two factors, while exhibiting similar expres-sion, are not interchangeable either in function or inregulation. The spatial expression and animal capexperiment data suggest that Dlx5 functions to de®nethe lateral boundary between the neural plate andepidermis in the trunk region by inhibiting theexpression of broadly-expressed early neural regula-tory genes such as ZicR1. In the anterior, Dlx5 may alsoact as a positive factor promoting the expression ofmarkers for cement gland, a tissue where Dlx5 isstrongly expressed in vivo (Fig. 4A; Papalopulu andKintner, 1993; Luo, et al., 2001).

It should be acknowledged that the expressionpatterns of Dlx genes are highly dynamic, and thatthe experiments discussed in this paper only deal withthe earliest phase of this gene activity. In particular,most if not all of the Dlx family members are activelytranscribed in the postmigratory cranial neural crest,in branchial arch mesenchyme, and epithelium (Robin-son and Mahon, 1994). Disruption of Dlx genes resultsin numerous defects in cranial neural crest derivatives

(Qiu et al., 1997; Thomas et al., 1997; Price et al., 1998;Acampora et al., 1999; Depew et al., 1999), so thistranscription is developmentally important. Presum-ably the early antagonism of neural crest induction byDlx3 gives way to a positive function later in develop-ment. It will be interesting to test these models for Dlx3and Dlx5 gene function in the early frog embryo, aproblem well suited to transgenic approaches that arebecoming more practical with this organism, and todetermine what is responsible for the differing regula-tion and functions of these two closely-related home-odomain genes.

ACKNOWLEDGMENTS

We thank E. De Robertis, M.G. Sargent, and J.B.Gurdon for providing the chordin, Xslug, and XtwistcDNA plasmids, respectively.

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