RESEARCH ARTICLE

Regulation of Melanoblast and RetinalPigment Epithelium Development by Xenopuslaevis MitfMayuko Kumasaka,1† Shigeru Sato,2 Ichiro Yajima,1† Colin R. Goding,3 and Hiroaki Yamamoto1*

Mitf is a central regulator of pigment cell development that is essential for the normal development of themelanocyte and retinal pigment epithelium (RPE) lineages. To understand better the role of Mitf, we haveused the Xenopus laevis experimental system to allow a rapid examination of the role of Mitf in vivo. Here,we report the function of XlMitf�-M on melanophore development and melanization compared with that ofSlug that is expressed in neural crest cells. Overexpression of XlMitf�-M led to an increase in melanophoresthat was partly contributed by an increase in Slug-positive cells, indicating that XlMitf�-M is a keyregulator of melanocyte/melanophore development and melanization. Moreover, overexpression of adominant-negative form of XlMitf� led to a decrease in the number of melanophores and induced abnormalmelanoblast migration. We also observed an induction of ectopic RPE and extended RPE by overexpressionof XlMitf�-M and possible interactions between XlMitf� and several eye-related genes essential for normaleye development. Developmental Dynamics 234:523–534, 2005. © 2005 Wiley-Liss, Inc.

Key words: melanin; melanocyte; melanophore; microphthalmia; Mitf; pigment cell; retinal pigment epithelium; Dct;Sox10; Slug; Xenopus; eye

Received 4 January 2005; Revised 1 May 2005; Accepted 18 May 2005

INTRODUCTION

In vertebrates, melanocytes (melano-phores in poikilotherms) are derivedfrom multipotent neural crest cells. Incontrast, the retinal pigment epithe-lium (RPE) is derived from monolayercells lying at the outer layer of theoptic cup. In some vertebrates, theepiphysis derived from the brain ves-icle also contains pigment cells, whichin humans does not produce melaninbut rather works as an endocrinegland secreting melatonin. Previous

studies have identified well over 100genes related to the development orfunction of pigment cells (reviewed byBennett and Lamoreux, 2003). Ofthese, the basic/helix–loop–helix/leucine zipper (bHLH-LZ) transcrip-tion factor Mitf (Hodgekinson et al.,1993; reviewed by Goding, 2000) is es-sential for the normal development ofall pigment cells.

In mice, at least 17 Mitf mutant al-leles have been identified and geneti-cally characterized (Green, 1989;

Tachibana et al., 1992; Steingrimssonet al., 1994). These mice exhibit re-duced numbers of or complete lack ofmelanocytes, leading to a white coatand sometimes to deafness, and mostalso develop microphthalmia (smalleye) due to the abnormal unpig-mented RPE (Hodgkinson et al., 1993;Opdecamp et al., 1997; Nakayama etal., 1998). In addition, some alleles inthe mouse Mitf gene affect the devel-opment of other cell types, includingosteoclasts resulting in osteoporosis,

1Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan2Division of Biology, Center for Molecular Medicine, Jichi Medical School, Minamikawachi, Tochigi, Japan3Signaling and Development Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey, United KingdomGrant sponsor: Ministry of Education, Culture, Sports, Science, and Technology, Japan.†Drs. Kumasaka and Yajima’s present address is Developmental Genetics of Melanocytes, UMR146 CNRS, Institute Curie, Bat 110 CentreUniversitaire, 91405 Orsay, Cedex, France.*Correspondence to: Hiroaki Yamamoto, Department of Developmental Biology and Neurosciences, Graduate School of LifeSciences, Tohoku University, 6-3 Aramaki-aza-Aoba, Aobaku, Sendai 980-8578, Miyagi, Japan.E-mail: hyamamot@mail.tains.tohoku.ac.jp

DOI 10.1002/dvdy.20505Published online 18 July 2005 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 234:523–534, 2005

© 2005 Wiley-Liss, Inc.

Fig. 1. Effects of XlMitf�-M and dominant-negative XlMitf�-M mRNA microinjection on the development of melanophores. A: Schematic represen-tation of the wild-type XlMitf�-M and the dominant-negative form of XlMitf� (dnXlMitf�). In the dnXlMitf� construct, the amino acid glutamic acid inthe basic region is converted to alanine. B: Control embryo. C: In XlMitf�-M-injected embryos, the number of melanophores was increased and themelanin granules in these cells dispersed much more extensively. D: Overexpression of dnXlMitf� induced a decrease in the number of melanophores,and a repression of melanin dispersal resulted in punctate-shaped melanophores. B–D: Stage 44, these images are the magnifications of the areasindicated in each inset therein.

Fig. 2. Fig. 3.

524 KUMASAKA ET AL.

and mast cells. When Mitf is ex-pressed ectopically in fish, ectopic pig-ment cells and expression of melano-genic genes can be induced (Lister etal., 1999), whereas ectopic expressionof Mitf in fibroblasts has been re-ported to lead to the adoption of amelanocyte-like phenotype (Tachi-bana et al., 1996). Mitf can regulatethe transcription of the melanogenesisgenes, such as Tyr, Tyrp1, Dct, andQNR-71 (Bentley et al., 1994; Ya-sumoto et al., 1994; Yavuzer et al.,1995), Kit (Tsujimura et al., 1996),Slug (Sanchez-Martin et al., 2002),and Tbx2 (Carreira et al., 1998, 2000),but also appears to regulate prolifera-tion through both p16INK4a (Lo-ercher et al., 2005) and p21Cip1 (Car-reira et al., 2005) expression. Thus,Mitf is a key regulator of proliferation,survival, and differentiation of twodifferent pigment cell derivatives, me-lanocytes/melanophores and the RPE.

Mitf expression is controlled by acombination of the Pax3, Sox10,CREB, and Lef1 transcription factors(reviewed by Goding, 2000) that to-gether appear to control Mitf expres-sion in the neural crest cells with theactivation of Mitf expression leadingto the commitment of a subset of neu-ral crest cells to the melanocyte lin-eages. Expression of Mitf subse-quently leads to regulation of genesimportant for the survival, prolifera-

tion, migration, and differentiation ofmelanocytes.

Intriguingly Mitf is expressed asmultiple isoforms each with a distinctdistribution. In humans and mice,multiple Mitf isoforms produced fromthe different promoters have been re-ported (Hallsson et al., 2000; Takedaet al., 2002; Watanabe et al., 2002)that differ at their N-termini but havethe same DNA-binding and dimeriza-tion domains. Of these, the melano-cyte-specific isoform Mitf-M is the bestcharacterized and is indispensable forthe development of neural crest-de-rived melanocytes but not for dien-cephalon-derived RPE (its develop-mental origin from the telencephalonhas been suggested recently; Fernan-dez-Garre et al., 2002). Therefore, itwas strongly suggested that Mitf iso-form(s) other than Mitf-M should havea key role for RPE development (Ya-jima et al., 1999). Although it was re-ported that Mitf-A is predominantlyexpressed in the RPE, this mRNA isubiquitous like the heart-type, Mitf-H(Amae et al., 1998; Udono et al., 2000).In addition, in humans, MITF-B,MITF-C, and MITF-D have been iso-lated (Yasumoto et al., 1998; Fuse etal., 1999; Takeda et al., 2002); andnow MITF-J/Mitf-J have been re-ported in human and mouse (Hersheyand Fisher, 2005).

Although much is known about the

role of Mitf in activating pigmentationgenes in cell lines in culture or thegeneral role of Mitf in melanocyte de-velopment based, for example, on thegenetics of Mitf in mice and humans,the limitations of these experimentalsystems have left many questions con-cerning Mitf function unresolved.Here, we have taken the first step toexamine the in vivo function of a Xe-nopus laevis Mitf homologue that wehave recently isolated (Kumasaka etal., 2004). In Xenopus, XlMitf� is ex-pressed in melanophore lineages, theRPE, and epiphysis, although wecould not identify isoform-specific ex-pression (Kumasaka et al., 2004). Theresults presented here obtained by in-jection of Xenopus laevis fertilized eggsindicate that XlMitf�-M (an orthologueof mammalian Mitf-M) can control bothmelanophore and RPE development.The results highlight the usefulness ofXenopus as a model system for the anal-ysis of genes involved in melanocyte de-velopment and function.

RESULTS

Overexpression of XlMitf�-Mand dnXlMitf� Influencesthe Number of Melanophoresand Their Dendricity

To gain an insight into the function ofXlMitf�-M during development, we

Fig. 2. A–N: Effects of overexpression of XlMitf�-M or dnXlMitf� on Dct, Sox10 and Slug expression. A-1, B-1 and C: At stage 31, Dct was normallyexpressed in melanoblasts on the dorsal side of the neural tube and in the RPE. A-2: On the XlMitf�-M-injected side, an increase in the number ofDct-positive melanophores (A-2, arrowheads) was observed ectopically covering the brown-colored melanized cells (A-2, arrows). B-2: On thednXlMitf�-injected side, Dct-positive melanoblasts/melanophores were detected on between the dorsal side of the neural tube and the surface of theyolk sac in the trunk (B-2, arrowheads) contrasting with the noninjected side (B-1). An arrow indicates the eye expressing no signal (B-2). F,I: At stage23, expression of Sox10 (F) and Slug (I) was detected in neural crest cells at the dorsal midline and in migrating cranial neural crest cells. D: InXlMitf�-M-overexpressed embryos, the number of Sox10-expressing premigratory neural crest cells was increased on the injected side (D, arrow-heads). G-1, G-2: Similarly, Slug-expressing premigratory neural crest cells was increased by XlMitf�-M overexpression (G-2, arrowheads), althoughSlug expression was not detected in ectopic melanized cells (G-1, arrow) contrasted with Dct expression in these cells (A-2,E,H-1). H-2: IndnXlMitf�-injected embryos, the expression patterns of Sox10 (E, black arrowheads) and Slug (H-1 and H-2, black arrowheads) were disturbed andthe signals shifted laterally contrasted with the noninjected side (H-1 and H-2, blue arrowheads). White arrows (E and H-1) and a black arrow (H-2) showthe midline of each embryo. Green arrowheads (H-2) show the regions between the strong Slug-expressing regions (H-2, black arrowheads). J,K,L:BrdU immunostaining detecting proliferating cells. M, N: single-strand DNA (ssDNA) immunostaining detecting apoptotic cells. These stainings wereperformed in transverse sections prepared from the same embryos (G-2 and H-2) after in situ hybridization. J: The number of BrdU-positive cells(shown by red cells) on the injected side was increased (black arrowheads, contrasting with those in the noninjected side pointed by blue arrowheads)in the region where Slug expression was promoted. In the Slug-expressing region shifted laterally on the dnXlMitf�-injected side (H-2, blackarrowheads), both the number of BrdU-positive cells (K, black arrowheads) and ssDNA-positive cells (M, black arrowheads) were larger than those inSlug-expressing area on the noninjected side (K and M, blue arrowheads). In the region where Slug signal became very weak on the injected side (H-2,green arrowheads), neither a decrease in the number of BrdU-positive cells (L, green arrowheads) nor an increase in the number of ssDNA-positivecells (N, green arrowheads) were observed, contrasted with Slug-expressing regions on the noninjected side (L and N, blue arrowheads). C,F,I:Noninjected embryos. A-1,A-2,B-1,B-2,C: Stage 32. D,E,F,H-1,I: Stage 23. G-1: Stage 21. G-2,H-2: Stage 25. NO, notochord; NT, neural tube.

Fig. 3. Effects of Slug overexpression on melanophore development. A,B: In Slug-injected embryos, the number of melanophores was increased (B)compared with noninjected embryos (A). C,D: Overexpression of Slug induced an increase in the number of XlMitf�-positive cells on the injected side(C,D, arrowheads). D: A transverse section of the trunk region of C, plane D. E: The number of Sox10-positive cells was also increased on the injectedside (E, arrowheads). Yellow arrows in C and E show the midline. NT, neural tube.

MITF AND XENOPUS LAEVIS PIGMENT CELLS 525

performed overexpression studies byinjecting XlMitf�-M or dominant-neg-ative XlMitf� (dnXlMitf�) mRNAssynthesized in vitro into one blas-tomere of two-cell stage embryos. AdnXlMitf� was generated by changingthe highly conserved amino acid glu-tamic acid within the basic domainnecessary for DNA binding to alanine(Fig. 1A). A similar strategy has beenused to generate a dominant-negativeform of other bHLH-LZ family mem-bers (Fisher and Goding, 1992; Krylovet al., 1997).

In XlMitf�-M-injected embryos, thenumber of melanophores seemed to beincreased and most melanophoreswere more dendritic compared withthose in control embryos (76% of 106embryos were affected, compare Fig.1B with C). In contrast, dnXlMitf�-injected embryos, the number of mela-nophores seemed to be decreased andthe melanophores exhibited a punc-tate shape compared with the normaldendritic form (71% of 42 embryoswere affected, compare Fig. 1B withD). These results suggest that XlMitf�controls not only melanophore num-ber but also their dendricity.

Overexpression of XlMitf�-Mand dnXlMitf� Influencesthe Expression ofMelanogenic Genes andNeural Crest Markers

We next analyzed the expression of amelanogenetic gene Dct (a marker formelanin-producing cells) and two neu-ral crest marker genes (Sox10 andSlug) in XlMitf�-M- or dnXlMitf�-in-jected embryos. At stage 31, normalDct expression is detected in melano-blasts located on the dorsal part of theneural tube in the trunk region andRPE in the eye (Fig. 2A-1, B-1, C).XlMitf�-M overexpression caused ec-topic Dct expression (Fig. 2A-2, arrow-heads) in and around the region whereectopic melanin production was ob-served (Fig. 2A-2, arrows). In con-trast, on dnXlMitf�-injected side, suchectopic melanin production was neverobserved, although ectopic Dct expres-sion in the trunk region was still de-tected (Fig. 2B-2, arrowheads). Nota-bly in the eye, its expression was lost(Fig. 2B-2, arrow). To understand atwhat developmental stage XlMitf�-M

caused an increase in the number ofmelanophores, we examined expres-sion of Sox10 and Slug that are usu-ally expressed in neural crest cells(Fig. 2F,I) and have been used as neu-ral crest markers (Aoki et al., 2003;Mayor et al., 1995). In XlMitf�-M-overexpressed embryos, the number ofSox10-positive cells was increasedalong the dorsal midline on the in-jected side (Fig. 2D) and similar re-sults were obtained for Slug expres-sion (Fig. 2G-2). Of interest,expression of Slug was not detected inthe ectopic melanized cells in contrastto the Dct expression in this region(compare Fig. 2A-2 with Fig. 2G-1).These results suggest that XlMitf�-Mhas the ability not only to increaseSlug-positive melanoblasts but alsoinduce ectopic melanization in Slug-negative non-neural crest cells. IndnXlMitf�-injected embryos, theSox10 and Slug signals were weak-ened in places on the injected side andthe expression was shifted slightly lat-erally (Fig. 2E, H-1, and H-2, blackarrowheads; note that the Slug-posi-tive area is shifted laterally).

To examine the effects of overex-pression of these two mRNAs on theproliferation of melanoblasts, we per-formed a bromodeoxyuridine (BrdU)incorporation assay. In this assay, weused the same embryos in Figure 2G-2and 2H-2, with sections being pre-pared from the region indicated by thewhite lines. In the region where Slugwas activated by overexpression ofXlMitf�-M, the number of BrdU-posi-tive cells was also increased (Fig. 2G-2and J, black arrowheads), although itwas not possible to determine whetherthe BrdU-positive cells expressedSlug. As for dnXlMitf�-injected em-bryos, we prepared transverse sec-tions from two different regions (Fig.2H-2, two white lines). In transversesections where strong Slug expressionwas observed (Fig. 2H-2 black arrow-heads), the number of BrdU-positiveproliferating cells was increased (Fig.2K, black arrowheads) compared withthe Slug-expressing regions on thenoninjected side (Fig. 2H-2 and K,blue arrowheads). In contrast, in theregions where the Slug signal wassubstantially weakened (Fig. 2H-2,green arrowheads), the number ofBrdU-positive cells (Fig. 2L, green ar-rowheads) did not seem to be influ-

enced compared with Slug-expressingregion on the noninjected side (Fig.2H-2 and L, blue arrowheads). In ad-dition, to determine whether this re-duction in Slug expression was re-lated to the activation of apoptosis orto any abnormal migration ability ofSlug-positive cells, we detected apo-ptotic cells by anti-single strand DNAantibody. Based on this assay, wecould not detect any activation of ap-optosis in the areas where Slug ex-pression was reduced (Fig. 2N, com-pare the region pointed by bluearrowheads with that indicated bygreen arrowheads). However, in theregions where Slug expression was ob-vious (Fig. 2H-2, black arrowheads),many apoptotic cells were detected(Fig. 2M, black arrowheads). This re-sult raised the possibility that the ap-pearance of the regions expressing re-duced levels of Slug was not related tothe activation of apoptosis but to theabnormal migration of neural crestcells, including melanoblasts result-ing in decreased numbers of melano-phores present at later developmentalstages.

Overexpression of SlugIncreases the Number ofXlMitf�-Positive CellsResulting in Increase in theNumber of Melanophores

Forced expression of XlMitf�-M in-duced an increase in the number ofSlug-positive cells resulting in an in-crease in melanophores. It has beenreported that Slug has the ability toenhance its own expression (LaBonneand Bronner-Fraser, 1998). Overex-pression of Slug induces an increasein the number of melanophores asshown in Fig. 3B (compare Fig. 3Awith B). However, it is unclearwhether the increase in melanophoresinduced by Slug overexpression iscaused by a direct induction of ectopicXlMitf� expression or by an indirectincrease in XlMitf�-positive cells afterinduction of Slug-positive neural crestcells by Slug itself. We therefore in-vestigated the influence of overexpres-sion of Slug on XlMitf� expression.We found that, in Slug-injected em-bryos, the number of XlMitf�-express-ing neural crest cells increased dra-matically on the injected side (Fig.

526 KUMASAKA ET AL.

3C,D, a yellow arrow indicates themidline); on the other hand, neitherectopic XlMitf�-positive cells nor ec-topic melanization that was observedin XlMitf�-M–injected embryos wasdetected. Similarly, Sox10 was also

up-regulated by Slug overexpression(Fig. 3E). These results suggest thatoverexpression of Slug does not induceectopic XlMitf�- and/or Sox10-positivecells and that the increase in melano-phores in Slug-overexpressed em-

bryos was the result of the increase inneural crest-derived XlMitf�-positivecells.

XlMitf�-M Induces Slug, ButIt Does Not Result in theInduction of NeuronalMarker Genes

To infer the differences between themolecular mechanism(s) underlyingan increase in melanophores causedby overexpression of XlMitf�-M andSlug, we performed reverse transcrip-tase-polymerase chain reaction (RT-PCR) analysis using animal cap ex-plants. In XlMitf�-M–overexpressedanimal cap explants, RT-PCR analy-sis using EF1� as a control, revealedthat Slug was already slightly in-duced at embryonic stage 12 (Fig. 4A).At embryonic stage 17, Sox10 (amarker for some neural crest lineagesincluding melanophores), Tyr andTyrp1 (melanin-producing cell mark-ers at later developmental stages com-pared with Dct), and Dct (a melanin-producing cell marker) were alsoinduced, whereas NeuroD and N-tu-bulin (neuronal marker genes), both ofwhich were inducible by Slug (Fig. 4C;also reported by LaBonne and Bron-ner-Fraser, 1998), were not (Fig. 4B).These results suggest that XlMitf�-Mhas an ability to induce Slug expres-sion, and in the cell population ofthese Slug-positive cells, melanoblastprecursors were included but neuro-nal precursor cells were not.

In Slug-injected animal cap ex-plants, Sox10 and N-tubulin and Neu-roD were induced at the time corre-sponding to embryonic stage 17, whenthese genes were normally detected,but in contrast, XlMitf� was not in-duced at this stage (Fig. 4C). How-ever, at the time corresponding tostage 22/23 when XlMitf� expressionwas detectable in vivo, it became de-tectable in Slug-injected animal caps(Fig. 4D). These results suggest thatforced expression of Slug inducesXlMitf� and/or Sox10-positive cellsbut does not have the ability to hastenthe onset of expression of these genesin contrast to induction of Slug ex-pression by the overexpression ofXlMitf�-M.

Fig. 4. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses using animal capexplants after injection of XlMitf�-M or Slug mRNAs. A,B: XlMitf�-M–injected animal cap explants(AC). A: Stage 12. B: Stage 17. XlMitf�-M injection induced neural crest markers (Slug and Sox10)and melanogenic genes (Tyr, Tyrp1, and Dct) but did not induce neuronal markers (NeuroD andN-tubulin) and Slug was induced earlier than the others. C,D: Slug-injected animal cap explants. C:Stage 17. D: Stage 22/23. C: In Slug-injected animal cap explants, Sox10, N-tubulin, and NeuroDwere induced at the time corresponding to stage 17 of embryos. D: In contrast, XlMitf� expressionwas not, but by the time corresponding to the stage 22/23, it became detectable. Dct expressionwas not detected in Slug-injected animal caps, although that was induced in XlMitf�-M–injectedanimal cap explants (compare B and D). EF1� was amplified to show the cDNA used were notdegraded.

MITF AND XENOPUS LAEVIS PIGMENT CELLS 527

Overexpression of XlMitf�-Mand dnXlMitf� InfluencesEye Development andExpressions of Eye MarkerGenes

At later developmental stages, wefound that some embryos (16% of 106embryos were affected) injected withXlMitf�-M also displayed severaleye-related defects (Fig. 5B–E). Inthe majority of embryos having suchdefects (11 of the 17 affected em-bryos), the optic fissure failed toclose properly, leaving a gap instructures of the eye such as theRPE (Fig. 5B) compared with thecontrol side (Fig. 5A). The phenotypeobserved most frequently was thatectopic RPE-like structures wereformed in the brain and along theoptic stalk in 15 of the 17 affectedembryos (Fig. 5C). Most embryosshowed combinations of these twodefects. A transverse section of thehead of this phenotype revealed thatthe abnormal RPE extends along theoptic stalk toward the ventral part ofthe brain (Fig. 5E), and this phenom-enon was observed in almost all theembryos with an open optic fissure.In some XlMitf�-M–injected em-bryos, the eye size was reduced (in 7of the 17 affected embryos; Fig. 5D).

Also in dnXlMitf�-injected embryos,the phenotypes of embryos with eyemalformations were observed (74% of42 embryos were affected). Most ofthem (28 of the 31 affected embryos)showed a combination of lack of theventral part of the RPE (Fig. 5F) andsmall eye (Fig. 5G), and an abnormaldistance between the brain and theeye on the injected side (Fig. 5G). Insevere cases, the size of the eye be-came very small (microphthalmia)and the eye fused to the brain (Fig.5H). In some embryos, the RPE wascompletely absent (4 of the 31 affectedembryos, Fig. 5I). Overexpression ofdnXlMitf� induced similar phenotypewith that induced by XlMitf�-M suchas partial lack of the RPE and smalleye but the percentage of these pheno-types were higher than those inducedby overexpression of XlMitf�-M. Dom-inant-negative XlMitf� never inducedectopic RPE or RPE extension, andthe reduction of the distance betweenthe brain and eye caused by the lack of

the optic stalk was specific in the dnX-lMitf�-injected embryos.

To understand better the cause ofthese eye-related abnormalities, westudied the expression of several eye-related genes. Both Pax6 and Pax2 en-code paired-box class transcriptionfactors and are essential for normaleye development. Pax6 in particular isa key regulator of eye developmentand is expressed in the entire opticvesicle. In contrast, Pax2 is expressedin the ventral part of the optic cup andin the optic stalk (Heller and Brandli,1997), although in mice, it is ex-pressed in the entire optic vesicle likePax6 (Nornes et al., 1990; Baumer etal., 2003). In XlMitf�-M–injected em-bryos, the eye field normally definedby Pax6 expression was reduced onthe injected side of several embryos(Fig. 6A), which might account for thereduced eye size. In addition, expres-sion of other eye-related genes, Rx1 (apaired-like homeobox gene, Fig. 6B),homeodomain-containing genes suchas Six6 (Fig. 6C), and Otx5b (a bicoid-class homeobox gene, Fig. 6D) werereduced similarly. In contrast, Pax2expression in the optic vesicle on theXlMitf�-M-injected side was up-regu-lated, although its expression in theotic vesicle and midbrain–hindbrainboundary was reduced (Fig. 6E).Otx5b is expressed not only in the op-tic vesicle but also the epiphysis innormal embryos, and expression inboth regions was repressed afterXlMitf�-M injection (Fig. 6D).

In dnXlMitf�-overexpressed em-bryos, expression of all genes exam-ined (Pax6, Rx1, Six6, Otx5b, andPax2) was also repressed in the opticvesicle (Fig. 6F–J) and Otx5b expres-sion in the pineal organ was alsodown-regulated on the injected side(Fig. 6I). These results were similarto those obtained from XlMitf�-M–overexpressed embryos, but expres-sion of Pax2 in the eye was regulatedto the contrary (compare Fig. 6Ewith J).

DISCUSSION

In this study, we have explored theuse of Xenopus as a system to analyzethe role of factors required for mela-nocyte development. Overexpressionof XlMitf�-M or dominant-negativeXlMitf� led to an altered number and

dendricity of melanophores, effects oneye development, and the expressionpatterns of several marker genes.

Function of XlMitf� onMelanophore Developmentand Melanogenesis

While overexpression of XlMitf�-M in-duced both ectopic melanization andan increase in the number of melano-phores, in contrast, Sox10-injected Xe-nopus embryos exhibited an increasein the number of Dct-positive cells andmelanophores but did not produce ec-topic melanin (Aoki et al., 2003). Inmice, Sox10 binds and activates Mitfpromoter (Bondurand et al., 2000; Leeet al., 2000; Potterf et al., 2000; Jiao etal., 2004) and it is likely that a failureto correctly activate the Mitf promoteraccounts for the pigmentation defectsobserved in Dominant megacolon(Dom) mice expressing a truncatedversion of Sox10. If a similar relation-ship occurs in Xenopus, it is likely thatSox10-mediated activation of Mitfmay be responsible for the elevatednumbers of melanophores observed inSox10-injected embryos. The inabilityof Sox10, unlike Mitf, to target pig-mentation genes such as Tyr andTyrp1 would also account for the factthat Mitf, but not Sox10, can induceectopic melanization. This interpreta-tion is supported by our in vitro anal-yses using animal cap explants, wherein XlMitf�-M–injected animal caps,melanogenic genes such as Tyr,Tyrp1, and Dct were induced beforethe initiation of normal Dct expressionin embryos, whereas in Sox10-injectedanimal caps (Aoki et al., 2003), Dctexpression was not induced until thecorresponding stage (stage 25) when itinitiates in embryos.

XlMitf� Mediates Increasesin the Number ofMelanophores

Overexpression of XlMitf�-M inducedan increase in melanophores, whereasmutations in Mitf in other organismsled to a loss of melanocytes/melano-phores (Steingrimsson et al., 1994;Lister et al., 1999; Yajima et al.,1999). Although it is clear that Mitf isessential for melanocyte/melanophoredevelopment, it is not yet evident pre-cisely which downstream genes medi-ate its effect. One candidate Mitf tar-

528 KUMASAKA ET AL.

Fig. 5.

Fig. 5. Effects of XlMitf�-M and dnXlMitf� mRNA microinjection on eye development. A: Control. B–E: XlMitf�-M-injected embryos. B: The opticfissure did not close (arrow). C: Ectopic retinal pigment epithelium (RPE, arrows) and RPE extension (arrowheads) were observed on the XlMitf�-M-injected side. D: The size of the eye on the injected side was small (arrow). E: A section of XlMitf�-M–injected embryos with an open optic fissure showsRPE extension toward the ventral part of the brain vesicle (arrow). F–I: dnXlMitf�-injected embryos. In dnXlMitf�-injected embryos, a lack of ventralRPE (F, black arrow), a reduction of eye size and a defect of optic stalk (G,H, black arrows) were observed. In severe cases, the eye became very smalland fused to the brain (H, black arrow). In addition, in some embryos, the phenotype with a lack of almost all the RPE was observed on the injectedside (I, black arrow). A–C: Stage 45. D,E: Stage 40. F–I: Stage 44. BR, brain vesicle.

Fig. 6. Effects of XlMitfM and dnXlMitf� mRNA microinjection on expression of eye-related genes. A–E: XlMitf�-M–injected embryos. A: The facialregion expressing Pax6 on the injected side (black arrow) was smaller than that on the noninjected side (blue arrow). Black dashes show the midline,and the eye region where Pax6 is normally expressed is shown by white dashes. B,C: Similarly, Rx1 (B) and Six6 (C) were repressed on the injectedside (black arrow). D: Expression of Otx5b was repressed both in the optic vesicle (black arrow) and the pineal body (white dashes) on the injectedside. Blue arrow shows normal Otx5b expression in the optic vesicle on the noninjected side, and black dashes show the midline. E: In contrast, Pax2expression was activated in the optic vesicle (black arrow), although those in the otic vesicle (green dashes) and midbrain–hindbrain boundary (pinkdashes) were repressed on the injected side compared with those on the noninjected side (blue, green, and pink arrows, respectively). F–J:dnXlMitf�-injected embryos; overexpression of dnXlMitf� induced the reduction of Pax6 (F), Rx1 (G), Six6 (H), Otx5b (I), and Pax2 (J) in the optic vesicleon the injected side (black arrows). Pax2 expression (J) in the otic vesicle (green dashes) and the midbrain–hindbrain boundary (pink dashes) and Otx5bexpression in the pineal body (I, white dashes) were also repressed on the injected side. A–C,E–H,J: Stage 23. D,I: Stage 25. CG, cement gland; MH,midbrain–hindbrain boundary; OT, otic vesicle; PB, pineal body.

Fig. 6.

MITF AND XENOPUS LAEVIS PIGMENT CELLS 529

get gene is Slug, which is a member ofthe Snail family of zinc-finger tran-scription factors that has an evolu-tionarily conserved function in meso-derm development and neural crestformation in vertebrates (LaBonneand Bronner-Fraser, 1998, 2000;Sefton et al., 1998; Del Barrio and Ni-eto, 2002). Slug is a useful marker forpremigratory neural crest cells (Nietoet al., 1994; Mayor et al., 1995), andSlug overexpression causes up-regula-tion of other neural crest markergenes and Slug expression itself (La-Bonne and Bronner-Fraser, 1998).

Significantly, overexpression ofXlMitf�-M induced Slug in vivo andin vitro, strongly suggesting thatSlug may play a role in the Mitf-mediated increase in melanophores.Recently, it was reported that Waar-denburg syndrome type 2 (WS2),which shows pigmentary abnormali-ties and sensorineural deafnesscaused by abnormalities of MITF ex-pression or function (Hughes et al.,1994; Tachibana et al., 1994; Tassa-behji et al., 1994, 1995; Goding,2000; and references therein), is alsocaused by deletions of human SLUG(Sanchez-Martin et al., 2002) andMITF that can bind the human SLUGpromoter and up-regulate its expres-sion in vitro (Sanchez-Martin et al.,2002). These reports complement ourobservations in the Xenopus systemand suggest that Mitf-mediated acti-vation of Slug may be an evolutionar-ily conserved event important for me-lanocyte/melanophore development.In agreement with this explanation,overexpression of Slug in Xenopus caninduce an increase in the number ofmelanophores (LaBonne and Bronner-Fraser, 1998) and inhibition of Slugleads to reduced melanophore forma-tion (Carl et al., 1999). Our resultsalso indicate that forced Slug expres-sion can induce an increase inXlMitf�-positive cells, raising the pos-sibility that, in addition to being adownstream target of Mitf, Slug mayalso act upstream, either by directlyactivating the Mitf promoter or indi-rectly by increasing the neural crestpopulation from which melanophoresoriginate. Thus, among Slug-positiveneural crest cells, some cells becomeXlMitf�-positive and are committed tobeing melanoblasts, and once commit-ted, these XlMitf�-positive cells up-

regulate Slug expression and as a re-sult activate the proliferation of Slug/XlMitf� double-positive committedmelanoblasts.

Decreased MelanophoresNumbers

Depending on the severity of the al-lele, mice with Mitf mutations show apartial or complete lack of melano-cytes, resulting in an entirely whitecoat or a white spotted coat, respec-tively (Steingrimsson et al., 2003).Moreover, in one of the zebrafish mu-tants nacre that has a mutation inMitfa corresponding to Mitf-M inother vertebrates, only melanophoresare absent, although xanthophoresand iridophores, both of which are alsoneural crest-derived pigment cells inpoikilotherms, exist (Lister et al.,1999). From these reports, Mitf ap-pears to be crucial for normal melano-cyte/melanophore development, al-though the mechanisms underlyingthe decrease or absence of melano-cytes/melanophores remain unknown.Our results using dnXlMitf� may pro-vide a hint as to the mechanisms in-volved.

In dnXlMitf�-injected embryos, adecrease in the number of melano-phores was observed and the expres-sion pattern of Dct was disturbed,whereas the numbers of Dct-positivecells appearing to be increased as inXlMitf�-M–injected embryos. Onepossible explanation is based on theobservation that, in dnXlMitf�-in-jected embryos, Sox10 and Slug ex-pression is shifted laterally precedingDct expression and that, as a result,Dct-positive cells abnormally devel-oped in this position instead of beingrestricted to their normal location.

Moreover, in dnXlMitf�-injectedembryos, the pattern of Slug andSox10 expression was disturbed withareas of weaker signals appearing be-tween regions with intense expres-sion. It was possible, therefore, thatoverexpression of dnXlMitf� causedapoptosis, repression of proliferation,and/or abnormal migration of neuralcrest cells. However, in the regionwhere the Slug signal was weaker, weobserved no significant down-regula-tion of proliferation or activation ofapoptosis compared with the Slug-ex-pressing region on the noninjected

side. On the other hand, at the regionwhere Slug expression was clearlyseen, the number of BrdU-positivecells and apoptotic cells was largerthan those on the noninjected side.These results may suggest that theweakening of the Slug signal arisesfrom the abnormal migration alongthe anteroposterior axis rather thanby the activation of apoptosis and therepression of proliferation of neuralcrest cells. In light of this possibility,the increase in BrdU-positive cellsand apoptotic cells in Slug-expressingregions on the dnXlMitf�-injected sidemay originate from the aggregation ofSlug-positive cells and neural crestcells with an abnormal migration abil-ity may die later, leading to a decreasein the number of melanophores atlater developmental stages. This pos-sibility remains to be elucidated.

XlMitf� Controls Dendricityof Melanophores

In addition to the role of XlMitf� inregulating melanophore numbers, ourresults also suggest a possible role forXlMitf� in melanophore dendricity.Thus, overexpression of dnXlMitf� ledthe surviving melanophores to adopt apunctate phenotype that appeared toreflect as change in cell shape ratherthan simply a redistribution of mela-nophores, while expression of normalXlMitf� appeared to increase dendric-ity. This result is consistent with ob-servations made using ectopic expres-sion of mammalian Mitf-M in culturedcells, which appears to increase den-dricity (Tachibana et al., 1996; Car-reira et al., 2005). However, althoughwe believe that Mitf is likely to regu-late genes intimately connected to reg-ulation of the cytoskeleton, we cannotrule out additional effects on regula-tion of melanin dispersal. Indeed theprocesses are closely connected be-cause melanin dispersal cannot occurwithout a compatible cytoskeleton or-ganization.

XlMitf� Is Essential forNormal Eye Development

In the assays used here, the XlMitf�used appears to be an orthologue ofthe mammalian Mitf-M that is a neu-ral crest-specific Mitf isoform. Previ-ously, we reported that XlMitf� is ex-

530 KUMASAKA ET AL.

pressed in melanophore lineages, aswell as the RPE and epiphysis (Ku-masaka et al., 2004). However, wewere unable to identify any isoform-specific expression, and it remains un-clear whether any isoform-specific or-thologue of Mitf-M is specificallyexpressed only in the neural crest-de-rived melanophore lineage as in mam-mals. In most of the XlMitf�-M–over-expressed embryos, ectopic RPE andRPE extensions were observed. Thus,two explanations are possible for thephenotype induced in the eye: (1) theXlMitf�-M used could function in RPEdevelopment as an alternative forother XlMitf� isoforms that are nor-mally specifically involved in RPE de-velopment, or (2) XlMitf�-M is nor-mally expressed in the RPE and forcedoverexpression of it induces multipleeye malformations.

Among XlMitf�-overexpressed em-bryos, a small number of embryos ex-hibited a small eye phenotype and areduction of Pax6 expression. Thesmall eye phenotype could arise eitheras a result of the capacity of XlMitf� toinhibit cellular proliferation, as sug-gested from results obtained usingmammalian Mitf-M (Carreira et al.,2005; Loercher et al., 2005) or alter-natively, but not mutually exclusively,by means of the suppression of Pax6expression or activity by XlMitf� over-expression. Several lines of evidencetend to support the latter interpreta-tion. First, Xenopus embryos injectedwith a dominant-negative form ofPax6 showed either complete loss ofeye structure or a reduction in eye size(Chow et al., 1999); second, infectionof chicken cultured RPE cells with aretrovirus-expressing Mitf, inhibitedPax6 expression (Mochii et al., 1998);and in quail, cultured RPE cells, theDNA-binding domains of Pax6 inter-act with the bHLH-LZ domain of Mitfand repress their respective targetpromoters (Planque et al., 2001).

A small eye phenotype was also ob-served by overexpression of dnX-lMitf�, a result similar to that ob-served in mice bearing Mitf mutations(Green, 1989; Tachibana et al., 1992;Steingrimsson et al., 1994). Loss ofXlMitf� function in the eye at earlydevelopmental stages could inducemorphological eye malformation aris-ing from repression of Pax6. In addi-tion, the dnXlMitf� used in this study

could potentially also interact withPax6 protein and repress its activityin the same manner of XlMitf�-Moverexpression. At present, we cannotdiscriminate between these possibili-ties.

The expression of other eye-relatedgenes we examined (Rx1, Six6, andOtx5b) were similarly repressed byforced expression of both XlMitf�-Mand dnXlMitf�. The mechanism un-derlying the repression of this set ofgenes remains unknown, although itis possible that they are indirectly reg-ulated by Pax6 and consequently anyinhibition of Pax6 function or expres-sion by XlMitf�-M and dnXlMitf�would affect their expression.

In contrast to Pax6, Rx1, Six6, andOtx5b expression, overexpression ofXlMitf�-M and dnXlMitf� had differ-ent effects on Pax2 expression. InXlMitf�-M-injected embryos, expres-sion of Pax2 at the ventral part of theeye vesicle was up-regulated, whereasforced expression of dnXlMitf� down-regulated the expression of this gene.Thus, Pax2 may be a candidate targetgene for XlMitf� in the optic vesicle.Consistent with this possibility, theectopic RPE and RPE extensions in-duced by XlMitf�-M overexpressionwere observed along the optic stalk inwhich Pax2 was strongly expressed.When XlMitf� is overexpressed in thisarea, reciprocal activation of XlMitf�and Pax2 may occur. Leading to ec-topic RPE and RPE extension. It isalso possible that the interactions be-tween Pax2 and Pax6 are one of thecauses of abnormal Pax2 expression inXlMitf� up-regulated and down-regu-lated embryos. Schwarz et al. (2000)postulated a model for the reciprocalinhibition between Pax2 and Pax6 atthe border of the optic stalk and opticcup for their spatial specification. InXlMitf�-M–overexpressed embryos,up-regulation of Pax2 expression ap-pears to be caused by both down-reg-ulation of Pax6 and up-regulation ofXlMitf�. However, in contrast, in dnX-lMitf�-overexpressed embryos, Pax6is down-regulated but the up-regula-tion of Pax2 has not been observed.These results suggest that Pax2 ex-pression could not be activated with-out XlMitf� even if Pax6 is down-reg-ulated.

CONCLUSION

In summary, we have demonstratedthat the Xenopus system provides arapid and efficient system for the anal-ysis of genes involved in melanocyteand RPE development. This study pro-vides new information for several am-biguous points about Mitf function inpigment cell development in vivo and invitro. From XlMitf�-M overexpressionanalyses, it is clear that XlMitf�-M con-tributes to an increase in the number ofmelanophores by increasing the num-ber of Slug-positive and Sox10-positivecells in vivo and this finding is sup-ported by the in vitro RT-PCR analysisusing animal cap explants. Further-more, XlMitf�-M has the ability to in-duce melanization in non-neural crestcells. Forced expression of dnXlMitf�suggests that XlMitf� is also necessaryfor the melanoblast migration. In addi-tion, overexpression of Slug shows thatSlug contributes to the specificationand proliferation of neural crest cells.As for eye development, the results in-dicate that XlMitf� has the ability toinduce RPE and that the interactionsbetween XlMitf� and other eye-relatedgenes are indispensable for the normaleye development. The results providean insight into the Mitf-dependentmechanisms underlying melanocyte/melanophore development, neural crestdifferentiation, and eye development.

EXPERIMENTALPROCEDURES

Embryos and Microinjection

Xenopus laevis adults purchased fromCOPACETIC Co., Ltd. (Aomori, Ja-pan) and HSK Co., Ltd. (Shizuoka, Ja-pan) were maintained in our labora-tory at 23°C. Fertilized eggs wereobtained from adults injected with250 U of human chorionic gonadotro-pin to induce egg laying. Embryoswere dejellied in 2% cysteine (pH 7.5)and were washed twice in 1� MMR(100 mM NaCl, 2 mM KCl, 1 mMMgCl2, 2 mM CaCl2, 0.1 mM ethyl-enediaminetetraacetic acid, 5 mMHEPES, pH 7.5). RNAs (XlMitf�-M,dnXlMitf� and Slug; 10 nl at 5 ng/�l)were injected into one blastomere atthe two-cell stage in 1� MMR�3% Fi-coll and were maintained for 5 hr at16°C. Embryos were transferred to

MITF AND XENOPUS LAEVIS PIGMENT CELLS 531

0.1� MMR�1% Ficoll and were main-tained overnight at 16°C. The follow-ing day, embryos were transferred to0.1� MMR and maintained until fix-ation. Staging was carried out accord-ing to Nieuwkoop and Faber (1967).Under the conditions we used, injec-tion of lower concentration ofXlMitf�-M or dnXlMitf mRNA (10 nlat 1 ng/�l) had no effect.

Plasmid Construction and InVitro RNA Synthesis

To generate pCS2 XlMitf�-M, positions1 to 852 and 640 to 1176 of the 1176-bpXlMitf�-M cDNA were PCR amplifiedusing primers XlMi-forward1 (5�-cgg-gatccctcgagATGCTGGAAATGCTTGA-CTATAACC-3�, flanked with BamHI andXhoI sites) and XlMi-reverse1 [5�-GTC-GAGCAGAGGCCAGTAGAAGG-3�],XlMi-forward2 (5�- atatgcggccgcAAA-AGCTTCGGTGGATTACATTCGC-3�,flanked with a NotI site), and XlMi-reverse2 [5�-ctacttgtcatcgtcgtccttgtagtc-ACAGTGATCATTTTCTTCCAT-3�, se-quences for FLAG epitope tag under-lined], respectively. The amplified frag-ments were subcloned using the TACloning Kit, excised by digesting withXhoI/SphI (position 758) and SphI (po-sition 758)/XbaI, respectively, and thencloned into the XhoI/XbaI sites of thepCS2.

Dominant-negative construct ofXlMitf� is converted LIER to LIAR inthe basic domain (Fig. 1A). To prepareXlMitf� dominant-negative construct,we first used PCR with primers XlMi-forward3 (5�-CAATTGGTTCAACCT-GTGAAAAAGAG-3�, corresponding toTIGSTCEKE) and dXlMi-reverse1 [5�-CTTCGCCTGCGCGCAATGAGG-3�,complementary to LIARRR andflanked with a BssHII site], the condi-tions for PCR were as follows: 94°C for5 min, 30 cycles of 94°C for 1 min,58°C for 1 min, and 72°C for 1 min,then 72°C for 10 min. dXlMi-forward1(5�-CCTCATTGCGCGCAGGCGAAG-3�, corresponding to LIARRR andflanked with a BssHII site) andXlMitf-reverse3 [5�-GTCGAGCAGAG-GCCGGTAGAAG-3�, complementaryto STGLCS], the conditions for PCRwere the same as those mentionedabove. Both fragments were cloned us-ing the TA Cloning System (Invitro-gen) and sequenced. The amplifiedfragments were subcloned using the

TA Cloning Kit (Invitrogen), excisedby digesting with BglII/BssHII andBssHII/SphI, respectively, and thencloned into the BglII/SphI sites of thepCS2 XlMitf�-M. To generate pCS2Slug, full-length Xenopus Slug cDNAwas PCR amplified using primersSlug-forward1 (5�-ATGCCCCGGT-CATTTCTGGTCAAG-3�, correspond-ing to MPRSFLVK) and Slug-reverse1(5�-ctcgagctacttgtcatcgtcgtccttgtagtcATGTGCTACACAGCAACCAGA-3�,complementary to SGCCVAH,flanked with a XhoI site and the se-quences for FLAG epitope tag under-lined). The amplified fragment wassubcloned using pGEM-T Easy Vec-tor System I (Promega) and se-quenced, and excised with EcoRI/XhoI and then cloned into EcoRI/XhoI sites of the pCS2 vector.Capped XlMitf�-M, dominant-nega-tive XlMitf�, and Slug mRNA weresynthesized with SP6 RNA polymer-ase using templates linearized withNotI using mMESSAGE mMA-CHINE High Yield Capped RNATranscription Kit (Ambion). �-galac-tosidase mRNA coinjected was syn-thesized from pCS2�n�-gal withSP6 RNA polymerase using templatelinearized with NotI in the sameway.

Red-Gal Staining and Whole-Mount In Situ Hybridization

Embryos were coinjected with �-ga-lactosidase mRNA (�-gal, 200 pg/em-bryo) and were fixed in MEMFA.Embryos were rinsed several timesin phosphate buffered saline (PBS)after fixation. �-Galactosidase stain-ing was carried out with 0.1 mM po-tassium ferricyanide, 0.1 mM potas-sium ferrocyanide, and 0.4% red-galin PBS at room temperature. Afterstaining, embryos were rinsed inPBS and dehydrated with 25%, 50%,75% ethanol/PBT (PBS, 0.1% Tween-20), and 100% ethanol at room tem-perature, and then stored in 100%ethanol until applying whole-mountin situ hybridization. Whole-mountin situ hybridization was carried outas described previously (Kumasakaet al., 2003).

For RNA probes, Slug, Sox10, Pax6,Pax2, Rx1, and Six6 were isolated by RT-PCR. Primers designed for this work

were: Slug-forward1 (5�-ATGCCCCGGT-CATTTCTGGTCAAG-3�, correspondingto MPRSFLVK) and Slug-reverse3 [5�-GC(GA)CCCAAGCT(CG)AC(AG)TAC-TCC-3�, complementary to KEYVSLGA],Sox10-foward1 (5�-TCAGGTCAAAGT-CATGGACCCCC-3�, corresponding toSGQSHGP) and Sox10-reverse1 [5�-GAGATGGAGGGAAATGCTGAACC-3�,complementary to GSAFLSIS], Pax6-forward (5�-AACCTGGCGAGCGAG-AAGCAGCAGA-3�, corresponding toNLASEKQQ) and Pax6-reverse [5�-TTCTTTTTCTAGCGCCTCTATTTG-3�complementary to QIEALEKE],Pax2-forward (5�-TCTGATGGTTCTGG-TCCAAATGG-3�, corresponding toSDGSGPNG) and Pax2-reverse[5�-GTGGCGGTCATAGGCAGTG-3�,complementary to ATAYDRH], Rx1-forward (5�-AGAAGAAACACAGAA-GGAACCGG-3�, corresponding toKKKHRRNR)andRx1-reverse[5�-CCA-AGGCTTGCCAATAAACTGGAT-3�,complementary to IQFIGKPW], Six6-forward (5�-ATGTTTCAGCTGCCT-ATTCTGAAC-3�, corresponding toMFQLPILN) and Six6-reverse [5�-TCAGATGTCACATTCACTGTCGC-3�, complementary to DSECDI]. Theconditions for these RT-PCR were asfollows: 94°C for 5 min; 40 cycles of94°C for 1 min, 56°C for 1 min, 72°Cfor 3 min; then 72°C for 10 min. Allthese fragments were cloned using theTA Cloning System (Invitrogen; Slug,Sox10, and Pax6) or the pGEM-T EasyVector System I (Promega; Pax2, Rx1,and Six6) and were verified by se-quencing. Digoxigenin-labeled anti-sense RNA probes for Dct, XlMitf�and Otx5b were described previously(Kumasaka et al., 2003) and for Slug,Sox10, Pax6, Pax2, Rx1, and Six6were synthesized with SP6 (Slug,Sox10, Pax6, Pax2, and Six6), or T7(Rx1) RNA polymerases using tem-plates linearized with NotI (Pax6 andSix6), SphI (Pax2), XhoI (Slug andSox10), or PstI (Rx1) using digoxige-nin RNA labeling mix (Roche Diagnos-tics).

Animal Cap Assay and RT-PCR Analysis

Dejellied embryos were injected withXlMitf�-M in the animal pole at thetwo-cell stage and incubated in1�MMR�3% Ficoll until at stage 8.5.After the removal of vitelline mem-

532 KUMASAKA ET AL.

branes, animal caps were cut out andcultured in 1� LCMR (66 mM NaCl,1.33 mM KCl, 0.33 mM CaCl2, 0.17mM MgCl2, 5 mM HEPES, pH 7.2)supplemented with 0.1% BSA andwere then harvested at the stages in-dicated in the figure legends. At eachstage, 10 animal cap explants wereharvested and RT-PCR performed. Weused RT-PCR with primers EF1� (for-ward primer, 5�-CAGATTGGTGCTG-GATATGC-3�; reverse primer, 5�-ACTGCCTTGATGACTCCTAG-3�; 28cycles), Tyr (forward, 5�-GCATC-CCGAGATGCCTTCATAGG-3�; reverse,5�-TGAAGTTGGCCGACCGATCCATG-3�; 28 cycles), Tyrp1 (forward, 5�-TC-CGTGAGCAAAACCTTTCTGGGC-3�;reverse, 5�-TGTGTCAAACATATTCA-CATCAAG-3�; 28 cycles), Dct (for-ward, 5�-CTGTCCGGGACACATTGC-TCG-3�; reverse, 5�-AGTGGAATTC-CTGAAAAAAGGAGG-3�; 28 cycles),Slug (forward, 5�-CAAACTTTCCG-ACTCGCATGCG-3�; reverse, 5�-CTA-ATGTGCTACACAGCAACCAGA-3�;28 cycles), Sox10 (forward, 5�-TTC-CACCTAATGGTCATGCTGGG-3�; re-verse, 5�-GAGATGGAGGGAAAT-GCTGAACC-3�; 28 cycles), N-tubulin(forward, 5�-ACACGGCATTGATC-CTACAG-3�; reverse, 5�-AGCTCCT-TCGGTGTAATGAC-3�; 28 cycles),XNeuroD (forward, 5�-CCATGGGA-CAGCAGCGCATGCTGAC-3�; re-verse, 5�-GTCCGCATTACTGCTCTC-CGGAACAACG-3�; 28 cycles), XlM-itf� (forward, 5�-GAAAGCCTCAGTG-GATTACATTCGCA-3�; reverse, 5�-GTGTATCGTCCATGAATATGTCT-TC-3�; 32 cycles).

BrdU Incorporation andImmunohistochemistry

BrdU was dissolved in 0.1� MMR at afinal concentration of 50 �g/ml. Em-bryos were incubated in this BrdU so-lution for 1 hr, transferred to0.1�MMR, and maintained for 2 hr.After fixation, these embryos wereused for whole-mount in situ hybrid-ization analysis and cryosections (10�m) were made from frozen blocks asdescribed previously (Kumasaka etal., 2003) for immunostaining. Immu-nostaining was performed using pri-mary antibody, anti-BrdU (Develop-mental Studies Hybridoma Bank),anti–single-strand DNA (DAKO) andfollowed by ENVISION Kit/HRP

(DAKO). Before primary antibody re-action, sections were incubated in 2 NHCl for 1 hr at 37°C to expose theDNA.

ACKNOWLEDGMENTSWe thank Drs. Hiroyuki Ide and KojiTamura for helpful discussions. Wealso thank all the members of theYamamoto and Ide laboratories fortechnical advice. H.Y. was funded by aGrant-in Aid from the Ministry of Ed-ucation, Culture, Sports, Science, andTechnology, Japan.

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