21. Ellis, R. H., Hong, T. D. & Roberts, E. H. A note on the development of a practical procedure for

promoting the germination of dormant seed of grape (Vitis spp.). Vitis 22, 211–219 (1983).

22. Peng, J. & Harberd, N. P. Transposon-associated somatic gai-loss sectors in Arabidopsis. Plant Sci. 130,

181–188 (1997).

23. Simpson, G. G., Gendall, A. R. & Dean, C. When to switch to flowering. Annu. Rev. Cell Dev. Biol. 15,

519–550 (1999).

24. Buttrose, M. S. Climatic factors and fruitfulness in grapevines. Hort. Abstr. 44, 319–326 (1974).

25. Boss, P. K., Davies, C. & Robinson, S. P. Analysis of the expression of anthocyanin pathway genes in

developing Vitis vinifera L. cv. Shiraz grape berries and the implications for pathway regulation. Plant

Physiol. 111, 1059–1066 (1996).

26. Frohman, M. A., Dush, M. K. & Martin, G. R. Rapid production of full-length cDNAs from rare

transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl Acad. Sci.

USA 85, 8998–9002 (1988).

27. Thomas, M. R., Matsumoto, S., Cain, P. & Scott, N. S. Repetitive DNA of grapevine: classes present

and sequences suitable for cultivar identification. Theor. Appl. Genet. 86, 173–180 (1993).

Supplementary Information accompanies the paper on Nature’s website

(http://www.nature.com).

AcknowledgementsWe thank R. King and A. Poole for the GA analyses by GC–MS; T. Franks for plantmaterial; S. McClure for help with the scanning electron mictroscopy; and D. MacKenzieand C. Reich for technical assistance. This work was supported in part by the Grape andWine Research and Development Corporation, Dried Fruits Research and DevelopmentCouncil and the Cooperative Research Centre for Viticulture.

Competing interests statement

The authors declare that they have no competing financial interests.

Correspondence and requests for materials should be addressed to M.R.T.

(e-mail: mark.r.thomas@csiro.au). The GenBank library accession number for VvGAI1 is

AF378125.

..............................................................

Emi1 is required for cytostatic factorarrest in vertebrate eggsJulie D. R. Reimann & Peter K. Jackson

Departments of Pathology, Microbiology and Immunology, Stanford UniversitySchool of Medicine, 300 Pasteur Drive, Stanford, California 94305-5324, USA.............................................................................................................................................................................

Vertebrate eggs are arrested at metaphase of meiosis II withstable cyclin B and high cyclin B/Cdc2 kinase activity. The abilityof the anaphase-promoting complex/cyclosome (APC), an E3ubiquitin ligase, to trigger cyclin B destruction and metaphaseexit is blocked in eggs by the activity of cytostatic factor (CSF)(reviewed in ref. 1). CSF was defined as an activity in matureoocytes that caused mitotic arrest when injected into dividingembryos2. Fertilization causes a transient increase in cytoplasmiccalcium concentration leading to CSF inactivation, APC acti-vation, cyclin B destruction and mitotic exit3. The APC activatorCdc20 is required for APC activation after fertilization4,5. Weshow here that the APCcdc20 inhibitor Emi1 (ref. 6) is necessaryand sufficient to inhibit the APC and to prevent mitotic exit inCSF-arrested eggs. CSF extracts immunodepleted of Emi1degrade cyclin B, and exit from mitosis prematurely in theabsence of calcium. Addition of Emi1 to these Emi1-depletedextracts blocks premature inactivation of the CSF-arrested state.Emi1 is required to arrest unfertilized eggs at metaphase ofmeiosis II and seems to be the long-sought mediator of CSFactivity.

CSF prevents parthenogenetic activation while animal eggs awaitfertilization. The molecular composition of CSF is unknown,although the MAP-kinase (MAPK) pathway is required to establishthe CSF state in maturing oocytes. Specifically, the proto-oncogeneMos, a MAPK kinase kinase, is involved in the activation of

maturation promoting factor (cyclin B–cdc2 kinase complex) inmeiosis I (MI) and during the transition from MI to meiosis II(MII) in Xenopus oocytes7,8. Unfertilized oocytes from Mos2/2 miceundergo parthenogenetic activation9,10. The Mos/MAPK pathwayalso prevents DNA replication between MI and MII—therebyallowing meiotic reduction in chromosome number—by prema-turely reactivating cyclin B/Cdc2 after metaphase11,12. However, theMAPK pathway is not sufficient for MII, because injection of theMos protein is not sufficient to prevent mitotic exit after anaphase Iin the presence of protein synthesis inhibitors8,11, indicating thatanother factor is required to progress to metaphase of MII. Theprotein kinases MAPK kinase (MEK), MAPK and p90rskare essentialdownstream mediators of Mos activity13–16. However, although Mos,MAPK and p90rsk each cause a CSF-like metaphase arrest wheninjected into dividing embryos, neither Rsk depletion nor MAPKinactivation in CSF-arrested egg extracts releases CSF arrest13 (seebelow). Thus, the MAPK pathway is required to establish, but not tomaintain, CSF arrest in MII.

Fertilization in vivo or the addition of calcium to extracts fromunfertilized Xenopus eggs (called CSF extracts) activates APC, theubiquitin ligase that mediates cyclin B destruction and mitoticexit3,17,18. The APC activator Cdc20 is required for the calcium-induced exit from CSF arrest, indicating that APCcdc20 is a target ofthe CSF inhibitory mechanism4,5. Cdc20 is also a target of thespindle-assembly checkpoint protein Mad2, which inhibits APCactivation by Cdc20 in response to spindle damage in the mitoticcell cycle19. Although Mad2 is present in CSF-arrested eggs20, Mad2and other spindle-assembly checkpoint proteins are not required forCSF arrest4,20–22.

We recently identified Emi1 as an APCcdc20 inhibitor in theXenopus embryo6. In the mitotic cell cycle, Emi1 accumulates beforemitosis and binds Cdc20 to inhibit its ability to activate the APC,thus allowing cyclin B to accumulate. In mitosis, Emi is ubiquiti-nated and destroyed independently of the APC. The microinjectionof Emi1 into cleaving embryos, like that of Mos23, constitutivelyactive MAPK15 or constitutively active p90rsk (ref. 14), causes a CSF-like mitotic arrest6. Given these results and the presence of Emi1 inthe unfertilized egg, we were interested in testing whether Emi1 is acomponent of CSF.

We first examined whether excess Emi1 protein was sufficient toprevent release from the MII metaphase arrest in the presence ofcalcium. Cyclin B and Mos are stable and MAPK is active in extractsprepared from metaphase of MII-arrested eggs (CSF extracts). InCSF extracts treated with control protein, cyclin B is destroyed, Mosis inactivated and destroyed, and MAPK is inactivated, after calciumaddition. The addition of purified maltose-binding protein (MBP)–Emi1 protein (1 mM, compared with ,300 nM endogenous Emi1(ref. 6)) to CSF extracts prevented the calcium-induced destructionof cyclin B and Mos and the inactivation of MAPK (Fig. 1a).Although Mos is not an APC substrate, Mos is stabilized whenthe APC is inhibited because Mos destruction requires cyclin B/cdc2inactivation24. Examination of DNA morphology revealed thatcontrol extracts exited from mitosis by 15 min, whereas Emi1-treated extracts remained arrested in metaphase after more than90 min (not shown). Inhibiting the APC by adding a destructionbox (D-box) peptide also blocked CSF release (Fig. 1b). Thus, theAPC is required for release from CSF arrest.

Mos inactivation and destruction occur later than cyclin Bdestruction after either egg activation or the addition of calciumto CSF extracts (Fig. 1a)25. Incubation of CSF extracts with Mosprotein before calcium addition did not prevent cyclin B destruc-tion even though MAPK remained activated (Fig. 1c), furtherindicating that Mos inactivation is not required for exit from CSFarrest. Addition of the MEK inhibitor U0126 to CSF extracts led tothe inactivation of MAPK and p90rsk (ref. 12) but did not releaseextracts from the CSF state (Fig. 1c), which is consistent withpreviously reported results on depletion of the MEK target p90rsk

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from CSF extracts13. These data indicate strongly that the MAPKpathway is not required for maintenance of CSF arrest. To testwhether Emi1 requires the MAPK pathway for inhibition ofcalcium-induced CSF release, we incubated CSF extracts withU0126 and found that Emi1 addition still prevented the destructionof cyclin B and Mos and exit from mitosis in the presence of calcium(Fig. 1c). Thus, Emi1’s APC inhibitory activity does not require theMAPK pathway.

Calmodulin-dependent protein kinase II (CaMKII) is requiredfor release of MII from metaphase, and constitutively active CaMKIIis sufficient to trigger cyclin B destruction and mitotic exit withoutfertilization or the addition of calcium26. Thus, the major calcium-sensitive mediator of CSF release is CaMKII. We found thatconstitutively active CaMKII did not trigger cyclin B destruction,Mos destruction or mitotic exit in CSF extracts in the presence ofexcess Emi1 (Fig. 1d), indicating that Emi1 acts downstream ofCaMKII. These results are consistent with the observation thatCdc20 depletion also prevents constitutively active CaMKII-induced release from CSF arrest4. Additionally, much like Cdc20depletion, excess Emi1 can block okadaic-acid-induced cyclin Bdestruction in CSF extracts in the absence of calcium (Supplemen-tary Information, Fig. S1).

We next examined whether the Emi1 protein accumulates inmaturing oocytes in sufficient time to account for CSF arrest. Emi1is first expressed to significant levels in stage VI G2/prophaseoocytes (Fig. 2a). After the addition of progesterone, Emi1 levelsincreased by germinal vesicle breakdown, similar to Cdc20 levels(Fig. 2a)27. Emi1 levels and phosphorylation state did not seem tofluctuate significantly during oocyte maturation, whereas Cdc20protein levels did (Fig. 2a)27. In mature oocytes, Emi1 is present insufficient amounts (,300 nM) to inhibit the endogenous pool of

Cdc20 (,40–100 nM (ref. 4)).To test whether Emi1 is required for the maintenance of the MII

metaphase arrest, we examined whether Emi1 depletion caused exitfrom CSF arrest. Incubation of CSF extracts with magnetic beadsprecoupled to Emi1 antibodies6 depleted more than 80% of Emi1(Fig. 2b). Even though a small amount of Cdc20 immunoprecipi-tated with the anti-Emi1 beads, most Cdc20 (,80%) remained inthe extracts (Fig. 2b, and data not shown). As shown in Fig. 2c–h,mock-depletion of CSF extracts did not affect cyclin B stability,metaphase arrest or sensitivity to calcium. In contrast, Emi1depletion reproducibly induced cyclin B destruction and mitoticexit (Fig. 2c–h). Emi1-depleted extracts also degraded Mos in theabsence of calcium (data not shown).

At time 0 in Emi1-depleted extracts, cyclin B levels were alreadylower than in mock-depleted extracts (Fig. 2c, f). Because cyclin Binteracts with Emi1 (ref. 6), we needed to exclude the possibility thatEmi1 depletion caused mitotic exit by depleting cyclin B as well. Wefound that only a small amount of cyclin B (,6.3%) was depletedtogether with Emi1 (Fig. 2b). Moreover, addition of the proteasomeinhibitor MG-132 prevented the cyclin B decrease and mitotic exitin Emi1-depleted extracts (Supplementary Information, Fig. S2).Thus, the decrease in cyclin B at time 0 in Emi1-depleted extracts ismost probably due to the occurrence of APC activation as Emi1 isdepleted.

To verify that the cyclin B destruction and mitotic exit observed inEmi-depleted extracts reflected the loss of Emi1’s APC inhibitoryfunction, we preincubated extracts with a stable carboxy-terminalfragment of Emi1 that is sufficient to inhibit APC activation in vitroand in vivo6. Pre-addition of this purified MBP–Emi1-CT proteinrescued cyclin B stability and CSF metaphase arrest (Fig. 2f–h).Furthermore, the addition of purified Emi1 protein to CSF extracts

Figure 1 Emi1 is sufficient to prevent release from CSF block in the presence of Ca2+.

a, b, Addition of Emi1 or a D-box peptide stabilizes cyclin B and Mos, and blocks MAP

kinase inactivation in the presence of calcium. CSF extracts were preincubated with MBP–

Emi1 (1 mM) or MBP (1 mM, control), D-box peptide (675 mM) or control peptide

(675 mM)6, and released with calcium. Samples were immunoblotted with antibodies

against cyclin B2, Mos (Santa Cruz Biotechnology) and active MAP kinase (NEB). Times

after calcium addition are indicated. c, Blocking by Emi1 of CSF release does not require

the MAP kinase pathway. CSF extracts were preincubated with MBP–Mos (1 mM), U0126

(50 mM) (Promega) or U0126 (50 mM) and then MBP–Emi1 (1 mM). Extracts were released

with calcium (except where indicated) and analysed as in a. Control extracts in a–c

re-entered mitosis by ,75 min. d, Constitutively active CaMKII cannot trigger calcium-

independent cyclin B destruction and mitotic exit in the presence of Emi1. Extracts were

analysed as in a. Times after CaMKII addition are indicated.

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after partial depletion of Emi1 stabilized the remaining cyclin B andprevented mitotic exit (Supplementary Information, Fig. S3).Although it is possible that additional Emi1-interacting proteinsimportant for CSF arrest are depleted together with Emi1, these datastrongly indicate that Emi1 is required for the maintenance of CSFarrest in Xenopus eggs.

If Emi1 acts in CSF-arrested eggs to inhibit APC activation byCdc20, then the addition of excess Cdc20 might be expected tooverwhelm the inhibitory effect of endogenous Emi1 and thus causeAPC activation and mitotic exit. To test this idea, we addedincreasing amounts of purified recombinant his-tagged Cdc20 toCSF-arrested extracts and examined whether these extracts exitedfrom CSF arrest in the absence of calcium. Indeed, Cdc20 inducedcyclin B degradation and mitotic exit in a dose-dependent fashionin CSF extracts. This effect was blocked by the addition of purifiedMBP–Emi1 protein but not by the addition of purified MBP–Mosprotein (Fig. 3a–c). The added Mos protein did prolong mitosis asdescribed previously28.

Cyclin B protein is destroyed by ,10 min after egg activation25 orrelease of CSF arrest in extracts (Fig. 1). Thus, the activation signalfrom CaMKII to the APC must be transduced quickly. Emi1 proteinlevels do not fluctuate significantly until the first mitosis afterfertilization6, and Cdc20 protein is stable in the early embryo (Fig.3d, e)4. How, therefore, is Emi1 prevented from inhibiting Cdc20after fertilization? One possibility is that CaMKII activation leads toa change in binding between Emi1 and Cdc20. To test this idea, weanalysed the binding of endogenous Emi1 and Cdc20 after calciumaddition. Emi1 interacts specifically with Cdc20 in vitro and in vivo6.Before calcium addition (time 0), Cdc20 immunoprecipitatedtogether with Emi1. By 2.5 min after calcium addition, only back-ground amounts of Cdc20 binding were detectable (Fig. 3d). Wesaw a similar loss of binding when 35S-labelled Cdc20 translated invitro was added to CSF extracts and immunoprecipitated togetherwith Emi1 (Fig. 3e).

The release of Cdc20 from Emi1 might result from a change inmodification in either protein. After calcium addition we observed a

Figure 2 Emi1 is required for CSF arrest. a, Emi1 accumulates in the maturing oocyte.

Samples from a maturation time course after progesterone addition (at time 0) and equal

aliquots of stage I–V oocytes were analysed for Emi1, Cdc20 and active MAP kinase by

immunoblotting and cyclin B-associated kinase activity by H1 kinase assay. GVBD, time at

which 100% germinal vesicle breakdown had occurred. b, Specificity of Emi1

immunodepletion (dpn). Extracts were assayed by immunoblotting for Orc1 as a loading

control, Emi1 and Cdc20. Beads from Emi1-depleted and mock-depleted extracts (from

the equivalent of 1 ml of extract) and CSF extract (1 ml) were assayed for cyclin B by

immunoblotting (right). c–h, Emi1 depletion causes cyclin B destruction and mitotic exit in

the absence of calcium. c–e, Emi1-depleted and mock-depleted extracts with (d, e) or

minus (c) sperm DNA were warmed to 23 8C. Calcium was added at 60 min. Samples

were taken at the indicated time after warming, then immunoblotted for cyclin B (c) or

assayed for DNA morphology (d). Mitotic figures were quantified (e). f–h, Similar to c–e,

except that one sample was preincubated with active non-degradable MBP–Emi1-CT

domain during the immunodepletion, and no calcium was added. A background band

(asterisks) served as a loading control; the exposure time was the same for all blots.

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rapid increase in the electrophoretic mobility of Cdc20 that wasconsistent with dephosphorylation (Fig. 3d, e). Alternatively, Emi1might be a target of CaMKII after its calcium-induced activation.Interestingly, Emi1 contains one consensus CaMKII phosphoryl-ation site, as well as three additional less conserved phosphorylationsites.

Our data demonstrate that Emi1 is essential for the maintenanceof CSF metaphase arrest. Masui originally proposed that CSF would(1) ‘appear’ (become activated) during oocyte maturation; (2)‘disappear’ (become inactivated) after fertilization; (3) be inacti-vated under the same conditions as those that cause egg activation;(4) be sufficient for the metaphase of MII (CSF) arrest in eggs; and(5) cause a reversible mitotic block in the zygote1. Unlike the Mos/MAPK pathway, which is not sufficient to maintain CSF arrest,Emi1 is both necessary and sufficient for metaphase of MII arrest.Thus, there seems to be a CSF biochemical pathway that acts toprevent cyclin B/Cdc2 inactivation until fertilization (Fig. 4). TheMos/MAPK pathway acts to keep Cdc2 active through positiveregulation and by increasing cyclin B synthesis during the MI–MIItransition (reviewed in ref. 29), whereas Emi1 acts to prevent cyclinB destruction through APC inhibition in MII. It remains to be seenhow Emi1 becomes activated during oocyte maturation, whetherEmi1 has a role earlier in oocyte maturation, and whether the Mos/MAPK pathway regulates Emi1 during this time. A

MethodsPlasmids and proteinsEmi1 and Mos constructs were as described6,8 and proteins were produced and purifiedwith the use of standard protocols. To prepare constitutively active CaMKII, residues 1–290 were subcloned from rat brain CaMKIIa (H. Schulman) into pCS2 vector.

Xenopus extracts and oocytesXenopus CSF extracts were prepared as described30 and 400 mM CaCl2 was used to releaseextracts from the CSF state. For addition experiments, CSF extracts were preincubated

Figure 3 Addition of exogenous Cdc20 to CSF extracts activates cyclin B destruction and

mitotic exit in the absence of calcium. a, CSF extracts were preincubated with buffer, 1 or

3 mM Cdc20, 3 mM Cdc20 preincubated with 3 mM Emi1, or 3 mM Cdc20 preincubated

with 1 mM Mos. Extracts were warmed to 23 8C and aliquots were analysed for cyclin B

levels by immunoblotting. A background band (asterisks) served as a loading control; the

exposure time was the same for all blots. b, c, Sperm DNA was added to extracts treated

as in a, and morphology was assessed (b) and quantified (c). d, e, Emi1 and Cdc20

dissociate after release from CSF arrest. Calcium was added at time 0 to CSF extracts (d)

or CSF extracts preincubated with 35S-labelled Cdc20 translated in vitro (IVT) (e), and the

immunoprecipitation (IP) of Emi1 together with Cdc20 was assayed by immunoblotting or

autoradiography. Unprecipitated extracts from specific time points were resolved by SDS–

PAGE and analysed as loading controls (lower panels).

Figure 4 Model. a, Perturbations that bypass the calcium requirement for APC activation

in the mature CSF-arrested oocyte: Emi1 depletion, excess Cdc20 protein, or the addition

of constitutively active CaMKII (ref. 26). Calcium-triggered APC activation is blocked by the

addition of Emi1 protein, Cdc20-neutralizing antibodies4 or CaMKII inhibitors26. b, Model

of how Emi1 and the Mos/MAPK pathway act to prevent release from metaphase of MII by

stabilizing cyclin B/Cdc2 activity. Mos promotes cyclin B/Cdc2 activity by negatively

regulating the Myt1 kinase and by increasing cyclin B synthesis during the MI–MII

transition27,29. Emi1 inhibits APC-dependent cyclin B destruction in MII, thereby stabilizing

cyclin B/Cdc2 activity.

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(15 min, 4 8C) with the indicated reagent. Extracts (with or without calcium addition attime 0) were warmed to 23 8C, and equal aliquots were taken at the indicated times.

For experiments using the addition of constitutively active CaMKII, CSF extracts werepreincubated (15 min, 4 8C) with buffer or MBP–Emi1 (300 nM). Constitutively activeCaMKII translated in vitro was added (1:14) at time 0, extracts were warmed to 23 8Cwithout the addition of calcium, and equal aliquots were taken at the indicated times.

For oocyte maturation experiments, stage VI oocytes were treated with 5 mg ml21

progesterone and equal aliquots were collected and lysed over 10 h as described31. Thecyclin-B-associated H1 kinase assay was as described30.

Depletion experimentsAffinity-purified anti-Emi1 rabbit antibodies (5 mM) or anti-Emi1 mouse polyclonalserum (0.2 ml serum per ml extract) were bound to magnetic protein A–dynabeads orprotein G–dynabeads, respectively (Dynal). Beads were washed (20 mM HEPES, pH 7.7,100 mM KCl) and incubated with CSF extracts (1 h, 4 8C), and samples were cleared ofbeads with a magnet. The process was repeated twice more (30 min, 4 8C) and the triple-depleted extracts were warmed to 23 8C for analysis. Mock-depletions were performed inexactly the same manner, except that purified rabbit IgG (5 mM) or mouse preimmuneserum (0.2 ml serum per ml extract) was used instead. For DNA morphology analysis,sperm DNA was added (1,000 ml21) and DNA was analysed by staining with Hoechst33258 (ref. 6). MBP–Emi1-CT domain (1 mM) was added for rescue experiments. Weperformed these Emi1 depletion experiments 10 different times with anti-Emi1-depletingantibodies (1) from several different rabbits; (2) against full-length Emi1 and an amino-terminal Emi1 fragment; (3) with affinity-purified versus crude sera; and (4) with anti-Emi1 sera from several different mice.

Immunoprecipitation assaysCalcium (400 mM) was added at time 0 to CSF extracts or CSF extracts preincubated(15 min, 4 8C) with 35S-labelled Cdc20 translated in vitro. Extracts were warmed to 23 8Cand samples were taken at the indicated times after calcium addition; they were then flash-frozen. Samples were diluted 1:10 in RIPB buffer6, precleared with protein G–Sepharose,incubated with anti-Emi1 serum (1 h, 4 8C), bound to protein G–Sepharose (45 min, 4 8C)and washed four times in NETN buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5%Nonidet P40, 1 mM dithiothreitol, 1 mM EDTA, 1% aprotinin); bound Cdc20 wasresolved by SDS–polyacrylamide gel electrophoresis and immunoblotting orautoradiography.

Received 10 October 2001; accepted 21 February 2002.

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Supplementary Information accompanies the paper on Nature’s website

(http://www.nature.com).

AcknowledgementsWe thank T. Hunt for Xcyclin B antibodies; E. Kramer and J. Peters for XCdc20 antibodiesand construct; H. Schulman for CaMKIIa cDNA; H. Yu for D-box peptide; Ferrelllaboratory members for technical assistance; and J. Peters and J. Ferrell for comments onthe manuscript.

Competing interests statement

The authors declare that they have no competing financial interests.

Correspondence and requests for materials should be addressed to P.J.

(e-mail: pjackson@stanford.edu).

..............................................................

Dominant role of the niche inmelanocyte stem-cell fatedeterminationEmi K. Nishimura*†‡, Siobhan A. Jordan§, Hideo Oshima‡k,Hisahiro Yoshida*, Masatake Osawa{, Mariko Moriyama{,Ian J. Jackson§, Yann Barrandonk, Yoshiki Miyachi†& Shin-Ichi Nishikawa*{

* Department of Molecular Genetics and † Department of Dermatology,Graduate School of Medicine, Kyoto University, Shogoin Kawaharacho 53,Sakyo-ku, Kyoto 606-8507, Japan§ MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU,UKk Ecole Nomale Superieure, 46 rue d’Ulm, Paris 75230, Cedex 05, France{Riken Center for Developmental Biology, Minatojima-minamicho 2-2-3,Chuo-ku, Kobe, 650-0047, Japan.............................................................................................................................................................................

Stem cells—which have the capacity to self-renew and generatedifferentiated progeny—are thought to be maintained in aspecific environment known as a niche1–3. The localization ofthe niche, however, remains largely obscure for most stem-cellsystems. Melanocytes (pigment cells) in hair follicles proliferateand differentiate closely coupled to the hair regeneration cycle4.Here we report that stem cells of the melanocyte lineage can beidentified, using Dct-lacZ transgenic mice5,6, in the lower perma-nent portion of mouse hair follicles throughout the hair cycle. Itis only the population in this region that fulfils the criteria forstem cells, being immature, slow cycling, self-maintaining and

‡ Present addresses: Division of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical

School, Dana 630A, 44 Binney Street, Boston, Massachusetts 02115, USA (E.K.N.); Department of Plastic

& Reconstructive Surgery, St Marianna University School of Medicine, 2-16-1 Sugao Miyamae-ku,

Kawasaki, 216-8511, Japan (H.O.).

letters to nature

NATURE | VOL 416 | 25 APRIL2002 | www.nature.com854 © 2002 Macmillan Magazines Ltd