nestin ironetin p75 s1 CNS SKPs Dermo-1 +ve SKPs CNS H2O SCGs Skin Di. SKPs TH SHOX2 GAPDH NCAM D H Figure S1, Immunoytohemil nlysis o SKP spheres ulture rom neontl mouse (nestin, ironetin, S-1) or rt (p75ntr) k skin, n pssge two or three times (top pnels) s ompre to neurospheres ulture rom the emryoni telenephlon n pssge similr numer o times (ottom pnels). In ll pnels, the immunostine ells re re, n the lue is rom Hoehst stining o the nulei to show ll ells. Note tht SKPs express ironetin n S-1 ut not p75ntr, wheres neurospheres o not express ironetin or S-1, ut o express p75ntr., RT-PCR or Dermo-1 n SHOX2, trnsription tors involve in erml n rnioil evelopment, in RNA rom SKPs n CNS neurospheres s esrie in Fig. 1. The positive ontrol (+ve) ws RNA rom E16 orelim., Western lot nlysis or NCAM n opmine-β-hyroxylse in murine SKP spheres versus SKPs ierentite or 14 ys in 10% serum supplemente with neurotrophins. The positive ontrol ws protein isolte rom ulture peripherl symptheti neurons rom the superior ervil gnglion (SCGs)., Immunostining o ierentite SKPs shows tht suset o ierentite ells express tyrosine hyroxylse (TH). WWW.NATURE.COM/NATURECELLBIOLOGY 1

200 000 /ml e nestin 100 000 /ml 25 000 /ml III tuulin g nestin / trp1 -kit h SKP Spheres Disso. ells Figure S2, Immunoytohemil nlysis or βiii-tuulin in primry E18 (mile pnel) or ult (right pnel) murine skin ells versus E18 murine CNS telenephli ells (let pnel). All ells were plte in FGF2 n 10% serum or 7 ys n ierentite in meium ontining 5% serum supplemente with neurotrophins., Phse ontrst mirogrphs o primry murine SKP spheres grown or 1 week t vrying onentrtions o strting ells (/ml)., Quntittion o the numer o ells tht give rise to primry SKP spheres in murine k skin isolte t vrious evelopmentl ges rnging rom emryoni y 13 to ulthoo. Cell numers were normlize to the totl numer o ells present in k skin isoltion., Immunoytohemil nlysis or nestin in primry murine SKP lone plte onto poly--lysine/lminin. e, Immunoytohemil nlysis or βiiituulin in primry murine SKPs lone grown in methylellulose n then ierentite., Primry SKP spheres generte y mixing limiting numers o YFP-positive ult virissl ells n E18 primry skin ells n ulturing in the presene o FGF2 n EGF or one week. The let pnel is phse s1/gfap s1/mbp s1/cnpse s1/-kit s1/trp1 s1/dct mirogrph n the right pnel luoresene mirogrph o the sme iel. The rrowhe inites YFP-positive sphere. g, Immunoytohemil nlysis o primry SKP spheres or mrkers o melnolsts n hemtopoieti stem ells. The two let pnels were immunostine or nestin (re, rrows) n or trp1 (green, rrowhes), with the top pnel eing totl issoite skin ells, n the ottom primry SKP sphere. The two right pnels were immunostine or -kit (green), with the top pnel eing ytospin o one mrrow spirte (positive ells re mrke with rrowhes), n the ottom pnel primry SKPs sphere. In ll pnels, the lue erives rom the Hoehst stining o ell nulei. h, Fluoresene mirogrphs o primry issoite orsl skin ells oule-lelle or the preursor ell mrker S-1 (re) n or the Shwnn ell mrkers GFAP, myelin si protein (MBP), n CNPse, or the melnolst/melnoyte mrkers -kit, trp1 n t (ll in green). The lue is Hoehst stining o the nulei. 2 WWW.NATURE.COM/NATURECELLBIOLOGY

s1 e DCT Kertin e III tuulin/cnpse g h III tuulin CEE + FGF Nestin III tuulin/nestin Serum + FGF III tuulin Figure S3, Photomirogrphs o virissl ppill issetion rom n ult pigmente mouse. The let piture shows the intt viriss, while the other pitures show the issetion. The rrowhe inites the ppill.,, Immunoytohemil nlysis o issete whisker ppille or S-1 n t.,e, Fluoresene photomirogrphs o issoite ppill ells tht were immunostine or kertin 18 () or βiii-tuulin n CNPse (e)., Immunoytohemil nlysis or nestin n βiii-tuulin in ult virissl ppill ells iretly ierentite or 7 ys in FGF2, 10% serum n neurotrophins. g, Phse photomirogrphs o issoite ppill ells tht were ulture or one week in the presene o FGF2 plus serum or hik emryo extrt (CEE). Note tht in the presene o CEE, loting spheres o ells were generte. h, Fluoresene photomirogrph o whisker ppill sphere tht ws ierentite on n herent sustrtum or 3 ys n then immunostine or βiii-tuulin. In pnels -e, n h, nulei were stine lue with Hoehst. WWW.NATURE.COM/NATURECELLBIOLOGY 3

P5 Pigmente Mouse E18 whisker p skin Nexin E18 k skin ells Phse Brightiel o X-gl stining E18 k skin Figure S4, Overlpping expression o β-gltosise n the erml ppill mrker, nexin, in postntl y 5 (P5) Wnt1Cre;Ros26R orsl skin rom pigmente mie. Pigmente melnin grnules re eposite in the eveloping hir sht y neurl rest-erive melnolsts resient n intersperse in the mtrix. A supopultion o ollile ppill ells express mrnas or oth β-gltosise (rrowhe) n nexin (rrowhe). Beyon the outer root sheth, osionl β-gltosise-positive ells n lso e seen in the erml sheth (lower rrow), struture losely ssoite with ollile ppille. Single β-gltosise-positive ells re seen emerging rom the upper portions o the ollile into the interollile epiermis n re likely melnolsts (upper rrow)., Expression o the β-gltosise trnsgene in E18 whisker p versus E18 k skin rom Wnt1Cre;Ros26R emryos, s etete y X-gl stining o ryostt setions. Similr results were otine t vriety o evelopmentl ges using oth X-gl stining n in situ hyriiztion or the β-gltosise trnsgene. Note tht very ew trnsgene-positive ells were etete in orsl skin, in spite o the t tht there re unnt melnolsts n Shwnn ells t this point, s emonstrte or neontl skin in Supplementry Fig. 2h., Photomirogrphs o issoite k skin ells rom neontl Wnt1Cre;R26R mie tht were stine with X-gl one y ter plting. The upper pnel is phse mirogrph while the lower is rightiel mirogrph o the sme iel showing tht neurl rest-erive pigmente melnoytes (oxe in re) were β-gltosise negtive. 4 WWW.NATURE.COM/NATURECELLBIOLOGY

Tle S1 Primer Sequenes Gene Primer Sequene Annel Temp ( C) Prout (BP) Px3 Ggggggttggggg 59 374 Cggtggtggttg Slug Cgtgggtttt 60 348 Tttgggggtt Twist Ctttgttttt 57 334 Gtgggtgttgtttt Snil Cggggtgtttt 61.5 398 Ggtggtggttttt Sox9 Cgtgtgt 59.5 544 Gttgtgttggtg P75 Gtgggggtgggtggt 62 422 Cggg SHOX2 Cgggg 63 355 Tgtt DBH Aggggggttt 59 353 Cggggggggg Peripherin Gggt 61.5 333 Gtggttttt MBP Tggggggtt 61.1 332 Ggtgt P0 Ctggtgtgtttttt 60.1 452 Cgttgt Dermo-1 Ggggtggt 61.4 356 Ctggggggg Nexin Cgggg 57.4 289 Gggtgttt Versin Tggggggttt 56.3 427 Ttgggtt Wnt-5 Ctgtggg 60.1 552 Cgggt GAPDH Gttttgggg 56 281 Gtgtggtggtgtggt WWW.NATURE.COM/NATURECELLBIOLOGY 5