Supplemental gure 1

Lin-Sca-1-

Marker Thymus Bone Skin

41% ±1 64% ±8 92% ±4 CD29 % of max % of max % of max

50% ±10 58% ±9 87% ±4 CD51 % of max % of max % of max

3% ±1 47% ±3 72% ±7 CD140a % of max % of max % of max

39% ±6 51% ±3 71% ±3 CD140b % of max % of max % of max

4% ±2 1% ±1 7% ±1 CD34 % of max % of max % of max

21% ±16 30% ±10 42% ±10 gp38 % of max % of max % of max

50% ±4 43% ±7 55% ±5 Ly51 % of max % of max % of max

49% ±3 8% ±1 73% ±6 CD90.2 % of max % of max % of max

36% ±10 35% ±10 77% ±5 CD105 % of max % of max % of max

2% ±2 47% ±8 78% ±6 CD73 % of max % of max % of max

9% ±8 8% ±3 38% ±2 CD44 % of max % of max % of max

48% ±3 27% ±1 52% ±4 CD146 % of max % of max % of max

17% ±13 11% ±8 28% ±7 Nestin % of max % of max % of max

6% ±5 23% ±29 37% ±25 CXCL12 % of max % of max % of max

SUPPLEMENTAL FIGURE 1. Flow cytometry analysis of Lin- Sca-1- cells from the thymus, bone and skin. Overlay histograms illustrate staining with the relevant antibody (in blue) and an isotype control (in red). Each overlay histogram is representative of three independent experiments (3-5 mice per biological replicate). Numbers represent the mean percentage of positive cells (+/- SD). Supplemental gure 2

A B 1000 250k M 100 tMC 98.2% K P bMC 200k R 10 sMC 1 A

- 150k 1 9 1 a 4 8 1 0 5 A 2 5 0 3 3 5 9 0 C S CD CD CD gp Ly CD CD1 FSC 100k CD14 CD140b 1000 tMC 50k bMC

M 100 sMC K

0 P Thymocytes R 10 -103 0 103 104 105 cTEC 1 mTEC SCA-1 t 5 e 1 m 1 i 4 3 3 n -k ca x c CD CD CD p o E F C tMC vs bMC tMC vs sMC bMC vs sMC 7 7 ) ) ) 6 695 6 6 365

5 426 sMC sMC bMC 5 5 4 4 4 3 3 3 2 2 2 (ReadCount (ReadCount 362 10 10 1 849 1 1 573 log log 0 log10(ReadCount 0 r = 0.476 0 r = 0.629 r = 0.607

0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 log (ReadCount tMC) log10(ReadCount bMC) log10(ReadCount tMC) 10

SUPPLEMENTAL FIGURE 2. Purity of cell populations used for RNA-Seq analyses. (A) Post-sort analysis of sorted Sca-1+ thymic MCs. (B) Upper panel, histogram depicting the expression levels (RPKM) of 10 typical MC markers in the Sca-1+ MCs from the thymus, bone and skin. Lower panel, histogram showing expression levels of expressed in thymocytes or TECs. (RPKM = 15; red line) (C) Scatterplots depicting the number of DEGs in each one-to-one comparison of MC populations. Numbers in bold represent the amount of DEGs associated to the respective MC population in each comparison. Pearson correlation value (r) is also depicted in the bottom right corner of each scatterplot. Supplemental figure 3

C1 - Thymus C2 - Bone Z-score Z-score 0 1 2 3 4 5 0 2 4 6 8 quantity of cells (113) quantity of blood cells (82) B migration of cells (145) quantity of leukocytes (77) leukocyte migration (125) cell movement (153) cell movement of lymphocytes (25) cell movement of leukocytes (107) cell movement of cell movement of myeloid cells (77) mononuclear leukocytes (28) Lymphocyte migration (23) cell movement of phagocytes (81) leukocyte migration (48) A cell movement of neutrophils (56) A cell movement of leukocytes (44) recruitment of phagocytes (42) migration of phagocytes (13) chemotaxis of leukocytes (53) migration of antigen presenting cells (10) chemotaxis of phagocytes (45) cell movement of antigen (21) cell movement of presenting cells mononuclear leukocytes (51) migration of phagocytes (37) orientation of cells (8) C infiltration of leukocytes (51) 0 5 10 15 20 infiltration of cells (56) - log P-value ( ) cell movement of antigen (38) 10 presenting cells

inflammatory response (95) D C3 - Skin proliferation of cells (183) E Z-score 0 1 2 3 4 angiogenesis (52) vascularization (25) B recruitment of myeloid cells (11) recruitment of cells (14) A internalization of cells (20) F recruitment of phagocytes (11) activation of myeloid cells (31) 2.4 2.6 2.8 3.0 3.2 3.4 C activation of neutrophils (12) - log10 P-value ( ) 0 10 20 30 40 50 ( ) - log10 P-value C4 - Thymus + Bone C6 - Bone + Skin

Z-score Z-score 0 5 10 15 0 5 10 15

size of body (27) differentiation of connective (21) B tissue cells B differentiation of cells (58) proliferation of cells (75) C transcription of DNA (29) development of (34) activation of DNA (26) C cardiovascular system endogenous promoter vasculogenesis (27) A angiogenesis (30) fertility (13) E

0 2 4 6 cell viability (21) - log10 P-value ( ) F

vasculogenesis (22) D C5 - Thymus + Skin angiogenesis (25) Z-score cell movement (45) 0 2 4 6 cell movement of myeloid cells (22) cell movement of phagocytes (18) migration of cells (20) A cell movement of cell movement of granulocytes (13) myeloid cells (8) A migration of myeloid cells (9) cell movement (8) of phagocytes migration of granulocytes (7)

size of body (13) B development of body axis (33) G

quantity of cells (21) C proliferation of epithelial cells (17) H

differentiation of cells (30) D adhesion of immune cells (10) I

2.0 2.5 3.0 3.5 4.0 0 1 2 3 4

- log10 P-value ( ) - log10 P-value ( ) SUPPLEMENTAL FIGURE 3. Core biological functions associated to the 6 DEG clusters. All functions depicted in histograms were signifcantly enriched (p < 0.05) and activated (Z-score > 2). Related functions were clustered (letters). Numbers in parentheses represent the number of genes per biological functions. Red/black dots represent p-values. Supplemental table 1

# symbol Gene name # Gene symbol Gene name

1 1700048O20Rik RIKEN cDNA 1700048O20 gene 40 Ccl7 chemokine (C-C motif) ligand 7, MCP3

2 2610524H06Rik RIKEN cDNA 2610524H06 gene 41 Cd248 CD248 antigen, endosialin

3 4930452B06Rik RIKEN cDNA 4930452B06 gene 42 Cdk18 cyclin-dependent kinase 18

4 A4galt alpha 1,4-galactosyltransferase 43 Cdkn1c cyclin-dependent kinase inhibitor 1C; p57Kip2

5 Acpl2 acid -like 2 44 Cdkn2b cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)

6 Adam12 a disintegrin and metallopeptidase domain 12 (meltrin alpha) 45 Cdyl2 chromodomain , Y -like 2

7 Adam19 a disintegrin and metallopeptidase domain 19 (meltrin beta) 46 Cep170b centrosomal protein of 170 kDa protein B

8 Adam8 a disintegrin and metallopeptidase domain 8 47 Chst1 carbohydrate (keratan sulfate Gal-6) sulfotransferase 1

a disintegrin-like and metallopeptidase (reprolysin type) with 9 Adamts12 48 Chst2 carbohydrate sulfotransferase 2 thrombospondin type 1 motif, 12 a disintegrin-like and metallopeptidase (reprolysin type) with 10 Adamts2 49 Cib2 calcium and integrin binding family member 2 thrombospondin type 1 motif, 2

11 Adcy3 adenylate cyclase 3 50 Cldn1 claudin 1

aminoglycoside phosphotransferase domain-containing 12 Agphd1 51 Col1a1 collagen, type I, alpha 1 protein 1

13 Ahr aryl hydrocarbon receptor 52 Col1a2 collagen, type I, alpha 2

14 Akap2 a-kinase anchor protein 2 53 Col3a1 collagen, type III, alpha 1

15 Akap6 akap6 protein 54 Col4a6 collagen, type IV, alpha 6

, tissue-nonspecific ; alkaline 16 Alpl 55 Col6a2 collagen, type VI, alpha 2 phosphatase, liver/bone/kidney

17 Angel1 protein angel homolog 1 56 Coro2a coronin-2A

18 Angptl4 angiopoietin-like 4 57 Crabp1 cellular retinoic acid binding protein I

19 Aoc3 amine oxidase, copper containing 3 58 Creb3l1 cAMP responsive element binding protein 3-like 1

20 Arc activity-regulated cytoskeleton-associated protein 59 Crip2 cysteine rich protein 2, CRP-2

21 Arhgap28 rac/Cdc42 guanine nucleotide exchange factor (GEF) 6 60 Csgalnact1 chondroitin sulfate N-acetylgalactosaminyltransferase 1

22 Arhgef28 rho-guanine nucleotide exchange factor 61 Ctsh cathepsin H

23 Arhgef6 rac/Cdc42 guanine nucleotide exchange factor (GEF) 6 62 Ctso cathepsin O

24 Arrb1 beta-arrestin-1 63 Cxcl12 chemokine (C-X-C motif) ligand 12

25 Atp10a ATPase, class V, type 10A 64 Cyp26b1 cytochrome P450, family 26, subfamily b, polypeptide 1

UDP-GalNAc:betaGlcNAc beta 1,3-galactosaminyltransferase, 26 B3galnt1 65 Cyp2j6 cytochrome P450 2J6 polypeptide 1

27 B4galnt1 beta-1,4 N-acetylgalactosaminyltransferase 1 66 Dclk2 serine/threonine-protein kinase DCLK2

28 Bach2 transcription regulator protein BACH2 67 Depdc7 DEP domain containing 7

29 Bmper BMP-binding endothelial regulator protein 68 Dhrs7 dehydrogenase/reductase (SDR family) member 7

30 Btbd2 protein Btbd2 69 Dock3 dedicator of cyto-kinesis 3

31 Cables1 CDK5 and Abl substrate 1 70 Dpysl3 dihydropyrimidinase-related protein 3

32 Calcrl -like 71 Dtx3l deltex 3-like (Drosophila)

33 Capn6 calpain 6 72 Duoxa1 dual oxidase maturation factor 1

34 Car13 carbonic anhydrase 13 73 Dynap protein Dynap

35 Car5b carbonic anhydrase 5b, mitochondrial 74 E130311K13Rik RIKEN cDNA E130311K13 gene

36 Casp12 caspase 12 75 Ebf3 early B cell factor 3

37 Cbr2 carbonyl reductase 2 76 Eda ectodysplasin-A

38 Ccdc80 coiled-coil domain containing 80 77 Efna5 ephrin A5

39 Ccl5 chemokine (C-C motif) ligand 5, RANTES 78 Ehd3 EH-domain containing 3 # Gene symbol Gene name # Gene symbol Gene name

eukaryotic translation initiation factor 2C, 4, Protein 79 Eif2c4 120 Il18rap interleukin 18 receptor accessory protein argonaute-4

80 Eif4e3 eukaryotic translation initiation factor 4E member 3 121 Il1r1 interleukin 1 receptor, type I

81 Elfn1 leucine rich repeat and fibronectin type III, extracellular 1 122 Il1rl1 interleukin 1 receptor-like 1

82 Ell2 elongation factor RNA polymerase II 2 123 Il4ra interleukin 4 receptor, alpha

83 Emb embigin 124 Inf2 inverted formin-2

84 En1 protein engrailed-1 125 Inpp4b inositol polyphosphate-4-phosphatase, type II

85 Epn3 epsin 3 126 Itgb3bp centromere protein R

86 Eya4 eyes absent 4 homolog (Drosophila) 127 Itm2a integral membrane protein 2A

87 Fabp4 fatty acid-binding protein, adipocyte 128 Ivd isovaleryl coenzyme A dehydrogenase

88 Fabp7 fatty acid binding protein 7, brain 129 Jak3 tyrosine-protein kinase JAK3

89 Fam110b protein FAM110B 130 Jup junction plakoglobin

90 Fam129a protein Niban 131 Kbtbd7 kelch repeat and BTB (POZ) domain containing 7

91 Fam20a protein FAM20A 132 Kcna4 potassium voltage-gated channel subfamily A member 4

92 Fat4 FAT tumor suppressor homolog 4 (Drosophila) 133 Kirrel3 kin of IRRE like 3 (Drosophila)

93 Fbln1 fibulin 1 134 Kitl kit ligand

94 Fgf10 fibroblast growth factor 10 135 Klra6 killer cell lectin-like receptor, subfamily A, member 6

95 Fgfr2 fibroblast growth factor receptor 2 136 Lamtor4 ragulator complex protein LAMTOR4

96 Fut8 alpha-(1,6)-fucosyltransferase 137 Lgalsl galectin-related protein A

97 Fyb FYN binding protein 138 Lgmn legumain

98 Fzd1 -1 139 Lhx9 LIM homeobox protein 9

99 Fzd8 frizzled homolog 8 (Drosophila) 140 Lonrf3 LON peptidase N-terminal domain and RING finger protein 3

UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- 100 Galnt13 141 Loxl1 lysyl oxidase homolog 1 acetylgalactosaminyltransferase 13

101 Gas2 growth arrest specific 2 142 Lpar1 receptor 1

102 Gata3 GATA binding protein 3 143 Lpar4 lysophosphatidic acid receptor 4

103 Gatsl3 GATS protein-like 3 144 Lphn2 2

104 Glrx glutaredoxin 145 Lpl lipoprotein

105 Gnao1 guanine nucleotide binding protein, alpha O 146 Lrig3 leucine-rich repeats and immunoglobulin-like domains 3

106 Gper1 G protein-coupled estrogen receptor 1 147 Lrp11 low-density lipoprotein receptor-related protein 11

107 Gria3 , ionotropic, AMPA3 (alpha 3) 148 Lrrc4c leucine rich repeat containing 4C

108 Gstt3 glutathione S-transferase T3 149 Ltbp2 latent transforming growth factor beta binding protein 2

109 H2-DMa class II histocompatibility antigen, M alpha chain 150 Ly6a lymphocyte antigen 6 complex, locus A

110 Hcn2 hyperpolarization-activated, cyclic nucleotide-gated K+ 2 151 Ly6f lymphocyte antigen 6 complex, locus F

avian musculoaponeurotic fibrosarcoma (v-maf) AS42 oncogene 111 Hoxb13 homeobox protein Hox-B13 152 Maf homolog c-Maf

112 Hoxc8 homeo box C8 153 Maged2 melanoma antigen, family D, 2

113 Hspb8 heat shock protein 8 154 Map9 microtubule-associated protein 9

114 Htra3 htrA serine peptidase 3 155 Marcks myristoylated alanine rich protein kinase C substrate

115 Ifi27l1 interferon, alpha-inducible protein 27 like 1 156 Matn2 matrilin 2

116 Ifit1 Interferon-induced protein with tetratricopeptide repeats 1 157 Medag mesenteric estrogen-dependent adipogenesis protein

117 Igf1 insulin-like growth factor 1 (somatomedin C) 158 Mest mesoderm specific transcript

118 Il13ra1 interleukin 13 receptor, alpha 1 159 Mid2 midline 2 , Probable E3 ubiquitin-protein ligase

119 Il15 interleukin 15 160 Mmp15 matrix metallopeptidase 15 # Gene symbol Gene name # Gene symbol Gene name

161 Mmp2 matrix metallopeptidase 2 202 Pear1 platelet endothelial aggregation receptor 1

162 Mmp23 matrix metalloproteinase-23 203 Phospho1 phosphatase, orphan 1

163 Mn1 meningioma 1 204 Plagl1 pleiomorphic adenoma gene-like 1

pleckstrin homology domain-containing, family A (phosphoinositide 164 Mogat2 monoacylglycerol O-acyltransferase 2 205 Plekha2 binding specific) member 2 pleckstrin homology domain containing, family G (with RhoGef 165 Morc4 microrchidia 4 206 Plekhg4 domain) member 4

166 Mrgprf MAS-related GPR, member F 207 Plxna2 plexin-A2

167 Mtss1l metastasis suppressor 1-like 208 Plxnd1 plexin-D1

168 Myo1d myosin ID 209 Pmp22 peripheral protein 22

169 Nadkd1 NAD kinase domain-containing protein 1 210 Pmvk phosphomevalonate kinase

170 Ncam1 neural cell adhesion molecule 1 211 Podxl podocalyxin-like

171 Nck2 cytoplasmic protein NCK2 212 Popdc3 popeye domain containing 3

172 Nckap5 peripheral clock protein 2 213 Porcn probable protein-cysteine N-palmitoyltransferase porcupine

173 Ndrg1 protein NDRG1 214 Postn periostin, osteoblast specific factor

174 Ndrg2 N-myc downstream regulated gene 2 215 Ppap2a phosphatidic type 2A

175 Nes nestin 216 Ppap2b phosphatidic acid phosphatase type 2B

176 Nfatc1 nuclear factor of-activated T-cells, cytoplasmic 1 217 Ppp1r13b 1, regulatory (inhibitor) subunit 13B

177 Nid2 nidogen 2 218 Prkar1b protein kinase, cAMP dependent regulatory, type I beta

178 Notch4 notch 4 219 Prkd1 protein kinase D1

179 Nova1 RNA-binding protein Nova-1 220 Prrg1 proline rich Gla (G-carboxyglutamic acid) 1

180 Npr2 natriuretic peptide receptor 2 221 Prune2 prune homolog 2 (Drosophila)

181 Npr3 natriuretic peptide receptor 3 222 Ptn pleiotrophin

182 Nrbp2 nuclear receptor-binding protein 2 223 Ptprf protein tyrosine phosphatase, receptor type, F

183 Nudt14 uridine diphosphate glucose pyrophosphatase 224 Ptx3 pentraxin related gene

184 Nxf3 nuclear RNA export factor 3 225 Pxmp4 peroxisomal membrane protein 4

185 Nynrin NYN domain and retroviral integrase containing 226 Pygo1 pygopus homolog 1

186 Oasl1 2-5 oligoadenylate synthetase-like 1 227 Rab3il1 guanine nucleotide exchange factor for Rab-3A

187 Oasl2 2-5 oligoadenylate synthetase-like 2 228 Rac3 RAS-related C3 botulinum substrate 3

188 Onecut2 one cut domain, family member 2 229 Ralgapa1 ral GTPase-activating protein subunit alpha-1

189 Osmr oncostatin M receptor 230 Rassf2 ras association (RalGDS/AF-6) domain family member 2

190 Oxr1 oxidation resistance 1 231 Rb1cc1 RB1-inducible coiled-coil protein 1

191 Pappa pregnancy-associated plasma protein A 232 Rbpms2 RNA binding protein with multiple splicing 2

192 Pax9 paired box gene 9 233 Rgs4 regulator of G-protein signaling 4

193 Pbxip1 pre-B-cell leukemia transcription factor-interacting protein 1 234 Rhobtb1 rho-related BTB domain-containing protein 1

194 Pcdhb22 protocadherin beta 22 235 Rnf150 RING finger protein 150

195 Pde1a 1A, calmodulin-dependent 236 Ror2 receptor tyrosine kinase-like 2

196 Pde1b phosphodiesterase 1B, Ca2+-calmodulin dependent 237 Rorb RAR-related orphan receptor beta

197 Pde4d cAMP-specific 3',5'-cyclic phosphodiesterase 4D 238 Rps6ka6 ribosomal protein S6 kinase polypeptide 6

198 Pdgfra platelet derived growth factor receptor, alpha polypeptide 239 Rspo2 R-spondin 2

199 Pdgfrb platelet derived growth factor receptor, beta polypeptide 240 Rtkn2 rhotekin-2

200 Pdk4 pyruvate dehydrogenase kinase, isoenzyme 4 241 Rtn4rl1 receptor-like 1

201 Pdlim4 PDZ and LIM domain 4 242 Rtp4 receptor transporter protein 4 # Gene symbol Gene name # Gene symbol Gene name

TAF9B RNA polymerase II, TATA box binding protein (TBP)-associated 243 S100a16 S100 calcium binding protein A16 278 Taf9b factor

244 S1pr1 sphingosine-1-phosphate receptor 1 279 Tbx20 T-box 20, hrt (zebrafish)

245 Scn1b sodium channel subunit beta-1 280 Tcf7l1 transcription factor 7-like 1, Tcf3

246 Sdc3 syndecan 3 281 Tex15 testis protein TEX15

247 Selp selectin, platelet 282 Tfap2a transcription factor AP-2, alpha

sema domain, immunoglobulin domain (Ig), short basic 248 Sema3f 283 Tgfbi transforming growth factor, beta induced domain, secreted, (semaphorin) 3F

249 Sepp1 selenoprotein P, plasma, 1 284 Thsd7a thrombospondin, type I, domain containing 7A

250 Serpinb1a serine (or cysteine) peptidase inhibitor, clade B, member 1a 285 Tiaf2 TGF-beta1-induced anti-apoptotic factor 2

251 Serpinb6b protein Serpinb6b 286 Tmeff2 tomoregulin-2

252 Serpinf1 pigment epithelium-derived factor 287 Tmem117 transmembrane protein 117

253 Sfrp2 secreted frizzled-related protein 2 288 Tmem121 transmembrane protein 121

254 Six1 homeobox protein SIX1 289 Tmem47 transmembrane protein 47

solute carrier family 1 (glial high affinity glutamate 255 Slc1a3 290 Tnfrsf23 tumor necrosis factor receptor superfamily, member 23 transporter), member 3

256 Slc27a3 solute carrier family 27 (fatty acid transporter), member 3 291 Tram1l1 translocation associated membrane protein 1-like 1

257 Slc37a2 sugar phosphate exchanger 2 292 Trim21 E3 ubiquitin-protein ligase TRIM21

258 Slc39a8 solute carrier family 39 (metal ion transporter), member 8 293 Tsc22d3 TSC22 domain family, member 3

259 Slc43a1 solute carrier family 43, member 1 294 Tspan6 tetraspanin-6

260 Slc4a4 solute carrier family 4 (anion exchanger), member 4 295 Tspan7 tetraspanin 7

261 Slfn9 schlafen 9 296 Tvp23a Family with sequence similarity 18, member A

262 Smoc2 SPARC related modular calcium binding 2 297 Ubash3b ubiquitin associated and SH3 domain containing, B

263 Snai2 protein SNAI2, Slug 298 Ube2l6 ubiquitin-conjugating enzyme E2L 6

264 Sorcs2 sortilin-related VPS10 domain containing receptor 2 299 Ust uronyl-2-sulfotransferase

265 Sox5 SRY-box containing gene 5 300 Wdr60 WD repeat-containing protein 60

266 Sp8 trans-acting transcription factor 8 301 Wisp2 WNT1 inducible signaling pathway protein 2

267 Speg SPEG complex locus 302 Wnt10b wingless related MMTV integration site 10b

268 Spon2 spondin-2 303 Zc2hc1a zinc finger C2HC domain-containing protein 1A

269 Spp1 secreted phosphoprotein 1 304 Zdhhc15 zinc finger, DHHC domain containing 15

270 Srpx2 sushi-repeat-containing protein, X-linked 2 305 Zdhhc2 palmitoyltransferase ZDHHC2

271 St3gal1 ST3 beta-galactoside alpha-2,3-sialyltransferase 1 306 Zfp518a zinc finger protein 518A

272 St6gal1 ST3 beta-galactoside alpha-2,3-sialyltransferase 1 307 Zfp618 zinc finger protein 618

ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N- 273 St6galnac2 308 Zfp639 zinc finger protein 639 acetylgalactosaminide alpha-2,6-sialyltransferase 2

274 Stat1 signal transducer and activator of transcription 1 309 Zfp709 zinc finger protein 709

275 Sulf2 2

sushi, von Willebrand factor type A, EGF and pentraxin domain 276 Svep1 containing 1

277 Syn1 synapsin I

SUPPLEMENTAL TABLE 1 : List of the 309 genes predicted to be operative in sites of hematopoiesis and upregulated in HSC-supportive mesenchymal cell lines (Charbord et al. 2014). Shaded boxes represent genes associated to "secreted", "extracellular" or "glycoprotein" functional annotation of DAVID online bioinformatic tool.