Supplementary Figure 1.

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1 Supplementary Figure 1. a 0.772% % CD4 2.93% Lineage CD44 CD8 CD25 c-kit b WT Ly5.1 Bone marrow 1:1 Ltbr +/- or Ltbr -/- Ly5.2 10Gy WT % of CD45.2+ ETP among total ETPs Ltbr +/- n.s. Ltbr -/- c 10Gy WT Ly5.1 Bone marrow WT Ly5.2 10Gy Ltbr -/- Ly5.2 %ETPs WT ** Ltbr -/- #ETPs ** WT Ltbr -/- d isotype anti-ltβr MFI=5721 MFI=361 LTβR Supplementary Figure 1. Radioresistant cell derived LTβR is required for thymic ETP population. (a) Gating strategy for the detection of thymic ETPs by flow cytometry. FACS plots show the successive gating strategy after standard forward/side scatter and live/dead 1

2 gating. A strict forward angle gate was used to exclude doublets. (b) Mixed bone marrow chimeric mice were generated as depicted. 8 weeks later, flow cytometric analysis shows comparable percentages of ETPs derived from Ltbr +/- and Ltbr -/- bone marrow cells. Results are mean ± SEM (n=6 or 7). (c) Bone marrow chimeric mice were generated as depicted. 8 weeks later, flow cytometric analysis shows dramatically reduced frequency and number of ETP population in Ltbr -/- hosts. Results are mean ± SEM (n=3). (d) Thymic tissues were digested as described in Methods. Cells were stained for flow cytometric analysis. The expression of LTβR on total thymic ECs is shown. Representative of two experiments. 2

3 Supplementary Figure 2. a Ltbr -/- Ltbr +/ % % CD45.2 CD45.1 b Ltbr fl/fl Ltbr fl/fl Tek Cre % % CD45.2 CD45.1 Supplementary Figure 2. Endothelial LTβR controls thymic progenitor homing. (a, b) Short-term thymic homing assay in Ltbr -/- (a), Ltbr fl/fl Tek Cre (b) and littermate control mice. Representative FACS plots are shown. The number indicates the percentage of gated dornor-derived Lin - cells among total thymocytes. 3

4 Supplementary Figure 3. a Blood % Donor Lin - cells n.s Lt br fl/fl Ltbr fl /f l Tek Cre # Donor Lin - cells per 1ml blood fl /fl Ltbr n.s. Ltbr fl/fl Tek Cre b 0.20 n.s n.s. Spleen % Donor Lin - cells # Donor Lin - cells c 0.00 Ltbr fl/fl Ltbr fl/fl Tek Cre 0 Ltbr fl/fl Ltbr fl/fl Tek Cre 0.20 n.s n.s. Bone marrow % Donor Lin - cells # Donor Lin - cells Ltbr fl/fl Ltbr fl/fl Tek Cre 0 Ltbr fl/fl Ltbr fl/fl Tek Cre Supplementary Figure 3. Endothelial L TβR d eficiency d oes not influence HPC distribution in the blood, spleen and bone marrow. (a, b, c ) Mice of indicated genotypes were injected intravenously with congenically marked bone marrow cells ( cells/mouse). Forty-eight hours later, the blood, spleen and bone marrow tissues were collected and cells were stained for flow 4

5 cytometric analysis. The percentage and number of donor-derived Lin - cells in each organ were calculated. (a) Blood; (b) Spleen; (c) Bone marrow. Graphs display the statistical analysis of the frequency (left) and number (right) of ETPs in total spleen, one tibia and femur bone marrow, and 1ml of blood. Results are mean ± SEM (n=3 or 5). Representative of two experiments. 5

6 Supplementary Figure 4. a Lta +/+ Lta +/ % % CD44 c-kit b %ETPs n.s. #ETPs n.s Lta +/+ Lta +/- 0 Lta +/+ Lta +/- Supplementary Figure 4. Comparable thymic ETP population in Lta +/+ and Lta +/- mice. (a, b) Flow cytometric analysis of ETPs (Lin - CD25 - CD44 + c-kit + ) in Lta +/+ and Lta -/- mice. (a) Representative dot plots are shown. (b) The graphs display the statistical analysis of the frequency and number of ETPs among total thymocytes. Results are mean ± SEM (n=7 or 8). 6

7 Supplementary Figure 5. a b WT > WT CD11c-DTR > WT *** 3.29% 0.392% #DCs CD11c MHC-II WT-WT CD11c-DTR-WT c d WT > WT CD11c -DTR > WT % % %ETPs n.s. #ETPs n.s. CD44 c-kit WT-WT CD11c-DTR-WT 0 WT-WT CD11c-DTR-WT e CD44 WT > WT μmt > WT % % c-kit f %ETPs WT-WT n.s. μmt-wt #ETPs WT-WT n.s. μmt-wt Supplementary Figure 5. DCs and B cells do not control thymic ETP population. (a, b ) Bone marrow chimeric mice were established in WT mice with WT or CD11c-DTR bone marrow cells. 6 weeks later, mice were treated with diphtheria toxin for 4 weeks. Thymic tissues were digested and cells were stained for flow cytometric analysis. MHC-II + CD11c hi DCs were analyzed. Representative dot plots are shown (a). Graphs display the statistical analysis of the number of DCs in total thymus (b). Results are mean ± SEM (n= 4). (c, d ) Bone marrow chimeric mice were established in WT mice with WT or 7

8 CD11c-DTR bone marrow cells. 6 weeks later, mice were treated with diphtheria toxin for 4 weeks before thymic ETP analysis. Representative dot plots are shown (c). Graphs display the statistical analysis of the frequency and number of ETPs among total thymocytes (d). Results are mean ± SEM (n=4). (e, f ) Bone marrow chimeric mice were established in WT mice with WT or μmt bone marrow cells. 8 weeks later, flow cytometric analysis of ETPs was performed. Representative dot plots are shown (e). Graphs display the statistical analysis of the frequency and number of ETPs among total thymocytes (f). Results are mean ± SEM (n= 3 or 5). 8

9 Supplementary Figure 6. a P-selectin/GAPDH (a.u) WT n.s. Ltbr -/- VCAM-1/GAPDH(a.u) WT n.s. Ltbr -/- ICAM-1/GAPDH (a.u) WT n.s. Ltbr -/- b n.s. # Endothelial cells Ltbr +/- Ltbr -/- Supplementary Figure 6. Gr ossly normal endothelial development and function in LTβR deficient mice (a) Quantitative real-time PCR analysis of the expression level of P-selectin, VCAM-1 and ICAM-1 in thymic endothelial cells from WT and Ltbr -/- mice. Results are mean ± SEM (n=3). (b) Normal number of thymic endothelial cells in Ltbr -/- mice. Results are mean ± SEM (n=4). 9

10 Supplementary Figure 7. a 0.256% 8.83% 12.0% CD % P-selectin 72.8% CD45 EpCAM Ly6C b Ctrl anti-gp % 0.026% 59% 0.027% gp % 2.46% 38.7% 2.23% CD31 Supplementary Figure 7. Thymic EC characterization by flow cytometry. (a) Gating strategy for the distinction and isolation of Ly6C + Sep -, Ly6C + Selp +, and Ly6C - Selp + thymic EC subsets by flow cytometry. FACS plots show the successive gating strategy after standard forward/side scatter and live/dead gating. A strict forward angle gate was used to exclude doublets. (b) Thymic tissues were digested as described in Methods. Cells were stained for flow cytometric analysis. The expression of podoplanin (gp38) on CD45 - EpCAM - thymic stromal cells is shown. Representative of two experiments. 10

11 Supplementary Figure 8. a Glycam-1/b-actin(a.u) n.d. n.d Ly6C+Selp+ Ly6C-Selp+ Lyve-1/b-actin(a.u) ** ** Ly6C+Selp+ Ly6C-Selp+ Pcdh7/b-actin(a.u) *** *** ** Ly6C+Selp+ Ly6C-Selp+ Ubd/b-actin(a.u) Ly6C+Selp- Ly6C+Selp- Ly6C+Selp- Ly6C+Selp- ** **** *** Ly6C+Selp+ Ly6C-Selp+ Bst1/b-actin(a.u) * ** Ly6C+Selp- Ly6C+Se lp+ Ly6C-Selp+ Supplementary Figure 8. Expression of selected genes in thymic EC subsets. (a) Quantitative real-time PCR analysis of the expression of selected HEC signature genes in different subsets of thymic ECs. Results are mean ± SEM of three independent experiments (n=3). ** C D 55/b-actin(a.u) ** **** ** Ly6C+Selp- Ly6C+Se + Ly6C-Selp+ VCAM-1/b-actin(a.u) ** *** * Ly6C+Selp- Ly6C+Selp+ Ly6C-Selp+ 11

12 Supplementary Figure 9. a b Ly6C Ly6C + - Selp + + Selp Correlation Ly6C Selp Ly6C - Selp + Ly6C + Selp + Ly6C + Selp - c d e TPEC HEC Supplementary Figur e 9. Ly6C - Selp + thy mic endothelia l ce lls is a specia lized population resembled LN HECs. (a) Unsupervised hierarchical clustering analysis based on the Pearson correlations of transcriptome among samples. Each terminal branch represents the result from a 12

13 single sequencing sample. (b) Hierarchical clustering of expression Z-score of sequencing samples (Ly6C + Selp -, Ly6C + Selp + and Ly6C - Selp + ) and published microarray samples (LN HECs and CAP ECs) with the list of genes significantly differentially expressed between Ly6C + Selp - and Ly6C - Selp + ECs as determined by Cuffdiff software. Z-score value was used to normalize the expression level of the same gene among sequencing samples and microarray samples, respectively. (c, d) Selected GO terms and pathways (right margin) showing significant enrichment (Benjamini < 0.05, analyzed by DAVID software) in the differential gene sets between Ly6C + Selp - and Ly6C - Selp + ECs (c), and between Ly6C + Selp + and Ly6C - Selp + ECs (d). Order and box color are based on log10 of the corrected P-value. (e) TPECs and HECs share significant number of signature genes. 13

14 Supplementary Figure 10. a P-selectin Ltbr fl/fl 13.8% 12.8% 68.6% Ly6C Ltbr fl/fl Tek Cre 4.18% 17.9% 73.8% b Endothelial cell subsets (%) Ly6C+Selp- * Ly6C+Selp+ ** Ly6C-Selp+ fl /fl Ltbr Ltbr fl /fl Tek Cre Supplementary Figur e 10. Endothelial L TβR s ignaling i s re quired f or t he development of Ly6C - Selp + thymic ECs. (a, b ) Flow cytometric analysis of thymic EC subsets in Ltbr fl/fl Tek Cre and their littermate control mice. ECs were gated on CD45 - EpCAM - CD31 + population. Representative dot plots are shown (a). The graph displays the statistical analysis of the frequency of each endothelial subset (b). Results are mean ± SEM (n=3). 14

15 Supplementary Figure 11. a % of Donor Lin - cells among total donor cells LN ** Thymus Supplementary Figur e 1 1. Pr eferential mi gration of pr ogenitor cells into the thymus than to the LNs compared to non-progenitor cells. (a) WT mice were injected intravenously with congenically marked bone marrow cells ( cells/mouse). Twenty-four hours later, the peripheral LNs and thymus were collected and cells were stained for flow cytometric analysis. The percentage of donor-derived Lin - cells among total donor-derived cells in each organ was calculated. Results are mean ± SEM (n=3). 15

16 Supplementary Figure 12. a Ltbr +/- Ltbr -/ % % CD44 c-kit b * 0.03 ** 3000 ** #thymocytes %ETPs #ETPs Ltbr +/- Ltbr -/ Ltbr +/- Ltbr -/- 0 Ltbr +/- Ltbr -/- Supplementary Figur e 12. L TβR contr ols thymic ETP population and total thymic cellularity at neonatal stage. (a, b ) Flow cytometric analysis of ETPs (Lin - CD25 - CD44 + c-kit + ) in Ltbr -/- and control mice at neonatal stage (postnatal day 1-2). (a) Representative dot plots are shown. (b) The graphs display the statistical analysis of the total thymoctes and the frequency and number of ETPs among total thymocytes. Results are mean ± SEM (n=3 or 4). 16

17 Supplementary Table 1: Comparision of thymic ETP defect in different conditions. Experimental condition Degree of thymic ETP integrity (%)* WT 100 Ltbr -/ ± 2.61 Ltbr fl/fl Tek Cre ± 1.60 Ltbr fl/fl K14 Cre ± 9.80 Lta -/ ± 8.21 Light -/ ± Lta -/- Light -/ ± 6.22 Tcra -/ ± 2.66 T cell-lta -/ ± 7.93 T cell-lta -/- Light -/ ± 5.94 DC deficient mice ± 5.22 B cell deficeint mice ± *, The integrity of thymic ETP population in WT or control mice is set as 100%; the percentage of thymic ETP among total thymic cells in different experimental conditions are normalized to that in WT or control mice. n=3-9. 1, CD11c-DTR bone marrow chimeric mice were treated with DT for 4 weeks. 2, μmt bone marrow chimeric mice. 17

18 Supplementary Table 2: FPKM of genes differentially expressed between thymic EC subset I and TPECs. * Gene Symble EC I-rep1 EC I-rep2 EC II-rep1 EC II-rep2 TPEC-rep1 TPEC-rep G04Rik O10Rik D01Rik M07Rik M08Rik K01Rik A4galt AU AW Abca Abca Abca Abcb1a Abcc Abhd17c Ablim Acer Ackr Acss Acta Actn Adamts Adamts Adamts Adarb Adcy Adk Adora2a Adra2a Aebp Afap1l Agap Ahdc Akap Akr1c Alpl Amigo Angpt Angptl Angptl Ano Anpep

19 Anxa Apln Aplnr Apoe Apol10b Aqp Arc Arhgap Arhgap Arhgap Arhgdib Arhgef Arhgef Arhgef Arl Arrb Arrdc Asah Asap Atoh Atp1b Atp2a Avpi Avpr1a B4galt B4galt Bace Basp Bcl11b Bcl2l Bcl6b Bcr Bend Bgn Birc Bmp Bmp Bst Btbd Btg C030046E11Rik C1qtnf C1ra Cables Cadm Cadps

20 Calcrl Camk2d Camk Car Car Carhsp Casd Cav Cav Ccdc Ccdc85a Ccdc85b Ccdc88a Ccdc88c Ccr Ccser Cd Cd209d Cd Cd Cd Cd Cd Cd Cd Cd Cd Cd Cdc42ep Cdc42ep Cdc42ep Cdh Cdh Cdk Cdkn1a Cdkn1c Cdr2l Ceacam Celf Celsr Cep85l Cept Ces2e Ces2g Cfb Cfh

21 Chp Ckb Clca Cldn Cldn Clec14a Clic Clstn Cmah Cmklr Cmpk Cnksr Col13a Col18a Col4a Col5a Col6a Col8a Colgalt Coro2b Cotl Cox6b Cp Cpe Cpeb Cpq Creb3l Creb3l Csf2rb Csf Csgalnact Csrp Ctgf Ctla2a Ctnnal Ctnnbip Ctsd Ctsh Ctsl Cx3cl Cxcl Cxcl Cxcl Cxx1a Cyb Cygb

22 Cyth Cyyr D8Ertd82e Daam Dab2ip Dach Daglb Dcbld Dclk Dcun1d Ddah Ddah Dennd Des Dgkh Dhh Disp Disp Dlg Dll Dll Dock Dock Dok Dpysl Dram Dsg Dusp Dusp Dusp Dysf E2f Edn Ednra Ednrb Eef1b Eepd Efhd Efnb Efnb Efr3b Egflam Egln Ehd Ehd Eln

23 Emp Enah Endou Enpep Enpp Entpd Eogt Epha Epsti Esm Etl Etv Extl F2r F F830016B08Rik Fabp Fabp Fads Fam101b Fam117b Fam124a Fam134b Fam13c Fam174b Fam198b Fam57b Fam65b Fas Fat Fblim Fbln Fgl Fhod Filip Fjx Fkbp Flt Flt Fmnl Fn Fnbp1l Fndc Foxc Foxf Foxp

24 Frmd Fryl Fscn Furin Fut Fxyd Fyb Gaa Gabarapl Galnt Galnt Gas Gas Gata Gata Gbp Gcnt Gem Gfod Gfpt Gja Gja Glipr Glul Glycam Gm Gm Gm2a Gm Gm Gm Gna Golm Gpc Gpihbp Gpr Gpr Gpr Gpr Gprc5b Gramd Gucy1a Gucy1b H2-Aa H2-Ab H2-Eb

25 Hamp Hbb-bt Hdac Hdc Hecw Hes Hexa Hey Heyl Hic Hid Hif1a Hip1r Hist1h4a Hs3st Hs3st3b Hspa12b Hspa1a Hspa Hspb Hsph Id Id Ier Ier5l Iffo Igf Igfbp Igfbp Igfbp Igfbp Ikzf Il13ra Il1r Il27ra Il2rg Il6st Impdh Inf Insr Iqgap Iqsec Irak Irf2bpl Irf Irs

26 Isyna Itga Itga Itga Itga Itga Itga Itgal Itgb Itih Itpkb Itpr Jag Jam Jdp Jup Kank Kank Kank Kazn Kcna Kcnb Kcne Kcnj Kcnj Kctd12b Kdr Khnyn Kifc Klf Klf Klf Klhl Klhl Klhl Klhl Ksr Lama Lama Lama Lamb Laptm4b Lbp Lck Lcp Lef

27 Lepr Leprel Lgals3bp Lgmn Lhfp Lifr Limch Lims Lipe Lipg Lmo Lnx Lox Loxl Lpl Lrg Lrmp Lrrc16a Lrrc3b Ltbp Lurap1l Ly6a Ly6c Lyve Lzts Man1a Man2b Map3k Map3k Map3k Map4k Map4k Map Marcksl Marveld Mast Mb21d Mbp Mcc Mcf2l Me Megf Mepce Mertk Met Mgll

28 Mgp Mical Micall Mlec Mllt Mme Mmrn Mnd Mpeg Mpzl Mr Mras Mt Mtss1l Mxd Myadm Myh Myo Myo1b Myo1e Myof Myrip N4bp N4bp Naaa Naglu Nav Nav Nbl Nck Nckap5l Neat Nes Net Nfatc Nid Nmt Nod Nos Notch Notch Notch Nov Nova Npc Nr2f

29 Nr4a Nrp Ntn Nuak Nxpe Odc Osbpl P2ry Palld Palm Pam Parp Pcdh Pcdh Pcdh Pcdh Pcp4l Pde7b Pdgfb Pdgfrb Pdia Pdlim Peak Per Per Pgm Phldb Phlpp Pi Piezo Pik3c2b Pik3cb Pik3r Pik3r Pitpnc Pkig Pkm Plcb Plekha Plekha Plekhg Plin Plscr Pltp Plxna Plxna

30 Plxnb Podxl Ppap2a Ppap2b Pparg Ppp1r Ppp1r13l Ppp1r14a Ppt Prcp Prdm Prex Prickle Prkag Prkcg Procr Prom Prr Prrg Prrx Psen Psme Ptgfrn Ptgs Ptgs Ptk2b Ptk Ptn Ptp4a Ptpn Ptprc Ptprg Pxdn Rab11fip Rab3il Rab8b Rai Ralgapa Ralgds Rapgef Rapgef Rapgef Rapgef Rarg Rasal Rasal

31 Rasd Rasgef1b Rasgrf Rasgrp Rasgrp Rassf Rbp Rbp Rcan Rcsd Rdh Ret Rfk Rftn Rgcc Rgma Rgs Rgs Rgs Rgs Rgs Rhbdf Rin Rnd Rnf144a Rnf19b Rps Rps6ka Rrad Rsad Rtp Rusc S100a S1pr Satb Scn1b Sdc Sec14l Sele Selp Sema3g Sema4c Sema7a septin Serpina1e Serpina3f

32 Serpina3g Serpinb1a Serpinb6b Serpinb Serpine Serpinh Sertad Sesn Setbp Sez6l Sgk Sh2b Sh2b Sh2d3c Sh3bgrl Sh3rf Sh3tc Shb Shroom Sipa Sipa1l Sirpa Slc12a Slc16a Slc16a Slc16a Slc1a Slc20a Slc26a Slc28a Slc2a Slc30a Slc37a Slc39a Slc6a Slc7a Slc9a3r Slco2a Slco2b Slco3a Smap Smtn Snai Sncg Snx Socs

33 Sod Sod Soga Sorbs Sorbs Sox Sox Sox Sparc Spata Spats Spint Spns Spred Spry Spry Spsb Ssu St3gal St St6gal St6galnac Stab Stard Steap Steap Stk Stk32c Sulf Sulf Susd Sybu Syne Syne Syngr Syt Tagln Tanc Tank Tbc1d Tbl1xr Tbx Tcf Tes Tet Tfpi