The following fluorochrome-conjugated antibodies were used: FITC and Alexa Fluor 700

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1 Flow cytometry analysis and cell sorting The following fluorochrome-conjugated antibodies were used: FITC and Alexa Fluor 700 anti-cd5. (), APC-Cy7 anti-epcam (G.; Biolegend, San Diego, CA), PE-Cy7 anti-cd31 (390; Biolegend), Alexa Fluor 7 anti-ly51 (C3; Biolegend), biotinylated anti-cd0 (3/3), PE-Cy7 anti-cd05 (05yekta; ebioscience, San Diego, CA), APC- Cy7 anti-cd (GK1.5), PE-Cy5 anti-cda (53-.7), FITC anti-tcrβ (H57), PE anti- CD5 (95; Cedarlane), PE-Cy7 anti-cd117 (c-kit, B). Biotin-labeled antibodies used in the lineage cocktail included: anti-cdα (53-.7), anti-nk1.1 (PK13), anti- TCRβ (H57), anti-cd11c (HL3), anti-tcrγδ (GL-3) and mouse lineage panel [CD3ε, CD11b (Mac-1), CD5R/B0, LyC/LyG (Gr-1), TER-119/erythroid cells (Ly-7)]. Biotinylated antibodies were detected with either APC or PE-TexasRed conjugated streptavidin, depending on the combination of fluorochromes used. All antibodies were purchased from BD Pharmingen (San Diego, CA) unless indicated otherwise. Cells were analyzed on a three laser BD LSRII flow cytometer using DiVa software and sorted on a three laser FACSAria (BD Biosciences, Mountain View, CA). Immunohistology For immunofluorescence staining, whole embryos at stage E1.5 were harvested and fixed overnight in 1% paraformaldehyde/pbs. After washing in PBS, the embryos were immersed in 30% sucrose/pbs and embedded in OCT compound (Sakura Finetech) for cryosectioning. Sections (µm) were postfixed in % paraformaldehyde for minutes

2 and antigen retrieval was accomplished by incubating sections in 0 mm Tris buffer (ph 9.0) for min. Following a blocking step, sections (3- per sample) were stained with monoclonal rat anti-k (Troma-1; developed by D.R. Soll and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology, Iowa City, IA) and either rabbit anti-k5 (Covance, Berkeley, CA), rabbit anti-k1 (Covance), or rabbit antiβ5t (MBL International, Woburn, MA). Staining was revealed with secondary reagents Alexa Fluor 555-conjugated polyclonal goat anti-rat IgG and Alexa Fluor 7-conjugated polyclonal donkey anti-rabbit IgG (Molecular Probes). Sections were observed with Zeiss LSM500 confocal microscope and contiguous images were acquired at a x magnification in order to cover the whole thymic lobe. The segmented images were reconstituted with Adobe Photoshop software. Quantitative analysis was performed using MetaMorph software (version 7., Molecular Device) to calculate the density of cytokeratin and β5t staining per thymic lobe. For the analysis of medullary surface area, medulla was defined as an agglomeration of K5+ cells in the absence of reticular/stellar K+ cells and with a low nuclear density.

3 Figure S1. Doxycycline-inducible Wnt deletion. A) Experimental design, indicating the time frame for Wnt deletion and the analysis of the phenotype with respect to normal kinetics of thymic cellularity. B) Efficiency of Wnt deletion in ROSA eyfp reporter mice in which eyfp expression is induced by Cre-mediated deletion of an upstream stop-codon. The events are gated on CD5 - EpCAM + TECs from ROSA WT mice (shaded), TetO-Cre/ROSA eyfp/rtta mice that have not received doxycycline (black line), and TetO-Cre/ROSA eyfp/rtta mice that were given four intraperitoneal injections of doxycycline (red line). C) Efficiency of Wnt deletion as detected by qrt-pcr from thymic stroma. The dashed line represents the mean Wnt expression (normalized on EpCAM expression) from Cre - control mice, with the shaded area depicting the 95% confidence interval. The black bars represent Wnt expression from five individual TetO- Cre/ROSA rtta/+ /Wnt lox/lox mice, indicating a deletion efficiency of over 90%. Figure S. Lack of Wnt does not result in major block in thymocyte differentiation. Percentages (left) and absolute numbers (right) of immature single positive (ISP; CD + Lin - TCRβ - ), DP (CD + CD + ) and CD + and CD + single-positive thymocytes from A) E1.5 Wnt +/+ and Wnt -/- mice, B) TetO-Cre/ROSA rtta/+ /Wnt lox/lox mice that were treated with doxycycline at birth, and C) mice that were given doxycycline at 5- weeks of age. All histograms represent the mean ± SEM (n=-7 per group). P < 0.05, P < 0.01, P <

4 Figure S3. Wnt haploinsufficiency does not accelerate the rate of thymic atrophy. Analysis comparing thymopoiesis in Wnt +/+ and Wnt +/- mice at E1.5, 1 month, 3 months and 1 months of age. A) Thymic cellularity. B) Number of CD5 - EpCAM + TECs/thymus. C) Ratio of Ly51 - mtecs over Ly51 + ctecs. D) Ratio of thymocytes over TECs. E) Relative thymic cellularity expressed as a percentage of that of 1-monthold mice. All histograms represent the mean ± SEM (n=- per group). P < Figure S. Wnt does not affect TEC proliferation, apoptosis, or early differentiation. A) E15.5 TECs (CD5 - EpCAM + ) from Wnt +/+ and Wnt -/- mice were sorted and kept in TRIzol reagent. RNA was extracted and qpcr was performed to assess the relative expression of TEC-associated genes in Wnt +/+ and Wnt -/- mice. B) E15.5 thymi were harvested and kept at 37 C for an overnight in vitro-labeling with BrdU. BrdU uptake of TECs was assessed by flow cytometry using a BrdU Flow Kit. C) TUNEL staining was performed to detect apoptosis of E15.5 TECs by flow cytometry. D) Flow cytometry analysis of early differentiation of cortical TECs. Representative dot plots (gated on CD05 - CD0 + mtecs, CD05 + CD0 lo and CD05 + CD0 + ctecs) are shown for E15.5 Wnt +/+ and Wnt -/- thymi. Histogram represents the percentages of each subtype over total EpCAM + cells (mean ± SEM; n=3 per genotype).

5 A Wnt deleted during post-natal expansion Wnt deleted in young adult - steady-state Thymic cellularity Dox p.o. 3 wk Analysis at -7 wk Thymic cellularity Dox i.p. d Analysis at 3.5 mo B RCN YFP Mouse age (mo) ROSA wt/wt ROSA rtta/eyfp -Dox ROSA rtta/eyfp +Dox C Relative Wnt expression 1 0,1 0,01 0,001 Mouse age (mo) 95% CI F fl/fl M fl/fl F fl/- F fl/- F fl/fl Mean Cre- Figure S1

6 A Percentage thymocytes Wnt +/+ Wnt -/- 5 3 Cells (x ) / thymus 0, 0, 0, 0, B Percentage thymocytes C 90 0 ISP DP CD+ ISP DP CD+ ISP DP CD+ Cre- Cre+ Cells (x ) ISP DP CD+ Percentage thymocytes 90 0 Cre- Cre+ Cells (x ) 0 p=0.03 ISP DP CD+ ISP DP CD+ Figure S

7 A Cells (x ) / thymus Wnt +/+ Wnt +/- B EpCAM+ cells (x 3 ) C Ratio (mtec/ctec) E1.5 1 mo 3 mo 1 mo D Ratio [T cell/tec (x 3 )] 0, 0, E1.5 1 mo 3 mo 1 mo E Relative thymic cellularity (% max) 1 mo 3 mo 1 mo Wnt +/+ Wnt +/- E1.5 1 mo 3 mo 1 mo age (mo) Figure S3

8 A B D % on EpCAM + c ells CD0 % of BrdU+ TECs Relative fold change (RQ ) KO / WT C Ctnb1 Cdh1 Cdh FoxN1 C % of TUNEL+ TECs Il7 Dll SCF (Kitl) Axin Myc CyclinD1.5 Wnt +/+ Wnt -/ Wnt +/+ Wnt -/- Wnt +/+ Wnt -/- 0 CD05 + CD05 + CD05 - CD0 - CD0 + CD0 - Figure S