Mice

Dr. Adam Glick (Penn State University) generously provided K5rtTA mice. TetO-Dkk1 K5rtTA and K14-Wnt7a mice have been described previously [37], [50], [51], as well as their use to inhibit Wnt signaling during epithelial development in other organs and tissues [36], [37], [50], [52]. C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). K5rtTA mice were bred to tetO-Dkk1 mice to generate tetO-Dkk1;K5rtTA double transgenic (DT) animals for study. Adult tetO-Dkk1;K5rtTA DT and littermate heterozygous control animals were fed mouse diet containing doxycycline (Dox) (2 g/kg, BioServe, NJ) for 4–8 weeks beginning at 6 weeks-of-age, unless otherwise indicated. For recovery experiments mice were fed Dox for 4 weeks and then food was changed to normal mouse chow for 2–4 weeks to allow recovery of thymic architecture. All mice were bred and maintained at the City College of New York animal facility and all experiments were performed with approval from the City College of New York institutional animal care and use committee.

Antibodies

The following primary antibodies were used for experiments: anti CD45-PE Cy7 (clone 30-F11, BD Biosciences), I-A/I-E-PE (clone M5/114.15.2, BD biosciences), I-A/I-E-FITC (clone M5/114.15.2, eBioscience), I-A/I-E-APC (clone M5/114.15.2, eBioscience), Ulex europaeus agglutinin-1 (UEA1)-Biotin (Vector), UEA1-PE (Vector), cytokeratin 5 (MK-5, Covance), cytokeratin 14 (MK-14, Covance), Troma I (Developmental Studies Hybridoma Bank, IA), Troma III (Developmental Studies Hybridoma Bank, IA), MTS10 (kindly provided by Dr. Richard Boyd from Monash University, Australia), DNp63 (clone N-16, Santa Cruz), Aire (clone M-300, Santa Cruz), EpCAM-PerCP (clone G8.8, Santa Cruz), Ki67-FITC (clone B56, BD Biosciences), Ki67 (clone SP6, LabVision), CD4-PE Cy7(clone RM4-5, BD Biosciences), CD8-PerCp Cy5.5 (clone 53–6.7, BD bioscience), CD25-PE (clone PC6I, BD Biosciences) and CD44-APC (clone IM7, BD Biosciences). The following secondary reagents were used for experiments: donkey anti rabbit IgG-TRITC, donkey anti rabbit IgG-Cy5, donkey anti rat IgG-TRITC, donkey anti goat IgG-FITC, goat anti rat IgM-TRITC (Jackson ImmnoResearch), donkey anti rabbit IgG-FITC (Santa Cruz), anti rat IgG2a-FITC, anti rat IgM-FITC, stretavidin-APC, stretavidin-APC Cy7 (BD Biosciences) and stretavidin-TRITC (Southern Biotechnology Associate).

Thymic Stromal Cell Preparation

Embryonic thymi were digested with collagenase D (1.2 mg/ml, Roche Diagnostics), DNase I (1.5 mg/ml, Roche Diagnostics), and Dispase (1.25 mg/ml, Invitrogen) at 37°C for 15 minutes with occasional gentle agitation with a glass pasture pipette. The resulting single cell suspension was washed with PBS and passed through 100 µm strainer (BD Biosciences) to remove any remaining undigested tissue. Adult thymi were cut into small pieces and majority of thymocytes were released by gentle agitation using a glass pasture pipette. The resulting tissue fragments were digested with collagenase D (1.2 mg/ml, Roche Diagnostics) and DNase I (1.5 mg/ml, Roche Diagnostics) for 15 minutes at 37°C followed by collagenase D (1.2 mg/ml, Roche Diagnostics), DNase I (1.5 µg/ml, Roche Diagnostics) and Dispase (1.25 mg/ml, Invitrogen) for 5 minutes at 37°C with occasional agitation using a glass pasture pipette. The single cell suspension was washed with PBS and passed through a 100 µm strainer (BD Biosciences).

DKK1 In Situ Hybridization and K5 K8 Immunofluorescence Staining to Demonstrate Transgene Expression

In situ hybridization to detect DKK1 expression was performed as previously described [53]. Briefly, TetO-Dkk1 double transgenic mice and ST littermate control animals were fed Dox for 5 days prior to tissue harvest to allow DKK1 transgene expression while minimizing DKK induced thymic architecture defects. 12 µm frozen sections were prepared, air dried and fixed in 4% paraformaldehyde. In situ hybridization was then performed using anti-sense and sense probe templates for Dkk1 synthesized by PCR of E14.5 mouse cDNA, using primers containing T7 RNA polymerase binding sites to amplify Dkk1: NM_010051. Following hybridization with DIG-labeled probes, bound probe was detected with anti-DIG Alkaline Phosphatase (AP) conjugate (Roche) followed by NBT/BCIP AP substrate (Roche). Following development of in situ signal, sections were counterstained with anti-K5 and anti-K8 antibodies followed by fluorochrome conjugated secondary reagents to allow co-localization of in situ signal with particular TEC subsets.

Flow Cytometry

Cells were suspended in 100 µl of FACS staining buffer (FSB-1% fetal bovine serum, 5 mM EDTA and 0.02% NaN₃ in PBS) with appropriately diluted primary antibodies for 20 minutes on ice in the dark. Secondary antibodies appropriately diluted in FSB were added cells were incubated for an additional 20 minutes on ice in the dark. After washing, cells were resuspended in 500 µl of FSB for data acquisition. Intracellular staining for Ki67 was performed using the FoxP3 staining kit (eBioscience) according to the manufacturer's instructions. TUNEL assays were performed using the In Situ Cell Death detection kit (Roche Diagnostics) according to the manufacturer's instructions. Live/dead discrimination was applied using ToPro3 (Invitrogen). Data acquisition was performed using an LSRII analyzer complete with three lasers (BD Biosciences) and cell sorting was performed using a FACS Aria (BD Biosciences). FACS data was analyzed using Flow Jo software (Tree Star).

Immunohistochemistry and Confocal Microscopy

Fresh tissues were embedded in OCT medium (Fisher); snap frozen and sectioned (8 µm) using a Leica CM1950 Cryostat. Sections were air dried on bond-rite slides and then fixed in 4% paraformaldehyde or 100% ice-cold acetone. Sections were washed with PBS and blocked with blocking buffer (1% BSA, 0.1%Triton-X, 5% normal serum in PBS) for 10 min. Sections were incubated with appropriately diluted primary antibodies in blocking buffer in a humidified chamber for 1 hour at 37°C followed by incubation with secondary reagents diluted in blocking buffer in humidified chamber for 30 minutes at 37°C, then mounted with ProLong gold anti-fade reagent with DAPI (Invitrogen). Isotype control staining was performed for all primary antibodies to ensure specificity of staining. Images were acquired using Zeiss LSM510 confocal microscope and analyzed using LSM software (Zeiss).

Quantification of Foxn1 Protein Expression In Vivo Using Confocal Microscopy

Frozen sections of thymic tissue were prepared and stained as described above. Confocal images were prepared by scanning each fluorochrome independently with a 40X lens with an optical slice thickness of 1.1 µm using a Zeiss LSM510 microscope. Cortical and medullary areas were defined by staining with anti-DEC205 and anti-K14, respectively, as well as TEC morphology in transitional zones. Thymic sections derived from control ST littermate and TetO-Dkk1 mice were placed on the same slide and stained with the same diluted antibody mix. Comparative analysis was always performed on sections in the same relative position on the slide and using identical laser power and detector voltage settings to ensure differences in Foxn1 fluorescence intensity were reflective of differences in protein expression. The Zeiss LSM software package was utilized to determine the Foxn1 fluorescence intensity of individual nuclei within either the cortex or medulla based on counterstaining. A total of 85 individual cells were scanned for each region of the thymus in both ST control and TetO-Dkk1 thymic tissues, following a 4-week induction of DKK1 with Dox. Tissues were derived from 3 independent experiments. Mean Foxn1 MFIs were determined for each condition and region. A students T test was performed to determine the significance of differences in Foxn1 fluorescence intensities calculated for each strain and TEC subset.

Quantification of the Relative Area of K5K8DP TECs as well as the Numbers of ΔNp63⁺ TECs and Aire⁺ TECs Using Confocal Microscopy

Frozen sections of thymic tissue were prepared and stained as described above. Confocal images were prepared by scanning each fluorochrome independently with a 20X lens with an optical slice thickness of 2 µm using a Zeiss LSM510 microscope. The line scan tool provided in the LSM software (Zeiss) was used to scan several areas of sections that were not stained with the appropriate antibodies to determine the background thresholds used for the co-localization tool in the calculation of the relative area of K5K8DP TECs. A mean background plus 5% was used as the minimum threshold for each specific fluorochrome. After calculating the thresholds the co-localization tool was set to colorize all cells within the entire scan which co-expressed both K5 and K8 proteins blue. The scan frame was always identical for ST control and TetO-Dkk1 sections and scans were oriented to have the cortico-medullary junction bisect the scan. Comparative analysis was performed on control and experimental sections on the same slide and stained with the same antibody preparation. The relative area of each section which co-expressed both K5 and K8 was calculated using the Zeiss LSM image analysis software. The mean relative area and standard deviation were calculated using a minimum of 6 sections of each thymus taken from various regions within each thymic lobe and derived from 5 independent experiments.

For calculations of the number of Aire ⁺ and ΔNp63 ⁺ TECs, sections were scanned as described above for Aire, ΔNp63 and UEA1. UEA1 and Aire staining were used to define the limits of the thymic medulla. An overlay box enclosing 10 mm² of thymic area was then sequentially moved over the entire image and the number of Aire+ and ΔNp63 ⁺ nuclei was counted within each 10 mm² area for both the cortex and medulla. Sections were prepared from the central area of the thymus to ensure inclusion of both cortical and medullary areas. A minimum of 6 sections from each thymus derived from at least 5 independent calculations was used to calculate the mean and standard deviation of the number of Aire ⁺ and ΔNp63 ⁺ TECs. DAPI staining was used to confirm that each fluorescent spot counted was indeed a nucleus, as partial nuclei were often encountered in the sections.

RNA Isolation and Real-Time PCR

Total RNA was isolated from sorted thymic epithelial cells using Trizol reagent (Invitrogen). RT-PCR was performed using SuperScript III first-strand synthesis system (Invitrogen). Real-Time PCR was performed using the TaqMan gene expression assay system with primer and probe sets for DKK1 and GAPDH on a 7500 real-time PCR system (Applied Biosystem). Relative expression values for each sample were normalized against endogenous control GAPDH and the 2^−DDCt method was used to calculate the relative level of target mRNA.

Fetal Thymic Organ Cultures

15.5 day-old fetal thymic lobes were dissected from C57BL/6 timed pregnant females, and incubated in 100 µl RPMI1640 complete media containing 10% fetal bovine serum supplemented with 100 µl Wnt3a conditioned medium harvested from L-cells transfected with a Wnt3a construct. Controls consisted of thymic lobes cultured in 100 µl of RPMI1640 complete medium supplemented with 100 µl of conditioned medium harvested from control non-transfected L-cells. FTOC cultures were performed in V-bottom 96-well plates (BD) in a high oxygen (70% O₂, 25% N₂, 5% C0₂) chamber at 37°C. After 72 hrs, thymi were washed in 10% complete RPMI and lobes were embedded in OCT and snap frozen for sectioning.

Statistical Analysis

Data comparison was performed using the Student's t-test function on the Excel software. A P-value of <0.01 was considered significant.

Inhibition of Canonical Wnt Signaling with DKK1 in TECs Results in Rapid Thymic Degeneration in Adult Mice

Several previous studies have demonstrated a role for canonical Wnt signaling in regulating early T cell development [46], [54], as well as the development of the thymic epithelium [38], [39], [47], [49] however, due to early death of the mice or early blocks in thymic development, analysis of a role for Wnt signaling in the maintenance of adult TEC microenvironments was not possible. In an effort to specifically address the contribution of canonical Wnt signaling in the maintenance of postnatal thymic epithelial microenvironments, mice containing the potent canonical Wnt inhibitor DKK1, controlled by a tetracycline-responsive regulatory element (TetO-Dkk1) [53] were crossed with mice harboring a K5 promoter-driven tet inducer-VP16 transgene (K5rtTA) [51]. We hypothesized that the resulting double transgenic tetO-Dkk1;K5rtTA mice would produce high levels of DKK1 in the K5-expressing TEC subsets in response to the Dox, including the dominant K5+ mTEC subset and the K5K8DP cTECs. To demonstrate that the Tet-inducible double transgenic system resulted in increased expression of the canonical Wnt signaling inhibitor within TECs, tetO-Dkk1;K5rtTA (TetO-Dkk1) and tetO-Dkk1-;K5rtTA+ single transgenic (ST) littermate mice were fed Dox for 4 weeks beginning at 6-weeks-of-age. Real-time PCR performed on FACS sorted TECs (CD45⁻MHCII⁺EpCAM⁺) revealed a dramatic 25 fold increase in DKK1 expression within the TECs in the TetO-Dkk1 mice, when compared to TECs derived from littermate ST controls (Figure 1A).

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Figure 1. Relative expression of Dkk1, Wnt target genes and Foxn1 in sorted TECs (CD45⁻ MHCII⁺EpCAM⁺) as well as TECs in vivo.

4-week old tetO-Dkk1;K5rtTA and tetO-Dkk1-ST littermate mice were fed Dox food for 4 weeks prior to harvest and enzymatic dissociation of thymic tissue. Viable CD45⁻MHCII⁺EpCAM⁺ TECs were sorted to >98%purity by FACS. Subsequent to mRNA isolation, real-time PCR was performed using primer and probe sets specific to Dkk1, Axin2, c-Myc, Foxn1 and GAPDH. Relative Dkk1, Axin2, c-Myc and Foxn1 expression were normalized against endogenous GAPDH expression. (A) Relative expression of the Dkk1 transgene following 4-week Dox induction in TECs. (B) Relative expression of the Wnt target genes Axin2, c-Myc and Foxn1 in sorted TECs following 4-week Dox induction of the Wnt inhibitor Dkk1. (C) Representative 400X confocal scans of the thymic cortex of ST (left) and TetO-Dkk1 (right) thymus following a 4-week Dox feeding to demonstrate the reduced Foxn1 protein expression resulting from DKK1 transgene induction. Sections were stained with K14 (pink) Foxn1 (green) and DEC205 (not shown for clarity of Foxn1 analysis). Red arrow in each scan shows the position and direction of a line scan used to generate the quantitative analysis of Foxn1 protein expression shown in the histograms below each image. White arrows show the edge of the thymic section. (D) Histogram comparing mean Foxn1 fluorescence intensity in cTECs vs. mTECs. The mean Foxn1 fluorescence intensity was calculated from both cTECs and mTECs derived from 4-week Dox treated ST or TetO-Dkk1 mice (based on their location within DEC205+ and K14+ regions of the thymus). Means for each location and strain were calculated from a total of 85 individual cells from 3 independent experiments. Error bars in the histograms represent standard deviation. **Statistical significance was determined using a T test and P<.001.

https://doi.org/10.1371/journal.pone.0009062.g001

To determine the specificity and spatial orientation of transgenic DKK1 expression within the thymus, in situ hybridization with a DKK1 specific probe was performed on thymic sections derived from either control ST or TetO-Dkk1 mice following 5 days of Dox induction. Following in situ hybridization, immunofluorescence staining was also performed with anti-Keratin 8 and anti-Keratin 5 antibodies to allow localization of the in situ signal with particular TEC subsets. The short induction time was utilized to ensure that the assessment of DKK1 transgene expression was performed before significant changes in TEC subsets or thymic epithelial organization were apparent. In situ hybridization of thymic sections derived from ST mice with a DKK1 specific probe, revealed a very low level endogenous expression of DKK1, with no apparent pattern of distribution within the K8 dominated cortex or K5 dominated medulla when viewed at 100x (Figure 2A–D) or 400x (Figure 2E–H). In contrast, in situ hybridization of thymic sections derived from TetO-Dkk1 mice revealed a distinct pattern of DKK1 transgene expression within both cortical and medullary areas of the thymus, with a greater abundance of DKK1 expression within the thymic cortex and cortico-medullary junction (Figure 2I–L). When viewed at higher magnification, it is clear that DKK1 transgene expression is restricted to keratin+ TECs; however, DKK1 expression is not restricted to K5-expressing cells. More abundant DKK1 expression was observed in the cortex within primarily K5K8DP cells however; expression was also detected within K8 expressing cTECs (Figure 2M–P). More limited expression of the DKK1 transgene was apparent within K5-expressing TECs in the medulla and within K5K8DP TECs at the cortico-medullary junction (Figure 2Q–T).

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Figure 2. Doxycycline-regulated expression of the DKK1 transgene is evident in both cTECs and mTECs in the adult thymus.

Following a 4-week Dox induction, 12 µm frozen thymic sections were prepared from ST control and TetO-Dkk1 mice and subjected to in situ hybridization with a DKK1 specific probe followed by detection with Sections were then counterstained with anti-Keratin 8 and anti-keratin 5 antibodies to allow localization the DKK1 expression to specific thymic regions and to cortical and medullary TEC subsets. Each row of photos shows in situ hybridization with a DKK1 specific probe followed by Immunofluorescent staining with K8 (green), K5 (red) and a merge of all three. K5rtTA-ST control lobes show little endogenous DKK1 expression at 100X (A–D) or 400X (E–H). In contrast, Dox induction resulted in widespread transgenic expression of DKK1 in the TetO-Dkk1 mice with positive TECs being more abundant in the K8 dominated cortex than the K5 dominated medulla when examined at 100X (I–L). High magnification examination of the thymic cortex of TetO-Dkk1 mice revealed that a high percentage of K5K8DP and a smaller number of K8+ cTECs exhibited strong DKK1 transgene expression. White arrows in all panels identify DKK1-expressing TECs, thus allowing characterization of the keratin profile (M–P). Surprisingly, more limited transgenic DKK1 expression was evident in the medulla, with a small percentage of the K5-expressing mTECs and a higher frequency of the K5K8DP TECs at the cortico-medullary junction expressing DKK1 (Q–T).

https://doi.org/10.1371/journal.pone.0009062.g002

Confirmation that transgenic expression of the Wnt inhibitor DKK1 resulted in a decrease in canonical Wnt signaling within the TECs was evident from the 30–60% decrease in the expression of the Wnt target genes Axin2 and c-Myc observed in TECs sorted from TetO-Dkk1 mice, when compared with identical populations sorted from Dox treated ST controls (Figure 1B). Similar reductions in Foxn1 expression were also observed (Figure 1B), supporting previous reports of a role of Wnt signaling in regulating Foxn1 expression in TECs in vitro [38].

Immunofluorescent staining of thymic sections with Foxn1 antibody allowed assessment of the level of Foxn1 protein expression within distinct TEC subsets in response to a 4-week Dkk1-mediated inhibition of Wnt signaling. Counterstaining these sections with anti-DEC205 and anti-K14 to define cortical and medullary areas of the thymus, respectively, revealed a lower Foxn1 staining intensity in both cTECs (Figure 1C) and mTECs (Figure S1). Quantitative confocal analysis was performed utilizing the Zeiss LSM software to scan individual CTEC and mTEC nuclei to determine the mean fluorescence intensity of Foxn1 antibody staining in ST control and TetO-Dkk1 sections. Representative scans shown for cortical areas in figure 1C (red arrow represents scan), together with the histograms showing the reduced Foxn1 staining intensity in the Dox treated TetO-Dkk1 thymus. A total of 85 cTEC and mTEC nuclei were scanned for each strain from 3 independent pairs of ST and TetO-Dkk1 mice and the resulting mean fluorescence intensities of Foxn1 were graphed in figure 1D. A significant reduction (P<0.001) in mean Foxn1 fluorescence intensity was detected in both cTECs and mTECs, with the most pronounced decrease apparent in cTECs which changed from a mean of 181 (ST) to a mean of 112 (TetO-Dkk1) representing a 40% decrease in Foxn1 expression. MTECs showed a more modest but still highly significant 20% reduction in Foxn1 expression. This difference could reflect a differential effect of DKK1 on cTECs and mTECs or might reflect the higher level of DKK1 transgene expression observed in the cortex.

Analysis of thymus size in response to Dox-driven DKK1 expression revealed a dramatic reduction in overall thymic size (Figure 3, A–C) with the most significant reduction in male mice (82% based on weight) while a 42% reduction was observed for females. This difference in size was representative of similar results obtained in 4 different experiments performed when Dox feeding was initiated at 6 weeks of age and carried out for a total of 4 weeks. Longer feeding of Dox, up to 8 weeks, resulted in almost a complete loss of the thymus, with the residual thymus remaining resembling a severely atrophied thymus that might be found in a mouse older than 1 year-of-age and making isolation of intact thymic tissue for analysis difficult.

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Figure 3. Effect of DKK1 expression on thymus size.

Upper row shows photographs of intact thymic lobes to demonstrate the dramatic thymic involution observed after 4 weeks of doxycycline driven Dkk1 expression. (A) Control Male K5rtTA-ST, (B) Male TetO-Dkk1 and (C) Female TetO-Dkk1. Lower row demonstrates the recovery of thymic size of doxycycline treated lobes following a 4 weeks chase period. (D) Control Female K5rtTA-ST, (E) Male TetO-Dkk1, (F) Female TetO-Dkk1. Female K5rtTA-ST mice exhibited a similar thymic size to male controls after Dox feeding, as did Male K5rtTA-ST mice after 4 weeks Dox feeding and 4 weeks chase (data not shown).

https://doi.org/10.1371/journal.pone.0009062.g003

To examine if DKK1-mediated inhibition of canonical Wnt signaling in TECs, impacted T cell development, total thymocyte numbers were counted. In addition, FACS analysis of thymocytes derived from TetO-Dkk1 and K5rtTA-ST mice following 4 weeks of Dox feeding using antibodies against CD4, CD8, TCRβ, CD25 and CD44 was used to assess differences in thymocyte subset frequency. FACS profiles representative of 4 independent experiments are shown in Figure 4A. Total thymocyte numbers were reduced by more than 50% in both male and female TetO-Dkk1 mice when compared with identically treated K5rtTA-ST littermate controls (Figure 4B). FACS analysis of thymocytes revealed no significant differences in the frequency of any thymocyte subsets (Figure 4C), suggesting that expression of DKK1 in TECs does not lead to stage specific blocks in T cell development but rather a general loss of thymic capacity maintain sufficient numbers of T cells, possibly due to a loss of epithelial niches.

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Figure 4. Effect of DKK1 on total thymocyte number and subset frequency.

A. Thymocyte FACS profiles representative of 4 independent experiments showing the frequency of thymocyte subsets including the DN1-SP subsets. No apparent differences in profile between Dox fed TetO-Dkk1 male and female when compared with similarly treated K5rtTA littermate controls. B. Total thymocyte number is dramatically reduced in both male and female TetO-Dkk1 mice following 4 weeks of Dox feeding, when compared with Dox fed ST littermate controls. C. Following 4 weeks of Dox feeding, FACS analysis of thymocytes derived from either male or female TetO-Dkk1 mice showed no changes in the frequency of individual thymocyte subsets. (The frequency of individual DN subsets represents the frequency of the total number of CD4-CD8- cells) Means for each subset were determined from 4 independent experiments with at least 3 mice of each sex and genotype.

https://doi.org/10.1371/journal.pone.0009062.g004

To determine the effect of DKK1-mediated inhibition of canonical Wnt signaling in TECs, on TEC development and organization, histological analysis of thymic sections derived from adult Dox treated TetO-Dkk1 mice and ST control mice, was performed with a panel of antibodies used to define thymic architecture. K8 is expressed in the majority of cTECs and a subset of mature mTECs, while K5 expression is found predominantly in immature mTEC subset as well as more rare cells scattered in the cortex. Cells that co-express both K5 and K8 are thought to contain a subset of TEC or cTEC progenitors [55], [56] and are primarily localized to the cortico-medullary junction (CMJ) with additional cells scattered through the medulla. Comparative analysis of female DKK1 transgenic mice stained with K8 and K5 antibodies at low magnification revealed a dramatic loss of both cortical and medullary TECs, with a pronounced thinning of the K8 expressing cTECs (Figure 5, F–I) when compared with Dox fed ST littermates (Figure 5, A–D). Both the control K5rtTA-ST and TetO-Dkk1 mice show abundant keratin negative areas in the medulla, which is a characteristic of CD1 background mice not normally found in inbred strains like C57BL/6. Similar K8 and K5 histological analysis of sections derived from Dox-treated male TetO-Dkk1 mice revealed almost a complete loss of normal cTEC organization and an abundance of aberrant cystic structures (Figure 5, J *), while identically treated ST male littermate mice showed a normal distribution and morphology of both cTECs and mTECs (Figure 5, E). Examination of thymic sections derived from Dox-treated female mice at higher magnification revealed almost a complete loss of K5K8DP TEC progenitors in TetO-Dkk1 mice, including cells at both the CMJ and within the medulla (Figure 5, O–R). Yellow arrows in identical positions within the individual color panels allow identification of K5K8DP TECs. The remaining K8SP TECs in the medulla of DKK1 mice (see white arrows) were primarily the mature UEA1 bright cells (data not shown). Comparative analysis of sections derived from Dox-treated K5rtTA-ST mice revealed abundant K5K8DP TEC progenitors at the CMJ as well as within the medulla (yellow arrows, Figure 5, K–N). Male mice showed similar reductions in K5K8DP TECs but more severe reductions in cTEC and mTECs and a more significant disruption of cTEC morphology.

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Figure 5. Effect of Dkk1 expression on the distribution of TEC subsets defined by K5 and K8 expression.

Frozen thymic sections prepared from K5rtTA-ST and TetO-Dkk1 mice after 4 weeks of doxycycline feeding to induce DKK1 expression. Low magnification images of thymic sections derived from female K5rtTA-ST mice (A–D) and TetO-Dkk1 mice (F–I) stained with anti-K8 (green) to identify the cortex and anti-K5 (red) to identify the medulla as well as DAPI. Merged images of K5 and K8 staining (D vs. I) reveal K5K8DP TECs thought to contain progenitors (yellow). Similar images of merged K5 and K8 staining from male K5rtTA-ST (E) and TetO-Dkk1 mice (J) reveal a more dramatic loss of cortical architecture and altered cTEC morphology. * -Identify cystic structures and abundant keratin negative areas (KNA) in the TetO-Dkk1 mice. Higher magnification images of female K5rtTA-ST mice stained with K5 and K8 antibodies reveal abundant K5K8DP TEC progenitors at the CMJ as well as within the medulla (K–N, yellow arrows). (White arrows show K8SP thought to be mature mTECs) Similar sections derived from littermate TetO-Dkk1 mice reveal an absence of the K5K8DP TEC progenitors at both the CMJ and within the medulla (O–R). Further evidence of the hypoplastic cortex and loss of the reticular cTEC network is revealed when TetO-Dkk1 mice with severe phenotypes are stained the cTEC specific DEC205 antibody (green) and UEA1 (red) to define medullary areas. (S). Compared with similar staining of K5rtTA-ST littermate mice (T). A dramatic loss of cortical area and the typical reticular cTEC morphology is apparent in the TetO-Dkk1 thymus. White arrows show the outer edge of the thymus.

https://doi.org/10.1371/journal.pone.0009062.g005

Staining with the cTEC specific DEC205 antibody revealed a severely hypoplastic cortex in Dox-treated TetO-Dkk1 mice. This is apparent from the reduced cortical area evidenced by the close proximity of the medulla, defined by the red UEA1⁺ mTECs, to the edge of the thymus (white arrow, Figure 5S). The cTECs also appear to have lost the normal reticular morphology and more expansive cortex, which are apparent in the control Dox-treated ST mice (Figure 5T). At 400× magnification, no UEA1⁺ medulla was visible when photographs were taken at the outer cortex of control mice. The reduced cortical area and loss of typical reticular morphology is similar to the phenotype described for the involuted thymus of aged mice [57], [58]. Low magnification DEC205 staining of TetO-DKK1 and K5rtTA-ST controls is provided in supplemental Figure 1 similarly demonstrating the reduced cortical area and disrupted cTEC organization in response to DKK1 expression.

In an effort to quantify the effect of transgenic expression of DKK1 on the frequency of total TECs and specific TEC subsets, thymic lobes from TetO-Dkk1 and K5rtTA-ST littermate mice were dissociated using Collagenase/Dispase/DNase digestion following 4 weeks of Dox feeding. A total of 5 thymi from each strain were pooled for each experiment to yield sufficient numbers of TECs for analysis and 3 independent experiments were performed. CD45 magnetic beads were then used to partially deplete CD45⁺ hematopoietic cells from the resulting single cell suspension, prior to staining with antibodies against CD45, MHCII, EpCAM, and CD80 together with UEA1 lectin. CD45⁻ MHCII+ EpCAM⁺ cells are defined as TECs, while immature and mature TECs within this population are defined as MHC^low EpCAM⁺ and MHC^hiEpCAM⁺ subsets, respectively. After gating on TECs further analysis with EpCAM versus UEA1 allowed separation of EpCAM⁺ UEA1⁺ mTECs and EpCAM⁺ UEA1⁻ cTECs. Analysis of the mTEC population with anti-CD80 allowed separation of the mature CD80hi and immature CD80 lo/neg mTEC subsets. A representative FACS TEC profile derived from 4-week Dox fed female TetO-Dkk1 and littermate ST mice is shown in Figure 6A. A total cell count, following CD45 depletion, was used together with the TEC subset frequencies to analyze the effect of DKK1 on total TEC and TEC subset numbers/thymus (Figure 6B). Following Dox feeding, TetO-Dkk1 mice showed a dramatic decrease in the frequency of CD45⁻ EpCAM⁺ MHCII⁺ TECs (mean 2.48+/−0.68%, representative freq. Figure 6A, upper left panel) when compared with K5rtTA-ST controls (8.3+/−1.94%, upper right panel) suggesting a loss of TECs within the smaller thymus. To confirm that this difference in TEC frequency was representative of an actual reduction in TEC numbers, the frequencies of TECs in each subset were used to calculate and compare absolute TEC numbers. Analysis of total TEC numbers revealed a significant decease (P<.005) in the number of TECs present in Dox treated TetO-Dkk1 mice (mean = 3.43+/− 0.35×10⁵/thymus) when compared to littermate ST controls (mean =  14.44+/−0.56×10⁵/thymus). Similar significant reductions in the number of all other TEC subsets analyzed was apparent in the Dox-treated TetO-Dkk1 mice, including cTECs, mTECs, total immature MHC^loTECs, total mature MHC^hiTECs, as well as both mature CD80^hiUEA1⁺ and immature CD80^lo/negUEA1⁺ mTECs (Figure 6B). While no other significant differences in the frequency of the various TEC subsets defined by differences in the expression of MHCII, UEA1 and CD80 were apparent, analysis of the CD45⁻EpCAM⁺ TECs using the mTEC specific lectin UEA1 revealed a small but repeatable reduction in the frequency of EpCAM⁺ UEA1⁻ cTECs in the Dox treated TetO-Dkk1 mice (23.3+/−2.12%) when compared with ST controls (31.4+/−5.3%) (Figure 6A, second set of panels). When the absolute numbers of cTECs and mTECs in Dox-treated TetO-Dkk1 mice were compared with identically treated ST littermate controls the ratio of mTEC to cTECs showed a moderately significantly decreased (P = .025) from 3.05+/− 0.52 in TetO-Dkk1 mice to 1.96+/−0.18 in ST controls (Figure 6C). A repeatable but not significant reduction in the ratio of immature EpCAM⁺MHC^lo to mature EpCAM⁺MHC^hi TECs was also observed in Dox-fed TetO-Dkk1 mice. Taken together these results suggest that K5 promoter-driven DKK1 expression in postnatal TECs leads to a dramatically reduced frequency and absolute number of TEC characterized by significantly reduced numbers of all TEC subsets with a slightly more significant reduction apparent in the cTECs. Given the higher expression of the DKK1 transgene within the cortex and specifically within the K5K8DP TEC subset, as well as the loss of K5K8DP TECs observed in Dox-fed TetO-Dkk1 mice in histology, we wanted to quantify the loss of K5K8DP TECs.

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Figure 6. FACS Analysis of TEC profile and quantification of TEC numbers in response to DKK1 mediated inhibition of Wnt signaling.

Following a 4-week Dox induction of DKK1 expression, thymic lobes from TetO-Dkk1 and K5rtTA-ST littermate mice were dissociated using Collagenase/Dispase/DNase digestion. CD45 magnetic beads were used to partially deplete CD45⁺ hematopoietic cells from the resulting single cell suspension, prior to staining with antibodies against CD45, MHCII, EpCAM, and CD80 together with UEA1 lectin. TetO-Dkk1 mice showed a dramatic decrease in the frequency of EpCAM⁺ MHCII⁺ CD45⁻ TECs (A, upper left) when compared with K5rtTA-ST controls (A, upper right panel). Further analysis of the CD45-EpCAM⁺ TECs using the MHCII and EpCAM to distinguish the MHCII^lo immature and MHC^hi mature TEC subsets, revealed no differences in the frequency of these subsets (A, third set of panels) Similarly, separation of the UEA1⁺ mTECs into UEA1⁺CD80^hi mature and UEA1⁺CD80^lo/neg immature mTEC subsets, revealed no affect of DKK1 induction on the frequency of mTEC subsets (A, lower set of panels). (B) Comparison of the mean total CD45⁻MHCII⁺EpCAM⁺ TEC numbers and the mean number of each TEC subset including EpCAM⁺UEA1⁻ cTECs, EpCAM⁺ UEA1⁺ mTECs, EpCAM⁺MHC^hi mature TECs, EpCAM⁺MHC^lo Immature TECs, UEA1⁺CD80^lo immature mTECs and UEA1⁺CD80^hi mature mTECs, following 4-weeks Dox feeding. Means represent the total cell number/thymus, calculated from 3 independent experiments utilizing 5-pooled dissociated thymi from each strain. Error bars show standard deviation. * P<.005 demonstrating significant reductions in TEC number for all subsets analyzed (C) Dox treatment of TetO-Dkk1 mice results in an increase in the mean mTEC/cTEC ratio (blue bar) when compared to identically treated K5rtTA-ST mice (red bar). Means calculated based on 3 independent experiments as described above. Error bars =  standard deviation. P = .025.

https://doi.org/10.1371/journal.pone.0009062.g006

Co-localization analysis of confocal images was utilized to quantify the decline in K5K8DP TEC, thought to contain or represent TEC progenitors, evident following 4 weeks of DKK1-mediated inhibition of canonical Wnt signaling. Male and female TetO-Dkk1 mice exhibited a 40% and 60% reduction in the mean relative area of K5K8 co-localization, respectively, when compared with littermate ST controls (Figure 7E, left two pairs of bars). These relative K5K8 co-localization values were means calculated based on the analysis of a minimum of 6 sections of each thymus derived from 5 independent experiments (N = 30). Representative sections used for analysis are shown in Figure 7A–B. The relative area of TECs exhibiting K5 and K8 co-localization in confocal images (blue colored cells) is shown in the lower left corner of each panel. This relative area was determined by setting an independent threshold for K5 and K8 staining at 5% above background fluorescence intensity. The Zeiss LSM image analysis software then colorizes all pixels, which exhibit fluorescence intensities above that threshold for both the K5 and K8 channels blue to indicate co-localization. The relative area of K5K8DP pixels is determined as a percentage of the total thymic area in the image. Note that the TECS co-expressing K5 and K8 and colorized blue that are affected by DKK1 expression, include a dominant population at the cortico-medullary junction, as well as a fairly large number of TEC within the medulla as well. Taken together these histological analyses suggest that canonical Wnt signaling is required to maintain the K5K8DP TEC subset thought to contain a TEC progenitor population or to control its development from an as yet undefined TEC stem cell population and that inhibition of Wnt signaling with DKK1 results in a premature thymic involution resulting in dramatic cortical defects.

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Figure 7. Inhibition of Wnt signaling leads to a decline in the number of K5K8DP TECs thought to contain a TEC progenitor population.

A dramatic reduction in the abundance of K5K8DP TECs is apparent in confocal images of thymic sections derived from TetO-Dkk1 (A) when compared with K5rtTA-ST littermate animals (B) following 4 weeks of doxycycline feeding. These K5K8DP TECs reappear in 4-wk Dox treated TetO-Dkk1 mice following a 4 weeks chase of doxycycline withdrawal (C) possessing a similar frequency of K5K8DP TECs to that of similarly treated K5rtTA-ST mice (D). TECs, which exhibited fluorescence intensities 5% above the mean fluorescent background for K5 and K8 and co-expressing both keratins, are colored blue using the Zeiss LSM image analysis software. The relative percentage of TECs co-expressing K5 and K8 is shown by the white numbers in the lower left corner of each image. (Magnification = 200×) (E) The mean relative area of K5K8DP TECs in male and female K5rtTA-ST mice (White bars) and TetO-Dkk1 mice (Gray bars) following 4 weeks of Dox feeding and in female mice following a 4 weeks Dox chase experiment. Error bars show standard deviation. * - P value <.005. ** - P value no longer significant following recovery period. Means were calculated based on 5 independent experiments, which analyzed a minimum of 6 sections cut from various locations within the thymus of each strain (TetO-Dkk1 versus ST littermate) and each sex (N = 30).

https://doi.org/10.1371/journal.pone.0009062.g007

The Number of ΔNp63⁺ TECs Is Reduced following Tet-Regulated DKK1 Induction

The P53 family member, P63, is required for proper development of thymic stroma and appears to influence epithelial stem cells, or TECs early in ontogeny as P63 KO mice exhibit a hypoplastic non-functional thymus, lacking in mature TEC subsets [59], [60]. In stratified epithelium, the N-terminal truncated isoform ΔNp63 is an early marker of epithelial fate and is expressed in stem cells in the basal layer, as well as the proliferating transit amplifying progeny, but is lost in more mature epithelial populations [60], [61], [62], [63], [64], [65]. While epithelial ontogeny in the thymus is less clearly defined, Aire and CD80 expression appear to be restricted to more mature mTEC subsets. Ontogeny of cTECs remains undefined, although evidence from fetal mice suggests that either bipotent TEC progenitors or cTEC progenitors are found within the K5K8DP TEC subset. In the thymus ΔNp63 expression is extremely abundant, however similar to stratified epithelium ΔNp63 is rarely co-expressed with Aire in the mature mTEC subset (see Figure 8F and [66]), suggesting that at least with respect to mTEC ontogeny, ΔNp63 expression follows a similar pattern in the thymus with expression dominating immature subsets. Since DKK1 inhibition of Wnt signaling appears to affect TEC subsets defined by K5 and K8 co-expression, the effect of tet-induced DKK1 expression on the number of potentially immature ΔNp63⁺ TECs, as well as the frequency of mature Aire⁺ mTECs was examined using Immunofluorescent staining of thymic sections. Low magnification images derived from Dox-treated TetO-Dkk1 mice revealed loss of ΔNp63⁺ TECs throughout the thymus (Figure 8A) when compared with Dox-treated ST controls (Figure 8B). Higher magnification images of distinct thymic functional microenvironments clearly reveal both a reduced number of ΔNp63⁺ TECs and a reduced expression level within both the cortex (Figure 8C) and the medulla (Figure 8E) of TetO-Dkk1 animals when compared with ST controls (Figure 8D, cortex & F, medulla). The frequency of mature mTECs defined by Aire expression (red nuclei) was also reduced in Dox-treated TetO-Dkk1 mice when compared to control ST animals. To quantify the changes in both ΔNp63⁺ immature and Aire⁺ mature TECs resulting from DKK1 inhibition of Wnt signaling, the number of TECs expressing ΔNp63 and Aire were counted in 10 mm² areas within confocal images prepared from either Dox-treated TetO-Dkk1 or ST control animals. Means were calculated from 4 independent experiments utilizing 3 animals from each strain and sex. The number of Aire+ or ΔNp63+ TECs was counted in a minimum of 50 (10 mm²) areas in sections derived from multiple thymic lobes for each mean. In male TetO-Dkk1 mice, the mean number of ΔNp63⁺ in the cortex was significantly reduced by >50% to 1.6 (+/−0.6) cells/10 mm², when compared with ST controls containing 3.4 (+/−0.5) ΔNp63⁺ cTECs/10 mm². Female TetO-Dkk1 mice showed a similar 50% reduction in the frequency of ΔNp63⁺ cTECs after Dox treatment (Figure 9A). The medulla of the thymus contains a higher density of ΔNp63⁺ TECs. Dox-treated TetO-Dkk1 female mice exhibited a 33% reduction in the mean number of ΔNp63⁺+ mTECs (11.4+/−3.8 cells/10 mm²) when compared to ST controls (16.8+/−5.1 cells/10 mm²) (Figure 9B middle bars). Male TetO-Dkk1 mice exhibited an even more significant 49% reduction in the frequency of ΔNp63⁺mTECs from 19.8+/−2.5 in ST controls to 10.1+/−5.0 in TetO-Dkk1 animals (Figure 9B, left bars). This more significant effect of inhibition of Wnt signaling on the number of ΔNp63+ TECs in male mice might contribute to the more pronounced thymic degeneration observed in male mice in response to DKK1 (see Figure 3 and 5). Similar ∼50% reductions in the frequency of mature Aire⁺ mTECs were also observed in TetO-Dkk1 mice when compared with ST controls in response to Dox feeding (Figure 9C). Again, the difference in the frequency of mature mTECs in males was slightly greater than that observed in females.

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Figure 8. DKK1 Expression results in reduced ΔNp63⁺ and Aire⁺ TECs.

Thymic sections derived from female TetO-Dkk1 (left column) and K5rtTA-ST mice (right column) stained with Anti-ΔNp63 (green), Anti-Aire (red) and UEA-1 (blue). A–E show thymic sections derived following 4 weeks of doxycycline feeding. Low magnification images of a large area of thymus from TetO-Dkk1 mice (A) compared with the K5rtTA-ST (B). Higher magnification images of the cortex (C & D) and medulla (E & F) in TetO-Dkk1 and K5rtTA-ST, respectively. Low magnification images of 4 week Dox treated thymic tissue derived from TetO-Dkk1 (G) or K5rtTA-ST animals following a 4-week recovery period.

https://doi.org/10.1371/journal.pone.0009062.g008

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Figure 9. Effect of inhibition of Wnt signaling in the adult thymus on the number of ΔNp63⁺ and Aire ⁺TECs.

(A) The mean number of ΔNp63 ⁺ nuclei/10 mm² area of cortex, calculated for K5rtTA-ST (white bars) and TetO-Dkk1 (gray bars) following 4 weeks of Dox feeding and after a 4 weeks Dox chase. (B) The mean number of ΔNp63 ⁺ nuclei/10 mm² area of medulla, calculated for K5rtTA-ST (white bars) and TetO-Dkk1 (gray bars) following 4 weeks of Dox feeding and after a 4 weeks Dox chase. (C) The mean number of Aire ⁺ nuclei/10 mm² area of medulla, calculated for K5rtTA-ST (white bars) and TetO-Dkk1 (gray bars) following 4 weeks of Dox feeding and after a 4 weeks Dox chase. *-P value <.01, **- no longer significant.

https://doi.org/10.1371/journal.pone.0009062.g009

Both immature and mature TEC subsets appear to be reduced after Dox-mediated DKK1 expression, suggesting that Wnt signaling may lead to reduced proliferation of TECs or death to TECs at an immature stage. Proper regulation of Wnt signaling has been shown to be necessary for maintenance of the regenerative capacity of stem cell populations as well as stem cell to TA cell transition in other epithelial tissues [23], [24], [48], [66], [67], [68]. Whether a similar stem cell mechanism is responsible for initial development and maintenance of adult TECs is yet to be determined.

DKK1 Induced Thymic Atrophy Is Reversible

To investigate whether progenitor cells in the thymus were permanently lost or simply blocked in development when Wnt signaling was inhibited, TetO-Dkk1 mice were treated with Dox for 4 weeks and then Dox food was withdrawn and replaced with normal mouse chow for a 4 week chase. The thymus size returned to normal in both male and female TetO-Dkk1 animals after a 4-week recovery period, when compared to similarly treated ST controls (Figure 3, lower row). This increase in total thymic size was accompanied by an expansion of the K5K8DP TEC subset at both the cortico-medullary junction and within the medulla (Figure 7C). A similar recovery of TECs was observed in male mice (data not shown). Quantitative analysis of TetO-Dkk1 mice showed that following a 4-week recovery period, the mean relative area of K5K8DP TECs recovered from the post-treatment 1.1+/− 0.2% to 2.8+/− 0.8%, which was no longer significantly different than identically treated ST controls, which contained a mean of 3.5+/−1.2% (Figure 7E). The frequency of both ΔNp63⁺ TECs (Figure 9, A&B) and mature Aire⁺ mTECs (Figure 9C) also returned to levels comparable with ST controls or non-Dox fed TetO-Dkk1 mice (data not shown). Representative fluorescent micrographs used for the quantitative analysis of TEC numbers, following the 4 weeks recovery period, are shown in Figure 8, G and H. The number of ΔNp63⁺ and Aire⁺ TECs clearly increases dramatically compared with TetO-Dkk1 sections obtained following 4 weeks of DKK1 mediated inhibition of Wnt signaling. Expression of the Wnt signaling inhibitor DKK1 in TECs localized primarily in the cortex leads to a rapid involution of the adult thymus. This involution is characterized by the loss of K5K8DP TECs as well as the loss of both ΔNp63⁺ and Aire⁺ TECs. This involuted thymus maintains the capacity to recover from the inhibition of Wnt signaling, as the thymus returns to a normal size with a normal distribution of TECs following inactivation of DKK1 expression (Figure 3D–F & Figure 7C–E).

DKK1 Expression Leads to a Reduction in Proliferation of Immature TECs but Has No Effect on Apoptosis

In an effort to understand the role of Wnt signaling in maintaining adult thymic epithelial microenvironments and to identify the mechanism responsible for the dramatic decrease in thymic size in response to transgenic expression of DKK1, both the number of cycling TECs and the frequency of apoptosis were examined. Immunofluorescent staining of thymic sections, derived from Dox-treated TetO-Dkk1 mice, revealed a significant reduction in total Ki67⁺ cells in both male and female animals (Figure 10A&B) with the most dramatic reduction in cycling cells and the most significant disruption in normal epithelial organization (detected with Pan-keratin staining), observed in male mice (Figure 10A). Normal TEC organization was apparent in identically Dox-treated ST control animals, which showed a typical pattern of abundant Ki67+ cells in the cortex (primarily thymocytes) and proportionally lower numbers of cycling cells in the medulla (Figure 10C). Due to the close association of TECs with the more abundant thymocytes and the complex three-dimensional organization of the thymic stroma, it is difficult ascertain whether the reduced frequency of cycling cells in the TetO-Dkk1 thymus observed by histology was limited to thymocytes or also included TECs.

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Figure 10. Inhibition of canonical Wnt signaling through transgenic expression of DKK1 leads to reduced proliferation of TECs.

Sections of thymus derived from (A) Male,TetO-Dkk1 (B) Female TetO-Dkk1 and (C) Male K5rtTA-ST littermate control animals stained with anti-Pan-keratin antibody (red) and anti-Ki67 antibody after 4 weeks of Dox feeding. FACS analysis of dissociated thymic tissue derived from similarly treated female TETO-DKK1 and control ST mice revealed only a slight reduction in Ki67 ⁺ cells within the mature CD45⁻ MHCII^hi EpCAM⁺ TEC subset in TetO-Dkk1 mice (D, upper panels). In contrast, a greater than 50% reduction in the frequency of cycling Ki67⁺ TECs was observed in the immature CD45⁻ MHCII^low EpCAM⁺ TEC subset in TetO-Dkk1 mice, when compared with Dox-treated ST littermate controls (D, lower panels). Positive gates for Ki67 staining within each sample were determined using rabbit isotype control antibody. The FACS data presented in D is derived from 5-pooled mice of each strain and the results are representative of 3 independent experiments.

https://doi.org/10.1371/journal.pone.0009062.g010

To specifically examine the impact of Tet-mediated DKK1 expression on the proliferation of TECs, flow cytometry was used to analyze Ki67 expression in dissociated thymic tissue derived from female mice after 4 weeks of Dox feeding. Female mice were chosen over male since the phenotype was less severe, ensuring the presence of all TEC subsets for analysis. Following enzymatic dissociation and partial magnetic depletion of CD45⁺ cells for TEC enrichment, cells were stained with CD45 EpCAM, MHCII and Ki67. Cells were initially gated for CD45⁻EpCAM⁺ TECs followed by separation of mature MHCII^hiEpCAM⁺ and immature MHCII^lowEpCAM⁺ subsets, as shown in Figure 6. Analysis of mature TECs derived from Dox-treated TetO-Dkk1 animals revealed only a slight but repeatable reduction in the frequency of Ki67⁺ cycling cells within CD45⁻MHCII^hiEpCAM⁺ TECs, when compared with Dox-treated ST animals (Figure 10D upper panels). In contrast, analysis of Ki67 expression within the immature MHCII^low EpCAM⁺ subset of TECs revealed a greater than 50% reduction in the number of cycling TECs derived from TetO-Dkk1 mice. The FACS profiles provided in Figure 10D are representative of the results obtained in 3 independent experiments.

Given the association between endogenous DKK1 expression and sites of apoptosis during limb development [31], [32], [33] it was reasonable to predict that the premature thymic involution observed in response to Tet-induced DKK1 expression in TECs could also result from an increase in apoptosis of TECs. To determine if inhibition of Wnt signaling by DKK1 was leading to increased apoptosis, a TUNEL assay was performed on dissociated thymic tissue derived from Dox-treated TetO-Dkk1 and ST mice after Dox treatment. No difference in the frequency of TUNEL⁺ cells was observed in the CD45⁻ EpCAM⁺ TEC subset (Figure 11). TUNEL and active caspase-3 staining of thymic sections counterstained to detect TECs confirmed that no difference in apoptosis of TECs in response to DKK1 expression was observed (data not shown).

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Figure 11. TEC TUNEL Assay: The effect of DKK1-mediated inhibition of canonical Wnt signaling on apoptosis in TECs.

The gating strategy used to analyze total CD45⁻EpCAM⁺ TECs derived from TetO-Dkk1 female mice (A) and K5rtTA-ST littermate female control mice (B) after 4 weeks of Dox feeding to induce DKK1 expression. An overlay of the TUNEL staining for TECs from TetO-Dkk1 and K5rtTA-ST mice, as well as TetO-Dkk1 TECs, stained in the absence of TDT as a negative control (C), reveals no difference in apoptosis with the TECs.

https://doi.org/10.1371/journal.pone.0009062.g011

Taken together these results suggest that inhibition of canonical Wnt signaling, through Tet-induced DKK1 expression within the majority of cTECs and a minor population of mTECs, results in a reduced number of cycling TECs, contributing to a decrease in frequency of K5K8DP putative TEC progenitors and premature involution of the thymus.