Fig 1.
Postnatal thymic epithelium contains Axin2-expressing cells in both cortex and medulla.
(A to B) P16 Axin2-lacZ thymus stained with X-gal. Arrowheads point to X-gal stained cells, and insets are magnified views of the boxed regions. (C to F) AXCT2;mTmG thymus traced with a high dose of tamoxifen (15mg tamoxifen/25g body weight) from P16-P18 and stained for K5 and K8 expression. Boxed regions in (D) are magnified in (E) and (F). (E) Cortical region showing clusters of AXCT2-labeled K8+ cTECs after two days (arrowhead). Some cells with circular morphology are observed, and are absent from the thymus after one month. (F) Medullary region showing a more dispersed pattern of AXCT2-labeled cells, which express K5 only (arrowhead), or both K5 and K8 (arrow). Scale bars for (A) and (B) represent 40μm, and 100μm for (D).
Fig 2.
Titration of tamoxifen dosage to achieve sparse labelling.
(A to E) Dosages of 2mg tamoxifen/25g body weight, 1mg/25g, 0.5mg/25g, 0.25mg/25g and 0.1mg/25g were titrated and the labeled clusters were examined 28 days post tamoxifen administration (P16-P44 trace). (F and G) Sparse labeling of TECs is observed in the cortex (F, K8 positive) and medulla (G, K5 K8 double positive) after 48 hours of inducing labeling. Insets are magnified views of the boxed regions showing labeled cells. Labeling frequency is sampled by imaging the entire thymus, and calculating the proportion of the total number of cells (DAPI-labeled) that are AXCT2-labeled (mGFP-labeled). Scale bars in (A) to (E) represent 100μm, and 20μm and (F) and (G).
Fig 3.
K8-only AXCT2-labeled clusters expand robustly over one year.
(A) Timeline for AXCT2;mTmG lineage tracing experiments. n = 3 or 4 mice per time point. (B) AXCT2-labeled clusters are grouped into four categories according to their K8 and K5 expression—K8 only (blue), K5 only (red), K5 K8 double positive (DP, orange) only and mixed (cluster with more than one type of cells, green). The graph shows the distribution of each cluster type category among all the labeled clusters observed per time point. (C) The average number of cells per cluster is plotted for K8-only cluster type at each time point. The differences in average cluster sizes between 48-hour trace and 6 month or 1 year trace are tested for significance. (D) The size distribution of K8-only clusters at each time point. (E) Representative images of mGFP-labeled K8-only clusters observed at 48 hours, 1 month, 3 months and 1 year after trace initiation. The left panels show co-staining with mGFP, K5 and the K8, and the right panels show the same clusters with DAPI. (F) AXCT2;mTmG mice were injected with corn oil at P16, and traced for two months, six months, and 1 year. Ectopic activation of AXCT2 in the AXCT2;mTmG thymus is absent throughout the period of trace. The mTomato signal is expressed constitutively by every cell in the absence of recombination. Graphs depict averages ± SEM, and two-tailed unpaired Student’s-t test was performed to obtain p values of significance. ns—not significant, ** p<0.01. Scale bars represent 20μm.
Fig 4.
K5-only and DP-only AXCT2-labeled clusters do not expand significantly over time.
(A) The average number of cells per cluster is plotted for K5-only (red), DP-only (yellow) and mixed (green) cluster types at each time point. The differences in average cluster sizes between 48 hour trace and 6 month or 1 year trace are tested for significance. (B and C) The size distribution of K5-only clusters (B) and DP-only clusters (C) at each time point. (D and E) Representative images of mGFP-labeled K5-only clusters (D) and DP-only clusters (E) observed at 48 hours, 1 month, 3 months and 1 year after trace initiation. The left panels show co-staining with mGFP, K5 and the K8, and the right panels show the same clusters with DAPI. Graphs depict averages ± SEM, and two-tailed unpaired Student’s t-test was performed to obtain p values of significance. ns—not significant, ** p<0.01. Scale bars represent 20μm.