The effects of miR-181a1 and miR-181b1 loss on TEC distribution.

<p>Fig 2A. Flow cytometric analysis of TEC (CD45⁻EpCAM⁺) subpopulations isolated in mice. The relative frequency of cTEC (UEA⁻1-Ly51⁺), mTEClo (UEA-1⁺Ly51⁻) mTEChi(UEA-1⁺Ly51⁺I-A^b+) are shown in both Mir181a1/b1^fl/fl mice compared to Foxn1-Cre::Mir181a1/b1^fl/fl. These are representative dot plots. This experiment was performed twice with at least 3 mice per group. Fig 2B. Flow cytometric analysis of TEC (CD45⁻EpCAM⁺) subpopulations isolated in mice. The relative frequency of cTEC (UEA⁻1-Ly51⁺), mTEClo (UEA-1⁺Ly51⁻) mTEChi(UEA-1⁺Ly51⁺I-A^b+) are shown as a percentage in the thymus in both Mir181a1/b1^fl/fl mice compared to Foxn1-Cre::Mir181a1/b1^fl/fl. This experiment was performed twice with at least 3 mice per group. Unpaired non-parametric t test was performed. * = P<0.05, ** = P<0.01.</p

Thymic profiles and numbers of cells are not altered due to loss of miR-181a1 and miR-181b1 in TECs.

<p>Flow cytometric analysis for the cell-surface expression of CD4 and CD8 on thymocytes isolated from Mir181a1/b1^fl/fl and Foxn1-Cre::Mir181a1/b1^fl/fl mice. Numbers denote the percentage of cells within the given gates in a representative experiment. B. Thymocytes were harvested at two different time points 6 weeks (B) and 4 months (C). Absolute numbers are shown of the thymic subsets. There were no significant differences between Mir181a1/b1^fl/fl (white) and Foxn1-Cre::Mir181a1/b1^fl/fl mice (filled).</p

miR-181a1 and miR-181b1 does not affect TECs based on absolute numbers.

<p>A. Flow cytometric analysis of TEC (CD45⁻EpCAM⁺MHC II⁺) subpopulations isolated in mice. The absolute number of total thymocytes, TEC, cTEC (UEA⁻1-Ly51⁺), mTEC (UEA1+),mTEClo (UEA-1⁺Ly51⁻), mTEChi(UEA-1⁺Ly51^-MCHII⁺) is shown in both Mir181a1/b1^fl/fl mice compared to Foxn1-Cre::Mir181a1/b1^fl/fl. This experiment was done two times with at least 3 mice per experiment.</p

Maturation of mTECs was not affected in Foxn1-Stat3-CKO.

<p>Cryostat sections of the thymus were stained with antibodies directed to K14 (green) and UEA1 (red) (A), and with antibodies to ERTR5 (red) and AIRE (green) (B). Sections were counterstained with DAPI (blue). Scale bars: 400 μm.</p

Medullary regions are severely affected in Foxn1-Stat3-CKO mice.

<p>(A) Macroscopy of the thymus derived from Stat3-flox/flox (Stat3f/f) and Foxn1Cre::Stat3f/f mice at 7 weeks of age. (B) Flowcytometric profiles of developing thymocytes derived from Stat3f/f and Foxn1Cre::Stat3f/f mice, at 7 weeks of age. (C) Cryostat sections of thymic tissue from Stat3f/f and Foxn1Cre::Stat3f/f mice (7 weeks of age) were stained with the mTEC specific antibody, ER-TR5 (red) and counterstained with DAPI (blue). Scale bars: 400 μm. (D) Cryostat sections of neonatal thymic tissue from Stat3f/f and Foxn1Cre::Stat3f/f mice were stained with antibody directed to K14 (green) and counterstained with DAPI (blue) Scale bars: 100 μm. (E) Total thymic cellularity of control (containing Cre-f/+ and f/f) and Foxn1Cre::Stat3f/f (Cre-f/f) mice at indicated ages. Bar stands for the average value of each experimental group. ns denotes a non-significant difference (P>0.1) in Student’s t test. (F) Changes in the proportional area of medullary regions in thymus tissue sections of control (containing Cre-f/+ and f/f, n = 8, 6, 7, 3 for neo, 3, 6, 12 week, respectively) and Foxn1Cre::Stat3f/f (Cre-f/f, n = 5, 4, 6, 5 for neo, 3, 6, 12 week, respectively) mice in the first 12 weeks of life. The area occupied by mTECs in thymus was quantitatively measured in sections stained with K14 antibody using Axiovision4 software (Carl Zeiss). Error bar stands for the standard deviation. ns denotes a non-significant difference (P>0.1) in Student’s t test. **;P<0.005, ***;P<0.0005. (G) Representative flow cytometric profiles showing frequencies of major TEC populations from 12 weeks old Stat3f/f and Foxn1Cre::Stat3f/f mice. EpCAM⁺CD45^- fraction represents whole TEC population, and UEA1 vs Ly51 profile was displayed for the cells gated on EpCAM⁺CD45^- fraction, where UEA1^highLy51^low and UEA1^lowLy51^high fraction were defined as mTEC and cTEC population, respectively. (H) Ratio of mTEC vs cTECs in flow cytometric analysis of control (containing Cre-f/+ and f/f, n = 4) and Foxn1Cre::Stat3f/f mice (n = 5) at 12 weeks of age is shown.</p

Medullary regions are severely affected while cTECs were normally regenerated in K5-Cre-Stat3-CKO mice.

<p>(A) Gross anatomical analysis of the thymus derived from 7 week old K5Cre::Stat3f/+ and K5Cre::Stat3f/f mice. Scale bars: 1 mm. (B) CD4 and CD8 cell surface expression on thymocytes derived from 7 week old Stat3f/f and K5Cre::Stat3f/f mice. (C) Cryostat sections of thymic tissues derived from either K5Cre::Stat3f/+ or K5Cre::Stat3f/f mice at 7 weeks of age. The tissues were stained with anti-K8 (red) and anti-K14 (green) antibody and counterstained with DAPI (blue). Scale bars: 400 μm. (D) Experimental design for data presented in panels (E) and (F). K5-Cre::Stat3-f/f mice were lethally irradiated and then rescued by hematopoietic stem cell transplantation (wild type C57BL/6). After 4 weeks, mice were sacrificed and the thymus was examined. (E) Gross anatomical analysis of the grafted thymus 4 weeks after hematopoietic stem cell transplantation. (F) Cryostat sections of the thymus were stained with anti-K14 antibody (green) and counterstained with DAPI (blue). Scale bars: 400 μm.</p

Regenerative potential of cTECs was not affected in Foxn1-Stat3-CKO mice.

<p>(A) Experimental procedure for (B) and (C). Foxn1-Cre::Stat3-f/f mice were lethally irradiated and then rescued by bone marrow transplantation from wild type mice. After 4 weeks, mice were sacrificed and thymic tissue was examined. (B) Macroscopy of the thymus 4 weeks after hematopoietic stem cell transplantation. (C) Cryostat sections of the thymus were stained with anti-K14 antibody (green) and counterstained with DAPI (blue). Scale bars: 400 μm. (D) Experimental design for data presented in panels (E) and (F). Foxn1-Cre::Stat3-f/f fetal thymic lobes (15 dpc) were cultured in vitro for 6 days in the presence of deoxyguanosine and subsequently transplanted under kidney capsule of wild type mice. After 4 weeks, mice were sacrificed and thymic grafts were examined. (E) Gross anatomical analysis of the thymic grafts 4 weeks after grafting into C57BL/6 wild type recipients. (F) Comparison of maximum cross-section area of thymic grafts of control (containing Cre-f/+ and f/f, n = 4) and Foxn1Cre::Stat3f/f mice (n = 4). (G) Cryostat sections of thymic grafts were stained with anti-K8 (red) and anti-K14 antibody (green). Sections were counterstained with DAPI (blue). Scale bars: 200 μm.</p

mTECs are reduced in Foxn1-EGF-R-CKO mice.

<p>(A) Gross anatomical analysis of the thymus derived from Foxn1Cre::EGF-Rf/+ mice (control) and Foxn1Cre::EGF-Rf/f mice at 6 weeks of age. (B) Quantitative analysis for the proportion of medullary regions in thymus of control (containing Cre-f/+ and f/f, n = 4) and mutant (Cre-f/f, n = 5) mice. (C) Cryostat sections of the thymus were stained with ER-TR5 antibody (green) and anti-AIRE antibody (red). Sections were counterstained with DAPI (blue). Scale bars: 400 μm.</p