1. Wnt4 regulates thymic cellularity through the expansion of thymic epithelial cells and early thymic progenitors
by Krista M. Heinonen, Juan Ruiz Vanegas, Sylvie Brochu, Jingdong Shan, Seppo J. Vainio, and Claude Perreault
Blood
Volume 118(19):
November 10, 2011
©2011 by American Society of Hematology

2. Wnt4 is predominantly expressed byTECs.
Wnt4 is predominantly expressed byTECs. Relative mRNA expression for Wnt4 in total TECs (CD45−EpCAM+), cTECs (Ly51+), immature mTECs (Ly51−MHCII−), mature mTECs (Ly51−MHCII+), endothelial cells (CD45−CD31+), fibroblasts (CD45−EpCAM−Ly51+), and in T-cell subsets (DN, DP, CD4 SP, and CD8 SP) of E18.5 (A) and 4-week-old adult (B) mice, expressed as mean 2−ΔCT standardized to the endogenous control GAPDH. (C) Relative mRNA expression of Fz receptors in adult TEC subsets is expressed as mean 2−ΔCT standardized to the endogenous control GAPDH. Histogram represents mean ± SEM of 3 mice.
Krista M. Heinonen et al. Blood 2011;118:
©2011 by American Society of Hematology

3. Wnt4 is needed for steady-state postnatal thymopoiesis.
Wnt4 is needed for steady-state postnatal thymopoiesis. (A) Thymic cellularity from E15.5 and E18.5 Wnt4+/+ and Wnt4−/− mice. (B) Thymic cellularity from mice in which Wnt4 deletion has been induced (Cre+) on E18.5 or in the adult, compared with Cre− littermate controls. (C-E) FACS analysis of lineage-negative thymocytes from E18.5 Wnt4+/+ and Wnt4−/− mice (C), mice in which Wnt4 has been deleted at birth (D), and mice in which Wnt4 has been deleted in adulthood (E). The graphs on the left show the proportion of ETP (Lin−cKithiCD25−), DN2 (Lin−cKithiCD25+), and DN3 (Lin−cKit−/loCD25+) subsets expressed as percentage of total thymocytes. The graphs in the middle show the absolute numbers of cells within each subset per thymus. Representative flow cytometry data are shown on the right. All histograms represent the mean ± SEM (n = 6-7/group; *P < .05, **P < .01, ***P < .005).
Krista M. Heinonen et al. Blood 2011;118:
©2011 by American Society of Hematology

4. Wnt4 has a direct effect on ETP and DN2 expansion.
Wnt4 has a direct effect on ETP and DN2 expansion. (A) Analysis of homing to the Wnt4-deficient and -sufficient adult thymus. The number and relative contribution of donor-derived (CD45.1+) Lin− cells was determined 36 hours after injection. The histograms represent mean ± SEM from 7 mice per group. (B) Expression of chemokines and P-selectin in the Wnt4-deficient and -sufficient adult thymus. The histograms represent mean ± SEM from 4 to 5 mice per group. (C) Analysis of T-cell development in culture in the presence of GFP+ OP9-DW4 or OP9-DL1 stromal cells. GFP− cells were analyzed by flow cytometry at indicated times and are expressed as number of cells/ETP seeded on day 0. The histogram represents mean ± SEM from a total of 8 replicates from 3 independent experiments. (D) Analysis of T-cell differentiation as defined by cKit and CD25 staining on day 5. The histogram on the left represents ETP and DN2 frequencies among GFP− cells. The histogram in the middle represents the number of cells with ETP and DN2 phenotype on d5 per ETP seeded (mean ± SEM; n = 8 for wild type, n = 5 for Tcf7−/−). Representative flow cytometry data gated on GFP− cells are shown on the right. (E) Expression of selected genes associated with DN2-DN3 progression and early T-cell development. Histogram represents the relative mRNA levels from sorted GFP− lymphocytes after 2 or 5 days of coculture (ratio OP9-DW4/OP9-DL1 from paired experiments; mean ± SEM; n = 3; *P < .05, **P < .01, ***P < .005).
Krista M. Heinonen et al. Blood 2011;118:
©2011 by American Society of Hematology

5. Wnt4 controls fetal thymic size through regulation of TEC numbers.
Wnt4 controls fetal thymic size through regulation of TEC numbers. (A) TEC numbers in E15.5 and E18.5 Wnt4+/+ and Wnt4−/− thymi dissociated by enzymatic digestions and analyzed by flow cytometry. (B) Thymocyte to TEC ratio in E15.5 and E18.5 thymi. (C) Hematoxylin- and eosin-stained paraffin sections of thymi from Wnt4+/+ and Wnt4−/− mice at E18.5. Original magnification ×40. Higher magnification views of selected regions (black boxes) are shown on the right. m indicates medulla; c, cortex. (D) Frozen thymic sections from Wnt4+/+ and Wnt4−/− mice stained with anti-K8 (i-viii), anti-K5 (i,v), anti-K 14 (iii,vii), and anti-β5t (iv,viii). Higher magnification views of selected regions (white boxes) are shown for K8/K5-stained thymic sections (ii,vi). 10×/0.3 NA (i,iii,v,vii), 40×/1.3 NA oil objective (iv,viii), and 63×/1.4 NA oil objective (ii,vi). (E) Quantitative analysis of β5t and K5 staining (left panel) and the area occupied by medullary regions (right panel) per lobe of E18.5 Wnt4+/+ and Wnt4−/− thymi. Histograms represent mean ± SEM of 8-10 mice (*P < .05, **P < .01, ***P < .005).
Krista M. Heinonen et al. Blood 2011;118:
©2011 by American Society of Hematology

6. Loss of Wnt4 suppresses post-natal TEC expansion.
Loss of Wnt4 suppresses post-natal TEC expansion. Flow cytometry analysis of thymi from doxycycline-treated tetO-Cre:ROSArtTA/+:Wnt4fl/fl (Cre+) Wnt4-deficient mice and ROSArtTA/+:Wnt4fl/fl (Cre−) control littermates. (A) Numbers of EpCAM+ cells per thymus. (B) Thymocyte/TEC ratio from the same mice. (C) Analysis of the cortical (CD205+Ly51+) and the medullary (CD205−Ly51−) TEC subsets. The graph on the left shows the mTEC/cTEC ratio from Cre+ and Cre− mice. Representative FACS data for MHCII and Ly51 staining are shown on the right. The numbers within the FACS plots represent mean percentages of the different epithelial subsets over total EpCAM+ cells. The asterisk (bottom right plot) denotes statistical significance for the proportion of cTECs. All histograms represent mean ± SEM from 6-7 mice from at least 4 independent experiments (*P < .05, **P < .01, ***P < .005).
Krista M. Heinonen et al. Blood 2011;118:
©2011 by American Society of Hematology

7. Wnt4-sufficiency in hematopoietic cells and extrathymic stromal cells does not rescue its absence in the thymic stroma.
Wnt4-sufficiency in hematopoietic cells and extrathymic stromal cells does not rescue its absence in the thymic stroma. (A) Total thymic cellularity in Wnt4+/+ and Wnt4−/− grafts. (B) H&E-stained paraffin sections of Wnt4+/+ and Wnt4−/− thymic grafts. Original magnification ×40. (C) Numbers of thymocytes in Wnt4+/+ and Wnt4−/− grafts are depicted. (D) Representative flow cytometry data for cKit (CD117) and CD25 (gated on Linlo/neg cells for ETP, DN2, and DN3) and CD4/CD8 cell staining. The numbers within cytometry plots represent mean percentages of total. (E) Mice were injected intraperitoneally with 1 mg BrdU in PBS to provide a labeling pulse. Four hours later, thymic grafts were harvested, and BrdU uptake was detected with a BrdU flow kit (BD Biosciences Pharmingen). (F) Annexin V staining was performed to detect apoptotic T cells by flow cytometric analysis. (G) Representative flow cytometry data for Wnt4-overexpressing and control thymic aggregates (left panel). Graphs show the fold expansion of total CD45+ cells, CD25−cKithi (ETPs), and TECs in Wnt4 aggregates compared with controls (right panel). Histograms represent the mean ± SEM of 5-10 mice per group (*P < .05, **P < .01, ***P < .005).
Krista M. Heinonen et al. Blood 2011;118:
©2011 by American Society of Hematology

8. Wnt4 regulates thymopoiesis at multiple stages.
Wnt4 regulates thymopoiesis at multiple stages. Working model depicting the different levels of Wnt4 action on T-cell development. (1) Wnt4 in the BM increases lymphoid-primed multipotential progenitor (LMPP) survival in a paracrine manner.23,25 (2) Wnt4 facilitates ETP/DN2 expansion in a paracrine manner (Figures 2–3). (3) Wnt4 facilitates TEC expansion, in particular that of mTECs in the fetal and early postnatal period, probably in an autocrine manner (Figures 4–5). (4) Wnt4 is likely to increase the clonal expansion capacity of precursor TECs (Figures 4,6). HSC indicates hematopoietic stem cell; CLP, common lymphoid precursor; CMP, common myeloid precursor; TESC, thymic epithelial stem/progenitor cell; CMJ, corticomedullary junction.
Krista M. Heinonen et al. Blood 2011;118:
©2011 by American Society of Hematology