Fig 2: Wnt4 enhances LSK expansion in culture via JNK-dependent mechanisms. A) Fetal liver cells were cultured on NIH3T3 or NIH3T3-Wnt4 cells for 24–72 h and the proportion and number of LSKs were analyzed by flow cytometry at different time points. Histograms on the left show absolute numbers of LSKs and the percentage of LSKs expressing high levels of Flt3 (mean ± SEM; n = 7). Representative flow cytometry data for both Wnt4 (▪) and control (□) cultures after 48 h are shown on the right. Numbers in the upper-right corners of the FACS panels represent the percentage of LSKs over total live events. *P≤0.05, **P≤0.005, Wnt4 vs control (paired). B) The effect of 3 µg/ml anti-Wnt4 antibody or an isotype control on LSK (□) and LMPP (▪) expansion after 48 h of culture. *P≤0.05, antibody-treated vs control. C) Analysis of the impact of Wnt4 on LSK apoptosis in culture. Histogram on the left shows the percentage of AnnexinV positive LSK over live (PI-negative) cells from Wnt4 (▪) and control (□) cultures after 48 h (mean ± SEM; n = 5). Histogram on the right shows the fold induction of Bcl2l mRNA in fetal liver LSKs by a five-hour stimulation with Wnt4 (ratio Wnt4/control, standardized to HPRT expression; n = 4). *P≤0.05, **P≤0.005, Wnt4 vs control (paired). D) Cell cycle analysis of LSKs after 24 h of culture. Representative ModFit analyses of Hoechst staining from control (top) and Wnt4 cultures (bottom) are shown together with the mean ± SEM of the percentages of LSKs in S vs G2/M fractions of the cycle. None of the differences were statistically significant (n = 4). E) Fetal liver cells were cultured as above for 48 h in the presence of the specific JNK-inhibitor sp600125. Histogram shows the fold expansion of LSK (□) and LMPP (▪) in Wnt4 cultures relative to controls (mean ± SEM; n = 7). *P≤0.05, **P≤0.005, treated vs untreated. F) Fetal liver cells were cultured as above for 48 h; a fixed fraction of harvested cells were subsequently seeded in growth factor supplemented methylcellulose for CFC assays. Histograms show the number of different colonies [granulocyte/erythrocyte/monocyte/megakaryocyte (GEMM) (blue); erythroid (E) (red); granulocyte, monocyte or granulocyte/monocyte (G+M) (yellow)] and the total number of colonies (black) counted on day 7 (mean ± SEM; n = 3). *P≤0.05, Wnt4 vs other conditions. G) Analysis of fetal liver LSK and LMPP expansion in the presence (□) and absence (▪) of β-catenin. Histogram shows the fold expansion in Wnt4 cultures relative to controls (mean ± SEM; n = 4).

Fig 3: Wnt4 signals are mediated through a PCP receptor, Frizzled 6. A) Real-time RT-PCR analyses on sorted fetal liver LSKs, showing the expression levels of different Frizzled family members (mean ± SEM; n = 4). Data were normalized against HPRT expression. B) Fz6+/+ (□) and Fz6−/− (▪) fetal liver cells were cultured for 48 h, as in Figure 2. Cells treated with the JNK inhibitor sp600125 (gray bars) are shown for comparison. Histogram shows the fold expansion in Wnt4 cultures relative to controls (mean ± SEM; n = 6). *P≤0.05, **P≤0.005, Fz6+/+ vs. Fz6−/−. C) Analysis of BM LSKs in Fz6+/+ (□) and Fz6−/− (▪) mice. Histogram shows the percentage of total LSKs as well as HSC (CD150+CD135−), MPP (CD150−CD135−) and LMPP (CD150−CD135+) subpopulations (mean ± SEM; n = 4). D) Expression of polarity-associated transcripts in Wnt4+/+ and Wnt4−/− fetal liver LSKs. Histogram represents the mean fold increase in the presence of Wnt4 (ratio Wnt4+/+/Wnt4−/−; n = 4–6). The dashed line is set at 1.5. *P≤0.05.

Fig 4: TFF2 is necessary for myeloid-derived non-canonical Wnt4 and Wnt16 expression.(a,b) Volcano plots showing results of a cDNA-based screen for Wnt pathway target genes expressed as the fold-difference between (a) WT Mφ exposed to damaged or quiescent epithelia and (b) between WT Mφ vs. TFF2−/− Mφ exposed to damaged epithelia. BMMφ were recovered at d4 of MERA for analysis. Each point represents mean of three biological replicates. (c) TER values during MERA with WT Mφ in the presence of anti-Wnt4a mAb or control IgG. (d, e) Flow-sorted AM, IM and CD103+DC from WT or TFF2 KO lungs at d4 after infection were analyzed for mRNA levels of (d) Wnt4 and (e) Wnt16. (f) Representative flow plots and (g) quantification of BrdU+SpC+ epithelia from distal lung digest cells pre-gated on the live, CD45−, EpCAM+ population for each designated genotype following i.n. administration of rWnt4/16+ R-Spondin 1 cocktail (1μg/mouse, 2 doses) or saline and analyzed by flow cytometry at d4 following N.b. infection (N=5/group). Graphs show Mean±SEM. *, p<0.05; **, p<0.01 and ***, p< 0.005 as determined by ANOVA or Student’s t-test.

Fig 5: Wnt4 activates JNK but not calcium-dependent non-canonical signaling in hematopoietic progenitor cells. A) Representative western blot showing kinetics of JNK phosphorylation in the bone marrow HPC line EML after stimulation with 30 ng/ml recombinant Wnt4. Calnexin was used as a loading control. Similar results were obtained in at least three independent experiments. B) Representative western blot showing pulldown of GTP-linked forms of Rac1 (top left) and Cdc42 (top right) after stimulation with 30 ng/ml recombinant Wnt4 for 15 min. Total Rac1 and Cdc42 are shown at the bottom. The numbers correspond to the relative amount of Rac1-GTP (Rac1 in the pull-down/Rac1 in total cell lysate), with the unstimulated condition set as one. Similar results were obtained in three independent experiments. C) Representative flow cytometry data for intracellular calcium levels in EML cells after stimulation with 30 ng/ml recombinant Wnt4 or ionomycin as a positive control. Similar results were obtained in three independent experiments and with a range of different concentrations of Wnt4 (10–300 ng/ml). D) Expression of β-catenin-associated and JNK-associated transcripts in fetal liver LSKs in the presence of Wnt4 and 10 µM cycloheximide. Histogram represents the mean fold induction by Wnt4 from paired experiments (ratio Wnt4/control, standardized to HPRT expression; n = 5–6). The dashed line is set at 1.5. *P≤0.05; †P≤0.005.