Contrasting responses of lymphoid progenitors to canonical and noncanonical Wnt signals

Abstract

The Wnt family of secreted glycoproteins has been implicated in many aspects of development, but its contribution to blood cell formation is controversial. We overexpressed Wnt3a, Wnt5a, and Dickkopf 1 in stromal cells from osteopetrotic mice and used them in coculture experiments with highly enriched stem and progenitor cells. The objective was to learn whether and how particular stages of B lymphopoiesis are responsive to these Wnt family ligands. We found that canonical Wnt signaling, through Wnt3a, inhibited B and plasmacytoid dendritic cell, but not conventional dendritic cell development. Wnt5a, which can oppose canonical signaling or act through a different pathway, increased B lymphopoiesis. Responsiveness to both Wnt ligands diminished with time in culture and stage of development. That is, only hematopoietic stem cells and very primitive progenitors were affected. Although Wnt3a promoted retention of hematopoietic stem cell markers, cell yields and dye dilution experiments indicated it was not a growth stimulus. Other results suggest that lineage instability results from canonical Wnt signaling. Lymphoid progenitors rapidly down-regulated RAG-1, and some acquired stem cell-staining characteristics as well as myeloid and erythroid potential when exposed to Wnt3a-producing stromal cells. We conclude that at least two Wnt ligands can differentially regulate early events in B lymphopoiesis, affecting entry and progression in distinct differentiation lineages.

Figure 1

Autocrine Wnt3a blocks hematopoiesis. Contour plots of flow cytometry results show potentials for (A) lymphopoiesis or (B) myelopoiesis from control (left) or Wnt3a transfected (right) LSK. Each population was held for 11 days in defined, stromal cell- free culture conditions, and (C) total cell yields per input progenitor, averaged with S.E. bars. One representative experiment of three is shown. Asterisks indicate statistical significance, * (p<0.05), ** (p<0.01).

Figure 2

Wnt family ligands differentially regulate the production of lymphoid-lineage cells. (A) Wnt3a, Wnt5a or Dkk1 were individually inserted into a IRES-GFP vector. Contour plots (B) show development of HSC after 11days of co-culture on the indicated Wnt family protein secreting cell lines. B-lineage cells are gated as CD19⁺ B220⁺, pDC (plasmacytoid dendritic cells) are within the CD11c⁺ CD11b⁻ gate of CD19⁻ B220⁺cells and cDC (conventional DC) are within the CD11c⁺ CD11b⁺ gate of CD19⁻ B220⁻ cells as indicated. (C) Total cell yields per input progenitor were calculated and given as averages with S.E. bars. Shown is one representative experiment of four that produced similar results. Asterisks indicate statistical significance, * (p<0.05), ** (p<0.01), *** (p<0.0001).

Figure 3

Wnt3a has minimal influence on B lymphopoiesis at long culture intervals. Contour plots (A) show hematopoietic development of HSC after 21 days of co-culture on the indicated Wnt family protein secreting cell lines. (B) Total cellular yields per input progenitor are given as averages with S.E. bars. Shown is one representative experiment of three. Asterisks indicate statistical significance, * (p<0.05), ** (p<0.01), *** (p<0.0001).

Figure 4

Wnt ligands preferentially affect early events. Contour plots (A) show B-lymphoid lineage development after 11 days of co-culture on the indicated Wnt family protein secreting cell line. (B) The same experimental results were calculated and shown as average yields per input progenitor with S.E. bars. Shown is one representative of 4 experiments. N.D. indicates not done. Asterisks indicate statistical significance, * (p<0.05), ** (p<0.01), *** (p<0.0001).

Figure 5

Wnt3a inhibits stem cell differentiation, slows their proliferation in co-cultures. (A) Contour plots show staining for freshly isolated lineage⁻ c-Kit^Hi Sca-1⁺ stem cells (left), or HSC after 2 days on OP9-Control (center) or after 2 days on OP9-Wnt3a stroma (right). (B) Overlay histogram shows acquisition of mature cell lineage markers by HSC after 11 days co-culture on OP9-Control (black solid line) or OP9-W3A (grey shaded overlay). (C) HSC were stained with the cytoplasmic DDAO-SE dye on day 0 (left panel) and evaluated after 3 days of co-culture on OP9-Control (center panel) or OP9-W3A (right panel). Dye dilution as a result of proliferation is illustrated in these flow cytometry histograms. Shown is one representative of four (A and B) or three experiments (C).

Figure 6

The canonical Wnt3a ligand extinguishes expression of RAG1 in lymphoid progenitors. (A) Semi-quantitative RT-PCR analyses of RAG1 transcripts were performed on HSC, ELP or ProL. Transcripts represent gene expression of either freshly isolated bone marrow cells (marrow) or cells that were co-cultured overnight on OP9-Control (Control) or on OP9-W3A (W3A) cells. (B) Overlay histograms show RAG1/GFP protein levels of freshly isolated GFP⁻ cells (blue) or ELP (red) compared to ELP that were cultured for two days on OP9-Control (black, solid) or OP9-W3A (grey shaded overlay) stromal cells. Shown is one representative of three experiments.

Figure 7

Wnt3a stimulates myeloid differentiation from lymphoid progenitors. (A) Total myeloid (CD19⁻ B220⁻ CD11b⁺ CD11c⁻) cell yields/input of HSC, ELP and ProL are shown for 11days of co-culture on OP9-Control or OP9-Wnt3a. (B) Myeloid colony forming efficiencies (CFU) are shown for HSC, ELP or ProL that were either freshly isolated or pre-stimulated on OP9-Control or OP9-Wnt3a for 3days and sorted before re-culture in methyl-cellulose medium. Shown is one representative experiment of three. Asterisks indicate statistical significance, * (p<0.05), ** (p<0.01), *** (p<0.0001).

Figure 8

Lymphoid committed progenitors acquire alternate lineage fates in response to Wnt3a. (A) HSC, ELP or ProL were placed in 11day co-cultures on OP9-Control or OP9-Wnt3a stromal cells. The recovered cells were then assessed for a stem cell (Lin⁻ c-Kit^Hi Sca-1⁺ Thy1.1^lo) phenotype. Results are shown as total HSC yields/input. (B) ELP were pre-cultured on OP9-Control or OP9-Wnt3a stromal cells for 3days and sorted before re-culture under stromal cell-free erythroid supportive conditions. The contour plots show erythroid (Ter119⁺ CD11b⁻ Gr-1⁻) differentiation potential in one representative of three experiments. Asterisks indicate statistical significance, ** (p<0.01), *** (p<0.0001).