c-Myb is essential for early T cell development

Abstract

The c-Myb transcription factor is important for fetal hematopoiesis and has been proposed to mediate later stages of lymphocyte development. Using homozygous null c-Myb/Rag1 chimeric mice, we have determined that c-Myb plays an important role in the differentiation of macrophages and lymphocytes from precursor stem cells. We also determine that deletion of c-Myb leads to a complete block in early T cell development just before the oligopotent thymocyte matures into the definitive T cell precursor. These data indicate that c-Myb plays an important role at multiple stages of hematopoiesis and is required at an early stage of T cell development.

Figure 1

Flow cytometric analyses of B and T lymphocytes from Rag1^−/− ↔ c-Myb^−/− mice. Splenocytes and thymocytes were isolated from Rag1^−/− ↔ c-Myb^−/−, Rag1^−/− ↔ c-Myb^+/−, Rag1^−/−, and 129 control mice and identified with combinations of B and T lineage-specific antibodies (see text for details). Chimera 2 was used as an example; similar results were obtained from all other chimeras (data not shown). (A) Splenic B cells and bone marrow B cell precursors; (B) splenic and thymic T cells.

Figure 1

Flow cytometric analyses of B and T lymphocytes from Rag1^−/− ↔ c-Myb^−/− mice. Splenocytes and thymocytes were isolated from Rag1^−/− ↔ c-Myb^−/−, Rag1^−/− ↔ c-Myb^+/−, Rag1^−/−, and 129 control mice and identified with combinations of B and T lineage-specific antibodies (see text for details). Chimera 2 was used as an example; similar results were obtained from all other chimeras (data not shown). (A) Splenic B cells and bone marrow B cell precursors; (B) splenic and thymic T cells.

Figure 2

Flow cytometric analysis of splenic macrophages.Splenocytes were isolated from Rag1^−/− ↔ c-Myb^−/−, Rag1^−/− ↔ c-Myb^+/−, Rag1^−/−, and 129 control mice and identified with antibodies against the macrophage lineage markers Mac-1 and Gr-1 and the c-Myb^−/− cell-specific marker Ly9.1 (see text ). The mature macrophage population was gated on and analyzed for Ly9.1 expression as shown. Chimera 2 is shown; similar results were obtained from all other chimeras (data not shown).

Figure 3

c-Myb^−/− HSCs can generate early double-negative thymocytes. (A) Thymocytes were isolated from representative Rag1^−/− ↔ c-Myb^+/−, Rag1^−/−, phenotype positive Rag1^−/− ↔ c-Myb^−/−, and 129 control mice and analyzed for surface expression of T-lineage-specific markers. Cells expressing any one of the CD8/CD3/B220/Mac-1/Gr-1/Ter-119 markers were excluded from the analysis and the remainder plotted based on their expression of CD25 (x-axis) and CD44 (y-axis). For the Rag1^−/− ↔ c-Myb^−/− mice, representative phenotype-positive data are shown. (B) Analysis gates drawn on the CD44^loCD25⁻ and CD44^+/loCD25⁺ cells for analysis of the expression of the 129 lineage-specific surface marker Ly9.1 are shown.

Figure 3

c-Myb^−/− HSCs can generate early double-negative thymocytes. (A) Thymocytes were isolated from representative Rag1^−/− ↔ c-Myb^+/−, Rag1^−/−, phenotype positive Rag1^−/− ↔ c-Myb^−/−, and 129 control mice and analyzed for surface expression of T-lineage-specific markers. Cells expressing any one of the CD8/CD3/B220/Mac-1/Gr-1/Ter-119 markers were excluded from the analysis and the remainder plotted based on their expression of CD25 (x-axis) and CD44 (y-axis). For the Rag1^−/− ↔ c-Myb^−/− mice, representative phenotype-positive data are shown. (B) Analysis gates drawn on the CD44^loCD25⁻ and CD44^+/loCD25⁺ cells for analysis of the expression of the 129 lineage-specific surface marker Ly9.1 are shown.

Figure 4

TCRβ-chain gene rearrangement in thymocytes from the Rag1^−/− ↔ c-Myb^−/− mice. DNA was purified from thymuses from the Rag1^−/− ↔ c-Myb^−/− and Rag1^−/− mice and from both whole thymus and sorted CD4⁻CD8⁻CD3⁻ thymocytes from +/+ 129 mice and analyzed them for TCR β-chain gene rearrangement as described previously (Anderson et al., 1992). Results obtained from the Rag1^−/− ↔ c-Myb^−/− chimeras 1, 2, 9, and 11 (left to right) are shown and are representative of the other chimeras. (Solid arrow) Germ-line band; (open arrow) Dβ–Jβ-rearranged bands.

Figure 5

Transcription factors important in early and intermediate T cell development; data are from Glimcher and Singh (1999) and references therein.