Article Figures & Data
Figures
- FIGURE 1.
RT-PCR analysis of expression of components of the BMP signaling pathway in mouse tissues. A, RT-PCR analysis of expression of RNA isolated from E15 head. HPRT (lane 1), BMP2 (lane 2), BMP4 (lane 3), BMPR-IA (lane 4), BMPR-IB (lane 5), BMPRII (lane 6), Smad-1 (lane 7), Smad-5 (lane 8), Smad-8 (lane 9), and Smad-4 (lane 10). The upper filled arrow indicates the position of the Smad-4 band in lane 10 and the lower semifilled arrow indicates the position of the Smad-8-specific band in lane 9. B, As above, but analysis made of RNA isolated from E15 thymus. C, Expression of HPRT in RNA isolated from E15 head (EH), E15 thymus (ET), and adult Rag1−/− thymus (RC). D, Expression of Noggin in RNA as described in C. The specific band is indicated with an arrow. E, Expression of Tsg in RNA as described in C. F, Expression of Chordin in RNA as described in C.
- FIGURE 2.
Determination of site of expression of BMP-4 and its receptor BMPR-IB in the thymus. Frozen sections of thymus were double stained with anti-BMP4 and anti-cytokeratin (A), or anti-BMP RIB and anti-CD2 (B). A, BMP-4 is found in cytokeratin-positive epithelial cells appearing both in the subcapsular (short arrows) and cortical (long arrows) regions. B, BMP RIB expression is found on most thymocytes (revealed with anti-CD2 Abs). Inset, A detail of BMP RIB-positive thymocytes in the cortical area: C, cortex; Sc, subcapsular area. Scale bars: 50 μm. C, RT-PCR analysis of expression of HPRT (lane 1), BMPR-IA (lane 2), BMPR-IB (lane 3), BMPR-II (lane 4), Smad-1 (lane 5), Smad-4 (lane 6), Smad-5 (lane 7), and Smad-8 (lane 8) in RNA isolated from sorted mouse DN thymocytes. D, RT-PCR analysis of expression of HPRT, Tsg, Chordin, and Noggin in deoxyguanosine-treated E14.5 thymic explants (lane 1) and thymocytes isolated from Rag−/− mice and sorted for expression of CD44 and CD25 to eliminate any contaminating epithelial cells.
- FIGURE 3.
BMP4 arrests thymocyte development at the CD44+CD25− DN stage in a dose-dependent manner. A, E14.5 FTOC were cultured for 3 days with 0.1 μg/ml BMP4 and analyzed by flow cytometry. Upper panel, Staining with anti-CD4 and anti-CD8. Lower panel, The composition of the DN subsets, stained with anti-CD44 and anti-CD25. BMP4 treatment increased the proportion of cells in the CD44+CD25− subset and decreased the proportion of cells in the CD44−CD25+ subset. B, Cells from the cultures in A were stained with anti-CD2. BMP4 treatment reduced cell surface CD2 expression. C, E14.5 FTOC were cultured for 5 days with 0.1 μg/ml BMP4 and analyzed by flow cytometry. Upper panel, Staining with anti-CD25 and anti-CD44, gated on DN cells. Lower panel, The composition of the DN subsets, stained with anti-CD117 and anti-CD25. BMP4 treatment increased the proportion of cells in the CD117+CD25− subset. Cell recoveries were 4.5 × 104 and 5.5 × 104 in control and BMP4-treated cultures, respectively. D, The scatter plot shows the cell recovery from E14.5 FTOC cultures for 5 days. The total number of cells recovered in each experiment from BMP4-treated cultures was divided by the number of cells recovered from the control cultures, to give the relative cell number from six individual experiments. E, The scatter plot shows the relative number of CD44+CD25− DN cells recovered from the same E14.5 FTOC used in the scatterplot of D. For each experiment, the number of CD44+CD25− DN cells recovered from BMP4-treated cultures was divided by the number of CD44+CD25− DN cells recovered from control cultures to give the relative number of CD44+CD25− DN cells from six different experiments. F, E14.5 FTOC were cultured for 6 days in diluting concentrations of BMP4 (0.1, 0.01, or 0.001 μg/ml) and analyzed by flow cytometry. Bars show the mean and SDs from six separate experiments. Left panel, The percentage of DN cells in the four DN subsets. Right panel, The ratio of CD44+CD25−:CD44−CD25+ cells. The ability of BMP4 treatment to arrest thymocyte differentiation at the CD44+CD25− DN stage was dose-dependent.
- FIGURE 4.
BMP4 treatment is not toxic. BMP4 enhances thymocyte survival and inhibits thymocyte proliferation. A, The effect of BMP4 treatment on DN thymocytes is neutralized by addition of the BMP4 antagonist Noggin. E14.5 FTOC were treated for 6 days with 0.1 μg/ml BMP4 alone (left dot plot), 0.1 μg/ml BMP4 and 0.5 μg/ml Noggin (middle dot plot), or no treatment (right dot plot). Cells were analyzed by flow cytometry, and anti-CD25 and anti-CD44 staining on DN cells are shown. Noggin inhibited the ability of BMP4 to arrest thymocyte differentiation, indicating that BMP4 is not nonspecifically toxic in the cultures. Cell recoveries were 1.3 × 104 for BMP4-treated cultures, 1.3 × 104 for BMP4 and Noggin treated cultures, and 1.8 × 104 for control cultures. B, BMP4 treatment enhances thymocyte survival. E14.5 FTOC were treated for 5 days with 0.1 μg/ml BMP4. Apoptosis was measured by annexin V staining. The graph shows the percentage of annexin-positive cells in the DN subsets and in the whole thymocyte population. Bars show data from six individual experiments. BMP4 treatment reduced the percentage of apoptotic cells in all subsets relative to control cultures. This reduction is statistically significant. C, BMP4 treatment inhibits thymocyte proliferation. Cells from the same cultures as in B were stained with PI and anti-CD44. The graph shows the percentage of cells in the S and G2 in all cells, the CD44+ fraction and the CD44− fraction. Data are from six individual experiments. BMP4 treatment significantly reduced the proportion of cells in S and G2 relative to the control.
- FIGURE 5.
Noggin accelerates the development of DN thymocytes. A–E, E14.5 FTOC were cultured for 6 days with 0.5 μg/ml Noggin and analyzed by flow cytometry. Cell recoveries in A, B, and D were 1.6 × 104 for the control cultures and 3 × 104 for the Noggin-treated cultures. A, CD4 and CD8 staining showed that Noggin increased the percentage of DP thymocytes. B, Composition of DN subsets, stained with anti-CD44 and anti-CD25. C, Proportion of DN cells in the four subsets defined by CD44 and CD25. Data are the mean and SD of six individual experiments. The increase in the percentage of CD25−CD44− DN cells in the Noggin-treated cultures is statistically significant, relative to the control. D, CD2 expression in Noggin-treated and control cultures. Noggin increased cell surface CD2 expression. E, The graph shows mean CD2 expression in Noggin-treated and control cultures. Data are from six individual experiments. F, The scatter plot shows the cell recovery from E14.5 FTOC cultures for 5 days. The total number of cells recovered in each experiment from Noggin-treated cultures was divided by the number of cells recovered from the control cultures to give the relative cell number from 13 individual experiments.
- FIGURE 6.
Noggin accelerates the development of DN thymocytes and enhances thymocyte survival. A–C, E13 FTOC were treated for 5 days with 0.5 μg/ml Noggin or 0.1 μg/ml BMP4 and analyzed by flow cytometry. A, Analysis of DN cells, stained with anti-CD25 and anti-CD44. B, Cell surface CD2 expression in the cultures. C, Cell surface CD4 and CD8 expression in the cultures. D and E, E14.5 FTOC were cultured for 5 days with 0.5 μg/ml Noggin. D, The graph shows the percentage of cells from these cultures in the S and G2 phase of the cell cycle in all cells, and in the CD44+ and CD44− fractions. There were no significant differences in cell cycle status between the Noggin-treated and control cultures. Data are derived from six individual experiments. E, Noggin treatment enhances thymocyte survival. Apoptosis was measured by annexin V staining. The graph shows the percentage of annexin-positive cells in the DN subsets and in the whole thymocyte population. Histograms show data from six individual experiments. Noggin treatment reduced the percentage of apoptotic cells, relative to control cultures. This reduction is statistically significant.
- FIGURE 7.
Noggin cannot promote differentiation of DN thymocytes in the absence of pre-TCR signaling. A, E17.5 TCRβ−/−δ−/− FTOC were cultured for 5 days with 0.5 μg/ml Noggin and analyzed by flow cytometry for CD4 and CD8 expression. Noggin did not induce differentiation to the DP stage. Cell recoveries were 1.3 × 103 for the control culture and 2 103 for the Noggin-treated culture. B–D, E14.5 TCRβ−/−δ−/− FTOC were cultured for 5 days with 0.1 μg/ml BMP4, both 0.1 μg/ml BMP4 and 0.5 μg/ml Noggin, or 0.5 μg/ml Noggin alone. B, DN cells were analyzed for CD25 and CD44 expression. C, Apoptosis in DN subsets was assessed by annexin V staining. The graph shows the percentage of annexin-positive cells in the DN subsets and in the whole thymocyte population. Bars show data from six individual experiments. Neither Noggin nor BMP4 treatment induced apoptosis. D, PI and anti-CD44 staining. The graph shows the percentage of cells from these cultures in the S and G2 phase of the cell cycle in all cells, and in the CD44+ and CD44− fractions. The decrease in the percentage of CD44+ cells in S and G2 in the BMP4-treated cultures was statistically significant compared with the other cultures. E, The scatter plot shows the cell recovery from 5-day FTOC cultures. The total number of cells recovered in each experiment from BMP4, BMP4 and Noggin, and Noggin-treated cultures was divided by the number of cells recovered from the control cultures, to give the relative cell number from nine individual experiments.
Tables
- Table I.
Expression of CD117 on CD44+CD25− DN cells from E14.5 FTOC treated with 0.1 μg/ml BMP4 for 5 daysa
Control BMP4 Percentage of CD44+CD25− DN cells that express CD117 97.3 96.9 -
a Cells were stained with Abs against CD3, CD4, CD8, and CD25 labeled with the same fluorochrome, and with anti-CD44 and anti-CD117. Cells that stained positive for CD3, CD4, CD8, and CD25 were excluded, and the percentage of CD44+ cells that stained with the anti-CD117 Ab in control and BMP4-treated cultures is given.
-
In this issue
