1. An Mll-Dependent Hox Program Drives Hematopoietic Progenitor Expansion
Patricia Ernst, Meghann Mabon, Alan J. Davidson, Leonard I. Zon, Stanley J. Korsmeyer 
Current Biology 
Volume 14, Issue 22, Pages (November 2004)
DOI: /j.cub

2. Figure 1 Mll−/− ES Cells Grow Normally and Differentiate into EBs
Embryonic stem cells of the genotypes indicated were allowed to differentiate and were photographed under phase contrast optics (A); alternatively, triplicate samples were harvested for cell enumeration from each clone tested (B). Wild-type, dashed line; Mll+/− EBs, blue lines; and Mll−/− EBs, red lines. Error bars reflect the standard deviation from the average of triplicate samples. (C) Flow cytometry analysis of Flk-1 expression. ES cells were allowed to differentiate for the indicated number of days (x axis), cells were dissociated with trypsin, and the percent Flk-1+ relative to isotype controls was determined by flow cytometry. Wild-type RW4 line, dashed line; average of two independent Mll+/− ES cell clones, blue lines; and average of two independent Mll−/− clones, red lines. Error bars reflect the standard deviation between duplicate samples for the Mll+/− and Mll−/− EBs.
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3. Figure 2
Development of Hematopoietic Populations in Mll-Deficient and Control EBs
(A) Development of the c-Kit+/CD41+ cells in Mll+/− (top row) and Mll−/− EBs (bottom row). ES cell clones were differentiated for the number of days indicated below the plots and dissociated with collagenase/dispase, and single-cell suspensions were analyzed by flow cytometry. Similar results were obtained with three different pairs of Mll+/− and Mll−/− clones.
(B) Percentages of c-Kit+ /CD41+cells in day 6 EBs. Analysis was performed as described above for eight independent experiments. Individual values are represented as black triangles, and red bars reflect the average of eight experiments.
(C) Hematopoietic-colony frequency is reduced within the Mll-deficient c-Kit+/CD41+ population. EB cultures were harvested as described in panel (B). c-Kit+/CD41+ cells were sorted, plated in duplicate, and scored as described in the Experimental Procedures. The graph represents the average of total colonies from duplicate cultures. Similar results were obtained with three different pairs of Mll+/− and Mll−/− clones. Mac, macrophage colonies; GM, granulocyte-macrophage colonies; and BFU-E, burst-forming units, erythroid.
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4. Figure 3 Gene Expression Differences during EB Differentiation
(A–L) Mll+/− (green bars) and Mll−/− EBs (red bars) were harvested daily for 10 days, and cDNA was prepared from total RNA. The resulting cDNA samples were subjected to real-time PCR analyses to detect the genes indicated above each graph. The values on the y axis represent relative transcript levels normalized to GAPDH for all reactions except those shown in panels (E), (J), and (L), for which actin served as the reference gene. For primer and probe sequences, see Figure S4. The values reflect averages of triplicate samples expressed in arbitrary units with the lowest normalized value defined as 1.
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5. Figure 4
Cdx4 Expression Rescues Hematopoietic-Colony Frequency in Mll−/− Progenitors
(A) Scheme for introducing genes into EB-derived hematopoietic cells and quantification of progenitor frequency. Day 6 embryoid body cells were enriched for CD41+ cells, plated on the OP9 stromal line, and infected with a GFP-expressing bicistronic retrovirus. GFP+ cells were then plated in methylcellulose cultures, and colonies were scored as described in the Experimental Procedures.
(B) Rescue of Mll−/− hematopoietic colonies by Cdx4. EB cells of the genotype shown below each set of bars were manipulated as described in (A). Five thousand GFP+ cells transduced with the virus indicated below each bar were plated as described above. Error bars represent the standard deviation for total colonies. GEMM, granulocyte-macrophage-erythroid-megakaryocyte colonies; Mac, macrophage colonies; GM, granulocyte-macrophage colonies; BFU-E, burst-forming units, erythroid; and Meg-Mix, mixed colonies containing megakaryocytes.
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6. Figure 5 Rescue of Mll−/− Hematopoietic-Colony Frequency
(A) Rescue of Mll−/− hematopoietic-colony frequency by Hox genes. The Mll+/−- and Mll−/−-GFP-transduced colony data are reproduced from Figure 4B. Colony abbreviations are as in Figure 4.
(B) Pitx2 expression does not rescue Mll−/− hematopoietic-colony frequency. Hoxb3 and Hoxb4 serve as positive controls for this experiment.
(C) BCL-2 expression does not rescue Mll−/− colony frequency. Hoxa9 serves as a positive control for this experiment. Cells were transduced and scored as described in Figure 4.
(D) Flow-cytometric analysis of cells from methylcellulose cultures. Both Mll+/− and Mll−/− EB cells were transduced with the viruses indicated above each column of FACS plots. Colonies quantified in the experiments above were allowed to grow for seven additional days after scoring, and cells were harvested and analyzed with anti-CD11b (Mac-1, y axis) and anti-Gr-1 (x axis) antibodies. All data shown are representative of at least five experiments performed in duplicate or triplicate.
(E) Model depicting the roles of Mll and Hox genes during hematopoietic development. The patterning of mesoderm by Hox gene expression is depicted as resulting in the production of hematopoietic progenitors indicated by the c-Kit+/CD41+ cells. Subsequently, the proliferative expansion of these cells is then dependent on the redundant Hox activity described in the Discussion; this is the point at which Mll is critical for maintaining appropriate levels of Hox gene expression (indicated by “Mll deficiency”). Subsequent differentiation is then influenced by the unique features of the particular Hox gene expressed in the studies described here.
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