Erythroblasts are Hh responsive in vitro and in vivo. (A) BM was cultured for 18 hours, control or treated with rDhh, anti-Hh mAb (5E1), rDhh and 5E1 together, and isotype control mAb. After culture, cells were stained with anti-CD71 and anti-Ter119 to confirm that the short treatment had not changed the relative distribution of the subsets (top panel dot plots), and with PI to confirm that survival/cell cycle status was not affected (middle panel, histograms) in erythroblasts. Erythroblasts II to IV were purified from each culture by magnetic bead purification for Ter119⁺ cells, and RNA prepared for quantitative RT-PCR analysis of Gli1 expression (bar chart). (B) Erythroblast population II was sorted from magnetic bead lymphocyte-depleted cells from BM (left dot plot) and spleen (right dot plot). Gates used for sorting are shown. Bar chart represents Gli1 expression, measured by quantitative RT-PCR, on RNA prepared from population II sorted from WT (filled bars) and Dhh^−/− (open bars) BM and spleen.