Fig. 2. Analysis of erythrocytic cell development in the spleen of TR^0/0 and Pax8^-/- mice. (A,B) Distributions of erythrocytic populations in TR^0/0 (A) and Pax8^-/- (B) mice. For each type of mouse, data from mutant, mutant treated with TH and littermate controls are presented. Freshly dissociated mouse spleen cells obtained from 15-day-old mice were counted and stained with anti-CD71 and anti-TER119 antibodies to distinguish the different developmental stages of erythroblast maturation (stages I to IV, as indicated) (Socolovsky et al., 2001). Percentages of each subset are indicated in the gates. Dead cells and enucleated red blood cells (with low forward scatter) were excluded from FACS analysis. (C) Benzidine-MGG-stained cytospin preparations of freshly isolated spleen cells from wild-type, Pax8^-/- and TH-treated Pax8^-/- mice, as indicated. The red arrowheads point to representative late basophilic erythroblasts. (D) Graphical representation of the distribution of the respective erythrocytic populations relative to the BFU-E population. For each tested mouse, the frequency of each cell population was divided by that of the BFU-Es. Numbers of animals analyzed were 5 for the control and Pax8^-/- and 4 for the Pax8^-/- +TH. (^*, P=0.003). (E,F) Total numbers of nucleated cells TER119⁺ KI67⁺ and TER119⁺ AnnexinV⁺, respectively, plotted as a cellularity index in control, Pax8^-/- and TH-treated Pax8^-/- mice. Spleen cells from 15-day-old mice were counted and analyzed by flow cytometry using anti-TER119 together with either anti-KI67 (E) or anti-Annexin V (F) antibody (^* P<0.01; n=3 for each genotype and treatment).