Anemia, reticulocytosis, and analysis of hematopoietic stem cells, MEPs, and erythroid cell populations in the bone marrow of HNF1A^−/− mice. (A) Flow cytometric reticulocyte quantification using thiazole orange in HNF1A^+/+ and HNF1A^−/− mice (n = 7 each). Representative images of Ter119⁺Thiazole orange⁺ reticulocytes (i), quantification of reticulocytes in percentage (ii) and total numbers per microliter of whole blood (iii). (B) Calculation of mature RBC counts per microliter of blood as performed by subtraction of reticulocyte counts from total RBC counts. (C) Representative images and gating strategy for LSK (Lin⁻Sca-1⁺c-Kit⁺IL-7R⁻) cells and MEPs (Lineage⁻IL-7R⁻Sca-1⁻c-Kit⁺CD34⁻CD16/32^−/low [FcγRII/III]) in bone marrow from HNF1A^−/− and HNF1A^+/+ mice (n = 6 each) as analyzed by flow cytometry (i). LSK and MEP cell populations were determined according to Kondo et al,²⁶  Pronk et al,²⁷  and Murphy et al²⁸  after eliminating lineage-positive cells (Lin⁻: CD11b, Gr-1, CD49b, CD11c, CD3ε, CD4, CD8, B220, Ter119). Percentage (ii) and total cell numbers per femur length (iii) of LSKs and MEPs as provided. Cell counts per femur were divided by femur length to normalize for the shorter HNF1A^−/− femura. (D) Representative fluorescence-activated cell sorter (FACS) plots and gating strategy of erythroid cell populations by combination of Ter119 and CD71 expression and determination of cell size in regions I to VI, respectively, as described.^29,30  Erythroid populations were distinguished in early proerythroblasts (Pro; Ter119^medFSC^highCD71^high), basophilic erythroblasts (Baso; Ter119^highFSC^highCD71^high), polychromatic erythroblasts (Poly; Ter119^highFSC^med/highCD71^med/high), orthochromatic erythroblasts (Ortho; Ter119^highFSC^medCD71^med), reticulocytes (Retic; Ter119^highFSC^lowCD71^low), and mature RBCs (Ter119^highFSC^lowCD71⁻) (i). Total cell numbers of proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts (ii), orthochromatic erythroblasts, reticulocytes, and mature RBCs (iii) per femur normalized for femur length are provided as indicated. All data are presented as mean ± SEM. *P < .05, **P < .01, and ***P < .001 for HNF1A^+/+ vs HNF1A^−/− (Student t test).