Gfi1 expression persists in miR-22^KOanimals throughout megakaryocytic differentiation. (A) Bone marrow from individual adult 129SV;miR-22 wildtype and miR-22 homozygous knockouts was stained according to surface markers listed in supplemental Table 3, DAPI for live/dead assessment, and sorted by FACS. Gene expression by qPCR for Gfi1 (left) and Gfi1b (right) in common myeloid progenitors through MK differentiation is shown. ActB was used as a housekeeping gene to quantify relative expression. Expression is shown relative to miR-22^+/+ CMP, with the dotted line showing Relative Expression = 1 (n = 2-3). Error bars represent standard error of the mean. (B) Ex vivo MK differentiation of primary miR-22^KO bone marrow cells. Bone marrow mononuclear cells were isolated from individual adult 129SV:miR-22 wildtype and miR-22^KO and were subjected to ex vivo MK differentiation by treatment with TPO. For gene expression analysis, MKs were enriched by 2-step bovine serum albumin gradient sedimentation. qPCR for Gfi1 in ex vivo differentiated MKs at 2 and 5 days after initiation of TPO treatment. ActB was used as a housekeeping gene to quantify relative expression. Expression is shown relative to day 2 miR-22^+/+ MKs, with the dotted line showing Relative Expression = 1 (n = 2-3). Error bars represent standard error of the mean. (C) Proposed model whereby repression of GFI1 by miR-22 permits megakaryopoiesis. miR-22 promotes megakaryopoiesis (dotted green line) through direct repression of GFI1 (solid red line). GFI1 represses miR-22 expression by binding at the promoter of the MIR22HG. BM, bone marrow; MkP, megakaryocyte progenitor; PreMegE, megakaryocyte-erythrocyte precursor. *P ≤ .05; **P < .01.