Loss of DOT1L results in the impairment of both primitive and definitive yolk sac erythropoiesis. (A-B) Identical numbers of dissociated E8.5 yolk sac cells were cultured for 7 days in media specifically supporting the growth of primitive erythroid colonies. Colonies were counted (A) and sizes were measured (B) with ImageJ software (National Institutes of Health). (C-D) Identical numbers of dissociated E10.5 yolk sac cells were cultured in methylcellulose growth medium, which supports both erythroid and granulocyte-macrophage progenitors. Colonies were scored and counted (C) after 10-12 days in culture. (D) The areas of multiple, single BFU-E and CFU-GM colonies were measured to estimate colony size with ImageJ software (National Institutes of Health). (E) Identical numbers of dissociated E10.5 yolk sac cells were plated in methylcellulose medium, which only supports the growth of erythroid progenitors (erythroid CFUs and mature BFU-Es). Colonies were identified and counted after 3 days of culture. (F) C-kit⁺ cells were isolated from individual yolk sacs by sorting. Identical numbers from each genotype were cultured in methylcellulose medium supporting both erythroid and myeloid lineages for 10 days. Total erythroid (BFU-E) and myeloid (CFU-GM) colonies were counted. (G-L) Representative images of primitive erythroid (G-H), BFU-E (I,J), and CFU-GM (K-L) colonies showing size and appearance differences between WT (G,I,K) and Dot1L KO (H,J,L). (G-H) Derived from experiments described in Figure 3A and B. (I-L) From the experiment described in Figure 3C and D. Cultures were viewed with a NIKON ECLIPSE TS100 microscope equipped with 10×-20× phase contrast, numerical aperture 1.2 objective lens. Images were taken with a LEICA DFC480 camera with the use of Leica Firecam software at room temperature. *Average number (A,C,E) and size (B,D,F) of KO colonies were significantly different from WT and heterozygotes (P < .01, t test). Error bars represent standard errors within each group.