Tmod3 is required in both erythroblasts and macrophages for erythroblast-macrophage island formation in the fetal liver. (A) Representative images of native islands isolated from fetal livers at E14.5. Cells were stained with F4/80-Alexa647 (red) for macrophages, Ter119-Alexa488 (green) for erythroblasts, and Hoechst (blue) for nuclei. (B) Numbers of erythroblasts bound per macrophage at E13.5 and E14.5. Islands were prepared from 3 independent litters and 30 to 90 islands were counted for each genotype. (C) Representative images of reconstituted islands from Tmod3^+/+ or Tmod3^−/− erythroblasts (Ery) incubated with Tmod3^−/− macrophages (Mac). (D) Numbers of erythroblasts bound per macrophage. For each cell combination, 3 independent litters with a total of 80 to 100 islands were counted (9.9 ± 0.7 Tmod3^+/+ erythroblasts, but only 2.5 ± 0.2 Tmod3^−/− erythroblasts bound to each wild-type macrophage). (E) Representative images of reconstituted islands from Tmod3^+/+ erythroblasts (Ery) incubated with Tmod3^+/+ or Tmod3^−/− macrophages (Mac). (F) Numbers of erythroblasts bound per macrophage. For each cell combination, 3 independent litters with a total of 70 to 80 islands were counted; 14.6 ± 0.9 wild-type erythroblasts were attached to each Tmod3^+/+ macrophage, while only 5.2 ± 0.5 were attached to each Tmod3^−/− macrophage. All values are means ± SD. ***P < .001. Panels A, C, and E were acquired using a Bio-Rad Radiance 2100 confocal microscope with a Zeiss 63× oil immersion objective (N.A. 1.4). Scale bar, 10 µm. ND, not determined.