Ectopic expression of C/EBPα induces myelopoiesis program in pluripotent embryonic cells. (A) Experimental setup: stage 8 animal caps were transplanted from cebpa and/or eGFP mRNA-injected embryos to unlabeled host embryos. Chimeras were recorded for live imaging (see supplemental Videos 1-9). (B) The boundaries between the eGFP⁺ transplants and nonfluorescent host embryos remain clear throughout embryonic development in the control eGFP transplants (i-ii). In contrast, blurred transplant margins and spotty pattern of eGFP⁺ cells were observed in the embryos containing cebpa expressing transplant cells (iii-iv). Confocal imaging at stage 40 showed that a subset of these scattered cells showed ramified morphology, reminiscent of macrophages (v). (C) Migration route of cebpa induced primitive myeloid cells before and after wounding. At stage 34, C/EBPα(+) graft derived migratory cells patrolled randomly before wounding (i, also see Video 4). Note that only a subset of GFP/CEBPα cells are migratory, whereas others retain the morphology of epidermal cells. After wounding, migratory GFP/CEBPα cells were immediately recruited to the wound site (ii; also see supplemental Video 5). Panels i and ii show the migratory paths of the cells before (i) and after (ii) wounding, as traced manually in supplemental Videos 4 and 5, respectively. Different colors were used to distinguish the migration routes of different cells. (D) Three-dimensional xyz confocal sections (20× objective, 1.0× zoom) of a macrophage (eGFP, green channel) that has phagocytosed bacteria (mCherry in red). Red bacteria are present in intracellular compartments surrounded by cytoplasm in the macrophages (green). For panels Bi-iv and C, images were obtained on a fluorescence stereoscope Leica MZ FLIII attached to a Sony CCD camera DXC-950 image capture system controlled by Northern Eclipse software 7.0 (Empix Image); 0.1× MMR was used as imaging medium. For panels B (v) and D, images were taken under Olympus IX81 FluoView FV1000 confocal microscope; 0.1× MMR, 2% methylcellulose (Sigma) 0.01% MS222 was used as imaging medium.