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Fig. 3 Ebf3 is required in lateral plate mesoderm-derived cells for sternum osteoblast differentiation. (A) Left: The generation of lateral plate mesoderm-specific Ebf3-KO mice embryos by crossing the Ebf3flox/+ Prx1-Cre line with Ebf3flox/flox Rosa26Tdtomato/Tdtomato mice. Right: The expression pattern of Prx1-Cre-activated Tdtomato in the sternum tissue and muscle connective tissues based on longitudinal sections of E15.5 embryos together with immunostaining for tenascin (green). (B) Ossification analysis based on Alizarin Red skeletal preparations of thoracic bones in Ebf3flox/flox Prx1-Cre embryos at E18.5. (C) Longitudinal sections of the sternum of Ebf3flox/flox Prx1-Cre and Ebf3flox/+ Prx1-Cre embryos at E18.5, immunostained for osteoblast (green, osterix) and pre-osteoblast (green, Runx2) markers. Upper panels show the distribution of lateral plate mesoderm-derived cells labeled with Prx1-Cre-activated TdTomato (red) in interstitial cells surrounding the sternum, which was decreased in Ebf3-KO embryos (n=7) compared with heterozygous mice (n=7). Lower-enlarged panels show much fewer osterix/Runx2-positive lateral plate mesoderm-derived cells in the periosteum of Ebf3-KO mice compared with heterozygous mice (white arrowheads). Scale bars: 100 μm.