Fig. 4. Analysis of dissociated frontal bone cells of HH39 chick embryos reveals distinct cell populations defined by the expression of skeletogenic markers. (A-D) Four populations of cells were identified: cells expressing only Coll IX (arrowheads in C), only Opn (broad arrows in C), both Coll IX and Opn (narrow arrows in C), and cells expressing neither marker (arrow in D). FISH revealed a similar situation with Opn and Runx2 (E-H), with Runx2 and Coll IX (I-L), and with PTHrP-R and Coll IX (M-P). (M-P) Despite the high degree of co-expression between Coll IX and PTHrP-R that was observed, some slides showed many cells expressing neither marker (arrows, P), suggesting that many of these cells were early osteoblasts not expressing Coll IX. (Q) Analysis of Col2::Cre3xRosa26::lacZ dentary bones with both alizarin red and ß-galactosidase staining revealed a considerable contribution of Coll II-expressing cells to the developing dentary bone. (R-T) Similar analysis of ß-galactosidase and H&E stainings of the developing frontal bone in 17.5 dpc Col2::Cre3xRosa26::lacZ embryos showed many ß-galactosidase-positive cells within this intramembranous bone. Db, dentary bone; FB, frontal bone. Scale bars: 50 µm.