Fig. 5. Comparison of osteogenic progenitor cell differentiation in the frontal primordium between the wild type and the Tgfbr2^fl/fl;Wnt1-Cre mutant samples. (A) At E13.5, definitive osteogenic differentiation marker Runx2 is expressed in the frontal primordium (arrowhead), indicating that these mesenchymal cells have committed to an osteogenic lineage. (B) Runx2 expression is also detected in the frontal bone primordium of Tgfbr2^fl/fl;Wnt1-Cre sample (arrowhead). (C,D) Type I collagen (ColI) is detected in the frontal primordium of both wild type and mutant samples. (E,F) Osterix expression is detected in the frontal primordium of both wild-type and Tgfbr2^fl/fl;Wnt1-Cre mutant sample (arrowhead). (G,H) Ibsp expression is present in the frontal primordium in both wild-type and Tgfbr2^fl/fl;Wnt1-Cre sample (arrowhead). (I) At E14.5, Runx2 is expressed throughout the frontal primordium, on both the calvarial (arrow) and the orbit (arrowhead) aspects. (J) In the E14.5 Tgfbr2^fl/fl;Wnt1-Cre sample, Runx2 expression is only detected in the orbital aspect of the frontal bone anlagen, suggesting that proper osteogenic lineage determination on the calvarial aspect of the frontal primordium (arrow) specifically requires TGFß signaling. (K,L) Type I collagen is detected in the frontal primordium (arrow) and the orbit region (arrowhead) of the frontal bone in both wild-type and mutant samples. (M,N) Osteonectin expression pattern is comparable within the frontal primordium (arrow and arrowhead) between the wild type and the Tgfbr2^fl/fl;Wnt1-Cre mutant sample. (O,P) Osterix expression is detected in the frontal primordium of the wild-type sample. There is a reduced osterix expression within the calvarial aspect of the frontal primordium (arrow), while normal osterix expression is present within the orbital surface region of the frontal bone in the Tgfbr2^fl/fl;Wnt1-Cre mutant sample (arrowhead). (Q-V) Bone matrix protein expression analysis shows that there is a defect in the production of bone matrix proteins, as indicated by reduced expression of osteopontin, Ibsp and osteocalcinin the calvarial aspect of frontal primordium of Tgfbr2^fl/fl;Wnt1-Cre mutant sample. Comparable expression of osteopontin, Ibsp and osteocalcin are detected in the orbit region of the frontal bone anlagen (arrowhead). (W,X) Alkaline phosphatase (ALPase) is present in the well-developed frontal bone primordium in the wild type, while its expression is greatly reduced in the Tgfbr2^fl/fl;Wnt1-Cre mutant, thus indicating compromised frontal bone matrix maturation. All insets show a low-power image (coronal-section) of the frontal region of the head at E14.5. The arrows in the insets indicate the frontal primordium region. All in situ probes were labeled with ³³P. Scale bars: 200 µm.