DEV002709 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Expression patterns of Coll II and Opn as revealed by double fluorescent in situ hybridizations on dermal bones. (A-D) Expression domains of Opn and Coll II in the dentary bone cells surrounding the Meckel’s cartilage. (E-H) High-magnification view of frontal bone cells near the sagittal suture. (C,G) Many cells of the developing dermal bones co-express Opn and Coll II (yellow; narrow arrows), while others express only Coll II (narrow arrowheads) or Opn (broad short arrows). (D,H) The same panels as in C,G overlaid over DAPI image. The large green spots in B,C,F,G are auto-fluorescing blood cells. Abbreviations as described in the manuscript. Scale bars: 200 μm in A; 100 μm in E.

• Supplemental Figure S2 -

  Fig. S2. Analysis of the cells from dissociated frontal bone of 17.5 dpc mouse embryos reveals distinct populations of cells as defined by expression and co-expression of skeletogenic markers. (A-D) Four populations of cells can be distinguished in double FISH analysis: cells expressing only Coll II (open arrowhead), cells expressing only Runx2 (broad arrow), cells expressing both Coll IX and Opn (narrow arrow), and cells expressing neither marker (open arrows in D). We find similar situation when we used double FISH with Coll II probe against PTHrP-R (E-H), Osx (I-L), and Osc (M-P). Note that we detected a high degree of co-expression between Coll II and PTHrP-R (E-H) but there was much less co-expression of Coll II and Osc, a mature osteoblast marker (M-P). Scale bars: 100 μm in A.

• Supplemental Figure S3 -

  Fig. S3. Molecular markers in the osteoblasts of the developing dentary bone. In the wild-type dentary (A-H) and frontal (I-P) bones, Ptc1 is broadly co-expressed with Coll IX (A-D; I-L) but has a more limited overlap with Opn expression (G-H; M-P). Scale bars: 200 μm in A.

• Supplemental Figure S4 -

  Fig. S4. Role of IHH in the developing dermal bone. (A-H) In situ hybridization analysis of the posterior frontal bone infected with RCAS::Ihh virus. Broad misexpression of RCAS::Ihh in and around the developing frontal bone (A,B) resulted in scattered expression of PTHrP (E-H). (I-P) Normally, cells of the developing frontal bone express Ptc1 in some overlap with expression of Opn.