DEV029355 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Evaluation of intramembranous ossification in the mandible at E15.5. Adjacent coronal sections of embryos from wild type (a-d) and Hand2^BA/BA (e-h) were stained with von Kossa (a,e) or hybridized with Bsp (b,f), osteocalcin (c,g) or Runx2 (d,h) probes. Hand2^BA/BA mandible shows more mineralized trabeculae (brown area; a,e) and broader expression of Bsp, osteocalcin and Runx2 (shown in white) than in wild-type mandible. Arrowheads indicate distal tip of the mandibles. md, mandibular bone. Scale bars: 200 µm.

• Supplemental Figure S2 -

  Fig. S2. Coronal sections of Hand2-lacZ transgenic embryos. E12.5 (a) and E14.5 (b). Labeled cells in a Hand2-lacZ transgenic embryo are observed in a similar pattern to Runx2-expressing cells at E12.5 (compare with Fig. 3Be); however, labeled cells are no longer detected in the mandibular bone or Meckel’s cartilage at E14.5 (arrow in b). mp, mandibular primordium; md, mandibular bone; mc, Meckel’s cartilage; t, tongue. Scale bars: 200 µm.

• Supplemental Figure S3 -

  Fig. S3. Interaction and regulation of Runx2 transcriptional activity by Hand1. (A) Schematic of Hand1 deletion mutants and functional domains. All mutants contained an N-terminal Myc-epitope tag. b, basic DNA-binding domain; HLH, helix-loop-helix; NLS, nuclear localization sequence; N, nucleus; N.D., not determined. (B) Mapping the Runx2-interaction domain of Hand1 by immunoprecipitation assays. COS cells were transfected with expression vectors encoding Myc-tagged wild-type Hand1 or Hand1 (Δ32-59) (as shown in A) and Flag-tagged Runx2. Myc-Hand1 was immunoprecipitated with an anti-Myc antibody, followed by western blot analysis to detect Flag-Runx2 binding. Five percent of input samples were immunoblotted with an anti-Flag or anti-Myc antibody. (C) Nuclear localization of wild-type and Hand1 mutant proteins detected with an anti-Myc antibody and a rhodamine-conjugated secondary antibody. (D) Identification of the Runx2-inhibition domain of Hand1. p6OSE2-luc reporter was transiently transfected along with Runx2 expression vectors and Hand1 deletion mutants as shown in A. Fold activity measured in the absence of Hand1 was normalized to 1.0. The data represent the mean ± s.e.m.