Fig. S3.

Hand2 directly interacts with Bmp signaling to regulate gene expression within the cranial neural crest. (A and B) Immunohistochemistry for phosphorylated SMAD1/5/8, reveals no difference between Control (A) and H2 CKO [H2^fx/-;Wnt1-Cre(+)] (B) embryos (white arrowheads denote area of expression). (C–L) In situ hybridization for a previously characterized set of BMP targets in the PAs shows that expression of Gata3 (C and D), Id1 (E and F), and Smad6 (G and H) was not detectably altered in H2 CKOs; however, expression of the tumor suppressor genes Growth arrest and DNA-damage-inducible beta (Gadd45β; I and J) and gamma (Gadd45γ; K and L) was markedly down-regulated specifically within the distal cap of H2 CKOs (outlined-white arrowheads denote area of expression). Published H2 ChIP-Seq data confirms significant peak enrichment within the Gadd45β and Gadd45γ loci (3, 4). (M) Coimmunoprecipitation experiments reveal that H2 protein interacts with SMAD1, but not SMAD4. Importantly, a DNA binding-deficient mutant (H2Δb) also interacts with SMAD1, indicating that Hand2 and Smad1 proteins interact without need for H2 DNA binding. (N) Reticulocyte lysates programmed with Myc+H2 and/or Myc+E12 were used in EMSAs (O–R) to confirm specific H2-E12 heterodimer binding, not to the 5′-most SMAD binding element (O), but to each of the three D-box–containing elements (P–R).