Fig. 3. Zfp64 is expressed in a broad range of mesenchymal tissues. (A,B) Whole-mount in-situ hybridization using a (A) mouse dpc 11 embryo and (B) embryonic day 3.5 (HH stage 22) chick embryo. (C) Chick Hey1 expression. (D) Section view of B. (E) Zfp64 expression in an E6 (HH stage 28-29) embryo. (F) Section view of E. (G,H,I) In-situ hybridization to embryonic day 18.5 mouse embryo. Zfp64 is expressed in (G) the basal layer of the dermis and subepithelial mesenchymal cells, (H) chondrocytes and (I) osteoblasts. (J,K,L) Immunohistochemical staining of an embryonic day 18.5 mouse embryo using anti-Zfp64 antibody shows nuclear staining in an expression pattern similar to that seen in G-I. (M) Zfp64 expression in adult organs. Ubiquitous expression of Zfp64 was observed by RT-PCR. (N) Zfp64 expression in mesenchymal cell lines, measured by real-time PCR. ^*, Since U2OS cells are a human cell line, the PCR primers were different from those used for the other cell lines. (O) Western blot of C2C12 or U2OS cell lysates using anti-Zfp64 antibody. At least two bands of different molecular mass were observed. The band of the higher molecular mass appears to correspond to isoform A and/or C, and the small protein appears to correspond to isoform B and/or D of Zfp64. (P) Immunocytostaining of C2C12 and U2OS cells using anti-Zfp64 antibody.