Xenbase Image ID: 125453

Figure 2. Shox2 regulates the expression of Bmp4. (A) Bmp4 expression in uninjected embryos at stage 10.5 (n=35). Black arrowhead indicates the dorsal lip. (B) Bmp4 expression is reduced or absent in 87% of LiCl-treated embryos (n=31). (C) Spots of Bmp4 expression in 77% of previously Shox2-injected, LiCl-treated embryos (n=30). Shox2 RNA was injected diagonally into two blastomers of a 4-cell stage embryo. Bmp4 staining in two areas of the embryo (marked by white arrows) indicates an upregulation of Bmp4 expression corresponding to the Shox2 injection. (D) Shox2 increases the activity of the human and Xenopus laevis Bmp4 promoter upon co-transfection of Shox2 expression plasmids (1 µg) and the indicated Bmp4 reporter-constructs (1 µg) into Cos-7 cells. Data using HEK-293 cells are not shown. (E) Electrophoretic mobility shift assay of the GST–SHOX2 fusion protein on a conserved promoter oligonucleotide sequence (BMP4). An excess of non-radioactively labelled oligonucleotides (homologous competitor) reduces the DNA-binding ability of SHOX2, whereas excess of a random oligonucleotide sequence (heterologous competitor) does not affect DNA-binding. 1, free oligonucleotide; 2, GST alone; 3, GST–SHOX2; 4–7, 10-fold, 50-fold, 75-fold and 150-fold molar excess of homologous competitor; 8, GST–SHOX2; 9–12, 10-fold, 50-fold, 75-fold and 150-fold molar excess of heterologous competitor. (F) Chromatin immunoprecipitation (ChIP) assay. HEK-293 cells were co-transfected with the FLAG-SHOX2a expression or empty control vector and with either the hBMP4 2013-Luc reporter or empty control vector as indicated. Formaldehyde-crosslinked DNA was immunoprecipitated using an anti-FLAG antibody (α-FLAG-Ab) or no antibody, as a negative control. Precipitated DNA fragments (ChIP) and DNA from lysate before immunoprecipitation (Input) were subjected to PCR using primer sets amplifying the putative SHOX2-binding element in the BMP4 gene (lower panel) or GAPDH (upper panel) as a control. Image published in: Puskaric S et al. (2010) © The Author 2010. Creative Commons Attribution-NonCommercial license Gene Synonyms Species Stage(s) Tissue bmp4 bmp-4, bmp2b, bmp2b1, bmp4-a, bmp4-b, DVR-4, ofc11, XBMP-4, xbmp4, zyme X. tropicalis Throughout NF stage 10.5 marginal zone Image source: Published Experiment + Assay Source Phenotypes and Disease Xtr Wt + LiCl + NF10.5 (in situ hybridization) Fig.2.B Expression Phenotype decreased amount bmp4 expression in marginal zone absent bmp4 expression in marginal zone Larger Image Printer Friendly View

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