Analysis of a limb-specific regulatory element in the promoter of the link protein gene

Supplemental to figure 1 of "Analysis of a limb-specific regulatory element in the promoter of the link protein gene." This supplemented figure 1 shows the details of the X-gal staining from embryos containing the -1020 and -690 nt Hapln-lacZ transgenes. Fig. 1. Link protein gene promoter constructs show in vivo expression patterns in either the skeleton or limb/genitalia. (A) Two different link protein promoter fragments consisting of the −1020 and −690 nt regions were tested in transgenic mice. (B) A representative E15.5 mouse transiently expressing the −1020 promoter region showed only X-gal staining in appendicular and axial cartilaginous skeletal tissues. A dark background was used to provide contrast to the photograph to allow easy visualization of the discrete expression in cartilage sites. (B-1) Sectioning showing staining in the tibia after X-gal and eosin counter-staining. (B-2) Immunofluorescence using antibody to Hapln1. (C) and (D) The shorter −690 promoter region demonstrated high-level expression in the limb and genitalia as shown in two representative mouse embryos. As shown, embryos showed variability of X-gal staining in the limb and hind paw regions. The embryos in C and D were dissected and either the paws (C1, D1; 5x) or genital tubercle (C2, D-2;20x) were examined by tissue-sectioning and counter-staining with eosin. In paws and genitalia mesenchymal cells show X-gal staining underneath the un-stained epithelial cells.

作为《分析连接蛋白基因启动子中特定于肢体调控元件》一文中图1的补充，本图1详细展示了含有-1020和-690碱基对Hapln-lacZ转座基因胚胎的X-gal染色细节。图1. 连接蛋白基因启动子构建体在骨骼或肢体/生殖器中的体内表达模式。（A）在转基因小鼠中测试了由-1020和-690碱基对组成的两种不同的连接蛋白启动子片段。（B）一个代表E15.5小鼠，瞬时表达-1020启动子区域，仅在肢体和轴向软骨组织中呈现X-gal染色。为了使照片中的软骨位点离散表达易于观察，采用了深色背景以提供对比。（B-1）X-gal和苏木精复染后对胫骨进行切片，以显示染色。（B-2）使用针对Hapln1的抗体进行的免疫荧光。（C）和（D）较短的-690启动子区域在肢体和生殖器中表现出高水平表达，如两个代表性小鼠胚胎所示。如图所示，胚胎在肢体和后爪区域表现出X-gal染色的变异性。C和D中的胚胎被解剖，通过组织切片和苏木精复染对爪（C1，D1；5倍）或生殖隆突（C2，D-2；20倍）进行了检查。在爪和生殖器中，间充质细胞在未染色的上皮细胞下方呈现X-gal染色。