Gene targeting and expression analysis of mouse Tem1/endosialin using a lacZ reporter Hsiang-Po Huang a , Chia-Lun Hong b , Chung-Yang Kao b , Shu-Wha Lin b,c,d , Shu-Rung Lin e , Hua-Lin Wu f,g , Guey-Yueh Shi f,g , Li-Ru You h , Chieh-Lin Wu a , I-Shing Yu b,d,⇑ a Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan b Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University Hospital, Taipei, Taiwan c Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan d Center of Genomic Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan e Department of Bioscience Technology, College of Science, Chung-Yuan Christian University, Taoyuan, Taiwan f Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan g Department of Biochemistry, College of Medicine, National Cheng Kung University, Tainan, Taiwan h Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan article info Article history: Received 4 November 2010 Received in revised form 27 February 2011 Accepted 5 March 2011 Available online 12 March 2011 Keywords: Tem1 Endosialin Estrous cycle Gene targeting lacZ knock-in mice Mesenchymal tissues Renal glomerulus Vascular wall abstract TEM1 (endosialin) expression is increased in the stroma and tumor vasculature of several common human cancers. The exact physiological role of TEM1 is still unknown since Tem1-deficient mice are viable and show only a lower rate of abdominal site-specific tumor invasion in tumor transplantation experiments. Previous studies have reported Tem1 expression in mouse embryos and adults, but did not determine the timing or location of the earliest expression, and did not examine all organ systems. Using the highly sen- sitive Bluo-Gal staining method for detecting temporal and spatial Tem1-lacZ activity in lacZ knock-in (+/ lacZ) mice, we found that Tem1 gene expression was initially detectable in the dorsal aortic wall, the heart, the umbilical vessels, the first branchial arch, and the cephalic mesenchyme at E9.5. From E10.5 to E14.5, Tem1 gene expression was additionally seen mainly in the genital tubercle, the mesonephros, the whisker follicles, the mesenchymal tissues around the eye, and the lung. Remarkably, the kidney expressed abundant Tem1-lacZ starting from E16.5. Postnatally, Tem1 expression decreased in most organs but elevated expression persisted in the renal glomerulus and the uterus, where the expression pattern varied at different estrous cycle stages. Co-localization studies indicated that most vimentin- positive cells co-expressed Tem1-lacZ, while a large portion of CD31- or desmin-positive cells were also positive for Tem1-lacZ. Taken together, our observations suggest that Tem1 is expressed throughout embryonic and adult development in several types of mesenchymal cells closely related to blood vessels. Ó 2011 Elsevier B.V. All rights reserved. Tumor endothelial marker 1 (TEM1), also known as endosialin or CD248, is a type I transmembrane glycoprotein initially identi- fied by a monoclonal antibody against human fetal fibroblasts (Ret- tig et al., 1992). TEM1 is expressed in the stroma of common epithelial cancers but is absent or expressed at low levels in normal tissues and benign epithelial tumors (Rettig et al., 1992). It was also isolated as one of the most differentially expressed genes in a serial analysis of gene expression that compared RNA from hu- man colon cancer epithelia to normal colon mucosa (St Croix et al., 2000). Upregulation of TEM1 expression in tumor vasculature has been confirmed (Brady et al., 2004; Dolznig et al., 2005; Huber et al., 2006; MacFadyen et al., 2005). The exact localization of TEM1 protein has been claimed in pericytes but not in endothelial cells (MacFadyen et al., 2005). Tem1-deficient mice develop normally and their angiogenesis and wound healing is comparable to wild-type mice (Nanda et al., 2006). When tumor cells are transplanted into abdominal sites of Tem1-deficient mice, a lower rate of tumor growth, perito- neal carcinomatosis, and liver metastasis is observed compared to wild-type mice. Tumor cells transplanted to subcutaneous tissues do not show the same effects. This suggests that abdominal site- specific tumor invasion requires Tem1, probably via the interaction of Tem1 with tumor cell proteins. However, whether Tem1-defi- ciency induces other phenotypes in other mouse tissues remain to be clarified. Previous studies using immunohistochemistry or in situ hybrid- ization have demonstrated that Tem1 expression changes dramat- ically in selected mouse embryo stages and in adult mouse tissues (Carson-Walter et al., 2001; MacFadyen et al., 2007; Opavsky et al., 1567-133X/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gep.2011.03.001 ⇑ Corresponding author at: Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University, College of Medicine, Taipei, Taiwan. Tel.: +886 2 23123456x66646; fax: +886 2 23817083. E-mail address: ishingyu@ntu.edu.tw (I-S. Yu). Gene Expression Patterns 11 (2011) 316–326 Contents lists available at ScienceDirect Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep