Again, the effect was dose-dependent, and at the highest levels of DR-KLF2 transcript injected (250 pg), 87% of embryos exhibited a reduction in expression (Table 1). vascular development. Mouse transgenic studies have shown that endothelial-specific expression of is regulated by an enhancer in the first intron, which contains binding elements for the transcription factors TAL1 (SCL) and members of the GATA and ETS families (Kappel et al., 1999). Mutation of the GATA or ETS motifs abolishes reporter expression in endothelial cells of transgenic mice, whereas mutation of the TAL1 site results LY278584 in reduced expression levels (Kappel et al., 2000). Additional ETS motifs are located in the promoter region of the mouse gene and these have been shown to function together with HIF-2 (EPAS1 – Mouse Genome Informatics) to regulate (Schlaeger et al., 1997) and VE-cadherin (cadherin 5) (Gory et al., 1999). Gain-of-function experiments have shown that ETS factors can upregulate endothelial gene expression in cultured cells (Birdsey et al., 2008; Hasegawa et al., 2004; Schwachtgen et al., 1997; Wakiya et al., 1996). Overexpression Rabbit Polyclonal to TPH2 (phospho-Ser19) of the ETS factor ERG in embryos is sufficient to activate ectopic transcription of the vascular marker and gene, which shows greatly reduced angioblast cell numbers and severe disruption of vascular development (Lee et al., 2008). Zebrafish studies have shown that knockdown of four vascular ETS genes results in a near complete loss of endothelial cells, whereas single knockdowns of individual genes exhibit less severe phenotypes (Pham et al., 2007). The Krppel-like factor (KLF) family of transcription regulators is also involved in the regulation of vascular gene expression (Atkins and Jain, 2007). KLFs bind a consensus recognition sequence of CACCC (Bieker, 2001; Dang et al., 2001), and three of the 17 family members, KLF2, KLF4 and KLF6, are expressed in the mouse embryonic vasculature (Kuo et al., 1997; Yet et al., 1998; Kojima et al., 2000; Botella et al., 2002; Lee et al., 2006). KLF proteins can act as either transcriptional activators or repressors and domain mapping of KLF2 has identified transactivating and transrepression domains within the protein (Conkright et al., 2001). Numerous endothelial genes have KLF binding sites in their promoter regions and cell culture studies have shown that KLF2 activates the expression of vascular genes including thrombomodulin (Lin et al., 2005) and (transcriptional regulation, both cell culture and microarray studies using adult endothelial cells have suggested that KLF2 functions as a repressor of expression (Bhattacharya et al., 2005; Dekker et al., 2006). Our investigation into the transcriptional regulation of embryo and that inhibition of KLF2 function results in the disruption of normal vascular development. Furthermore, we show that ETS and KLF proteins physically interact and synergistically activate embryonic expression of the gene. MATERIALS AND METHODS Preparation of in situ probes and mRNAs The insert from a full-length clone (“type”:”entrez-nucleotide”,”attrs”:”text”:”BC043732″,”term_id”:”27695176″,”term_text”:”BC043732″BC043732) was isolated using sequences were inserted into pGEM T-easy, linearized with and in situ hybridization probes has been described previously (Cleaver et al., 1997; Baltzinger et al., 1999; Devic et al., 1996). The KLF2 coding region was PCR amplified from “type”:”entrez-nucleotide”,”attrs”:”text”:”BC043732″,”term_id”:”27695176″,”term_text”:”BC043732″BC043732 with Pfu polymerase, subcloned into pT7TS and the sequence verified. For synthesis of mRNA, antisense MO (MO, 5-ATCCGAATCAGATTGTCAGCAAAAC-3) was targeted to the 5 untranslated region (UTR) of transcripts. MO effectively blocked translation of test transcripts containing the 5 UTR plus a portion of the coding region sequences of fused to the coding region of EGFP (see Fig. 3D,E). For in vivo experiments, 12.5, 25 or 50 ng of MO or control antisense MO (5-GGTAGTAATAGATGCTGTGATCTAT-3) was microinjected into the mediolateral region of one cell of two-cell staged embryos and later assayed at stage 34 for transcripts by whole-mount in situ LY278584 hybridization. For measuring transcript levels, or control MO was injected at the one-cell stage. Open in a separate window Fig. 3. Inhibition of KLF2 function results in reduced expression in the embryo. (A-C) Whole-mount in situ hybridization analysis of and expression in embryos (stage 34, lateral view). For each gene, expression is observed in the endothelial cells of the major developing vessels, including the posterior cardinal vein (PCV), intersomitic vessels (IS), aortic arches (AA) and in the forming plexus on the flank of the embryo LY278584 (PL). (D,E) MO effectively blocks translation of a control transcript. (D) Bright-field and fluorescent images of embryos injected with a control transcript in which the 5 UTR of transcript plus MO (25 ng). Note that.