The Notch and Mef2-binding sites in the 200-bp region were mutated from GTGAGAA to ACACAGG and CTAAAAATA to AGGGGGGGC, respectively

The Notch and Mef2-binding sites in the 200-bp region were mutated from GTGAGAA to ACACAGG and CTAAAAATA to AGGGGGGGC, respectively. genetic screens in as our tool. Such synergies have been uncovered before in (Moberg et al, 2005; Ferres-Marco et al, Nestoron 2006; Vallejo et al, 2011), suggesting the presence of Nestoron several factors that can influence proliferation through synergistic or additive effects with Notch signals. The screen we carried out resulted in the identification of Mef2 (myocyte enhancer factor 2) as a crucial synergistic partner of Notch in triggering massive proliferation and an invasive metastatic phenotype. (Go et al, 1998; Baonza and Garcia-Bellido, 2000) and in vertebrates (Fre et al, 2005; van Es et al, 2005). Constitutively activated Notch is usually oncogenic in several distinct contexts, often in combination with other factors (Radtke and Raj, 2003; Ranganathan et al, 2011). In order to identify these factors, we sought to carry out a genetic screen Nestoron for modifiers of a large eye’ phenotype induced by the overexpression of a constitutively active, ligand-independent form of the Notch receptor (Nact), that has been shown to be oncogenic in mammals (Kiaris et al, 2004). Ectopic expression of Nact in the eye, using the eye-specific driver genome (Kankel et al, 2007), we identified two individual Gal4-driven mutations, d06622 and d03191, that strongly enhanced the large eye phenotype caused by Nact (Physique 1C). Open in a separate window Physique 1 Synergy between Notch and Mef2 in the eye. (A) Wild-type travel eye. (B) Activated Notch (Nact) in the eye results in a large eye when driven by the eye-specific possesses a single Mef2 gene that codes for an 57 kilodalton hJAL protein (Nguyen et al, 2002), whereas vertebrates harbour four genes (Mef2 A, B, C, and D) (Potthoff and Olson, 2007). Western blot and immunofluorescence analyses of both alleles revealed elevated expression levels of the Mef2 protein compared with wild type, proving the nature of the mutations (Supplementary Physique S1). Neither of the Exelixis Mef2 alleles on their own affected the adult eye morphology and the corresponding eye discs appeared wild type (Physique 1F). In contrast, coexpression of a single copy of either of these two Mef2 alleles with Nact resulted in massively overgrown discs, showing excessive EdU incorporation (Physique 1G). Expressing an Mef2 transgene caused an even stronger synergy with Nact (Supplementary Physique S1). Costaining with the neuronal differentiation marker Elav revealed that this hyperproliferative cell compartment was restricted to the anterior of the morphogenetic furrow harbouring the undifferentiated cells of the eye disc (Physique 1G). To examine whether the observed synergy is usually confined to the eye or is usually a more general phenomenon, we extended our analysis to the developing wing and coexpressed Nact and Mef2 under two different wing-specific drivers, have been previously reported and associated with invasive and metastatic behaviour (Ferres-Marco et al, 2006; Palomero et al, 2007). Open in a separate window Physique 2 Notch and Mef2 synergize to induce MMP1 expression and invasiveness. (A) An adult travel coexpressing Nact and the Exelixis Mef2 stock d06622 under E1Gal4 displays an ectopic eye in the abdomen (arrow). (BCG) Wing discs expressing various UAS constructs (as indicated) under domain name. The A/P boundary is usually marked with a dotted line. Low magnification images showing the topology of the wing disc are shown in K, L, M, and N. (KCK, LCL) Both control discs and discs expressing Nact alone show restricted expression of (Uhlirova and Bohmann, 2006). Therefore, we used MMP1 as a molecular marker to further characterize the Nact and Mef2 invasive phenotype. Wild-type wing discs show endogenous MMP1 in the trachea and a small region of the notum, consistent with previously published data (Physique 2B) (Page-McCaw et al, 2003). Expression of Nact using the wing pouch-specific driver tumour suppressor gene, causes an upregulation of MMP1, resulting in invasive cellular behaviour (Uhlirova and Bohmann, 2006). Furthermore, upregulation of JNK signalling has been linked to the control of both epithelial integrity and proliferation. We were thus led to examine whether the mechanism underlying the invasive, hyperproliferative behaviour of Nact and Mef2 cells is related to JNK signalling. To monitor JNK signal activation, we used the transgenic reporter, (is usually a transcriptional target as well as a feedback inhibitor of JNK. Activating Nact alone in the D/V boundary cells using (Physique 4B). In contrast, Mef2 alone led to a clear upregulation of (green), a marker for JNK activity. Note that Mef2 induces expression in the D/V boundary (C), which is usually enhanced many-fold when Nact is usually coexpressed (D). (ECI) DIC images of (L, M; green) to near normal levels. All wing discs are oriented with dorsal to the top and posterior to.