An RNAi screen for targets of the Notch signaling pathway

During the development of the hermaphrodite vulva, six vulval precursor cells (VPCs, P3.p through P8.p) adopt an invariant pattern of three cell fates. The anchor cell signal induces the primary vulval fate in P6.p. P6.p then sends a lateral signal to specify the secondary vulval fate in the neighboring VPCs P5.p and P7.p. The VPCs that receive neither inductive nor lateral signals (P3.p, P4.p and P8.p) adopt the tertiary, non-vulval cell fate. The lateral signal is received in P5.p and P7.p by the LIN-12 Notch receptor, which is necessary and sufficient to specify the secondary fate. LIN-12 Notch signaling induces the expression of secondary fate-specific genes via CSL transcription factors. Known LIN-12 Notch target genes contain clusters of CSL binding sites (RTGGGAA) in their 5 regulatory region. In order to identify new LIN-12 Notch target genes, we searched the C. elegans genome sequence for genes containing clusters of at least four CSL sites in their upstream region (Thomas Berset and Marc Sohrmann, unpublished results). All 106 candidate LIN-12 Notch target genes were knocked-down by feeding dsRNA to test if they play a role in secondary cell fate specification. As a read-out we examined the EGL-17::YFP expression pattern in L4 larvae as molecular for secondary cell fate specification. (In wild-type L4 larvae, EGL-17::YFP is only expressed in the secondary VulC and VulD cells.) In addition, dsRNA treated L4 larvae were inspected for defects in vulval morphogenesis. RNAi against 17 of the 106 candidate genes caused altered EGL-17::YFP marker expression and/or vulva morphogenesis defects. Details about the genes identified in this screen will be presented at the meeting.