Figure 1. Genetic screen for activators of
egl-1 expression using RNA-mediated interference:A) Flowchart of genetic screens for activators of
egl-1 expression. (B) Primary (positive) screen for activators of
egl-1 expression. After RNAi-mediated knockdown of RBP genes, candidates were identified by screening for a decrease in GFP::HIS-58 signal in oocytes of animals carrying the reporter Pmai-2gfp::
his-58::
egl-1 3' UTR (bcSi26). White arrows point to GFP::HIS-58 signal in oocyte nuclei. (C) Secondary (negative) screen. Nonspecific candidates were eliminated by screening for a decrease in GFP::HIS-58 signal in embryos of animals carrying the reporter Pmai-2gfp::
his-58::
mai-2 3' UTR (bcSi25). (D) NSM sister cell (NSMsc) survival screen. (Top) Schematics showing the NSM lineage in wild-type (+/+) and
ced-3(717) animals (Ellis and Horvitz, 1986). The two bilaterally symmetric neurosecretory motoneuron (NSM) neuroblasts (NSMnb) (left and right) divide ~410 minutes after the first zygotic cleavage (at 20°C), each generating one NSM neuron, which is programmed to survive, and one NSM sister cell (NSMsc), which is programmed to die ('unwanted' daughter cell) (Sulston et al., 1983). In wild-type (+/+) animals, the NSMsc undergoes apoptotic cell death, resulting in one NSM from each NSM neuroblast. When apoptosis is blocked, the NSMsc inappropriately survives, resulting in an extra 'NSM-like' cell. The NSM and the 'undead' NSMsc can be identified in the anterior pharynx of L3/L4 larvae using the reporter Ptph-1gfp::
his-24 (Yan et al., 2013). (Bottom) RNAi knockdown of
eif-3.H or
hrpr-1 causes NSMsc survival. To enhance RNAi efficiency in the NSM lineage, RNAi experiments were also performed in the
nre-1(
hd20)
lin-15b(
hd126) background (Schmitz et al., 2007). The percentage of NSMsc survival is enhanced in the background of
n2427, a weak loss of function mutation of
ced-3 (Shaham et al., 1999). The sample size (n) is shown in the table. The complete genotypes of strains used are provided in Table 1.