Figure 3. Regulation of the Initiation of
ttx-3 Expression by REF-2, HLH-2, HLH-3, and HLH-16(A) Left: expression of
ttx-3 (
ttx-3p::gfp) in embryos at epidermal enclosure just after division of the NBSMDD/AIY neuroblast (ventral view, anterior is left; scale bar,10 um; note that the expression of
ttx-3 in the AIN lineage (AINm, an unrelated neuronal lineage that also expresses
ttx-3) is affected by loss of
ref-2 and
hlh-2function but not by loss of
hlh-3 and
hlh-16 function). Right: percentage of NBSMDD/AIY lineages that display expression of
ttx-3 (
ttx-3p::gfp) at epidermal enclosure(n > 100, error bars show SE of proportion). See also Figure S3.(B) EMSA using in vitro (reticulocyte lysate) produced REF-2 or POP-1 proteins on probes containing the wild-type or mutated Zic site from the
ttx-3 promoter.See also Figure S4.(C) Coimmunoprecipitation from C. elegans embryos expressing the REF-2::VENUS (otEx3091) translational fusion (+) or not (), using an anti-POP-1 antibody orcontrol anti-HA antibody for immunoprecipitation and an anti-VENUS antibody for western blot. (Note that in addition to the REF-2::VENUS band [62 kDa] anadditional band corresponding to the antibody used for immunoprecipitation [50 kDa] is also detected. The same quantity of anti-POP-1 and anti-HA antibodies was used in the immunoprecipitations; differences of intensity of the antibody band between anti-POP-1 and anti-HA reflects difference of affinity of theseantibodies with the western blot antibodies.)(D) Left: expression of the translational fusions REF-2::VENUS (otEx3091), HLH-2::GFP (nIs47), HLH-3::YFP (otEx4140), HLH-16::GFP (otEx4503) in theNBSMDD/AIY (Ant.) and NBSIAD/SIBV (Post.) neuroblasts at epidermal enclosure (scale bar, 2 mm). Right: percentage of lineages expressing the translational fusionHLH-3::YFP in both NBSMDD/AIY and NBSIAD/SIBV at similar level (black), in both NBSMDD/AIY and NBSIAD/SIBV with higher level in NBSMDD/AIY (gray), or only inNBSMDD/AIY (white). Two independent HLH-3::YFP lines (#1 otEx4140, #2 otEx4142) were analyzed. Expression in the NBSMDD/AIY and NBSIAD/SIBV neuroblasts wasscored early (during interphase) or late (when entering mitosis as monitored by chromosome condensation using
hlh-16p::his::mCherry and by the appearance ofthe cleavage furrow). The effects of control and
sys-1 RNAi were tested on otEx4140. (n > 30, error bars show SE of proportion).