Fig. 1. Expression of
bed-3 reporter is regulated by
blmp-1. A. Expression of
bed-3::gfp reporter (syEx962) is reduced when
blmp-1 is knocked down by RNAi. Corresponding Nomarski (top) and fluorescence (bottom) images are shown. Both worms are at the mid-L4 stage. B. Enhancer activity of DNA fragments F1 through SF3. F1, F2 and SF2 fragments drove expression of
pes-10::gfp in the vulva and the hypodermis. Percentages of animals showing detectable GFP expression in the vulva and hypodermis are shown. syEx962 containing the NspI fragment fused to the
bed-3 promoter is shown for comparison. The expression was significantly reduced in the
blmp-1(
tm548) mutant background. Error bars indicate 95% confidence interval (not shown for NspI reporter). Between 70 and 130 animals were assayed for each fragment. C. Mapping of intron 3 enhancer element. Fragments which exhibited enhancer activity are shown in green whereas fragments lacking enhancer activity are shown in gray. From these it can be inferred that multiple regions are potentially important (orange). One of these, IR2 is present in all enhancer-containing fragments identified and coincides with the BLMP-1 binding site established using EMSA (red). D. Enhancer activity of SF2 sub-fragments. Between 70 and 170 animals were assayed for each fragment. Error bars indicate 95% confidence interval. E.
blmp-1 mutants exhibit phenotypes similar to
bed-3 mutants.
blmp-1(-) animals have reduced vulval cell number in the mid-L4 stage, presumably because like
bed-3,
blmp-1 mutation causes loss of terminal cell division. The number of vulval cell nuclei descended from P5.p or P7.p vulval precursor cell is shown. Numbers of animals examined are indicated on the right. F. qPCR analysis of
bed-3 mRNA levels in control,
blmp-1 and
jmjd-3.2 RNAi treated animals (Materials and methods). Average and standard deviation from two repeated experiments are shown. Results were normalized to the wild-type level of
bed-3 and the
act-1 actin gene. *p b 0.05, Student's t-test. G. Regulation of SF2-9 and SF2-4 by
blmp-1. The expression of the SF2-4 reporter in the hypodermis was not obviously affected by the
blmp-1 mutation.