A synthetic HMG-Helper site reporter reveals a novel POP-1 function in rhythmic defecation behaviour.

Abstract

<p>(A–F) Nomarski images of animals with stably integrated <i>POPHHOP</i> (<i>6× HMG-Helper::GFP</i>) and <i>POPTOP</i> (<i>7× HMG::mCherry</i>) reporters showing GFP (A, D) and mCherry (B, E) fluorescence. Live L1 larvae have overlapping expression of GFP and mCherry in some tail neurons (A–C) and live L3 larvae display overlapping DTC expression (D–F). In addition, POPHHOP displayed strong expression in the int9 intestinal cells of early L1 Larvae (A) onward through adulthood (not shown). (G–H) Stably integrated <i>POPHHOP</i> animals in a wild-type (G) or <i>pop-1(hu9)</i> background (H). The reporter expression seen in the int9 cells, tail neurons, and occasionally in the VC neurons is low or undetectable in the <i>pop-1</i> mutants. Scale bars = 10 µm. (I) Box-whisker plot showing the median (line inside the box), third quartile (upper box), first quartile (lower box), longest pBoc cycle time (upper whisker limit) and shortest pBoc cycle time (lower whisker limit) for N2 controls and two <i>pop-1</i> alleles at the L2 stage. A statistically significant increase was seen in the pBoc cycle time based on a Student's two-tailed <i>t</i> test (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen-1004133-t001" target="_blank">Table 1</a>). (J) 8 individual pBocs (X-axis) were monitored (n = 26, each color representing one larva) for each genotype and plotted against time between each pBoc (y-axis). <i>pop-1(q645)</i> mutants have greater variability between pBocs than the wild-type N2 control. (K) Box-whisker plot showing the pBoc period of <i>pop-1</i> depleted worms compared to ctrl RNAi worms using the OLB11 strain, which allows intestine-specific RNAi <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen.1004133-McGhee1" target="_blank">[65]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen.1004133-Pilipiuk1" target="_blank">[66]</a>. Animals were assayed at the young adult stage. A statistically significant increase was seen in the pBoc cycle time based on a Student's two-tailed <i>t</i> test (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen-1004133-t001" target="_blank">Table 1</a>). (L) 8 individual pBocs (X-axis) were monitored in young adults (n = 24, each color representing one adult) for each genotype and plotted against time between each pBoc (y-axis). <i>pop-1 RNAi</i> leads to a high variability in the cycle time in <i>pop-1</i> depleted adults compared to controls.</p