A single E-box in the <i>Cel-lin-3</i> CRM is not sufficient for <i>lin-3</i> expression in the anchor cell of <i>C</i>. <i>elegans</i>.

<p><b>(A)</b> New <i>cis</i>-regulatory <i>lin-3</i> alleles with deleted E-box_L and NHR or NHR and E-box_R. <b>(B)</b> Quantification of vulval induction in these new mutants. Note the complete absence of any induction in the recovered <i>lin-3</i> alleles (n>30). Scorings of <i>lin-3(1417)</i> animals are the same as those reported in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006278#pgen.1006278.g005" target="_blank">Fig 5</a> and are used here to indicate that this mutation leads to vulval hypo-induction rather than no induction at all. <b>(C-D)</b> smFISH in <i>lin-3(mf72)</i> <b>(C)</b> and N2 <b>(D)</b> animals. Green spots correspond to <i>lin-3</i> transcripts and red spots to <i>lag-2</i> that is used as an anchor cell marker. Blue is DAPI staining of nuclei. Note the absence of <i>lin-3</i> expression in the anchor cell in the <i>lin-3(mf72)</i> mutant animal. Absence of <i>lin-3</i> signal in the anchor cell was also confirmed for the other <i>lin-3</i> alleles.