Figure 2. ptk7 acts with wntP-2 and ndl-3 to suppress trunk identity in a context-dependent manner.
(A) ptk7, wntP-2, and ndl-3 dsRNAs were fed to animals individually or in pair-wise combinations prior to amputation to remove heads and tails, fixation 25 days later, staining with a laminin riboprobe and Hoechst dye. (B) Scoring information for pharynx and mouth duplication phenotypes. Animals were scored for presence of ectopic mouth (defined as a superficial circle of laminin+ cells which was always present posterior to the original mouth, arrow), and ectopic pharynx (defined as having pharynx morphology by laminin+ and Hoechst+ staining, double arrows) and its orientation with respect to the A-P body axis. Animals with an ectopic mouth but not a fully formed ectopic pharynx often had varying degrees of internal laminin expression suggestive of a growing pharynx primordium and were scored as having an ectopic mouth only. Co-inhibition of any pairwise combination of the three genes enhanced the penetrance and expressivity of the ectopic pharynx phenotypes. Note that combined pairwise inhibition of ptk7, wntP-2 and ndl-3 enhanced the trunk duplication phenotype and that dual inhibition of ptk7 and wntP-2 produced the strongest effects. (C) Analysis of ectopic pharynx orientation, measured at the proximal end of the ectopic pharynx. In many cases, the ectopic pharynx was oriented at an oblique angle with respect to the body axis, perhaps as a result of ectopic mouth placement nearby the original mouth, and ectopic pharynges were observed with fully inverted polarity. In all animals that formed 2 ectopic pharynges (derived from pairwise combinations of dsRNAs), both structures were oriented toward a common ectopic mouth located along the posterior midline. (D) Images of live animals showing the ectopic pharynx in ptk7(RNAi);wntP-2(RNAi) animals (red arrow) can be functional for feeding. (E) Prolonged inhibition of ptk7 and wntP-2 in uninjured animals for at least 36 days (64 days shown) caused formation of an ectopic pharynx (6/8 animals) and multiple posterior mouths (8/8 animals). (F) Inhibition of ptk7 and wntP-2 or ptk7 and ndl-3 caused head and tail fragments to regenerate only a single pharynx like control animals. Therefore, the effects of ptk7, ndl-3 and wntP-2 in body patterning are context dependent. Anterior, left. Bars, 300 (A,E) or 500 (F) microns.
Figure 2—figure supplement 1. Verification of RNAi knockdown for ptk7, wntP-2 and ndl-3.
