Figure 3. ndl-3 and wntP-2 restrict trunk positional identity.

(A) Live, ventral images of ectopic pharynges and mouths in 20–30 day post-amputation (dpa) RNAi animals. Right top, cartoon depicts esophagus, pharynx, and mouth. Left, pharynges (yellow arrows) and ectopic mouths without a protruding pharynx (white arrows). Scale bar, 500 μm. Right bottom, mouths (white arrows) in 7 dpa RNAi animals. Anterior, left. Total number of animals were pooled from at least 2 independent RNAi experiments. (B) Increased numbers of para-pharyngeal mmp1⁺ cells in RNAi animals. NB.22.1e labels mouths. Graph below shows mean ± SD (n>8 animals/condition, 2 pooled experiments, One-way ANOVA). (C) Esophagus-gut connection in 20 dpa trunk fragments, region in dotted rectangle is shown at higher magnification below. FISH: mat (gut), mhc-1 (pharynx), and NB.22.1e (esophagus). Bracket, pharyngeal cavity length. (D) Time-lapse images of an ndl-3; wntP-2 RNAi animal eating liver through both pharynges (yellow arrows), see Video 1.

DOI: http://dx.doi.org/10.7554/eLife.12845.011

Figure 3—figure supplement 1. ndl-3 and wntP-2 restrict the number of mouths and pharynges in the trunk region.

(A) Ectopic posterior mouths are observed in regenerating trunk pieces of ndl-3, wntP-2, and ndl-3; wntP-2 RNAi animals 7 dpa. Number of animals showing ectopic mouths are described in Figure 3A. Yellow arrows point to mouths. Anterior, left; ventral, up. Scale bar, 100 μm. (B) DAPI stainings of RNAi animals show pharynges in the different RNAi conditions. Bracket indicates pharyngeal cavity length. Anterior, up; Scale bar, 100 μm. Images are representative of n>10 animals per condition. (C) Graph shows the percentage of intact RNAi animals with a total of two or more mouths after 8 RNAi feedings. p-values, Fisher’s exact test. 3 independent RNAi experiments are pooled in this analysis. Number of animals with ectopic mouths out of total animals (n) are indicated. (D) FISH using RNA probes for ndl-3 and wntP-2 show a decrease in the expression of those genes following their RNAi demonstrating the efficiency of the inhibition (total of 8 RNAi feedings). Graphs below (mean ± SD) show quantification of the mRNA levels by qRT-PCR. Student's t-test, * p<0.05, ** p<0.01. Cartoons show the region from where the mRNA was extracted. (E) Graph shows the percentage of intact RNAi animals with a total of two or more mouths after different number of RNAi feedings. The first five RNAi feedings were performed with only ndl-3; wntP-2 dsRNA. β-catenin-1 or control dsRNA was added in addition to ndl-3; wntP-2 starting on feeding six.

DOI: http://dx.doi.org/10.7554/eLife.12845.012