Figure 5. Trunk control genes likely signal independently of β-catenin-1.
(A) In situ hybridizations show reduced expression of ptk7 (11/11 animals), wntP-2 (6/6 animals), and ndl-3 (6/6 animals) after 8 days (wntP-2, ndl-3) or 19 days (ptk7) of β-catenin-1 RNAi in uninjured animals. (B) In situ hybridizations showing reduction of axin-B expression after 11 days of β-catenin-1 RNAi (14/14 animals) but not after inhibition of wntP-2 and ptk7 (14/14 animals) or ndl-3 and ptk7 (14/14 animals). Bars, 400 microns.
Figure 5—figure supplement 1. Examining the effect of APC RNAi on expression of ptk7, wntP-2, and ndl-3.
APC(RNAi) regenerating animals formed a domain of ectopic ptk7 (8/8 animals) and wntP-2 (9/9 animals) expression in the anterior tail. The ectopic tail appears to have a domain expressing wntP-2 and not ptk7 at the terminus and a domain expressing both ptk7 and wntP-2 near the amputation site. Such animals form an ectopic anterior pharynx likely as the consequence of tail formation, and this expressed ndl-3, ptk7, and wntP-2. Bars, 400 microns.
Figure 5—figure supplement 2. fzd1/2/7 and dvl-2 inhibition causes ectopic pharynx and mouth formation in the posterior.
The indicated dsRNA was delivered to animals by injection prior to amputation, 23 days of regeneration and staining for laminin expression to detect pharyngeal tissues. fzd1/2/7 inhibition (4/6 animals) caused formation of an ectopic pharynx similar to ptk7, wntP-2 and/or ndl-3 inhibition. dvl-2 dsRNA enhanced the ptk7 ectopic pharynx phenotype (9/9 animals). Bars, 300 microns.
Figure 5—figure supplement 3. Testing planar cell polarity genes for involvement in trunk patterning.
Regenerating trunk fragments undergoing RNAi as indicated and stained by FISH with a laminin riboprobe to detect the pharynx and mouth. Inhibition of ptk7 along with vangl1 (8/8 animals), vangl2 (4/4 animals), DAAM1 (6/6 animals) and ROCK (6/6 animals) did not suppress or enhance defects due to ptk7 inhibition alone. Bars, 300 microns.
Figure 5—figure supplement 4. ptk7, wntP-2, and ndl-3 inhibition do not influence axin-B expression and are not modified by APC inhibition.
(A) qPCR detecting expression of axin-B normalized to ubiquilin on RNA purified from day 10 regenerating animals after the indicated dsRNA treatments. β-catenin-1 inhibition reduced relative axin-B mRNA abundance, but ptk7+wntP-2 dsRNA and ptk7 + ndl-3 dsRNA had no effect. (B) ptk7+ wntP-2 RNAi did not alter expression of the β-catenin-1 target gene teashirt in animals 18 days after head and tail amputation (3/3 animals). (C) APC inhibition did not detectably affect the frequency of ectopic pharynx formation due to ptk7 and wntP-2 RNAi (p=1.000, Fisher’s exact test) (animals that regenerated an ectopic pharynx: 0/10 control fragments, 0/10 APC(RNAi) fragments, 7/9 ptk7(RNAi);wntP-2(RNAi);control(RNAi) fragments and 7/10 ptk7(RNAi);wntP-2(RNAi);APC(RNAi) fragments). Bars, 200 microns. Anterior, top.