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Science 326 (5958): 1406-1410

Copyright © 2009 by the American Association for the Advancement of Science

Planarian Hh Signaling Regulates Regeneration Polarity and Links Hh Pathway Evolution to Cilia

Jochen C. Rink*, Kyle A. Gurley*, Sarah A. Elliott, and Alejandro Sánchez Alvarado{dagger}

Department of Neurobiology and Anatomy, Howard Hughes Medical Institute, University of Utah School of Medicine, 401 MREB, 20 North 1900 East, Salt Lake City, UT 84103, USA.


Figure 1
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Fig. 1. Planarian Hedgehog signaling. (A) Gene expression in intact animals. Boxes magnified on right. 1, Epifluorescence image, hh (green), CNS (magenta, {alpha}-tubulin antibody). 2, Confocal image, ventral head, ptc (green). CNS (magenta, {alpha}-tubulin antibody). 3, Root of pharynx, ptc (green). Nuclei [blue, 4',6'-diamidino-2-phenylindole (DAPI)]. 4, sufu expression near root of pharynx. 5, gli-1 (green), gut epithelium (magenta, Smed-porcn-1 and Smed-sialin). Scale bar, 0.5 mm. Arrowheads, ventral nerve cord. Arrow, root of pharynx. (B) ptc expression in RNAi-treated intact animals. The seemingly paradoxical up-regulation of ptc upon ptc(RNAi) results from the massive promotion of Hh signaling by ptc(RNAi). Scale bar, 0.5 mm. (C) ptc expression confocal images in sufu(RNAi) intact animals. Scale bar, 0.2 mm.

 

Figure 2
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Fig. 2. Misregulated Hh signaling results in A/P patterning defects. (A to F) Live images of regenerating trunk fragments 14 days after amputation. Decreased Hh signaling achieved by hh(RNAi) (A to B''), and increased signaling by ptc(RNAi) (D to F''). Phenotypes are arranged according to severity. (A' to F') Expression of anterior marker [Smed-sFRP-1 (31, 33)]. (A'' to F'') Expression of posterior marker [Smed-fz-4 (31)].

 

Figure 3
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Fig. 3. Hh specifies A/P fate through interaction with Wnt/β-catenin signaling. (A and B) Quantification of double-RNAi experiments scored for anterior or posterior regeneration defects. Relative frequency of indicated phenotypes in a cohort of trunk fragments scored 14 days after amputation. (A) N > 15 animals per condition. (B) N > 20 animals per condition. (C) wntP-1 expression at 1 day after amputation. White arrowheads, expression at both anterior (upper panels of each set) and posterior (lower panels of each set) wounds in control animals. Black arrows, up-regulated expression in ptc(RNAi). Scale bars, 0.2 mm. (D and E) Quantification of double-RNAi experiments scored for (D) anterior and (E) posterior regeneration defects. Relative frequency of indicated phenotypes in trunk fragment cohort scored 14 days after amputation. N > 21 animals per condition.

 

Figure 4
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Fig. 4. Hh signaling is cilia-independent, and kif27, fused, and iguana are cilia genes in planarians. (A) Mean translocation speed quantified from movies of animals having received the indicated RNAi treatments. Error bars, SEM. *, P < 0.01 versus control (one-way analysis of variance). Data is from ≥ 4 movies with N ≥ 5 animals per movie. (Inset) Example of tissue edema caused by IFT172(RNAi). Scale bars, 0.5 mm. (B) Ventral cilia confocal projections (green, acetylated tubulin antibody), overlayed with nuclei (blue, DAPI) to demonstrate epithelial integrity. RNAi treatments as indicated. Insets, zoom. Punctate pattern, remaining cilia stumps. Scale bars, 50 µm. (C) Translocation speed calculated as in (A). (D) Tissue edema in iguana(RNAi) animal 14 days after amputation. Scale bar, 0.5 mm. (E) Ventral cilia confocal projections (green, acetylated tubulin antibody) overlayed with nuclei (blue, DAPI) as in (B). Arrows, remaining tufts of cilia on cells not yet affected by RNAi. Residual staining in fused(RNAi), iguana(RNAi), and kif27(RNAi) animals due to non–cilia-related staining of subepithelial structures. Scale bar, 50 µm.

 


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