Despite myriad challenges, clinicians see room for progress.
Image credit: Shutterstock/David Tadevosian.
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Edited by Brigid L. M. Hogan, Duke University Medical Center, Durham, NC, and approved October 30, 2009 (received for review July 8, 2009)
Article Figures & SI
Figures
- Fig. 1.
“Janus-tails” phenotype is caused by Djptc RNAi. Anterior end is placed at the top in all panels. Rg, regeneration. (A–L) Djptc(RNAi) caused abnormal anterior blastema formation in trunk fragment regenerants at 7–9 days postamputation. (A and G) Live animals. (B–F and H–L) Characterization by in situ detection of DjPC2 (CNS marker; B and H), Djndk (a head marker; C and I), DjAbd-Ba (a tail marker; D and J), and DjfzT (a tail marker; E and K), and by immunostaining for DjAADCA (digestive gut branch staining; F and L). Arrowheads indicate the single anterior and two posterior gut branches (F), and the bifurcated branching of the main gut tract in the anterior region as posterior fate (L). Arrow indicates an extra pharynx (H). (Scale bars, 500 μm.) (M–R) Posteriorized anterior end in Djptc(RNAi) trunk regenerants 1 day after amputation. Expression patterns of DjAbd-Ba (M and P), DjZicB (anterior marker; N and Q), and Djndk (O and R) in the anterior ends. (Scale bar, 200 μm.) (S) Time course of relative gene expression (RGE) level reveals gradual posteriorization of anterior blastema after Djptc(RNAi) during early regeneration (1 and 3 days). The expression levels of target genes (DjAbd-Ba, DjZicB, DjotxA, and Djndk) in anterior or posterior blastemas were normalized and expressed relative to the level in the anterior end of control trunk regenerants at day 0 taken as 1.0 (white column of “Anterior” bars; indicated by dashed line). Mean of quadruplicates.
- Fig. 2.
Djptc functions upstream of Wnt/β-catenin signaling. (A–D) Djndk expression in anterior (Upper) and posterior (Lower) regions of RNAi trunk regenerants at day 7. Djptc(RNAi) caused Janus-tails formation (B), whereas Djβ-cateninB(RNAi) caused Janus-heads formation (C) with or without simultaneous Djptc(RNAi) (D). Numbers represent the fraction of tested regenerants showing the indicated phenotype. (Scale bar, 200 μm.) (E) RGE levels of Wnt genes in anterior and posterior blastemas of RNAi trunk regenerants at day 1, with the anterior (DjZicB) and posterior (DjfzT) marker genes shown as positive controls for RNAi phenotype. ND, not detected.
- Fig. 3.
Hh signaling is involved in the establishment of AP polarity. (A–D) Loss of Hh signaling activity caused Janus-heads or loss of the tail identity in regeneration. DjfzT expression in head fragments at day 11 of the first round of regeneration (A–D), and trunk fragments at day 7 of the second round of regeneration (A′–D′) after strong inhibition of the positive regulators of the Hh signaling cascade (RNAi of Djhh or Djgli). Note that the generation of ectopic eyes in the posterior blastemas (arrowheads) and extra eye formation (arrow; Inset) near the posterior end (7%; 1/14, D′). (Scale bars, 300 μm.) (E–M) Janus-tails formation after RNAi of negative regulators of Hh signaling cascade (Djptc or Djsufu) and its suppression by simultaneous RNAi of positive regulators. Djndk expression in trunk fragments at regeneration day 9. (Scale bar, 300 μm.) (N) RGE index of Hh signaling component genes in anterior and posterior blastemas after Djptc(RNAi) as in Fig. 1S.
- Fig. 4.
Hh signaling regulates transcription of Wnt genes. (A–H) DjwntP-1 expression was lost by RNAi of Djhh or Djgli in the posterior end of both head (A–D) and trunk (E–H) regenerants on day 1 of the second round of regeneration. (Scale bars, 200 μm.) (I) RGE quantification of Wnt genes in anterior and posterior blastemas of trunk fragments at regeneration day 1 after RNAi of Hh signaling components genes, with the anterior (DjZicB) and posterior (DjfzT) marker genes as positive controls for RNAi phenotype. ND, not detected.
- Fig. 5.
Posteriorizing signal originates in differentiated cells (neuron). (A–D) Expression of Djhh was observed in X-ray-resistant differentiated cells of the VNCs. In situ detection of Djhh in nonirradiated (A) and irradiated (C) planarians, followed by labeling of the CNS with anti-DjSynaptotagimin antibody (neural marker; Syt; B and D). (Scale bar, 1 mm.) (E) RGE index of Wnt genes in the posterior blastema of head fragments from X-ray-irradiated animals 1 day after amputation. (F-I) Confocal images of expression patterns of Djhh, DjwntP-1, or Djsyt by dual in situ detection in the posterior (F–H) and anterior (I) end in regenerants from head and trunk fragments, respectively, at 18 h after amputation. Panels show individual expression patterns (red or green color) and their merged image with the image of nuclear staining (Hoechst; blue). The rightmost panel is a magnified view of each boxed area. Arrow indicates VNC (F). Note that Djhh was expressed in neural cells of the VNCs, which were labeled with Djsyt probe (arrowheads; F), but not in the posterior blastema (F and G). Also, DjwntP-1 was predominantly expressed in the posterior end surrounding the VNCs (G and H), but was also weakly expressed in the anterior end (I). (Scale bars, 50 μm.)
- Fig. 6.
Models for two aspect of the establishment of AP polarity by Hh signaling in planarians. (A) The signaling cascade model for posterior specification in planarian regeneration inferred from the present study. Hh signaling triggers posteriorization by inducing transcription of Wnt genes in differentiated cells (yellow-shaded box), from which the signal is transmitted to stem cells (green-shaded box) through Wnt/β-cateninB signaling for tail regeneration. Positive and negative regulators of posterior specification are written with red and blue letters, respectively. (B) A possible model for the establishment of AP polarity by Hh signaling in planarians. DjwntP-1-expressing cells (red dots) at the posterior end suggests that Hh signaling is exclusively activated there (pink gradients on the left), although Djhh-expressing cells (neural cells; blue dots) are localized all along the VNCs (CNS is indicated by light green). In this model, Djhh gene products would be transported posteriorly via VNCs (pink arrows), leading to the induction of posterior specification at the posterior end.
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Planarian Hedgehog/Patched establishes anterior–posterior polarity by regulating Wnt signaling
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