Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
BACKGROUND: Appendage regeneration in amphibians is regulated by the combinatorial actions of signaling molecules. The requirement of molecules secreted from specific tissues is reflected by the observation that the whole process of regeneration can be inhibited if a certain tissue is removed from the amputated stump. Interestingly, urodeles and anurans show different tissue dependencies during tail regeneration. The spinal cord is essential for tail regeneration in urodele but not in anuran larva, whereas the notochord but not the spinal cord is essential for tail regeneration in anuran tadpoles. Sonic hedgehog is one of the signaling molecules responsible for such phenomenon in axolotl, as hedgehog signaling is essential for overall tail regeneration and sonic hedgehog is exclusively expressed in the spinal cord. In order to know whether hedgehog signaling is involved in the molecular mechanism underlying the inconsistent tissue dependency for tail regeneration between anurans and urodeles, we investigated expression of hedgehog signal-related genes in the regenerating tail of Xenopus tadpole and examined the effect of the hedgehog signal inhibitor, cyclopamine, on the tail regeneration.
RESULTS: In Xenopus, sonic hedgehog is expressed exclusively in the notochord but not in the spinal cord of the regenerate. Overall regeneration was severely impaired in cyclopamine-treated tadpoles. Notochord maturation in the regenerate, including cell alignment and vacuolation, and myofiber formation were inhibited. Proliferation of spinal cord cells in the neural ampulla and of mesenchymal cells was also impaired.
CONCLUSION: As in the axolotl, hedgehog signaling is required for multiple steps in tail regeneration in the Xenopus tadpole, although the location of the Shh source is quite different between the two species. This difference in Shh localization is the likely basis for the differing tissue requirement for tail regeneration between urodeles and anurans.
Expression of hedgehog signal-related genes in regenerating tail. (A) Semi-quantitative gene expression analysis by reverse transcriptionâpolymerase chain reaction (RTâPCR) was carried out. RNA was isolated from amputated tails at the indicated day. Specific primer pairs and PCR conditions are indicated in Additional file 2: Table S1. (B-E) Whole mount (B, D, E) and section (C) in situ hybridization of regenerated tail. The spatial expression of shh(B, C)ptc-1(D) and ptc-2(E) was analyzed on the regenerating tail at day 4 (C-E) or day 5 (B). Arrows indicate hybridization signal in the regenerating notochord (B, C) or spinal cord (D, E). A pair of arrowheads marks the amputation plane. Black dashed lines indicate the shapes of the regenerating spinal cord (C) or notochord (D, E). Bars, 100 μm.
Effect of cyclopamine on the tail regeneration. The tail-amputated tadpole was maintained in the presence of the indicated compound. ET, 0.1% ethanol. TO, 2.5 μM tomatidine. CP, 2.5 μM cyclopamine. CP + PM, 2.5 μM cyclopamine and 0.25 μM purmorphamine. (A-D) Regenerated tails were observed under a stereoscopic microscope at day 7. Bar, 500 μm. (E-H) Histological analysis of the regenerated tail at day 7. Sagittal sections were stained with hematoxylin and eosin. nc, notochord. Bar, 100 μm. A pair of arrowheads marks the amputation plane. (I, J) The length of the regenerated tail and notochord at day 5. Mean length of the regenerated tail (I) or the notochord (J) was indicated with the standard deviation. Detail is shown in Tables 1 and ​and2.2. *, p-value < 0.05. **, p-value <0.01. (K) Gene expression analysis with RT-PCR. RNA from the control or cyclopamine-treated tails was analyzed at day 2.5 or day 5.
Cyclopamine affects cell differentiation in the regenerating tail. (A-C) Histological analysis of the regenerating tail at day 3. Sagittal sections were stained with hematoxylin and eosin. Cyclopamine suppressed the elongation and the vacuolation of the regenerating notochord cells. It also suppressed the formation of the neural ampulla (C). nc, notochord. na, neural ampulla. sc, spinal cord. Bar, 200 μm. (D-I) Immunohistochemical detection of nerve cells and myofibers in the regenerating tail at day 5. Whole mount sample was immunostained with an anti-NCAM monoclonal antibody (4d) and a monoclonal antibody 12/101 to detect the nerve cells (D-F) and the myofibers (G-I), respectively. Arrows indicate the regenerated spinal cord (D-F) and the regenerated myofibers (G, H). Bar, 500 μm. (J-K) Immunofluorescent detection of muscle cells. The frontal cryosection was immunostained with a monoclonal antibody 12/101 (J, K) or an anti-PAX7 monoclonal antibody (L, M) to detect myofibers (white arrows in J, K) or myoblasts (white arrows in L, M) in the regenerated tail at day 5. Bar, 200 μm. A pair of black and white arrowheads marks the amputation plane.
Cyclopamine affects cell proliferation in the regenerating tail. Tail-amputated tadpoles maintained in the presence of the indicated compound were incubated with BrdU for 12 h before fixation. (A-F) Immunohistochemical detection of proliferation cells at day 2.5. BrdU-incorporated cells (brown) were detected on sagittal (A-C) or frontal (D-F) section. Un-labeled nuclei were counter-stained with hematoxylin (blue). Dashed lines indicate the shapes of the regenerating notochords (A-C) and the mesenchymal regions (D-F) containing the myoblasts. nc, notochord. na, neural ampulla of the regenerating spinal cord. m, muscle. A pair of arrowheads marks the amputation plane. Bar, 100 μm. (G) Proliferation rate of cells in the regenerating tail. Mean rate of the BrdU-labeled cells was determined for the indicated tissue and shown with standard deviation. Detail is shown in Table 3. **, p-value <0.01.
Figure 1. Expression of hedgehog signal-related genes in regenerating tail. (A) Semi-quantitative gene expression analysis by reverse transcriptionâpolymerase chain reaction (RTâPCR) was carried out. RNA was isolated from amputated tails at the indicated day. Specific primer pairs and PCR conditions are indicated in Additional file 2: Table S1. (B-E) Whole mount (B, D, E) and section (C) in situ hybridization of regenerated tail. The spatial expression of shh(B, C)ptc-1(D) and ptc-2(E) was analyzed on the regenerating tail at day 4 (C-E) or day 5 (B). Arrows indicate hybridization signal in the regenerating notochord (B, C) or spinal cord (D, E). A pair of arrowheads marks the amputation plane. Black dashed lines indicate the shapes of the regenerating spinal cord (C) or notochord (D, E). Bars, 100 μm.
Figure 2. Effect of cyclopamine on the tail regeneration. The tail-amputated tadpole was maintained in the presence of the indicated compound. ET, 0.1% ethanol. TO, 2.5 μM tomatidine. CP, 2.5 μM cyclopamine. CP + PM, 2.5 μM cyclopamine and 0.25 μM purmorphamine. (A-D) Regenerated tails were observed under a stereoscopic microscope at day 7. Bar, 500 μm. (E-H) Histological analysis of the regenerated tail at day 7. Sagittal sections were stained with hematoxylin and eosin. nc, notochord. Bar, 100 μm. A pair of arrowheads marks the amputation plane. (I, J) The length of the regenerated tail and notochord at day 5. Mean length of the regenerated tail (I) or the notochord (J) was indicated with the standard deviation. Detail is shown in Tables 1 and 2. *, p-value < 0.05. **, p-value <0.01. (K) Gene expression analysis with RT-PCR. RNA from the control or cyclopamine-treated tails was analyzed at day 2.5 or day 5.
Figure 3. Cyclopamine affects cell differentiation in the regenerating tail. (A-C) Histological analysis of the regenerating tail at day 3. Sagittal sections were stained with hematoxylin and eosin. Cyclopamine suppressed the elongation and the vacuolation of the regenerating notochord cells. It also suppressed the formation of the neural ampulla (C). nc, notochord. na, neural ampulla. sc, spinal cord. Bar, 200 μm. (D-I) Immunohistochemical detection of nerve cells and myofibers in the regenerating tail at day 5. Whole mount sample was immunostained with an anti-NCAM monoclonal antibody (4d) and a monoclonal antibody 12/101 to detect the nerve cells (D-F) and the myofibers (G-I), respectively. Arrows indicate the regenerated spinal cord (D-F) and the regenerated myofibers (G, H). Bar, 500 μm. (J-K) Immunofluorescent detection of muscle cells. The frontal cryosection was immunostained with a monoclonal antibody 12/101 (J, K) or an anti-PAX7 monoclonal antibody (L, M) to detect myofibers (white arrows in J, K) or myoblasts (white arrows in L, M) in the regenerated tail at day 5. Bar, 200 μm. A pair of black and white arrowheads marks the amputation plane.
Figure 4. Cyclopamine affects cell proliferation in the regenerating tail. Tail-amputated tadpoles maintained in the presence of the indicated compound were incubated with BrdU for 12 h before fixation. (A-F) Immunohistochemical detection of proliferation cells at day 2.5. BrdU-incorporated cells (brown) were detected on sagittal (A-C) or frontal (D-F) section. Un-labeled nuclei were counter-stained with hematoxylin (blue). Dashed lines indicate the shapes of the regenerating notochords (A-C) and the mesenchymal regions (D-F) containing the myoblasts. nc, notochord. na, neural ampulla of the regenerating spinal cord. m, muscle. A pair of arrowheads marks the amputation plane. Bar, 100 μm. (G) Proliferation rate of cells in the regenerating tail. Mean rate of the BrdU-labeled cells was determined for the indicated tissue and shown with standard deviation. Detail is shown in Table 3. **, p-value <0.01.
Beck,
Beyond early development: Xenopus as an emerging model for the study of regenerative mechanisms.
2009, Pubmed,
Xenbase
Beck,
Beyond early development: Xenopus as an emerging model for the study of regenerative mechanisms.
2009,
Pubmed
,
Xenbase
Beck,
Temporal requirement for bone morphogenetic proteins in regeneration of the tail and limb of Xenopus tadpoles.
2006,
Pubmed
,
Xenbase
Beck,
Molecular pathways needed for regeneration of spinal cord and muscle in a vertebrate.
2003,
Pubmed
,
Xenbase
Caubit,
Possible roles for Wnt genes in growth and axial patterning during regeneration of the tail in urodele amphibians.
1997,
Pubmed
Caubit,
Reactivation and graded axial expression pattern of Wnt-10a gene during early regeneration stages of adult tail in amphibian urodele Pleurodeles waltl.
1997,
Pubmed
Chen,
Control of muscle regeneration in the Xenopus tadpole tail by Pax7.
2006,
Pubmed
,
Xenbase
Chiang,
Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function.
1996,
Pubmed
Elia,
Sonic hedgehog promotes proliferation and differentiation of adult muscle cells: Involvement of MAPK/ERK and PI3K/Akt pathways.
2007,
Pubmed
Gargioli,
Cell lineage tracing during Xenopus tail regeneration.
2004,
Pubmed
,
Xenbase
Géraudie,
Gene expression during amphibian limb regeneration.
1998,
Pubmed
Ho,
TGF-beta signaling is required for multiple processes during Xenopus tail regeneration.
2008,
Pubmed
,
Xenbase
Kawakami,
Distributions of PAX6 and PAX7 proteins suggest their involvement in both early and late phases of chick brain development.
1997,
Pubmed
Kintner,
Monoclonal antibodies identify blastemal cells derived from dedifferentiating limb regeneration.
,
Pubmed
,
Xenbase
Koleva,
Pleiotropic effects of sonic hedgehog on muscle satellite cells.
2005,
Pubmed
Lin,
Transgenic analysis of signaling pathways required for Xenopus tadpole spinal cord and muscle regeneration.
2012,
Pubmed
,
Xenbase
Lin,
Requirement for Wnt and FGF signaling in Xenopus tadpole tail regeneration.
2008,
Pubmed
,
Xenbase
Mochii,
Tail regeneration in the Xenopus tadpole.
2007,
Pubmed
,
Xenbase
Schnapp,
Hedgehog signaling controls dorsoventral patterning, blastema cell proliferation and cartilage induction during axolotl tail regeneration.
2005,
Pubmed
Slack,
The Xenopus tadpole: a new model for regeneration research.
2008,
Pubmed
,
Xenbase
Sugiura,
Xenopus Wnt-5a induces an ectopic larval tail at injured site, suggesting a crucial role for noncanonical Wnt signal in tail regeneration.
2009,
Pubmed
,
Xenbase
Sugiura,
Differential gene expression between the embryonic tail bud and regenerating larval tail in Xenopus laevis.
2004,
Pubmed
,
Xenbase
Takabatake,
Hedgehog and patched gene expression in adult ocular tissues.
1997,
Pubmed
,
Xenbase
Tanaka,
The cellular basis for animal regeneration.
2011,
Pubmed
,
Xenbase
Taniguchi,
Spinal cord is required for proper regeneration of the tail in Xenopus tadpoles.
2008,
Pubmed
,
Xenbase
Watanabe,
Topography of N-CAM structural and functional determinants. I. Classification of monoclonal antibody epitopes.
1986,
Pubmed
,
Xenbase
Zhang,
Differential regulation of fibroblast growth factor receptors in the regenerating amphibian spinal cord in vivo.
2002,
Pubmed
Zhang,
FGF-2 Up-regulation and proliferation of neural progenitors in the regenerating amphibian spinal cord in vivo.
2000,
Pubmed
