Taniguchi Y et al. (2014), Notochord-derived hedgehog is essential for tai...

XB-ART-49152

BMC Dev Biol 2014 Jun 18;14:27. doi: 10.1186/1471-213X-14-27.

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Notochord-derived hedgehog is essential for tail regeneration in Xenopus tadpole.

Taniguchi Y , Watanabe K , Mochii M .

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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.

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Species referenced: Xenopus laevis
Genes referenced: ncam1 pax7 ptch2 shh tbx2
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	Expression of hedgehog signal-related genes in regenerating tail. (A) Semi-quantitative gene expression analysis by reverse transcription–polymerase chain reaction (RT–PCR) 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 transcription–polymerase chain reaction (RT–PCR) 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.

References [+] :

Beck, Temporal requirement for bone morphogenetic proteins in regeneration of the tail and limb of Xenopus tadpoles. 2006, Pubmed, Xenbase