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Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae.

Rodrigues AM, Christen B, Martí M, Izpisúa Belmonte JC - BMC Dev. Biol. (2012)

Bottom Line: In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate.Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation.In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Regenerative Medicine of Barcelona, 08003 Barcelona, Spain.

ABSTRACT

Background: Mammals are not able to restore lost appendages, while many amphibians are. One important question about epimorphic regeneration is related to the origin of the new tissues and whether they come from mature cells via dedifferentiation and/or from stem cells. Several studies in urodele amphibians (salamanders) indicate that, after limb or tail amputation, the multinucleated muscle fibres do dedifferentiate by fragmentation and proliferation, thereby contributing to the regenerate. In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate. We set out to study whether dedifferentiation was present during muscle regeneration of the tadpole limb and zebrafish larval tail, mainly by cell tracing and histological observations.

Results: Cell tracing and histological observations indicate that zebrafish tail muscle do not dedifferentiate during regeneration. Technical limitations did not allow us to trace tadpole limb cells, nevertheless we observed no signs of dedifferentiation histologically. However, ultrastructural and gene expression analysis of regenerating muscle in tadpole tail revealed an unexpected dedifferentiation phenotype. Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation. In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres.

Conclusions: Our results show that complete skeletal muscle dedifferentiation is less common than expected in lower vertebrates. In addition, the discovery of incomplete dedifferentiation in muscle fibres of the tadpole tail stresses the importance of coupling histological studies with in vivo cell tracing experiments to better understand the regenerative mechanisms.

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Expression of mature muscle genes decreases with regeneration in tail and limb. (a) Real Time PCR analysis of tail regeneration. myod gene expression does not significantly change in the distal stump between 0 and 3 dpa, while cardiac α actin (car) and myosin heavy chain 4 (myh4) have 5-fold lower expression at 3 dpa. n = three independent experiments of 10 tail samples per time point. (b) Real Time PCR analysis of stage 54 limb regeneration. Three samples were used: 0 dpa zeugopod; 3 dpa zeugopod; growth control (GC)-not amputated, three days older zeugopod. myod, myogenin and car levels are the same between 0 and 3 dpa. However, comparing 3 dpa with GC, we observed a significant lower expression of myod, myogenin and car in the regenerating limb. n = four independent experiments of 10 or 20 limb samples per time point. Results were normalized for ornithine decarboxylase (odc) expression and relativized to the time points with highest expression (0 dpa for tail and GC for limb). Error bars: standard error. Asterisk: p value < 0.01; Student's t test.
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Figure 4: Expression of mature muscle genes decreases with regeneration in tail and limb. (a) Real Time PCR analysis of tail regeneration. myod gene expression does not significantly change in the distal stump between 0 and 3 dpa, while cardiac α actin (car) and myosin heavy chain 4 (myh4) have 5-fold lower expression at 3 dpa. n = three independent experiments of 10 tail samples per time point. (b) Real Time PCR analysis of stage 54 limb regeneration. Three samples were used: 0 dpa zeugopod; 3 dpa zeugopod; growth control (GC)-not amputated, three days older zeugopod. myod, myogenin and car levels are the same between 0 and 3 dpa. However, comparing 3 dpa with GC, we observed a significant lower expression of myod, myogenin and car in the regenerating limb. n = four independent experiments of 10 or 20 limb samples per time point. Results were normalized for ornithine decarboxylase (odc) expression and relativized to the time points with highest expression (0 dpa for tail and GC for limb). Error bars: standard error. Asterisk: p value < 0.01; Student's t test.

Mentions: During myogenesis, the expression of muscle genes changes from early or progenitor markers (myod and myf5) to intermediate or differentiation markers (myogenin and MRF4) and finally to late or structural markers (actins, myosins and others) [42,43]. However, during muscle dedifferentiation, the expression of many muscle genes, mainly the structural ones, is expected to decrease [28,44-47]. Our Real Time PCR analysis showed that the expression of the late muscle genes car (alpha-cardiac actin) and myh4 (myosin heavy chain 4) was 5-fold lower in the 3 dpa distal tail stump compared to the 0 dpa distal stump (Figure 4a). This indicates that, after tail amputation, muscle fibres decrease the expression of structural proteins, or, in other words, that the myofibres dedifferentiate. Since we later observed that a considerable number (but no more than half) of myofibres located in the analysed region die after amputation, we repeated the tail amputations but collected 3 dpa stump samples 1 mm away from the amputation plane, a region with negligible fibre loss. With these samples, we observed a downregulation of car to 53.7% ± 4.5% (standard error, p value = 49 × 10-5) compared with 0 dpa, confirming the dedifferentiation of muscle fibres. In fact, we also observed a visual decrease in car promoter activity as the expression of GFP under the Car promoter decreased in a wide region of the distal stump during tail regeneration (not shown).


Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae.

Rodrigues AM, Christen B, Martí M, Izpisúa Belmonte JC - BMC Dev. Biol. (2012)

Expression of mature muscle genes decreases with regeneration in tail and limb. (a) Real Time PCR analysis of tail regeneration. myod gene expression does not significantly change in the distal stump between 0 and 3 dpa, while cardiac α actin (car) and myosin heavy chain 4 (myh4) have 5-fold lower expression at 3 dpa. n = three independent experiments of 10 tail samples per time point. (b) Real Time PCR analysis of stage 54 limb regeneration. Three samples were used: 0 dpa zeugopod; 3 dpa zeugopod; growth control (GC)-not amputated, three days older zeugopod. myod, myogenin and car levels are the same between 0 and 3 dpa. However, comparing 3 dpa with GC, we observed a significant lower expression of myod, myogenin and car in the regenerating limb. n = four independent experiments of 10 or 20 limb samples per time point. Results were normalized for ornithine decarboxylase (odc) expression and relativized to the time points with highest expression (0 dpa for tail and GC for limb). Error bars: standard error. Asterisk: p value < 0.01; Student's t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3313851&req=5

Figure 4: Expression of mature muscle genes decreases with regeneration in tail and limb. (a) Real Time PCR analysis of tail regeneration. myod gene expression does not significantly change in the distal stump between 0 and 3 dpa, while cardiac α actin (car) and myosin heavy chain 4 (myh4) have 5-fold lower expression at 3 dpa. n = three independent experiments of 10 tail samples per time point. (b) Real Time PCR analysis of stage 54 limb regeneration. Three samples were used: 0 dpa zeugopod; 3 dpa zeugopod; growth control (GC)-not amputated, three days older zeugopod. myod, myogenin and car levels are the same between 0 and 3 dpa. However, comparing 3 dpa with GC, we observed a significant lower expression of myod, myogenin and car in the regenerating limb. n = four independent experiments of 10 or 20 limb samples per time point. Results were normalized for ornithine decarboxylase (odc) expression and relativized to the time points with highest expression (0 dpa for tail and GC for limb). Error bars: standard error. Asterisk: p value < 0.01; Student's t test.
Mentions: During myogenesis, the expression of muscle genes changes from early or progenitor markers (myod and myf5) to intermediate or differentiation markers (myogenin and MRF4) and finally to late or structural markers (actins, myosins and others) [42,43]. However, during muscle dedifferentiation, the expression of many muscle genes, mainly the structural ones, is expected to decrease [28,44-47]. Our Real Time PCR analysis showed that the expression of the late muscle genes car (alpha-cardiac actin) and myh4 (myosin heavy chain 4) was 5-fold lower in the 3 dpa distal tail stump compared to the 0 dpa distal stump (Figure 4a). This indicates that, after tail amputation, muscle fibres decrease the expression of structural proteins, or, in other words, that the myofibres dedifferentiate. Since we later observed that a considerable number (but no more than half) of myofibres located in the analysed region die after amputation, we repeated the tail amputations but collected 3 dpa stump samples 1 mm away from the amputation plane, a region with negligible fibre loss. With these samples, we observed a downregulation of car to 53.7% ± 4.5% (standard error, p value = 49 × 10-5) compared with 0 dpa, confirming the dedifferentiation of muscle fibres. In fact, we also observed a visual decrease in car promoter activity as the expression of GFP under the Car promoter decreased in a wide region of the distal stump during tail regeneration (not shown).

Bottom Line: In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate.Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation.In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Regenerative Medicine of Barcelona, 08003 Barcelona, Spain.

ABSTRACT

Background: Mammals are not able to restore lost appendages, while many amphibians are. One important question about epimorphic regeneration is related to the origin of the new tissues and whether they come from mature cells via dedifferentiation and/or from stem cells. Several studies in urodele amphibians (salamanders) indicate that, after limb or tail amputation, the multinucleated muscle fibres do dedifferentiate by fragmentation and proliferation, thereby contributing to the regenerate. In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate. We set out to study whether dedifferentiation was present during muscle regeneration of the tadpole limb and zebrafish larval tail, mainly by cell tracing and histological observations.

Results: Cell tracing and histological observations indicate that zebrafish tail muscle do not dedifferentiate during regeneration. Technical limitations did not allow us to trace tadpole limb cells, nevertheless we observed no signs of dedifferentiation histologically. However, ultrastructural and gene expression analysis of regenerating muscle in tadpole tail revealed an unexpected dedifferentiation phenotype. Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation. In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres.

Conclusions: Our results show that complete skeletal muscle dedifferentiation is less common than expected in lower vertebrates. In addition, the discovery of incomplete dedifferentiation in muscle fibres of the tadpole tail stresses the importance of coupling histological studies with in vivo cell tracing experiments to better understand the regenerative mechanisms.

Show MeSH
Related in: MedlinePlus