Genetic labelling of muscle fibres for the in vivodetection of dedifferentiation
While we have a fair understanding of how muscle regenerates in the Xenopus tadpole tail, there is a lack of knowledge about tadpole limb muscle regeneration. To obtain more information on this process, specifically whether limb muscle fibres dedifferentiate/fragment and contribute to the new muscle, we attempted to genetically label limb muscle fibres with GFP and to follow them during regeneration. We first confirmed that the alpha-cardiac actin (Car) promoter was able to drive strong and specific GFP expression in muscle fibres of the limb and tail (Figure 1a-c) [29]. However, these simple Car-GFP transgenics are not useful for cell tracing experiments, since the unlabeled myogenic progenitors activate GFP expression during regeneration and become indistinguishable from any possible unicellular fragments derived from labelled dedifferentiating myofibres (Figure 1d, arrows) [8].
To avoid myogenic progenitors from getting labelled de novo during regeneration and to unambiguously identify myofibre fragments, an inducible labelling system was needed. We used the Cre-lox recombination system, where Cre recombinase can remove (or invert) genomic sequences lying between two loxP sites [30]. This allows constitutive labelling of any desired cell lineage when using a tissue specific promoter driving cre expression and a ubiquitous promoter followed by lox-STOP-lox and a fluorescent protein gene like GFP. To make this system inducible and to avoid Cre (and GFP) activation during de novo myogenesis, we used the well established tamoxifen-inducible Cre system, where Cre is fused to two mutated estrogen receptors (ERT2) that inhibit Cre activity in the absence of tamoxifen [31, 32]. We, therefore, constructed the "Car-ERCreER/CALNL-GFP" vector containing a tamoxifen-inducible Cre under the control of the Car promoter and the lox-neo-lox-GFP cassette under control of the constitutive CAG promoter [32, 33] (Figure 2a). After Cre induction in founder animals, all we ever achieved were a few randomly labelled muscle fibres in the tail without any positive fibres in the limb. Similar results were obtained with F1 and F2 offspring from two different transgenic lines, even after two weeks of daily intraperitoneal injections of tamoxifen. We observed very few and dispersed labelled myofibres in the tail (Figure 2b) and, more importantly, only one animal (from hundreds) had three faintly labelled fibres in the limb (Figure 2c, arrows), which did not allow us to study limb regeneration with these transgenics.
As an alternative to the ERT2CreERT2 system we tested the alpha complementation of Cre subunits. This system is based on the fact that the Cre protein can be split into two inactive fragments that, when co-expressed, re-associate and restore Cre activity [34]. For higher re-association efficiency, Cre fragments can be fused with dimerizing peptides [35, 36]. We reasoned that putting one fragment under the control of an inducible promoter (e.g. the Heat-shock promoter Hsp70) and the other under the Car promoter, the result would be the desired inducible and muscle-specific expression of Cre recombinase. Therefore, we built two new constructs (Figure 2d), one containing the N-terminal Cre fragment (nCre) under the control of the Car promoter and the second with the C-terminal Cre fragment (nlcCre) under the control of the Hsp70 promoter. We injected these two constructs together with the reporter construct, pCALNL-GFP, into Xenopus eggs to obtain triple transgenics. After heat-shock induction, some founder transgenics had many labelled tail muscle fibres, but still no labelled limb fibres. Similarly, the offspring of a founder male frog occasionally showed strong expression of GFP in trunk muscles (compare Figure 2e with 2e, arrows) but no labelling in the hindlimbs (compare Figure 2e with 2e, arrowheads). Labelling was very dispersed close to the tail tip (Figure 2f), like in "CreER" transgenics, however, the middle of the tail was reasonably labelled (Figure 2g). The inability to label limb muscle with these constructs precluded us from studying limb muscle dedifferentiation by in vivo cell tracing, so we proceeded to study it histologically.
Tail, but not limb myofibres, show signs of dedifferentiation after amputation
One of our main goals in this study was to detect the presence or absence of muscle dedifferentiation during tadpole limb regeneration. Since we could not obtain results with the cell tracing experiments, we decided to study limb muscle regeneration histologically. It has been known for a long time that Xenopus limb regeneration capacity decreases during development: an immature limb bud regenerates perfectly, while a fully differentiated limb does not [37]. Therefore, we analysed stage 54 limbs (staging according to Nieuwkoop & Faber [38]), which lay halfway in between the two extremes, i.e., they are not too immature and still regenerate to some extent. Tail amputations were used as a negative control for muscle dedifferentiation, as it was shown by Gargioli & Slack that tail muscle fibres do not contribute to the regenerate [8]. Surprisingly, we observed a dedifferentiation phenotype in the tail (Figure 3a-j), mainly at 3 days post-amputation (dpa). 0 dpa control tails showed the expected sarcomeric striations occupying basically all the sarcoplasm of the myofibres (Figure 3a, b). At 1 dpa, many myofibres had a less elongated shape with fairly regular sarcomeric striations (Figure 3d, e, arrows) while some fibres had small regions without sarcomeres (Figure 3d, arrowhead) and only in rare cases did myofibres have severe sarcomeric loss (Figure 3e, arrowhead). At 3 dpa, many more stump myofibres showed loss of sarcomeric organization and had irregular shapes, both close and farther from the amputation plane (Figure 3f-h, arrowheads). Alpha sarcomeric actin (ASA) staining also showed evidence of a lack of sarcomeric organization (Figure 3i, arrowhead). Electron microscopy observations showed that myofibrils (Figure 3j, arrow) were detaching from each other, leading to the loss of sarcomeric striations (Figure 3j, arrowheads), a phenotype previously described as dedifferentiation [19, 20, 39–41].
In the limb the situation was less clear (Figure 3k-r). Stage 54 hindlimbs have very immature distal muscle, grouped in small patches and composed mainly of myoblasts and small myofibres. These small and thin myofibres have few myofibrils and sarcomeres inside their cytoplasm (Figure 3k, l, arrows). At 1 dpa, the myofibres continued to present aligned sarcomeres with no signs of dedifferentiation (Figure 3m, n, arrows). At 3 dpa, the distal muscle patches were still very immature and similar to the ones identified at 0 and 1 dpa. We continued to observe small myofibres with normal striations and no obvious dedifferentiation phenotype (Figure 3o-r), but we recognize that due to tissue immaturity, sarcomeric disorganization might be hard to identify. To obtain more information on limb and tail muscle regeneration, we performed gene expression analysis by quantitative Real Time PCR.
Tail muscle dedifferentiation is corroborated by gene expression analysis
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).
In the limb the situation was more complex to analyse. Because the stage 54 limb is growing quickly and more muscle is being added every day, we needed a "growth control" to compare with the 3 dpa distal stump. The growth control (GC) came from stage 54 animals that were not amputated on day 0 but were left to grow for three more days, when the proximal half of the zeugopod was collected. Analogous regions were collected for 0 dpa and 3 dpa. Comparing gene expression between 0 dpa and 3 dpa zeugopods, we found no differential expression of myod, myogenin and car (Figure 4b), indicating the absence of dedifferentiation. The expression of myh4 slightly increased, but the change was not statistically significant (p value = 0.34). However, when we compared the 3 dpa tissue with the GC, we observed that the regenerating tissue has a 2- to 3-fold lower expression of myod, myogenin and car, indicating that myogenesis is halted by the amputation.
In vivotracing confirms that myonuclei do not contribute to the tail regenerate
The observed signs of tail muscle dedifferentiation were unexpected since it was previously shown by Gargioli and colleagues that tail muscle fibres did not contribute to the regenerate [8]. To confirm Gargioli's results, we returned to our inducible Cre transgenics ("CreER" and "Cre fragments", Figure 2) to follow labelled tail muscle during regeneration. As already stated, a common characteristic of the induced "Cre fragments" transgenics (and to a lesser extent in "CreER" transgenics) was that many of the labelled myofibres were concentrated in the trunk, close to the head and hindlimbs (Figure 2e'), while less labelling was found in the middle of the tail (Figure 2g) and little labelling was present close to the tip of the tail (Figure 2b, f). This allowed us to do two kinds of experiments. One was to amputate through the middle of the tail ("proximal amputations"; only in "Cre fragments" transgenics) and follow the overall changes of the group of labelled fibres during regeneration. The other experiment was to amputate closer to the tip, at around 2/3 of the tail ("distal amputations"; in both "CreER" and "Cre fragments" transgenics), and follow, in detail, individually labelled myofibres. The latter experiment is analogous to one performed by Echeverri et al. (2001), where they showed that axolotl tail myofibres fragment to proliferative mononuclear cells. Fibre fragmentation (or cellularization) and re-entry into the cell cycle are independent mechanisms expected to conclude the process of muscle dedifferentiation [5, 48].
After proximal amputations of reasonably well labelled tails, we observed that many myofibres located close to the amputation plane were missing at 1 dpa, indicating the death of these fibres (Figure 5a, b, arrows). Treatment of wild-type tadpoles with the vital stain Propidium iodide at 0 dpa showed many positive myonuclei next to the amputation plane, confirming the death of distal-most myofibres (not shown). Over the following days, the number and position of GFP labelled fibres barely changed (please compare 1 dpa with 6 dpa and 12 dpa in both Figure 5a and 5b), indicating that these myofibres were stable and not fragmenting. In addition, no labelling was observed in the tail regenerates after careful examination with higher magnifications. These observations indicate that myofibre cellularization and contribution to the regenerate was not occurring during tail regeneration, confirming Gargioli's results [8].
Distal myofibres retract after amputation but do not fragment
Taking advantage of the low number of labelled myofibres in the distal part of the tail, we were able to follow individually labelled myofibres during regeneration. A similar experiment done by Echeverri et al. in the axolotl tail showed synchronous myofibre fragmentation a few days after the labelled muscle fibre was clipped by the amputation [4]. In our hands, clipping the tip of a myofibre always led to its death (n = 14). On the contrary, amputations that did not directly touch the myofibres led to much higher fibre survival. A common characteristic of the surviving myofibres was that they frequently showed a compact or retracted shape at 1 dpa (n = 94, Figure 5c, d). In some situations, a slight retraction could already be observed a few minutes after amputation. Between two to four days post-amputation, these myofibres started to recover their size (Figure 5c, d) and sometimes they formed irregular cytoplasmatic projections (Figure 5d, arrowheads). By 12 days post-amputation, these myofibres had generally recovered their initial length (Figure 5c) or were even longer (Figure 5d). So, in contrast to what is described in the axolotl [4], we did not observe myofibre fragmentation in the tadpole tail.
Interestingly, different types of muscle fibres responded differently to amputation: "short" myofibres (around 2/3 the size of a myotome) were much more prone to retract than "long and thin" myofibres (length of a myotome). In fact, all the "short" fibres retracted after distal amputation (n = 83, with variable degrees), while only 11 out of 32 "long and thin" fibres slightly retracted after amputation. Therefore, not all the myofibres traced during regeneration showed an obvious retraction phenotype. In several cases, muscle fibres maintained their shape and size from the day of amputation till the end of observations and they generally belonged to three different groups: "long and thin" myofibres (n = 21, see example below); fibres from "proximal amputations" (n > 100, see below and Figure 5a, b); fibres far from the amputation plane (n = 7, not shown).
Dedifferentiating myofibres do not enter the cell cycle
To analyse the extent of dedifferentiation, specifically if dedifferentiating myofibres entered the cell cycle, we did a BrdU pulse to label cells synthesizing DNA. BrdU was injected intraperitoneally at 3 dpa (when dedifferentiation is most frequent) and tail samples were fixed 3, 24 and 72 hours later. All myonuclei were BrdU negative in the samples fixed 3 and 24 hours after injection (Figure 6, arrows), showing that no dedifferentiating myofibre entered S-phase at 3 dpa (n = 76 BrdU- myonuclei at 3 h + 100 BrdU- myonuclei at 24 h). On the other hand, many cells were proliferating at 3 dpa and 12% of them were found to be activated satellite cells (Pax7+; n = 34 BrdU+/Pax7+ of 294 BrdU+ nuclei; not shown). As a result, the samples fixed 72 hours after the pulse had many muscle fibres with BrdU positive myonuclei (n = 33 BrdU+ myonuclei; not shown). These labelled nuclei most likely derived from satellite cells that were proliferating at 3 dpa and later fused with new or pre-existing myofibres. In addition, PCNA and pHH3 staining confirmed the absence of cell cycle in dedifferentiating myofibres at various days post-amputation (not shown). These results, together with the in vivo tracing, indicate that tadpole tail myofibres experience an incomplete dedifferentiation, lacking cell cycle re-entry and fragmentation.
Dedifferentiation is associated with myofibre retraction
Several reasons led us to believe that the dedifferentiation phenotype observed histologically (Figure 3c-j) was related to the muscle fibre retraction observed in vivo (Figure 5c, d). Firstly, the dedifferentiating fibres had irregular and compact shapes that resembled the retracted fibres observed in vivo (Figure 7a-j). As described previously, myofibres had an elongated appearance before amputation (Figure 7a) with well organized sarcomeres (Figure 7f, arrowheads). At 1 dpa, many myofibres showed a more compact shape in vivo (Figure 7b and 5c, d) as well as histologically, with occasional loss of sarcomeric striations (Figure 7g, arrows. See also Figure 3c-e, arrowheads). At 3 dpa, the retracted fibres still had a compact shape in vivo (Figure 7c), but now, a more frequent dedifferentiation phenotype was visible in the retracted/irregularly-shaped myofibres, histologically (Figure 7h, arrow). At 6 dpa, the retracted myofibres were recovering their elongated shape in vivo (Figure 7d) and, histologically, the myofibres showing signs of dedifferentiation also had a more elongated shape (Figure 7i, arrows). The dedifferentiation phenotype was now less frequent, since more muscle fibres had normal sarcomeric striations (Figure 7i, arrowheads). Later in regeneration, the previously retracted myofibres regained an elongated shape (Figure 7e and 5c, d (at 12 dpa)) and only rare dedifferentiation remnants were observed histologically (Figure 7j, arrow (at 14 dpa)). The majority of myofibres close to the amputation plane showed normal sarcomeric striations at 14 dpa (Figure 7j, arrowheads).
Secondly, retraction and dedifferentiation phenotypes were specific to one type of myofibres. Sasaki described that the tadpole tail has two types of muscle fibres: "red" fibres, with a small diameter and located externally, very close to the skin (Figure 7m, arrowheads); and "white" fibres, which are wider and located internally, below the red fibres (Figure 7m, arrows) [49]. By shape and size comparison, we concluded that the "short" fibres observed in vivo (Figure 7k, fibres "a" and "c") are white (internal) fibres, while the "long and thin" (Figure 7k, fibre "b") are red (external) fibres. These myofibres behaved differently after distal amputation, since the short and internal myofibres always retracted after amputation (Figure 7k, l; compare size and shape of fibre "c" between 0 dpa and 3 dpa) and showed the dedifferentiation phenotype at 3 dpa (Figure 7n, arrows). In contrast, the long and thin myofibres, generally, did not contract (Figure 7k, l. Compare fibre "b" between 0 dpa and 3 dpa) and never showed the histological dedifferentiation phenotype after amputation (Figure 7n, arrowheads).
Finally, we also observed a relationship between retraction and dedifferentiation when we compared proximal and distal amputations. Although retraction was commonly observed after distal amputations, it was much less frequent and noticeable after proximal amputations (Figure 7o-q and Figure 5a, b). Accordingly, semithin sections revealed that myofibres frequently showed regular sarcomeric structure after proximal amputations (Figure 7s, t, arrowheads) with some myofibres showing very small regions of sarcomeric disorganisation (Figure 7s, t, arrows).
Larval zebrafish myofibres do not dedifferentiate after tail amputation
Very little is known about skeletal muscle regeneration in fish. Recent reports have shown that dedifferentiation is very common during regeneration of zebrafish tissues [6, 40, 50, 51], but it remains unknown if this is true for skeletal muscle regeneration. To study muscle regeneration in the zebrafish, we cloned the Car-ERT2CreERT2 construct (without the reporter part) into a Tol2 vector and made transgenics in a Tg(eab2:[EGFPTmCherry]) background [52] (Figure 8a). These transgenics ubiquitously expressed GFP (Figure 8b), but no mCherry (red fluorescence) (Figure 8c). When tamoxifen was added, Cre became active and GFP was switched for mCherry expression specifically in muscle fibres (Figure 8d).
For the regeneration studies, we treated embryos with tamoxifen and amputated two week old larvae proximally to the base of the fin fold, removing about 1/5 of the tail musculature (Figure 8e, f). Six days after amputation, we could observe a small tail regenerate proximal to the regenerated caudal fin, generally smaller than the length of two myotomes, without mCherry labelling (Figure 8g, arrowhead). Although the new caudal fins continued to grow with time, the small tail regenerates stayed short in length and free of mCherry labelling (Figure 8h, arrowhead (14 dpa)). We further confirmed that the regenerated tail indeed contained new muscle (Figure 8i). The absence of mCherry labelling in the regenerate indicated that the new muscle did not derive from dedifferentiated stump fibres. Although the number of mCherry+ fibres stayed approximately the same during regeneration, we observed that de novo myogenesis occurring in the stump (due to the overall growth of the animal) was depositing new GFP+/mCherry- fibres next to the old mCherry+ ones. This led to a dispersion of mCherry+ fibres, resulting in stumps with many GFP+ fibres and a few mCherry+ fibres at 21-28 dpa, impeding clear and illustrative pictures at these later time points.
The absence of muscle dedifferentiation was corroborated by histological observations. At 0 dpa and 1 dpa, we observed that the myofibres closest to the amputation plane showed a damaged phenotype, characterized by the alteration and darkening of the sarcomeric striations (Figure 9a, b, arrowheads). A similar phenotype was observed in Xenopus tail myofibres damaged by the amputation, at 0 dpa (not shown) [53]. The remaining fibres, although much smaller than the Xenopus tail myofibres, showed clear and regular sarcomeric striations (Figure 9a, b, arrows). At 3 dpa (and 2 dpa, not shown) damaged fibres were no longer visible. Instead, a region with few muscle fibres was present, implying that the majority of damaged fibres had degenerated (Figure 9c, asterisk). Moreover, we did not observe signs of myofibre dedifferentiation. Between 4 and 5 dpa no significant changes were observed (not shown) and at 6 dpa we could confirm that the small regenerate was being populated by new muscle cells (Figure 9d). These results, together with the cell tracing experiments, indicate that muscle dedifferentiation does not occur during zebrafish skeletal muscle regeneration.