Ferchault de Réaumur, in 1712, said that during regeneration “Nature gives back to the animal precisely and only that which it has lost, and she gives back to it all that it has lost”. Since then, we are amazed of seeing that if we amputate, for example, the limb of an axolotl, it regrows up to the same size and shape as the original limb. Moreover, this regenerated limb is as functional as the original one. Similarly, if we cut a planarian in ten pieces, the new ten flatworms that are formed in few days look identical to the original planarian. So, it seems that what Réaumur said 300 years ago, mainly stands true. But, what happens if we analyze the regenerated structures in deep detail? Are they really identical to the original one? In other words, how perfect is regeneration?
Now, a recent paper from the laboratories of Rui Diogo and Elly Tanaka describes the exact pattern of regeneration of 35 muscles that form the arm, forearm and hand of the axolotl’s forelimb as well as a pectoral muscle, the coracoradialis (http://www.ncbi.nlm.nih.gov/pubmed/24692358). This study is part of a larger project to characterize the development, homologies and evolution of many body muscles of all major vertebrate clades. Here, they used transgenic axolotls that expressed GFP in all muscle fibers. They analyzed the pattern of regeneration of these muscles in forelimbs that were experimentally amputated at the level of the arm as well as forelimbs that were amputated “naturally” from bites by other axolotls. In total they analyzed 23 regenerated forelimbs and found out that there were muscle anomalies in 10 of them (43%). This is surprisingly high under the assumption of the precision of regeneration. However, the total number of anomalies in these 23 regenerated forelimbs was only 20, so in average each forelimb had anomalies in only 2,5% of the total number of muscles (n=36) examined. Moreover, the percentage of anomalous muscle was much higher in those regenerates after bite-induced “natural amputations” (3,9% of muscles affected) than in the experimentally amputated regenerates (1,5%). This could be explained by recurrent aggressions affecting different regions compared with the precise and unique experimental amputation.
Another interesting observation was that none of the muscle defects seen in these regenerates was seen in the original limbs analyzed. Remarkably, one of the most common anomalies seen in 35% of the regenerated forelimbs (8 of the 23 regenerated forelimbs analyzed) was the presence of a fleshy coracoradialis at the level of the arm. Usually, this muscle only has a thin tendon at the level of the arm. This anomaly does not necessarily change the main function of this muscle (to flex the forearm). From an evolutionary perspective this anomaly is really interesting because this fleshy configuration resembles very much the fleshy biceps brachii of amniotes, which suggests a parallel between a regeneration defect and a major phenotypic change that occurred during tetrapod limb evolution. In fact, it has been suggested that at least part of the biceps brachii corresponds to the amphibian coracoradialis and, therefore, it could mean that at some point in evolution the tendinous part of the coracoradialis of amphibians had to become associated with fleshy fibers. The authors discuss then that, as suggested by other authors, there is often a parallel between anomalies occurring because of natural or experimental reasons and the normal phenotype found in their closest relatives. This could be interpreted because evolution is highly constrained and, as a consequence, similar phenotypes are often created. In fact, some other of the anomalies in the pattern of the regenerated muscles found in this study are normally found in other non-urodele taxa.
Finally, the authors also investigated the similarities in muscle morphogenesis between regeneration and embryonic development. In both cases, muscles appear to differentiate following a proximodistal and a radioulnar gradient. However, they found here that there is also a ventro-dorsal gradient of differentiation at least for the forearm muscles, that has not been previously seen during embryogenesis, and that would be worthy to further characterize in the future.
In summary, this study addresses how precise is the regeneration of the axolotl limb. On one side they observed that a large percentage of the regenerated limbs showed anomalies in the muscle pattern. But, on the other side, the average number of muscles affected in each limb was really low. Remarkably, they found a common anomaly seen in most of the limbs, which could have further implications for the evolution of muscles in tetrapods.
In this week’s post just let me present to you some of our own recent results on planarian regeneration. After amputation different events must take place in the right order and at the exact time to achieve a successful regeneration. In the case of planarian regeneration one of the first event after wound healing is the establishment of polarity, that is, the animal must know whether to regenerate a head or a tail. Then, once the polarity of the forming blastema is determined other events occur; these include patterning, growth and differentiation of those blastemas. In recent years several genes have been shown to play an important role in the re-establishment of polarity and pole formation in planaria. However, less is known about how for example, a new brain differentiates within an anterior blastema. Now, in a paper from my laboratory, we have characterized a gene that is important for head regeneration in planarians, mainly through its role on the differentiation of the brain primordia (http://www.ncbi.nlm.nih.gov/pubmed/24700819).
In a previous work we had reported that Smed-egfr-3, a homologue of the epidermal growth factor receptor family was important for blastema growth most probably by affecting cell differentiation. Then, we decided to do some DGE analyses to identify putative downstream targets of Smed-egfr-3. One of the isolated genes was Smed-egr-4 a homologue of the early growth response gene family of zinc finger transcription factors.
In situ hybridizations showed that egr-4 was expressed in the CNS, especially the brain, and the mesenchyme in irradiation-insensitive cells (that is, in post mitotic differentiated cells). As expected, the expression of egr-4 was downregulated after egfr-3 RNAi. However, we found out that egr-4 went through two phases of expression, an early expression immediately after amputation or wounding and up to two days, that was independent of egfr-3 and a second phase from the second day of regeneration that depends on egfr-3. The silencing of egr-4 blocked specifically anterior regeneration without affecting the regeneration of posterior regions. In egr-4 RNAi animals, anterior blastemas were very small compared to controls and they failed to regenerate a normal brain or the proper pattern of several other anterior markers. On the other side, the silencing of egr-4 did not seem to largely affect the number of neoblasts (planarian pluripotent stem cells) and although there was a slight decrease in the number of mitotic cells, this did not seem to explain by itself the lack of regeneration. Moreover, other markers suggested that the silencing of egr-4 could be affecting the differentiation of neoblast late progeny.
Recently, other genes have been reported to show a similar phenotype. In those cases, the inhibition of regeneration has been associated to the failure of re-establishing a proper polarity or pole as the expression of polarity determinants is inhibited. Therefore, we wanted to determine whether the impairment of head regeneration observed after egr-4 RNAi was due to defects in the re-establishment of the anterior polarity. However, that did not seem to be the case, after analyzing the expression of several polarity determinants, which suggests that egr-4 might be affecting an event downstream of anterior polarity re-establishment.
As egr-4 was expressed in the mature and differentiated cephalic ganglia we next investigated if egr-4 was required for CNS regeneration. By performing combinatorial RNAi of egr-4 together with an APC homologue we found out that egr-4 appeared to be required for the differentiation of the brain primordia. Given the fact that egr-4 RNAi inhibited anterior but not posterior regeneration and the Wnt/b-catenin pathway mediates the specification of head versus tail regeneration (the silencing of b-catenin1 transforms any blastema into an anterior blastema that will differentiate into a head) we further investigated the relationship between egr-4 and this pathway. Surprisingly double RNAi of egr-4 and b-catenin1 resulted in normal anterior regeneration, which suggested that in a normal situation egr-4 may antagonize b-catenin activity to allow head regeneration. Finally, experiments in which egr-4 was silenced at different time points after amputation and other experiments in which some brain tissues were left after amputation indicated that: (1) the action of egr-4 would be required during the first 2-3 days of regeneration, being dispensable for regeneration after that stage, and (2) the presence of brain tissues was able to rescue the defects observed after the silencing of egr-4.
Overall our results suggest that egr-4 plays a key role in the differentiation of the brain primordia by antagonizing b-catenin function downstream of polarity determinants.
An interesting question raised from these results is that if egr-4 is required for the early differentiation of the cephalic ganglia, how does inhibition of CNS differentiation blocks head regeneration? What we hypothesize is that the brain primordia could send some signal(s) to promote the proliferation, migration and/or differentiation of the neoblasts to allow blastema growth and head regeneration. In the absence of proper brain primordia after egr-4 RNAi, the lack of such putative inducing signal would explain the inhibition of head regeneration. Further experiments are needed to explore this putative relationship between brain differentiation and head regeneration in planarians, which would support the view of an evolutionarily conserved role of the nervous system in animal regeneration.
Several signaling pathways have been shown to play important functions during regeneration in different invertebrate and vertebrate models. Many of these pathways including activin, BMP, Hh, Igf, Notch, retinoic acid (RA) and Wnt/b-catenin have been shown to be required for the successful regeneration of the zebrafish fin. However, little is known yet on the tissue-specific roles of theses pathways as well as on how they interact to orchestrate fin regeneration. Now, a recent paper from the laboratory of Gilbert Weidinberg has described the mechanisms through which the Wnt/b-catenin pathway regulates blastema growth and regeneration (http://www.ncbi.nlm.nih.gov/pubmed/24485658).
Upon fin amputation, wound healing leads to the formation of a multilayered wound epidermis. Then, a blastema is formed within 48 hours and a subsequent regenerative outgrowth phase concludes in the complete regeneration of the fin in about 3 weeks. This blastema is compartmentalized in 4 main domains: (1) a non-proliferative distal part, (2) a highly proliferative proximal medial region, (3) highly proliferative bilateral zones containing osteoblast progenitors, and (4) domains directly medial to these osteoblast progenitors. In this paper, the authors have used transgenic pathway reporters and elegant tissue-specific pathway manipulations to finely characterize the function of the Wnt/b-catenin pathway. It had been previously shown that this signaling pathway is required for blastema formation and cell proliferation; however, it was not known how does it exert this function.
First, the authors used transgenic reporters of b-catenin-dependent transcription to define the sites of endogenous b-catenin activity. One first surprise was to see that the epidermis was devoid of Wnt/b-catenin signaling although lef1, a well-known Wnt target, is expressed in the epidermis. These results suggested that this pathway must indirectly regulate lef1 expression. Also, and as Wnt/b-catenin signaling is required for blastemal cell proliferation the authors wondered whether Wnt activity correlated with sites of blastema cell proliferation. Surprisingly, they found that this pathway was strongly activated in the most distal part of the blastema (a non-proliferative domain) and weaklier in lateral domains of the proximal blastema (in actinotrichia-forming cells adjacent to osteoblast progenitors). However, this pathway was not active neither in most part of the highly proliferative proximal medial blastema or in committed osteoblasts. Therefore, and because the inhibition of the Wnt/b-catenin pathway strongly reduced proliferation in the proximal blastema, it seems that this pathway regulates cell proliferation indirectly. The specific inhibition of this pathway in the highly proliferative proximal blastema slowed down regeneration but without blocking it, in contrast to the strong regeneration blockade observed when the pathway was also inhibited in the most-distal blastema. This further supported that Wnt/b-catenin activity in the distal blastema indirectly regulates proliferation in the proximal blastema. Next, the authors investigated about the function of Wnt/b-catenin activity in the actinotrichia-forming cells and found out that the activation of this pathway here was necessary to regulate (also indirectly) the commitment and differentiation of the adjacent osteoblast progenitors.
As all this data suggested that the Wnt/b-catenin pathway regulates fin regeneration largely indirectly the authors sought to find putative downstream target signals by performing gene expression profiling after inhibition of this pathway. They found that upon inhibition of the Wnt/b-catenin pathway the expression of many genes related to other signaling pathways was also significantly reduced. These putative targets included several elements of the Hh, BMP, RA, Igf, Notch and FGF signaling. However, the silencing of those pathways appeared to have very little (if any) effect on Wnt/b-catenin activity, suggesting that Wnt signaling would act upstream of a network of pathways during fin regeneration.
Finally, the authors characterized how these different pathways could interact to regulate different aspects of fin regeneration. Thus, they found that epidermal lef1 expression would be regulated by BMP and FGF signaling downstream of b-catenin activity. Similarly, they found that Wnt/b-catenin signaling acted upstream of Hh and RA to regulate blastema cell proliferation. In addition to this indirect regulation, the authors found that the expression of fgf3 and aldh1a2 (a key enzyme for RA synthesis), in the distal blastema was directly regulated by b-catenin signaling, which suggest that these 2 genes function as signals that mediate the effect of Wnt/b-catenin signaling in the distal blastema on regenerative growth and patterning of the surrounding tissues. For example, RA formed in the distal blastema would diffuse to regulate proliferation in the proximal blastema.
In summary, the results presented here suggest that within the fin blastema, Wnt/b-catenin signaling may define organizing centers that would control regeneration by regulating the function of several downstream signaling pathways that would mediate the effects of these organizers on surrounding tissues.