That some crustaceans are able to regenerate their limbs is something known since the 18th century. Limb regeneration has been also observed in other arthropods like some insects. In all these animals, regeneration is closely associated to the molting process. Unfortunately, for many of these species we do not have the proper tools to analyze regeneration at the molecular and/or gene expression level. Therefore, it is important to develop those tools in these species so they can become good models to study the regeneration process. In a paper from the laboratory of David S. Durica the authors applied for the first time RNAi to study regeneration in a brachyuran crab, the fiddler crab Uca pugilator (http://www.ncbi.nlm.nih.gov/pubmed/23142248).
Upon predation or injury these animals can cast off their legs at a predetermined breakage plane by a process called autotomy. After autotomy, the wound is sealed and a scab is formed. Two days following autotomy the epidermal cells underlying the scab divide to originate a blastema. The exact origin of the cells that form the blastema is not completely clear yet although it seems that migrating epidermal cells dedifferentiate and proliferate to give rise to it (however, it is not known the degree of dedifferentiation that those epidermal cells suffer). Seven to nine days after autotomy a blastema emerges; this protuberance is called papilla. This papilla (limb bud) will grow and differentiate into limb segments. When the animal gets ready to molt, this limb goes through a second growth phase called proecdysial growth marked by hypertrophy.
Ecdysteroids are steroid hormones that play pivotal roles during growth, development and reproduction in arthropods. Previous studies had shown how the level of ecdysteroids varies along the different phases of limb growth and development before and during the molt and, therefore, the authors wanted to analyze the function of this signaling during limb regeneration. Ecdysteroids bind to nuclear receptors that are transcription factors that bind to conserved Hormone Response Element (HRE) sequences. Here, the authors used RNAi to silence EcR and RXR the genes encoding the ecdysteroid receptor heterodimer. These genes had been shown to be expressed in the blastema and limb buds at the proecdysial growth phase during regeneration, which further supported the hypothesis of a role of this signaling pathway during blastema formation.
The authors checked the efficiency of their RNAi experiments and used different injection protocols to deliver the dsRNA of these two genes at different time points after autotomy. In general, what they observed was that the emergence of the blastema in form of a papilla was significant reduced compared to controls. Moreover, these defects were more penetrant when dsRNA was delivered earlier after autotomy. Somehow this suggests that the ecdysteroid receptor signaling may be especially important during the first days after autotomy. After autotomy, epidermal cells migrate underneath the scab, proliferate and secrete also a very thin cuticle. The first segment of the limb is formed by the invagination of this cuticle (around 7 days following autotomy). In the dsEcR/dsRXR-injected animals no cuticular invaginations were seen and, compared to controls, a much thicker cuticle was secreted from the epidermal cells that migrate underneath the scab.
In order to check whether the lack of papilla formation and blastema arrest could be due to defects in cell proliferation the authors performed some BrdU labellings. After EcR and RXR RNAi a significant decrease of proliferating cells was observed. In contrast, in controls they found division of epidermal cells underneath the scab as well as in cells along the nerve. However, the silencing of these receptors did not seem to affect the normal migration of epidermal cells towards the wound. Overall, these experiment suggest that the silencing of EcR and RXR resulted in the failure of epidermal cells to proliferate and give rise to a normal blastema. Finally, those animals failed to molt and died which suggested that the RNAi of EcR and RXR might result in the inability to correctly respond to hormonal signaling at the end of the molt cycle.
In summary the ecdysteroid receptor signaling appears to be required for the proliferation and differentiation of the blastema cells during fiddler crab limb regeneration.
A general property of animal development is that all organs and appendixes grow up to specific proportions relative to the body size of the organism. Remarkably, in those species capable of regenerating, for example a complete limb de novo, the regenerated structure grows up to its original size restoring the normal body proportions. How this coordinated growth is regulated is not much known. Now a recent paper from the laboratory of Christopher Antos (http://www.ncbi.nlm.nih.gov/pubmed/24561038) describes the role of calcineurin on the coordination of growth during zebrafish fin regeneration.
Isometric growth refers to the situation where growth occurs at the same rate for all parts of an organism. Any deviation from this situation for a specific organ or structure is refereed as an allometry. During the development of the zebrafish fin, this appendix grows initially allometrically respect to the rest of the body until it approaches its final size when it switches to an isometrical growth. Similarly, during fin regeneration, the initial stages are characterized by an allometric growth that ends up to an isometric growth when the fin is regenerated. This paper describes the role of calcineurin on regulating this fin growth rate.
Calcineurin is a protein phosphatase that activates the T cells of the immune system. Because the immune response after an injury can modulate the regenerative output the authors wanted to check whether using the immunosuppressant Tacrolimus (FK506) had any effect on zebrafish fin regeneration. By treating regenerating fins with this drug they observed how those amputated fins grew much larger than the controls, giving rise to large fins disproportionate to the body size. The growth rate of the FK506-treated fins started to differ from the control rate around day 7 post-amputation and was kept higher beyond 4 weeks. In controls, the growth rate of the regenerated fin initially increased respect to the body growth rate but then decreased around 3 weeks post-amputation as the regenerated fin reached its original size. The authors show that after FK506 treatment the increased growth rate in the regenerating fin occurred in the distal regenerate and not in the stump region. In fact, FK506 binds to the so-called FK506-binding proteins (FKBPs) and fkbp1Aa, fkbp10 and fkbp14 genes were expressed in the blastema, further suggesting that FK506 acted on blastema cells. At the histological level this enlarged fins showed normal organization, normal tissue morphometry and there were no evidences of tumorous growth. The authors also checked that the expression of several genes that are normally reactivated during regeneration was maintained further after FK506 treatment, suggesting that the enlarged outgrowths were due to the maintenance of this proregenerative transcriptional program.
Remarkably, FK506 treatment of uninjured adult limbs also induced their allometric growth respect to the body resulting in enlarged fins. Here, again several proregenerative genes were upregulated in those uninjured fins. In juvenile zebrafish the development of the fins involves also an initial allometric growth. FK506 treatment of those juveniles also resulted in the maintenance of an allometric growth and enlarged fins. In all these experiments it should be noticed that the effects of FK506 were restricted to the growth rate of fins and not the rest of the body. Overall, these experiments suggested that FK506 affected a mechanism that regulates a switch between isometric and allometric growth. Not all immunosuppressant yielded the same effects pointing out to a FK506-specific target. It is known that the FK506-FKBP complex binds calcineurin inhibiting its activity. Using another drug that also inhibits calcineurin the authors found the same effects on fin outgrowth, suggesting that calcineurin could be regulating this allometric growth. Accordingly to this hypothesis the authors found that during early stages of regeneration when the regenerating fin is growing allometrically the activity of calcineurin was decreased compared to an uninjured fin. Conversely, this activity increased as the regenerating fin was achieving its final size and switching to an isometric growth. Therefore, calcineurin appeared to be involved in regulating allometric growth.
It is also known that amputated fins at more proximal regions regenerate at higher rates that those fins amputated more distally. Thus, for example if one lobe of the bilobed fin is amputated at a proximal region and the other lobe is amputated more distally, both lobes reaches their final size at the same time because the lobe amputated proximally regenerates faster. This suggests that the positional information along the proximodistal axis can determine the growth rate. If repeating this experiment of amputating the two lobes of the fin at different levels along the proximodistal axis and allowing them to regenerate in the presence of FK506 the authors found that both lobes grew at a high rate characteristic of the proximal regenerate of controls. The growth rate in the FK506-treated fins never converged so they regenerated enlarged and disproportionate lobes with the one amputated more distally larger than the one amputated more proximal (and both larger than controls). The authors concluded then that this enhanced growth rate was due to a proximalization either of the regenerating tissue or the growth program. This was further supported by the observation that the FK506-treated fins showed a distal shift of the bifurcation point of each fin ray.
Finally, as the retinoic acid (RA) pathway has been shown to regulate positional growth by proximalizing the outgrowth the authors analysed this pathway after FK506 treatment. FK506 induced the expression of raldh2 and crabp2b (proteins that increase RA signalling) as well as induced the downregulation of enzymes that degrade RA, suggesting that calcineurin inhibition promoted the expression of RA signalling during fin regeneration that lead to the proximalized allometric growth rate.
In summary, the authors describe the role of calcineurin as a molecular regulator of the switch between isometric and allometric growth during fin development and regeneration.
Few months ago I informed you about “Regeneration”, the first scientific journal dedicated exclusively to our field. Now, the first issue is already out online. You can check the first publications at: (http://onlinelibrary.wiley.com/doi/10.1002/reg2.2014.1.issue-1/issuetoc)
Congratulations to everybody working for the succeed of this journal. I wish we can all collaborate to make it a reference journal
Heart failure is one of the major causes of mortality worldwide. After myocardial infarctions the human cardiomyocytes are incapable of proliferating to give rise to new cardiac muscle and, instead, non-contractile scar tissue is formed. On the other hand, vertebrates as zebrafish can fully regenerate their heart after amputation, cryoinjury or genetic ablation of cardiomyocytes. Also, 1-day old mice can regenerate their hearts after amputation, ability that is lost around one week after birth. Importantly, although the adult mammalian heart does not regenerate a low rate of cardiomyocyte proliferation has been recently reported. Therefore, these examples provide hope that science can find the way to enhance the very poor regenerative abilities of the human heart.
A first step is to fully understand how zebrafish, for example, regenerate their hearts. Now a recent paper from the laboratory of Geoffrey and Caroline Burns reports on the function of Notch signalling during zebrafish heart regeneration (http://www.ncbi.nlm.nih.gov/pubmed/24474765). Previous reports had indicated that some components of the Notch signalling were upregulated after heart amputation, however the functional relevance of such upregulation remained to be determined. In this paper the authors show first how of the four zebrafish Notch receptors, notch1a, notch1b and notch2 were upregulated in the endocardium after amputation, especially in regions close to the wounds. On the other hand, notch1a and notch2 were also upregulated in the epicardium covering the wound. Finally, no expression of any notch receptor was detected in the myocardium in un-injured and injured hearts.
Because of a possible redundancy of the several notch genes, the authors used a dominant negative isoform of the MAML protein, a pan-Notch pathway inhibitor, under the control of a heat shock promoter. After amputation the animals were heat shocked daily for 30 days and then they checked the amount of regeneration. Notch inhibition resulted in failed myocardium regeneration and instead a fibrotic scar was seen at the site of amputation. Because notch genes were upregulated in the endocardium and epicardium the authors analysed then what happened to those tissues after Notch inhibition. Remarkably, the silencing of this pathway did not affect the normal response of these tissues to amputation. Thus, in normal conditions, after amputation epicardial cells go through an epithelial-to-mesenchymal transition to produce epicardial derived cells (EPDCs) that at the end become smooth muscle cells that support the regenerating coronary network. This activating response to form EPDCs was normally observed after Notch inhibition. On the other hand, after amputation endocardial cells upregulate the expression of raldh2 and shift from flattened to rounded morphology. This change in the morphology persists at the wound site several days after amputation. Animals in which the Notch pathway had been silenced showed a normal activation of the endocardium after amputation.
Also, although the treated animals lacked regenerated myocardium they had endothelial tubes in the wound area suggesting that Notch signalling seemed dispensable for initiating coronary artery regeneration. In contrast when they checked what happened to the myocardium after amputation the authors found out that whereas the cardiomyocyte dedifferentiation process triggered by the amputation was not affected in a Notch signalling-inhibited background, their proliferation was severely impaired. These results suggest that Notch signalling would be required for cardiomyocytes proliferation.
The authors wanted then to check whether overactivating Notch signalling could have a beneficial effect on heart regeneration. Surprisingly, however, they found that the hyperactivation of Notch signalling resulted in collagen deposition at the wound region and a significant decrease of cardiomyocyte proliferation. These results indicate that cardiomyocyte proliferation would depend on the fine regulation of the Notch signalling. In fact such fine regulation of this pathway has been also proposed during zebrafish fin regeneration in which, again, both inhibition and overactivation of Notch signalling impairs regeneration (although by affecting either proliferation or differentiation).
In parallel to the characterization of the Notch pathway during heart regeneration the authors describe also in this paper that the new coronary endothelium derives, at least partially, from pre-existing endothelium. This agrees with previous observations in fish as well as in salamanders in which new cell types during regeneration appear to derive from the same pre-existing cell types.
In summary this paper shows how Notch signalling is activated in the endocardium and epicardium upon ventricular apex amputation. Remarkably, its silencing does not affect endocardium and epicardium activation but blocks regeneration by impairing cardiomyocyte proliferation in a non-cell autonomous manner. Further experiments should help to better understand how overactivating Notch signalling inhibits also cardiomyocyte proliferation in the zebrafish heart.