Patients of neurodegenerative diseases such as Alzheimer and Parkinson as well as people that suffer spinal cord injuries would surely benefit if we would manage to enhance somehow the very limited regenerative capacities of our central nervous system. But whereas mammals show almost no regeneration potential in their CNS, other vertebrates such as amphibians and zebrafish are capable of regenerating their spinal cord. Also, invertebrates such as planarians can regenerate a complete CNS de novo from any small piece of their bodies. So, how do amphibians and fish do to regenerate their spinal cord? What prevents mammals from doing the same? Because of the great potential benefits of enhancing mammalian CNS regeneration a lot of research is being done in this field; however, the results are not being really very promising yet, at least as fast as demanded by the society.
In a recent review from the laboratory of Karen Echeverri the authors discuss the current state-of-the-art of vertebrate CNS regeneration (http://www.ncbi.nlm.nih.gov/pubmed/23581406). When comparing the regenerative response of mammals to that from amphibians and fish important aspects that need to be considered include the cellular response to injuries and the formation and role of the glial scar. In zebrafish, upon injury, glial cells amplify and migrate to the lesion where they elongate creating a glial bridge that axons use as a scaffold to grow across the lesion. A very interesting observation in zebrafish is that the re-wiring of the regenerated CNS does not need to be one hundred per cent accurate (in terms of re-establishing the original connections) to yield a functional recovery.
Amphibians, both anurans (tailless amphibians) and urodeles (tailed amphibians) regenerate the spinal cord during tail regeneration. In Xenopus it has been described a population of putative neural stem cells positive for Sox-2 that, upon injury, proliferate and migrate forming a substrate for axonal regeneration. In urodeles, also, fibroblasts and glial cells migrate to create a substrate for axonal regeneration. Thus, in all these models once the spinal cord is injured certain cell types are able to create a regeneration-permissive environment. Another thing that is shared by amphibians and zebrafish refers to the upregulation of signalling pathways such as Wnt, BMP and FGF during spinal cord regeneration.
In contrast, CNS regeneration in mammals is inhibited by both extrinsic and intrinsic factors. One of the most important inhibitors is the glial scar that acts as a physical and chemical barrier to regeneration. In zebrafish and amphibians, however, no inhibitory glial scar is formed. Recently it is emerging the view that in mammals the glial scar although inhibiting axonal regeneration may have an important role as a protective agent during early stages after injury. In this sense it becomes interesting to further analyse whether the inhibitory action of the glial scar on regeneration is an unavoidable trade-off of its beneficial role on stabilizing the injury site preventing further damage. If this is true maybe it means that there is a kind of evolutionary constraint that really works against the possibility of enhancing CNS regeneration in mammals. Another field to develop is the study of the inhibitory action on regeneration by myelin components such us Nogo, myelin-associated protein (MAG) and oligodendrocyte-myelin glycoprotein (OMgp). Different studies have shown contradictory results in terms of enhancing the regenerative capabilities after inhibiting these factors. Therefore more data and comparative analyses are needed from animals that can and cannot regenerate their CNS.
In summary, and as it has been pointed out in previous posts on this blog, it is important not only to understand as deeply as possible how regeneration takes place at both cellular and molecular level, but also to try to determine why the regeneration potential has been lost in certain lineages. In this sense spinal cord regeneration provides with a very attractive model in which direct comparisons between regenerating and non-regenerating models (phylogenetically close, such as amphibians, fish and mammals) can be beneficial for the field as it may help, for example, to determine what really makes that some species are able to create a regeneration-permissive environment whereas others, in which similar cell types and molecular pathways are present and even upregulated after injury, cannot do it.
Among vertebrates zebrafish display amazing regenerative capabilities as they can regrow fins, the tail and even the heart. Also, and in contrast to other vertebrates such as mammals, zebrafish can regenerate their retinal cells. Zebrafish retinal regeneration depends upon the activation of Müller glia. After damage in the retina, Müller glia dedifferentiate and re-enter the cell cycle giving rise to a cycling population of multipotent progenitors that will differentiate into the required retinal cell types.
A recent paper from the laboratory of David R. Hyde reports on the role of Tumor Necrosis Factor-Alpha 1 in this process (http://www.ncbi.nlm.nih.gov/pubmed/23575850). Previous studies had shown that following a light-induced photoreceptor cell death there is an increase in the number of cycling Müller glia. This suggested that maybe a factor from the dying cells could activate that Müller glia re-enter the cell cycle. In fact, the injection of homogenates from light-damaged retinas into undamaged eyes is able to increase the number of cycling Müller glia. In contrast, no such increase is observed when injecting homogenates from undamaged retinas. In order to find out proteins present in those homogenates from light-damaged retinas that could activate Müller glia, the authors carried out a comparative proteomic approach. By doing this they identified more than 50 proteins expressed >2-fold in the light-damaged retinal homogenates. One of those proteins was TRAP1 (TNF receptor associated protein 1), which led to the authors check the role of the TNFa signalling pathway in the activation of Müller glia.
Upon light-induced damage the expression of TNFa increases first in apoptotic photoreceptors and later in Müller glia at the time they start to proliferate. Then, the authors carried out several experiments with morpholinos against tnfa delivered at different time points before or after the light-induced damaging of the retinas. A first conclusion of those experiments is that tnfa does not appear to be required for the initial light-induced cell death but is necessary for the activation of Müller glia at later stages of regeneration. Next the authors show that the expression of tnfa in the damaged retina is also necessary for the upregulation of Ascl1a and Stat3 in the Müller glia, which in fact appear also required for a successful regeneration.
Based on previous reports that suggested the existence of different cell subpopulations among Müller glia, the authors propose here a model in which TNFa secreted from apoptotic retinal neurons activates the expression of Ascl1a in PPMg (Primary Proliferating Müller glia). This expression of Ascl1a makes PPMg to re-enter the cell cycle and express Stat3. Then Stat3 induces the expression of tnfa in the PPMg. This new TNFa is secreted and activates the expression of Ascl1 in SPMg (Secondary Proliferating Müller glia), which, in turn, activates the proliferation of those cells. However, the exact relationships between all these different factors within the different types of Müller glia remain to be clearly elucidated.
In summary, this study identifies TNFa signalling produced by apoptotic retinal neurons as the signal that would induce Müller glia proliferation at the initiation of zebrafish retinal regeneration. These results are in agreement with those obtained from other models in which it has been shown that signals coming from apoptotic cells are required for a proper regenerative output (i.e. Hydra). Further studies should try to elucidate the exact role of apoptosis in triggering regeneration in different models and cellular and tissue contexts.
In the field of animal regeneration an important and recurrent issue is how pivotal is the nervous system to the process. That is, does regeneration depend on any neural factor or neural influence? Based on what we know from many regenerative models it is generally assumed that the nervous system is largely required for regeneration, however, very little is known about how exactly the nervous system controls this process. What are the molecules involved? Do these neural factors control cell proliferation? Or differentiation? Or migration? Or patterning? Or polarity? One thing that appears more or less clear is that the nature of such neural influence will not be probably electrical or transmitter-based (at least from current knowledge in amphibians).
In freshwater planarians, classical and more recent data suggest a role of the nervous system on regeneration. Thus, old experiments from Bondi (1959) and Lender and Gripon (1962) showed that during head regeneration, planarians with a very shortened ventral nerve cord on one side formed symmetrical blastemas, but the eye corresponding to the side of the longer ventral nerve cord appeared earlier than the eye of the shorter nerve cord. Other experiments by Kishida and Kurabuchi (1978) reported a delay in regeneration in fragments deprived of nerve cords. Studies from Lender (1961, 1964) and Sauzin-Monot (1972) showed the presence of an increased number of neurosecretory cells after amputation. More recently, it has been described that the silencing of Smed-roboA results in the new cephalic ganglia being disconnected of the underlying ventral nerve cords, with this disconnection giving rise to the differentiation of ectopic pharynges and dorsal outgrowths. A proposed explanation was that in the absence of proper connectivity between the brain and the ventral nerve cords, putative neurally-derived signals could be present in the surrounding tissues, altering the behaviour of the neoblasts and inducing the morphogenetic defects observed (http://www.ncbi.nlm.nih.gov/pubmed/17251262). Also, it has been shown how the disruption of the ventral nerve cords continuity results in altered fate and axial polarity of the regenerating planarians (http://www.ncbi.nlm.nih.gov/pubmed/20026026).
One of the laboratories that has worked more on nerve-dependence in regeneration providing the most clear molecular evidences so far is the laboratory of Jeremy Brockes. In a recent review (http://www.ncbi.nlm.nih.gov/pubmed/22989534) they discuss the current state-of-the-art of nerve-dependence regeneration. The authors revise some examples of nerve-dependence regeneration in annelids, planarians, Hydra, sea stars and mammals. In the case of the amphibian limb it is well-known since the 1950s that denervation prior to amputation blocks regeneration. However, it was only in 2007 that the laboratory of Brockes found the possible molecular basis of such nerve-dependence (http://www.ncbi.nlm.nih.gov/pubmed/17975060). After amputation the secreted newt anterior gradient (nAG) protein is upregulated in the Schwann cells of axons near the stump. Later nAG is expressed in gland cells below the wound epithelium. Denervation prevents the expression of nAG Remarkably, ectopic expression of nAG in a denervated limb induces the expression of nAG in gland cells below the wound epithelium and is able to rescue the regenerative capability. Although it is not known how exactly nAG promotes regeneration and whether or not this rescue is directly caused by nAG or instead by any other factor regulated b y nAG, in vitro experiments suggest that nAG may promote cell proliferation.
The fact that amphibian limb regeneration requires innervation is in contrast with what happens during embryonic development where the outgrowth of the limb does not depend on nerves. Remarkably, in amphibians it is possible to make them develop a so-called “aneurogenic” limb. For that, a section of the neural tube can be removed from the embryos so the limb develops without any innervation. Interestingly, those aneurogenic limbs are able to regenerate despite their lack of innervation. However, when those aneurogenic limbs are transplanted to a normal host, they become innervated and, from that time, their regeneration becomes nerve-dependence. In a paper published few months ago the laboratory of Brockes showed that the aneurogenic limb maintains high expression of nAG in gland cells and how, after transplantation and innervation, there is a marked downregulation of nAG expression in the epidermis (http://www.ncbi.nlm.nih.gov/pubmed/21825124). Thus, it would be the persistent expression of nAG in the aneurogenic limbs what would allow their regeneration. On the other side, it would be then the downregulation of nAG what probably mediates the establishment of nerve-dependence for the regeneration of these aneurogenic limbs once they are innervated.
However, nAG seems to work through Prod-1, a membrane GPI-anchored protein that appears salamander-specific. Therefore, and as the authors point out, nerve dependence will be most probably based in a variety of mechanisms. The final goal could be to ensure that the regenerated structured becomes functionally innervated. In any case, it seems clear that we need much more work, especially in non-amphibian models, first to identify the molecular basis of such nerve-dependence phenomenon and then, to be able to put it in a evolutionary context as well as in relation with other developmental programs that operate during embryogenesis or asexual reproduction.
In many regenerating systems there are three pivotal processes that need to occur: i) cell proliferation to provide a cellular source for regeneration (except in those cases in which there can be a direct transdifferentiation or dedifferentiation and redifferentiation in the absence of proliferation); ii) these new cells will need to differentiate into the missing cell types; and iii) the new tissues, organs and structures derived from those cells will need to be proper patterned and integrate within the pre-existing tissues, organs or structures. Even though is relatively known how these different processes occur and are regulated in different systems, less known is how these 3 events are coordinated with each other.
Two recent papers in Development from the laboratories of José Luis de la Pompa (http://www.ncbi.nlm.nih.gov/pubmed/23344707) and Gilbert Weidinger (http://www.ncbi.nlm.nih.gov/pubmed/23462472) report on the role of Notch signalling pathway in coordinating cell proliferation and differentiation within the blastema during fin regeneration in zebrafish. In these animals fin regeneration goes through 3 stages: i) wound healing, ii) blastema formation (by about 48h post amputation), and iii) regenerative outgrowth (from 48hpa). During the growth of the new fin, the blastema (defined as a mass of proliferating undifferentiated cells) restricts to the distal tip (below the wound) of the new fin whereas a proximo-distal gradient of cell differentiation is established. Thus, the most proximal region close to the amputation plane contains differentiated cells with a very reduced proliferation (“differentiation zone”), whereas the distal proliferative blastema bears mainly precursor cells. Following loss-of-function and gain-of-function of Notch signalling both papers nicely show how this pathway maintains blastema cells in an undifferentiated, proliferative state
After amputation Notch pathway is activated in the proliferative blastema (2dpa) and, as regeneration proceeds, it is mainly restricted in this distal undifferentiated region (as opposed to the proximal “differentiation zone”). The inhibition of the Notch pathway (mediated by different drugs or using specific morpholinos against some elements of this pathway) impairs fin regeneration. Apparently, wound healing and the initial blastema formation are not affected after inhibiting Notch pathway, however from the regenerative outgrowth stage the proliferation of the blastema cells significantly decreases. This suggests that Notch is required to regulate proliferation within the fin blastema. Moreover, Notch seems also necessary to maintain blastemal cells.
Next, both papers used transgenic lines and the Gal4-UAS system to ectopically express the Notch1a intracellular domain (NICD), which translocates to the nucleus and modifies the expression of its target genes. Remarkably, the overexpression of Notch activity also impairs fin regeneration. During the first 3 days after amputation no significant differences with controls are observed, however, from day 5 the regenerative outgrowth is significantly reduced. Also, and compared to controls, treated blastemas appear wider and with obvious alterations in the density and organization of cells in their mesenchymal regions. By checking the proliferative rates along the regenerated fin as wells as the expression of different markers specific of blastema cells, both studies show how proliferation is significantly increased in the proximal differentiation zone and there is also a proximal expansion of the blastema. Interestingly, this proximal expansion of the proliferative blastema goes together with a significant decrease in cell differentiation. Both papers analyse bone regeneration after the overactivaton of Notch pathway by using specific markers of the skeletogenic lineage: pre-osteoblasts, committed osteoblasts and differentiated osteoblasts. In controls these different cell populations are differentially located along the proximo-distal axis of the regenerated fins with differentiated cells in proximal regions and precursors cells more distal. After activating the Notch pathway there is a significant decrease in the number of differentiated osteoblasts at the same time that early precursors, normally restricted to distal regions, are expanded also to more proximal regions.
Thus, Notch signalling appears to be necessary to maintain blastema cells into an undifferentiated proliferative state. The function of Notch in supressing bone differentiation during fin regeneration in zebrafish parallels the role of Notch during bone development in mammalian embryos in which this pathway maintains osteoblast progenitors undifferentiated. In summary, both studies show how the Notch pathway plays a pivotal role in coordinating cell proliferation and differentiation in a regenerative context.