regeneration in nature

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Yearly Archives: 2013

Happy New Year 2014!

This is a short post just to wish all of you a happy new year. I hope 2014 will be an exciting year for regeneration and it will bring us tens of papers reporting novel data on how our beloved friends are able to regrow injured or lost organs, structures and body parts. I will stay here blogging about basic research focussed on understanding this fascinating biological process.

Autophagy in zebrafish fin regeneration

Autophagy is a biological process through which cells self-degrade unnecessary or dysfunctional components. Autophagy may be important in certain contexts such as for example nutrient starvation where a minimum level of energy is required to maintain the basic functions that warranty the survival of the cells. Another context in which autophagy can be important is during the remodelling of the cytoplasm that takes place in the process of cell reprogramming. In animal models in which regeneration depends upon an initial dedifferentiation of differentiated cells into proliferative cells, an extensive cytoplasmic remodelling must occur. However, few data is available about the role that autophagy may play during regeneration. Now, a recent paper from the laboratories of DJ Klionsky and T Vellai reports on the role of autophagy in zebrafish fin regeneration (http://www.ncbi.nlm.nih.gov/pubmed/24317199).

The authors use first a transgenic line carrying a GFP reporter under the control of Lc3 promoter, a specific marker for autophagosomes and autolysosomes. They found that Lc3 was upregulated within the tail fin blastema at 2 days after amputation (dpa). From that time the levels of Lc3 gradually decreased until reaching basal levels around 6 dpa. Lc3 was expressed in newly differentiated cells within the blastema. As those cells come from dedifferentiation, Lc3 expression could be reflecting an autophagic activity during this dedifferentiation-redifferentiation process. This increase in autophagy during regeneration was further corroborated by observations of electron microscopy. Thus, an increase of autophagic features was detected in epidermal cells, osteocytes and pigment cells of 2-day blastemas.

In order to see whether this increase in autophagy had a role during regeneration the authors used different methods to block this process. They either injected an antisense morpholino oligonucleotide against atg5 or a drug that acts as an autophagy inhibitor. Silencing of atg5 at 2 dpa blocked regeneration and induced the degeneration of the existing blastema. Similar results were obtained after using the inhibitory drug. Because in other models the inhibition of autophagy induces apoptosis the authors checked next whether the defects in regeneration after blocking autophagy could be explained in terms of increased apoptosis. Indeed, an increase of apoptotic cells within the blastemas was observed. Not only apoptosis was missregulated but also they observed a significant reduction of proliferative cells as well as problems with cell differentiation. All these data lead the authors to conclude that autophagy would promote cell survival and proliferation and would mediate cell differentiation during regeneration.

Finally, the authors looked for upstream regulators of autophagy in this context. It is well known that the FGF signalling is required for fin regeneration and it silencing inhibits cell dedifferentiation. MAPK/ERK is one of the downstream effectors of FGF signalling and has been involved with the regulation of autophagy. Therefore, the authors checked whether the blocking of MAPK/ERK itself influenced fin regeneration. Using an inhibitory drug they found a complete impairment of fin regeneration. Remarkably, they also observed a decrease of autophagy in those treated animals, suggesting that MAPK/ERK activity was required for the upregulation of autophagy during regeneration. From all that data the authors propose a model in which Fgf signalling would lead to the phosphorylation of MAPK/ERK that would then regulate autophagy.

In summary, the authors conclude that autophagy would act as a prerequisite for the regeneration of the zebrafish tail fin through the regulation of the reorganization and remodelling of the cytoplasmic compartment during cell dedifferentiation.

Joint formation during zebrafish fin regeneration

Zebrafish fins are composed of multiple bony rays each of which is comprised of multiple bony segments separated by joints. As in all vertebrates these joints must be formed at precise positions to ensure the flexibility of the skeletal system. During regeneration (as well as during growth), there is a sequential addition of new bony segments and joints in a proximal to distal direction. Therefore, the youngest tissue is located most distally. Not much is known about the genes involved in joint morphogenesis in this context. Now a paper from the laboratory of M. Kathryn Iovine (http://www.ncbi.nlm.nih.gov/pubmed/24278401) has characterized a pathway involved in joint regeneration during zebrafish fin regeneration.

Previous reports have suggested that the transcription factor even-skipped 1 (evx1), an eve-related homeobox gene, is required for joint formation during regeneration. Genes that are expressed during joint formation are expressed in discrete groups of cells located within the lateral population of skeletal precursor cells. Based on the expression pattern the authors found that three genes dlx5a (distal-less homeobox-5a), mmp9 (matrix metalloproteinase 9) and col10a1b showed a similar expression than evx1 in this lateral mesenchymal compartment of the ray. In order to characterize the relationship between these three genes and evx1, they first checked how their expression was affected in evx1 mutants. Whereas the expression of col10a10b was not affected in evx1 mutants, dlx5 and mmp9 were significantly reduced, although not completely abolished. These results suggested that dlx5 and mmp9 were expressed downstream of evx1. Next, the authors characterized the function of dlx5 and mmp9 on fin regeneration. Morpholino-mediated knockdowns of these genes resulted in increased ray segment length suggesting that dlx5 and mmp9 are necessary for correct joint placement.

During fin ray regeneration genes required at early stages of differentiation are expressed in more distal regions, whereas proximal regions include late differentiation genes. Evx1 was expressed in the most distal domain of the skeletal precursor cells, consistent with this gene acting earlier. On the other side, dlx5a was expressed more proximally than evx1 and mmp9 was expressed even more proximally than evx1 and dlx5a. These results suggested a linear pathway initiated by evx1 followed by dlx5a and then followed by mmp9. This was further supported by the observation that mmp9 expression was reduced in dlx5a knockdowns. In contrast dlx5a expression was not affected after mmp9 knockdown. Similarly evx1 expression was not affected in dlx5a or mmp9 knockdowns.

This same laboratory had previously reported that the activity of Cx43 (a gap junction protein) suppresses joint formation.  Cx43 is expressed in the medial compartment adjacent to the lateral population of skeletal precursor cells. Mutants for this gene are characterized by short segments because of premature joints. Accordingly, and as predicted, the expression of evx1, dlx5 and mmp9 was initiated more distally (meaning sooner) in regenerating fins of Cx34 mutants. This premature expression of these genes would account for the short segments observed in Cx34 mutants.

In summary, the authors propose a model in which Cx43 would inhibit evx1 which is itself required to sequentially activate dlx5a and mmp9 for proper joint formation.

Newts and axolotls show a different cellular origin for the regenerated limb skeletal muscle

Salamanders display amazing regenerative capacities that include the ability to regrow new limbs. Previous results have indicated that blastema cells are originated mainly by the dedifferentiation of pre-existing tissues into cells that re-enter the cell cycle and proliferate to form the missing structures. However, and because salamanders possess muscle satellite cells positive for Pax7 it was not clear the relative contribution of mature myofibers and those satellite cells into the regenerated limb skeletal muscle. Now, a recent paper from the laboratories of András Simon and Elly Tanaka (http://www.ncbi.nlm.nih.gov/pubmed/24268695) provides more clear evidence of the cellular origin of the regenerated muscle fibers in two different salamanders: Notophtalmus viridescens (newt) and Ambystoma mexicanun (axolotl).

Remarkably, by using a CreloxP-based genetic fate mapping of muscle fibers during regeneration they found fundamental differences in the cellular origin of the regenerated muscle in these two species of salamanders. Two weeks after co-electroporating the upper arm of newts with the different constructs needed to specifically label the mature muscle fibers (positive for myosin heavy chain (MHC)) with YFP (without labelling the Pax7+ satellite cells), those limbs were amputated.  The authors found YFP+ cells all along the regenerated limb except the digit tips, indicating a direct contribution of the pre-existing muscle into the regenerated myofibers. Detailed analyses of those blastemas showed that in their distal regions they contained YFP+ cells that were negative for MHC pointing out to a dedifferentiation of the muscle cells to form mononuclear blastema cells. Moreover those blastema cells had re-entered the cell cycle as indicated by labelling with PCNA and the incorporation of the nucleotide analog Edu. Importantly, Edu was never incorporated in YFP+/MHC+ or MHC+ cells suggesting that cell cycle re-entry occurred after the fragmentation and dedifferentiation of muscle fibers into blastema cells.

In contrast, when they used the same strategy to label the mature muscle fibers of the axolotl and trace them upon amputation, no YFP+ cells were found within the blastema or in the regenerated limb. Although some morphological changes of the pre-existing fibers were seen at the amputation plane, these results suggested that the pre-existing muscle fibers did not contribute to muscle regeneration in axolotls. Previous results from the laboratory of Elly Tanaka had shown that GFP+ muscle fibers and satellite cells contribute to muscle regeneration in axolotls, but without being able to distinguish their relative contributions. Here, they found that of 834 GFP+ positive blastema cells, 809 expressed Pax7. Also, these GFP+ were cycling as they incorporated EdU. Taking in account these two results (lack of YFP+ cells and presence of GFP+/Pax7+ cells within the blastema) the authors conclude that were the Pax7+ satellite cells from the mature limb the ones that gave rise to the proliferative muscle progenitors required for regeneration.

Finally, they analysed whether those differences between newts and axolotls could be explained by the neotenic nature of axolotls. They metamorphosed axolotls and analysed limb regeneration in them, following the same methodological approach. Similarly, muscle regeneration in this context did not seem to depend on the dedifferentiation of pre-existing muscle fibers. Also, and complementarily, Pax7+ cells were not found either in larval newt limb blastemas  (in agreement with the results obtained in adult newt blastemas).

In summary, this study characterizes at the cellular level the origin of the regenerated muscle fibers in two different species of salamanders. Remarkably, these two species use very different strategies to achieve the same final goal: skeletal muscle regeneration. These results are a beautiful and clear example of how different animals (even relatively closed phylogenetically) use different strategies to form the same cell type within the regenerative blastema. This flexibility and diversity of successful strategies may be of special relevance for the field of regenerative medicine.

Genome reprogramming during zebrafish retina regeneration

In previous posts in this blog I have discussed how zebrafish are able to regenerate their retinas from the dedifferentiation of quiescent supportive Müller glia (MG) that re-enter the cell cycle and give rise to a cycling population of multipotent progenitors (MGPC) that differentiate into the different retina cell types. Previous studies have suggested that dynamic changes in the DNA methylation landscape can have a function in the transition from MG to MGPC. How much similar is this cellular reprogramming that occurs during regeneration to the reprogramming required to transform somatic cells into induced pluripotent stem cells (iPS) is an interesting question for the field of regenerative medicine. During iPS generation there is an increased DNA demethylation of the promoter regions of pluripotency genes that correlates with an increase in their expression. Similarly, the expression of pluripotency genes as well as other regeneration-associated genes increases during the transition from MG to MGPC.

In a recent paper from the laboratory of Daniel Goldman the authors wonder up to what extend changes in the DNA methylation landscape are important for the transition from MG to MGPC (http://www.ncbi.nlm.nih.gov/pubmed/24248357). First, they checked how the expression of several regulators of DNA methylation changed in MGPCs  (from injured retinas) compared to MG (from uninjured retinas). They found an increased expression of genes associated with both DNA demethylation and methylation, suggesting that the regulation of DNA methylation may be important for MGPC formation. Next, they showed how the induction of DNA demethylation perturbed the migration and differentiation of MGPC-derived progeny. They took then a genomic approach to compare the DNA methylation landscapes between MG from uninjured animals and MGPCs from injured retinas 4 dpi (between 2 and 7 days post-injury there is an asymmetric division and proliferative amplification of MGPCs). They compared the methylation levels of 611,434 individual cytosines within the CpG context and found 9,554 differentially methylated bases (DMBs) that represented an overall difference of 1,54%. Of those changes, 54% corresponded to increased methylation events and 46% to decreased ones. Most DMBs were localized in intergenic and intronic regions with few of them localized in promoter regions.

From 2 to 4 dpi the DNA methylation landscape shifted from one that was predominantly driven by demethylation to one with increasing levels of methylation, which could correlate to the MGPCs getting ready to enter into differentiation. As expected, a decrease methylation in the promoter regions correlated with an increased gene expression. However, and surprisingly, no DMBs were found in the promoters of pluripotency and retina regeneration-associated genes. Even more remarkably was the fact that when the authors checked the levels of methylation of the promoters of those genes in quiescent MG they found that they were also hypomethylated. So, these data made the authors hypothesize that pluripotency and regeneration-associated genes might be poised for activation in quiescent MG, implying that MG would require only limited reprogramming and would be more stem-like than thought before. Because the regenerative capacity of mouse MG is very limited the authors checked the methylation state of mouse MG. Remarkably, they found that the promoters of pluripotency and regeneration-associated genes showed low levels of methylation, as observed in zebrafish.

In summary, this study shows how the global DNA methylation landscape changes during the transition from MG to MGPCs pointing out an important regulatory function during this reprogramming. However, pluripotency and other genes required for regeneration appeared to be hypomethylated already in MG suggesting that they could be already preprogrammed for a regenerative response. These genes could be then regulated by other events such as histone modifications or transcription factors, indicating that changes in the DNA methylation status would be required but it would not be sufficient for MG reprogramming. The fact that mouse MG share this hypomethylated landscape in pluripotency genes opens the door to search for strategies to facilitate their reprogramming into progenitor cells that could enhance the poor regenerative abilities of the mammalian retina.

Expression of Hox genes during polychaete regeneration

Hox genes code for transcription factors that play an essential role in the regionalization of the anterior-posterior axis in animals. Thus, they function by providing specific identities to the different segments of the body plan, which translates in the differentiation of the correct structures in each segment. Well-known and astonishing examples of homeotic transformations include those that transform Drosophila antennas into legs or flies with two pairs of wings. Hox genes have been highly conserved through evolution and, for example, Hox genes from flies can replace the function of their vertebrate orthologs.

The function of Hox genes has been (and still is) largely studied during the embryonic development of a large number of invertebrate and vertebrate species. During regeneration, specially in those cases in which large structures or body regions need to be re-grown, it is easy to imagine that Hox genes should have an important function in the re-establishment of the positional identity within the regenerate. However, and in contrast to the observations that conserved signalling pathways such us the BMP, Wnt/beta-catenin, Hedgehog and FGFs play important functions during regeneration, many less functional studies have been reported on the role of Hox genes during regeneration. Thus, whereas several families of genes containing an homeobox domain (pbx, prox, pitx, six, Rx, msx,…) have been functionally characterized during regeneration in planarians, tunicates or amphibians, most of the studies on Hox genes and regeneration focus on their changes in expression during this process. Some of those studies were important for example to show how Hoxc10L is not expressed during axolotl forelimb development but it is upregulated during regeneration, pointing to a “regeneration-specific” expression of this gene (http://www.ncbi.nlm.nih.gov/pubmed/11150241). Also, few years ago it was reported that Hoxc13 orthologs are important for zebrafish tail regeneration where are required for blastema cells proliferation and growth (http://www.ncbi.nlm.nih.gov/pubmed/17437127).

Now, a recent paper from the laboratory of Elena Nokikova and Milana Kulakova reports on the expression of 10 Hox genes during regeneration in the polychaete Alitta (Nereis) virens (http://www.ncbi.nlm.nih.gov/pubmed/23638687). During postlarval development the Hox genes are mainly expressed in a posterior-to-anterior gradient mode. These annelids can regenerate their posterior body end. After amputation the terminal pygidial structures and prepygidial growth zone (GZ) form first and then the new segments appear sequentially. The authors divided the Hox genes into four groups depending on their changes in expression during regeneration. Three genes (early genes: Nvi-Lox5, Nvi-Lox2 and Nvi-Post2) were rapidly upregulated in the nervous system near to the amputation plane by 4 hpa (hours post amputation). Then, Nvi-Hox5 and Nvi-Hox7 (middle genes) expression patterns in the nervous system were reorganized by 10 and 18 hpa, respectively, before active proliferation in the GZ started. Next, two Hox genes, Nvi-Hox2 and Nvi-Hox3 (middle genes), that are not expressed in a graded manner but specifically found in the GZ were upregulated de novo by 10 hpa. Nvi-Hox2 first appeared in two bilateral domains at the amputation plane. By 2 dpa (days post amputation) this gene was expressed in the mesoderm and ectoderm of the area between the forming pygidium and the last body segment. By 7 dpa it was detected in the mesoderm of the GZ and the mesoderm and ectoderm of the newly formed segments.  Nivi-Hox3 was also first seen by 10 hpa and as regeneration proceeds got restricted to the ectoderm of the GZ. Finally, there are three genes (late genes: Nvi-Hox1, Nvi-Hox4 and Nvi-Lox4) whose expression patterns changed at late stages of regeneration once proliferation and organogenesis were under their way.

Based on all these expression patterns and dynamics the authors divided the regeneration process in two phases: during the first 48 hpa the expression patterns were reorganized inside the new body boundaries. The second phase (that overlaps with the first one) started around 24 hpa when the blastema was formed. Most of the Hox genes were highly expressed within the blastema and the rudiment of the terminal structures that were evident by 3 dpa.

Because during postlarval development Hox genes are expressed in a anterior-to-posterior gradient in segments that are morphologically similar, the authors suggest that Hox genes at this stage provide the positional information needed to determine the position of body parts. Thus, after amputation the expression of most of the Hox genes of the early and middle groups is reorganized so the last body segments adjacent to the amputation plane acquire the Hox pattern typical of the posterior body end. Remarkably the blastema seems to be formed after the Hox genes have been reorganized to the new body proportions.

Future functional experiments should help to determine the exact role of the Hox genes during not only the re-patterning of the body during polychaete regeneration but also in other models such as planarians or amphibians.

 

Homeostatic signalling interferes with fin regeneration in male zebrafish

There are several cases in which regenerative capabilities vary depending on the regenerative stage of the organisms.  Some examples of this loss of regeneration include that of limbs in post-metamorphic amphibians and the heart in one week-old mice. Now a recent paper from the laboratory of Kenneth Poss describes how the pectoral fins in zebrafish show a sexually dimorphic response to amputation and links that to the maintenance of male-specific structures and the regulation of the Wnt signalling pathway (http://www.ncbi.nlm.nih.gov/pubmed/24135229).

In a previous paper the same laboratory had found that pectoral fin regeneration is often impaired in males whereas it proceeds normally in females (http://www.ncbi.nlm.nih.gov/pubmed/22079110), being such impairment partially rescued by overactivating the Wnt/b-catenin pathway. Regeneration failure correlates with sexual maturity indicating an age- and sex-dependent loss of regenerative abilities for the male zebrafish pectoral fins. In this new paper the authors characterize in more detail the molecular basis of such sexual dimorphism and focus on the study of Dkk1, an inhibitor of the Wnt/b-catenin signalling pathway, which is well-known for being required for a successful regeneration in many contexts and species, including zebrafish fins. dkk1 was found to be expressed at higher levels in male pectoral fins that in females. By using a transgenic line the authors found that dkk1 displayed a sexual dimorphic expression being detected in what they called epidermal tubercles (ETs) in the anteromedial rays of the pectoral fins of males. Because similar structures had been described in other fishes and associated to mating and spawning, the authors first addressed the role of ETs in spawning. What they found was that zebrafish males used the pectoral fin to grasp the female abdomen to stimulate egg laying. In fact, males with amputated pectoral fins appeared mostly incapable of stimulating an efficient spawning.

Next, they analysed the role of androgens in the development of ETs. Remarkably, an androgenic factor was able to induce the development of ETs in the pectoral fins of females; conversely, the application of an androgen inhibitor in males decreased the number and definition of ETs. These changes were accompanied with an increase or decrease of dkk1 expression, respectively. In order to characterize the role of the Wnt/b-catenin pathway in the formation of ETs they crossed a transgenic line bearing a Wnt signalling reporter with the line with labelled dkk1-expressing cells. Following the maturation of ETs they found 3 distinct types of ETs: immature structures where Wnt signalling is active and dkk1 is not expressed, intermediate cells expressing dkk1 and active Wnt signalling and mature ETs expressing dkk1 and no active Wnt signalling. Because the balance of activated/inhibited Wnt signalling is important for the induction and/or patterning of epidermal appendages the authors further analysed the role of Wnt/b-catenin on ETs formation. Experiments inhibiting or overactivating Wnt signalling suggested that that this pathway has a positive role in the formation of new ETs. In fact, ETs go through a continuous cell turnover from a basal layer cell population(s) between adjacent ET units, being this renewal dependent on local dkk1 regulation and Wnt activation.

During fin regeneration, at 2 days post amputation (dpa) Wnt signalling is activated in early blastema cells. As regeneration proceeds Wnt pathway keeps active in the proliferative distal blastema, whereas dkk1 is expressed in more proximal differentiating cells. This dynamics was observed in different fins in both females and males, including female pectoral fins and posterior regions of male pectoral fins. Defects in male pectoral fin regeneration mostly appeared when amputation was performed across a region containing ETs. Further analyses on male pectoral fin regeneration revealed that the wound was capped by epidermal cells expressing high levels of dkk1 with little proliferative activity underneath. This was in contrast to females in which dkk1 was not expressed in the blastema cells at this stage (2 dpa). Also, whereas in females axin2, a known target of active Wnt signalling was detected in blastema cells, no expression was found in males clearly indicating a failure in Wnt activation in those regenerating males. As regeneration proceeded males appeared to achieve different degrees of recovery. After several weeks of regeneration males were then classified in three groups depending on whether the defects in their regenerated pectoral fins were mild or severe. Remarkably, males with severe defects were incapable of stimulating spawning during mating.

In summary, this paper describes the molecular basis of the sexual dimorphism shown by regenerating pectoral fins in female and male zebrafish. Thus, a fine balance between active and inactive Wnt signalling is pivotal for proper regeneration. In males, the high level of dkk1 required in ETs to maintain these basic structures for a successful mating appears to come at the expense of reducing their regenerative potential.

Neural progenitor cells in planarians

Planarians are really amazing creatures as they can regenerate a whole animal from a tiny piece of their bodies. Planarians can do that because they possess a population of adult pluripotent stem cells, called neoblasts. For many years one of the debates within the field has been how homogeneous or heterogeneous is this neoblast population. Thanks to a study from the laboratory of Peter Reddien we know now that at least a proportion of those neoblasts are real totipotent stem cells. But how do these neoblasts, the only proliferative cells in planarians, differentiate into the different cell-lineages? Are there cell type-specific progenitors in these animals? Recently, some studies from Peter Reddien clearly indicate that at least for some cell types, such as photoreceptors and the excretory system, specific progenitors exist.

One of most astonished abilities of planarians is that they can regenerate a complete central nervous system de novo from those undifferentiated neoblasts. For long time people have wondered whether neural progenitor cells exist in these animals and how they would behave during regeneration. Two very recent papers from the laboratories of Kerstin Bartscherer (http://www.ncbi.nlm.nih.gov/pubmed/24131630) and Bret Pearson (http://www.ncbi.nlm.nih.gov/pubmed/23903188) have shown that the transcription factors lhx175-1 and pitx were required for the regeneration of the serotonergic lineage. Importantly, these transcription factors were co-expressed in cells expressing Smedwi-1, a homologue of the PIWI proteins and a marker of planarian neoblasts, suggesting the existence of progenitor cells for the serotonergic lineage.

Now these findings have been further corroborated and expanded by the laboratory of Ricardo Zayas (http://www.ncbi.nlm.nih.gov/pubmed/24173799). In this study they carried out a genome-wide analysis of bHLH transcription factors in planarians. In other models bHLH factors play pivotal roles in neurogenesis, from fate commitment to cell migration. In planarians, the authors identified 44 genes predicted to code for a bHLH domain, of which 12 were expressed in the CNS and neoblasts. Because of their specific expression patterns they mainly focussed on three of them: coe (collier/olfactory-1/early B-cell factor), hesl-3 (hairy/enhancer of split) and sim (single-minded). By double labelling they first checked how these factors were co-expressed with different markers of specific neuronal populations: cholinergic, GABAergic, octopaminergic, dopaminergic and serotonergic neurons. Next, in intact non-regenerating animals and through elegant BrdU pulse-chase experiments, they detected proliferating cells expressing coe or sim close to the nervous system, which could be traced to the brain or ventral nerve cords. Over time, the number of such double-labelled cells increased, especially in the head region. Similarly, during anterior regeneration these populations of progenitor cells expressing coe or sim, seems to contribute to the regenerative blastema. Therefore, it seems that these transcription factors would be labelling progenitor cells for distinct neuronal populations.

In the second part of the paper the authors performed RNAi analyses of this set of bHLH transcription factors in order to determine their functions during regeneration. Remarkably, they did not find any strong phenotype after knocking-down proneural bHLH genes such as neuroD or acheate-scute. However, silencing of coe, hesl-3 and sim resulted in clear defects of the regenerating brain either in terms of its gross morphology and/or the number or localization of specific neuronal populations. On the other hand, in intact animals the silencing of hesl-3 and sim did not produced any detectable defect in the nervous system.  This was different for coe, as after RNAi in intact animals these animals displayed an aberrant external morphology and they lost specific neuropeptidergic neurons (also lost in regenerating animals after coe RNAi).

In summary, these results suggest that coe, sim and hesl-3 may define progenitor cells committed to distinct neural fates and their function would be required for the differentiation of some neuronal cell types. As coe and sim are co-expressed with specific markers of different neuronal populations, their expression could be defining a set of multipotent progenitors.

Regeneration Journal

This week I am not going to comment on any specific paper on regeneration but rather publicize the launch of a new journal dedicated to this fascinating field of regeneration. The new journal is called Regeneration and will be published by John Wiley & Sons (http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)2052-4412). The Editor-in-Chief is Susan V. Bryant a prestigious researcher in the field of limb regeneration in amphibians. The associate editors include Kiyokazu Agata, Enrique Amaya, Cheng-Ming Chuong, Ken Muneoka, Ken Poss and Elliot Meyerowitz. Regeneration is an open access journal and will be the first journal to be dedicated exclusively to regeneration and tissue repair in animals and plants. Therefore the regeneration community have now an excellent tool to disseminate our results in a more compact way that should make more visible our advances and research and, at the same time, attract more people to this field. Hopefully this journal will be useful also to establish bridges between naturally occurring regeneration and the need to enhance assisted regeneration. I really hope that we can all contribute to the success of this journal. I will keep you posted when the first issue is available.

miRNAs in newt heart regeneration

microRNAs (miRNAs) are small non-coding RNA molecules (21-23 nucleotides) that have been identified as important conserved regulators of gene expression. In animals miRNAs usually bind to a complementary region within the 3’UTR of target mRNA molecules resulting either in target degradation o translational repression.  miRNAs have been associated to a variety of diseases, cancer and the development of the nervous system, among other processes. Related to regeneration, miRNAs have been involved in liver and cardiac regeneration.

Now a recent paper from the laboratory of Jamie Ian Morrison has characterized the role of miRNAs during heart regeneration in newts (http://www.ncbi.nlm.nih.gov/pubmed/24055866). Contrary to mammals, newts (similarly to zebrafish) are capable of regenerating their heart. The authors divide newt heart regeneration into three main stages: early wounding response (7-14 dpi), hyperplasia (21-30 dpi) and reverse remodelling (45-60 dpi). They performed a microarray screen to identify miRNAs differentially expressed during heart regeneration. Initially, they identified 37 miRNAs that showed a significant expression pattern between 7 and 21 dpi. Then they decided to concentrate in miR-128 that showed a very high expression at a time-point when cardiac hyperplasia is elevated. In situ hybridizations experiment showed that in uninjured hearts miR-128 was expressed at very low levels in sporadic cardiomyocytes lining the epicardial border. In contrast, at 21 dpi there was a significant increase of miR-128 expression around the regenerative zone, not only in cardiomyocytes but also in non-cardiomyocytes. This peak of expression of miR-128 coincides with the peak of hyperplasia during heart regeneration.

In other models miR-128 has been described as a tumour suppressor gene but it has not been related to heart biology. Using an antagomir (miRNAs inhibitors that prevent the binding of miRNAs to their targets) knockdown strategy the authors found first a significant increase in the proliferation of non-cardiomyocyte cells in the regenerating heart, whereas no clear effect on the proliferation of cardiomyocytes was observed. These results suggest that during heart regeneration miR-128 would act as an inhibitor of proliferation of non-cardiomoycyte cells. Another consequence of miR-128 inhibition was that as regeneration proceeds there is a persistent presence of extracellular matrix and fibrin tissue within the regeneration zone. This excessive fibrin and collagen matrix deposition after miR-128 inhibition (in clear contrast to the little scarring observed in control regenerating hearts), resulted in a delayed regeneration.  Therefore, miR-128 appears to have an important role regulation extracellular matrix deposition during heart regeneration.

Finally, the authors followed a bioinformatics approach to identify putative targets of miR-128 and found the transcription factor islet1 as a potential gene to be regulated by miR-128. These finding agree with previous results of the same laboratory that had identified islet1 as a factor differentially expressed during newt heart regeneration. Here, the authors report that, compared to uninjured hearts, the expression of islet1 significantly decreases at 21 dpi when the expression of miR-128 is maximum. Importantly, the expression of islet1 increased by a 60-fold when miR-128 was inhibited. As islet1 has been described as a chordate cardiac progenitor cell marker these results would fit with a model in which miR-128 would act, by inhibiting islet1, as a negative regulator of progenitor cell activity and would promote cell differentiation during heart regeneration.

In summary, this is the first report of miRNAs being involved in heart regeneration in newts and provide new actors and tools that can help to understand in the near future how these animals regenerate their heart.

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