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A support cell subpopulation maintains robust regeneration of adult hair cells in zebrafish

In contrast to other vertebrates, mammals cannot regenerate the mechanosensory hair cells in the epithelia of their adult ears after age-related, disease or trauma-induced cell death. Zebrafish can definitely regenerate their hair cells located not only in the inner ear but also within the sensory lateral line. This lateral line consists in rather regularly spaced sensory organs called neuromasts formed by hair and support cells. It is well known that in larval zebrafish hair cells show a strong regenerative ability and, after their ablation, are regenerated from the symmetrical division of the surrounding support cells. Now, a recent paper from the laboratory of David W. Raible reports for the first time on the robust regeneration of these hair cells on aged adult zebrafish and characterizes a slow-dividing subpopulation of support cells that could explain this robustness in hair cells regeneration (http://www.ncbi.nlm.nih.gov/pubmed/25869855).

In this study the authors used several transgenic lines that allowed them to easily visualize and track both hair cells and the surrounding support cells. After neomycin treatment on sexually matured animals, they showed first that 75% of hair cells were ablated by 2 hr and then normal numbers were recovered by 72 hr, a rate of recovery similar to that observed in larval zebrafish. Next, they analyzed whether this regenerative capacity was diminished with age by comparing 1-year and 3-year-old animals. Their results show that 3-year-old zebrafish were still capable of fully regenerating their hair cells after neomycin treatment although it took a little bit longer compared to 1-year-old animals (5 days instead of 3 days). Remarkably, these adult zebrafish were capable of properly regenerating their hair cells after each of 10 sequential rounds of hair cells ablation and regeneration.

In larval zebrafish, regenerated hair cells derive from the symmetrical division of support cells. The authors then checked whether the number of these support cells changed after repeated rounds of regeneration in adults. However, the number of support cells in each neuromasts remained about constant after those experiments suggesting that support cell renewal was tightly regulated during hair cell regeneration. Then the authors combined a transgenic line with labeled hair cells and BrdU staining and found out that the number of BrdU positive hair cells decreased 12 days after BrdU exposure but at the same time the number of hair cells remained quite constant indicating that in normal conditions adult hair cells go through constantly loss and replacement. These results were further corroborated by the use of transgenic lines carrying the photoactivatable fluorescent protein Eos.

Finally the authors tried to understand how support cells are capable of dividing symmetrically to give rise to hair cells during multiple rounds of ablation and regeneration without being depleted themselves. They hypothesized the existence of a subpopulation of slow dividing support cell progenitors. Therefore, they used a transgenic line expressing Eos in all support cells and their rationale was that after multiple rounds of regeneration the red Eos signal present in dividing support cells originating hair cells would be diluted. In contrast, support cells that would not divide or did it much less frequently would retain higher levels of red Eos. Interestingly, they found a population of label-retaining support cells at the anterior end of the neuromasts as well as smaller population with similar characteristics at the posterior end. These results suggested that support cells behave differently depending on their localization within the neuromast. Remarkably, these anterior support cells were much less probable to give rise to hair cells during regeneration.

In summary the authors described here how adult and aged zebrafish are still capable of robustly regenerating the hair cells from their lateral lines. More importantly, they characterized a subpopulation of slow dividing support cells that could originate the hair cells precursors during regeneration and cell turnover. Future experiments should corroborate these findings and further analyze whether the environment around these distinct anterior support cells might have a role as a niche to maintain these support cells in a more quiescent state.

Neuregulin1 induces the endogenous heart regeneration program in zebrafish

Contrary to mammals, zebrafish can regenerate their hearts after a significant loss of their cardiomyocytes. Taking into account the high incidence of myocardial infarction in humans understanding heart regeneration in zebrafish might provide clues to enhance our limited regenerative abilities. Now, a recent paper from the laboratory of Ken Poss reports on the important role of neuregulin1 (nrg1) t o induce the endogenous heart regeneration program in zebrafish (http://www.ncbi.nlm.nih.gov/pubmed/25830562).

Nrg1 had been previously identified as having multiple roles in cardiovascular biology in both mammals and zebrafish, although some contradictory reports existed respect to the role of nrg1 on cardiomyocytes proliferation. Therefore, in this paper, the authors addressed the role of nrg1 on the strong regenerative response of the zebrafish heart. Whereas nrg1 expression was rarely detected in uninjured hearts, after the genetic ablation of about 50% of the cardiomyocytes there was a significant upregulation of nrg1 that peaked at 7 days post amputation (dpa), a stage in which cardiomyocytes proliferation also peaks. Double stainings determined that most nrg1 positive cells in the postnatal ventricular wall came from the tcf21+ epicardial derived perivascular cell compartment. Rare overlap of nrg1 and markers of vascular endothelial cells and cardiomyocytes was observed.

Nrg1 signals through the Erbb2 and Erbb4b receptors. The pharmacological inhibition of these receptors decreased cardiomyocytes proliferation during regeneration. On the other hand, gain-of-function experiments showed that overexpressing nrg1 in cardiomyocytes resulted in a strong increase of cardiomyocyte proliferation near the injury site, clearly suggesting an important role of nrg1 signalling on cardiomyocyte proliferation during heart regeneration. Next, the authors sought to determine the effects of overexpressing nrg1 in uninjured hearts. After 7 days of treatment (dpt) they observed a marked increase in cardiomyocyte proliferation especially within the ventricular wall. Over time, this increased proliferation resulted in obvious changes in cardiac anatomy. Thus, the ventricular wall thickened by 76% at 7 dpt, by 265% at 14 dpt and by 459% after 30 dpt. Measurements of cardiomyocyte cell number and size suggested that this enormous thickening of the ventricular wall was mainly due to an increase in cell number. The authors referred to it as nrg1-induced cardiac hyperplasia (iCH). Next, the authors analysed the long-term effects of iCH on uninjured adult hearts. On average after 6 months of treatment those hearts showed a 2.1-fold greater ventricular section area. An increased cardiomyocyte proliferation was still evident at this late stage although the authors also observed some mild fibrin and collagen deposition in some regions of the ventricular wall that were not seen just after 30 dpt.

By measuring physiological parameters and cardiac function the authors did not find any cardiac dysfunction at 3 months of iCH. However, at 8-9 months the animals showed a reduction in swimming endurance, a test based on measuring the ability of the fish to swim against increasing water currents, that is an assay of cardiac function sensitive to cardiac damage or heart failure. Therefore, these results indicated a deleterious effect of nrg1 continuous overexpression over a long period of time.

In a last set of experiments, the authors investigated whether iCH depended on similar mechanisms to those triggered by injury-induced regeneration. During normal development the cortical muscle in the ventricular wall form from a small number of large cardiomyocyte clones with discernable boundaries between them. In contrast, during regeneration there is an increase of cardiomyocyte proliferation at the injury site that generates a mixed conglomeration of small clones in the regenerated ventricular wall. The authors observed that the thickening of the ventricular wall during iCH in uninjured hearts contained mixed small clones something reminiscent of an injury-induced regenerative response. When analysing the molecular signatures of iCH the authors first saw an upregulation of gata4 (an embryonic cardiogenic transcription factor) in the outermost layer of cortical muscle at 7dpt. Previous results had shown that after apex amputation gata4 is upregulated in cardiomyocytes near the injury and is essential for regeneration. Moreover, they observed that after iCH there was a reduction of cmlc2 (cardiomyocyte marker) expression in the cortical muscle and a sarcomere disorganization, which would resemble the dedifferentiation process that occurs during regeneration. Another genes implicated in regeneration, such as tgfb3, fn1 (fibronectin synthesis) and raldh2 (retinoic acid signalling), were also upregulated after iCH. Finally, iCH also induced the formation of a major vascular network throughout the expanded ventricular wall. Overall, all these results suggest that nrg1 is sufficient to induce and maintain a global heart regeneration program involving several cell types.

In summary, the authors report here nrg1 as a potent activator of the heart regeneration program in zebrafish. In the future, it will important to determine how is nrg1 upregulated during regeneration as well as its downstream targets.

Med14 is required for the maintenance of stem cell populations in vivo in planarians and zebrafish

An important aspect of stem cell biology is to determine the genes responsible for the maintenance of the different stem cell populations as well as to understand both how they are regulated and how they exert their function. Now, a paper from the laboratories of Ian C. Scott and Bret J. Pearson reports on the identification of the Mediator subunit Med14 as an important regulator of stem cell maintenance in zebrafish and planarians (http://www.ncbi.nlm.nih.gov/pubmed/25772472).

The Mediator complex was first identified in yeasts and its core consists of three modules (head, middle and tail) with an additional kinase module present sometimes. Because in yeast Mediator is located at the promoters of nearly all protein coding genes it has been suggested that it may be part of the general transcription machinery. However, several reports in some plant and animal models do not seem to fully support this model. Interestingly, several subunits of this Mediator complex have been identified as regulators of pluripotency of mouse ESCs. However little is known about the putative role of Mediator on stem cells in vivo.

In this paper the authors characterized the function of the subunit Med14 in stem and progenitor cells and regeneration in two different models: zebrafish and freshwater planarians. First they found that the zebrafish mutant logelei (log) corresponds to mutation of med14. Pleiotropic effects that suggest a developmental arrest characterize the phenotype of this mutant. Accordingly, morpholinos of med14 recapitulate many of these phenotypes, which can be then rescued by the injection of wild-type med14. Analyses of genome-wide transcript levels and mRNA levels did not support a general affectation of transcription in these mutants. In contrast, when the authors focused on stem and progenitor cells and regeneration in the log mutants they observed specific defects. Thus, using different markers they saw that retinal, hematopoietic and gut stem cells were reduced in the log mutant and that med14 seemed to be important in heart progenitor cells. Also, when the tail fin of these animals was amputated it did not regenerate.

In order then to better characterize the putative function of med14 on stem cells and regeneration the authors studied it in planarians, an attractive model in which to study stem cells in vivo. In planarians, as in zebrafish, med14 was ubiquitously expressed including in neoblasts (planarian totipotent stem cells and the only proliferative cells in them). The silencing of med14 by RNAi in intact planarians resulted in a phenotype resembling that obtained after depletion of the neoblasts. That is, the animals curled ventrally, lost their heads and finally lysed. On the other hand, the silencing of med14 also blocked regeneration. After med14 silencing there was a gradual loss of cells expressing the neoblast marker Smedwi-1 (a piwi homologue). There was also a loss of proliferative activity detected with different markers of mitosis and S-phase such as phosphorylated histone H3, histone h2b and pcna. Using a marker of neoblast early progeny the authors found that this cell population was also gradually depleted. In contrast, med14 RNAi did not affect the expression of specific markers for differentiated cell types including neurons, gut cells, muscle, pharynx and eye. Moreover they found a significant increase in apoptosis all throughout the treated animals. These results suggested that med14 silencing affected specifically the neoblasts and their progeny and that transcription in general was not compromised.

In summary, the results presented here indicate that the Mediator complex has an important role in stem cell maintenance in two distant models such as zebrafish and planarians. Further analyses should help determining how the Mediator exerts its function, maybe through establishing the epigenetic landscape essential for pluripotency and stem cell maintenance in these animals.

A hematopoietic cell-derived signal is required for cell survival during zebrafish fin regeneration

During zebrafish fin regeneration different genes and signalling pathways are locally upregulated in the wound area and play important roles to trigger a successful regenerative response. However, less is known about the existence and function that putative factors provided by tissues away from the wound could have during these regeneration events. A recent paper from the laboratory of Atsushi Kawakami reports on a diffusible signal derived from hematopoietic cells that would support cell survival and proliferation during zebrafish fin regeneration (http://www.ncbi.nlm.nih.gov/pubmed/25533245).

The authors studied a mutant called cloche (clo) previously identified from a phenotype that lacks most hematopoietic and endothelial cells. In wild-types (fish larvae), the fin fold was normally regenerated (in size and shape) by 3 days after amputation. In contrast, clo mutants displayed aberrant regeneration with the formation of tissues with a blister-like appearance. The authors thought that this might represent dying cells so they checked with TUNEL assay to find out a large number of dying cells in this region, compared to wild-types. Uncut clo mutants did not display higher levels of cell death so it seemed that this increase in cell death in clo mutants was regeneration-dependent. This was further confirmed because in a wound healing assay there was no increase in cell death in these mutants. This increase in cell death was accompanied by a decrease in cell proliferation as assayed by BrdU labelling. Again, here, the cell proliferation levels in the head and trunk (away from the wound) were normal compared to wild-types.

Immediately after amputation (0,5 hours), necrotic cell death was similarly induced in the injured region of wild-types and clo mutants. This necrotic cell death disappeared by 6 hours in wild-types and clo mutants, but then at 12 hours after amputation clo mutants displayed a high number of TUNEL positive cells. This cell death was mostly suppressed when overexpressing Bcl2 (a negative regulator of apoptosis) in these mutants, suggesting that cell death was most probably caused by a Bcl2-regulated apoptosis pathway.

In clo mutants this increased apoptosis coincided with the stage in which regenerative cell proliferation begins. The authors checked then the expression of junb and junbl early markers induced in the wound epithelium and the blastema, respectively. These two genes appeared to be normally induced in the clo mutants, suggesting that the initial regenerative response might be normal in them. Next, they carried out some transplantation experiments between clo mutants and wild-types to investigate the cell autonomy of Clo protein function during regeneration, concluding that Clo function was non-cell autonomous for the survival of the regenerative cells. These results prompted them to wonder whether the lack of hematopoietic cells in clo mutants was responsible for these reduced cell survival during regeneration. Thus, they analysed tal1, another hematopoietic mutant, and found out similar phenotypes: impaired fin fold regeneration with increased apoptosis and reduced cell proliferation. As the hematopoietic lineage contains several cell types they wanted to identify the cell types required for the survival of the regenerative cells in these mutants. In red blood and endothelial cell mutants they did not observed apoptosis of the regenerative cells. However, when they knocked down the myeloid lineage through the silencing of pu.1, a transcription factor required for myeloid cell differentiation, they obtained similar results to those observed in clo and tal1 mutants, suggesting that myeloid cells would have an important function on the survival of the regenerative cells.

Finally, they wanted to investigate how this putative signal was mediated from the hematopoietic tissues to the amputation site. They developed an in vitro assay for a tail explant cultured for 12 hours post-amputation. Wild-type and clo and tal1 mutant explants normally induced the expression of junb and junbl. Whereas in wild-types no apoptosis was observed, explants from clo and tal1 mutants displayed an induced cell death. These results suggest that the defects observed in clo mutants could not be caused by an impaired circulation. Remarkably, when the mutant explants were treated with body extracts prepared from wild-types there was a rescue of the apoptosis. On the other hand, body extracts from the mutants did not rescue the apoptosis in the tail explants from those mutants. These results suggested that the survival of the regenerative cells might be supported by a diffusible factor in the wild-type body.

In summary, this study suggests that a diffusible, and still unknown, factor derived from the hematopoietic cells would support the survival and proliferation of primed regenerative cells during fin fold regeneration in zebrafish.

A dual mode of heart regeneration in zebrafish

Mammals cannot regenerate their hearts despite that some studies have reported that newborn mice can regenerate the heart apex if it is amputated during their first days of life. Also, in some special pathological or physiological conditions adult cardiomyocytes can be forced to re-enter the cell cycle and/ or dedifferentiate. However, these responses cannot sustain the proper regeneration that should take place after, for example, an acute myocardial infarction. In contrast, zebrafish cardiomyocytes retain their ability to dedifferentiate and re-enter the cell cycle throughout their lives, which explain the amazing capacity of these animals to regenerate a large portion of their hearts. Experimentally, regeneration studies in the zebrafish heart are carried out after either amputation or cryoinjury.

Now a recent paper from the laboratory of Anna Jazwinska reports on the spatial and temporal dynamics of cell proliferation and differentiation during cryoinjury-induced heart regeneration (http://www.ncbi.nlm.nih.gov/pubmed/25557620). The authors first used MCM5 as a marker of the G1/S phase of the cell cycle and phospho histone H3 (PH3) to label mitotic cells in G2/M. Moreover, they used these markers on transgenic zebrafish expressing GFP under the control of the cardiac specific promoter cmlc2. During regeneration the initial fibrotic tissue was progressively replaced with new myocardium, which resulted in the decrease of the infarcted area relative to the entire ventricle. As expected, regeneration was characterized by an increase in cardiomyocytes (CM) proliferation, quite evident by 7 dpci (days post-cryoinjury). During the progression phase at 17 dpci the number of proliferating cells declined and at 30 dpci (termination phase) the mitotic levels had declined up to the levels in uninjured fish. However, at this late stage, the number of MCM5-positive CM still remained significantly higher compared to uninjured animals.

Then, the authors determined the spatial distribution of the dividing CMs relative to the injury border. During the initiation phase (7 dpci) there was a graded distribution of PH3 mitotic cells. Thus, 50% of the PH3 cells were within the first 100 mm adjacent to the cryoinjury border; 20% of them were found between 100 and 200 mm from the border, and the remaining 30% were distributed from the 200 to 1000 mm. Thus, most of the mitotic cells were located at the close vicinity of the wounded area. This graded distribution of mitotic cells was not detected during the progression phase (at 17 dpci) when the PH3 cells were nearly evenly spaced within the entire myocardium. It is important to point out that at this later stage the number of PH3 cells was still significantly higher compared to an uninjured heart. Next the authors performed some BrdU labelling and found out, as expected, that the majority (65%) of CMs located in the regenerated myocardium were BrdU-positive, indicating that they derive from newly generated cells. In the adjacent region (0-100 mm from the regenerated myocardium) about 30% of the CMS were BrdU-positive. Beyond this zone and in all the subregions that the authors checked they found an even percentage of BrdU-positive CMs (about 10%), that again was higher compared to uninjured hearts. Overall, these results suggest that in addition to a local rapid and strong proliferative response to cryoinjury there was also a systemic response all throughout the pre-existing heart.

Finally, the authors analysed the formation of immature CMS during regeneration. To do that they took advantage of an antibody raised against a mammalian neonatal muscle and that binds the N-terminal end of slow b/cardiac myosin heavy chain. In zebrafish this antibody seemed to label embryonic undifferentiated CMs. During regeneration (at 7-10 dpci) the authors found a significant number of CMs positive for this antibody at the injury border within a distance up to 100 mm from the post-infarcted tissue. No labelled cells were found in regions beyond this zone. During the progression stage (17 dpci) these positive CMs lost their initial homogenous alignment along the injury border and intermingled with the regenerated mature cardiac tissue. At 30 dpci almost no labelling was detected. Altogether, the authors concluded that during the initial phase of regeneration, undifferentiated CMS appear at the leading edge of the dedifferentiating adult myocardium and would provide a source of new CMs during regeneration.

In summary, the authors propose that upon cryoinjury a local epimorphic regenerative response is triggered within the 100-200 mm adjacent to the injury border and in which a significant increase in immature and proliferating CMs was detected. This initial response in this blastema-like region would be further reinforced by a systemic compensatory regenerative response within the entire pre-existing myocardium.

teashirt, a target of Wnt/β-catenin pathway, regulates AP polarity during planarian regeneration

What are the exact mechanisms that control the re-establishment of axial polarity remains as an important question in many regeneration models. In freshwater planarians several studies in the last years have identified the Wnt/β-catenin signalling pathway as pivotal for the establishment and maintenance of the anteroposterior (AP) axis. Thus, several Wnts and Wnt receptors are expressed in the posterior regions whereas different inhibitors of Wnt signalling are expressed at the anterior end. Consequently, the inhibition of this pathway by silencing either β-catenin or some Wnt genes (such as Wnt1) results in the regeneration of heads instead of tails. Even in intact animals the silencing of these genes transforms the tail region into a head with a proper brain. Conversely, the activation of the Wnt/β-catenin pathway in anterior regions leads to the transformation of those heads into tails. Therefore, it seems quite clear that Wnt/β-catenin is required to specify posterior fates in these animals.

Now, a paper from the laboratory of Kerstin Bartscherer has aimed to identify genes regulated by this pathway (http://www.ncbi.nlm.nih.gov/pubmed/25558068) that could be involved in controlling tissue polarity. In order to identify target genes the authors carried out RNAseq experiments after silencing Smed-β-catenin1 in intact animals. Initially they identified 440 downregulated and 348 upregulated transcripts. Among the downregulated genes they found previously described posterior genes and, conversely, upregulated genes included some known anterior genes. Then, they selected 70 genes downregulated after β-catenin silencing and analysed their expression patterns by in situ hybridization. Of them, 35% displayed differential expression along the AP axis with high expression in the tail. Among the other genes, 21 of them were expressed within or around the digestive system. When they analysed the expression of most of these genes after β-catenin RNAi, they found that their expression was strongly inhibited, validating thus the RNAseq data. Also, they found that many of the upregulated genes after β-catenin RNAi were mainly expressed in anterior regions, as expected.

Next, they selected some of these genes that showed a graded expression along the AP axis and analysed in more detail their expression by FISH. These experiments clearly showed this graded expression, high in the tail and lower towards more anterior regions, strengthening the hypothesis that a β-catenin activity gradient may control gene expression. Recently, it has been described that collagen-positive subepidermal muscle cells express several position control genes (PCGs), which has lead to propose that these muscle cells could somehow provide positional information along the AP axis. Here, the authors show that some of the putative novel targets of β-catenin are also expressed in those posterior muscle cells. In addition, some of these genes were expressed in intestinal muscle cells, which suggests that the gut musculature could be also a source of PCGs. Then, and in order to determine whether planarian neoblasts (totipotent stem cells) differentially expressed the candidate genes sp5 and abdBa along the AP axis they isolated different FACS-sorted cell populations from anterior and posterior regions and quantified their expression in those cell fractions. Interestingly, they found that sp5 and abdBa transcripts were higher in the stem cells from posterior regions compared to anterior neoblasts, suggesting that planarian neoblasts may respond to graded β-catenin activity along the AP axis.

Notum is a secreted Wnt inhibitor that, in planarians, is expressed at the anterior tip of the animal and whose silencing leads to the regeneration of heads instead of tails. It has been proposed that notum regulates early polarity decisions through the inhibition of β-catenin activity at anterior-facing wounds. As the authors reasoned that notum inhibition might induce β-catenin-dependent genes they carried out RNAseq experiments after notum RNAi at 18 hours of regeneration, a stage in which polarity decisions are supposed to occur. By comparing the two sets of genes from the two different RNAseq experiments, they found that 38 of the downregulated genes after β-catenin RNAi were induced after notum silencing. Conversely, 16 of the upregulated genes after β-catenin RNAi were inhibited after notum RNAi. Thus, these 54 genes could represent the genes involved in early polarity decisions during planarian regeneration. Remarkably, 33 out of these 54 genes were also present in a previously generated set of Wnt/β-catenin target genes in zebrafish, which suggests that the common requirement of the Wnt/β-catenin pathway during regeneration in planarians and zebrafish may include a shared set of target genes.

One of the novel candidate target genes identified here was teashirt, a Tsh-related zinc finger protein, a gene that appears to act as modulator of Wnt signalling during Drosophila and Xenopus development. In planarians, teashirt (tsh) was expressed all throughout the central nervous system as well as in a graded pattern in the mesenchyme with highest expression in the tail. Tsh expression was induced upon amputation and, as predicted, it was inhibited after β-catenin RNAi and induced after β-catenin overactivation. Remarkably, a tsh homolog in zebrafish was detected in fin regenerates at 3 days after amputation, being this expression also dependent on the Wnt/β-catenin pathway further supporting an evolutionarily conserved role of Wnt/β-catenin in ensuring correct tissue identity and/or patterning during regeneration. Going back to planarians, the silencing of tsh resulted in two-headed planarians, a phenotype reminiscent of β-catenin and Wnt1 RNAi. These results indicated that tsh would be required to suppress anterior fate at a posterior-facing wound. During posterior regeneration Wnt1 is rapidly induced in the stump. Tsh co-localized with wnt1-positive cells both in intact and regenerating tails. Morevover, tsh was also expressed in neoblasts and that expression seemed to be higher in neoblasts from posterior regions.

In summary, the authors show here that tsh is expressed in wnt-positive cells, probably subepidermal muscle cells, as well as in a subpopulation of neoblasts in both cases in a β-catenin-dependent manner. Tsh could be then a downstream transducer of Wnt signalling important to regulate AP polarity.

Schwann cells and DCC guide regenerating axons towards their original path

In contrast to the central nervous system the axons of peripheral nerves are able to regenerate in many vertebrates, including mammals. How this regeneration is achieved and the cellular processes involved have been described in many studies. An important point here is that the cellular environment plays an important role in promoting this axonal regrowth. Among these elements, Schwann cells have been described as required for peripheral axons regeneration. Now, a recent paper from the laboratory of Michael Granato reports on the observation in vivo of axonal regeneration in zebrafish as well as the requirement of Schwann cells to direct those axons towards their original paths (http://www.ncbi.nlm.nih.gov/pubmed/25355219).

Here, the authors use transgenic zebrafish expressing GFP in motor neurons and RFP in Schwann cells to follow in vivo the regeneration of fully transected motor nerves in larvae at 5dpf (days post fertilization). The first thing they describe is how upon transection the amputated distal axons were degraded and fragmented leaving behind axonal debris and denervated Schwann cells. Then, Schwann cells suffered dramatic changes from a tube-like morphology to a shorter and more rounded one. As motor axons regrew into their original paths, Schwann cells membranes also reverted to their normal thinner appearance.

In order to define in vivo the role of Schwann cells the authors used different mutant strains that lack differentiated Schwann cells (mutants for sox10, erbb2, erbb3 and nrg1 type III). In all these mutants, motor axons developed normally through 5 dpf. However, upon transection, axon regeneration was severely affected in all mutants lacking Schwann cells. In them, in 50-80% of the transected nerves the regenerated axons failed to target their original paths and instead ectopically grew into more lateral territories. These results suggest that Schwann cells directed regenerating axons. One possibility could be that proper axonal regeneration would depend on some factor produced by Schwann cells during development. To address this question the authors used a transgenic line in which Schwann cells develop normally but can be eliminated at the requested time point of development. Then, the authors killed the Schwann cells at 3,5 dpf and transected the motor nerves at day 5 dpf. In this context, the regenerated axons also failed to find their original targets as previously described for the sox10 mutants, suggesting that Schwann cells must be present during regeneration to direct re-growing axons. In another set of experiments the authors show how in sox10 mutants the regenerative growth cones initially sprouted and extended from the proximal stump in multiple directions as it happens in wild-type larvae. However, and in contrast to wild-type, axons continued to extend in all directions without going back to their original paths.

Distal denervated Schwann cells could exert this guiding role either by providing a physical substrate for the regenerating axons or by producing some factors that would direct the re-growing axons towards their original paths. To try to distinguish between these two possibilities the authors used a transgenic line in which the degeneration of the amputated distal nerves was delayed by more than 1 week. They crossed this line with the sox10 mutant line so an axonal scaffold distal to the transection gap completely lacking Schwann cells was formed. In mutants for axonal degeneration but with normal Schwann cells, motor axons regenerated normally indicating that those distal amputated axons had no inhibitory action on regeneration. However, in the double mutant with sox10, the regenerating axons failed to find their original paths. In other experiments, the authors transected only half of the axons in the motor nerve to create a continuous axonal scaffold devoid of Schwann cells (in sox10 mutants). There, the presence of this axonal scaffold could only partially compensate for the role of Schwann cells as 50% of the transected nerves showed aberrant abnormal projections compared to the 90% observed in fully transected nerves lacking Schwann cells. Overall, these results suggest that Schwann cells provide more than just a permissive substrate for regeneration.

Finally, the authors analysed the role of dcc (deleted in colorectal cancer) a receptor for netrin, a well-known attractive guidance cue for axonal growth. At 5 dpf dcc was expressed in motor neurons as well as in Schwann cells, whereas netrin1b was expressed in Schwann cells before and after transection. Also, dcc was expressed in motor neurons during the first stages of regeneration. Then the authors used a mutant line for dcc in which, interestingly, motor axons and Schwann cells develop normally. However, upon transection, about 40% of the nerves extended axons not only to their original paths but also ectopically into more lateral territories.

In summary, the authors show here the process of motor axon regeneration in vivo in zebrafish. Schwann cells are required for guiding the regenerated axons to their original paths and do it probably not only by providing some physical substrate but actively producing some factor. Future experiments should determine if netrin could be one of such factors.

Macrophages in zebrafish tail regeneration

In previous posts I have discussed the important role of macrophages and the immune system in triggering a successful regeneration in contrast to a scarring wound healing, by modulating the inflammatory response. Thus, for example, I commented on the requirement of macrophages to induce blastema formation during limb regeneration in salamanders probably by activating cell dedifferentiation and the proliferation of progenitor cells needed to rebuild the missing tissues (https://regenerationinnature.wordpress.com/2013/06/06/macrophages-and-limb-regeneration-in-salamanders/). Now, a paper from the laboratory of Timothy Petrie and Randall T. Moon reports on the need of macrophages also during tail regeneration in zebrafish (http://www.ncbi.nlm.nih.gov/pubmed/24961798).

            Zebrafish tail regeneration may be divided into three main stages: 1) wound healing (0-1 days of regeneration); 2) blastema formation (1-3 days of regeneration); and 3) regenerative outgrowth and patterning of the new tissue (>3 days of regeneration). Previous studies in zebrafish larvae had reported that upon injury neutrophils and macrophages accumulate at the wound region suggesting a similar role of these cells compared to their mammalian counterparts. However, the role of these inflammatory cells in adult zebrafish regeneration remains mainly unknown.

            In this study, the authors used several transgenic lines to track neutrophils and macrophages upon amputation. For neutrophils, these cells rapidly accumulated at the wound from 6 hours post amputation (hpa). A maximum peak was achieved by 3 days post amputation (dpa) and then their number declined from 5 dpa until reaching pre-amputation levels of neutrophils by 7 dpa. It is known that regeneration rates are different along the proximo-distal axis of the tail. Thus, proximal amputations result in faster regeneration compared to slower growth upon distal amputations. Interestingly, proximal amputations recruited over twice the number of neutrophils as distal ones. Whereas few neutrophils were detected in uninjured tails, macrophages were found in higher density. Upon amputation macrophages began accumulating in the wound region by 3-4 dpa, reaching a peak by 6-8 dpa. As neutrophils, macrophages also accumulated faster and at greater densities in proximal amputations.

            Upon injury, neutrophils accumulation appeared to depend on their migration from the vasculature near the wound. The authors inhibited neutrophil recruitment into the wound but this reduced accumulation did not result in any difference in the rate of regeneration compared to controls. This is in contrast to what happens in larval tails as previous reports have suggested that neutrophil deficiency increases the regeneration rates. Next, the authors sought to determine the consequences of macrophage ablation during regeneration. They used a transgenic line bearing the enzyme nitroreductase (NTR) downstream of a macrophage-specific promoter. NTR converts the pro-drug metronidazole (MTZ) into a cytotoxic agent that kills the cell. Upon 36 h of MTZ treatment there was a strong reduction of around 80-90% of the macrophages in the tail. In control animals, MTZ by itself did not affect either the inflammatory response or the regeneration rate and success. Then, the authors amputated the tail and treated the animals with MTZ for 14 dpa. They saw that macrophage ablation resulted in a significant decrease of the extent of new tissue growth. Moreover, these fish also showed aberrant tissue growth along the regenerated tail. The zebrafish tail is composed, among other tissues, of segmented bony rays. Macrophage ablation resulted in a reduction in the average number of segments in the regenerated ray. Also, the degree of mineralization was reduced in NTR+MTZ fish, indicating that macrophage depletion impaired bone ray patterning and the quality of bone formation.

            Then, the authors analyzed in more detail which events required for regeneration could be affected upon macrophage ablation. They observed that although the loss of macrophages did not significantly affect gross blastema morphology and size, there was a significant decrease in cell proliferation. Also, at 4 dpa there was a strong reduction in the expression of regeneration-associated and injury-response genes. On the other hand, neutrophils normally accumulated upon macrophage ablation. In order to determine at what stage of regeneration are macrophages required the authors ablated them at two distinct time points. In one set of experiments they ablated them from 2 days before amputation through 3 dpa, corresponding to the stages of wound healing and blastema formation. The authors observed the same defects on regenerative rate and aberrant morphologies of the regenerated tails as when MTZ treatment was applied for 14 dpa. On the other side, and in order to analyze the requirement of macrophages during the outgrowth stage, the authors ablated the macrophages from 3 dpa through 14 dpa. In those experiments, the regeneration rate was not significantly affected; however, they still observed a higher occurrence of aberrant morphologies of the regenerated tails. So, it seems that during the early stages of regeneration macrophages would be required for blastema formation, whereas during tissue outgrowth macrophages would be also required to modulate tissue patterning.

            Finally, the authors wanted to study the relationship between the Wnt/b-catenin pathway and inflammation during tail regeneration as it has been shown that this pathway is required for blastema formation and outgrowth in zebrafish tail regeneration. Moreover, Wnt/b-catenin modulates several inflammatory processes in other models. Again, they used several transgenic lines to track the activation of this signaling pathway. As described above for neutrophils and macrophages, a greater density of cells with activated Wnt/b-catenin signaling were found in proximal amputations compared to distal ones. Flow cytometry analyses showed that less than 1% of neutrophils and 3% of macrophages exhibited activated Wnt/b-catenin signaling. Interestingly, macrophage accumulation at the wound was almost completely inhibited after inhibiting Wnt/b-catenin. Also, the inhibition of this pathway resulted in delayed neutrophil resolution and prolonged neutrophil number in the wound region, suggesting that the Wnt/b-catenin pathway might be required for the progression of the injury response after amputation. Thus, Wnt signaling might mitigate the initial inflammatory response and function as a molecular switch from neutrophil resolution to macrophage enrichment.

            In summary, this study reports on the requirement of macrophages for zebrafish tail regeneration providing a functional link between inflammation and regeneration.

Wnt and BMP pathways coordinate bone regeneration in zebrafish

Several weeks ago I commented on a study from the laboratory of Gilbert Weidinberg in which they had characterized an organizing center defined by the Wnt/b-catenin pathway within the distal blastema of regenerating zebrafish fin, that would control regeneration by regulating the function of several downstream signaling pathways that would mediate the effects of this organizer on surrounding tissues. Here, I comment on a study from the laboratory of Scott Stewart and Kryn Stankunas that describe how the Wnt/b-catenin and BMP signaling pathways work together and in opposite directions to coordinate bone regeneration during zebrafish fin regeneration (http://www.ncbi.nlm.nih.gov/pubmed/24485659).

In zebrafish, bone regenerates through dedifferentiation and re-differentiation of lineage-restricted osteoblasts. Osteoblasts are the responsible of depositing the osteoid, a unique extracellular matrix that form the mature bone. Although previous reports have implicated several signaling pathways, including Wnt/b-catenin and BMP receptor, in this process, how they act at the cellular and molecular levels to drive a successful regeneration is not completely known. Here, the authors first analyzed the expression of Runx2 and sp7, two transcription factors with well-known roles on bone formation. Early after amputation Runx2 was upregulated in osteoblasts lining preexisting bone adjacent to the amputation plane. Later, some Runx2+ mesenchymal cells expressed also sp7. Then, Runx2-/sp7+ cells first appeared near the amputation plane. By 72h of regeneration the osteoblast lineage was highly organized along the proximo-distal axis of the blastema: Runx2+ cells were located in most distal regions while sp7+ cells were mainly found near the amputation plane. In between Runx2+/sp7+ cells were found. In terms of proliferation, more Runx2+ cells incorporated EdU compared to sp7+ cells, suggesting that sp7+ cells near the amputation plane would be non-proliferative osteoblasts that append to progressively elongating bone.

After amputation, osteoblasts dedifferentiate to give rise to Runx2+ preosteoblasts. The authors showed that osteoblasts have epithelial-like properties as they were labeled with antibodies against catenins (a- and b-) that are found in adherens junctions that interconnect epithelial sheets. During regeneration, distal Runx2+ did not express a-catenin in contrast to Runx2+/sp7+ and sp7+ cells in close proximity to new bone, that were positive for membrane-localized a-catenin. At 24h after amputation osteoblasts rapidly lost a-catenin expression as they dedifferentiate into a progenitor state. Moreover, as they became Runx2+ and Runx2+/sp7+ cells they changed their shape from long and thin to a more compact, polygonal morphology. These results suggested that osteoblast went through an epithelial-to-mesenchymal transformation (EMT) during regeneration. This was further supported by the observation that twist2, a well-known transcription factor that directs EMT, and runx2a were rapidly induced in tissue adjacent to the amputation plane. Later, distal Runx2+ cells co-expressed twist2. Therefore, the authors concluded that Runx2+ cells originated from EMT of differentiated osteoblasts and distal Runx2+ preosteoblasts were maintained in a mesenchymal twist2-expressing state.

Next, the authors analyzed the Wnt/b-catenin signaling in regenerating fins. At 24h of regeneration, Runx2+ cells had nuclear b-catenin staining. By 72h post amputation, strong nuclear b-catenin was observed in distal Runx2+ preosteoblasts with much less staining in sp7+ differentiating osteoblasts near the amputation plane, suggesting that downregulation of Wnt signaling correlates to osteoblast maturation. It is known that Wnt signaling can initiate EMT and induce twist expression during mouse bone development. Here, the authors used IWP-2, an inhibitor of Wnt signaling. IWP-2 treatment arrested regeneration by interfering with osteoblast EMT and the induction of twist2 expression, indicating an important role of Wnt signaling in osteoblast EMT. Moreover, IPW-2 treatment from 48h to 72h post-amputation also blocked regeneration by depleting osteoblast-lineage cells distal to the amputation plane, suggesting a role of this pathway in maintaining the preosteoblast population.

Finally, the authors analyzed the BMP pathway as it has been also implicated in bone formation. The activation of the BMP signaling leads to the phosphorylation of the transcription factor Smad1/5/8, that can then go to the nucleus and activate its downstream target genes. Here, pSmad1/5/8 was detected in differentiating sp7+ cells but not in Runx2+ or Runx2+/sp7+ preosteoblasts. The inhibition of BMPR resulted in a pronounce decrease in the extend and levels of sp7 expression and reduced bone formation. These treated fins were able to form a blastema but failed to produce mineralized bone, suggesting a role of the BMP pathway in osteoblast maturation. Remarkably, BMPR inhibition resulted also in an increase in the number of Runx2+ cells and a decrease of the Runx2+/sp7+ and sp7+ populations. Osteoblast proliferation and cell death were not affected by this treatment suggesting that BMP would drive osteoblast differentiation.

As BMP inhibition expanded proximally the distal Runx2+ population the authors hypothesized that BMP activity in proximal regions would normally inhibit Wnt/b-catenin activity in those proximal domains. This was supported by the observation that BMPR inhibition reduced the expression of Dkk proteins, well-known negative regulators of Wnt activity. In agreement with the idea of distal Wnt active and proximal BMP active populations, wnt5a and wnt5b were mainly expressed at the distal tip of the blastema whereas bmp2 was expressed in differentiating proximal osteoblasts.

In summary, the authors have shown that zebrafish bone regeneration is mainly regulated by the antagonistic and coordinated function of the Wnt and BMP signaling pathways in order to provide a precise balance between cell plasticity and differentiation. In their proposed model, Wnt activity drives EMT of osteoblasts to give rise to dedifferentiated Runx2+ preosteoblasts. Sustained levels of Wnt activity in the distal blastema maintain these Runx2+ proliferative cells. Then, as these preosteoblasts are located to more proximal regions they upregulate bmp2 and activate autocrine BMP activity that promotes osteoblast differentiation by inducing the expression sp7 and dkk1b, that inhibits Wnt activity to prevent the overexpansion of the progenitor pool.

V-ATPase activity during adult zebrafish fin regeneration

In recent years several papers have uncovered the importance of ion channels during regeneration in different models. Thus, for example, cellular hyperpolarization is essential for Xenopus tadpole tail regeneration and cellular depolarization is required to specify anterior polarity in planarians. Now a recent paper from the laboratories of Ana Certal and Joaquín Rodríguez-León has reported for the first time the requirement of V-ATPase activity for fin regeneration in adult zebrafish (http://www.ncbi.nlm.nih.gov/pubmed/24671205).

After any wounding an electric current is generated as a response; however, only in those cases in which a regenerative process is triggered these endogenous electric currents are maintained beyond wound closure. Here, the authors first analysed the contribution of different ions (K+, Na+, H+, Ca2+ and Cl) to the electric current during adult zebrafish fin regeneration. Of all these ions H+ was the only one for which the authors found that 24 hpa (hours post-amputation), during blastema formation, there was still an efflux that was 14-fold higher than the one detected in intact fins. Previous microarray experiments had detected V-ATPase, a main H+ transporter, as being upregulated after 24h during fin regeneration. Thus, the authors checked the expression of several V-ATPase subunits during regeneration. Two of them, atp6v1a and atp6ve1b, were not expressed in intact fins but were upregulated in the blastema by 24 hpa. At 72 hpa some expression was still detected at the distal part of the blastema. Next, the authors assessed the role of V-ATPase during regeneration. They blocked the pump’s activity either by using concA or morpholinos (MO) against atp6v1e1b. Both approaches delayed fin regeneration suggesting a role for this H+ pump in the regenerative process.

As it happens for amphibian limb regeneration, proximal amputations of the caudal fin resulted in higher regeneration rates compared to distal stumps. Interestingly, the expression of atp6v1e1b was already visible by 12 hpa, whereas in distal stumps the first expression of this gene was observed at 24 hpa. Not only the expression appeared earlier but it also covered a wider region. By 48 hpa these differences were not so obvious anymore. In agreement with this earlier and stronger upregulation of atp6v1e1b in proximal stumps, the authors found out that the H+ efflux started earlier in those proximal stumps (3 hpa instead of 12 hpa in distal ones) and was higher at any time point measured than in distal stumps. These results clearly indicate a relationship between V-ATPase and H+ efflux and the regeneration rate along the PD (proximo-distal) axis. Further supporting this, atp6v1e1b knockdown significantly decreased the H+ efflux. This MO-mediated silencing of atp6v1e1b also resulted in a decreased regenerated area, being this inhibition higher in proximal stumps, suggesting that those proximal stumps with higher regenerative rates are more dependent on V-ATPase activity. Remarkably, the inhibition of V-ATPase activity did not seem to affect the regeneration of the larval fin fold.

Finally, the authors studied the effects of inhibiting the V-ATPase activity on cell proliferation and gene expression. Although no differences in proliferation were observed at 24 hpa, by 48 hpa atp6v1e1b knockdowns showed a significant reduced number of proliferative cells within the blastema, compared to controls. Different signalling pathways, including FGF, Wnt/B-catenin and Retinoic acid (RA), have been shown to regulate cell proliferation during regeneration. In controls, the expression of mkp3 (FGF signalling) and aldh1a2 (RA signalling) was detected in wider domains in proximal stumps compared to distal ones, similarly to the differences observed for V-ATPase activity. The silencing of atp6v1e1b resulted in the inhibition of the expression of mkp3 and aldh1a2 indicating that V-ATPase was required for the expression of these two genes during regeneration. Last, the authors reported that V-ATPase seems to be also necessary for the normal innervation of the regenerating fin.

In summary, this study reports for the first time the requirement of V-ATPase for adult zebrafish fin regeneration. The authors propose that the regulated H+ efflux generates pH and/or voltage domains within the regenerating tissue that, directly or indirectly (for example, via innervation), would act on FGF and RA signalling pathways to regulate cell proliferation during regeneration.

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