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.
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.
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.
In general vertebrates have very limited regenerating capabilities. Among invertebrate chordates however, amphioxus and tunicates show remarkable regenerative abilities. Studying regeneration in these animals is important, as tunicates appear to be the closest living relatives of the vertebrates. Colonial ascidians are capable of whole body regeneration by the activation of stem cells in their vascular system or the epicardium. Solitary ascidians, such as Ciona, show more restricted regenerative capabilities. In addition, it is not clear whether regeneration in these animals depend also on stem cells. Now a recent paper from William R. Jeffery reports on the existence of stem cells in the brachial sac of Ciona, required for regeneration (http://onlinelibrary.wiley.com/doi/10.1002/reg2.26/abstract).
Previous studies had shown that after bisection only basal parts are able to regenerate the distal parts, whereas distal parts are not able to regenerate the missing basal ones. Among the distal parts that can be regenerated researchers have focused mainly on the neural complex and the oral siphon, a muscular tube leading into the pharynx and that contains orange-pigmented sensory organs (OPOs). For both structures regeneration involves the formation of a blastema although the origin of the regenerative cells is not clear.
In this paper, the author focused on the regeneration of the distal oral siphon and its OPOs. First, the author confirmed that only the basal parts were capable of regenerating complete animals. The distal parts did not regenerate any of the proximal (basal) regions and eventually disintegrated. Also, middle body parts were able to regenerate the distal oral siphons with OPOs even in the absence of the basal parts. After the amputation of the oral siphon a blastema containing proliferating cells is formed about 4 days after amputation. Then, he investigated the role of cell proliferation in OPO regeneration. Remarkably, blocking cell division did not affect OPO regeneration. Next, and in order to determine the source of stem cells for distal regeneration two different approaches were used. In a first set of experiments the author labelled dividing cells with EdU for 3, 8, 24 and 48 h after bisection. Control (intact) animals showed EdU labelling in the stomach, intestines and basal stalk. Regenerating animals showed similar levels of EdU labelling in those organs. However, in those regenerating animals a strong EdU labelling was observed from 3 h of regeneration in the transverse vessels of the branchial sac. The labelling was most prominent in the lymph nodes, known to be involved in blood cell renewal. On the other side, the regenerating oral siphon did not show EdU labelling until 48 h of regeneration. Secondly, the location of stem cells was analysed by using alkaline phosphatase (AP) and PIWI markers. In adults, the most intense AP activity was seen in transverse vessels of the branchial sac, where PIWI was also immunodetected. Altogether these results suggest that the transverse vessels of the branchial sac are a potential source of stem cells for distal regeneration.
Next, he carried out several EdU chase experiments to determine the source of the blastema cells. Control and regenerating animals were exposed to EdU for 24 h and then chased without EdU for 5-10 days. Regenerating animals showed an intense labelling in the distal blastema, which was not labelled after 2 days of EdU pulse, which suggests a source of proliferating cells outside the blastema. Then, he transplanted EdU labelled branchial sacs into control hosts that were afterwards bisected at a level leaving the transplanted tissues in the basal part. After 10-15 days of regeneration EdU positive cells were detected in the distally regenerating neural complex and oral siphon. Therefore, it seems that proliferating cells from the branchial sac migrate into the blastema during distal regeneration. Respect to the cells that give rise to the OPOs, experiments with regenerating oral siphons explants suggested that AP labelled stem cells original for the branchial sac would invade the distal areas and differentiate into OPOS during the early stages of regeneration.
The regenerative abilities of Ciona decline with age. So, in a final set of experiments the author wanted to determine whether this decline was related to changes in the stem cells of the branchial sac. He compared the regeneration of young (6 months) and old (12 months) animals. As expected old animals were either unable to regenerate or regenerated partial siphons. Remarkably, the distribution of proliferating cells and the structure of the branchial sacs appeared disorganized in old animals. Moreover, old animals showed very few AP and PIWI labelled cells in the transverse vessels, suggesting that stem cells may be depleted in the branchial sac during aging, being this responsible of their reduced regenerative potential.
In summary, this paper reports on the role of stem cells from the branchial sac in the regeneration of distal structures in Ciona. In these animals the blastema appears to be formed by at least two types of progenitor cells: 1) a subset of branchial sac cells that incorporate EdU very early during regeneration but that they are detected in the blastema after few days (once they migrate there), and 2) another subset of branchial sac cells that migrate to the blastema very early during regeneration and differentiate into the OPOs without undergoing cell division.
I just posted an annual report of this blog prepared by WordPress. I am very happy to see how the number of visits to this site has increased compared to 2013. So thank you very much to all of you that regularly or occasionally read my posts. And also, special thanks to those of you (very few yet) that comment on some of the posts. Unfortunately I couldn’t write any new post the last weeks of December as I got a lot of teaching. But I will be back very soon. So keep ready to read more about regeneration
The WordPress.com stats helper monkeys prepared a 2014 annual report for this blog.
Here’s an excerpt:
The concert hall at the Sydney Opera House holds 2,700 people. This blog was viewed about 13,000 times in 2014. If it were a concert at Sydney Opera House, it would take about 5 sold-out performances for that many people to see it.