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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.
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.