regeneration in nature

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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.

Axonal regeneration in zebrafish is promoted after a first lesion

In contrast to the central nervous system, peripheral axons are capable of regeneration up to different extend in many vertebrates, including mammals. Zebrafish offer the opportunity to follow this process of axonal regeneration in vivo. A recent paper from the laboratory of Alain Ghysen (http://www.ncbi.nlm.nih.gov/pubmed/24474787) describes how axonal regeneration in the posterior lateral line (PLL) occurs throughout adulthood with high fidelity, although the latency before the nerve regenerates increases with age. Noticeably, they found that regeneration is promoted after a first lesion at any age.

            The PLL is formed by neuromasts, superficial mechanosensory organs innervated by afferent neurons. Previous studies had shown how axons innervating the neuromasts were efficiently regenerated within 24h in young fishes. Here, the authors analysed whether the PLL nerve regenerates in adults. They used a reporter line in which all neurons were labelled and took advantage that PLL axons run right under the skin, so they are quite accessible for an easy imaging. They first checked axonal regeneration in juveniles at 1 mpf (month post fertilization). Remarkably, the branching pattern of the regenerated axons mimicked the original pattern. Even when the original (pre-amputated) pattern displayed some irregularity (as for example, nerve branches leaving the lateral nerve half a somite anterior or posterior to the normal branching point), these irregularities were reproduced by the regenerated axons with a high fidelity.

            Next, they studied axonal regeneration at 1, 3, 6 and 15 mpf by following how neuromasts were reinnervated after a nerve cut. The onset of reinnervation appeared to be delayed with age. This latency between nerve transection and neuromast reinnervation increased steadily with age. But on the other side, and in terms of speed of reinnervation, no differences were found between 1 and 6 mpf, as in all cases there was an almost complete regeneration within 5 days after the onset of reinnervation. However, this was not the case for fishes at 15 mpf, as in them only about 25% of the neuromasts were reinnervated in the same period. Therefore, there is an increase in latency from 1 to 15 mpf, whereas the speed of regeneration is only diminished in 15 mpf fishes.

            The authors then analysed the dynamics of regeneration after a second cut. What they did was to cut again the PLL nerve immediately posterior to the first cut and after a complete reinnervation had been achieved (at days 6, 7, 9 and 14 after the first nerve cut in 1, 3, 6 and 15 mpf fishes, respectively). They found that regeneration was successful after this second cut. Remarkably, whereas the speed of regeneration was not significantly different between the first and the second regeneration event, they observed how the latency of reinnervation (from amputation to complete regeneration) was dramatically reduced after the second cut. That is, after a second cut the reinnervation of the neuromasts was achieved faster than the first time.

            These results suggested that there could be some regeneration-promoting factor induced or derived from the first cut that would still be present by the time of the second cut, accelerating this second regenerative event. However, this acceleration of the second regeneration event took place only at a certain time after the first cut. Thus, for example, in 3 mpf fishes, regeneration was faster when the second cut was made 1 week after the first cut, but not when it was done after 3 weeks. In this last case, the latency was the same as for the first regeneration.  Next, the authors tried to characterize better the nature of this regeneration-promoting factor. They found that when the second cut was done just posterior to the first cut a faster regeneration was observed. However, when the second cut was done 2-3 somites anterior to the first one, no reduction of the latency of reinnervation was observed. These results suggested a local origin of this regeneration-promoting factor. The fact that this promoting effect was observed when the second cut was done distal rather than proximal to the first cut could suggest that it was caused by an increased attractiveness of the distal Schwann cells. However, the presence of Schwann cells distal to the cut was not strictly required for nerve regeneration (although they serve as a preferred substrate and guidance cue for the regenerating axons). Also, Schwann cells did not seem to play a role in the reduction of latency after a second cut, because a faster regenerative event was observed after a second cut even in the absence of Schwann cells over 2-3 somites distal to the first cut. Therefore, this promoting effect appeared to be most likely intrinsic to the axons.

            In summary, the authors provided evidence that PLL nerves regenerate all throughout adulthood although from 15 mpf this process slows down. Also, they showed how the latency before complete reinnervation increases steadily with age but is reduced when a second cut is made distal to the first one. This promoting effect probably involves a local change in the properties of the axons. Future experiments should help to identify the nature of this regeneration-promoting factor.

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