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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.
The extracellular matrix (ECM) does not only provide with a physical or structural support to cells and tissues but also it is a source of signaling and regulatory functions that impact most biological functions. Consequently, regeneration is also a process in which the ECM may obviously play an important role. Thus, for instance, just think on freshwater planarians. During regeneration and homeostasis stem cells proliferate and migrate towards the tissues or structures in which new cells are needed (i.e. the blastema or an old organ). Also, during these processes, the planarians may go through an extensive remodeling of the preexisting tissues. Considering the relevance that the ECM appears to have in regulating cell adhesion, cell migration and stem cell niche, self-renewal or differentiation in other systems, it is surprising how little we know about the role of ECM during planarian regeneration, or even how it changes during the required remodeling associated to regeneration or homeostasis. Now, a recent paper by Isolani and collaborators from the laboratory of Renata Batistoni reports the functional characterization of four matrix metalloproteinases (MMPs) in these animals (http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0055649).
Surprisingly, the silencing of two of these MMPs (mmp1, a zinc-dependent proteinase and mt-mmpA, a membrane-anchored proteinase) results in very severe (leading to lethality) phenotypes affecting normal homeostasis in intact planarians, but without apparently impairing regeneration in a significant way. This is true especially for mmp-1, whereas the knockdown of mt-mmpA delays the normal growth and differentiation of the blastema. The silencing of mmp-1 in non-regenerating animals results in tissue disorganization, breaking of the basal lamina and multilayered epidermis. At the cellular level, and compared to controls, the number of proliferating neoblasts appear to increase at 2-3 days of RNAI but then it goes down to the same level as in controls, whereas there is an increase in the expression of post-mitotic markers. On the other hand there is a significant decrease in apoptotic cell death which the authors interpret as mmp-1 being a positive regulator of apoptosis in planarians. In the case of mt-mmpA its silencing in intact non-regenerating animals also leads them to die. However, here, neoblast proliferation is not affected at any time-point, the expression of post-mitotic markers is reduced and authophagy is significantly increased. From BrdU labeling the authors conclude that mt-mmpA may mediate cell migration during homeostasis.
Further experiments are required to better characterize how the silencing of these MMPs alters the ECM as well as any ECM-cell interaction to explain the severe phenotypes observed. It will be also important to determine which cell types (neoblasts or differentiated cells or both) are involved and why these defects do not appear during regeneration. Still, this work may represent a stimulating starting point to characterize the function of ECM during planarian regeneration and homeostasis.