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Heterogeneity in ependymoglia cells in intact and regenerating newt brain

Even though active neurogenesis in adult mammals is well known and occurs in some regions of our brain, our capacity to replace neurons after an injury or lost is quite limited. In contrast, other vertebrates such as zebrafish and salamanders can regenerate their central nervous system much better. In these animals radial glia like cells (GFAP+) function as neuronal progenitors during regeneration. In the newt brain there are regions (hot spots) in which active neurogenesis is observed during homeostasis in intact animals. However, it is also possible to trigger neuronal regeneration in regions in which neurogenesis is not detected in normal conditions. A recent paper from the laboratory of András Simon (http://www.ncbi.nlm.nih.gov/pubmed/24749074) addresses the heterogeneity of the ependymoglia cells within and outside of the constitutively active niches in the newt telencephalon.

As a first step to isolate neural stem cells (NSCs) the authors tested whether brain cells from different regions were able to form neurospheres in vitro, a typical assay to determine the NSC nature. Neurospheres were indeed formed and included GFAP+ cells that proliferated. Upon media changes cells expressing differentiated neuronal markers were found in those neurospheres indicating that GFPA+ cells have stem cell properties.

In a previous study these authors showed that proliferating ependymoglia cells were mainly localized in hot spots in the newt brain. Here, they addressed the characterization of the heterogeneity of these cells. By analyzing the expression of glutamine synthetase (GS) they could distinguish two different population s of ependymoglia cells. Type 1 GFAP+ cells were positive for GS whereas type 2 GFAP+ cells did not express GS. Type 2 (GS-) cells were found in clusters in hot spots and represent about 32% of the ependymoglia cells. Most of the proliferating ependymoglia cells in hot spots (about 85%) are type 2 cells. The remaining proliferating cells in hot spots (about 15%) are type 1 (GS+). In contrast, in non-hot spots type 2 cells represent only 0,3% of the ependymoglia cells, whereas most of the proliferating cells in these regions are of type 1 (about 90% of the proliferating ependymoglia cells). Thus, type 1 and 2 are found in hot spots but type 2 is practically absent from non-hot spots.

As Notch signaling has a well-known role in the regulation of neural stem and progenitor cells the authors characterized then the expression Notch receptor in these 2 populations of ependymoglia cells in hot spots and non-hot spots. In hot spots their observations are consistent with type 1 cells being GFAP+/GS+/Notch1+ and type 2 GFAP+/GS-/Notch1-. In agreement with this, they found that 94% of proliferating cells in hot spots were Notch1-, whereas most of the proliferating ependymoglia cells in non-hot spots were Notch1+.

Next, as type 2 cells are mainly localized in hot spots the authors hypothesized that they could have a stem cell nature. However, what they found was that a majority of stem cells were in fact type 1 ependymoglia cells. By doing pulse chase experiments with BrdU to detect the long-term label retention characteristic of stem cells together with treatments with AraC (a method to selectively eliminate transit-amplifying cells that divide more frequently than slowly dividing stem cells) the authors concluded that type 1 ependymoglia cells (found in most of the ventricle wall of the telencephalon) have stem cell properties whereas type 2 cells in the hot spots would be transit-amplifying cells.

Then, the authors analyzed how these distinct cell populations responded to the ablation of cholinergic neurons in hot spots and non-hot spots. Upon ablation they found an increase in the proliferation of type 1 and type 2 cells in the hot spots. Remarkably, they also found that in non-hot spots there appeared type 2 cells as well as cells positive for PSA-NCAM, a marker of immature neurons that is not detected in homeostasis in these regions. These last results suggested that neuronal ablation gave rise to the appearance of new neurogenic regions.

Finally, the authors analyzed how interfering with Notch signaling affected the behavior of type 1 and type 2 cells in homeostasis and during regeneration. Upon treatment with the Notch inhibitor DAPT, they concluded that in homeostasis type 1 (stem cell) proliferation is not sensitive to Notch signaling whereas type 2 (transit-amplifying) proliferation is Notch sensitive. During regeneration, type 1 cells in the hot spots are still insensitive to Notch signaling; however, the increase in proliferation of stem cells in non-hot spots is dependent on Notch signaling.

In summary, the authors report here the identification of two distinct subpopulations among the ependymoglia cells in the newt telencephalon: type 1 with stem cell properties and type 2 with transit-amplifying ones. Remarkably, although in the newt telencephalon there are active hot spots, type 1 cells are also found in most of the ventricle wall, which could account for the high neuroregenerative capacity of these animals. In fact, neuronal ablation leads to the appearance of new neurogenic niches in non-hot spots, in which there is an increase in the proliferation of type 1 cells (Notch dependent) as well as the appearance of type 2 and neuronal precursors.

Hedgehog signaling from the notochord during Xenopus tail regeneration

There has been a high level of evolutionary conservation in the function that several signaling pathways play during regeneration. Thus, it is well known the role that pathways such us BMP/TGF-b, Wnt/b-catenin, notch, Hedgehog and RTKs have during the regeneration of different structures in a variety of animals from axolotls and zebrafish to Hydra and planarians. There are some cases in which the ability to regenerate in different species depends on different tissue dependencies. An example is the regeneration of the tail in axolotls and Xenopus tadpoles. In axolotls the spinal cord is necessary for tail regeneration, whereas in Xenopus tadpoles is the notochord and not the spinal cord what is required. In axolotls this dependency seems to be determined by Hedgehog signaling as shh (sonic hedgehog) is exclusively expressed in the spinal cord. Now, a recent paper from the laboratory of Yuka Taniguchi and Makoto Mochii reports that in Xenopus tadpoles shh is expressed in the notochord and required for tail regeneration (http://www.ncbi.nlm.nih.gov/pubmed/24941877).

Whereas in axolotls shh is expressed in the spinal cord during tail regeneration, the authors show here that shh was exclusively expressed in the notochord in the entire regeneration region in Xenopus tadpoles. On the other side, shh receptors patched 1 and 2 (ptc-1 and ptc-2) were expressed in the spinal cord in them. In order to determine the function of Hh signaling on tail regeneration the authors used cyclopamine a widely used inhibitor for Hh signaling. Upon cyclopamine treatment tail regeneration was severely impaired as well as the length of the regenerating notochord. These effects were rescued by the treatment of pumorphamine, an agonist for the Hh pathway. Cyclopamine treatment did not result in an increase in apoptotic cells, but a significant down-regulation of genes related to the Hh pathway such as ptc-1, ptc-2, gli-1 and smo was observed in those treated animals.

During normal tail regeneration, undifferentiated notochord cells accumulate at the distal edge of the amputated notochord by day 2. Then these cells align perpendicular respect to the AP axis and differentiate into cells containing large vacuoles after day 3. In contrast, upon cyclopamine treatment, undifferentiated notochord cells normally accumulated at the edge of the amputated structure and proliferated; however, their posterior alignment and final differentiation was inhibited. Interestingly, cyclopamine treatment impaired the growth of the regenerating spinal cord as well as the formation of myofibers. In fact, the expression of myoD, a well-known myogenic transcription factor, was suppressed upon treatment. Also, the authors observed a strong reduction in the number of cells positive for Pax-7, another myogenic marker. These results suggest a strong dependence of muscle regeneration on Hh signaling, being this in agreement with previous results in mouse and chicken in which shh has a positive effect on the proliferation and differentiation of the satellite cells (muscle stem cells).

Overall, the results presented here uncover a pivotal role of shh in tail regeneration in Xenopus tadpoles as its inhibition impairs regeneration by affecting several processes such as the final differentiation of new notochord cells as well as the proliferation of progenitors for the spinal cord and muscle fibers. Future experiments should determine whether the defects observed here in the differentiation of the notochord are due to a direct autocrine action of shh (as it is expressed in the notochord) or an indirect effect of shh function on spinal cord regeneration.

In summary, this work describes how the differential expression of shh either in the notochord or the spinal cord could account for the different tissue dependency of tail regeneration in Xenopus tadpoles and axolotls, respectively.

EMBO Regeneration Conference 2014

Back from the 2014 EMBO Conference on The Molecular & Cellular Basis of Regeneration & Tissue Repair held at St. Feliu de Guixols. This was an excellent meeting mainly focussed on basic aspects of regeneration in several animal models. The role of ROS signalling and the inflammatory response on wound healing and the triggering of a regenerative response were some of the hottest topics of the meeting, as well as neural and heart regeneration. Also, there were some talks and posters on new (or not so popular yet) models for regeneration such as the cnidarian Nematostella vectensis, several echinoderms and the crustacean Parhyale hawaiensis. It is also clear that large transcriptomic analyses and the enormous amount of data currently generated from RNAseq experiments in all different models and regenerative contexts will help to identify the molecular and cellular bases of key events of regeneration such us wound healing, blastema formation and growth, patterning and polarity, among others. Thus, comparative analyses might uncover novel conserved and taxon-specific elements required for regeneration. I hope we can discuss all this data in the near future in this blog.

EMBO_regeneration_2014

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.

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