regeneration in nature

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Monthly Archives: February 2014

The role of the Hippo pathway in Xenopus limb bud regeneration

As I have discussed before in this blog, the Hippo signalling pathway has a conserved function in controlling organ size and patterning through the regulation of the balance between cell proliferation and cell death. During regeneration these processes must be tightly regulated so the regenerated organs and structures attain proper sizes. However, not much is known yet about the role of Hippo signalling during regeneration. Previous reports have shown that this pathway is required for proper insect leg as well as Macrostomum (flatworm) regeneration. In fact, for Macrostomum, I discussed those results in this blog (https://regenerationinnature.wordpress.com/2013/06/27/the-hippo-pathway-in-macrostomum-homeostasis-and-regeneration/).

Now a recent paper from the laboratory of Hitoshi Yokoyama reports a role for Yap1 during regeneration of the Xenopus limb bud (http://www.ncbi.nlm.nih.gov/pubmed/24491818). The transcriptional co-factor Yap1 (Yorkie in invertebrates) is the canonical effector of the Hippo signalling pathway. When the Hippo pathway is activated, Yap1 gets phosphorylated and becomes inactive as it gets trapped in the cytoplasm. On the other side, active dephosphorylated Yap1 can enter the nucleus where it activates the expression of its target genes. In other systems it has been shown how the over activation of Yap1 (or Yorkie) leads to an increase of proliferation and a decrease of apoptosis. Conversely, the inactivation of Yap1 results in decreased cell proliferation and higher levels of apoptosis.

In this study, the authors analysed the regeneration of amputated limb buds of Xenopus tadpoles at stage 52. In a first set of experiments they found that all the main core components of the Hippo pathway, including some regulators and target genes, were expressed in 5-day blastemas. As Yap1 is the effector of the pathway they focussed on this gene. In situ hybridizations showed that Yap1 was upregulated within the blastema. Taking advantage of the cross-reaction of an antibody against human Yap1 they could see how in intact limb buds Yap1 was in the cytoplasm whereas, upon amputation, Yap1 was localized in the nuclei of blastema cells. These results suggested that Yap1 was translocated to the nuclei during regeneration where it could activate its target genes. Next, the authors characterized the function of Yap1 during regeneration by using a dominant-negative form of this gene (dnYap) under control of a heat shock promoter. Overexpression of dnYap resulted in impaired limb regeneration characterized by a reduction in the size and number of skeletal elements, as well as a reduction in the number of digits in those regenerated limbs. Interestingly, the overexpression of dnYap did not apparently affect the normal development of the uncut contralateral limb bud.

In addition to these external defects, the authors checked the expression of several patterning genes with well-defined and region-specific patterns. The overexpression of dnYap impaired the expression of hoxa13 and hoxa11 so both genes partially overlapped in contrast to their expression in well-separated domains in controls. Also, the expression of shh, fgf8 and mkp3 was clearly downregulated. Moreover, the small blastemas of dnYap animals contained much less differentiated muscle and innervation. As the Hippo pathway controls cell proliferation and apoptosis the authors checked how these events were affected after overexpressing dnYap. The number of mitotic cells significantly decreased within the blastema as well as in the stump. On the other side, ectopic apoptotic cells were found the stump. These results suggested that the impaired regeneration phenotype was associated to missregulation of cell proliferation and apoptosis. Again, the overexpression of dnYap did not affect cell proliferation, apoptosis or differentiation in uncut limb buds and they developed into normal limbs.

In summary, this paper shows how Yap1 is required for limb bud regeneration in Xenopus. In this context Yap1 appears to have a conserved role in the regulation of cell proliferation and apoptosis. Future experiments should determine whether other elements of this pathway as well as its upstream regulators are equally conserved between vertebrates and invertebrates and are also required for regeneration.

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.

A novel (and unknown) mechanism for dendrite regeneration in Drosophila

A successful functional regeneration of the nervous system could have a major social impact. Unfortunately, most animals, including mammals, do not regenerate well the central nervous system. However, some regenerative abilities are seen in the peripheral nervous system of flies, nematodes and mammals. After axon injury a cascade of signals travels from the injury site to the cell body triggering a regenerative response that results in axonal regrowth. An important aspect that is mainly unknown is whether dendrites (also quite sensitive to different damage situations) are able to respond to injury as axons do. Now, a paper from the laboratory of Melissa Rolls reports that, in Drosophila, dendrites can efficiently regenerate and do it through an unknown molecular mechanism different from the one used by axons (http://www.ncbi.nlm.nih.gov/pubmed/24412365).

            Previous studies had suggested that dendrites regenerated only in certain neurons at specific developmental stages. Here, the authors used a pulsed UV laser to remove all dendrites from different types of dendritic arborization (da) neurons. First, they removed all the dendrites from larval ddaE neurons. After 48h a newly regenerated branched dendrite arbor was found in almost half of the samples. Although the final area of the body wall covered by the regenerated dendrites was smaller than in controls, the complexity of those regenerated dendrite arbors in terms of the number of dendrite branch points was equivalent to controls. Next, they checked ddaC neurons. Similarly to what was observed for ddaE neurons, after 24h some processes started to grow. After 96h regeneration was completed and the new dendritic arbors covered the same area of the body wall as in controls.

In order to see if these regrown processes were real dendrites the authors followed several approaches. First, they checked microtubule polarity in those regrown neurites.  Whereas axons contain plus-end-out microtubules, dendrites are distinguished by the presence of minus-end-out microtubules. This has been observed in Drosophila, C. elegans and mammals. After 48h of regeneration the neurites regrown from severed ddaC neurons contained minus-end-out microtubules, suggesting that those neurites were real dendrites. Additionally, the authors used an Apc2-GFP marker, specific for dendrite branch points and found that this marker was localized in the regrown processes. In a final experiment the authors checked the effect of dynein on dendrite regeneration. It is known that dynein is required for the development of dendrites. Consequently, and as predicted, dynein RNAi affected dendrite regeneration. Taking in account all these results the authors concluded that after dendrite removal these different neurons were able to regenerate new dendrites. Moreover, and importantly, the authors also show how dendrite regeneration was successful not only at early larval stages but also in late larval and adult neurons.

After axonal injury an important player to trigger a regenerative response is DLK, a dual leucine zipper kinase. In several models (flies, nematodes, mammals) DLK (a MPAKKK) is activated after injury and subsequently activates cJun N-terminal kinase (JNK), p38 and the transcription factor fos. In order to see if this conserved signalling cascade required for axonal regeneration played a role also during dendrite regeneration the authors used RNAi and mutants to inhibit the levels of DLK in their model. Surprisingly, whereas axonal regeneration was impaired as expected, dendrite regeneration was not affected in those same da neurons. Finally, the authors show how dominant-negative forms of JNK or fos did not block dendrite regeneration. Thus, DLK/JNK pathway was not required for dendrite regeneration. Remarkably, this pathway was not even activated after dendrite removal.

In summary, this paper shows how dendrite from different neuronal types can regenerate in both larva and adult flies. This dendrite regeneration takes place using a novel unknown mechanism different from the conserved pathway activated during axonal regeneration in different models. Future experiments should try to determine how dendrite regeneration is triggered at the molecular level and whether this mechanism has been conserved in other animals.      

A novel model uncovers striking similarities during limb regeneration between arthropods and vertebrates

In previous posts I have mentioned the importance of studying how regeneration takes place at both cellular and molecular levels in a large variety of animals. This will help us not only to understand how the process of regeneration itself occurs in different animals but will provide us with basic data for comparative analyses that can unravel common and specific regenerative strategies throughout phylogeny.

The laboratory of Michalis Averof reports now on the regeneration capacities of the crustacean Parhyale hawaiensis and shows striking similarities during limb regeneration in these animals compared to vertebrates (http://www.ncbi.nlm.nih.gov/pubmed/24385602). Parhyale can regenerate all their appendages throughout their lifetime. Here, the authors used morphological, cellular and genetic markers to describe limb regeneration. After amputation, wound closure takes place within a day. Then a blastema consisting of proliferative cells is formed around day 2 and 3. By day 4-6 the distal tip of the regenerated limb is visible by the expression of Distal. Finally, the muscles regenerate within a week from moulting.

An important question that the authors wanted to address here was about the origin of the regenerative cells in Parhyale. In order to distinguish between pluripotent and lineage-restricted progenitor cells they marked different cell lineages (at the 8-blastomere stage) and followed their contribution during limb regeneration in adults. At this stage, 3 blastomeres (El, Er and Ep) are fated to produce the ectoderm, 3 more (ml, mr, Mav) to mesoderm, 1 (en) to endoderm and 1 (g) to the germline. They injected those embryos with a transposon carrying a fluorescence marker driven by a ubiquitous promoter activated after heat-shock. After injecting about 4,000 embryos they got 79 individuals in which specific lineages were labelled. Limbs from these animals were then amputated and allowed to regenerate. Remarkably, descendants of blastomeres El, Er and Ep gave rise exclusively to ectodermal derivatives (epidermis and neurons) whereas descendants of ml, mr and Mav gave rise only to muscle. No contributions from the endoderm or germline lineages were found in the regenerated limb. Moreover none labelled lineage contributed to both ectodermal and mesodermal derivatives suggesting that neither pluripotent progenitors nor trans-differentiation across ectoderm and mesoderm appears to occur in Parhyale. Therefore, and similarly to what happens in the axolotl limb, in Parhyale, new regenerated ectodermal cells appear to originate from the pre-existing ectodermal lineage whereas new mesodermal cells originate from the mesodermal one. Further experiments should determine whether these lineage-restricted progenitor cells for regeneration derive from stem cells or differentiated cells that re-enter the cell cycle.

Because the authors were able to specifically label ectodermal and mesodermal lineages of the left or right sides of the body, they found out that the descendants of blastomeres El and ml, for instance, contribute to the regeneration of the appendages of the left side (descendants of Er and mr contribute only to regeneration in the right side). Thus, regenerative cells have a local origin respect to the amputated limb. Finally, the authors found some cells closely associated to the muscle fibers that reminded the satellite cells in vertebrates. Satellite cells are stem cells for muscle regeneration and are recognized by the expression of Pax3/7. Interestingly, these cells in Parhyale also expressed Pax3/7 and had a mesodermal origin. In order to study the function of these satellite-like cells (SLCs) the authors transplanted individual labelled SLCs from the limbs of transgenic animals into the amputated limbs of control animals and found out that in some cases these GFP-labelled cells gave rise to regenerated muscle fibers. Thus, SLCs might function as muscle progenitor cells.

In summary, this study introduces Parhyale as a regeneration model and points out several similarities between crustacean and vertebrate limb regeneration such as lineage-restricted progenitor cells, local origin of the regenerative cells and the presence of Pax3/7 positive muscle progenitors.

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