regeneration in nature

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Monthly Archives: January 2014

Attenuated RA signaling during Xenopus lens regeneration

Different vertebrate species such as newts, salamanders, fish and Xenopus are capable of regenerating their lenses. In newts and salamanders lens regeneration occurs through transdifferentiation of the pigmented iris epithelium. In newts transdifferentiation occurs from cells in the dorsal epithelium whereas in axolotls, both dorsal and ventral pigmented iris epithelium cells can regenerate the lens. On the other hand, in Xenopus, lenses are regenerated from the cornea. The factors that make the cornea competent for lens regeneration are not very well known yet. Recently, it has been suggested that in Xenopus, lens regeneration may occur not from cornea transdifferentiation but from a population of multipotent corneal stem cells (http://www.ncbi.nlm.nih.gov/pubmed/23274420). Now, a paper also from the laboratory of John Henry reports on the role of retinoic acid (RA) during Xenopus lens regeneration (http://www.ncbi.nlm.nih.gov/pubmed/24384390).

RA plays many important roles during the development and regeneration of a large variety of organs. In newts, RA signaling is required for proper lens regeneration. Here, the authors investigated the role of this signaling in Xenopus. They first analyzed the expression of aldh1a1, aldh1a2 and aldh1a3, required for the synthesis of endogenous RA. They also examined the expression of cyp26a1, cyp26b1 and cyp26c1. CYP26 is an enzyme of the cytochrome P450 family that regulates RA by metabolizing it. They found that all these 6 transcripts, except cyp26c1, were expressed in the cornea of control and regenerating tissues. To analyze the role of RA signaling in Xenopus lens regeneration they cultured lentectomized eyecups in the presence of different agonists and antagonists of this pathway. When they used Liarozole, an antagonist of CYP26, lens regeneration was strongly inhibited. These results suggested that CYP26 activity was required for regeneration pointing out the possibility that RA levels were needed to be reduced for a successful regeneration. This was further supported when they used TTNPB, an RA analog that is resistant to CYP26. Treatment with TTNPB also inhibited lens regeneration. Finally, the exogenous application of RA at a 20mm concentration also impaired lens regeneration (lower doses of exogenous RA did not affect regeneration).

Cyp26 genes are upregulated in response to RA. Therefore, to check whether Liarozole and TTNPB affected RA signaling they checked the expression of cyp26a1 in drug-treated tissues. In all cases the expression of cyp26a1 was strongly upregulated suggesting an activation of RA signaling after those treatments that would correlate with the inhibition of lens regeneration.

However, the application of exogenous RA at 1mm also induced cyp26a1 expression without inhibiting regeneration. Therefore, cyp26a1 over-expression can be seen as a readout of RA activity but by itself would not be the responsible of the inhibition of lens regeneration. Next, the authors used inhibitors of RA signaling and they observed how their application did not inhibit lens regeneration, further supporting the idea that RA signaling does not seem to be required for regeneration.

Finally, the authors checked that treatment with Liarozole reduced cell proliferation but TTNPB and exogenous RA did not have a significant impact on cell proliferation, suggesting that Liarozole effects on cell division may not result from increased RA levels. Also, the authors checked the expression of three putative corneal stem cell markers (sox2, oct60 and p63). What they saw was that Liarozole treatment, although inhibiting lens regeneration, did not affect the expression of these genes. On the other hand, exogenous RA at 1mm did not inhibit regeneration but altered the expression of these three genes. Therefore, the fact that RA activity appears to inhibit lens regeneration does not seem to be related to the expression of these genes.

In summary, although further studies are required to better understand the exact function of RA signaling on lens regeneration in Xenopus it seems clear that in contrast to what happens in newts, RA activity needs to be attenuated for a successful regeneration in these frogs.

Regulation of stem cell mRNAs during planarian regeneration and homeostasis

Freshwater planarians are truly amazing animals as they can regenerate the whole body from a tiny piece of them based on the presence of a unique population of adult pluripotent somatic stem cells. These cells, named neoblasts, are the only proliferative cells and the source of all regenerative cells. In recent years, several genes have been reported to be necessary for proper neoblast function and properties such as proliferation and self-renewal. However, we are still far to fully understand how gene expression is regulated in these stem cells. Now a recent paper from the laboratories of Aziz Aboobaker and Nikolaus Rajewsky reports on a post-transcriptional regulatory mechanism that would control neoblast differentiation during planarian regeneration and homeostasis (http://www.ncbi.nlm.nih.gov/pubmed/24367277).

mRNA degradation is an important process to control gene expression at a post-transcriptional level. A first step during mRNA degradation is deadenylation (shortening of the poly(A) tail) mediated by the CCR4-NOT complex. Little is know, however, about the function of this complex and deadenylation itself in stem cell regulation. Therefore, the authors analysed the function of Smed-not1, a planarian homologue of Not1, a central protein of the CCR4-NOT complex. Smed-not1 is expressed in neoblasts, their postmitotic progeny as well as in differentiated cells of the central nervous system (CNS). Upon the silencing of Smed-not1 the animals were able to start the regenerative process and made small blastemas; however, after few days these blastemas stopped growing, did not differentiate, regressed and finally all the planarians died. A similar observation was found in intact non-regenerating animals: Smed-not1 RNAi lead to head regression starting after 15-20 days and, eventually, all treated planarians died. Because this phenotype of head regression and lethality had been previously associated to depletion of the neoblast population the authors checked first cell proliferation after silencing Smed-not1 in intact animals. Remarkably, cell proliferation was not significantly affected in these animals, suggesting that the primary function of Smed-not1 could be related to neoblast differentiation more than to neoblast maintenance and proliferation.

Next, the authors analysed in more detail the neoblasts and their progeny after silencing Smed-not1. Normal numbers of Smedwi-1 positive cells (neoblasts) were found up to 15 days after RNAi, whereas a significant decrease was apparent by day 20. Smed-nb21.11e cells (early progeny) were normally present by day 10 but started to decline in number after day 15. Finally, Smed-agat-1 cells (late progeny) increased by day 10 and from that point started to decline in number. By day 20 a significant reduction in the presence of these cells was observed. Overall, these results somehow support the idea that the silencing of Smed-not1 primarily affected cell differentiation rather than neoblast maintenance, as changes in the proper number of neoblast progeny and a visible head regression preceded the decrease of neoblasts. An interesting and puzzling result was obtained when the authors quantified the levels of the transcripts of these genes after Smed-not1 RNAi. Thus, for Smed-nb21.11e the levels of mRNA were the same as controls after 15 days, despite the reduced number of positive cells observed by in situ hybridizations. On the other hand, the levels of Smed-agat-1 mRNA in Smed-not-1(RNAi) animals were almost two-fold higher than in controls after 15 days, in huge contrast with the observation that these animals had similar numbers of Smed-agat-1 cells than controls. The authors concluded that Smed-agat-1 transcripts were accumulated in a decreasing number of Smed-agat-1 cells.

As the CCR4-NOT complex regulates deadenylation previous to mRNA degradation one possibility could be that the increase in the levels of mRNA observed after Smed-not1 RNAi could be due to an impaired deadenylation of such transcripts. If this is true then, an increased frequency of poly(A) tail lengths should be observed.  And this is exactly what the authors found for Smed-agat-1 and Smed-nb21-11e after Smed-not1 RNAi. Then, they performed similar experiments for the neoblast markers Smedwi-1, Smedtud-1, Smed-vasa-1 and Smed-pcna and in all cases they found an increased in the average length of their poly(A) tails together with increased transcript levels. Importantly, these changes were not observed for genes expressed specifically in differentiated cells.

As Smedtud-1 and Smed-vasa-1 are expressed also in the CNS the authors wondered whether the increased in their mRNAs levels after Smed-not1 was found in all the cells expressing these genes or only in the neoblasts or the CNS. To solve this question they combined RNAi with irradiation, as it is well known that 24 hours after a certain dose of irradiation all neoblasts are effectively eliminated.  The rationale here was that if Smed-not1 action on deadenylation was limited to the transcripts present in the neoblasts, then after irradiation no differences should be observed between control and Smed-not1 RNAi animals in terms of both transcript levels and poly(A) length. And this is precisely what they observed, confirming that the effects of Smed-not1 on the mRNAs of these genes were confined to neoblasts.

In summary, the results presented here offer novel insights into the post-transcriptional regulation of neoblasts. As the authors propose, after Smed-not1 RNAi the transcripts of several genes important for neoblast function would accumulate without being degraded and subsequently preventing somehow neoblast differentiation. Further studies will help to better characterize the function of the CCR4-NOT complex in regulating stem cells not only in planarian but also in other animal models.

One year of “Regeneration in nature”

Just one year ago I started this blog on regeneration in nature. After 20 years working on planarian regeneration (since I was an undergraduate in 1993), writing papers, attending to meetings and fulfilling my academic obligations (at both levels, teaching and administrative, that since 2006) I felt myself with the need to do something different. The idea of writing a blog had been in my mind since long ago; however, I was undecided on what to write about: science, politics, social issues or personal thoughts (whatever that means). At the end, I decided to write about regeneration because on one side, the study of regeneration has made me a scientist and, on the other, it has offered me the opportunity to go through important vital experiences such as living in Okayama (Japan) and Urbana (USA) which have influenced somehow how I am nowadays. So, in many aspects, and as it happens to most of us, what we do influences on how we are and, the other way around, how we are influences on what we do. And at some point it is difficult to establish what is a cause and what is a consequence. Therefore, I felt in debt with regeneration so I decided to start this blog to share my passion for this biological phenomenon with as many people as possible. One year later I feel really happy with this blog. As many of you know I post my comments on Thursdays and I must say that many weeks I am really eager that that day arrives.

But a blog is nothing without its readers so I would really like to thank all of you that regularly visit this blog (80 people are already subscribed and therefore receive an automatic e-mail every time a new post is published) or just hit it by chance and stay few minutes reading some of the posts. And I really appreciate readers such as Oné Pagan (follow his blog at http://baldscientist.wordpress.com) and Jaume Baguñà for their often comments to my posts. I hope that you keep visiting this blog, enjoying the posts, learning about regeneration and, if you have few minutes do not be shy and just comment on any of the posts you like or you do not like. It is only with your help and implication that this blog can become a two-direction or multi-direction communication and discussion channel.

Here you can see some stats of the 12,383 views that this blog has had during its first year.

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Thank you very much again and as Oné often says in his blog: “Stay tuned”.

Proximodistal specification during axolotl limb regeneration

An important question during limb regeneration is how the identity along the proximodistal axis is re-specified. An often-assumed model mainly based on grafting experiments suggested that in axolotls this re-specification occurred mainly by cell intercalation. Under this model the first blastema cells that appear acquire the most distal identity and then the missing identities intercalate between the stump and this most distal part. This intercalation is observed, for instance, when grafting distal leg fragments to more proximal ones in insects, or grafting a head and tail planarian pieces, or grafting a distal limb blastema to an upper arm stump in amphibians. But the question that remains is whether this intercalation model is also followed during normal regeneration.

Now, a recent paper from the laboratory of Elly Tanaka indicates that during axolotl limb regeneration there is a proximal-to-distal sequence of segment specification (http://www.ncbi.nlm.nih.gov/pubmed/24337297). During axolotl limb development (and other vertebrates) HoxA9, HoxA11 and HoxA13 are expressed in a spatial and temporal sequence. HoxA9 is first expressed in the limb bud before HoxA11 and HoxA13. Then, HoxA11 is expressed with a more distal expression boundary compared to HoxA9, whereas HoxA13 is expressed at the most distal part. These results suggest that progenitor cells are specified in a proximal-to-distal sequence. So, here the authors sought to determine whether this spatial and temporal colinearity for HOXA proteins was also observed during regeneration. They used axolotls in which the connective tissue was labelled with GFP. At day 1 of regeneration no blastema cells (derived from the GFP positive connective tissue) expressed any of the HOXA proteins. At day 6 (early-bud stage) HOXA9 and HOXA11 but not HOXA13 were expressed in the blastema. Between days 8 and 12 (medium- and late-bud stages) a nested expression pattern was observed with HOXA9 found broadly in the blastema, HOXA11 in a medial band and HOXA13 at the distal tip. Thus, these results indicate that during regeneration HOXA proteins are expressed colinearly, as it happens during limb development.

The association of HOXA13 expression with hand identity was further confirmed by: i) the observation that after hand amputation HOXA13 was expressed in the blastema cells at 4 days well before the onset of HOXA13 expression in an upper arm blastema, and ii) the fact that treatment with retinoic acid (that convert hand blastemas into upper arm blastemas) silenced the expression HOXA13 at 4 and 6 days after hand amputation. Finally, the authors performed a series of transplantation experiments to assess the order of blastema cell specification. In all those experiments the host was always an unlabelled 6-day upper arm blastema (a stage in which HOXA13 is not yet expressed). The donor cells were blastema cells derived from GFP positive connective cells from different stages and amputation levels. When 8-day hand blastema cells were transplanted into the donor they only contributed to hand structures (as expected based on the rule of distal transformation in which blastema cells derived from connective tissue can only form segments more distal to their original identity). When 8-day upper arm blastema cells from the distal tip were transplanted they contributed also only to hand structures, indicating that at 8 days the transplanted distal blastema cells were already committed to hand identity. On the other side, when 8-day proximal upper arm blastema cells were transplanted they contributed to lower arm and hand structures, corresponding to the HOXA9+HOXA13- upper-arm progenitor cell potential. Finally, when the most distal tip cells of 4-day upper arm blastemas (a stage at which they do not express yet HOXA13) were transplanted they contributed to lower arm and hand in the regenerated limb indicating that those transplanted cells had not been committed yet to hand-identity.

Overall these transplantations indicate that blastema cells are specified in a proximal-to-distal sequence during normal regeneration similarly to what happens during embryonic development.

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