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Transdifferentiation during zebrafish heart regeneration

Because of the high incidence of deaths and diseases related to heart failure may research is being done to try to promote the differentiation of new cardiomyocytes in the human heart. However, and as it happens for many other vital organs, humans are quite bad regenerators.  However, at foetal stages mammals conserve some degree of heart regeneration. In fact, newborn mice are able to regenerate their heart if a part of it gets amputated during the first week. After that initial period the capacity of heart regeneration gets lost. Other vertebrates, such as zebrafish are able to regenerate their hearts as adults. In those animals what it has been reported is that upon amputation cardiomyocytes are able to dedifferentiate into a proliferative stage, divide and re-differentiate again into new cardiomyocytes.

A recent paper from the laboratory of Neil C. Chi has reported that in zebrafish embryos atrial cardiomyocytes can transdifferentiate into ventricular cardiomyocytes to contribute to successful heart regeneration (http://www.ncbi.nlm.nih.gov/pubmed/23783515). Specifically, the authors addressed the role of atrial cardiomyocytes that has been shown to divide in adult vertebrate hearts after a ventricular injury. The authors used a series of fluorescence reporter transgenes to distinctively label atrial and ventricular cardiomyocytes as well as a specific cell-ablation system to ablate ventricular cardiomyocytes from zebrafish embryos and follow their regeneration.

By doing it, the authors described how after cell ablation, pre-existing atrial cardiomyocytes migrate to the ventricle and transdifferentiate into ventricular cardiomyocytes. Along this pathway atrial cardiomyocytes appear to go through an intermediate progenitor-like stage. In addition to characterizing this transdifferentiation event at the molecular (gene expression) level the authors also reported that electrophysiological recordings showed how the newly transdifferentiated ventricular cardiomyocytes exhibited electrical features similar to endogenous ventricular cardiomyocytes (and distinct to those from atrial cardiomyocytes).

It has been previously suggested that endocardial activation is required for heart regeneration. Here, the authors showed how Notch signalling is activated in atrial endocardial cells upon ventricular ablation. However, no evidence of reprogramming of those endocardial cells into cardiomyocytes was observed.  Remarkably, DAPT-mediated inhibition of Notch signalling resulted in impaired ventricular regeneration with a reduced migration of atrial cardiomyocytes into the injured ventricle. Thus, these results open the possibility that Notch signalling may be required to regulate heart regeneration through a non-cell autonomous mechanism.

In summary, this paper shows how a ventricular injury is able to induce in vivo the reprogramming of atrial cardiomyocytes into a progenitor-like stage from which ventricular cardiomyocytes differentiate. One of the issues to analyse in the future is whether these transdifferentiation potential is age-dependent because when ventricular ablations were done in adult zebrafish the authors found a decrease in the number of transdifferentiated atrial cardiomyocytes that contribute to ventricular regeneration. Also, future analyses should determine whether atrial cells in mammals retain this transdifferentiation potential to provide a cellular source during repair and regeneration.

Notch signalling in Hydra head regeneration

Hydra is one of the most basal animals and a classical model of regeneration. These polyps display an amazing plasticity as they can regenerate a whole animal from a tiny piece of their bodies, can reproduce asexually by budding and, amazingly, dissociated cells can re-aggregate to form a new individual. Hydra has a well-established oral-aboral axis. In the head (oral) region there is a hypostome and a ring of tentacles. Many studies have reported that the tip of the head has an organizing activity. Thus, if this head tip is transplanted into the body column it is able to induce a new head and a secondary axis. This organizing activity appears to be controlled by the Wnt/Beta-catenin pathway. HyWnt3 is expressed at the tip of the head in intact and regenerating animals. Ectopic induction of Wnt signalling in the body column induces secondary axes all along it.

In a recent paper from the laboratory of Angelika Böttger the authors describe how Notch signalling plays a key role in maintaining and re-establishing the Hydra head (http://www.ncbi.nlm.nih.gov/pubmed/24012879). Notch signalling is a well-known and highly conserved pathway that plays a role, among other, in establishing well-defined boundaries as it can specify different cell fates in two adjacent cells. Once Notch transmembrane receptor is bound by a ligand the intracellular domain of the Notch receptor (NICD) is, then, cleaved and can go to the nucleus where it works as a co-activator of target genes. DAPT is a well-known inhibitor of the Notch pathway as it prevents cleavage of the active NICD and it is usually used in different animal models.

Upon culturing intact Hydras in DAPT for 48h the authors saw how the tentacles were shortened (beginning after 24h of treatment). After 48h of treatment DAPT was removed and in the following days these polyps displayed different levels of abnormalities in the patterning of their tentacles and heads, and even a new secondary hypostome appeared in some of them.  At the molecular level, the expression pattern of HyWnt3 at the tip of the hypostome was not affected by DAPT treatment. However, the expression of HyAlx (a marker or tentacle boundary) was severely affected. Previous studies had suggested that HyAlx might play a role in the specification of tissue for the formation of the tentacles. The results presented here support this view and point out Notch signalling as required for proper HyAlx expression and, therefore, differentiation of tentacles.

Next, the authors analysed the function of Notch signalling during head regeneration. In most of their experiments the animals were treated with DAPT for 24h prior to amputation. After 24h of regeneration in DAPT, this inhibitor was then removed from the medium and the animals were analyzed at different time points of further regeneration. After 60h only 18% of the animals had regenerated their heads. This lack of head regeneration can be explained because of the lack of re-establishment of a new head organizer. Thus, whereas in controls HyWnt3 is initially expressed in the regenerating tip in a broad cap-like pattern and then it becomes restricted to the tip of the new hypostome (around 36hpa), DAPT prevented the expression of HyWnt3 for up to 48h of regeneration. A similar result was observed for HyB-catenin. To check that DAPT really inhibits the formation of a head organizer the authors transplanted 24h regenerating tips of DAPT treated animals into the body column of host polyps. Only in 25% of the transplants a secondary axis developed in sharp contrast to the 100% of secondary axes observed when transplanting the regenerating tips of control animals.

Finally, the authors checked the expression of HyAlx during regeneration. In controls HyAlx is initially expressed in the whole regenerating tip (at 24h). At 48h this gene gets restricted to 4 or 5 rings at the sites where the tentacles will emerge. In DAPT-treated polyps, the initial expression of HyAlx appears normal but it never gets confined to 4 or 5 rings and it remains in the whole regenerating tip and gets expanded, suggesting that the whole tip had characteristics of tentacle precursors. Finally, the authors checked that upon DAPT treatment the expression of HyNotch in the regenerating tip is not affected but that of HyHes, a target of Notch signalling is abrogated.

Whereas in intact Hydra the authors suggest that Notch signalling is required to define the tentacle boundary, during regeneration Notch would be required to separate tentacle and hypostome cell fate. In their model, the Notch signal-receiving cells suppress the tentacle fate and become hypostomal cells by stabilizing the expression of HyWnt3 and HyB-catenin. Consequently, the inhibition of Notch signalling would allow the observed increased in tentacle cell fate and the lack of the Wnt/beta-catenin-mediated re-establishment of the head organizer.

Positional information in planarian muscle

In my las post before the summer break I commented on three papers that had reported how the silencing of a single gene, B-catenin1, was able to rescue head regeneration in three different species of planarians that usually do not regenerate their heads, when amputated post-pharyngeally. Now, in this first post after the holidays I go back to planarians to comment on the recent findings by the laboratory of Peter Reddien on positional information (http://www.ncbi.nlm.nih.gov/pubmed/23954785). As the authors state in their paper, during regeneration, in addition to new cells required for rebuilding the missing structures, these cells must obey very strict instructions in order to be able to form the proper tissues and organs in the appropriate territories. In this sense, no much is known about how positional identities are maintained and re-established during planarian regeneration. Previous studies from the laboratory of Kiyokazu Agata had suggested that such positional information resided in differentiated cells (http://www.ncbi.nlm.nih.gov/pubmed/11319861). Now, the new data from the Reddien’s lab points out that are the muscle cells that would provide such instructions during regeneration.

 First, the authors define the position control genes (PCGs), as genes that (i) display regionalized expression along one or more body axes, and (ii) either their RNAI-mediated silencing results in a patterning defect or encode a protein related to the Wnt, BMP or FGF signalling pathways that are involved in is patterning. These PCGs include: notum, sfrp-1,sfrp-2, several wnts, fz-4, prep, ndk, ndl-3, ndl-4, netrin-2, bmp, admp, ngl-7, ngl-8, nog-1, nog-2 and tolloid. The expression patterns and functions of these genes have been reported in recent years. Interestingly, all these PCGs are expressed in a population of uncharacterized subepidermal differentiated cells. So, the authors wondered whether those cells could represent the source of positional information in planarians.

By doing in situ hybridization with multiple combinations of all the PCGs they found first that many of them co-localized in those subepidermal cells. Next, they found that subepidermal muscle cells that co-expressed collagen, troponin and tropomyosin displayed a similar distribution of those expressing PCGs. Remarkably, every PCG tested was co-expressed with collagen or troponin, suggesting that muscle cells may provide instructive signalling during planarian regeneration. Quantitatively, between 95,7% and 99,8% of all muscle cells analyzed from different body regions co-express PCGs. Although most PCGs are expressed in the subepidermal body wall musculature, others are also expressed in the muscle cells that surround the digestive system or the pharyngeal muscle.

 Finally, the authors analyzed whether the expression of these PCGs was regulated in muscle cells after amputation. Thus, for example, the polarity determinants notum and wnt1 are rapidly induced after amputation in muscle cells. Importantly, this expression occurs in irradiated (neoblast-depleted) animals, suggesting that the existing muscle cells are able to dynamically change the expression of PCGs in them as response to amputation. Moreover, muscle cells are able to re-adjust the profile of the PCGs that express to the one corresponding to the new region along the body axes in which they are placed after amputation.

 Overall the authors propose a model in which changes in the expression of PCGs in muscle cells at the wound regions would influence neoblast cell fate according to their new positions. In the future it would be interesting to test this model by trying to analyze the regenerative capabilities of muscle-deficient planarians (if that is really possible). 

Back to blogging

Dear all

I hope you all have had nice holidays. After a long summer break I am ready to continue my blogging activity on animal regeneration. As I have been doing in the last several months I will publish a new post every Thursday, starting this week. I hope you will enjoy them. And please, feel free to comment on any aspect of them in order that we all can promote some helpful and interesting discussions.

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