Contrary to mammals, zebrafish can regenerate their hearts after a significant loss of their cardiomyocytes. Taking into account the high incidence of myocardial infarction in humans understanding heart regeneration in zebrafish might provide clues to enhance our limited regenerative abilities. Now, a recent paper from the laboratory of Ken Poss reports on the important role of neuregulin1 (nrg1) t o induce the endogenous heart regeneration program in zebrafish (http://www.ncbi.nlm.nih.gov/pubmed/25830562).
Nrg1 had been previously identified as having multiple roles in cardiovascular biology in both mammals and zebrafish, although some contradictory reports existed respect to the role of nrg1 on cardiomyocytes proliferation. Therefore, in this paper, the authors addressed the role of nrg1 on the strong regenerative response of the zebrafish heart. Whereas nrg1 expression was rarely detected in uninjured hearts, after the genetic ablation of about 50% of the cardiomyocytes there was a significant upregulation of nrg1 that peaked at 7 days post amputation (dpa), a stage in which cardiomyocytes proliferation also peaks. Double stainings determined that most nrg1 positive cells in the postnatal ventricular wall came from the tcf21+ epicardial derived perivascular cell compartment. Rare overlap of nrg1 and markers of vascular endothelial cells and cardiomyocytes was observed.
Nrg1 signals through the Erbb2 and Erbb4b receptors. The pharmacological inhibition of these receptors decreased cardiomyocytes proliferation during regeneration. On the other hand, gain-of-function experiments showed that overexpressing nrg1 in cardiomyocytes resulted in a strong increase of cardiomyocyte proliferation near the injury site, clearly suggesting an important role of nrg1 signalling on cardiomyocyte proliferation during heart regeneration. Next, the authors sought to determine the effects of overexpressing nrg1 in uninjured hearts. After 7 days of treatment (dpt) they observed a marked increase in cardiomyocyte proliferation especially within the ventricular wall. Over time, this increased proliferation resulted in obvious changes in cardiac anatomy. Thus, the ventricular wall thickened by 76% at 7 dpt, by 265% at 14 dpt and by 459% after 30 dpt. Measurements of cardiomyocyte cell number and size suggested that this enormous thickening of the ventricular wall was mainly due to an increase in cell number. The authors referred to it as nrg1-induced cardiac hyperplasia (iCH). Next, the authors analysed the long-term effects of iCH on uninjured adult hearts. On average after 6 months of treatment those hearts showed a 2.1-fold greater ventricular section area. An increased cardiomyocyte proliferation was still evident at this late stage although the authors also observed some mild fibrin and collagen deposition in some regions of the ventricular wall that were not seen just after 30 dpt.
By measuring physiological parameters and cardiac function the authors did not find any cardiac dysfunction at 3 months of iCH. However, at 8-9 months the animals showed a reduction in swimming endurance, a test based on measuring the ability of the fish to swim against increasing water currents, that is an assay of cardiac function sensitive to cardiac damage or heart failure. Therefore, these results indicated a deleterious effect of nrg1 continuous overexpression over a long period of time.
In a last set of experiments, the authors investigated whether iCH depended on similar mechanisms to those triggered by injury-induced regeneration. During normal development the cortical muscle in the ventricular wall form from a small number of large cardiomyocyte clones with discernable boundaries between them. In contrast, during regeneration there is an increase of cardiomyocyte proliferation at the injury site that generates a mixed conglomeration of small clones in the regenerated ventricular wall. The authors observed that the thickening of the ventricular wall during iCH in uninjured hearts contained mixed small clones something reminiscent of an injury-induced regenerative response. When analysing the molecular signatures of iCH the authors first saw an upregulation of gata4 (an embryonic cardiogenic transcription factor) in the outermost layer of cortical muscle at 7dpt. Previous results had shown that after apex amputation gata4 is upregulated in cardiomyocytes near the injury and is essential for regeneration. Moreover, they observed that after iCH there was a reduction of cmlc2 (cardiomyocyte marker) expression in the cortical muscle and a sarcomere disorganization, which would resemble the dedifferentiation process that occurs during regeneration. Another genes implicated in regeneration, such as tgfb3, fn1 (fibronectin synthesis) and raldh2 (retinoic acid signalling), were also upregulated after iCH. Finally, iCH also induced the formation of a major vascular network throughout the expanded ventricular wall. Overall, all these results suggest that nrg1 is sufficient to induce and maintain a global heart regeneration program involving several cell types.
In summary, the authors report here nrg1 as a potent activator of the heart regeneration program in zebrafish. In the future, it will important to determine how is nrg1 upregulated during regeneration as well as its downstream targets.
An important aspect of stem cell biology is to determine the genes responsible for the maintenance of the different stem cell populations as well as to understand both how they are regulated and how they exert their function. Now, a paper from the laboratories of Ian C. Scott and Bret J. Pearson reports on the identification of the Mediator subunit Med14 as an important regulator of stem cell maintenance in zebrafish and planarians (http://www.ncbi.nlm.nih.gov/pubmed/25772472).
The Mediator complex was first identified in yeasts and its core consists of three modules (head, middle and tail) with an additional kinase module present sometimes. Because in yeast Mediator is located at the promoters of nearly all protein coding genes it has been suggested that it may be part of the general transcription machinery. However, several reports in some plant and animal models do not seem to fully support this model. Interestingly, several subunits of this Mediator complex have been identified as regulators of pluripotency of mouse ESCs. However little is known about the putative role of Mediator on stem cells in vivo.
In this paper the authors characterized the function of the subunit Med14 in stem and progenitor cells and regeneration in two different models: zebrafish and freshwater planarians. First they found that the zebrafish mutant logelei (log) corresponds to mutation of med14. Pleiotropic effects that suggest a developmental arrest characterize the phenotype of this mutant. Accordingly, morpholinos of med14 recapitulate many of these phenotypes, which can be then rescued by the injection of wild-type med14. Analyses of genome-wide transcript levels and mRNA levels did not support a general affectation of transcription in these mutants. In contrast, when the authors focused on stem and progenitor cells and regeneration in the log mutants they observed specific defects. Thus, using different markers they saw that retinal, hematopoietic and gut stem cells were reduced in the log mutant and that med14 seemed to be important in heart progenitor cells. Also, when the tail fin of these animals was amputated it did not regenerate.
In order then to better characterize the putative function of med14 on stem cells and regeneration the authors studied it in planarians, an attractive model in which to study stem cells in vivo. In planarians, as in zebrafish, med14 was ubiquitously expressed including in neoblasts (planarian totipotent stem cells and the only proliferative cells in them). The silencing of med14 by RNAi in intact planarians resulted in a phenotype resembling that obtained after depletion of the neoblasts. That is, the animals curled ventrally, lost their heads and finally lysed. On the other hand, the silencing of med14 also blocked regeneration. After med14 silencing there was a gradual loss of cells expressing the neoblast marker Smedwi-1 (a piwi homologue). There was also a loss of proliferative activity detected with different markers of mitosis and S-phase such as phosphorylated histone H3, histone h2b and pcna. Using a marker of neoblast early progeny the authors found that this cell population was also gradually depleted. In contrast, med14 RNAi did not affect the expression of specific markers for differentiated cell types including neurons, gut cells, muscle, pharynx and eye. Moreover they found a significant increase in apoptosis all throughout the treated animals. These results suggested that med14 silencing affected specifically the neoblasts and their progeny and that transcription in general was not compromised.
In summary, the results presented here indicate that the Mediator complex has an important role in stem cell maintenance in two distant models such as zebrafish and planarians. Further analyses should help determining how the Mediator exerts its function, maybe through establishing the epigenetic landscape essential for pluripotency and stem cell maintenance in these animals.