Unfolding the secrets of the zebrafish’s regenerative capacity…
Hope comes in many forms, but hope in the form of a fish? It’s definitely not a fishy affair! Though small in size, the zebrafish holds tremendous potential to unlock some of the deepest mysteries of Biology. The latest take on this small organism is its capability to undergo organ regeneration – particularly heart regeneration. Scientists are studying heart regeneration in this small fish to unlock secrets which may help “repair” human hearts.(2) In fact, the British Heart Foundation has started a campaign to conduct research on zebrafish to study their heart development and regeneration processes and the mechanisms involved in these processes, so they may be able to “teach” the human heart cells to do the same.(1, 2)
Regeneration of the liver had previously been studied in mice. It has been found that hepatic progenitors are activated in a damaged liver, and further studies in this direction could form the basis for engineering liver cells in vitro for cell transplantation to sustain patients with liver failure. Zebrafish is an ideal model for studying liver regeneration because they produce hundreds of offspring with every mating and their transparent embryos develop outside of the mother, allowing constant visualization and easy manipulation. Also, organogenesis is underway in nearly all systems by 24 hours post fertilization.(3)
Cardiac injury in humans (mammals) leads to scarring and there is no regeneration of the lost cardiac tissue. This makes the heart vulnerable to further injury and is one of the leading causes of death arising from myocardial infarction. Zebrafish can fully regenerate hearts within 2 months of ventricular resection. Recent studies in this direction have revealed several factors which contribute this regeneration process and how these may help in understanding similar molecular mechanisms in human beings.
In a study conducted by Poss et al. (2002), they resected 20% of the ventricular myocardium of adult zebrafish and observed the regeneration of the resected portion. They found that by 30 days post amputation (dpa) there was partial recovery of ventricular section surface area, and by 60 dpa, the surface area was completely recovered. The contractile properties of the beating hearts at 60 dpa also appeared normal. It was also found that a new layer of compact myocytes (which are usually present externally and penetrated by blood vessels) was re-established and expanded after amputation. Proliferation of cardiomyocytes was established by incorporation of Bromodeoxyuridine (BrdU) in the nucleus of the cells, during the cell cycle. BrdU is a marker of DNA synthesis (More info about protocol at: http://openwetware.org/wiki/Bromodeoxyuridine_%28BrdU%29). It was seen that BrdU incorporation peaked at 14 dpa, with most cycling myocytes localized to compact muscle at the lateral edges of the wound. (4, 5)
Cardiomyocyte proliferation accompanies zebrafish heart regeneration. (A) Confocal image of a heart section of an unamputated fish labeled for 7 days with BrdU, stained for myosin heavy chain to identify cardiomyocytes (red), and stained with BrdU to detect cycling cells (green) and with 4 ,6-diamidino- 2-phenylindole (DAPI) to detect nuclei (blue). A low percentage of compact myocytes incorporate BrdU over this period (arrowhead). (B) 7 dpa (0- to 7-dpa BrdU labeling). BrdU incorporation occurs in trabecular myocytes at the amputation plane (arrowheads). Most hearts also showed labeling in compact myocytes adjacent to the wound area at this stage. (C) 14 dpa (7- to 14-dpa BrdU labeling). Many cardiomyocytes incorporate BrdU during this period, largely in compact muscle adjacent to the wound. (D) 30 dpa (23- to 30-dpa BrdU labeling). Myocyte BrdU incorporation continues as the compact muscular layer expands. Labeling is usually limited to the most epicardial myocytes of the apex (arrowheads).,6-diamidino-2-phenylindole (DAPI)to detect nuclei (blue).A low percentage ofcompact myocytes incorporateBrdU overthis period (arrowhead)(5). (B) 7 dpa (0- to7-dpa BrdU labeling).BrdU incorporation occursin trabecular myocytesat the amputationplane (arrowheads).Most hearts also showedlabeling in compactmyocytes adjacent tothe wound area at thisstage (not shown). (C)14 dpa (7- to 14-dpaBrdU labeling). Manycardiomyocytes incorporateBrdU during thisperiod, largely in compactmuscle adjacent tothe wound. (D) 30 dpa(23- to 30-dpa BrdU labeling).Myocyte BrdUincorporation continuesas the compact muscularlayer expands. Labelingis usually limited to the most epicardial myocytes of the apex (arrowheads).
Fibrosis is the most dominant response to injury in mammalian hearts. Yet, zebrafish hearts show minimal deposition of collagen, post injury. The authors proposed a model in which scarring complements regeneration, and the vigor of myocyte proliferation within a given species is what determines the predominant response. This model predicts that inhibition of myocyte proliferation leads to scarring. To support this model, the authors used a mutant zebrafish mps1, which has a temperature sensitive mutation in the Mps1 gene, which is a cell cycle regulator (mitotic checkpoint kinase). It was found that the mps1 mRNA was induced in myocytes near the wound. This mutation prevents cell proliferation at restrictive temperatures (particularly prevents fin regeneration). When ventricular resection was performed on these mutant fish, they developed large scars and retained fibrin deposits. This showed that Mps1 was required for cardiomyocyte proliferation, which thereby leads to cardiac regeneration.(4)
Cardiomyocyte proliferation is required for scarless regeneration. 14-dpa (A) and 60- dpa (B) ventricles stained with acid fuchsin–orange G (AFOG; fibrin, orange/red; collagen, blue), which is highly sensitive for collagen (arrowheads). Regenerated ventricles contain minimal collagen. (C and D) 17-dpa ventricles double-stained for myosin heavy chain and with aniline blue (muscle, brown; fibrin and collagen, grayish blue). Wild-type ventricles display new compact muscular wall formation (C), whereas mps1 mutant ventricles demonstrate no evidence of new muscle (D). (E and F) 26-dpa hearts stained with AFOG. The wild-type cardiac injury response includes minor fibrin retention and collagen deposition (E), whereas extensive fibrosis (arrowheads) is observed in cardiac wounds of mps1 mutants (F).
The studies of heart regeneration indicate that adult hearts contain epicardial tissue and cardiac progenitors. However, ways to activate and utilize these towards successful regeneration have not yet been found in mammals. The finding that, regeneration and fibrosis, are competing events in the vertebrate heart have interesting implications. It may be possible in the future, to identify pathways that may stimulate regeneration in the mammalian heart and thereby restrict scarring, in injuries characteristic of myocardial infarction.(4)
Though the study is not very recent, it was one of the pioneering research work conducted in this direction.
MENDING, NOT JUST THE HEART!
Zebrafish are not just capable of regenerating their hearts, but also other organs like the liver. In short, zebrafish are capable of being “reborn”, just by regenerating injured/damaged body parts! A similar approach, as described above, was taken to study the liver regenerating ability of the zebrafish (Sadler et al. 2007). Partial hepatectomy (PH) was performed on adult zebrafish to remove the caudal portion of the ventral lobe of the liver, which accounts for ~ 20-40% of the total liver mass. After seven days of partial hepatectomy, the liver of the operated animals was indistinguishable from that of the control animals. This showed that partial hepatectomy can lead to complete liver regeneration within a week. In order to show that this regeneration was due to proliferation of hepatocytes, immunohistochemical staining for proliferating cell nuclear antigen (PCNA), as a proliferating cell marker, was done in the hepatocytes (More info on PCNA at: http://en.wikipedia.org/wiki/Proliferating_cell_nuclear_antigen). PCNA expression peaked at 48 hr post PH, showing that regeneration was mainly due to hepatocyte proliferation. PCNA positive cells were concentrated at the site of resection, showing that the lobe was growing back. (6)
Uhrf1 is a gene which is required for physiologic liver growth It was surmised that factors which are required for physiological growth would be upregulated post partial hepatectomy and during recovery. Using Q-PCR, it was found that the levels of uhrf1 were drastically upregulated post hepatectomy, during regeneration. Transcription of Top2a is directly regulated by Uhrf1 and the levels of top2a were also increased during regeneration. A heterozygous mutation in uhrf1 was created and its effect on the activation of top2a was studied, during regeneration. It was observed that the transcript levels of uhrf1 in the heterozygous mutants (uhrf+/-) was half of that present in the wild type (carrying 2 copies of the gene) and the levels of top2a post PH was suppressed. Also, to test whether regeneration was affected in the heterozygous mutants, PH was performed in these mutants. It was seen that none of the mutants showed significant lobe regrowth at the end of 5 days post PH as compared to the WT. WT animals had complete lobe regrowth by this time. (More info on Uhrf1 at: http://en.wikipedia.org/wiki/UHRF1)
These data illustrate the importance of cell-cycle regulators (Uhrf1 and Mps1) in the proliferation of cells affecting organ regeneration. Top2a is required for cell cycle progression and the relationship between Top2a and Uhrf1 suggests a mechanism wherein injury can trigger cell cycle progression of proliferating cells. It is of interest to study Uhrf1 and how it acts to control cell proliferation since it has been proposed as a prognostic marker for some human tumors. (6)
To cut a long story short…
Several questions, yet, remain to be answered: How do different cell types interact during development? Do the pathways regulating cell proliferation in the embryo also control regeneration in adults? What is the link between the genes contributing to development and those contributing to pathology? How are the genes deregulated during disease and can they be manipulated as a means of treatment? Zebrafish are ideal models of Organ Repair and Regeneration and uncovering the mysteries of the mechanisms involved may help engineer human organs to do the same!
- URL: http://www.labnews.co.uk/laboratory_article.php/6309/2/Unlocking-theecrets-of-the-zebrafish
- URL: http://speakingofresearch.com/2011/02/16/a-fish-named-hope/
New School in Liver Development: Lessons from Zebraﬁsh. Jaime Chu, Kirsten C. Sadler, Hepatology Vol 50 (5) (July 2009) Link: http://onlinelibrary.wiley.com/doi/10.1002/hep.23157/pdf
- Heart regeneration in Zebrafish. Kenneth D Poss, et al. , Science 298, 2188 (2002) Link: http://www.ncbi.nlm.nih.gov/pubmed?term=Heart%20regeneration%20in%20Zebrafish.%20Kenneth%20D%20Poss%2C%20et%20al.%20%2C%20Science%20298%2C%202188%20(2002)
- Getting to the heart of regeneration in zebrafish. Kenneth D Poss, Seminars in Cell & Developmental Biology 18 (2007) 36–45 Link: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WX0-4MDP846-2&_user=10&_coverDate=02%2F28%2F2007&_rdoc=1&_fmt=high&_orig=gateway&_origin=gateway&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=9b92d9ea77ed1793bc3eb0a537019e5a&searchtype=a
- Liver growth in the embryo and during liver regeneration in zebrafish requires the cell cycle regulator, uhrf1. Kirsten C Sadler, et al. , PNAS Vol 104 (5) (Jan 2007) 1570 – 75 Link: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1785278/?tool=pubmed