When his team published their groundbreaking article, Poss says he would have predicted it would take at least 40 years before regenerative techniques would be ready for application in humans. But, since then, findings in the field have accumulated at a surprisingly rapid pace. "Just in the last year or two the rate of discovery in heart regeneration has been spectacular. I would now predict that in the next several years we'll see some of the findings from our lab and others being used to develop regenerative applications for humans."
It has always been the case that the greatest advances in treating human disease stand on the shoulders of basic research. Dr. Poss has spent the last ten years building one of the largest zebrafish colonies in the United States and a dedicated team of scientists who work with him to uncover biological mechanisms by which adult zebrafish re-grow tissues after damage. This unassuming little fish with its characteristic black-and-white stripes provides a surprisingly elegant model for regenerative research. Even after very severe injuries, the zebrafish is able to quickly regenerate a wide variety of complex structures including fins, retina, spinal cord, and heart. Poss explains his choice to study zebrafish this way, "You can visualize (zebrafish) from the one-cell stage all the way up through several days as all the organ systems form – so you can pretty much see cells migrating and vessels growing ... The ability to visualize it is (the zebrafish model's) greatest strength besides its strength in numbers." In other words, not only is it relatively easy to image these miraculous animals, but they are also conveniently compact allowing researchers to keep large populations in limited space. Poss's team maintains 5,300 tanks of 10 to 30 fish each in a 3,000 square foot facility that will more than double in size by next year when two new locations are complete.
While most zebrafish research focuses on the initial, embryonic development of the heart and other organs in the first days of life, Poss's team is one of a few looking at cardiac regeneration in later life. To do this, they developed new tools for studying adult fish including dozens of transgenic strains to help visualize different cell types. Vikas Gupta, a graduate student in Poss’s lab, was the first to adapt Brainbow technology (originally developed for mouse studies) to color-code zebrafish cardiomyocytes – allowing offspring cells to be tracked as they divide from the parent cell and migrate around the heart. Gupta developed a technique for expressing various amounts of red, blue, and yellow proteins inside the cardiomyocytes, labeling them in more than 20 distinct colors. This break-through allowed the team to image cardiac development and the regenerative process in fine detail because each cell permanently retains its color and passes it on to new cells that divide from it. Using microscopic imaging, the team could clearly document patterns of cell division and migration within the fish hearts from embryo to adulthood and as repairs occurred after cardiac injury. In fact, Poss's researchers were the first to use Brainbow to define how cells behave during organ development.
Dr. Poss attributes their most surprising findings to this ability to microscopically distinguish and trace dozens of cell types by color. He explains, "It is possible for us to retrospectively watch as particular cell-types divide and multiply out during the formation of the heart in a zebrafish embryo. On average, we found that there are usually only eight cardiomyocytes that replicate to form the large patches of tissue that eventually make the entire wall of the heart. That was really surprising to us." This and other findings have turned turned researchers' attention, which had become increasingly focused on using stem cells to spur regeneration, toward other sources of new cells. As Poss says, "What we've found in fish is that the heart muscle cells - the cardiomyocytes – are not only the source of heart muscle cells during the initial formation of the embryonic heart, but that they are also the source of new heart muscle cells during regeneration. They do behave like stem cells in these situations and expand much more than we originally thought individual cardiomyocytes could. We haven't found out yet how or why that happens. But this finding in fish has directed more attention to looking at muscle cells and not just focusing on stem cells as the pathway for how to make regeneration happen. Because, in these fish, we have been able to clearly image how the heart muscle cells themselves are central to the regenerative process."
Another unexpected finding was the extent of response to a localized injury. Poss says, "We expected a very local response in the injured area. We expected all the regeneration and signaling to occur right at that injured location. What we found was that it is a much more dynamic process. If you injure the heart in a very small area, you get signals and activation of pathways far from the injury site. We injured the ventricle and found signaling responses in the atrium within hours of the injury. There's some mechanism that when the heart is injured very locally there is an organ-wide response to it in the first day. Then the tissue recognizes that the injury is localized and that's where signaling and responses begin to concentrate. It's much more dynamic than we expected. Also, it's not gradual – the very early set of organ-wide responses occur within an hour and we can see that a certain set of genes in the injured heart are turned on everywhere not just in the damaged area. Within a couple of days those genes are turned on only at the injury site... We did not expect to find this and think it's important for the ability to regenerate. This process of 'finding' the injury is probably critical to target signaling that supports tissue reformation at that particular site."
The team also continues to be surprised by how quickly zebrafish recover from injuries that would be fatal in most other vertebrates. "Our most severe injury is to genetically ablate or kill over two-thirds of all the muscle cells throughout the heart. It's an incredibly severe injury. They go into heart failure with all the classic signs of very late stage heart failure in humans: edema, sensitivity to exercise, shock, rapid respiration, lethargy. ... In humans, there is very little we can do for this level of heart failure. These fish can grow the muscle back and reverse the symptoms! In a couple of weeks you can't even tell they were injured. They are darting around in the tank. The regenerative capacity itself still surprises me." Poss says with a smile.
These important and unexpected findings underscore the importance of basic research, which has recently struggled to maintain funding. As Poss puts it, "When you're looking at that fundamental level of how something works – how it happens - you have the opportunity to find anything. That's why many of us are really adamant that we need to keep supporting basic research. We need to study basic developmental biology – how embryos grow and how organs form. What happens as an animal goes from the juvenile to the adult stage – we don't know that much about that. And what happens when any tissue regenerates – fin or heart, retina, muscle, liver, or pancreas – generally these things, as far as we know now, share a subset of signaling pathways that they use in different ways. There are going to be tissue specific mechanisms as well. It all - the body of work – all joins together ... That's how science is supposed to work."
And there is still much work to be done before cardiac regeneration is fully understood. Key questions remain such as how do newly generated cells know what they are, where to go, and how to function? This is a particularly confounding question in the heart which consist of electrically coupled muscle tissue. How do new cardiac cells position themselves and incorporate into an injured, beating heart without causing an arrhythmia? What is the mechanism that prevents over-growth by telling the regenerative process when to stop?
There are now hundreds of labs around the world studying zebrafish and developing new genetic tools to manipulate them which help accelerate findings across the entire field. Improvements in microscope speed and sensitivity – particularly the ability to use fluorescent stereo imaging of protein markers and improvements in confocal color separation and customizing wavelengths – are also contributing to the fast pace of discovery in regenerative medicine.
For their parts, Poss and his team at Duke continue working to unravel the fundamental mechanisms of regeneration. "The ultimate goal is to figure out how regeneration happens in these zebrafish systems and apply those concepts to mammalian contexts. To look at a mouse, rat, or human heart that normally scars after any type of injury and begin to apply factors that we've identified in the fish to support tissue regeneration in another animal." With new discoveries in regeneration being made faster than ever before, there is hope that we might reach this goal sooner than anyone once thought possible.
- Choi WY, Gemberling M, Wang J, Holdway JE, Shen MC, Karlstrom RO and Poss KD: In vivo monitoring of cardiomyocyte proliferation to identify chemical modifiers of heart regeneration. Development 140: 660–66 (2013).
- Kikuchi K and Poss KD: Cardiac regenerative capacity and mechanisms. Annual Review of Cell and Developmental Biology 28: 719–41 (2012).
- Nachtrab G. and Poss KD: Toward a blueprint for regeneration. Development 139: 2639–42 (2012).
- Gupta V and Poss KD: Clonally dominant cardiomyocytes direct heart morphogenesis. Nature 484: 479–84 (2012).
- Singh SP, Holdway, JE and Poss KD: Regeneration of amputated zebrafish fin rays by de novo osteoblasts. Developmental Cell 22: 879–86 (2012).
- Choi WY and Poss KD: Cardiac regeneration. Current Topics in Developmental Biology 100: 319–44 (2012).
- Yin VP, Lepilina A, Smith A and Poss KD: Regulation of zebrafish heart regeneration by miR-133. Developmental Biology 365: 319–27 (2012).
- Nachtrab G, Czerwinski M and Poss KD: Sexually dimorphic fin regeneration in zebrafish controlled by androgen/GSK3 signaling. Current Biology 21: 1912–17 (2011).
- Wang J, Panáková D, Kikuchi K, Holdway JE, Gemberling M, Burris JS, Singh SP, Dickson AL, Lin YF, Sabeh, Werdich AA, Yelon D, MacRae CA and Poss KD: The regenerative capacity of zebrafish reverses cardiac failure caused by genetic cardiomyocyte depletion. Development 138: 3421–30 (2011).
- Kikuchi K, Gupta V, Wang J, Holdway JE, Wills AA, Fang Y and Poss KD: tcf21+ epicardial cells adopt non-myocardial fates during zebrafish heart development and regeneration. Development 138: 2895–2902 (2011).
- Kikuchi K, Holdway JE, Major RJ, Blum N, Dahn RD, Begemann G and Poss KD: Retinoic acid production by endocardium and epicardium is an injury response essential for zebrafish heart regeneration. Developmental Cell 20: 397–404 (2011).
- Shin D, Lee Y, Poss KD and Stainier DYR: Restriction of hepatic competence by Fgf signaling. Development 138: 1339–48 (2011).
- Liu J, Bressan M, Hassel D, Huisken J, Staudt D, Kikuchi K, Poss KD, Mikawa T and Stainier DYR: A dual role for ErbB2 signaling in cardiac trabeculation. Development 137: 3867–75 (2010).
- Poss KD: Advances in understanding tissue regenerative capacity and mechanisms in animals. Nature Reviews Genetics 11: 710–22 (2010).
- Lee Y, Nachtrab G, Klinsawat PW, Hami D and Poss KD: Ras controls melanocyte expansion during zebrafish fin stripe regeneration. Disease Models and Mechanisms 3: 496–503 (2010).
- Yin C, Kikuchi K, Hochgreb T, Poss KD and Stainier DYR: Hand2 regulates extracellular matrix remodeling essential for gut-looping morphogenesis in zebrafish. Developmental Cell 18: 973–84 (2010).
- Kikuchi K, Holdway JE, Werdich AA, Anderson RM, Fang Y, Egnaczyk GF, Evans T, MacRae CA, Stainier DYR and Poss KD: Primary contribution to zebrafish heart regeneration by gata4+ cardiomyocytes. Nature 464: 601–5 (2010).
- Gonzalez-Quevedo R, Lee Y, Poss KD and Wilkinson DG: Neuronal regulation of the spatial patterning of neurogenesis. Developmental Cell 18: 136-47 (2010).