Q&A: What is regeneration, and why look to planarians for answers?
© Alvarado; licensee BioMed Central Ltd. 2012
Received: 12 October 2012
Accepted: 2 November 2012
Published: 8 November 2012
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© Alvarado; licensee BioMed Central Ltd. 2012
Received: 12 October 2012
Accepted: 2 November 2012
Published: 8 November 2012
Historically, philosophers, naturalists and biologists alike have referred to the restoration of missing body parts after traumatic injury as regeneration. While still valid today, the concept of regeneration has expanded through the years to include a diverse set of phenomena. For instance, August Weisman considered physiological cell renewal to be regeneration and wrote so in a chapter dedicated to regeneration in his seminal 1893 book The Germ Plasm: 'the functions of certain organs depend on the fact that their parts continually undergo destruction, and are then correspondingly renewed. In this case it is the process of life itself, and not an external enemy, that destroys the life of a cell' . Soon after, TH Morgan would also attempt to refine the precision of the concept of regeneration by coining terms that distinguish between regeneration requiring cell proliferation (epimorphosis) and regeneration effected by tissue remodeling (morphallaxis) . Presently, regeneration is used to include multiple restorative processes manifested either as a result of physiological turnover (for example, the renewal of blood, skin and gut epithelial cells) or injury, and more recently has been used to define a branch of medical practice referred to as 'regenerative medicine'. Thus, rather than becoming more specific, the concept of regeneration has become much more general. This peculiarity can be attributed in great part to the fact that presently, and not unlike previous centuries, little unambiguous molecular, cellular, and evolutionary evidence exists to support a common or divergent mechanism controlling physiological and traumatic regeneration within and between species. That such diverse biological phenomena as adult neurogenesis and limb regeneration can be catalogued under the same umbrella is indicative of our limited mechanistic understanding of regenerative processes, and thus underscores how much more discovery research remains to be done.
The emerging field of regenerative medicine aims to identify strategies to repair tissues, organs, and human body parts that cannot be naturally replaced when damaged by either trauma or disease. Examples are spinal cord injury, loss of limbs and the loss of neurons to stroke and degenerative diseases like Parkinson's and Alzheimer's. Given that natural regeneration of such tissues occurs with frequency across vertebrate and invertebrate organisms alike, it stands to reason that if we can understand these processes, we should be able to extrapolate this knowledge to human health matters. If regeneration is evolutionarily ancestral and its mechanisms conserved across all animals with regenerative capacities, then it should be possible to coax mammalian tissues to launch a regenerative response by modulating pre-existing repair and regenerative mechanisms. On the other hand, if regeneration is an attribute invented independently multiple times in evolution, understanding which aspects of this process are unique, species-dependent inventions will also have an impact on how to apply the knowledge derived from animal regeneration studies to human health. For instance, understanding why a particular regenerative process takes place in a model system but not in human tissues may help identify new molecular pathways and cellular activities that could be extended to human cells and tissues to stimulate regeneration should endogenous mechanisms not be readily available. Either way, deciphering the modes and mechanisms driving regeneration in multiple model systems will not only help us resolve a long-standing question in biology and evolution, but also have clear ramifications for our understanding of wound healing and regeneration in humans.
There are many reasons why we chose planarians as a model system for the molecular and cellular dissection of animal regeneration . Our decision was driven in great part by a need to bridge experimental gaps left exposed by traditional genetic model systems. The pronounced limitations of somatic tissue turnover and regenerative properties in standard invertebrate models such as Drosophila and nematodes, coupled with the difficulties of studying adult vertebrate somatic stem cells in vivo, were compelling reasons to examine and test the suitability of planarians, free-living members of the phylum Platyhelminthes, to inform both regeneration and stem cell biology. Planarians, which are non-parasitic flatworms, display remarkable regenerative capacities for all of their tissues, irrespective of whether these were derived from endoderm, mesoderm or ectoderm. Because of their evolutionary position, these bilaterally symmetric, triploblastic organisms were expected to share with vertebrates a large number of the molecular and cellular processes that make form and function possible in animals. We now know that this is indeed the case, as planarians share with vertebrates all of the major developmental signaling pathways responsible for the establishment of the bilateral body plan [8, 9]. In addition to their remarkable powers of regeneration, and in contrast to vertebrate regeneration model systems, planarians are small (about the size of a toenail clipping), and rather easy and relatively inexpensive to rear in great numbers in the lab, allowing for genome-wide functional studies of regeneration. Planarians were also very attractive as a model system because an extensive body of literature spanning over two centuries exists, which describes in great detail the remarkable developmental plasticity of these animals . This exquisite body of knowledge has, for the most part, just begun to be examined using the rigors and methods of modern molecular and cellular biology.
Across multiple species and phyla, the stimulus for regeneration is amputation. Planarians are no exception. Wounding and amputation in this organism leads to a coordinated cellular and molecular response that can be measured and is currently under intense investigation. We know, for example, that upon amputation, the body wall musculature undergoes depolarization, which in turn results in the contraction of the muscle fibers near the amputation plane, effectively reducing the surface area of the wound. This is followed by a loss of columnar morphology of the epidermal cells adjacent to the wound and their subsequent migration over the wound, until the exposed tissues are completely covered by a monolayer of these cells. This amputation-induced epithelial-mesenchymal interaction is likely involved in the signaling that triggers regeneration, as one of the earliest genes induced in response to wounding is part of the ancient, broadly conserved Wnt/β-catenin signaling pathway [11–13], which also plays a key role in wound healing .
All of them - that is why planarians are so attractive for the study of regenerative mechanisms. As such, it becomes possible to study how the differentiated derivatives of all embryonic germ layers (ectoderm, mesoderm and endoderm) can be restored in an adult context after they have been lost to amputation.
This often-asked question was answered by TH Morgan in 1898 . He reported that a fragment equivalent to 1/279th the size of the original animal was sufficient to produce a complete animal. He arrived at this number by first measuring the animal using eye-micrometers in his microscope, for which each division was 1/53 mm and 1/28 mm. After measuring the worm, Morgan would then draw, cut, and weigh a thin but larger cardboard scale replica of the intact animal. He would then cut the animal into the smallest possible pieces, measure each piece, and then cut an equivalent sized fragment form the cardboard replica. He followed the regeneration of the cut fragments, and then measured the weight of the cardboard pieces corresponding to the animal fragments that completed regeneration successfully. In other words, the cardboard replica was measured to weigh 279.5 mg, and the smallest planarian fragment that could regenerate corresponded to a cardboard piece weighing 1 mg, thus resulting in the 1/279th value for the smallest piece capable of regenerating a complete worm.
Yes. Large numbers of small, undifferentiated cells populating the body plan of many flatworms were noticed towards the end of the 19th century . These cells were also noted to be mitotically active, and their role in regeneration was confirmed by the pioneering work of Bardeen and Baetjer . These investigators reported in 1904 that animals exposed to ionizing radiation lost their regenerative capacities. When the worms were inspected histologically, Bardeen and Baetjer reported a complete absence of both mitotic activity and undifferentiated cells. These specialized cells are referred to as neoblasts.
Neoblasts are pluripotent, somatic stem cells that are broadly distributed across the planarian anatomy. In asexual animals they are the only cells capable of undergoing cell division and as such can be readily eliminated by gamma-irradiation to produce an animal that can survive for several weeks, but is incapable of mounting a regenerative response upon wounding. Neoblasts are small (approximately 5 µm in diameter) and by morphology alone correspond to approximately 25% of all cells in the organism. They share with other stem cells the characteristic of having a large nucleus containing highly decondensed chromatin and a scant, basophilic cytoplasm . Molecular markers and genes affecting the function of neoblasts and their progeny have been identified [18–20], providing the field with novel molecular tools to characterize their biological functions in vivo.
While the in vitro culture of neoblasts has yet to be established, single stem cell transplantation into adult planarians is possible, making the animal itself a tissue culture chamber in which to grow these cells. Recent experiments have unambiguously demonstrated that, with some frequency, single, transplanted neoblasts can restore viability and rescue many of the morphological defects of lethally irradiated adult animals . Interestingly, under these conditions, the rescue of the irradiated animals occurs through a clonal expansion rather than migration of the injected cell, followed by expansion of the resulting colony of stem cells. These data would indicate that neoblasts are not migratory cells, a somewhat surprising result given how many niches (that is, the cellular microenvironment capable of supporting the maintenance of stem cells in plants and animals) were left vacant by the irradiation that would have been expected to promote stem cell mobilization.
Regeneration remains one of the last untamed frontiers of developmental biology. It is amongst the oldest biological problems known to humankind, dating back to antiquity in many cultures and, perplexingly, still awaiting a satisfactory mechanistic explanation. It is my firm belief that the time to plumb the molecular depths of regeneration is now. Tremendous strides have been made in the study of regeneration in Hydra, planarians, zebrafish, newts, and salamanders. Hence, a critical mass of knowledge is accruing that would permit a systematic interspecies comparison of regenerative capacities across very distant and diverse phyla. Equally important, a systematic and formal exploration of how the mechanisms of regeneration compare to embryogenesis can now begin in earnest. Such a comparison would help address the long-standing question of whether regeneration is simply a recapitulation of development or made possible by independent mechanistic innovations. In the case of planarians, are their embryonic stem cells functionally different from neoblasts? When during embryogenesis are neoblasts specified? To what extent are embryonic axes formation and organogenesis mechanisms similar or dissimilar between planarian embryogenesis and regeneration? Is the genetic toolkit required to organize body axes and facilitate organogenesis during embryogenesis the same as during regeneration? This is a particularly important question because many organisms such as the mouse, fruitfly, and frog can recover from ablation of numerous blastomeres or substantial injury to embryonic organs, yet display limited regenerative capacities as adults. Therefore, testing whether regulative development occurs in planarian embryos, for example, may help us identify key differences crucial to preserving regenerative abilities into adulthood. These and many more fascinating questions abound , so it is clear that when it comes to regeneration, we have but just begun to scratch the surface.
ASA is an investigator of the Howard Hughes Medical Institute and the Stowers Institute for Medical Research and is a recipient of NIH NIGMS MERIT award R37GM057260.
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