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Tissue Homeostasis, Regeneration, and Stem Cells

Research Summary

Alejandro Sánchez Alvarado has established a powerful new model system to study the molecular mechanics of regeneration, using the freshwater flatworm Schmidtea mediterranea. Sánchez Alvarado's lab has developed the molecular tools needed to reveal how regeneration works in this flatworm.

Despite the importance of regenerative processes to human biology and health, the molecular and cellular mechanisms driving the restoration of body parts lost to physiological turnover and/or injury remain largely unexplored. The dearth of molecularly accessible animal model systems largely explains why we presently have such a rudimentary mechanistic understanding of regeneration. To develop a molecularly tractable organism in which to study regeneration, I selected planarians, a classic developmental system that, for historical rather than biological reasons, fell out of favor early in the 20th century. In the past seven years, my laboratory has endeavored to develop molecular tools to dissect the remarkable biology of these animals.

With funding from the National Institute of General Medical Sciences, we have established loss-of-function assays and large collections of cDNAs and recently completed a large-scale RNA interference (RNAi)-based screen. In addition, we have begun sequencing the genome of S. mediterranea at the Washington University Genome Sequencing Center (with funding from the National Human Genome Research Institute). These advances have allowed us to commence systematic cellular and molecular genetic studies on animal regeneration and the attendant stem cells driving this phenomenon. Our efforts are beginning to offer insights into biological properties that are not only crucial to regeneration but also essential for the normal development of higher organisms, including humans.

Figure 1: Regenerative capacities of the planarian...

Why Study Regeneration?
Besides the obvious and important practical ramifications of improving human health, the study of regeneration also provides fertile and largely unexplored grounds to obtain a greater understanding of the fundamental molecular and cellular activities governing biological function. Unfortunately, the numerous superficial similarities that exist between regeneration and embryogenesis have engendered the misleading view that regeneration is merely a recapitulation of embryonic events, i.e., a redundant problem of development that will eventually be resolved by studying the embryo.

Like embryogenesis, regeneration does involve the self-assembly of new tissues. Yet, very much unlike embryogenesis, regeneration also entails the anatomical and functional integration of newly made parts into older, pre-existing tissues. Consequently, many regenerated organs and organ systems are out of proportion with the body size of the animal, resulting in asymmetries that must be corrected for the organism to regain its proper function and proportion. Moreover, not all animals can replace structures lost to damage or amputation, even though all organisms share a finite, pleiotropic set of developmental pathways. If regeneration merely recycles such pathways in the adult form, why can some animals regenerate while others cannot? Mechanisms that sense perturbations of homeostasis must exist that are capable of reactivating and regulating the pleiotropic activities of developmental pathways to achieve the specificity required to restore only the missing body parts and re-establish homeostatic balance. Therefore, multiple, uncharacterized (and perhaps unpredicted) postembryonic regulators of development must exist, and their identification and characterization will largely depend on the availability of varied and experimentally accessible biological contexts. Because regeneration exposes developmental pathways to conditions not found during embryogenesis, its study may uncover mechanisms by which pathway specificity is regulated. Irrespective of what we may learn about the molecular nature of regeneration, it is clear that its study will also provide insights into many fundamental mechanisms and unresolved aspects of metazoan biology.

Why Planarians?
I followed two basic criteria in selecting a suitable model system to study regeneration. First, the animal should be one of the simplest metazoans in which regeneration is patently manifested. Second, the organism should be relatively easy to manipulate experimentally. Of the several animals considered, planarians fulfilled these criteria best, as they are one of the simplest bilaterians known to display robust regenerative capacities, and more than 100 years of scientific literature exists reporting planarian experimentation.

Movie 1: Because planarians are not particularly fond of bright light, they are said to display negative phototaxis. The time-lapse movie illustrates the speed at which planarians move away from the light, which is detected by the "eye-like" structures found on the dorsal anterior surface of these animals. Light detection is then relayed to the brain, resulting in a stereotypic avoidance behavior.

Credit: Alejandro Sánchez Alvarado

Planarians are best known for their capacity to regenerate complete individuals from minuscule body parts. Such extraordinary tissue plasticity is in direct contrast to the lack of pliancy displayed by most somatic tissues of adult Caenorhabditis elegans and Drosophila melanogaster. The difference lies in a population of adult somatic stem cells called neoblasts that are distributed throughout the planarian body. As we have shown, neoblasts are the only mitotically active cells in planarians, and their division progeny generate the ~40 different cell types found in the adult organism. In intact planarians, neoblasts replace cells lost to normal physiological turnover, while giving rise in amputated animals to the regeneration blastema, the structure in which missing tissues are regenerated. The pronounced absence of somatic tissue turnover and regenerative properties in current invertebrate models, coupled with the difficulty of studying vertebrate somatic stem cells in vivo, are compelling reasons to examine and test the suitability of planarians to inform both regeneration and stem cell biology at large.

Why Schmidtea mediterranea?
To exploit the biological potential of planarians, we had to identify a suitable species. There are thousands of different known species, but only several dozen have been characterized in some detail. Of these, the free-living, freshwater hermaphrodite Schmidtea mediterranea emerged as a likely candidate because it displays robust regenerative properties and, unlike most other planarians, it is a stable diploid (2n = 8) with a genome size nearly half that of other common planarians (8 x 10 8 bp). Moreover, a Robertsonian translocation between chromosomes 1 and 3 produced an exclusively asexual biotype. Both sexual and asexual forms have proved easy to rear in the laboratory. By serially amputating individual worms and allowing the fragments to regenerate, we expanded single animals into clonal colonies of thousands of individuals. We then succeeded in breeding clonal lines of the sexual strain that produce fertile progeny, overcoming previous limitations in the sexual propagation of planarians in captivity. This has allowed us to generate clonal inbred lines for genomic and genetic analyses, and to begin a molecular and morphological characterization of the embryogenesis of freshwater planarians. As alluded to earlier, the belief that embryogenesis and regeneration can be likened to each other has persisted in the absence of direct tests. Access to thousands of S. mediterranea embryos, coupled to the regenerative capacities of this species, allows, for the first time, systematic comparative and functional studies of embryogenesis and regeneration, a task essential for understanding their true relationship.

Using Schmidtea mediterranea to Solve the Problem of Regeneration
Why does regeneration happen? What are the factors that determine the extent and varied manifestations of this metazoan attribute? Answering these questions requires us to dissect the genetics, cell biology, and physiological aspects that make regeneration possible. By studying S. mediterranea, we overcome a number of important experimental limitations in the study of regeneration. For example, an effort has been made to genetically dissect regeneration in the adult zebrafish. However, to avoid potential embryonic lethality that could mask the identification of postembryonic gene function, it was necessary to devise a temperature-sensitive screen in this organism. Like all conditional screens, such an approach could not saturate the zebrafish genome and was thus significantly limited in its scope.

In contrast, RNAi screens can be carried out and analyzed in the adult S. mediterranea, bypassing the need for recovering conditional mutations. By identifying genes and genetic activities associated with regeneration and tissue homeostasis, our recently completed RNAi-based screen proved the practicality and effectiveness of this approach. In fact, S. mediterranea provides us with the only regeneration model system in which it is possible to saturate the genome with loss-of-function phenotypes to uncover genomic activities associated with regenerative properties. Larger and/or more cell/tissue-specific RNAi screens to cover a wider representation of the planarian genome are likely to yield further insight into regeneration and will provide new paradigms for investigating metazoan gene function.

As of May 30, 2012

Scientist Profile

Investigator
Stowers Institute for Medical Research
Cell Biology, Developmental Biology