The ability of organisms to regenerate missing body parts is one of the great mysteries of biology. Planarians are freshwater invertebrates that have been a classic regeneration model for over a century. The regenerative abilities of planarians astound: following decapitation, a new head can be regenerated in under a week and an entire animal can be regenerated from a body fragment approximately 1/300th the size of the original animal. Regeneration involves formation of an outgrowth of tissue at wound sites (a blastema) that produces missing tissues. Because planarian regeneration involves a population of adult pluripotent stem cells (the cNeoblasts), planarians are excellent organisms for in vivo studies of how stem cells can be regulated to replace aged, damaged, and missing tissues. Because more than 50% of examined planarian genes have human counterparts, genetic study of neoblasts stands to impact our understanding of human stem cell biology.
The development of planarian RNA interference (RNAi) screening methodologies has set the stage for molecular, genetic characterization of regeneration. We aim to understand how neoblasts are regulated to bring about the regeneration of missing tissues. Our approach involves study of cellular events of regeneration and attendant roles for regulatory genes that control regeneration steps. We utilize an array of methodologies, including high-throughput sequencing, RNAi screening, and numerous assays and tools for phenotypic analysis to identify and characterize regeneration regulatory genes.
The planarian Schmidtea mediterranea
The source of new cells: adult pluripotent stem cells promote regeneration
A cellular explanation for planarian regeneration has been sought since the late 1800s, when a population of proliferative cells (neoblasts) was identified. A key unanswered question has been whether planarian regeneration is explained by a cell type displaying pluripotency (capacity to produce all cell types of the soma) at the single cell level, or by the collective action of multiple dividing cell types that each has limited and different potential. To address this question we developed single cell assays and determined that certain individual cells could divide to clonally generate many similar dividing cells and displayed broad differentiation potential. We named such cells clonogenic neoblasts (cNeoblasts). Single cNeoblasts were able to restore regenerative capacity to lethally irradiated hosts lacking all other cell division, ultimately converting hosts to the genotype of the donor. We conclude that pluripotent stem cells persist into adulthood providing a cellular basis for the remarkable regenerative abilities of planarians.
The hallmark attributes of stem cells are the capacity for self-renewal and the ability to produce one or more differentiated cell types. We aim to understand these stem cell features, as well as the molecular basis of pluripotency, by molecular genetic study of planarian cNeoblasts. Planarians cNeoblasts and their progeny cells are regulated by contextual cues (wounding, growth, homeostatic replacement of aged cells) and are capable of replacing essentially every cell type in the animal. This rich stem cell biology can now be studied with emerging tools: any gene desired can be inhibited and the effects on neoblasts in vivo assessed, we can isolate >100,000 neoblasts in a day by flow cytometry, and assays exist for assessing neoblast differentiation. Together, these attributes and tools present a powerful new approach to stem cell biology.
The initiation of regeneration
How does regeneration start? We have developed efficient and quantitative assays for measuring the response of neoblasts to wounds. There exist two peaks in mitotic neoblast numbers following wounding; the first peak occurs following many injury types. We therefore propose an initial step in regeneration involves a generic sensation of wounding. By contrast, the second mitotic peak occurs locally and only following injuries that involve missing tissue, requiring blastema formation for repair. In response to tissue absence, neoblasts migrate to wounds and are induced to differentiate. We are exploring the molecular control of regeneration initiation by seeking genes required for the process utilizing RNAi screening.
Specification of missing tissue type
How are animals capable of specifying missing tissue types to initiate correct regeneration programs? We have studied the head-versus-tail regeneration choice made at transverse wounds (regeneration polarity) as a paradigm for addressing this question. We discovered that RNAi of bcatenin-1 causes the striking phenotype of head regeneration in place of tails. bcatenin proteins mediate Wnt signaling, and we determined that a wound-induced Wnt expression program is required for regeneration polarity. We next determined that the regeneration polarity choice is mediated by selective feedback inhibition of Wnt signaling at anterior-facing wounds by the action of the gene notum (encoding a secreted hydrolase), such that wound-induced WNT1 is selectively active at posterior-facing wounds. We have also characterized roles of dorsal-ventral patterning factors in guiding regeneration decisions, such as bmp4 and admp. This work on regeneration polarity and taken together with comparison of our data to that from embryos in other species, suggests that posterior Wnt activation is a unifying principle in the formation of the primary body axis of animals. Furthermore, regeneration could in principle have involved re-scaling of tissue gradients involving autonomous attributes of the signaling circuitry involved. By contrast, our data suggest that wounds have active input in the initiation of appropriate regeneration programs by induction of signaling proteins that specify regional tissue identity.
Planarians as a model system for metazoan biology
Planarians provide a new experimental platform for uncovering basic functions for conserved metazoan genes. The RNAi screening approach, coupled with amputation and observation of the steps of regeneration, can allow study of the function of essentially any gene in regeneration. Prominent features of planarian biology--abundant stem cells, robust regeneration, restoration of missing patterns and cell types, and homeostatic replacement of aged cells--can thus be studied using a molecular genetic approach. Planarians therefore present the opportunity for the discovery of roles for genes in fundamental and understudied biological processes that are critical to the life of metazoans.
Jessica N. Witchley, Mirjam Mayer, Daniel E. Wagner, Jared H. Owen, and Peter W. Reddien. Muscle cells provide instructions for planarian regeneration. Cell Reports, 4(4): 633-41 (2013)
Sylvain W. Lapan and Peter W. Reddien. Transcriptome analysis of a simple eye identifies ovo as a specific regulator of eye regeneration. Cell Reports 2(2): 294-307 (2012)
Elly M. Tanaka and Peter W. Reddien. The cellular basis for animal regeneration. Developmental Cell 21, 172-185 (2011)
Daniel E. Wagner1, Irving E. Wang1, and Peter W. Reddien. Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration. Science 332, 811-816 (2011)
Christian P. Petersen and Peter W. Reddien. Polarized notum activation at wounds inhibits Wnt function to promote planarian head regeneration. Science 332, 852-855 (2011)
Christian P. Petersen and Peter W. Reddien. Smed-bcatenin-1 is required for anteroposterior blastema polarity in planarian regeneration. Science 319, 327-30 (2008)