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 more than 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 at wound sites of an outgrowth of tissue (a blastema) that produces missing tissues. Because planarian regeneration involves a population of adult pluripotent stem cells (the neoblasts), planarians are excellent organisms for in vivo studies of how stem cells can be regulated to replace aged, damaged, and missing tissues. Because greater than 50 percent of examined planarian genes have human counterparts, genetic study of neoblasts stands to impact our understanding of human stem cell biology.
I previously performed the first planarian RNA interference (RNAi) screen, which helps set the stage for molecular genetic characterization of regeneration. The central focus of my research program is to understand how neoblasts are regulated to bring about the regeneration of missing tissues. Our approach involves study of our identified regeneration regulatory genes and continued usage of our developed RNAi screening approaches.
Specification of Missing Tissue Type
How do wounded planarians specify the identity of missing tissues? To address this question, we are studying genes required for the decision to regenerate heads, tails, sides, and other specific regions of the body. For example, we have characterized a BMP signal transduction pathway that is needed for the regeneration of dorsal-ventral polarity in blastemas and for the formation of blastemas during lateral, asymmetric regeneration.
Planarians regenerate heads at anterior-facing wounds and tails at posterior-facing wounds throughout the body. How this regeneration polarity is specified has been a classic problem for more than a century. We performed an RNAi screen for regeneration regulatory genes in the planarian S. mediterranea and identified a gene, Smed-β-catenin-1, that controls regeneration polarity. Posterior-facing blastemas regenerated heads instead of tails in β-catenin-1(RNAi) animals, resulting in two-headed animals. β-catenin-1 was required after wounding and at any posterior-facing wound for polarity. Additionally, intact β-catenin-1(RNAi) animals displayed anteriorization during tissue turnover. β-catenin proteins are found throughout the Metazoa and mediate canonical Wnt signal transduction to regulate many developmental events. We found that Wnt genes and a secreted Frizzled-related protein (sFRP) Wnt antagonist gene are expressed in domains along the anteroposterior axis. These Wnt genes are expressed during regeneration near wound sites and are involved in making posterior-facing wounds distinct from anterior-facing wounds. Our data suggest Wnt signaling specifies the posterior character of the anteroposterior axis throughout the Bilateria and can act at wounds to specify regeneration polarity.
Our work suggests that morphogen signaling specifies and maintains the body plan in the adult animal and that changes in the site of expression of these morphogens following wounding are instructive to blastema formation and identity.
Stem Cell Regulation
The hallmark attributes of stem cells are the capacity for self-renewal and the ability to produce one or more differentiated cell types. To understand these stem cell features, we are studying planarian neoblasts. Planarians are full of neoblasts, the neoblasts are regulated by contextual cues (wounding, growth, homeostatic replacement of aged cells), and the neoblasts are capable of replacing essentially every cell type in the animal. This rich stem cell biology can now be studied with our emerging tools: we can inhibit any gene desired and assess the effects on neoblasts in vivo, isolate >100,000 neoblasts in a day by flow cytometry, and use existing assays to assess neoblast differentiation. These tools and neoblast attributes present a powerful new approach to stem cell biology.
Because neoblast-expressed genes have a recognizable pattern of expression in whole-mount in situ hybridizations, it has been possible to identify neoblast-expressed genes. We have identified dozens of genes that are expressed in neoblasts and that are associated with neoblast dysfunction following inhibition with RNAi. A SMEDWI-2 (PIWI-like) protein is required for neoblast progeny function and numerous additional genes are required for neoblast maintenance. Neoblasts normally divide during homeostatic maintenance of the body and respond to wounds to result in an increase in numbers of mitotic cells. We are utilizing our RNAi screening approach to identify genes involved in this process.
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.