RIKEN Center for Developmental Biology
2003 Annual Report
Laboratory for Evolutionary Regeneration Biology
Its position on an important but scientifically under-explored branch of the evolutionary tree and its remarkable biology combine to make the planarian flatworm a fascinating and invaluable model for basic research in fields from evolutionary development to regenerative medicine. Kiyokazu Agata has adopted the freshwater planarian, Dugesia japonica, as a model species in his study of the origins and properties of stem cells, which are prevalent and experimentally accessible in these worms and which he believes hold the key to understanding the development, organization and maintenance of the cellular diversity that characterizes metazoan life.
Isolating planarian stem cells
Agata's laboratory has developed a method for identifying subsets of planarian stem cells, also called neoblasts. These somatic stem cells are the only mitotically active cells in the planarian body, making them susceptible to X-ray irradiation. Examination of cell populations shown to be vulnerable to elimination by X-rays, revealed that planarian neoblasts, like stem cells in other species, seem to exist in proliferating and resting states. Real-time PCR analysis indicates that each of these sub-populations expresses discrete sets of genes. One fraction, X1, expresses genes specific to actively cycling cells, while a second fraction, X2, lacks this expression profile and is thought to comprise stem cells in a state of quiescence. Interestingly, many X1 cells also express signal receptor molecules which are switched off in the X2 fraction, while the corresponding ligands are expressed in a variety of differentiated cells resistant to X-ray irradiation.
Brain regeneration by stages
A planarian can re-grow a fully functional brain within five days following the amputation of its entire head, an extraordinary feat of self-healing that involves recapitulating the development of the worm's entire nervous system. Researchers in the Agata group studied this process and identified patterns of gene expression that indicate the regeneration of the brain comprises five distinct stages. In the first stage, at about eight hours after wound closure, a noggin-like gene (Djnlg) is activated in the stump prior to the formation of a blastema, a mass of proliferating undifferentiated cells that serves as the frontline of regeneration. Soon thereafter, the brain-specifying gene nou-darake is switched on in the anterior fringe of the blastema, allowing the brain rudiment to be formed. These first two steps occur within 24 hours of decapitation, and set the stage for the third phase in which brain patterning is established by the expression of a set of three otd/Otx-related genes (relatives of which also function to pattern brain development in many other taxa, including vertebrates) followed by that of the planarian homolog of Wnt at about 48 hours into the regenerative process. In the fourth step, which occurs by day four of regeneration, a homolog of the axon-guidance gene netrin is triggered. At this stage the discrete components of the regenerating brain begin to form connections and higher-order organizations; chiasmata cross-linking eyes and brain develop, and connections are set up between the brain and the ventral nerve cords, which serve as a rudimentary peripheral nervous system. Thus, the basic components and connections of the planarian neural network are in place by the fourth day, but the functional recovery of the brain is not completed until a final stage in which two newly-identified genes are expressed is entered.
A parallel study revealed that these two genes, 1020HH and eye53, are necessary for planarians to regain their normal light avoidance behavior, known as negative phototaxis. Knockdown of these genes by RNAi had no discernible effects on brain or eye morphology, but the worms failed to respond to light stimuli in the normal manner, even after five days of regeneration, when the structure of the brain has been fully reinstated.
Evolution of the central nervous system
The planarian is one of the lowest forms of animal known to possess a central nervous system, making it an apt model for the study of the evolution of this most complex and elaborately organized biological system. Using clones of over 3,000 expressed sequence tags (ESTs) isolated from the planarian head region, the team was able to identify 116 clones with significant similarities to genes closely linked to the nervous system in other species, including genes involved in neurotransmission, the neural network, brain morphogenesis and neural differentiation. These results point toward a shared evolutionary origin for many nervous system genes, indicating that the nervous system is likely to have arisen in a single common ancestor of bilaterian animals. Intriguingly, nearly 30% of the genes identified also have homologous sequences in yeast and the plant species, Arabidopsis thaliensis, both of which entirely lack neural development, which suggests that a significant number of genes that predate the advent of the nervous system have been co-opted to perform functions specific to neurobiology.
The Agata lab is also now participating in an international collaboration established to sequence and annotate a set of more than 10,000 planarian ESTs, which promises to provide an invaluable resource for the study of evolutionary biology, comparative genomics, and the genetics underlying the unique characteristics of these organisms. The availability of an annotated database of planarian cDNAs may provide keys to the understanding of stem cell biology, tissue plasticity and maintenance and other fundamentally important processes.
Agata K. Regeneration and Gene Regulation in Planarians. Curr Opin Genet Dev 13:492-6 (2003).
Mineta K, Nakazawa M, Cebria F, Ikeo K, Agata K and Gojobori T. Origin and Evolutionary Process of the CNS Elucidated by Comparative Genomics Analysis of Planarian ESTs. Proc Natl Acad Sci U S A 100:7666-71 (2003).
Nakazawa M, Cebria F, Mineta K, Ikeo K, Agata K and Gojobori T. Search for the Evolutionary Origin of a Brain: Planarian Brain Characterized by Microarray. Mol Biol Evol 20:784-91 (2003).
Agata K, Tanaka T, Kobayashi C, Kato K and Saitoh Y. Intercalary Regeneration in Planarians. Dev Dyn 226:308-16 (2003).
Saito Y, Koinuma S, Watanabe K and Agata K. Mediolateral Intercalation in Planarians Revealed by Grafting Experiments. Dev Dyn 226:334-40 (2003).
Cebria F, Kobayashi C, Umesono Y, Nakazawa M, Mineta K, Ikeo K, Gojobori T, Itoh M, Taira M, Sanchez Alvarado A and Agata K. FGFR-related gene nou-darake restricts brain tissues to the head region of planarians. Nature 419:620-4 (2002).