Elly Tanaka - Cellular mechanisms of regeneration in vertebrates

Previous and current research

Molecular genetics of the Axolotl
Regeneration has traditionally been considered a difficult research topic due to the complex nature of adult tissue. The amputation site in the limb or tail contains numerous different tissue types. How each one responds to the injury, and how the tissues interact in order to produce a pool of progenitor cells competent to undergo regeneration is a daunting task.

To untangle this complex web, we have focused on developing the molecular genetics of the Axolotl. Having developed an efficient transgenesis protocol for the animal (Sobkow et al, 2005), we are refining these techniques to label specific cell and tissue types, and to perturb gene expression in these respective cells. To do this we have implemented site-specific recombination. We have generated lines of transgenic animals harboring site-specific recombination sites that allow us to turn on gene expression in a tissue-dependent, or time dependent manner by controlling expression of the recombinase.

Molecular control of blastema formation from different cell sources
Limb and tail regeneration occur by producing a group of progenitor cells called the blastema. Recently we have been characterizing the make-up of the limb blastema to determine if it is a homogeneous pool of highly multipotent stem cells, or a collection of lineage restricted cells. In the limb we have tracked dermal cells, Schwann cells and cartilage cells during regeneration and find limited abilities of cells to transdifferentiate into other tissues. This means that the blastema is a heterogeneous pool of progenitor cells from the start.

Using our tissue and cell-specific cell tracking methods, we are currently undertaking a molecular analysis to determine the genetic programs that are activated in specific tissues during blastema formation.

Molecular control of spinal cord regeneration
Regeneration of the tail is a particularly interesting topic because it involves regeneration of the spinal cord, and overall reconstruction of the primary body axis. We have shown that irrespective of where the tail is cut, the stem cells in a 500μm length of the spinal cord is recruited to regenerate the missing portion. The progenitor cells in this part of the spinal cord increase their divisions to form an elongating neuroepithelial tube. The cells at the end of this growing tube form a closed bulb called the terminal vesicle. Interestingly, cells escape the terminal vesicle into the surrounding blastema.

We are currently characterizing the genes that determine this 500μm growth zone and how wounding signals to the spinal cord to activate its progenitor cells. We are also studying the cell biological properties of the cells that allows them to reconstruct the spinal cord.

We have found that one important property of the axolotl spinal cord that allows it to regenerate so well is the retention of an embryonic spatial coordinate information system in the stem cells of the adult tissue—a property that is lost early in development in the mammalian embryo. This spatial information includes the local production of extracellular signalling molecules such as sonic hedgehog, as well as the downstream response genes in the stem cells. Interestingly, during tail regeneration, sonic hedgehog is required not only to determine the dorsal versus ventral domains of the spinal cord as in embryogenesis, but is also required for the growth of the whole blastema.

Application to mammalian systems
We seek to apply the knowledge that we gain in the axolotl system to the mammal, both in order to develop prospective therapeutic strategies, as well as novel insights into the basic mechanisms of tissue formation. In terms of muscle dedifferentiation, we are investigating whether the cell-cycle stimulating factor from serum and other blastema factors elicit a full or partial response in mammalian myotubes, and how a partial response may be boosted by genetic interventions such as via gene knockdown using siRNAs.

In the spinal cord, we are using our knowledge to engineer mouse neural stem cells so that they will form a regenerative neural tube in vitro. We are growing different types of neural stem cells in three-dimensional culture conditions that mimic as closely as possible, the growth and patterning conditions of the regenerating spinal cord.

Future prospects and goals

Evolution of regenerative ability
We are focussing our efforts more and more toward comparing the properties of the axolotl system to the mammal in order to understand the cellular and molecular basis of regenerative ability. This comparison necessarily interfaces with the evolutionary considerations of how regeneration arose in some animals, or more likely, was lost in many organisms. By understanding the fundamental cellular properties that underlie regenerating systems and the molecular pathways that control them, we hope in future to understand how the these molecular modules have evolved in different organisms to account for these properties.

Size control of regenerating structures
We are also interested in how the regenerating structure determines the extent to which it should grow. Tail amputation of a 2 cm long larvae versus a 20 cm long adult results in a regenerate that is proportionally scaled to the main body of the animal. This issue will clearly have implications in attempts to engineer tissues for replacement therapy, as the tissues must be scaled to the host.

Environmental influences on regeneration
It is clear that regenerative ability depends not only in the cell intrinsic properties of the axolotl cells, but also to the environmental conditions that the cells find themselves in after injury. We are interested in determining what distinguishes a permissive environment versus a hostile environment for regeneration.

Selected publications

Lööf S., Straube WL., Drechsel D., Tanaka EM., Simon A. (2007): Plasticity of mammalian myotubes upon stimulation with a thrombin-activated serum factor. Cell Cycle; 6:1096-101

Mchedlishvili L., Epperlein HH., Telzerow A., Tanaka EM.(2007): A clonal analysis of neural progenitors during axolotl spinal cord regeneration reveals evidence for both spatially restricted and multipotent progenitors. Development; 134:2083-2093

Sobkow L., Epperlein HH., Herklotz S., Straube WL., Tanaka EM.(2006): A germline GFP transgenic axolotl and its use to track cell fate: dual origin of the fin mesenchyme during development and the fate of blood cells during regeneration. Dev Biol; 290:386-97

Mercader N., Tanaka EM., Torres M.(2005): Proximodistal identity during vertebrate limb regeneration is regulated by Meis homeodomain proteins. Development; 132:4131-42

Schnapp E., Kragl M., Rubin L., Tanaka EM.(2005): Hedgehog signaling controls dorsoventral patterning, blastema cell proliferation and cartilage induction during axolotl tail regeneration. Development; 132:3243-53

Echeverri E., Tanaka EM.(2005): Proximodistal patterning during limb regeneration Dev Biol; 279:391-401

Schnapp E., Tanaka EM.(2005): Quantitative evaluation of morpholino-mediated protein knockdown of GFP, MSX1 and PAX7 during tail regeneration in Ambystoma mexicanum Dev Dyn; 232:162-70

Elly Tanaka

1993: PhD at University of California in San Francisco, Department of Biochemistry

1994-1999: Post-doctoral fellow, Ludwig Institute for Cancer Research, London and University College, London, Department of Biochemistry and Molecular Biology

since 1999: Group Leader at the Max Planck Institute of Molecular Cell Biology and Genetics