Limb Regeneration David M. Gardiner University of California Irvine

Regeneration is wide-spread and common Hydra Crustacean Planarian Drosophila Worm salamander Fish frog Mouse There is variability in the ways that animals regenerate, suggesting that regeneration has evolved multiple times. Therefore there likely will be multiple strategies to consider, and each model system has something to teach us. This view begs the question of why humans have not evolved the ability to regenerate organs.

What do you need to regenerate an organ (like a limb)? - Regeneration-competent cells that can do what needs to be done - Migrate - Proliferate - Differentiate - Regeneration-permissive environment (the niche) - Regeneration-instructive environment

The regeneration blastema is equivalent to a developing limb bud Apical Epithelial Cap (AEC) is equivalent to the AER Blastema mesenchyme is equivalent to the limb bud mesenchyme Zone of “dedifferentiation” is unique to regeneration Mature (differentiated) proximal tissues are the source of the blastema The source of regeneration-competent cells

Up to 80% (nearly 50% on average) of the early blastema cells come from dermal fibroblasts. Dermal fibroblasts migrate and proliferate to form the early regeneration blastema

Epidermis  Wound Epithelium  AEC Stump Cells  Blastema - Contribution to the Blastema - Dermis (Fibroblast): 19%  43% Muscle (Myofibers): 55%  17% Skeleton (cartilage/bone): 6%  2% What is the source of the blastema cells? - Tissues in the stump all contribute to the blastema - Tissues in the stump do not contribute in proportion to their availability - Progenitor cells are lineage restricted (except for fibroblasts)

There are three challenges: 1- Non-regenerating models are like playing the Lotto 2- Amputation studies in regenerating models are inherently loss-of-function analyses 3- Amputations create noise (extraneous signals) that obscures the regeneration signals The goal is to reduce the complexity of regeneration and design gain-of-function experiments for the identification of the signals that are necessary and sufficient for regeneration

The Accessory Limb Model Studies of ectopic limb formation tell us that all you need are: - specialized epidermis - a nerve - fibroblast-derived blastema cells

- Make a wound - Deviate a nerve to the wound site to stimulate formation of an ectopic blastema How to Make an Ectopic Blastema

How to Make an Ectopic Arm Graft a piece of skin (epidermis + dermis) from the opposite side of the limb to the wound site

From Akira Satoh, 2010

Regeneration is a step-wise process Regeneration No Regeneration Regeneration is a complex, integrated sequence of events (“epimorphic regeneration”), and if you isolate any one of these steps it looks like “tissue regeneration”. Some models can do it all and some can do only a little bit; however, it is all a part of the whole, just to varying degrees

Understanding pattern formation (the blueprint function) is essential to understanding regeneration An organ has emergent properties – a blueprint

AP AP A A P P Connective tissue fibroblasts are the progenitors of the early blastema cells - Fibroblasts have positional information - Positional interactions between fibroblasts create the blueprint for limb regeneration

The outcome of regeneration is dependent on positional information Growth and Pattern formation are controlled by local cell-cell interactions

Akira Satoh Katrina Llewellyn Kate McCusker Jason Lee Anne Phan Cristian Aguilar Jeff Lehrberg Kim Liuzzi Gillian Cummings Ryan Lank Veronica Aguilar Sue Bryant Defense Advance Research Projects Agency – DOD Army Research Office - DOD Ambystoma Genetic Stock Center – NSF Organogenesis, Inc. Ken Muneoka Tetsuya Endo