Salamander limb regeneration is a nerve-dependent process. In the absence of nerves, blastema cells (i.e. cells that give rise to the new structure) will not proliferate. A major question in regeneration biology is how the nerve exerts its growth promoting effect on blastema cells. To better understand the cellular and molecular signals responsible for growth and pattern formation during regeneration, I have adapted the use organotypic slice culture for axolotl blastemas. Using microarray, I have identified BMP2 as a possible nerve derived factor responsible for growth and proliferation during regeneration. Here I will demonstrate how organotypic slice cultures are responsive to culture conditions and the candidate neurotrophic factor BMP2 in both terms of levels of proliferation and gene expression.

The formation of a blastema during regeneration of an axolotl limb involves important changes in the behavior and function of cells at the site of injury. One of the earliest events is the formation of the wound epithelium and subsequently the apical epidermal cap, which involves in vivo dedifferentiation that is controlled by signaling from the nerve. I have investigated the role of epigenetic modifications to the genome as a possible mechanism for regulating changes in gene expression patterns of keratinocytes of the wound and blastema epithelium that are involved in regeneration. I report a modulation of the expression DNMT3a, a de novo DNA methyltransferase, within the first 72 hours post injury that is dependent on nerve signaling. Treatment of skin wounds on the upper forelimb with decitabine, a DNA methyltransferase inhibitor, induced changes in gene expression and cellular behavior associated with a regenerative response. Furthermore, decitabine-treated wounds were able to participate in regeneration while untreated wounds inhibited a regenerative response. Elucidation of the specific epigenetic modifications that mediate cellular dedifferentiation likely will lead to insights for initiating a regenerative response in organisms that lack this ability. To this end, a novel RNA seq experiment has been designed to compare the transcription profiles of tissues that have undergone dedifferentiation to uninjured tissues and tissues that participate in simple wound healing. In addition, tissues treated with a DNA methyltransferase inhibitor will be evaluated for changes in gene expression that are a result of DNA methylation dynamics specifically. As the mechanisms behind in vivo cellular dedifferentiation are elucidated and applied, it will be important to use an appropriate assay in order to determine the success and extent of dedifferentiation. I include here a developed protocol designed to assay for cellular dedifferentiation called the Excisional Repair Model (ERM) model. Briefly, treated cells or tissues are grafted into a cavity in the lower forearm created by the excision of a segment of the radius. The ability of the treated cells or tissue to differentiate and replace the missing bone fragment, and incorporate into the existing bone reveals their regenerative capacity.

Salamanders are unique among adult vertebrates in their ability to regenerate complex body structures after traumatic injury. Axolotl limb regeneration is a stepwise sequence of three requisite processes: (1) scarless wound healing to generate a regenerative wound epithelium, (2) blastema formation by migration, proliferation and dedifferentiation to create a mass of multipotent regeneration-competent progenitor cells, and (3) induction of pattern formation by interaction of cells with opposing positional information. The challenge to understanding how to induce and enhance regenerative ability in humans is to discover which of these processes fail in humans and why they fail.

In axolotl regeneration, cells maintain a memory of their original position and use this `positional information' to accurately regenerate the lost pattern. The goal of this project was to elucidate the molecular mechanism of positional information in axolotl limb regeneration and determine if it is conserved in mammals.

I used an in vivo gain-of-function assay for the requisite processes of axolotl limb regeneration (Accessory Limb Model) to determine the minimal components required to mediate positional information and induce de novo limb pattern.

I found that cell-free anterior and posterior extracellular matrix (ECM) have position-specific signaling properties that either inhibit regeneration or induce de novo limb pattern. The position-specific functionality of the ECM is established by differential expression of heparan sulfate sulfotransferases during regeneration resulting in position-specific growth factor regulation. My findings indicate that heparan sulfates are both necessary and sufficient to mediate positional information in axolotl limb regeneration. Similarly, mouse ECM was also capable of inducing limb pattern in a position-specific, heparan sulfate-dependent, age-dependent manner. Using mammalian-derived, commercially available factors, I synthetically induced axolotl limb regeneration, thus demonstrating that the factors required for regeneration are not salamander-specific.

This study demonstrates a specific novel mechanism for positional information in axolotl limb regeneration and establishes a crucial functional link between salamander regeneration and mammals. The ability to regulate axolotl regeneration with mammalian ECM has allowed identification of target mechanisms to induce regeneration in humans. In addition to the fields of regeneration, development, and glycobiology, this novel mechanism of positional information has implications to the fields of cancer biology and aging.