Urodele amphibians, such as the axolotl, have the unique ability to regenerate their limbs throughout their adult life. The process of limb regeneration proceeds after amputation with migration of epidermal cells across the amputation plane to form the regeneration epithelium (RE). Direct epithelial-mesenchymal interaction is essential between the RE and the underlying stump tissue to promote the formation of a mass of dedifferentiated cells called the blastema. The blastema redevelops into the missing structures of the limb. Redevelopment requires positional identity of the mesenchymal cells so that the appropriate structures regenerate with proper patterning.
Despite decades of research, it is unclear how the regeneration process is induced to proceed past the wound healing stage. This thesis presents the Positional Discontinuity Model, which hypothesizes that the RE is a specialized wound epithelium that provides an instructive signal for the induction of regeneration as a result of its formation by contact of epidermal cells with different positional identities. There is no literature precedent for epidermal positional identity so microarray analysis was used to profile gene expression in four positions of axolotl epidermal tissue. A list of 25 genes that are more highly expressed in one position than the opposing position are discussed.
Furthermore, there are few markers for the RE during the earliest stages of limb regeneration when induction is hypothesized to occur. Microarray analysis was again used to profile gene expression of the RE. A list of 125 genes and expressed sequence tags (ESTs) were identified. A subset of these genes was analyzed over the time course of regeneration to understand their expression profiles. One gene in particular, sodefrin precursor-like factor (axSPF), was analyzed further using functional assays. Knockdown of axSPF at the wound healing stage caused the blastema to be smaller as compared to the control at the medium-bud stage. The mechanism of action of axSPF is still unknown.
Finally, in order to begin to understand the differences in regeneration ability between axolotls and humans, microarray expression profiling on human epidermal tissue was used to identify a list of 277 genes that were differentially expressed between dorsal and ventral positions.
This study provides the first description of gene expression profiling in epidermis samples from the axolotl. The results will provide useful markers for further research as well as an opportunity to compare epidermal biology in humans to that of the axolotl, a regeneration-competent animal.
|Advisor:||Crews, Craig M.|
|School Location:||United States -- Connecticut|
|Source:||DAI-B 73/05, Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology, Developmental biology|
|Keywords:||Epimorphic regeneration, Limb regeneration, Regeneration epithelium|
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