Exploring the Role of Kazald2 in Axolotl Limb Regeneration through Computational Approaches

Abstract

Regenerative medicine aims to restore tissues and functions lost to damage. The Mexican axolotl (Ambystoma mexicanum) serves as an ideal model organism for regeneration because it has limbs that are anatomically similar to humans and it is also able to fully regenerate a limb after an amputation throughout its life. In a 2017 paper, the Whited lab identified Kazald2 as a gene critical to axolotl limb regeneration. Building upon these results, this current project was designed to explore differential expression between Kazald2-/- (knockout) organisms and Kazald2+/+ (wild type) organisms. Using computational genomic tools such as kallisto and DESeq2, we identified a subset of transcripts with significant expression changes between the two experimental conditions. Of these, we were particularly interested in the differential expression of a host of early stress response genes (Fos, Junb, Atf3, Egr1) and a transcript with connections to mTOR (Eif4ebp2). We next employed pathfindR to perform a pathway enrichment analysis in order to elucidate pathways impacted by the loss of Kazald2 gene. From these results, we identified the mitogen-activated protein kinase (MAPK) pathway as significantly enriched, and were able to experimentally validate the connection between KAZALD2 and MAPK at the protein level through a western blot. These findings support the hypothesis that Kazald2 plays a key role in cellular regulation during regeneration. Beyond this immediate project, this research hopes to contribute to the comprehensive understanding of axolotl limb regeneration as a model for potential future applications in human limb regeneration.