Limb loss is a major health concern in the U.S. with nearly two million patients living with the consequences of a major limb amputation. This number is expected to rise with increases in key risk factors, and no biological therapeutics have been devised to address this problem. While humans have exceedingly limited regenerative abilities in limbs and other key structures, axolotl salamanders can regenerate entire limbs throughout their lives. Axolotl limbs are anatomically similar to human limbs, and they develop by similar mechanisms. Gaining a thorough understanding of the molecular mechanisms that enable axolotl limb regeneration stands to offer critical insights into future approaches that may be taken in regenerative medicine, which could in turn revolutionize the treatment options offered to patients facing amputation. This thorough mechanistic understanding has evaded researchers to date because of a paucity of tools available for experimentally manipulating gene expression in axolotls. However, within the last eight years, we-and others-have developed powerful molecular genetic tools that are operational in vivo in axolotls. We propose to leverage these developments to take a fresh look at the longstanding and important question of vertebrate limb regeneration. Axolotls regenerate limbs by elaborating a spatially- and temporally-controlled progenitor cell pool, the blastema. Blastemas are critical for limb regeneration, but their creation and growth are poorly understood. Additionally, information to create the new limb, such as positional information and tissue composition, is encoded within blastemas, and the molecular logic underlying this information-encoding is unknown. Through a massive RNA-sequencing effort, we have identified candidate genes whose expression is greatly upregulated in regenerating axolotl blastema cells relative to all other tissue types sampled. Most of these genes have known human orthologs, while some are likely novel. We further analyzed these blastema enriched transcripts at the single-cell level by performing single-cell RNA-seq and differential gene expression analysis. Using this pipeline, we identified a gene, AxRBP-1, whose robust expression across all blastema cells we experimentally determined to be required for proper limb regeneration. Here we propose to perform a secondary, experimental analysis, on the existing RNA-seq/single-cell dataset to determine if other blastema- enriched genes also perform functions essential for limb regeneration. In Specific Aim 1, we will test the necessity of 6 blastema-enriched genes by knocking down gene expression using morpholinos and then by gene editing for selected genes. In Specific Aim 2, we will test the requirement for temporal, spatial, and/or lineage restriction of the expression of these genes by misexpression using our retrovirus. These secondary analyses will allow us to prioritize specific genes for future studies aimed at elucidating the precise mechanisms whereby they operate to control limb regeneration while testing the feasibility of this approach.