Enhancing regenerative capacities is a fundamental goal in medicine. As yet, the principles of salamander regeneration to augment mammalian healing are not directly applicable. Here we propose using lizards, more closely related to mammals yet exhibiting remarkable regenerative capabilities, as model organisms in a set of studies aimed at manipulating skeletal regeneration capacities. While both salamanders and lizards regenerate their tails, salamanders regenerate near-perfect copies of original tails, while regenerated lizard tails are known as an “imperfect replicates” with several key anatomical differences compared to originals. The most striking of these “imperfections” concerns the lack of dorsoventral patterning and segmentation in regenerated lizard tail skeletons. Progress made under our original proposal identified the signals regulating regenerated skeletal tissue induction and patterning, creating the first dorsoventrally-patterned regenerated lizard tails. This renewal proposal focuses on later stages of skeletal maturation that, given the proper signals, culminate in segmentation. Our recent comparative analyses indicate that regenerated skeleton segmentation is dependent upon three distinct milestones: (1) perichondrium patterning, (2) cartilage hypertrophy, and (3) periosteum formation. Both salamander and lizard regenerated tail skeletons begin as unsegmented cartilage elements. Our preliminary findings suggest a novel role for spinal cord meningeal tissues in regulating skeleton segmentation. Regenerated salamander, but not lizard, meninges contain specialized cell populations capable of recreating embryonic segmentation signals in adjacent perichondrium and initiating a signaling cascade that transforms the entire regenerated skeleton. Some of these signals induce cartilage hypertrophy in salamander cartilage before anti- ossification processes that dominate lizard skeletons have the chance to stagnate cartilage maturation. Other signals missing in lizard tails allow salamander bone cells to survive and promote periosteum development. Based on this comparative analysis, we hypothesize the feasibility of mechanistically based interventions to shift the “imperfectly” regenerating lizard tail to phenocopy the “perfectly” regenerating salamander tail. The Aims are: (1) Introduce patterning to regenerated lizard tail perichondrium by supplementing adult spinal cord meninges with embryonic segmenting cells; (2) Induce regenerated lizard cartilage hypertrophy by interrupting anti- maturation signals that result in dysregulated cartilage development; and (3) Promote periosteum formation within regenerated lizard tails by inducing bone cell survival and recruitment. An integrated approach is proposed, incorporating a unique, asexually reproducing lizard species with in vivo surgical manipulations to deliver cells and bioactive agents toward manipulating skeletal development. We believe that this approach will produce the first regenera...