Cellular regulation of pattern in the regenerating limb

About This Grant

PROJECT SUMMARY Human limb amputations can occur at any position along the limb axis, yet clinical strategies to regenerate partial limb structures remain limited. The Mexican axolotl provides a powerful model to study this challenge, as it can fully regenerate limb segments regardless of amputation level. During regeneration, anterior (A) and posterior (P) limb cells express distinct molecular signals that are essential for organizing the spatial pattern of the new tissue. However, how these cells direct the formation of precisely the missing structures, no more, no less, remains a critical unanswered question. Recent work has revealed that A and P cells are distributed asymmetrically along the proximal-distal (Pr/Di) axis of the axolotl limb. Preliminary spatial transcriptomic and qRT-PCR data show that A- and P-specific gene expression closely correlates with these asymmetric cellular distributions in both uninjured and regenerating limbs. Prior studies suggest that changing the dosage of A or P cells can shift regenerative outcomes, but the effect of these changes on gene expression domains and final tissue patterning has not been systematically examined. This project tests the hypothesis that A/P cell composition acts as a key regulator of regenerative patterning by influencing both the molecular landscape and anatomical output. Specifically, the research will address three major knowledge gaps: (1) how varying A/P cell ratios alter the expression and spatial localization of A/P and Pr/Di patterning genes; (2) how A/P composition controls the Pr/Di positioning of regenerated structures; and (3) how A/P asymmetry is reestablished in the regenerating tissue. Aim 1 will use spatial transcriptomics and in vivo regenerative assays to define the natural spatial expression patterns of A/P and Pr/Di genes and cell populations. Aim 2 will use targeted grafting approaches to manipulate A/P cell ratios and assess resulting effects on gene expression and regenerated limb morphology. Aim 3 will determine whether A-to-P cell conversion contributes to the restoration of asymmetry using lineage tracing, single-cell and spatial omics, and pharmacological perturbation of P-cell-specific signals. By defining how A/P cell composition directs the location and identity of regenerated structures, this project will provide a foundational mechanistic framework for understanding patterning in complex tissue regeneration. These insights will inform future efforts to guide partial limb regeneration in humans and accelerate the development of regenerative therapies for limb loss