Mechanisms of Skeletal Patterning During Limb Development and Regeneration

Limb musculoskeletal defects are among the most common causes of disability in the United States affecting 0.8/1000 children born each year. Considering the importance of skeletal function in human disease, gaining an understanding of skeletal patterning will undoubtedly enable strategies for alleviating musculoskeletal diseases. In this proposal, we aim to provide insight into skeletal formation by studying a clinically relevant signaling molecule, retinoic acid, in regeneration. The vertebrate limb is a premier model for studying developmental mechanisms because it is accessible, has a morphologically complex skeletal pattern, and is subject to developmental and age-related pathologies. A long-term goal of medicine is to leverage this knowledge to regenerate skeletal elements. Of particular use are animals that can regenerate naturally after limb amputation such as axolotl salamanders (Ambystoma mexicanum). Salamanders regenerate the limb by generating a heterogeneous mass of lineage-restricted progenitors called a blastema. The blastema uses positional information retained in connective tissue cells to regenerate the proper skeletal elements. The goal of this proposal is to study how positional information is established and maintained in connective tissue cells, which is critical to our understanding of limb regeneration. We will use unbiased spatial transcriptomic approaches to compare gene expression at single-cell resolution to understand the endogenous role of retinoic acid signaling. Furthermore, we will determine the genomic targets of retinoic acid specifically in limb connective tissue cells that are undergoing reprogramming from distal to proximal cell identity. Lastly, manipulation of spatial identity will be performed in connective tissue cells in order to “engineer” connective tissue cells in vivo. In the end, the major outcome of this project will be to know how a salamander limb knows what skeletal structures to grow back.