PROJECT SUMMARY Human patients who lose limbs due to injury or disease are faced with profound challenges for the rest of their lives. Prostheses, while increasingly sophisticated, lack key functionalities, and amputees report dissatisfaction with current prosthetic options. A long-term goal of regenerative medicine is to develop therapeutic limb regeneration strategies. Yet, this goal remains in the distant future because of a fundamental lack of scientific understanding of how to stimulate and guide a patient’s own cells to create a new limb. Animal models are likely to be key in building this missing foundation in scientific understanding of how to regenerate a limb. While mice offer many advantages in genetic studies, they are extremely limited in their limb regeneration abilities—like humans, mice are only naturally capable of regenerating the extreme distal tip of their digits. Full, natural limb regeneration has not been reported in any mammal to date. Frogs can naturally regenerate full limbs but only as tadpoles before development is complete. In contrast, many (if not all) salamander species studied to date can regenerate full limbs following amputation throughout life. Salamander limbs are remarkably similar in tissue composition and anatomy to human limbs. Thus, salamanders are ideal models for elucidating the fundamental biological processes required to regenerate limbs. Among salamanders, axolotls have emerged as a premier model because many genetic and experimental tools have now been developed for axolotl. An important missing piece of the overall puzzle is how stem cells are specified to form during axolotl embryological development and how these relate to the axolotl’s ability to regenerate limbs later in life. Here, we propose to use modern molecular genetic tools to build a map of transcriptional control of axolotl embryogenesis with single-cell resolution. This resource will enable us to identify the origin and nature of stem cells in axolotl embryos, and it will enable other researchers to develop hypotheses about other cell types as well. We will identify stem cells as they arise during development, along with the key transcripts that distinguish these cells from other cells, which will be essential for future studies, including those directed at understanding how limb regeneration may use stem cells. In parallel, in a complementary strategy, we will interrogate the embryological origins of 8 putative fibroblast stem cell types we recently isolated from axolotl limbs and demonstrated become activated to proliferate by amputation. We will extend this analysis to developing limbs. This work is essential for understanding the contribution of stem cells to the diverse tissues of the regenerate limb. It will also pave the way to experimentally manipulate specific stem cell populations in future studies in order to rigorously define their activities and the factors they use to execute these functions.