Newts can regrow their entire limbs upon amputation, including when challenged with multiple rounds of removal and regeneration. However, it is not clear whether this extraordinary capacity is supported by specific subpopulations recruited from the limb stump. My goal is to develop an unbiased method that combines clonal lineage tracing and single cell RNA sequencing to investigate the regenerative capacity of Iberian ribbed newts (Pleurodeles waltl). DNA barcodes are genomically encoded unique sequences that mark individual clones and can be retrieved alongside each cells’ transcriptome. I will take two approaches to create barcoded newt cells. The first one is to deliver directly to the limbs a barcode library [1] that can be integrated via MMLV retroviral activity [2]. This library is currently being built by the lab. The second approach employs a germline-encoded cassette – Polylox [3] – that can be randomly recombined following inducible Cre activity. Lineage tracing using either of these approaches will be followed by retrieval of the barcoded cells and single-cell RNA sequencing. The analyzed data will include information about both cell state (transcriptome library) and cell lineage (barcode library). This way, it will be possible to identify at clonal level which cells can support repeated events of limb regeneration. This data can help finding key mechanisms for regeneration in newts, including multigenerational stem cell markers, as well as give clues on how to stimulate a latent regenerative potential of other species under a therapeutic perspective. References: [1] Ratz M, von Berlin L, Larsson L, Martin M, Westholm JO, La Manno G, Lundeberg J, Frisén J. Clonal relations in the mouse brain revealed by single-cell and spatial transcriptomics. Nat Neurosci. 2022 Mar;25(3):285-294. doi: 10.1038/s41593-022-01011-x. [2] Whited JL, Tsai SL, Beier KT, White JN, Piekarski N, Hanken J, Cepko CL, Tabin CJ. Pseudotyped retroviruses for infecting axolotl in vivo and in vitro. Development. 2013 Mar;140(5):1137-46. doi: 10.1242/dev.087734. [3] Pei W, Shang F, Wang X, Fanti AK, Greco A, Busch K, Klapproth K, Zhang Q, Quedenau C, Sauer S, Feyerabend TB, Höfer T, Rodewald HR. Resolving Fates and Single-Cell Transcriptomes of Hematopoietic Stem Cell Clones by PolyloxExpress Barcoding. Cell Stem Cell. 2020 Sep 3;27(3):383-395.e8. doi: 10.1016/j.stem.2020.07.018.