Progenitor cell formation from connective tissue during Axolotl limb regeneration
Final Report Abstract
Our goal was to implement transgenic reporter animals to track the fate of cartilage and fibroblast cells during limb regeneration and determine if these cells become a multipotent skeletal progenitor during regeneration. We also sought to identify molecules involved in inducing these connective tissue cells to become blastema cells. This was performed by generating transgenic reporter animals. On one hand, we generated brainbow transgenics that allowed us to live image digit regeneration where we concluded that cells do become multipotent during regeneration, and that cell migration, controlled by PDGF signalling is essential for blastema cell formation. Interestingly, cells migrated into the blastema in waves, and the timepoint of migration was related to the ultimate fate of the cells. On the other hand, we performed long-term genetic fate mapping using a series of tissue-specific cre drivers. Here we came to the surprising finding that the periskeletal cells are involved in elongating an existing bone but do not contribute to new limb segments while the soft connective tissue regenerates all cells of the lower limb skeleton/connective tissue. By molecular profiling we could show that connective tissue cells pass through a unique state while dedifferentiating into an embryonic limb bud-like progenitor. Long-term clonal fate mapping showed that cells form a multi-potent skeletal progenitor during regeneration. We also identified several candidate genes involved in connective cell dedifferentiation, and developed a baculovirus-expression system that allows us to test the function of these genes.
Publications
- (2018) Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration. Science (New York, N.Y.) 362 (6413)
Gerber, Tobias; Murawala, Prayag; Knapp, Dunja; Masselink, Wouter; Schuez, Maritta; Hermann, Sarah; Gac-Santel, Malgorzata; Nowoshilow, Sergej; Kageyama, Jorge; Khattak, Shahryar; Currie, Joshua D.; Camp, J. Gray; Tanaka, Elly M.; Treutlein, Barbara
(See online at https://doi.org/10.1126/science.aaq0681) - (2013). Connective tissue cells, but not muscle cells, are involved in establishing the proximo-distal outcome of limb regeneration in the axolotl. Development. 140:513-8
Nacu E, Glausch M, Le HQ, Damanik FF, Schuez M, Knapp D, Khattak S, Richter T, Tanaka EM
(See online at https://doi.org/10.1242/dev.081752) - (2014). Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, metamorphosis, transgenesis and tamoxifen-mediated recombination. Nat Protoc. 9:529-540
Khattak, S., P. Murawala, H. Andreas, V. Kappert, M. Schuez, T. Sandoval-Guzman, K. Crawford, and E.M. Tanaka
(See online at https://doi.org/10.1038/nprot.2014.040) - (2016) Live Imaging of Axolotl Digit Regeneration Reveals Spatiotemporal Choreography of Diverse Connective Tissue Progenitor Pools. Dev Cell. 39:411-423
Currie JD, Kawaguchi A, Traspas RM, Schuez M, Chara O, Tanaka EM
(See online at https://doi.org/10.1016/j.devcel.2016.10.013) - The Molecular and Cellular Choreography of Appendage Regeneration. (2016). Cell. 165:1598-608
Tanaka EM
(See online at https://doi.org/10.1016/j.cell.2016.05.038) - (2017) Pseudotyped baculovirus is an effective gene expression tool for studying molecular function during axolotl limb regeneration. Dev Biol. S0012-1606(17)30438-4
Oliveira CR, Lemaitre R, Murawala P, Tazaki A, Drechsel DN, Tanaka EM
(See online at https://doi.org/10.1016/j.ydbio.2017.10.008)
