Characterization of regeneration initiating cells during Xenopus laevis tail regeneration [spatial transcriptomics]

In this experiment, we revealed the critical steps for regeneration initiation. We discovered Regeneration Initiating Cells (RICs) using single cell and spatial transcriptomics of the regenerating Xenopus laevis tail. RICs are formed transiently from the basal epidermal cells and are critical for the modification of the surrounding extracellular matrix to allow for migration of other cell types that promote regeneration. Absence or deregulation of RICs leads to excessive extracellular matrix deposition and regeneration defects. Overall design: X. laevis tadpoles at stage 40 (regenerative) and stage 46 (refractory) were analysed. Amputation was performed manually (removal of ~30% of tail tissue) using tricaine anesthetized tadpoles, with 6 replicates for refractory and 8 for regenerating embryos. Together we analyzed: 1) regenerative 6 hours post amputation (hpa) and 1 days post amputation (dpa), 2) refractory 6 hpa and 1 dpa, 3) regenerative and refractory 3 dpa. Dissected tissues were embedded and oriented in 50% optimal cutting temperature (OCT) medium, rapidly frozen using dry ice and then transferred to -80°C for a maximum of six weeks of storage. Samples were sectioned sagitally (20 µm thickness) using Leica CM1950 cryostat (Leica Microsystems). Sections collected for the 10X Visium Spatial Gene Expression processing were then stored at -80°C.