Single cell gene expression profiling using 10x v3

Regeneration is a widespread phenomenon that often requires formation of a new tissue outgrowth at wounds called a blastema. Identifying the key signals that trigger blastema formation is therefore fundamental for understanding the mechanistic basis of regeneration. We uncovered a role for the wound epidermis in regeneration initiation in planarians. The gene equinox is expressed within hours of injury in the planarian wound epidermis and encodes a secreted protein conserved in many phyla. Following equinox inhibition, animals failed to form blastemas, reset positional information, and upregulate stem cell (neoblast) proliferation at wounds. Associated with these defects was an inability to maintain wound-induced gene expression. We propose that activation of equinox in the wound epidermis initiates planarian regeneration. Overall design: FACS sorted cells were isolated from post-pharyngeal wound fragments at 0, 6, and 18 hours post-amputation (hpa) from planarians. One condition was fed control RNAi four times over two weeks prior to the sort. Only x-insensitives were collected. All conditions except for the 6 hour were sorted based on hoeschst high, propidium iodide low. The 6 hour time point was sorted on hoechst high, calcein medium to hight to allow for more damaged cells to be captured.