CellCousin2: an optimized system for partial ablation and tracing of regenerative lineages

Regeneration can rely on multiple cellular sources, including stem cells, self-duplicating cells, and transdifferentiating cells. A central question in regenerative biology is how these distinct lineages contribute to repair and interact within a functionally regenerated tissue. We previously developed the CellCousin system to study cellular plasticity using inducible recombination and nitroreductase-mediated ablation in zebrafish. Here, we present CellCousin2, which introduces two key improvements for the long-term tracking of spared and regenerating cells. First, to reduce background recombination, we developed a Dihydrofolate Reductase (DHFR)-CreER system with dual control: DHFR-mediated degradation in the absence of trimethoprim, and tamoxifen-dependent activation. This combination minimizes leakiness while maintaining high recombination efficiency. Second, we replaced the original nitroreductase with NTR2.0, enabling effective ablation with a tenfold lower metronidazole concentration, reducing off-target effects on the liver. Together, these enhancements make CellCousin2 a robust platform for dissecting the dynamics and interactions of regenerative lineages.