Probe Sequencing Analysis of Regenerating Lizard Tails Indicates Crosstalk Among Osteoclasts, Epidermal Cells, and Fibroblasts

Lizards are distinguished as the only amniotes, and closest relatives of mammals, capable of multilineage epimorphic regeneration. Tail blastemas of green anole lizards (<i>Anolis carolinensis</i>) consist of <i>col3a1⁺</i> fibroblastic connective tissue cells enclosed in <i>krt5⁺</i> wound epidermis (WE), both of which are required for regeneration. Blastema and WE formation are known to be closely associated with phagocytic cell populations, including macrophages and osteoclasts. However, it remains unclear what specific phagocytic cell types are required to stimulate regeneration. Here, we explicitly assess the roles of osteoclast activity during blastema and WE formation in regenerating lizard tails. First, probe sequencing was performed at regenerative timepoints on fibroblasts isolated based on <i>col3a1</i> expression toward establishing pathways involved in stimulating blastema formation and subsequent tail regrowth. Next, treatments with osteoclast inhibitor zoledronic acid (ZA) were used to assess the roles of osteoclast activity in lizard tail regeneration and fibroblast signaling. ZA treatment stunted lizard tail regrowth, suggesting osteoclast activity was required for blastema formation and regeneration. Transcriptomic profiling of fibroblasts isolated from ZA-treated and control lizards linked inhibition of osteoclast activity with limitations in fibroblasts to form pro-regenerative extracellular matrix and support WE formation. These results suggest that crosstalk between osteoclasts and fibroblasts regulates blastema and WE formation during lizard tail regeneration.