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

Abstract: Lizards are distinguished as the only amniotes, and closest relatives of mammals, capable of multilineage epimorphic regeneration. Tail blastemas of green anole lizards (Anolis carolinensis) consist of col3a1+ fibroblastic connective tissue cells enclosed in krt5+ 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 perfomed at regenerative timepoints on fibroblasts isolated based on col3a1 expression toward establishing pathways involved in stimulating blastema formation and subsequent regeneration. Next, treatments with osteoclast inhibitor zoledronic acid (ZA) was used to assess roles of osteoclast activity in lizard tail regeneration and fibroblast signlaing. 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 oseolat activity with limitations in fibroblats to form extracellular matrix and support WE formation. These results suggests that crosstalk between osteoclasts and fibroblasts regulate blastema and WE formation during lizard tail regeneration. Overall design: RNA-seq profiling of fixed, FISH sorted anolis carolinensis fibroblasts at 7,14, and 28 days post amputation as well as D21 treated with zoledronic acid (za) or a PBS control.