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. 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.

摘要：蜥蜴是目前已知唯一具备多谱系插层再生（multilineage epimorphic regeneration）能力的羊膜动物（amniotes），同时也是与哺乳动物亲缘关系最近的类群。绿色安乐蜥（Anolis carolinensis）的尾芽基（tail blastemas）由col3a1阳性的成纤维结缔组织细胞构成，外层被krt5阳性的创伤表皮（wound epidermis, WE）包裹，二者均为再生过程所必需。已知芽基与创伤表皮的形成与巨噬细胞、破骨细胞等吞噬细胞群体密切相关，但目前仍不清楚具体哪些吞噬细胞类型可刺激再生进程。本研究明确评估了破骨细胞活性在蜥蜴尾再生过程中芽基与创伤表皮形成阶段的作用。首先，本研究针对基于col3a1表达分离得到的成纤维细胞，在再生相关时间节点开展探针测序（probe-sequencing），以解析参与诱导芽基形成及后续再生的信号通路。随后，通过破骨细胞抑制剂唑来膦酸（zoledronic acid, ZA）处理蜥蜴，以此探究破骨细胞活性在蜥蜴尾再生及成纤维细胞信号传导中的功能。实验结果显示，ZA处理会阻滞蜥蜴尾的再生生长，表明破骨细胞活性是芽基形成与再生所必需的。对经ZA处理及对照组蜥蜴分离得到的成纤维细胞进行转录组分析（transcriptomic profiling），结果表明破骨活性抑制会限制成纤维细胞形成细胞外基质并支持创伤表皮形成的能力。本研究结果提示，在蜥蜴尾再生过程中，破骨细胞与成纤维细胞之间的信号串扰（crosstalk）可调控芽基与创伤表皮的形成。此外，本研究还对截肢后7、14、28天以及经ZA或磷酸盐缓冲液（phosphate buffered saline, PBS）处理的第21天的安乐蜥成纤维细胞，进行了固定、荧光原位杂交（Fluorescence in situ hybridization, FISH）分选后的RNA测序（RNA-seq）分析。