Identification of TOR-responsive slow-cycling neoblasts in planarians. Identification of TOR-responsive slow-cycling neoblasts in planarians

Epimorphic regeneration commonly relies on the activation of quiescent stem cells to drive new cell production. The planarian Schmidtea mediterranea is among the best regenerators in nature, thanks to its large population of adult stem cells, called neoblasts. While neoblasts have long been known to drive regeneration, whether a subset of neoblasts is reserved for this purpose is unknown. Here, we revisited the idea of reserved neoblasts by approaching neoblast heterogeneity from a regulatory perspective. By implementing a new fluorescence-activated cell sorting strategy in planarians, we identified a population of neoblasts defined by low transcriptional activity. These RNAlow neoblasts are relatively slow-cycling at homeostasis and undergo a morphological regeneration response characterized by cell growth at 48 hours post-amputation. At this time, RNAlow neoblasts proliferated in a TOR-dependent manner. Additionally, knockdown of the tumour suppressor Lrig-1, which is enriched in RNAlow neoblasts, resulted in RNAlow neoblast growth and hyperproliferation at homeostasis, and ultimately delayed regeneration. We propose that slow-cycling RNAlow neoblasts represent a regeneration-reserved neoblast population. Overall design: Bulk or single cells from wild type animals were collected from FACS gates set based on DNA (Hoechst) and RNA (pyrnonin Y) content and libraries were prepared and sequenced.

后生再生（Epimorphic regeneration）通常依赖于静息干细胞的激活以驱动新细胞生成。地中海真涡虫（Schmidtea mediterranea）是自然界中再生能力最为强悍的物种之一，其体内拥有大量被称为新胚细胞（neoblasts）的成体干细胞。尽管长期以来学界已知新胚细胞可驱动再生过程，但其中是否存在专门留存以服务于再生的亚群，目前仍未明确。
本研究从调控视角解析新胚细胞的异质性，以此重新探讨“留存性新胚细胞”这一假说。我们通过在涡虫中建立全新的荧光激活细胞分选（fluorescence-activated cell sorting, FACS）策略，鉴定出一类以低转录活性为特征的新胚细胞亚群。这类RNAlow新胚细胞（RNAlow neoblasts）在稳态条件下增殖速度相对缓慢，并在截肢后48小时进入以细胞生长为特征的形态学再生应答过程。在此阶段，RNAlow新胚细胞以雷帕霉素靶蛋白（TOR, target of rapamycin）依赖的方式进行增殖。此外，在RNAlow新胚细胞中富集表达的肿瘤抑制因子Lrig-1，当其被敲低后，会导致稳态条件下RNAlow新胚细胞的异常生长与过度增殖，并最终延缓再生进程。我们据此提出：增殖缓慢的RNAlow新胚细胞即为负责再生的留存性新胚细胞亚群。
实验整体设计：本研究从基于DNA（Hoechst）与RNA（吡咯啉Y, pyrnonin Y）染色设置的荧光激活细胞分选门中，收集野生型涡虫的批量细胞或单细胞，随后构建测序文库并进行测序。