Cellular responses in the FGF10‐mediated improvement of hindlimb regenerative capacity in Xenopus laevis revealed by single‐cell transcriptomics

Xenopus laevis tadpoles possess regenerative capacity in their hindlimb buds at early developmental stages (stages ~52–54); they can regenerate complete hindlimbs with digits after limb bud amputation. However, they gradually lose their regenerative capacity as metamorphosis proceeds. Tadpoles in late developmental stages regenerate fewer digits (stage ~56), or only form cartilaginous spike without digits or joints (stage ~58 or later) after amputation. Previous studies have shown that administration of fibroblast growth factor 10 (FGF10) in late‐stage (stage 56) tadpole hindlimb buds after amputation can improve their regenerative capacity, which means that the cells responding to FGF10 signaling play an important role in limb bud regeneration. In this study, we performed single‐cell RNA sequencing (scRNA‐seq) of hindlimb buds that were amputated and administered FGF10 by implanting FGF10‐soaked beads at a late stage (stage 56), and explored cell clusters exhibiting a differential gene expression pattern compared with that in controls treated with phosphate‐buffered saline. The scRNA‐seq data showed expansion of fgf8‐expressing cells in the cluster of the apical epidermal cap of FGF10‐treated hindlimb buds, which was reported previously, indicating that the administration of FGF10 was successful. On analysis, in addition to the epidermal cluster, a subset of myeloid cells and a newly identified cluster of steap4‐expressing cells showed remarkable differences in their gene expression profiles between the FGF10‐ or phosphate‐buffered saline‐treatment conditions, suggesting a possible role of these clusters in improving the regenerative capacity of hindlimbs via FGF10 administration.

非洲爪蟾蝌蚪在发育早期阶段（约52 - 54期）的后肢芽具有再生能力；它们在肢芽截断后能够再生出带有趾的完整后肢。然而，随着变态发育的进行，它们逐渐失去再生能力。发育后期的蝌蚪在截断后再生的趾数量减少（约56期），或者在截断后仅形成没有趾或关节的软骨刺（约58期或更晚）。先前的研究表明，在后期（56期）蝌蚪后肢芽截断后施用成纤维细胞生长因子10（FGF10）能够提高它们的再生能力，这意味着对FGF10信号作出反应的细胞在肢芽再生中起重要作用。在这项研究中，我们对在后期（56期）截断并通过植入浸泡有FGF10的珠子施用FGF10的后肢芽进行了单细胞RNA测序（scRNA - seq），并探索了与用磷酸盐缓冲盐水处理的对照组相比表现出差异基因表达模式的细胞群。scRNA - seq数据显示，在FGF10处理的后肢芽顶端表皮帽细胞群中，表达fgf8的细胞增多，这与先前的报道一致，表明FGF10的施用是成功的。经分析，除了表皮细胞群外，一部分髓样细胞和一个新鉴定的表达steap4的细胞群在FGF10或磷酸盐缓冲盐水处理条件下的基因表达谱存在显著差异，这表明这些细胞群可能在通过施用FGF10提高后肢再生能力方面发挥作用。