How the axolotl makes a new limb Unlike most vertebrate limbs, the axolotl limb regenerates the skeleton after amputation. Dermal and interstitial fibroblasts have been thought to provide sources for skeletal regeneration, but it has been unclear whether preexisting stem cells or dedifferentiation of fibroblasts formed the blastema. Gerber et al. developed transgenic reporter animals to compare periskeletal cell and fibroblast contributions to regeneration. Callus-forming periskeletal cells extended existing bone, but fibroblasts built new limb segments. Single-cell transcriptomics and Brainbow-based lineage tracing revealed the lack of a preexisting stem cell. Instead, the heterogeneous population of fibroblasts lost their adult features to form a multipotent skeletal progenitor expressing the embryonic limb program. Science, this issue p. eaaq0681 Deconstructing cell composition, reconstructing lineage relationships, and tracing tissue reprogramming in limb regeneration are explored. INTRODUCTION Axolotls (Ambystoma mexicanum) and other salamanders are the only tetrapods that can regenerate whole limbs. During this complex process, changes in gene expression regulate the outgrowth of a new appendage, but how injury induces limb cells to form regenerative progenitors that differentiate into diverse cell fates is poorly understood. Tracking and molecular profiling of individual cells during limb regeneration would resolve distinct differentiation pathways and provide clues for how cells convert from a mature resting state into regenerative cell lineages. RATIONALE Axolotl limbs are composed of many different cell types originating from neural, myogenic, epidermal, and connective tissue (CT) lineages. Upon limb amputation, cells from nearby the amputation plane accumulate in a distinctive tissue called the blastema, which serves as a progenitor cell source to build the new limb. Transgenic axolotl strains in which descendants of distinct adult cell types can be labeled, tracked, and isolated during the regenerative process provide an opportunity to understand how particular cell lineages progress during blastema formation and subsequent limb regrowth. Combining transgenic axolotl strains with single-cell RNA sequencing (scRNA-seq) enables the tracking of individual cell types, as well as the reconstruction of the molecular steps underlying the regeneration process for these particular cell lineages. CT cells, descendants of lateral plate mesoderm, are the most abundant lineage contributing to the blastema and encompass bone and cartilage, tendons, periskeleton, and dermal and interstitial fibroblasts. These cells detect the position of the amputation site, leading to the regeneration of appropriate limb parts and making the CT a key cell lineage for deciphering and understanding molecular programs of regeneration. RESULTS We used an inducible Cre-loxP fluorescence system to establish genetically marked transgenic axolotl strains for isolating CT cells from adult limb tissue as well as CT descendants in the blastema. We used scRNA-seq to molecularly profile CT cells along a dense time course of blastema formation and the outgrowth of the regenerated arm, as well as stages of embryonic limb development. This profiling indicated that CT cells express adult phenotypes that are lost upon the induction of regeneration. The heterogeneous population of CT-derived cells converges into a homogeneous and transient blastema progenitor state that at later stages recapitulates an embryonic limb bud–like program. Notably, we did not find evidence of CT stem cells or blastema-like precursors in the mature arm. We found that CT subtypes have spatially restricted contributions to proximal and distal compartments in the regenerated limb. Specifically, a particular CT subtype—periskeletal cells—extended the severed skeleton at the amputation site whereas fibroblastic CT cells de novo regenerated distal skeletal segments. By using high-throughput single-cell transcriptomics and Brainbow axolotl-based clonal lineage tracing, we could follow the redifferentiation trajectories of CT lineages during the final stages of regeneration. These findings established the formation of a multipotent skeletal progenitor cell that contributed to tendons, ligaments, skeleton, periskeleton, and fibroblasts. CONCLUSION CT cells are a key cell type for understanding regeneration because they form the patterned limb skeleton that guides the regeneration of the other limb tissues, such as muscle. Because of the cells’ heterogeneity and intermingling with other cell types, it had been difficult to study how CT forms regenerative blastema cells. The use of these newly generated transgenic reporter strains combined with single-cell transcriptomic analysis and clonal tracing have allowed us to determine that CT cells with diverse molecular features traverse through a distinctive molecular state as they dedifferentiate into a common, multipotent progenitor resembling an embryonic limb bud cell. In the future, it will be important to test which components of the transition state are required for the dedifferentiation process. Furthermore, this work opens the possibility to examine how regeneration-associated genes and their associated chromatin structure are regulated during this transition. Lastly, the work raises the possibility that the limited regeneration seen among mammals is due to an inability to reprogram CT to such embryonic states. Regeneration of the axolotl upper arm CT. Transgenic axolotl strains were used to isolate CT cells at different stages during limb regeneration. Single-cell transcriptomes were generated, and transcriptional signatures were used to track the fate of cells during regeneration. We found that heterogeneous CT cell types in the adult upper arm funnel into a homogeneous multipotent skeletal progenitor state that recapitulates axolotl limb development, before their redifferentiation into the heterogeneous CT cell subtypes. Amputation of the axolotl forelimb results in the formation of a blastema, a transient tissue where progenitor cells accumulate prior to limb regeneration. However, the molecular understanding of blastema formation had previously been hampered by the inability to identify and isolate blastema precursor cells in the adult tissue. We have used a combination of Cre-loxP reporter lineage tracking and single-cell messenger RNA sequencing (scRNA-seq) to molecularly track mature connective tissue (CT) cell heterogeneity and its transition to a limb blastema state. We have uncovered a multiphasic molecular program where CT cell types found in the uninjured adult limb revert to a relatively homogenous progenitor state that recapitulates an embryonic limb bud–like phenotype including multipotency within the CT lineage. Together, our data illuminate molecular and cellular reprogramming during complex organ regeneration in a vertebrate.

中文翻译：

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单细胞分析揭示了蝾螈肢体再生过程中细胞身份的趋同

蝾螈如何制造新肢体 与大多数脊椎动物的肢体不同，蝾螈肢体在截肢后会再生骨骼。真皮和间质成纤维细胞被认为是骨骼再生的来源，但尚不清楚是预先存在的干细胞还是成纤维细胞的去分化形成了胚基。格伯等人。开发了转基因报告动物来比较骨周细胞和成纤维细胞对再生的贡献。形成愈伤组织的骨周细胞延伸了现有的骨骼，但成纤维细胞构建了新的肢体节段。单细胞转录组学和基于 Brainbow 的谱系追踪揭示了缺乏预先存在的干细胞。相反，异质的成纤维细胞群失去了其成年特征，形成了表达胚胎肢体程序的多能骨骼祖细胞。科学，本期第 14 页。eaaq0681 探索了肢体再生中解构细胞组成、重建谱系关系和追踪组织重编程。简介 蝾螈（Ambystoma mexicanum）和其他蝾螈是唯一可以再生整个肢体的四足动物。在这个复杂的过程中，基因表达的变化调节新附属物的生长，但损伤如何诱导肢体细胞形成分化成不同细胞命运的再生祖细胞却知之甚少。在肢体再生过程中对单个细胞进行跟踪和分子分析将解决不同的分化途径，并为细胞如何从成熟的静息状态转变为再生细胞谱系提供线索。基本原理 蝾螈四肢由许多不同的细胞类型组成，这些细胞类型源自神经、肌源、表皮和结缔组织 (CT) 谱系。肢体截肢后，截肢平面附近的细胞会积聚在一种称为胚基的独特组织中，该组织作为构建新肢体的祖细胞来源。转基因蝾螈品系可以在再生过程中标记、追踪和分离不同成体细胞类型的后代，这为了解特定细胞谱系在芽基形成和随后的肢体再生过程中如何进展提供了机会。将转基因蝾螈品系与单细胞 RNA 测序 (scRNA-seq) 相结合，能够追踪单个细胞类型，并重建这些特定细胞谱系再生过程背后的分子步骤。CT 细胞是侧板中胚层的后代，是对胚基最丰富的谱系，包括骨和软骨、肌腱、外骨骼以及真皮和间质成纤维细胞。这些细胞检测截肢部位的位置，从而导致适当肢体部位的再生，并使 CT 成为破译和理解再生分子程序的关键细胞谱系。结果我们使用诱导型 Cre-loxP 荧光系统建立了基因标记的转基因蝾螈品系，用于从成体肢体组织以及胚基中的 CT 后代中分离 CT 细胞。我们使用 scRNA-seq 对 CT 细胞沿芽基形成和再生臂生长以及胚胎肢体发育阶段的密集时间过程进行分子分析。该分析表明 CT 细胞表达在诱导再生后丢失的成体表型。CT 衍生细胞的异质群体会聚成同质且短暂的胚基祖细胞状态，在后期阶段重现胚胎肢芽样程序。值得注意的是，我们没有在成熟臂中发现 CT 干细胞或胚基样前体细胞的证据。我们发现 CT 亚型对再生肢体的近端和远端区室的贡献在空间上受到限制。具体来说，一种特定的 CT 亚型——骨周细胞——在截肢部位延伸了切断的骨骼，而成纤维细胞 CT 细胞则从头再生了远端骨骼段。通过使用高通量单细胞转录组学和基于 Brainbow 蝾螈的克隆谱系追踪，我们可以追踪 CT 谱系在再生最后阶段的再分化轨迹。这些发现证实了多能骨骼祖细胞的形成，该祖细胞有助于肌腱、韧带、骨骼、外骨骼和成纤维细胞。结论 CT 细胞是理解再生的关键细胞类型，因为它们形成有图案的肢体骨架，指导其他肢体组织（例如肌肉）的再生。由于细胞的异质性以及与其他细胞类型的混合，研究 CT 如何形成再生胚基细胞非常困难。使用这些新生成的转基因报告菌株，结合单细胞转录组分析和克隆追踪，我们能够确定具有不同分子特征的 CT 细胞在去分化为类似于胚胎肢体的常见多能祖细胞时，会经历独特的分子状态芽细胞。将来，测试去分化过程需要过渡态的哪些组成部分将很重要。此外，这项工作为研究再生相关基因及其相关染色质结构在这一转变过程中如何受到调节提供了可能性。最后，这项工作提出了一种可能性，即哺乳动物的再生能力有限是由于无法将 CT 重新编程为胚胎状态。蝾螈上臂 CT 再生。使用转基因美西螈品系来分离肢体再生过程中不同阶段的CT细胞。生成了单细胞转录组，并使用转录特征来追踪细胞在再生过程中的命运。我们发现，成人上臂中的异质 CT 细胞类型在重新分化为异质 CT 细胞亚型之前，会进入同质多能骨骼祖细胞状态，从而概括蝾螈肢体的发育。蝾螈前肢的截肢会导致胚基的形成，胚基是一种短暂的组织，在肢体再生之前祖细胞会在其中积累。然而，之前由于无法识别和分离成体组织中的胚基前体细胞，阻碍了对胚基形成的分子理解。我们结合使用 Cre-loxP 报告基因谱系追踪和单细胞信使 RNA 测序 (scRNA-seq) 来分子追踪成熟结缔组织 (CT) 细胞异质性及其向肢体芽基状态的转变。我们发现了一个多相分子程序，在未受伤的成年肢体中发现的 CT 细胞类型恢复到相对同质的祖细胞状态，再现了胚胎肢芽样表型，包括 CT 谱系内的多能性。总之，我们的数据阐明了脊椎动物复杂器官再生过程中的分子和细胞重编程。