RESUMEN
We present a multiomic cell atlas of human lung development that combines single-cell RNA and ATAC sequencing, high-throughput spatial transcriptomics, and single-cell imaging. Coupling single-cell methods with spatial analysis has allowed a comprehensive cellular survey of the epithelial, mesenchymal, endothelial, and erythrocyte/leukocyte compartments from 5-22 post-conception weeks. We identify previously uncharacterized cell states in all compartments. These include developmental-specific secretory progenitors and a subtype of neuroendocrine cell related to human small cell lung cancer. Our datasets are available through our web interface (https://lungcellatlas.org). To illustrate its general utility, we use our cell atlas to generate predictions about cell-cell signaling and transcription factor hierarchies which we rigorously test using organoid models.
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Feto , Pulmón , Humanos , Diferenciación Celular , Perfilación de la Expresión Génica , Pulmón/citología , Organogénesis , Organoides , Atlas como Asunto , Feto/citologíaRESUMEN
Understanding the genetic and molecular drivers of phenotypic heterogeneity across individuals is central to biology. As new technologies enable fine-grained and spatially resolved molecular profiling, we need new computational approaches to integrate data from the same organ across different individuals into a consistent reference and to construct maps of molecular and cellular organization at histological and anatomical scales. Here, we review previous efforts and discuss challenges involved in establishing such a common coordinate framework, the underlying map of tissues and organs. We focus on strategies to handle anatomical variation across individuals and highlight the need for new technologies and analytical methods spanning multiple hierarchical scales of spatial resolution.
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Variación Anatómica , Diagnóstico por Imagen/normas , Examen Físico/normas , Diagnóstico por Imagen/métodos , Humanos , Examen Físico/métodos , Estándares de ReferenciaRESUMEN
Metastasis constitutes the primary cause of cancer-related deaths, with the lung being a commonly affected organ. We found that activation of lung-resident group 2 innate lymphoid cells (ILC2s) orchestrated suppression of natural killer (NK) cell-mediated innate antitumor immunity, leading to increased lung metastases and mortality. Using multiple models of lung metastasis, we show that interleukin (IL)-33-dependent ILC2 activation in the lung is involved centrally in promoting tumor burden. ILC2-driven innate type 2 inflammation is accompanied by profound local suppression of interferon-γ production and cytotoxic function of lung NK cells. ILC2-dependent suppression of NK cells is elaborated via an innate regulatory mechanism, which is reliant on IL-5-induced lung eosinophilia, ultimately limiting the metabolic fitness of NK cells. Therapeutic targeting of IL-33 or IL-5 reversed NK cell suppression and alleviated cancer burden. Thus, we reveal an important function of IL-33 and ILC2s in promoting tumor metastasis via their capacity to suppress innate type 1 immunity.
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Eosinófilos/inmunología , Células Asesinas Naturales/inmunología , Neoplasias Pulmonares/inmunología , Pulmón/inmunología , Linfocitos/inmunología , Animales , Línea Celular Tumoral , Citotoxicidad Inmunológica , Humanos , Tolerancia Inmunológica , Inmunidad Innata , Interleucina-33/metabolismo , Interleucina-5/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Células Th2/inmunologíaRESUMEN
The major and essential objective of pre-implantation development is to establish embryonic and extra-embryonic cell fates. To address when and how this fundamental process is initiated in mammals, we characterize transcriptomes of all individual cells throughout mouse pre-implantation development. This identifies targets of master pluripotency regulators Oct4 and Sox2 as being highly heterogeneously expressed between blastomeres of the 4-cell embryo, with Sox21 showing one of the most heterogeneous expression profiles. Live-cell tracking demonstrates that cells with decreased Sox21 yield more extra-embryonic than pluripotent progeny. Consistently, decreasing Sox21 results in premature upregulation of the differentiation regulator Cdx2, suggesting that Sox21 helps safeguard pluripotency. Furthermore, Sox21 is elevated following increased expression of the histone H3R26-methylase CARM1 and is lowered following CARM1 inhibition, indicating the importance of epigenetic regulation. Therefore, our results indicate that heterogeneous gene expression, as early as the 4-cell stage, initiates cell-fate decisions by modulating the balance of pluripotency and differentiation.
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Proteínas Adaptadoras de Señalización CARD/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción SOXB2/metabolismo , Animales , Blastocisto/metabolismo , Factor de Transcripción CDX2 , Epigénesis Genética , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Proteínas de Homeodominio/genética , Ratones , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/metabolismo , Factores de Transcripción SOXB1/metabolismo , Análisis de la Célula Individual , Factores de Transcripción/genéticaRESUMEN
The recent flood of single-cell data not only boosts our knowledge of cells and cell types, but also provides new insight into development and evolution from a cellular perspective. For example, assaying the genomes of multiple cells during development reveals developmental lineage trees-the kinship lineage-whereas cellular transcriptomes inform us about the regulatory state of cells and their gradual restriction in potency-the Waddington lineage. Beyond that, the comparison of single-cell data across species allows evolutionary changes to be tracked at all stages of development from the zygote, via different kinds of stem cells, to the differentiating cells. We discuss recent insights into the evolution of stem cells and initial attempts to reconstruct the evolutionary cell type tree of the mammalian forebrain, for example, by the comparative analysis of neuron types in the mesencephalic floor. These studies illustrate the immense potential of single-cell genomics to open up a new era in developmental and evolutionary research.
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Evolución Biológica , Biología Evolutiva , Genómica , Análisis de la Célula Individual/métodos , Animales , Epigénesis Genética , Humanos , Células Madre/metabolismoRESUMEN
How cells respond to myriad stimuli with finite signaling machinery is central to immunology. In naive T cells, the inherent effect of ligand strength on activation pathways and endpoints has remained controversial, confounded by environmental fluctuations and intercellular variability within populations. Here we studied how ligand potency affected the activation of CD8+ T cells in vitro, through the use of genome-wide RNA, multi-dimensional protein and functional measurements in single cells. Our data revealed that strong ligands drove more efficient and uniform activation than did weak ligands, but all activated cells were fully cytolytic. Notably, activation followed the same transcriptional pathways regardless of ligand potency. Thus, stimulation strength did not intrinsically dictate the T cell-activation route or phenotype; instead, it controlled how rapidly and simultaneously the cells initiated activation, allowing limited machinery to elicit wide-ranging responses.
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Linfocitos T CD8-positivos/fisiología , Citotoxicidad Inmunológica , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Animales , Línea Celular , Genoma , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , ARN/genética , Transducción de Señal , Análisis de la Célula IndividualRESUMEN
Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months1. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common2. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.
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To mechanistically characterize the microevolutionary processes active in altering transcription factor (TF) binding among closely related mammals, we compared the genome-wide binding of three tissue-specific TFs that control liver gene expression in six rodents. Despite an overall fast turnover of TF binding locations between species, we identified thousands of TF regions of highly constrained TF binding intensity. Although individual mutations in bound sequence motifs can influence TF binding, most binding differences occur in the absence of nearby sequence variations. Instead, combinatorial binding was found to be significant for genetic and evolutionary stability; cobound TFs tend to disappear in concert and were sensitive to genetic knockout of partner TFs. The large, qualitative differences in genomic regions bound between closely related mammals, when contrasted with the smaller, quantitative TF binding differences among Drosophila species, illustrate how genome structure and population genetics together shape regulatory evolution.
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Evolución Molecular , Ratones/clasificación , Ratones/genética , Factores de Transcripción/genética , Animales , Drosophila/genética , Hígado/metabolismo , Ratones/metabolismo , Ratones Endogámicos , Ratones Noqueados , Ratas/genética , Factores de Transcripción/metabolismoRESUMEN
Early organogenesis represents a key step in animal development, during which pluripotent cells diversify to initiate organ formation. Here, we sampled 300,000 single-cell transcriptomes from mouse embryos between E8.5 and E9.5 in 6-h intervals and combined this new dataset with our previous atlas (E6.5-E8.5) to produce a densely sampled timecourse of >400,000 cells from early gastrulation to organogenesis. Computational lineage reconstruction identified complex waves of blood and endothelial development, including a new programme for somite-derived endothelium. We also dissected the E7.5 primitive streak into four adjacent regions, performed scRNA-seq and predicted cell fates computationally. Finally, we defined developmental state/fate relationships by combining orthotopic grafting, microscopic analysis and scRNA-seq to transcriptionally determine cell fates of grafted primitive streak regions after 24â h of in vitro embryo culture. Experimentally determined fate outcomes were in good agreement with computationally predicted fates, demonstrating how classical grafting experiments can be revisited to establish high-resolution cell state/fate relationships. Such interdisciplinary approaches will benefit future studies in developmental biology and guide the in vitro production of cells for organ regeneration and repair.
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Gastrulación , Organogénesis , Ratones , Animales , Diferenciación Celular , Organogénesis/genética , Línea Primitiva , Endotelio , Embrión de Mamíferos , MamíferosRESUMEN
The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.
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Movimiento Celular , Rastreo Celular , Células/citología , Biología Evolutiva/métodos , Embrión de Mamíferos/citología , Feto/citología , Difusión de la Información , Organogénesis , Adulto , Animales , Atlas como Asunto , Técnicas de Cultivo de Célula , Supervivencia Celular , Visualización de Datos , Femenino , Humanos , Imagenología Tridimensional , Masculino , Modelos Animales , Organogénesis/genética , Organoides/citología , Células Madre/citologíaRESUMEN
The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.
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Envejecimiento , Sistema Nervioso Entérico/citología , Feto/citología , Salud , Intestinos/citología , Intestinos/crecimiento & desarrollo , Ganglios Linfáticos/citología , Ganglios Linfáticos/crecimiento & desarrollo , Adulto , Animales , Niño , Enfermedad de Crohn/patología , Conjuntos de Datos como Asunto , Sistema Nervioso Entérico/anatomía & histología , Sistema Nervioso Entérico/embriología , Sistema Nervioso Entérico/crecimiento & desarrollo , Células Epiteliales/citología , Femenino , Feto/anatomía & histología , Feto/embriología , Humanos , Intestinos/embriología , Intestinos/inervación , Ganglios Linfáticos/embriología , Ganglios Linfáticos/patología , Ratones , Ratones Endogámicos C57BL , Organogénesis , Receptores de IgG/metabolismo , Transducción de Señal , Análisis Espacio-Temporal , Factores de TiempoRESUMEN
The balance between self-renewal and differentiation in human foetal lung epithelial progenitors controls the size and function of the adult organ. Moreover, progenitor cell gene regulation networks are employed by both regenerating and malignant lung cells, where modulators of their effects could potentially be of therapeutic value. Details of the molecular networks controlling human lung progenitor self-renewal remain unknown. We performed the first CRISPRi screen in primary human lung organoids to identify transcription factors controlling progenitor self-renewal. We show that SOX9 promotes proliferation of lung progenitors and inhibits precocious airway differentiation. Moreover, by identifying direct transcriptional targets using Targeted DamID, we place SOX9 at the centre of a transcriptional network, which amplifies WNT and RTK signalling to stabilise the progenitor cell state. In addition, the proof-of-principle CRISPRi screen and Targeted DamID tools establish a new workflow for using primary human organoids to elucidate detailed functional mechanisms underlying normal development and disease.
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Pulmón , Factor de Transcripción SOX9 , Células Madre , Humanos , Diferenciación Celular/fisiología , Pulmón/embriología , Transducción de Señal , Factor de Transcripción SOX9/metabolismo , Células Madre/metabolismoRESUMEN
Droplet-based single-cell assays, including single-cell RNA sequencing (scRNA-seq), single-nucleus RNA sequencing (snRNA-seq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), generate considerable background noise counts, the hallmark of which is nonzero counts in cell-free droplets and off-target gene expression in unexpected cell types. Such systematic background noise can lead to batch effects and spurious differential gene expression results. Here we develop a deep generative model based on the phenomenology of noise generation in droplet-based assays. The proposed model accurately distinguishes cell-containing droplets from cell-free droplets, learns the background noise profile and provides noise-free quantification in an end-to-end fashion. We implement this approach in the scalable and robust open-source software package CellBender. Analysis of simulated data demonstrates that CellBender operates near the theoretically optimal denoising limit. Extensive evaluations using real datasets and experimental benchmarks highlight enhanced concordance between droplet-based single-cell data and established gene expression patterns, while the learned background noise profile provides evidence of degraded or uncaptured cell types.
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ARN Nuclear Pequeño , Programas Informáticos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Perfilación de la Expresión Génica/métodosRESUMEN
Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.
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Tratamiento Basado en Trasplante de Células y Tejidos , Atención a la Salud/métodos , Atención a la Salud/tendencias , Medicina/métodos , Medicina/tendencias , Patología , Análisis de la Célula Individual , Inteligencia Artificial , Atención a la Salud/ética , Atención a la Salud/normas , Diagnóstico Precoz , Educación Médica , Europa (Continente) , Femenino , Salud , Humanos , Legislación Médica , Masculino , Medicina/normasRESUMEN
Effector cytotoxic T lymphocytes (CTLs) are critical for ridding the body of infected or cancerous cells. CTL T cell receptor (TCR) ligation not only drives the delivery and release of cytolytic granules but also initiates a new wave of transcription. In order to address whether TCR-induced transcriptomic changes impact the ability of CTLs to kill, we asked which genes are expressed immediately after CTLs encounter targets and how CTL responses change when inhibiting transcription. Our data demonstrate that while expression of cytokines/chemokines and transcriptional machinery depend on transcription, cytotoxic protein expression and cytolytic activity are relatively robust to transcription blockade, with CTLs lysing nearby target cells for several hours after actinomycin D treatment. Monitoring CTL movement among target cells after inhibiting transcription demonstrates an infiltration defect that is not rectified by provision of exogenous cytokine/chemokine gradients, indicating a cell-intrinsic transcriptional requirement for infiltration. Together, our results reveal differential molecular control of CTL functions, separating recruitment and infiltration from cytolysis.
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Citocinas , Linfocitos T Citotóxicos , Linfocitos T Citotóxicos/metabolismo , Citocinas/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Biochemical reactions are intrinsically stochastic, leading to variation in the production of mRNAs and proteins within cells. In the scientific literature, this source of variation is typically referred to as 'noise'. The observed variability in molecular phenotypes arises from a combination of processes that amplify and attenuate noise. Our ability to quantify cell-to-cell variability in numerous biological contexts has been revolutionized by recent advances in single-cell technology, from imaging approaches through to 'omics' strategies. However, defining, accurately measuring and disentangling the stochastic and deterministic components of cell-to-cell variability is challenging. In this Review, we discuss the sources, impact and function of molecular phenotypic variability and highlight future directions to understand its role.
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Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.
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Diferenciación Celular/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Gastrulación , Organogénesis , Análisis de la Célula Individual , Animales , Linaje de la Célula/genética , Quimera/embriología , Quimera/genética , Quimera/metabolismo , Endodermo/citología , Endodermo/embriología , Endodermo/metabolismo , Endotelio/citología , Endotelio/embriología , Endotelio/metabolismo , Femenino , Gastrulación/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Hematopoyesis/genética , Masculino , Mesodermo/citología , Mesodermo/embriología , Ratones , Mutación/genética , Células Mieloides/citología , Organogénesis/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Línea Primitiva/citología , Línea Primitiva/embriología , Proteína 1 de la Leucemia Linfocítica T Aguda/deficiencia , Proteína 1 de la Leucemia Linfocítica T Aguda/genéticaRESUMEN
Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes1-5. Global epigenetic reprogramming accompanies these changes6-8, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.
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Metilación de ADN , Epigénesis Genética , Gástrula/citología , Gástrula/metabolismo , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , ARN/genética , Análisis de la Célula Individual , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Cromatina/genética , Cromatina/metabolismo , Desmetilación , Cuerpos Embrioides/citología , Endodermo/citología , Endodermo/embriología , Endodermo/metabolismo , Elementos de Facilitación Genéticos/genética , Epigenoma/genética , Eritropoyesis , Análisis Factorial , Gástrula/embriología , Gastrulación/fisiología , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , ARN/análisis , Factores de Tiempo , Dedos de ZincRESUMEN
Absence of a specialized wound epidermis is hypothesized to block limb regeneration in higher vertebrates. However, the factors preventing its formation in regeneration-incompetent animals are poorly understood. To characterize the endogenous molecular and cellular regulators of specialized wound epidermis formation in Xenopus laevis tadpoles, and the loss of their regeneration competency during development, we used single-cell transcriptomics and ex vivo regenerating limb cultures. Transcriptomic analysis revealed that the specialized wound epidermis is not a novel cell state, but a re-deployment of the apical-ectodermal-ridge (AER) programme underlying limb development. Enrichment of secreted inhibitory factors, including Noggin, a morphogen expressed in developing cartilage/bone progenitor cells, are identified as key inhibitors of AER cell formation in regeneration-incompetent tadpoles. These factors can be overridden by Fgf10, which operates upstream of Noggin and blocks chondrogenesis. These results indicate that manipulation of the extracellular environment and/or chondrogenesis may provide a strategy to restore regeneration potential in higher vertebrates.