RESUMEN
Salamander limb regeneration is an accurate process which gives rise exclusively to the missing structures, irrespective of the amputation level. This suggests that cells in the stump have an awareness of their spatial location, a property termed positional identity. Little is known about how positional identity is encoded, in salamanders or other biological systems. Through single-cell RNAseq analysis, we identified Tig1/Rarres1 as a potential determinant of proximal identity. Tig1 encodes a conserved cell surface molecule, is regulated by retinoic acid and exhibits a graded expression along the proximo-distal axis of the limb. Its overexpression leads to regeneration defects in the distal elements and elicits proximal displacement of blastema cells, while its neutralisation blocks proximo-distal cell surface interactions. Critically, Tig1 reprogrammes distal cells to a proximal identity, upregulating Prod1 and inhibiting Hoxa13 and distal transcriptional networks. Thus, Tig1 is a central cell surface determinant of proximal identity in the salamander limb.
Asunto(s)
Extremidades , Urodelos , Amputación Quirúrgica , Animales , Extremidades/fisiología , Tretinoina/farmacología , Urodelos/genéticaRESUMEN
In multicellular organisms, the specification, coordination, and compartmentalization of cell types enable the formation of complex body plans. However, some eukaryotic protists such as slime molds generate diverse and complex structures while remaining in a multinucleate syncytial state. It is unknown if different regions of these giant syncytial cells have distinct transcriptional responses to environmental encounters and if nuclei within the cell diversify into heterogeneous states. Here, we performed spatial transcriptome analysis of the slime mold Physarum polycephalum in the plasmodium state under different environmental conditions and used single-nucleus RNA-sequencing to dissect gene expression heterogeneity among nuclei. Our data identifies transcriptome regionality in the organism that associates with proliferation, syncytial substructures, and localized environmental conditions. Further, we find that nuclei are heterogenous in their transcriptional profile and may process local signals within the plasmodium to coordinate cell growth, metabolism, and reproduction. To understand how nuclei variation within the syncytium compares to heterogeneity in single-nucleus cells, we analyzed states in single Physarum amoebal cells. We observed amoebal cell states at different stages of mitosis and meiosis, and identified cytokinetic features that are specific to nuclei divisions within the syncytium. Notably, we do not find evidence for predefined transcriptomic states in the amoebae that are observed in the syncytium. Our data shows that a single-celled slime mold can control its gene expression in a region-specific manner while lacking cellular compartmentalization and suggests that nuclei are mobile processors facilitating local specialized functions. More broadly, slime molds offer the extraordinary opportunity to explore how organisms can evolve regulatory mechanisms to divide labor, specialize, balance competition with cooperation, and perform other foundational principles that govern the logic of life.
Asunto(s)
Células Gigantes/fisiología , Physarum polycephalum/metabolismo , Análisis de la Célula Individual , Transcriptoma , Regulación de la Expresión Génica , RNA-SeqRESUMEN
Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR-Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.
Asunto(s)
Corteza Cerebral/citología , Genes Reporteros , Células Madre Pluripotentes Inducidas/citología , Organoides/citología , Análisis de la Célula Individual/métodos , Sistemas CRISPR-Cas , Linaje de la Célula , Humanos , Microscopía/métodos , Mutación , Neuronas/citología , Neuronas/fisiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ARN , Proteína 2 del Complejo de la Esclerosis Tuberosa/genéticaRESUMEN
Conversion/alloy active materials, such as ZnO, are one of the most promising candidates to replace graphite anodes in lithium-ion batteries. Besides a high specific capacity (qZnO = 987 mAh g-1), ZnO offers a high lithium-ion diffusion and fast reaction kinetics, leading to a high-rate capability, which is required for the intended fast charging of battery electric vehicles. However, lithium-ion storage in ZnO is accompanied by the formation of lithium-rich solid electrolyte interphase (SEI) layers, immense volume expansion, and a large voltage hysteresis. Nonetheless, ZnO is appealing as an anode material for lithium-ion batteries and is investigated intensively. Surprisingly, the conclusions reported on the reaction mechanism are contradictory and the formation and composition of the SEI are addressed in only a few works. In this work, we investigate lithiation, delithiation, and SEI formation with ZnO in ether-based electrolytes for the first time reported in the literature. The combination of operando and ex situ experiments (cyclic voltammetry, X-ray photoelectron spectroscopy, X-ray diffraction, coupled gas chromatography and mass spectrometry, differential electrochemical mass spectrometry, and scanning electron microscopy) clarifies the misunderstanding of the reaction mechanism. We evidence that the conversion and alloy reaction take place simultaneously inside the bulk of the electrode. Furthermore, we show that a two-layered SEI is formed on the surface. The SEI is decomposed reversibly upon cycling. In the end, we address the issue of the volume expansion and associated capacity fading by incorporating ZnO into a mesoporous carbon network. This approach reduces the capacity fading and yields cells with a specific capacity of above 500 mAh g-1 after 150 cycles.
RESUMEN
Limb regeneration, while observed lifelong in salamanders, is restricted in post-metamorphic Xenopus laevis frogs. Whether this loss is due to systemic factors or an intrinsic incapability of cells to form competent stem cells has been unclear. Here, we use genetic fate mapping to establish that connective tissue (CT) cells form the post-metamorphic frog blastema, as in the case of axolotls. Using heterochronic transplantation into the limb bud and single-cell transcriptomic profiling, we show that axolotl CT cells dedifferentiate and integrate to form lineages, including cartilage. In contrast, frog blastema CT cells do not fully re-express the limb bud progenitor program, even when transplanted into the limb bud. Correspondingly, transplanted cells contribute to extraskeletal CT, but not to the developing cartilage. Furthermore, using single-cell RNA-seq analysis we find that embryonic and adult frog cartilage differentiation programs are molecularly distinct. This work defines intrinsic restrictions in CT dedifferentiation as a limitation in adult regeneration.
Asunto(s)
Diferenciación Celular , Fibroblastos/citología , Regeneración/fisiología , Ambystoma mexicanum , Animales , Tipificación del Cuerpo , Cartílago/citología , Reprogramación Celular , Células del Tejido Conectivo/citología , Dermis/citología , Embrión no Mamífero/citología , Larva , Xenopus laevis/embriologíaRESUMEN
In the version of this protocol originally published, the recipe for CAS9 buffer was incorrectly identified as a recipe for sodium acetate solution, and vice versa. These errors have been corrected in the PDF and HTML versions of the paper.
RESUMEN
Genomic manipulation is essential to the use of model organisms to understand development, regeneration and adult physiology. The axolotl (Ambystoma mexicanum), a type of salamander, exhibits an unparalleled regenerative capability in a spectrum of complex tissues and organs, and therefore serves as a powerful animal model for dissecting mechanisms of regeneration. We describe here an optimized stepwise protocol to create genetically modified axolotls using the CRISPR-Cas9 system. The protocol, which takes 7-8 weeks to complete, describes generation of targeted gene knockouts and knock-ins and includes site-specific integration of large targeting constructs. The direct use of purified CAS9-NLS (CAS9 containing a C-terminal nuclear localization signal) protein allows the prompt formation of guide RNA (gRNA)-CAS9-NLS ribonucleoprotein (RNP) complexes, which accelerates the creation of double-strand breaks (DSBs) at targeted genomic loci in single-cell-stage axolotl eggs. With this protocol, a substantial number of F0 individuals harboring a homozygous-type frameshift mutation can be obtained, allowing phenotype analysis in this generation. In the presence of targeting constructs, insertions of exogenous genes into targeted axolotl genomic loci can be achieved at efficiencies of up to 15% in a non-homologous end joining (NHEJ) manner. Our protocol bypasses the long generation time of axolotls and allows direct functional analysis in F0 genetically manipulated axolotls. This protocol can be potentially applied to other animal models, especially to organisms with a well-characterized transcriptome but lacking a well-characterized genome.
Asunto(s)
Ambystoma mexicanum/genética , Sistemas CRISPR-Cas , Edición Génica/métodos , Técnicas de Sustitución del Gen/métodos , Técnicas de Inactivación de Genes/métodos , Animales , Animales Modificados Genéticamente/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Mutación del Sistema de Lectura , Fenotipo , ARN Guía de Kinetoplastida/genéticaRESUMEN
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.
Asunto(s)
Reprogramación Celular/fisiología , Células del Tejido Conectivo/fisiología , Miembro Anterior/fisiología , Regeneración/fisiología , Ambystoma mexicanum , Animales , Linaje de la Célula , Rastreo Celular , Genes Reporteros , Integrasas , ARN Mensajero/genética , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual , Células Madre/fisiologíaRESUMEN
Ectopic expression of defined transcription factors can force direct cell-fate conversion from one lineage to another in the absence of cell division. Several transcription factor cocktails have enabled successful reprogramming of various somatic cell types into induced neurons (iNs) of distinct neurotransmitter phenotype. However, the nature of the intermediate states that drive the reprogramming trajectory toward distinct iN types is largely unknown. Here we show that successful direct reprogramming of adult human brain pericytes into functional iNs by Ascl1 and Sox2 encompasses transient activation of a neural stem cell-like gene expression program that precedes bifurcation into distinct neuronal lineages. During this transient state, key signaling components relevant for neural induction and neural stem cell maintenance are regulated by and functionally contribute to iN reprogramming and maturation. Thus, Ascl1- and Sox2-mediated reprogramming into a broad spectrum of iN types involves the unfolding of a developmental program via neural stem cell-like intermediates.
Asunto(s)
Linaje de la Célula/fisiología , Reprogramación Celular/fisiología , Células-Madre Neurales/fisiología , Neuronas/fisiología , Pericitos/fisiología , Adulto , Anciano , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Células-Madre Neurales/citología , Neuronas/citología , Pericitos/citología , Factores de Transcripción SOXB1/genética , Adulto JovenRESUMEN
Single-cell transcriptomics has been used for analysis of heterogeneous populations of cells during developmental processes and for analysis of tumor cell heterogeneity. More recently, analysis of pseudotime (PT) dynamics of heterogeneous cell populations has been established as a powerful concept to study developmental processes. Here we perform PT analysis of 3 melanoma short-term cultures with different genetic backgrounds to study specific and concordant properties of PT dynamics of selected cellular programs with impact on melanoma progression. Overall, in our setting of melanoma cells PT dynamics towards higher tumor malignancy appears to be largely driven by cell cycle genes. Single cells of all three short-term cultures show a bipolar expression of microphthalmia-associated transcription factor (MITF) and AXL receptor tyrosine kinase (AXL) signatures. Furthermore, opposing gene expression changes are observed for genes regulated by epigenetic mechanisms suggesting epigenetic reprogramming during melanoma progression. The three melanoma short-term cultures show common themes of PT dynamics such as a stromal signature at initiation, bipolar expression of the MITF/AXL signature and opposing regulation of poised and activated promoters. Differences are observed at the late stage of PT dynamics with high, low or intermediate MITF and anticorrelated AXL signatures. These findings may help to identify targets for interference at different stages of tumor progression.
RESUMEN
Conventional two-dimensional differentiation from pluripotency fails to recapitulate cell interactions occurring during organogenesis. Three-dimensional organoids generate complex organ-like tissues; however, it is unclear how heterotypic interactions affect lineage identity. Here we use single-cell RNA sequencing to reconstruct hepatocyte-like lineage progression from pluripotency in two-dimensional culture. We then derive three-dimensional liver bud organoids by reconstituting hepatic, stromal, and endothelial interactions, and deconstruct heterogeneity during liver bud development. We find that liver bud hepatoblasts diverge from the two-dimensional lineage, and express epithelial migration signatures characteristic of organ budding. We benchmark three-dimensional liver buds against fetal and adult human liver single-cell RNA sequencing data, and find a striking correspondence between the three-dimensional liver bud and fetal liver cells. We use a receptor-ligand pairing analysis and a high-throughput inhibitor assay to interrogate signalling in liver buds, and show that vascular endothelial growth factor (VEGF) crosstalk potentiates endothelial network formation and hepatoblast differentiation. Our molecular dissection reveals interlineage communication regulating organoid development, and illuminates previously inaccessible aspects of human liver development.
Asunto(s)
Comunicación Celular , Diferenciación Celular , Linaje de la Célula , Hígado/citología , Hígado/embriología , Organogénesis , Técnicas de Cultivo de Tejidos/métodos , Anciano , Hipoxia de la Célula , Movimiento Celular , Endotelio/citología , Células Epiteliales/citología , Matriz Extracelular/metabolismo , Femenino , Feto/citología , Hepatocitos/citología , Humanos , Masculino , Persona de Mediana Edad , Organoides/citología , Células Madre Pluripotentes/citología , Análisis de Secuencia de ARN , Transducción de Señal , Análisis de la Célula Individual , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adulto JovenRESUMEN
DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly degraded DNA, especially ancient DNA, library preparation has been found to be more efficient if each of the two DNA strands are converted into library molecules separately. We present a new method for single-stranded library preparation, ssDNA2.0, which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of random bases hybridized to a 3Î biotinylated donor oligonucleotide. A thorough evaluation of this ligation scheme shows that single-stranded DNA can be ligated to adapter oligonucleotides in higher concentration than with CircLigase (an RNA ligase that was previously chosen for end-to-end ligation in single-stranded library preparation) and that biases in ligation can be minimized when choosing splinters with 7 or 8 random nucleotides. We show that ssDNA2.0 tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and better compatible with automation. We also provide an in-depth comparison of library preparation methods on degraded DNA from various sources. Most strikingly, we find that single-stranded library preparation increases library yields from tissues stored in formalin for many years by several orders of magnitude.
Asunto(s)
ADN Ligasas/genética , ADN de Cadena Simple/genética , Biblioteca de Genes , Animales , Huesos/química , ADN/genética , ADN/metabolismo , ADN Ligasas/metabolismo , Cartilla de ADN/química , Cartilla de ADN/metabolismo , ADN de Cadena Simple/metabolismo , Fósiles , Secuenciación de Nucleótidos de Alto Rendimiento , Caballos , Humanos , Hígado/química , Hibridación de Ácido Nucleico , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , PorcinosRESUMEN
Recent technological advances in single-cell genomics make it possible to analyze cellular heterogeneity of tumor samples. Here, we applied single-cell RNA-seq to measure the transcriptomes of 307 single cells cultured from three biopsies of three different patients with a BRAF/NRAS wild type, BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant melanoma metastasis, respectively. Analysis based on self-organizing maps identified sub-populations defined by multiple gene expression modules involved in proliferation, oxidative phosphorylation, pigmentation and cellular stroma. Gene expression modules had prognostic relevance when compared with gene expression data from published melanoma samples and patient survival data. We surveyed kinome expression patterns across sub-populations of the BRAF/NRAS wild type sample and found that CDK4 and CDK2 were consistently highly expressed in the majority of cells, suggesting that these kinases might be involved in melanoma progression. Treatment of cells with the CDK4 inhibitor palbociclib restricted cell proliferation to a similar, and in some cases greater, extent than MAPK inhibitors. Finally, we identified a low abundant sub-population in this sample that highly expressed a module containing ABC transporter ABCB5, surface markers CD271 and CD133, and multiple aldehyde dehydrogenases (ALDHs). Patient-derived cultures of the BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant metastases showed more homogeneous single-cell gene expression patterns with gene expression modules for proliferation and ABC transporters. Taken together, our results describe an intertumor and intratumor heterogeneity in melanoma short-term cultures which might be relevant for patient survival, and suggest promising targets for new treatment approaches in melanoma therapy.
Asunto(s)
Biomarcadores de Tumor , Mapeo Cromosómico , Heterogeneidad Genética , Predisposición Genética a la Enfermedad , Melanoma/genética , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Adulto , Anciano , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Biología Computacional/métodos , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Epigénesis Genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes ras , Humanos , Masculino , Melanoma/metabolismo , Melanoma/mortalidad , Melanoma/patología , Persona de Mediana Edad , Metástasis de la Neoplasia , Piperazinas/farmacología , Pronóstico , Proteínas Proto-Oncogénicas B-raf/genética , Piridinas/farmacología , Análisis de Secuencia de ARN/métodos , Transducción de Señal , Análisis de la Célula Individual/métodos , TranscriptomaRESUMEN
Cerebral organoids-3D cultures of human cerebral tissue derived from pluripotent stem cells-have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Neocórtex/embriología , Organoides/fisiología , Diferenciación Celular , Linaje de la Célula , Humanos , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Técnicas de Cultivo de TejidosRESUMEN
Although great progress has been made in improving methods for generating DNA sequences from ancient biological samples, many, if not most, samples are still not amenable for analyses due to overwhelming contamination with microbial or modern human DNA. Here we explore different DNA decontamination procedures for ancient bones and teeth for use prior to DNA library preparation and high-throughput sequencing. Two procedures showed promising results: (i) the release of surface-bound DNA by phosphate buffer and (ii) the removal of DNA contamination by sodium hypochlorite treatment. Exposure to phosphate removes on average 64% of the microbial DNA from bone powder but only 37% of the endogenous DNA (from the organism under study), increasing the percentage of informative sequences by a factor of two on average. An average 4.6-fold increase, in one case reaching 24-fold, is achieved by sodium hypochlorite treatment, albeit at the expense of destroying 63% of the endogenous DNA preserved in the bone. While both pretreatment methods described here greatly reduce the cost of genome sequencing from ancient material due to efficient depletion of microbial DNA, we find that the removal of human DNA contamination remains a challenging problem.