RESUMO
Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and resume growth remains poorly understood. Here, we visualized budding yeast cells undergoing meiosis by cryo-electron tomography (cryoET) and discovered elaborate filamentous assemblies decorating the nucleus, cytoplasm, and mitochondria. To determine filament composition, we developed a "filament identification" (FilamentID) workflow that combines multiscale cryoET/cryo-electron microscopy (cryoEM) analyses of partially lysed cells or organelles. FilamentID identified the mitochondrial filaments as being composed of the conserved aldehyde dehydrogenase Ald4ALDH2 and the nucleoplasmic/cytoplasmic filaments as consisting of acetyl-coenzyme A (CoA) synthetase Acs1ACSS2. Structural characterization further revealed the mechanism underlying polymerization and enabled us to genetically perturb filament formation. Acs1 polymerization facilitates the recovery of chronologically aged spores and, more generally, the cell cycle re-entry of starved cells. FilamentID is broadly applicable to characterize filaments of unknown identity in diverse cellular contexts.
Assuntos
Gametogênese , Mitocôndrias , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Aldeído Desidrogenase/metabolismo , Aldeído Desidrogenase/química , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Coenzima A Ligases/metabolismo , Microscopia Crioeletrônica , Citoplasma/metabolismo , Tomografia com Microscopia Eletrônica , Meiose , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Esporos Fúngicos/metabolismo , Modelos Moleculares , Estrutura Quaternária de ProteínaRESUMO
Multiple sclerosis (MS) is a neurological disease characterized by multifocal lesions and smoldering pathology. Although single-cell analyses provided insights into cytopathology, evolving cellular processes underlying MS remain poorly understood. We investigated the cellular dynamics of MS by modeling temporal and regional rates of disease progression in mouse experimental autoimmune encephalomyelitis (EAE). By performing single-cell spatial expression profiling using in situ sequencing (ISS), we annotated disease neighborhoods and found centrifugal evolution of active lesions. We demonstrated that disease-associated (DA)-glia arise independently of lesions and are dynamically induced and resolved over the disease course. Single-cell spatial mapping of human archival MS spinal cords confirmed the differential distribution of homeostatic and DA-glia, enabled deconvolution of active and inactive lesions into sub-compartments, and identified new lesion areas. By establishing a spatial resource of mouse and human MS neuropathology at a single-cell resolution, our study unveils the intricate cellular dynamics underlying MS.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Medula Espinal , Animais , Humanos , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Camundongos , Análise da Expressão Gênica de Célula Única , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Neuroglia/metabolismo , Neuroglia/patologiaRESUMO
Paired mapping of single-cell gene expression and electrophysiology is essential to understand gene-to-function relationships in electrogenic tissues. Here, we developed in situ electro-sequencing (electro-seq) that combines flexible bioelectronics with in situ RNA sequencing to stably map millisecond-timescale electrical activity and profile single-cell gene expression from the same cells across intact biological networks, including cardiac and neural patches. When applied to human-induced pluripotent stem-cell-derived cardiomyocyte patches, in situ electro-seq enabled multimodal in situ analysis of cardiomyocyte electrophysiology and gene expression at the cellular level, jointly defining cell states and developmental trajectories. Using machine-learning-based cross-modal analysis, in situ electro-seq identified gene-to-electrophysiology relationships throughout cardiomyocyte development and accurately reconstructed the evolution of gene expression profiles based on long-term stable electrical measurements. In situ electro-seq could be applicable to create spatiotemporal multimodal maps in electrogenic tissues, potentiating the discovery of cell types and gene programs responsible for electrophysiological function and dysfunction.
Assuntos
Eletrônica , Análise de Sequência de RNA , Humanos , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/metabolismo , Análise de Célula Única , Transcriptoma , Eletrônica/métodosRESUMO
Ductal carcinoma in situ (DCIS) is a common precursor of invasive breast cancer. Our understanding of its genomic progression to recurrent disease remains poor, partly due to challenges associated with the genomic profiling of formalin-fixed paraffin-embedded (FFPE) materials. Here, we developed Arc-well, a high-throughput single-cell DNA-sequencing method that is compatible with FFPE materials. We validated our method by profiling 40,330 single cells from cell lines, a frozen tissue, and 27 FFPE samples from breast, lung, and prostate tumors stored for 3-31 years. Analysis of 10 patients with matched DCIS and cancers that recurred 2-16 years later show that many primary DCIS had already undergone whole-genome doubling and clonal diversification and that they shared genomic lineages with persistent subclones in the recurrences. Evolutionary analysis suggests that most DCIS cases in our cohort underwent an evolutionary bottleneck, and further identified chromosome aberrations in the persistent subclones that were associated with recurrence.
Assuntos
Neoplasias da Mama , Carcinoma Ductal de Mama , Carcinoma Intraductal não Infiltrante , Feminino , Humanos , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/patologia , Progressão da Doença , Genômica/métodos , Análise da Expressão Gênica de Célula Única , Linhagem Celular TumoralRESUMO
Cryo-electron microscopy (cryo-EM) continues its remarkable growth as a method for visualizing biological objects, which has been driven by advances across the entire pipeline. Developments in both single-particle analysis and in situ tomography have enabled more structures to be imaged and determined to better resolutions, at faster speeds, and with more scientists having improved access. This review highlights recent advances at each stageof the cryo-EM pipeline and provides examples of how these techniques have been used to investigate real-world problems, including antibody development against the SARS-CoV-2 spike during the recent COVID-19 pandemic.
Assuntos
COVID-19 , Pandemias , Microscopia Crioeletrônica/métodos , Humanos , SARS-CoV-2 , Imagem Individual de MoléculaRESUMO
Understanding the basis for cellular growth, proliferation, and function requires determining the roles of essential genes in diverse cellular processes, including visualizing their contributions to cellular organization and morphology. Here, we combined pooled CRISPR-Cas9-based functional screening of 5,072 fitness-conferring genes in human HeLa cells with microscopy-based imaging of DNA, the DNA damage response, actin, and microtubules. Analysis of >31 million individual cells identified measurable phenotypes for >90% of gene knockouts, implicating gene targets in specific cellular processes. Clustering of phenotypic similarities based on hundreds of quantitative parameters further revealed co-functional genes across diverse cellular activities, providing predictions for gene functions and associations. By conducting pooled live-cell screening of â¼450,000 cell division events for 239 genes, we additionally identified diverse genes with functional contributions to chromosome segregation. Our work establishes a resource detailing the consequences of disrupting core cellular processes that represents the functional landscape of essential human genes.
Assuntos
Sistemas CRISPR-Cas , Genes Essenciais , Humanos , Células HeLa , Técnicas de Inativação de Genes , FenótipoRESUMO
Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 µm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.
Assuntos
Região Hipotalâmica Lateral/metabolismo , Hibridização in Situ Fluorescente , Animais , Biomarcadores/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Região Hipotalâmica Lateral/citologia , Imageamento Tridimensional , Masculino , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA/metabolismo , RNA-Seq , Análise de Célula Única , Transcrição GênicaRESUMO
We present deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq) for co-mapping of mRNAs and proteins in a formaldehyde-fixed tissue slide via next-generation sequencing (NGS). Parallel microfluidic channels were used to deliver DNA barcodes to the surface of a tissue slide, and crossflow of two sets of barcodes, A1-50 and B1-50, followed by ligation in situ, yielded a 2D mosaic of tissue pixels, each containing a unique full barcode AB. Application to mouse embryos revealed major tissue types in early organogenesis as well as fine features like microvasculature in a brain and pigmented epithelium in an eye field. Gene expression profiles in 10-µm pixels conformed into the clusters of single-cell transcriptomes, allowing for rapid identification of cell types and spatial distributions. DBiT-seq can be adopted by researchers with no experience in microfluidics and may find applications in a range of fields including developmental biology, cancer biology, neuroscience, and clinical pathology.
Assuntos
Código de Barras de DNA Taxonômico , Genômica , Especificidade de Órgãos/genética , Animais , Automação , Encéfalo/embriologia , Análise por Conglomerados , DNA Complementar/genética , Embrião de Mamíferos/metabolismo , Olho/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Microfluídica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Análise de Célula Única , Transcriptoma/genéticaRESUMO
Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are anchored to cells via lipopolysaccharide. Here, we report an electron cryomicroscopy structure of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide. Using native mass spectrometry and molecular dynamics simulations, we deduce the length of the O-antigen on cells and show how lipopolysaccharide binding and S-layer assembly is regulated by calcium. Finally, we present a near-atomic resolution in situ structure of the complete S-layer using cellular electron cryotomography, showing S-layer arrangement at the tip of the O-antigen. A complete atomic structure of the S-layer shows the power of cellular tomography for in situ structural biology and sheds light on a very abundant class of self-assembling molecules with important roles in prokaryotic physiology with marked potential for synthetic biology and surface-display applications.
Assuntos
Proteínas da Membrana Bacteriana Externa/ultraestrutura , Caulobacter crescentus/metabolismo , Glicoproteínas de Membrana/ultraestrutura , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Caulobacter crescentus/ultraestrutura , Microscopia Crioeletrônica/métodos , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana/metabolismo , Tomografia/métodosRESUMO
Although complex inflammatory-like alterations are observed around the amyloid plaques of Alzheimer's disease (AD), little is known about the molecular changes and cellular interactions that characterize this response. We investigate here, in an AD mouse model, the transcriptional changes occurring in tissue domains in a 100-µm diameter around amyloid plaques using spatial transcriptomics. We demonstrate early alterations in a gene co-expression network enriched for myelin and oligodendrocyte genes (OLIGs), whereas a multicellular gene co-expression network of plaque-induced genes (PIGs) involving the complement system, oxidative stress, lysosomes, and inflammation is prominent in the later phase of the disease. We confirm the majority of the observed alterations at the cellular level using in situ sequencing on mouse and human brain sections. Genome-wide spatial transcriptomics analysis provides an unprecedented approach to untangle the dysregulated cellular network in the vicinity of pathogenic hallmarks of AD and other brain diseases.
Assuntos
Doença de Alzheimer/patologia , Análise de Sequência de DNA/métodos , Transcriptoma , Doença de Alzheimer/genética , Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Estresse Oxidativo/genéticaRESUMO
Understanding neural circuits requires deciphering interactions among myriad cell types defined by spatial organization, connectivity, gene expression, and other properties. Resolving these cell types requires both single-neuron resolution and high throughput, a challenging combination with conventional methods. Here, we introduce barcoded anatomy resolved by sequencing (BARseq), a multiplexed method based on RNA barcoding for mapping projections of thousands of spatially resolved neurons in a single brain and relating those projections to other properties such as gene or Cre expression. Mapping the projections to 11 areas of 3,579 neurons in mouse auditory cortex using BARseq confirmed the laminar organization of the three top classes (intratelencephalic [IT], pyramidal tract-like [PT-like], and corticothalamic [CT]) of projection neurons. In depth analysis uncovered a projection type restricted almost exclusively to transcriptionally defined subtypes of IT neurons. By bridging anatomical and transcriptomic approaches at cellular resolution with high throughput, BARseq can potentially uncover the organizing principles underlying the structure and formation of neural circuits.
Assuntos
Córtex Auditivo/metabolismo , Rede Nervosa/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Mapeamento Encefálico , Humanos , Integrases/genética , Camundongos , Neuritos/metabolismo , Células Piramidais/metabolismo , Tratos Piramidais/metabolismoRESUMO
Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.
Assuntos
Testes Genéticos , Genômica , NF-kappa B/genética , Fator de Transcrição RelA/genética , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Humanos , Complexo Mediador/genética , RNA Guia de Cinetoplastídeos/genéticaRESUMO
The process of cardiac morphogenesis in humans is incompletely understood. Its full characterization requires a deep exploration of the organ-wide orchestration of gene expression with a single-cell spatial resolution. Here, we present a molecular approach that reveals the comprehensive transcriptional landscape of cell types populating the embryonic heart at three developmental stages and that maps cell-type-specific gene expression to specific anatomical domains. Spatial transcriptomics identified unique gene profiles that correspond to distinct anatomical regions in each developmental stage. Human embryonic cardiac cell types identified by single-cell RNA sequencing confirmed and enriched the spatial annotation of embryonic cardiac gene expression. In situ sequencing was then used to refine these results and create a spatial subcellular map for the three developmental phases. Finally, we generated a publicly available web resource of the human developing heart to facilitate future studies on human cardiogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Miócitos Cardíacos/metabolismo , Análise de Célula Única , Transcriptoma , Feminino , Humanos , Masculino , Morfogênese , Miócitos Cardíacos/citologia , RNA-SeqRESUMO
Ductal carcinoma in situ (DCIS) is an early-stage breast cancer that infrequently progresses to invasive ductal carcinoma (IDC). Genomic evolution has been difficult to delineate during invasion due to intratumor heterogeneity and the low number of tumor cells in the ducts. To overcome these challenges, we developed Topographic Single Cell Sequencing (TSCS) to measure genomic copy number profiles of single tumor cells while preserving their spatial context in tissue sections. We applied TSCS to 1,293 single cells from 10 synchronous patients with both DCIS and IDC regions in addition to exome sequencing. Our data reveal a direct genomic lineage between in situ and invasive tumor subpopulations and further show that most mutations and copy number aberrations evolved within the ducts prior to invasion. These results support a multiclonal invasion model, in which one or more clones escape the ducts and migrate into the adjacent tissues to establish the invasive carcinomas.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Evolução Clonal , Adulto , Idoso , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Movimento Celular , Exoma , Feminino , Humanos , Pessoa de Meia-Idade , Mutação , Invasividade Neoplásica , Análise de Sequência de DNA , Análise de Célula ÚnicaRESUMO
N6-methyladenosine (m6A) RNA modification plays important roles in the governance of gene expression and is temporally regulated in different cell states. In contrast to global m6A profiling in bulk sequencing, single-cell technologies for analyzing m6A heterogeneity are not extensively established. Here, we developed single-nucleus m6A-CUT&Tag (sn-m6A-CT) for simultaneous profiling of m6A methylomes and transcriptomes within a single nucleus using mouse embryonic stem cells (mESCs). m6A-CT is capable of enriching m6A-marked RNA molecules in situ, without isolating RNAs from cells. We adapted m6A-CT to the droplet-based single-cell omics platform and demonstrated high-throughput performance in analyzing nuclei isolated from thousands of cells from various cell types. We show that sn-m6A-CT profiling is sufficient to determine cell identity and allows the generation of cell-type-specific m6A methylome landscapes from heterogeneous populations. These indicate that sn-m6A-CT provides additional dimensions to multimodal datasets and insights into epitranscriptomic landscape in defining cell fate identity and states.
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Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies.
Assuntos
Subpopulações de Linfócitos B/imunologia , Epitopos/imunologia , Vacinas Antimaláricas/imunologia , Malária/imunologia , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/imunologia , Vacinas de DNA/imunologia , Transferência Adotiva , Animais , Anticorpos Antiprotozoários/metabolismo , Modelos Animais de Doenças , Epitopos/genética , Engenharia Genética , Humanos , Evasão da Resposta Imune , Imunogenicidade da Vacina , Camundongos , Camundongos SCID , Proteínas de Protozoários/genética , Relação Estrutura-Atividade , VacinaçãoRESUMO
Extension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatiotemporal coordination of telomerase and telomeres and remains poorly understood at the cellular level. To understand how cancer cells execute this process, we combine CRISPR genome editing and MS2 RNA tagging to image single molecules of telomerase RNA (hTR). Real-time dynamics and photoactivation experiments of hTR in Cajal bodies (CBs) reveal that hTERT controls the exit of hTR from CBs. Single-molecule tracking of hTR at telomeres shows that TPP1-mediated recruitment results in short telomere-telomerase scanning interactions, and then base pairing between hTR and telomere ssDNA promotes long interactions required for stable telomerase retention. Interestingly, POT1 OB-fold mutations that result in abnormally long telomeres in cancers act by enhancing this retention step. In summary, single-molecule imaging unveils the life cycle of telomerase RNA and provides a framework to reveal how cancer-associated mutations mechanistically drive defects in telomere homeostasis.
Assuntos
Corpos Enovelados/metabolismo , DNA de Cadeia Simples/metabolismo , RNA/metabolismo , Imagem Individual de Molécula/métodos , Telomerase/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA de Cadeia Simples/genética , Edição de Genes , Células HeLa , Humanos , Mutação , RNA/genética , Complexo Shelterina , Telomerase/genética , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Ebola viruses (EBOVs) assemble into filamentous virions, whose shape and stability are determined by the matrix viral protein 40 (VP40). Virus entry into host cells occurs via membrane fusion in late endosomes; however, the mechanism of how the remarkably long virions undergo uncoating, including virion disassembly and nucleocapsid release into the cytosol, remains unknown. Here, we investigate the structural architecture of EBOVs entering host cells and discover that the VP40 matrix disassembles prior to membrane fusion. We reveal that VP40 disassembly is caused by the weakening of VP40-lipid interactions driven by low endosomal pH that equilibrates passively across the viral envelope without a dedicated ion channel. We further show that viral membrane fusion depends on VP40 matrix integrity, and its disassembly reduces the energy barrier for fusion stalk formation. Thus, pH-driven structural remodeling of the VP40 matrix acts as a molecular switch coupling viral matrix uncoating to membrane fusion during EBOV entry.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Humanos , Doença pelo Vírus Ebola/metabolismo , Fusão de Membrana , Proteínas do Core Viral/metabolismo , Endossomos/metabolismo , Proteínas da Matriz ViralRESUMO
Multiplexed spatial profiling of mRNAs has recently gained traction as a tool to explore the cellular diversity and the architecture of tissues. We propose a sensitive, open-source, simple and flexible method for the generation of in-situ expression maps of hundreds of genes. We exploit direct ligation of padlock probes on mRNAs, coupled with rolling circle amplification and hybridization-based in situ combinatorial barcoding, to achieve high detection efficiency, high throughput and large multiplexing. We validate the method across a number of species, and show its use in combination with orthogonal methods such as antibody staining, highlighting its potential value for developmental and tissue biology studies. Finally, we provide an end-to-end computational workflow that covers the steps of probe design, image processing, data extraction, cell segmentation, clustering and annotation of cell types. By enabling easier access to high-throughput spatially resolved transcriptomics, we hope to encourage a diversity of applications and the exploration of a wide range of biological questions.
RESUMO
Embryonic development is a complex and dynamic process that unfolds over time and involves the production and diversification of increasing numbers of cells. The impact of developmental time on the formation of the central nervous system is well documented, with evidence showing that time plays a crucial role in establishing the identity of neuronal subtypes. However, the study of how time translates into genetic instructions driving cell fate is limited by the scarcity of suitable experimental tools. We introduce BirthSeq, a new method for isolating and analyzing cells based on their birth date. This innovative technique allows for in vivo labeling of cells, isolation via fluorescence-activated cell sorting, and analysis using high-throughput techniques. We calibrated the BirthSeq method for developmental organs across three vertebrate species (mouse, chick and gecko), and utilized it for single-cell RNA sequencing and novel spatially resolved transcriptomic approaches in mouse and chick, respectively. Overall, BirthSeq provides a versatile tool for studying virtually any tissue in different vertebrate organisms, aiding developmental biology research by targeting cells and their temporal cues.