RESUMO
Spatial transcriptomics (ST) methods unlock molecular mechanisms underlying tissue development, homeostasis, or disease. However, there is a need for easy-to-use, high-resolution, cost-efficient, and 3D-scalable methods. Here, we report Open-ST, a sequencing-based, open-source experimental and computational resource to address these challenges and to study the molecular organization of tissues in 2D and 3D. In mouse brain, Open-ST captured transcripts at subcellular resolution and reconstructed cell types. In primary head-and-neck tumors and patient-matched healthy/metastatic lymph nodes, Open-ST captured the diversity of immune, stromal, and tumor populations in space, validated by imaging-based ST. Distinct cell states were organized around cell-cell communication hotspots in the tumor but not the metastasis. Strikingly, the 3D reconstruction and multimodal analysis of the metastatic lymph node revealed spatially contiguous structures not visible in 2D and potential biomarkers precisely at the 3D tumor/lymph node boundary. All protocols and software are available at https://rajewsky-lab.github.io/openst.
Assuntos
Imageamento Tridimensional , Transcriptoma , Animais , Camundongos , Humanos , Transcriptoma/genética , Imageamento Tridimensional/métodos , Software , Perfilação da Expressão Gênica/métodos , Linfonodos/patologia , Linfonodos/metabolismo , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Encéfalo/metabolismo , Camundongos Endogâmicos C57BL , Metástase Linfática , FemininoRESUMO
The generation of distinct messenger RNA isoforms through alternative RNA processing modulates the expression and function of genes, often in a cell-type-specific manner. Here, we assess the regulatory relationships between transcription initiation, alternative splicing, and 3' end site selection. Applying long-read sequencing to accurately represent even the longest transcripts from end to end, we quantify mRNA isoforms in Drosophila tissues, including the transcriptionally complex nervous system. We find that in Drosophila heads, as well as in human cerebral organoids, 3' end site choice is globally influenced by the site of transcription initiation (TSS). "Dominant promoters," characterized by specific epigenetic signatures including p300/CBP binding, impose a transcriptional constraint to define splice and polyadenylation variants. In vivo deletion or overexpression of dominant promoters as well as p300/CBP loss disrupted the 3' end expression landscape. Our study demonstrates the crucial impact of TSS choice on the regulation of transcript diversity and tissue identity.
Assuntos
Processamento Alternativo , Isoformas de RNA , Sítio de Iniciação de Transcrição , Humanos , Poliadenilação , Regiões Promotoras Genéticas , Isoformas de RNA/metabolismo , RNA Mensageiro/metabolismoRESUMO
The endoribonuclease Dicer is known for its central role in the biogenesis of eukaryotic small RNAs/microRNAs. Despite its importance, Dicer target transcripts have not been directly mapped. Here, we apply biochemical methods to human cells and C. elegans and identify thousands of Dicer-binding sites. We find known and hundreds of additional miRNAs with high sensitivity and specificity. We also report structural RNAs, promoter RNAs, and mitochondrial transcripts as Dicer targets. Interestingly, most Dicer-binding sites reside on mRNAs/lncRNAs and are not significantly processed into small RNAs. These passive sites typically harbor small, Dicer-bound hairpins within intact transcripts and generally stabilize target expression. We show that passive sites can sequester Dicer and reduce microRNA expression. mRNAs with passive sites were in human and worm significantly associated with processing-body/granule function. Together, we provide the first transcriptome-wide map of Dicer targets and suggest conserved binding modes and functions outside of the miRNA pathway.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , RNA Helicases DEAD-box/metabolismo , Ribonuclease III/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Imunoprecipitação da Cromatina , Humanos , MicroRNAs/metabolismo , Fotoquímica , RNA/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , RNA Mitocondrial , Proteínas de Ligação a RNA/metabolismo , Sítio de Iniciação de Transcrição , TranscriptomaRESUMO
The global rise in obesity has revitalized a search for genetic and epigenetic factors underlying the disease. We present a Drosophila model of paternal-diet-induced intergenerational metabolic reprogramming (IGMR) and identify genes required for its encoding in offspring. Intriguingly, we find that as little as 2 days of dietary intervention in fathers elicits obesity in offspring. Paternal sugar acts as a physiological suppressor of variegation, desilencing chromatin-state-defined domains in both mature sperm and in offspring embryos. We identify requirements for H3K9/K27me3-dependent reprogramming of metabolic genes in two distinct germline and zygotic windows. Critically, we find evidence that a similar system may regulate obesity susceptibility and phenotype variation in mice and humans. The findings provide insight into the mechanisms underlying intergenerational metabolic reprogramming and carry profound implications for our understanding of phenotypic variation and evolution.
Assuntos
Modelos Animais de Doenças , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epigênese Genética , Obesidade/genética , Animais , Metabolismo dos Carboidratos , Dieta , Embrião não Mamífero/metabolismo , Cor de Olho , Feminino , Predisposição Genética para Doença , Heterocromatina/metabolismo , Humanos , Masculino , Camundongos , Obesidade/metabolismo , Espermatozoides/metabolismoRESUMO
Knockout of the ubiquitously expressed miRNA-17â¼92 cluster in mice produces a lethal developmental lung defect, skeletal abnormalities, and blocked B lymphopoiesis. A shared target of miR-17â¼92 miRNAs is the pro-apoptotic protein BIM, central to life-death decisions in mammalian cells. To clarify the contribution of miR-17â¼92:Bim interactions to the complex miR-17â¼92 knockout phenotype, we used a system of conditional mutagenesis of the nine Bim 3' UTR miR-17â¼92 seed matches. Blocking miR-17â¼92:Bim interactions early in development phenocopied the lethal lung phenotype of miR-17â¼92 ablation and generated a skeletal kinky tail. In the hematopoietic system, instead of causing the predicted B cell developmental block, it produced a selective inability of B cells to resist cellular stress; and prevented B and T cell hyperplasia caused by Bim haploinsufficiency. Thus, the interaction of miR-17â¼92 with a single target is essential for life, and BIM regulation by miRNAs serves as a rheostat controlling cell survival in specific physiological contexts.
Assuntos
Linfócitos B/citologia , Proteína 11 Semelhante a Bcl-2/metabolismo , Sobrevivência Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Hematopoese/genética , MicroRNAs/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Linfócitos B/patologia , Proteína 11 Semelhante a Bcl-2/genética , Técnicas de Inativação de Genes , Pulmão/embriologia , Camundongos , MicroRNAs/genética , Mutação , Estresse FisiológicoRESUMO
Organoids derived from stem cells have become an increasingly important tool for studying human development and modeling disease. However, methods are still needed to control and study spatiotemporal patterns of gene expression in organoids. Here we combined optogenetics and gene perturbation technologies to activate or knock-down RNA of target genes in programmable spatiotemporal patterns. To illustrate the usefulness of our approach, we locally activated Sonic Hedgehog (SHH) signaling in an organoid model for human neurodevelopment. Spatial and single-cell transcriptomic analyses showed that this local induction was sufficient to generate stereotypically patterned organoids and revealed new insights into SHH's contribution to gene regulation in neurodevelopment. With this study, we propose optogenetic perturbations in combination with spatial transcriptomics as a powerful technology to reprogram and study cell fates and tissue patterning in organoids.
Assuntos
Proteínas Hedgehog , Optogenética , Humanos , Proteínas Hedgehog/metabolismo , Organoides/metabolismo , Diferenciação Celular , Expressão GênicaRESUMO
The circular RNA (circRNA) Cdr1as is conserved across mammals and highly expressed in neurons, where it directly interacts with microRNA miR-7. However, the biological function of this interaction is unknown. Here, using primary cortical murine neurons, we demonstrate that stimulating neurons by sustained depolarization rapidly induces two-fold transcriptional upregulation of Cdr1as and strong post-transcriptional stabilization of miR-7. Cdr1as loss causes doubling of glutamate release from stimulated synapses and increased frequency and duration of local neuronal bursts. Moreover, the periodicity of neuronal networks increases, and synchronicity is impaired. Strikingly, these effects are reverted by sustained expression of miR-7, which also clears Cdr1as molecules from neuronal projections. Consistently, without Cdr1as, transcriptomic changes caused by miR-7 overexpression are stronger (including miR-7-targets downregulation) and enriched in secretion/synaptic plasticity pathways. Altogether, our results suggest that in cortical neurons Cdr1as buffers miR-7 activity to control glutamatergic excitatory transmission and neuronal connectivity important for long-lasting synaptic adaptations.
Assuntos
Ácido Glutâmico , MicroRNAs , Neurônios , Transmissão Sináptica , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Neurônios/metabolismo , Camundongos , Ácido Glutâmico/metabolismo , Transmissão Sináptica/genética , Plasticidade Neuronal/genética , RNA Circular/genética , RNA Circular/metabolismo , Sinapses/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Regulação da Expressão Gênica , Células CultivadasRESUMO
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.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Atenção à Saúde/métodos , Atenção à Saúde/tendências , Medicina/métodos , Medicina/tendências , Patologia , Análise de Célula Única , Inteligência Artificial , Atenção à Saúde/ética , Atenção à Saúde/normas , Diagnóstico Precoce , Educação Médica , Europa (Continente) , Feminino , Saúde , Humanos , Legislação Médica , Masculino , Medicina/normasRESUMO
Circular RNAs (circRNAs) are formed in all domains of life and via different mechanisms. There has been an explosion in the number of circRNA papers in recent years; however, as a relatively young field, circRNA biology has an urgent need for common experimental standards for isolating, analyzing, expressing and depleting circRNAs. Here we propose a set of guidelines for circRNA studies based on the authors' experience. This Perspective will specifically address the major class of circRNAs in Eukarya that are generated by a spliceosome-catalyzed back-splicing event. We hope that the implementation of best practice principles for circRNA research will help move the field forward and allow a better functional understanding of this fascinating group of RNAs.
Assuntos
RNA Circular , RNA , RNA/genética , RNA/metabolismo , Splicing de RNARESUMO
The impact of epigenetics on the differentiation of memory T (Tmem) cells is poorly defined. We generated deep epigenomes comprising genome-wide profiles of DNA methylation, histone modifications, DNA accessibility, and coding and non-coding RNA expression in naive, central-, effector-, and terminally differentiated CD45RA+ CD4+ Tmem cells from blood and CD69+ Tmem cells from bone marrow (BM-Tmem). We observed a progressive and proliferation-associated global loss of DNA methylation in heterochromatic parts of the genome during Tmem cell differentiation. Furthermore, distinct gradually changing signatures in the epigenome and the transcriptome supported a linear model of memory development in circulating T cells, while tissue-resident BM-Tmem branched off with a unique epigenetic profile. Integrative analyses identified candidate master regulators of Tmem cell differentiation, including the transcription factor FOXP1. This study highlights the importance of epigenomic changes for Tmem cell biology and demonstrates the value of epigenetic data for the identification of lineage regulators.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Epigênese Genética/imunologia , Epigenômica/métodos , Memória Imunológica/imunologia , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica/métodos , Humanos , Aprendizado de Máquina , Reação em Cadeia da Polimerase , TranscriptomaRESUMO
Multiplexed RNA sequencing in individual cells is transforming basic and clinical life sciences1-4. Often, however, tissues must first be dissociated, and crucial information about spatial relationships and communication between cells is thus lost. Existing approaches to reconstruct tissues assign spatial positions to each cell, independently of other cells, by using spatial patterns of expression of marker genes5,6-which often do not exist. Here we reconstruct spatial positions with little or no prior knowledge, by searching for spatial arrangements of sequenced cells in which nearby cells have transcriptional profiles that are often (but not always) more similar than cells that are farther apart. We formulate this task as a generalized optimal-transport problem for probabilistic embedding and derive an efficient iterative algorithm to solve it. We reconstruct the spatial expression of genes in mammalian liver and intestinal epithelium, fly and zebrafish embryos, sections from the mammalian cerebellum and whole kidney, and use the reconstructed tissues to identify genes that are spatially informative. Thus, we identify an organization principle for the spatial expression of genes in animal tissues, which can be exploited to infer meaningful probabilities of spatial position for individual cells. Our framework ('novoSpaRc') can incorporate prior spatial information and is compatible with any single-cell technology. Additional principles that underlie the cartography of gene expression can be tested using our approach.
Assuntos
Expressão Gênica , Animais , Drosophila melanogaster , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Análise de Sequência de RNA , Análise de Célula Única , SoftwareRESUMO
Circular RNAs (circRNAs) are abundant and evolutionarily conserved RNAs of largely unknown function. Here, we show that a subset of circRNAs is translated in vivo. By performing ribosome footprinting from fly heads, we demonstrate that a group of circRNAs is associated with translating ribosomes. Many of these ribo-circRNAs use the start codon of the hosting mRNA, are bound by membrane-associated ribosomes, and have evolutionarily conserved termination codons. In addition, we found that a circRNA generated from the muscleblind locus encodes a protein, which we detected in fly head extracts by mass spectrometry. Next, by performing in vivo and in vitro translation assays, we show that UTRs of ribo-circRNAs (cUTRs) allow cap-independent translation. Moreover, we found that starvation and FOXO likely regulate the translation of a circMbl isoform. Altogether, our study provides strong evidence for translation of circRNAs, revealing the existence of an unexplored layer of gene activity.
Assuntos
Proteínas de Drosophila/biossíntese , Drosophila melanogaster/metabolismo , Proteínas Nucleares/biossíntese , Biossíntese de Proteínas , RNA/metabolismo , Ribossomos/metabolismo , Animais , Linhagem Celular , Códon de Iniciação , Códon de Terminação , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Fatores de Transcrição Forkhead/metabolismo , Genótipo , Cabeça , Espectrometria de Massas , Camundongos , Mutação , Proteínas Nucleares/genética , Conformação de Ácido Nucleico , Estado Nutricional , Fenótipo , RNA/química , RNA/genética , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Circular , Ratos , Ribossomos/química , Ribossomos/genética , Inanição/genética , Inanição/metabolismo , Relação Estrutura-Atividade , TransfecçãoRESUMO
Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and still largely unknown functions. Their biogenesis, which proceeds via a back-splicing reaction, is fairly well characterized, whereas their role in the modulation of physiologically relevant processes is still unclear. Here we performed expression profiling of circRNAs during in vitro differentiation of murine and human myoblasts, and we identified conserved species regulated in myogenesis and altered in Duchenne muscular dystrophy. A high-content functional genomic screen allowed the study of their functional role in muscle differentiation. One of them, circ-ZNF609, resulted in specifically controlling myoblast proliferation. Circ-ZNF609 contains an open reading frame spanning from the start codon, in common with the linear transcript, and terminating at an in-frame STOP codon, created upon circularization. Circ-ZNF609 is associated with heavy polysomes, and it is translated into a protein in a splicing-dependent and cap-independent manner, providing an example of a protein-coding circRNA in eukaryotes.
Assuntos
Proliferação de Células , Desenvolvimento Muscular , Proteínas Musculares/biossíntese , Distrofia Muscular de Duchenne/metabolismo , Mioblastos Esqueléticos/metabolismo , Biossíntese de Proteínas , RNA/metabolismo , Animais , Genótipo , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Camundongos , Proteínas Musculares/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/fisiopatologia , Mioblastos Esqueléticos/patologia , Fases de Leitura Aberta , Fenótipo , RNA/genética , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Interferência de RNA , Splicing de RNA , RNA Circular , Análise de Sequência de RNA/métodos , Transdução de Sinais , TransfecçãoRESUMO
RNA abundance is tightly regulated in eukaryotic cells by modulating the kinetic rates of RNA production, processing, and degradation. To date, little is known about timedependent kinetic rates during dynamic processes. Here, we present SLAMDropseq, a method that combines RNA metabolic labeling and alkylation of modified nucleotides in methanolfixed cells with dropletbased sequencing to detect newly synthesized and preexisting mRNAs in single cells. As a first application, we sequenced 7280 HEK293 cells and calculated genespecific kinetic rates during the cell cycle using the novel package Eskrate. Of the 377 robustcycling genes that we identified, only a minor fraction is regulated solely by either dynamic transcription or degradation (6 and 4%, respectively). By contrast, the vast majority (89%) exhibit dynamically regulated transcription and degradation rates during the cell cycle. Our study thus shows that temporally regulated mRNA degradation is fundamental for the correct expression of a majority of cycling genes. SLAMDropseq, combined with Eskrate, is a powerful approach to understanding the underlying mRNA kinetics of singlecell gene expression dynamics in continuous biological processes.
Assuntos
Ciclo Celular , RNA Mensageiro , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ciclo Celular/genética , Cinética , Análise de Sequência de RNA/métodos , HumanosRESUMO
The Lupus autoantigen La is an RNA-binding protein that stabilizes RNA polymerase III (Pol III) transcripts and supports RNA folding and has in addition been implicated in the mammalian microRNA (miRNA) pathway. Here, we have analyzed effects of La depletion on Argonaute (Ago)-bound small RNAs in human cells. We find that in the absence of La, distinct tRNA fragments are loaded into Ago proteins. Thus, La functions as gatekeeper ensuring correct tRNA maturation and protecting the miRNA pathway from potentially functional tRNA fragments. However, one specific isoleucin pre-tRNA produces both a functional tRNA and a miRNA even when La is present. We demonstrate that the fully complementary 5' leader and 3' trailer of the pre-tRNA-Ile form a double-stranded RNA molecule that has low affinity to La. Instead, Exportin-5 (Xpo5) recognizes it as miRNA precursor and transports it into the cytoplasm for Dicer processing and Ago loading.
Assuntos
Autoantígenos/metabolismo , MicroRNAs/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Transferência de Isoleucina/metabolismo , Ribonucleoproteínas/metabolismo , Células A549 , Proteínas Argonautas/metabolismo , Autoantígenos/genética , Sítios de Ligação , RNA Helicases DEAD-box/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Carioferinas/metabolismo , Células MCF-7 , MicroRNAs/genética , Conformação de Ácido Nucleico , Ligação Proteica , Interferência de RNA , RNA Polimerase III/metabolismo , Precursores de RNA/química , Precursores de RNA/genética , RNA de Transferência de Isoleucina/química , RNA de Transferência de Isoleucina/genética , RNA Viral/genética , RNA Viral/metabolismo , Ribonuclease III/metabolismo , Ribonucleoproteínas/genética , Relação Estrutura-Atividade , Transfecção , Antígeno SS-BRESUMO
To explore the role of Dicer-dependent control mechanisms in B lymphocyte development, we ablated this enzyme in early B cell progenitors. This resulted in a developmental block at the pro- to pre-B cell transition. Gene-expression profiling revealed a miR-17 approximately 92 signature in the 3'UTRs of genes upregulated in Dicer-deficient pro-B cells; a top miR-17 approximately 92 target, the proapoptotic molecule Bim, was highly upregulated. Accordingly, B cell development could be partially rescued by ablation of Bim or transgenic expression of the prosurvival protein Bcl-2. This allowed us to assess the impact of Dicer deficiency on the V(D)J recombination program in developing B cells. We found intact Ig gene rearrangements in immunoglobulin heavy (IgH) and kappa chain loci, but increased sterile transcription and usage of D(H) elements of the DSP family in IgH, and increased N sequence addition in Igkappa due to deregulated transcription of the terminal deoxynucleotidyl transferase gene.
Assuntos
Diversidade de Anticorpos , Linfócitos B/citologia , Sobrevivência Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Regiões 3' não Traduzidas/química , Regiões 3' não Traduzidas/metabolismo , Animais , Northern Blotting , Perfilação da Expressão Gênica , Rearranjo Gênico do Linfócito B , Imunoglobulinas/genética , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonuclease III , Organismos Livres de Patógenos EspecíficosRESUMO
Circular RNAs (circRNAs) are an endogenous class of animal RNAs. Despite their abundance, their function and expression in the nervous system are unknown. Therefore, we sequenced RNA from different brain regions, primary neurons, isolated synapses, as well as during neuronal differentiation. Using these and other available data, we discovered and analyzed thousands of neuronal human and mouse circRNAs. circRNAs were extraordinarily enriched in the mammalian brain, well conserved in sequence, often expressed as circRNAs in both human and mouse, and sometimes even detected in Drosophila brains. circRNAs were overall upregulated during neuronal differentiation, highly enriched in synapses, and often differentially expressed compared to their mRNA isoforms. circRNA expression correlated negatively with expression of the RNA-editing enzyme ADAR1. Knockdown of ADAR1 induced elevated circRNA expression. Together, we provide a circRNA brain expression atlas and evidence for important circRNA functions and values as biomarkers.
Assuntos
Encéfalo/metabolismo , RNA/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Drosophila melanogaster , Humanos , Camundongos , Dados de Sequência Molecular , Neurogênese , Especificidade de Órgãos , RNA/genética , RNA Circular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Sinapses/metabolismoRESUMO
BACKGROUND: During their lifetime, animals must adapt their behavior to survive in changing environments. This ability requires the nervous system to undergo adjustments at distinct temporal scales, from short-term dynamic changes in expression of neurotransmitters and receptors to longer-term growth, spatial and connectivity reorganization, while integrating external stimuli. The nematode Caenorhabditis elegans provides a model of nervous system plasticity, in particular its dauer exit decision. Under unfavorable conditions, larvae will enter the non-feeding and non-reproductive stress-resistant dauer stage and adapt their behavior to cope with the harsh new environment, with active reversal under improved conditions leading to resumption of reproductive development. However, how different environmental stimuli regulate the exit decision mechanism and thereby drive the larva's behavioral change is unknown. To fill this gap and provide insights on behavioral changes over extended periods of time, we developed a new open hardware method for long-term imaging (12h) of C. elegans larvae. RESULTS: Our WormObserver platform comprises open hardware and software components for video acquisition, automated processing of large image data (> 80k images/experiment) and data analysis. We identified dauer-specific behavioral motifs and characterized the behavioral trajectory of dauer exit in different environments and genetic backgrounds to identify key decision points and stimuli promoting dauer exit. Combining long-term behavioral imaging with transcriptomics data, we find that bacterial ingestion triggers a change in neuropeptide gene expression to establish post-dauer behavior. CONCLUSIONS: Taken together, we show how a developing nervous system can robustly integrate environmental changes activate a developmental switch and adapt the organism's behavior to a new environment. WormObserver is generally applicable to other research questions within and beyond the C. elegans field, having a modular and customizable character and allowing assessment of behavioral plasticity over longer periods.
Assuntos
Proteínas de Caenorhabditis elegans , Nematoides , Neuropeptídeos , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Larva , Neuropeptídeos/metabolismoRESUMO
Acute kidney injury (AKI) is a major health issue, the outcome of which depends primarily on damage and reparative processes of tubular epithelial cells. Mechanisms underlying AKI remain incompletely understood, specific therapies are lacking and monitoring the course of AKI in clinical routine is confined to measuring urine output and plasma levels of filtration markers. Here we demonstrate feasibility and potential of a novel approach to assess the cellular and molecular dynamics of AKI by establishing a robust urine-to-single cell RNA sequencing (scRNAseq) pipeline for excreted kidney cells via flow cytometry sorting. We analyzed 42,608 single cell transcriptomes of 40 urine samples from 32 patients with AKI and compared our data with reference material from human AKI post-mortem biopsies and published mouse data. We demonstrate that tubular epithelial cells transcriptomes mirror kidney pathology and reflect distinct injury and repair processes, including oxidative stress, inflammation, and tissue rearrangement. We also describe an AKI-specific abundant urinary excretion of adaptive progenitor-like cells. Thus, single cell transcriptomics of kidney cells excreted in urine provides noninvasive, unprecedented insight into cellular processes underlying AKI, thereby opening novel opportunities for target identification, AKI sub-categorization, and monitoring of natural disease course and interventions.