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1.
Nat Cell Biol ; 25(2): 351-365, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36646791

RESUMO

The lung contains numerous specialized cell types with distinct roles in tissue function and integrity. To clarify the origins and mechanisms generating cell heterogeneity, we created a comprehensive topographic atlas of early human lung development. Here we report 83 cell states and several spatially resolved developmental trajectories and predict cell interactions within defined tissue niches. We integrated single-cell RNA sequencing and spatially resolved transcriptomics into a web-based, open platform for interactive exploration. We show distinct gene expression programmes, accompanying sequential events of cell differentiation and maturation of the secretory and neuroendocrine cell types in proximal epithelium. We define the origin of airway fibroblasts associated with airway smooth muscle in bronchovascular bundles and describe a trajectory of Schwann cell progenitors to intrinsic parasympathetic neurons controlling bronchoconstriction. Our atlas provides a rich resource for further research and a reference for defining deviations from homeostatic and repair mechanisms leading to pulmonary diseases.


Assuntos
Embrião de Mamíferos , Perfilação da Expressão Gênica , Humanos , Diferenciação Celular/genética , Pulmão , Células-Tronco
2.
Cell Rep ; 37(7): 110015, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34788611

RESUMO

Previous large-scale studies have uncovered many features that determine the processing of microRNA (miRNA) precursors; however, they have been conducted in vitro. Here, we introduce MapToCleave, a method to simultaneously profile processing of thousands of distinct RNA structures in living cells. We find that miRNA precursors with a stable lower basal stem are more efficiently processed and also have higher expression in vivo in tissues from 20 animal species. We systematically compare the importance of known and novel sequence and structural features and test biogenesis of miRNA precursors from 10 animal and plant species in human cells. Lastly, we provide evidence that the GHG motif better predicts processing when defined as a structure rather than sequence motif, consistent with recent cryogenic electron microscopy (cryo-EM) studies. In summary, we apply a screening assay in living cells to reveal the importance of lower basal stem stability for miRNA processing and in vivo expression.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/biossíntese , MicroRNAs/genética , Animais , Humanos , Plantas/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/genética
3.
Commun Biol ; 3(1): 565, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037292

RESUMO

The field of spatial transcriptomics is rapidly expanding, and with it the repertoire of available technologies. However, several of the transcriptome-wide spatial assays do not operate on a single cell level, but rather produce data comprised of contributions from a - potentially heterogeneous - mixture of cells. Still, these techniques are attractive to use when examining complex tissue specimens with diverse cell populations, where complete expression profiles are required to properly capture their richness. Motivated by an interest to put gene expression into context and delineate the spatial arrangement of cell types within a tissue, we here present a model-based probabilistic method that uses single cell data to deconvolve the cell mixtures in spatial data. To illustrate the capacity of our method, we use data from different experimental platforms and spatially map cell types from the mouse brain and developmental heart, which arrange as expected.


Assuntos
Biologia Computacional , Perfilação da Expressão Gênica , Análise de Célula Única , Transcriptoma , Animais , Biologia Computacional/métodos , Biologia Computacional/normas , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos , Especificidade de Órgãos , Organogênese/genética , Análise de Célula Única/métodos , Análise de Célula Única/normas
4.
Bioessays ; 42(10): e1900221, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32363691

RESUMO

Recent advances in spatially resolved transcriptomics have greatly expanded the knowledge of complex multicellular biological systems. The field has quickly expanded in recent years, and several new technologies have been developed that all aim to combine gene expression data with spatial information. The vast array of methodologies displays fundamental differences in their approach to obtain this information, and thus, demonstrate method-specific advantages and shortcomings. While the field is moving forward at a rapid pace, there are still multiple challenges presented to be addressed, including sensitivity, labor extensiveness, tissue-type dependence, and limited capacity to obtain detailed single-cell information. No single method can currently address all these key parameters. In this review, available spatial transcriptomics methods are described and their applications as well as their strengths and weaknesses are discussed. Future developments are explored and where the field is heading to is deliberated upon.


Assuntos
Transcriptoma , Transcriptoma/genética
5.
Cell ; 179(7): 1647-1660.e19, 2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31835037

RESUMO

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-Seq
6.
Sci Rep ; 9(1): 3179, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30816197

RESUMO

Heart failure affects 2-3% of adult Western population. Prevalence of heart failure with preserved left ventricular (LV) ejection fraction (HFpEF) increases. Studies suggest HFpEF patients to have altered myocardial structure and functional changes such as incomplete relaxation and increased cardiac stiffness. We hypothesised that patients undergoing elective coronary bypass surgery (CABG) with HFpEF characteristics would show distinctive gene expression compared to patients with normal LV physiology. Myocardial biopsies for mRNA expression analysis were obtained from sixteen patients with LV ejection fraction ≥45%. Five out of 16 patients (31%) had echocardiographic characteristics and increased NTproBNP levels indicative of HFpEF and this group was used as HFpEF proxy, while 11 patients had Normal LV physiology. Utilising principal component analysis, the gene expression data clustered into two groups, corresponding to HFpEF proxy and Normal physiology, and 743 differentially expressed genes were identified. The associated top biological functions were cardiac muscle contraction, oxidative phosphorylation, cellular remodelling and matrix organisation. Our results also indicate that upstream regulatory events, including inhibition of transcription factors STAT4, SRF and TP53, and activation of transcription repressors HEY2 and KDM5A, could provide explanatory mechanisms to observed gene expression differences and ultimately cardiac dysfunction in the HFpEF proxy group.


Assuntos
Insuficiência Cardíaca/genética , Miocárdio/metabolismo , Transcriptoma/genética , Função Ventricular Esquerda/genética , Idoso , Idoso de 80 Anos ou mais , Biópsia , Diástole , Ecocardiografia , Feminino , Regulação da Expressão Gênica/genética , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Contração Miocárdica/genética , Miocárdio/patologia , Volume Sistólico/genética
7.
Sci Rep ; 7(1): 12941, 2017 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-29021611

RESUMO

Heart failure is a major health problem linked to poor quality of life and high mortality rates. Hence, novel biomarkers, such as fetal marker genes with low expression levels, could potentially differentiate disease states in order to improve therapy. In many studies on heart failure, cardiac biopsies have been analyzed as uniform pieces of tissue with bulk techniques, but this homogenization approach can mask medically relevant phenotypes occurring only in isolated parts of the tissue. This study examines such spatial variations within and between regions of cardiac biopsies. In contrast to standard RNA sequencing, this approach provides a spatially resolved transcriptome- and tissue-wide perspective of the adult human heart, and enables detection of fetal marker genes expressed by minor subpopulations of cells within the tissue. Analysis of patients with heart failure, with preserved ejection fraction, demonstrated spatially divergent expression of fetal genes in cardiac biopsies.


Assuntos
Biomarcadores/metabolismo , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Miocárdio/metabolismo , Adulto , Idoso , Humanos , Masculino , Pessoa de Meia-Idade
8.
Science ; 353(6294): 78-82, 2016 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-27365449

RESUMO

Analysis of the pattern of proteins or messengerRNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics. This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call "spatial transcriptomics," that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics.


Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Transcriptoma , Animais , Encéfalo/metabolismo , Neoplasias da Mama/metabolismo , DNA Complementar/biossíntese , Feminino , Humanos , Camundongos , Especificidade de Órgãos , RNA Mensageiro/metabolismo
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