RESUMO
Nephrons are the functional units of the kidney. During kidney development, cells from the cap mesenchyme-a transient kidney-specific progenitor state-undergo a mesenchymal to epithelial transition (MET) and subsequently differentiate into the various epithelial cell types that create the tubular structures of the nephron. Faults in this transition can lead to a pediatric malignancy of the kidney called Wilms' tumor that mimics normal kidney development. While human kidney development has been characterized at the gene expression level, a comprehensive characterization of alternative splicing is lacking. Therefore, in this study, we performed RNA sequencing on cell populations representing early, intermediate, and late developmental stages of the human fetal kidney, as well as three blastemal-predominant Wilms' tumor patient-derived xenografts. Using this newly generated RNAseq data, we identified a set of transcripts that are alternatively spliced between the different developmental stages. Moreover, we found that cells from the earliest developmental stage have a mesenchymal splice-isoform profile that is similar to that of blastemal-predominant Wilms' tumor xenografts. RNA binding motif enrichment analysis suggests that the mRNA binding proteins ESRP1, ESRP2, RBFOX2, and QKI regulate alternative mRNA splicing during human kidney development. These findings illuminate new molecular mechanisms involved in human kidney development and pediatric kidney cancer.
Assuntos
Neoplasias Renais , Tumor de Wilms , Humanos , Criança , Processamento Alternativo , RNA Mensageiro/genética , Tumor de Wilms/genética , Tumor de Wilms/patologia , Neoplasias Renais/patologia , Rim/patologia , Células Cultivadas , Fatores de Processamento de RNA/genética , Proteínas Repressoras/genéticaRESUMO
BACKGROUND: During mammalian kidney development, nephron progenitors undergo a mesenchymal-to-epithelial transition and eventually differentiate into the various tubular segments of the nephron. Recently, Drop-seq single-cell RNA sequencing technology for measuring gene expression from thousands of individual cells identified the different cell types in the developing kidney. However, that analysis did not include the additional layer of heterogeneity that alternative mRNA splicing creates. METHODS: Full transcript length single-cell RNA sequencing characterized the transcriptomes of 544 individual cells from mouse embryonic kidneys. RESULTS: Gene expression levels measured with full transcript length single-cell RNA sequencing identified each cell type. Further analysis comprehensively characterized splice isoform switching during the transition between mesenchymal and epithelial cellular states, which is a key transitional process in kidney development. The study also identified several putative splicing regulators, including the genes Esrp1/2 and Rbfox1/2. CONCLUSIONS: Discovery of the sets of genes that are alternatively spliced as the fetal kidney mesenchyme differentiates into tubular epithelium will improve our understanding of the molecular mechanisms that drive kidney development.
Assuntos
Rim/embriologia , Mesoderma/embriologia , Organogênese/genética , Urotélio/embriologia , Animais , Técnicas de Cultura de Células , Camundongos , Isoformas de RNA , Análise de Sequência de RNARESUMO
The origins and functions of kidney macrophages in the adult have been explored, but their roles during development remain largely unknown. Here we characterise macrophage arrival, localisation, heterogeneity, and functions during kidney organogenesis. Using genetic approaches to ablate macrophages, we identify a role for macrophages in nephron progenitor cell clearance as mouse kidney development begins. Throughout renal organogenesis, most kidney macrophages are perivascular and express F4/80 and CD206. These macrophages are enriched for mRNAs linked to developmental processes, such as blood vessel morphogenesis. Using antibody-mediated macrophage-depletion, we show macrophages support vascular anastomoses in cultured kidney explants. We also characterise a subpopulation of galectin-3+ (Gal3+) myeloid cells within the developing kidney. Our findings may stimulate research into macrophage-based therapies for renal developmental abnormalities and have implications for the generation of bioengineered kidney tissues.
Assuntos
Galectina 3/genética , Rim/crescimento & desenvolvimento , Néfrons/crescimento & desenvolvimento , Organogênese/genética , Animais , Proteínas de Ligação ao Cálcio/genética , Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento , Rim/metabolismo , Lectinas Tipo C/genética , Macrófagos/metabolismo , Receptor de Manose , Lectinas de Ligação a Manose/genética , Camundongos , Néfrons/metabolismo , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G/genética , Células-Tronco/metabolismoRESUMO
In recent years, there has been an effort to develop new technologies for measuring gene expression and sequence information from thousands of individual cells. Large data sets that were obtained using these 'single cell' technologies have allowed scientists to address fundamental questions in biomedicine ranging from stems cells and development to cancer and immunology. Here, we provide a brief review of recent developments in single-cell technology. Our intention is to provide a quick background for newcomers to the field as well as a deeper description of some of the leading technologies to date.