Characterization of alternative mRNA splicing in cultured cell populations representing progressive stages of human fetal kidney development.

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.

Single-Cell RNA Sequencing Reveals mRNA Splice Isoform Switching during Kidney Development.

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.

Ex Vivo Expanded 3D Human Kidney Spheres Engraft Long Term and Repair Chronic Renal Injury in Mice.

End-stage renal disease is a worldwide epidemic requiring renal replacement therapy. Harvesting tissue from failing kidneys and autotransplantation of tissue progenitors could theoretically delay the need for dialysis. Here we use healthy and end-stage human adult kidneys to robustly expand proliferative kidney epithelial cells and establish 3D kidney epithelial cultures termed "nephrospheres." Formation of nephrospheres reestablishes renal identity and function in primary cultures. Transplantation into NOD/SCID mice shows that nephrospheres restore self-organogenetic properties lost in monolayer cultures, allowing long-term engraftment as tubular structures, potentially adding nephron segments and demonstrating self-organization as critical to survival. Furthermore, long-term tubular engraftment of nephrospheres is functionally beneficial in murine models of chronic kidney disease. Remarkably, nephrospheres inhibit pro-fibrotic collagen production in cultured fibroblasts via paracrine modulation, while transplanted nephrospheres induce transcriptional signatures of proliferation and release from quiescence, suggesting re-activation of endogenous repair. These data support the use of human nephrospheres for renal cell therapy.

Chromatin occupancy and epigenetic analysis reveal new insights into the function of the GATA1 N terminus in erythropoiesis.

Mutations in GATA1, which lead to expression of the GATA1s isoform that lacks the GATA1 N terminus, are seen in patients with Diamond-Blackfan anemia (DBA). In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in Gata1s mice. Defects in yolks sac and fetal liver hematopoiesis included impaired terminal maturation and reduced numbers of erythroid progenitors. RNA-sequencing revealed that both erythroid and megakaryocytic gene expression patterns were altered by the loss of the N terminus, including aberrant upregulation of Gata2 and Runx1. Dysregulation of global H3K27 methylation was found in the erythroid progenitors upon loss of N terminus of GATA1. Chromatin-binding assays revealed that, despite similar occupancy of GATA1 and GATA1s, there was a striking reduction of H3K27me3 at regulatory elements of the Gata2 and Runx1 genes. Consistent with the observation that overexpression of GATA2 has been reported to impair erythropoiesis, we found that haploinsufficiency of Gata2 rescued the erythroid defects of Gata1s fetuses. Together, our integrated genomic analysis of transcriptomic and epigenetic signatures reveals that, Gata1 mice provide novel insights into the role of the N terminus of GATA1 in transcriptional regulation and red blood cell maturation which may potentially be useful for DBA patients.

A brief review of single-cell transcriptomic technologies.

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.