Single-cell RNA sequencing reveals mRNA splice isoform switching during kidney development

Wineberg, Yishay; Bar-Lev, Tali Hana; Futorian, Anna; Ben-Haim, Nissim; Armon, Leah; Ickowicz, Debby; Oriel, Sarit; Bucris, Efrat; Yehuda, Y.; Pode‐Shakked, Naomi; Gilad, Shlomit; Schwessinger, Benjamin; Hohenstein, Peter; Dekel, Benjamin; Urbach, Achia; Kalisky, Tomer

doi:10.1101/688564

Cited by 6 publications

(12 citation statements)

References 63 publications

(8 reference statements)

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“…Compared to other reports identifying specific targets of ESRPs using RNA-seq (58, 59), we found several targets in common, including well-known genes such as CD44, CTNND1, SCRIB and SLK , as well as over 600 genes that had not been previously described as ESRP2 targets (figure 7C). An additional comparison with a recent study of MET-associated alternative splicing changes during kidney development (60) also revealed overlap with some of our target genes (figure 7D). Interestingly, the two lists of genes identified in figures 7C and D as overlapping our ESRP2 targets, included five genes in common ( CTNND1, CTTN, FLNB, MAO3K7, MPRIP ; shown in bold in figures 7C and D).…”

Section: Resultssupporting

confidence: 59%

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The epithelial splicing regulatorESRP2is epigenetically repressed by DNA hypermethylation in Wilms tumour and acts as a tumour suppressor

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Moriarty

et al. 2020

Preprint

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Wilms tumour (WT), a childhood kidney cancer with embryonal origins, has been extensively characterised for genetic and epigenetic alterations, but a proportion of WTs still lack identifiable abnormalities. To uncover DNA methylation changes critical for WT pathogenesis, we compared the epigenome of fetal kidney with two WT cell lines, using methyl-CpG immunoprecipitation. We filtered our results to remove common cancer-associated epigenetic changes, and to enrich for genes involved in early kidney development. This identified four candidate genes that were hypermethylated in WT cell lines compared to fetal kidney, of which ESRP2 (epithelial splicing regulatory protein 2), was the most promising gene for further study. ESRP2 was commonly repressed by DNA methylation early in WT development (in nephrogenic rests) and could be reactivated by DNA methyltransferase inhibition in WT cell lines. When ESRP2 was expressed in WT cell lines, it acted as an inhibitor of cellular proliferation in vitro and in vivo it suppressed tumour growth of orthotopic xenografts in nude mice. RNA-seq of the ESRP2-expressing WT cell lines identified several novel splicing targets, in addition to well-characterised targets of ESRP2. One of these targets, LEF1, is a component of the Wnt signalling pathway that is essential for kidney development and commonly disrupted in WT. We propose a model in which the Wnt pathway can be disrupted in early kidney development to generate WT, either by genetic abnormalities such as WT1 mutations, or by epigenetic defects, such as ESRP2 methylation.

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Section: Resultssupporting

confidence: 59%

“…D: Venn diagram comparing unique genes identified in this study (SE+MXE+RI) with a RNA-seq analysis of MET-associated splicing changes in the developing kidney (60). …”

Section: Resultsmentioning

confidence: 99%

The epithelial splicing regulatorESRP2is epigenetically repressed by DNA hypermethylation in Wilms tumour and acts as a tumour suppressor

Legge

Moriarty

et al. 2020

Preprint

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“…Ideally, the best way to characterize alternative splicing throughout the different developmental stages is by applying full transcript length single-cell RNA sequencing [42][43][44][45]. We have recently performed full transcript length single-cell RNA sequencing in the developing mouse fetal kidney [46] and identified a set of transcripts -similar to those found here -that undergo splice isoform switching during the transition between mesenchymal and epithelial cellular states that takes place in the course of mouse fetal kidney development. Likewise, RNA binding motif enrichment analysis suggested that Esrp1/2 and Rbfox1/2 are splicing regulators of the Mesenchymal to Epithelial Transition (MET) that occurs during mouse kidney development, also similar to what we found here.…”

Section: Discussionmentioning

confidence: 62%

Characterization of splice isoform switching during human kidney development

Wineberg

Kanter

Ben-Haim

et al. 2019

Preprint

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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 kidney development has been characterized at the gene expression level, a comprehensive characterization of alternative splicing is lacking. We therefore 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. 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' tumors. RNA binding motif enrichment analysis suggests that the mRNA binding proteins ESRP1, ESRP2, RBFOX2, and QKI regulate mRNA splice isoform switching during human kidney development. These findings illuminate new molecular mechanisms involved in kidney development and pediatric kidney tumors. KEYWORDSKidney development, RNA sequencing, Alternative splicing, Mesenchymal to epithelial transition (MET), Wilms' tumor HIGHLIGHTS During fetal kidney development, kidney progenitor cells undergo a mesenchymal to epithelial transition (MET) and subsequently differentiate into the various epithelial cell types that create the tubular structures of the nephron.  RNA sequencing identifies a set of transcripts that undergo splice isoform switching during the mesenchymal to epithelial transition (MET) that occurs in the course of human fetal kidney development.  Cells in the early stages of kidney development have a mesenchymal spliceisoform profile that is similar to that observed in blastemal-predominant Wilms' tumor patient-derived xenografts (WT-PDX) that represent an aggressive subtype of Wilms' tumors.  RNA binding motif enrichment analysis indicates that the mRNA binding proteins ESRP1, ESRP2, RBFOX2, and QKI regulate splice isoform switching during human kidney development.

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“…They are likely to be useful for a wide number of future applications. Another important aspect of these data is that it adds information on alternative splicing differences along the renal tubule, adding to other recent evidence 70 . We believe these data together with previous RNA-seq 17 and more recent proteomic data 61 from rat tubules will enhance our understanding of renal function.…”

Section: Discussionmentioning

confidence: 81%

Targeted single-cell RNA-seq identifies minority cell types of kidney distal nephron that regulate blood pressure and calcium balance

Chen

Chou

Knepper

2020

Preprint

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A major objective in modern biology is generation of comprehensive atlases of various organs identifying all cell types and their expressed genes. In kidney, extensive data exists for proximal tubule and collecting duct cells, but not for non-abundant intermediate epithelial cell types. Here, we coupled a FACS-enrichment protocol with single-cell RNA-seq analysis to profile the transcriptomes of 9099 cells from the nephron region adjacent to the macula densa. Clusters containing Slc12a3+/Pvalb+ and Slc12a3+/Pvalb- cells were identified as DCT1 and DCT2 cells. The DCT1 cells appear to be heterogeneously associated with variable expression of Slc8a1, Calb1, and Ckb among other mRNAs. No DCT2-specific transcripts were found. The analysis also identified two distinct cell types in the Slc12a1+ portion of Henle's loop as well as Nos1+/Avpr1a+ macula densa cells. Thus, we identify unexpected cell diversity in the intermediate region of the nephron and create a web-based data resource for these cells.

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Single-cell RNA sequencing reveals mRNA splice isoform switching during kidney development

Cited by 6 publications

References 63 publications

The epithelial splicing regulatorESRP2is epigenetically repressed by DNA hypermethylation in Wilms tumour and acts as a tumour suppressor

The epithelial splicing regulatorESRP2is epigenetically repressed by DNA hypermethylation in Wilms tumour and acts as a tumour suppressor

Characterization of splice isoform switching during human kidney development

Targeted single-cell RNA-seq identifies minority cell types of kidney distal nephron that regulate blood pressure and calcium balance

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