BASIC RESEARCH www.jasn.org Single-Cell RNA Sequencing Reveals mRNA Splice Isoform Switching during Kidney Development Yishay Wineberg,1 Tali Hana Bar-Lev,1 Anna Futorian,2 Nissim Ben-Haim,1 Leah Armon,2 Debby Ickowicz,2 Sarit Oriel,1 Efrat Bucris,1 Yishai Yehuda,1 Naomi Pode-Shakked ,3,4,5 Shlomit Gilad,6 Sima Benjamin,6 Peter Hohenstein,7 Benjamin Dekel,3,4,5 Achia Urbach,2 and Tomer Kalisky1 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT 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 indi- vidual 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 tran- sition 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. JASN 31: ccc–ccc, 2020. doi: https://doi.org/10.1681/ASN.2019080770 Kidney development is a complex process that in- population. Next, cells from the CM undergo a mes- volves multiple interacting cell types.1–5 It starts at enchymal-to-epithelial transition (MET) and the embryonic stage and continues from week 5 to progressively differentiate into early epithelial struc- week 36 of gestation in humans and from embry- tures: pretubular aggregates, renal vesicles, and onic day (E) 10.5 to approximately day 3 after birth comma and S-shaped bodies. The S-shaped bodies in mice. The process is initiated by signaling inter- actions between two lineages originating from the intermediate mesoderm: the ureteric duct and the Received August 2, 2019. Accepted May 23, 2020. metanephric mesenchyme (Figure S1 in Supple- Y.W., T.H.B.-L., A.F., and N.B.-H. are cofirst authors and B.D., A. mental Appendix 1). These interactions invoke U., and T.K. are cosenior authors. the ureteric duct to invade the metanephric mesen- Published online ahead of print. Publication date available at chyme and create a tree-like structure. Around the www.jasn.org. tip of each branch of this tree, the ureteric tip, cells Correspondence: Dr. Tomer Kalisky, Department of Bio- from the metanephric mesenchyme are induced engineering, Bar-Ilan University, Ramat Gan, Israel 52900. Email: to condense and form the cap mesenchyme (CM), [email protected] which is a transient nephron progenitor cell Copyright © 2020 by the American Society of Nephrology JASN 31: ccc–ccc, 2020 ISSN : 1046-6673/3110-ccc 1 BASIC RESEARCH www.jasn.org further elongate and differentiate to form the various epithelial Significance Statement tubular segments of the fully developed nephron, whose main constituents are the podocytes (PODOs), the proximal tubule, Kidney development is a complex process involving multiple in- the loop of Henle (LOH), and the distal tubule. At an early stage teracting and transitioning cell types. Drop-seq single-cell tech- in their differentiation the distal tubules connect to the ureteric nology, which measures gene expression from many thousands of individual cells, has been used to characterize these cellular differ- tips that form the collecting duct system for draining the neph- entiation changes that underlie organ development. However, the rons. Meanwhile, the uninduced cells of the metanephric mes- alternative splicing of many genes creates an additional layer of enchyme differentiate into other supporting cell types of the cellular heterogeneity that Drop-seq technology cannot measure. kidney such as interstitial fibroblasts, pericytes, and mesangial Therefore, in this study, full transcript length single-cell RNA se- cells (Figure S1 in Supplemental Appendix 1). In the past few quencing was used to characterize alternative splicing in the mouse embryonic kidney, with particular attention to the identification of years, the various cell populations of the developing kidney genes that are alternatively spliced during the transition from – were characterized,6 8 mainly using the Drop-seq single-cell mesenchymal to epithelial cell states, as well as their splicing reg- RNA sequencing protocol that enables measuring of gene ex- ulators. These results improve our understanding of the molecular pression levels from many thousands of individual cells.9 mechanisms that underlie kidney development. A central process in kidney development is the MET that occurs during the differentiation of cells from the metanephric heterogeneous organ that is composed of numerous cell mesenchyme to the CM and then to nephron tubules. Similar populations of widely varying proportions,6–8,24 it is diffi- transitions from mesenchymal to epithelial states and vice cult to isolate pure populations of mesenchymal and epi- versa (epithelial-to-mesenchymal transition [EMT]) are thelial states. Moreover, typical sequencing depths from a thought to play a central role in development, as well as in single cell are not sufficient for splicing analysis. We there- 10 pathologic processes such as cancer metastasis and organ fore performed single-cell RNA sequencing on 576 indi- fi 11 fi brosis. There are signi cant structural and functional vidual cells from the kidneys of E18.5 mouse embryos differences between mesenchymal and epithelial cells. Mesen- using the Smartseq2 protocol for sequencing full transcript chymal cells are typically loosely associated with each other, lengths.25,26 We first identified the main cell lineages that surrounded by an extracellular matrix, and have migratory coexist in the nephrogenic zone of the fetal developing capabilities, whereas epithelial cells are tightly interconnected kidney: the uninduced metanephric mesenchyme, the by junctions and are polarized with distinct apical and baso- CM, podocytes, epithelial cells, endothelial cells, and in- lateral membranes. Thus, epithelial cells can create two- filtrating immune cells (e.g., macrophages). We then dimensional surfaces and tubes with a clear in/out distinction merged the raw reads from all cells belonging to each pop- that are capable of absorption and secretion. There are also ulation in order to create “bulk” in silico transcriptomes large differences in gene expression: mesenchymal cells typi- that represent each cell population. These bulk transcrip- cally express Fibronectin (Fn1), Vimentin (Vim), and tomes had sufficient sequencing depth to allow us to char- 12 N-cadherin (Cdh2), as well as the transcription factors acterize splice isoform switching and to identify putative Snai1/2, Zeb1/2, and Twist1/2, whereas epithelial cells typi- splicing regulators. cally express other genes such as E-cadherin (Cdh1) and Epcam. It was recently discovered that mesenchymal and epithelial METHODS cells also express alternative splice isoforms of genes that are expressed in both cell states. For example, in many systems Tissue Collection, Dissociation, and Flow Cytometry both in vivo and in vitro, the genes Enah, Cd44, Ctnnd1, and A wild-type female mouse was crossed with a male that was Fgfr2 were found to be expressed in both mesenchymal and heterozygous for the Six2-GFP transgene.27 The female was epithelial states, but with unique isoforms specifictoeach euthanized at E18.5 of pregnancy and kidneys from 11 em- state.13–18 It was also found that RNA binding proteins, such bryos were dissected, placed in PBS on ice, and examined un- as ESRP1/2, RBFOX1/2, RBM47, QKI, act as splicing regula- der a fluorescence microscope to check for the presence of tors that promote splicing of specific mesenchymal or epithe- GFP. Kidneys from transgenic embryos showed a clear fluo- lial variants.12,17,19,20 mRNA splicing creates an additional rescent pattern marking the CM (Figure 1A), whereas those layer of heterogeneity that, apart from specific genes,21–23 from the nontransgenic embryos showed uniform back- has not yet been comprehensively studied in the developing ground fluorescence. Seven out of the 11 embryos contained kidney. the Six2-GFP transgene. Keeping the transgenic and nontrans- Therefore, in this study we set to characterize the splice genic kidneys separate, each kidney was then cut into two to isoform switching events that occur during the transition four small pieces using a surgical razor blade and placed in between the mesenchymal and epithelial cellular states in 1mloftrypsin0.25%(03–046–1A, Trypsin Solution B the course of kidney development by comparing gene ex- [0.25%]; Biologic Industries) using forceps. Initial tissue trit- pression and alternative splicing in the various mesenchy- uration was performed using a tissue grinder (D8938, Dounce, mal and epithelial cell populations. Because the kidney is a large clearance pestle) followed