Identification Of Novel Essential Genes Of Mouse Glomerular Cells Based On Single-cell RNA-sequencing

Identification of genes that are essential for a given cell type is critical for understanding of the biology of the cell type.However,there is a lack of approaches that are capable of identifying such genes efficiently.It is known that gene expression differs substantially among individual cells of the same type.We speculate that genes that are expressed in all but a portion of cells in a given cell type would be likely essential and required for either the cells to survive(house-keeping)or for the cell type's unique structure and function that enable the organism to survive or reproduce(cell type specific-essential).Whereas,those expressed only in a portion of cells of a cell type would be likely dispensable.Here,we performed RNA-seq of 14 mouse glomerular mesangial cells and 20 podocytes based on the Fluidigm C1TM Single-Cell Auto Prep System.Single-cell RNA-seq analysis of the 14 mesangial cells showed that gene expression profile in individual mesangial cells was tremendously heterogeneous,with correlation coefficients of 0.20 ± 0.045(Mean±SD).Most of the genes were actually expressed in only a portion of mesangial cells and therefore presumably dispensable.We found that 1,045 genes were expressed in each of the 14 mesangial cells(cutoff>0 RPKM)and were considered mesangial cell essential gene(MCEG)candidates.The Gene Ontology analysis revealed a significant enrichment of genes associated with the endothelium,supporting the inference that mesangial cells function as pericytes.Among 58 endothelium-associated genes,18 encode proteins that are secreted and may be directly involved in endothelial homeostasis.Importantly,among the 18 genes,11(ANGPT2,ANXA5,AXL,ECM1,ENG,FN1,MFGE8,MSN,NRP1,SERPINE2,AND SPARC)were upregulated,while 2(APOE and FGF1)were downregulated in various glomerulopathies.The enrichment of genes associated with other reported functions of mesangial cells was found as well.Furthermore,we identified 173 genes that are specifically expressed in every mesangial cell in glomeruli from the 1,045 MCEG candidates above.Finally,we found that the commonly used markers of podocytes and glomerular endothelial cells,including WT1,TEK/TIE2,KDR/FLK1,FLT1/VEGFR1,and CD34,were also expressed in some the 14 mesangial cells,indicating that these markers are actually not podocytes or endothelial cells specific.In summary,based on the single-cell RNA-seq analysis of 14 mesangial cells,we found a significant enrichment of genes associated with endothelium and other reported functions of mesangial cells,thus having provided the first transcriptomic evidence that mesangial cells act as pericytes,and having revealed the molecular basis underlying the other known functions of mesangial cells.These results have also proven our speculation that genes expressed in every single cell of a given cell type are essential for that type of cells.As mentioned above,essential genes in a cell can be divided into two categories,house-keeping genes and the cell type-specific essential genes.We speculate that it is feasible to separate these two categories of essential genes by comparing with the genes expressed in other cell types,i.e.,the genes commonly expressed in other cell types are house-keeping genes,while genes expressed only in the given cell type are the cell type-specific essential genes.To prove it,single-cell RNA-seq analysis of 20 podocytes was carried out.Similar to mesangial cells,a great heterogeneity in gene expression among individual podocytes was also found with correlation coefficients of 0.27±0.035(Mean±SD).We identified 335 genes that were expressed in all of the 20 podocytes with a more stringent criterion(cutoff>0.5 RPKM in contrast with>0 for mesangial cells).Among the 335 genes,239 were also expressed in mesangial cells and endothelial cells and are involved in energy metabolism,protein synthesis,etc.,according to GO analysis,thus being house-keeping genes.In contrast,92 genes were preferentially expressed in podocytes and are therefore the essential gene candidates that are specific for podocytes.Assessments by bioinformatics,conserved expression in human podocytes,and association with injury/disease all support their essentiality for podocytes.We functionally analyzed 37 novel genes by siRNA silencing in cultured podocytes and examined the cytoskeletons of the cells.Podocyte cytoskeletons determine podocyte unique structure that is critical glomerular filtration.The result showed that a deficiency of 30(81.1%)genes led to podocyte injury.These genes include FGFR1,AOX1 AIF1L,HAUS8,RAB3B,LPIN2,GOLIM4,CERS6,ARHGEF18,ARPC1A,SRGAP1,ITGB5,ILDR2,MPP5,TSC22D1,DNAJC11,SEPT10,MOCS2,FNBP1L,TMOD3,IFT80,MYOM2,ANXA4,CYB5R4,GPC1,ZNF277,NSF,ITGAV,CRYAB,and MTSS1.In summary,this work has greatly expanded the list of the genes that are essential for podocyte cytoskeletons;it has also demonstrated that most of the podocyte-specific essential genes are associated with cytoskeletons,consistent with podocytes' major function that is to produce an unique structure to form an crictial part of glomerular filtration barrier.In conclusion,we have performed single-cell RNA-seq analysis of mouse glomerular mesangial cells and podocytes and analyzed the results based on our innovated approach,thereby having identified a large number of mesangial cell or podocyte-specific essential genes.Our work has provided insights into the biology of mesangial cells and podocytes.Meanwhile,this work has also demonstrated the feasibility and effectiveness of our research strategy in identifying essential genes of a cell type.Finally,this high throughput approach can certainly be applied to any other cell types.