Validating single-cell genomics for the study of renal development.
Single-cell genomics will enable studies of the earliest events in kidney development, although it is unclear if existing technologies are mature enough to generate accurate and reproducible data on kidney progenitors. Here we designed a pilot study to validate a high-throughput assay to measure the expression levels of key regulators of kidney development in single cells isolated from embryonic mice. Our experiment produced 4608 expression measurements of 22 genes, made in small cell pools, and 28 single cells purified from the RET-positive ureteric bud. There were remarkable levels of concordance with expression data generated by traditional microarray analysis on bulk ureteric bud tissue with the correlation between our average single-cell measurements and GUDMAP measurements for each gene of 0.82-0.85. Nonetheless, a major motivation for single-cell technology is to uncover dynamic biology hidden in population means. There was evidence for extensive and surprising variation in expression of Wnt11 and Etv5, both downstream targets of activated RET. The variation for all genes in the study was strongly consistent with burst-like promoter kinetics. Thus, our results can inform the design of future single-cell experiments, which are poised to provide important insights into kidney development and disease.
1] Renal Division, Department of Internal Medicine, Washington University School of Medicine, St Louis, Missouri, USA [2] Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri, USA.
Sanjay
S
Jain
Department of Genetics, Washington University, School of Medicine, St Louis, Missouri, USA.
Michiel J
MJ
Noordam
Renal Division, Department of Internal Medicine, Washington University School of Medicine, St Louis, Missouri, USA.
Masato
M
Hoshi
1] Department of Genetics, Washington University, School of Medicine, St Louis, Missouri, USA [2] Center for Genome Sciences and Systems Biology, Washington University School of Medicine, Washington University School of Medicine, St Louis, Missouri, USA.
Francesco L
FL
Vallania
1] Department of Genetics, Washington University, School of Medicine, St Louis, Missouri, USA [2] Center for Genome Sciences and Systems Biology, Washington University School of Medicine, Washington University School of Medicine, St Louis, Missouri, USA.
Donald F
DF
Conrad
D000818:Animals; D002469:Cell Separation / Q000379:methods; D016000:Cluster Analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental; D023281:Genomics / Q000379:methods; D005865:Gestational Age; D059014:High-Throughput Nucleotide Sequencing; D007668:Kidney / Q000196:embryology / Q000378:metabolism*; D008822:Mice, Transgenic; D020411:Oligonucleotide Array Sequence Analysis; D038081:Organogenesis; D010865:Pilot Projects; D011401:Promoter Regions, Genetic; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014513:Ureter / Q000196:embryology / Q000378:metabolism*; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; C513242:Etv5 protein, mouse; D014157:Transcription Factors; D051153:Wnt Proteins; C508957:Wnt11 protein, mouse; D051096:Proto-Oncogene Proteins c-ret; C494999:Ret protein, mouse