Pseudotemporal Ordering of Single Cells Reveals Metabolic Control of Postnatal β Cell Proliferation.

Pancreatic β cell mass for appropriate blood glucose control is established during early postnatal life. β cell proliferative capacity declines postnatally, but the extrinsic cues and intracellular signals that cause this decline remain unknown. To obtain a high-resolution map of β cell transcriptome dynamics after birth, we generated single-cell RNA-seq data of β cells from multiple postnatal time points and ordered cells based on transcriptional similarity using a new analytical tool. This analysis captured signatures of immature, proliferative β cells and established high expression of amino acid metabolic, mitochondrial, and Srf/Jun/Fos transcription factor genes as their hallmark feature. Experimental validation revealed high metabolic activity in immature β cells and a role for reactive oxygen species and Srf/Jun/Fos transcription factors in driving postnatal β cell proliferation and mass expansion. Our work provides the first high-resolution molecular characterization of state changes in postnatal β cells and paves the way for the identification of novel therapeutic targets to stimulate β cell regeneration.

[1]  Blaz Zupan,et al.  Stage prediction of embryonic stem cell differentiation from genome-wide expression data , 2011, Bioinform..

[2]  Giuseppe Jurman,et al.  Promoter-level expression clustering identifies time development of transcriptional regulatory cascades initiated by ErbB receptors in breast cancer cells , 2015, Scientific Reports.

[3]  Jason D. Ray,et al.  Nkx6.1‐mediated insulin secretion and β‐cell proliferation is dependent on upregulation of c‐Fos , 2016, FEBS letters.

[4]  G. Crabtree,et al.  Neonatal β cell development in mice and humans is regulated by calcineurin/NFAT. , 2012, Developmental cell.

[5]  M. Permutt,et al.  Conditional ablation of Gsk-3β in islet beta cells results in expanded mass and resistance to fat feeding-induced diabetes in mice , 2010, Diabetologia.

[6]  S. Bonner-Weir,et al.  Dynamics of β-cell Mass in the Growing Rat Pancreas: Estimation With a Simple Mathematical Model , 1995, Diabetes.

[7]  Lorenz Wernisch,et al.  Pseudotime estimation: deconfounding single cell time series , 2015, bioRxiv.

[8]  Sean C. Bendall,et al.  Single-Cell Trajectory Detection Uncovers Progression and Regulatory Coordination in Human B Cell Development , 2014, Cell.

[9]  Hongkai Ji,et al.  TSCAN: Pseudo-time reconstruction and evaluation in single-cell RNA-seq analysis , 2016, Nucleic acids research.

[10]  Sinisa Hrvatin,et al.  Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3 , 2012, Nature Biotechnology.

[11]  Blaz Zupan,et al.  Interest Propagation for Knowledge Extraction and Representation , 2013, EFMI-STC.

[12]  Caleb Webber,et al.  Laplacian eigenmaps and principal curves for high resolution pseudotemporal ordering of single-cell RNA-seq profiles , 2015, bioRxiv.

[13]  Sean C. Bendall,et al.  Wishbone identifies bifurcating developmental trajectories from single-cell data , 2016, Nature Biotechnology.

[14]  J. Kushner,et al.  Very Slow Turnover of β-Cells in Aged Adult Mice , 2005 .

[15]  Ning Li,et al.  Mitochondrial dysfunction in pancreatic β cells , 2012, Trends in Endocrinology & Metabolism.

[16]  Cole Trapnell,et al.  The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells , 2014, Nature Biotechnology.

[17]  Amitabh Sharma,et al.  Rat neonatal beta cells lack the specialised metabolic phenotype of mature beta cells , 2011, Diabetologia.

[18]  J. Boonstra,et al.  Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells. , 2004, Gene.

[19]  T. Richmond,et al.  The B‐box dominates SAP‐1–SRF interactions in the structure of the ternary complex , 2001, The EMBO journal.

[20]  J. Sage,et al.  PDGF signalling controls age-dependent proliferation in pancreatic β-cells , 2011, Nature.

[21]  R Bellazzi,et al.  Supporting Regenerative Medicine by Integrative Dimensionality Reduction , 2012, Methods of Information in Medicine.

[22]  James D. Johnson,et al.  Reduced Insulin Production Relieves Endoplasmic Reticulum Stress and Induces β Cell Proliferation. , 2016, Cell metabolism.

[23]  Senta Georgia,et al.  β cell replication is the primary mechanism for maintaining postnatal β cell mass , 2004 .

[24]  C. Donaldson,et al.  The transcriptional landscape of mouse beta cells compared to human beta cells reveals notable species differences in long non-coding RNA and protein-coding gene expression , 2014, BMC Genomics.

[25]  Fabian J Theis,et al.  Computational analysis of cell-to-cell heterogeneity in single-cell RNA-sequencing data reveals hidden subpopulations of cells , 2015, Nature Biotechnology.

[26]  J. Schug,et al.  Single-Cell Transcriptomics of the Human Endocrine Pancreas , 2016, Diabetes.

[27]  T. Prolla,et al.  Mitochondrial Oxidative Stress Mediates Angiotensin II–Induced Cardiac Hypertrophy and G&agr;q Overexpression–Induced Heart Failure , 2011, Circulation research.

[28]  K. Kaestner,et al.  Pancreatic beta cells require NeuroD to achieve and maintain functional maturity. , 2010, Cell metabolism.

[29]  Olle Korsgren,et al.  Identification of proliferative and mature β-cells in the islets of Langerhans , 2016, Nature.

[30]  A. Murphy,et al.  Single-Cell RNAseq Reveals That Pancreatic β-Cells From Very Old Male Mice Have a Young Gene Signature. , 2016, Endocrinology.

[31]  T. Kietzmann,et al.  Antioxidant responses and cellular adjustments to oxidative stress , 2015, Redox biology.

[32]  W. Vale,et al.  Urocortin 3 Marks Mature Human Primary and Embryonic Stem Cell-Derived Pancreatic Alpha and Beta Cells , 2012, PloS one.

[33]  Jonathan Schug,et al.  Human islets contain four distinct subtypes of β cells , 2016, Nature Communications.

[34]  N. Neff,et al.  Reconstructing lineage hierarchies of the distal lung epithelium using single cell RNA-seq , 2014, Nature.

[35]  S. Itzkovitz,et al.  The Genetic Program of Pancreatic β-Cell Replication In Vivo , 2016, Diabetes.

[36]  S. Bonner-Weir,et al.  Mafa expression enhances glucose-responsive insulin secretion in neonatal rat beta cells , 2011, Diabetologia.

[37]  J. Kushner,et al.  Cyclins D2 and D1 Are Essential for Postnatal Pancreatic β-Cell Growth , 2005, Molecular and Cellular Biology.

[38]  P. Reynolds,et al.  Insulin demand regulates β cell number via the unfolded protein response. , 2015, The Journal of clinical investigation.

[39]  Jeffrey T. Leek.  svaseq: removing batch effects and other unwanted noise from sequencing data , 2014 .

[40]  Dirk Trauner,et al.  Beta Cell Hubs Dictate Pancreatic Islet Responses to Glucose , 2016, Cell metabolism.

[41]  R. Evans,et al.  ERRγ Is Required for the Metabolic Maturation of Therapeutically Functional Glucose-Responsive β Cells. , 2016, Cell metabolism.

[42]  Evan Z. Macosko,et al.  Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets , 2015, Cell.

[43]  Joseph L. Herman,et al.  Characterizing transcriptional heterogeneity through pathway and gene set overdispersion analysis , 2015, Nature Methods.

[44]  M. Atkinson,et al.  Formation of a human β-cell population within pancreatic islets is set early in life. , 2012, The Journal of clinical endocrinology and metabolism.

[45]  I. Artner,et al.  MafA and MafB Regulate Genes Critical to β-Cells in a Unique Temporal Manner , 2010, Diabetes.