Decoding the Regulatory Network for Blood Development from Single-Cell Gene Expression Measurements

Reconstruction of the molecular pathways controlling organ development has been hampered by a lack of methods to resolve embryonic progenitor cells. Here we describe a strategy to address this problem that combines gene expression profiling of large numbers of single cells with data analysis based on diffusion maps for dimensionality reduction and network synthesis from state transition graphs. Applying the approach to hematopoietic development in the mouse embryo, we map the progression of mesoderm toward blood using single-cell gene expression analysis of 3,934 cells with blood-forming potential captured at four time points between E7.0 and E8.5. Transitions between individual cellular states are then used as input to develop a single-cell network synthesis toolkit to generate a computationally executable transcriptional regulatory network model of blood development. Several model predictions concerning the roles of Sox and Hox factors are validated experimentally. Our results demonstrate that single-cell analysis of a developing organ coupled with computational approaches can reveal the transcriptional programs that underpin organogenesis.

[1]  Lingsong Zhang,et al.  STATISTICAL METHODS IN BIOLOGY , 1902, Nature.

[2]  J. Mcwhir,et al.  A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency. , 1992, Nucleic acids research.

[3]  T. Davies,et al.  Staging of gastrulating mouse embryos by morphological landmarks in the dissecting microscope. , 1993, Development.

[4]  C. Begley,et al.  Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Janet Rossant,et al.  Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice , 1995, Nature.

[6]  S. Orkin,et al.  Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL , 1995, Nature.

[7]  Y Fujiwara,et al.  Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[8]  A. Ullrich,et al.  Flk-1 expression defines a population of early embryonic hematopoietic precursors. , 1997, Development.

[9]  Janet Rossant,et al.  A Requirement for Flk1 in Primitive and Definitive Hematopoiesis and Vasculogenesis , 1997, Cell.

[10]  T. Gu,et al.  Cbfa2 is required for the formation of intra-aortic hematopoietic clusters. , 1999, Development.

[11]  G. Daley,et al.  HoxB4 Confers Definitive Lymphoid-Myeloid Engraftment Potential on Embryonic Stem Cell and Yolk Sac Hematopoietic Progenitors , 2002, Cell.

[12]  W. Vainchenker,et al.  Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells. , 2002, Development.

[13]  S. Orkin,et al.  Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo. , 2003, Blood.

[14]  J. Downing,et al.  Role of RUNX1 in adult hematopoiesis: analysis of RUNX1-IRES-GFP knock-in mice reveals differential lineage expression. , 2004, Blood.

[15]  Ann B. Lee,et al.  Geometric diffusions as a tool for harmonic analysis and structure definition of data: diffusion maps. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[16]  K. Sachs,et al.  Causal Protein-Signaling Networks Derived from Multiparameter Single-Cell Data , 2005, Science.

[17]  S. Orkin,et al.  Tie2Cre-mediated gene ablation defines the stem-cell leukemia gene (SCL/tal1)-dependent window during hematopoietic stem-cell development. , 2005, Blood.

[18]  Stéphane Lafon,et al.  Diffusion maps , 2006 .

[19]  Berthold Göttgens,et al.  Gata2, Fli1, and Scl form a recursively wired gene-regulatory circuit during early hematopoietic development , 2007, Proceedings of the National Academy of Sciences.

[20]  S. Nishikawa,et al.  Cell tracing shows the contribution of the yolk sac to adult haematopoiesis , 2007, Nature.

[21]  Giovanni De Micheli,et al.  Synchronous versus asynchronous modeling of gene regulatory networks , 2008, Bioinform..

[22]  Elizabeth A. Kruse,et al.  The transcription factor Erg is essential for definitive hematopoiesis and the function of adult hematopoietic stem cells , 2008, Nature Immunology.

[23]  Shuo Lin,et al.  Interplay among Etsrp/ER71, Scl, and Alk8 signaling controls endothelial and myeloid cell formation. , 2008, Blood.

[24]  Geoffrey E. Hinton,et al.  Visualizing Data using t-SNE , 2008 .

[25]  K. McGrath,et al.  All primitive and definitive hematopoietic progenitor cells emerging before E10 in the mouse embryo are products of the yolk sac. , 2008, Blood.

[26]  R. Huang,et al.  Epithelial-Mesenchymal Transitions in Development and Disease , 2009, Cell.

[27]  V. Kouskoff,et al.  Sox7-sustained expression alters the balance between proliferation and differentiation of hematopoietic progenitors at the onset of blood specification. , 2009, Blood.

[28]  Elaine Dzierzak,et al.  Runx1 is required for the endothelial to hematopoietic cell transition but not thereafter , 2009, Nature.

[29]  Mark A. Dawson,et al.  The transcriptional program controlled by the stem cell leukemia gene Scl/Tal1 during early embryonic hematopoietic development. , 2009, Blood.

[30]  Mikael Huss,et al.  Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst. , 2010, Developmental cell.

[31]  Berthold Göttgens,et al.  ERG promotes T-acute lymphoblastic leukemia and is transcriptionally regulated in leukemic cells by a stem cell enhancer. , 2011, Blood.

[32]  Yosuke Tanaka,et al.  Etv2/ER71 induces vascular mesoderm from Flk1+PDGFRα+ primitive mesoderm. , 2011, Blood.

[33]  W. Alexander,et al.  ERG dependence distinguishes developmental control of hematopoietic stem cell maintenance from hematopoietic specification. , 2011, Genes & development.

[34]  Milos Pekny,et al.  Defining cell populations with single-cell gene expression profiling: correlations and identification of astrocyte subpopulations , 2010, Nucleic acids research.

[35]  F. Tang,et al.  Development and applications of single-cell transcriptome analysis , 2011, Nature Methods.

[36]  Fabian J Theis,et al.  Hierarchical Differentiation of Myeloid Progenitors Is Encoded in the Transcription Factor Network , 2011, PloS one.

[37]  Yosuke Tanaka,et al.  Early ontogenic origin of the hematopoietic stem cell lineage , 2012, Proceedings of the National Academy of Sciences.

[38]  Carsten Peterson,et al.  Inferring rules of lineage commitment in haematopoiesis , 2012, Nature Cell Biology.

[39]  V. Kouskoff,et al.  Origin of blood cells and HSC production in the embryo. , 2012, Trends in immunology.

[40]  Changya Chen,et al.  Dynamic HoxB4-regulatory network during embryonic stem cell differentiation to hematopoietic cells. , 2012, Blood.

[41]  B. Göttgens,et al.  The Flk1‐Cre‐Mediated Deletion of ETV2 Defines Its Narrow Temporal Requirement During Embryonic Hematopoietic Development , 2012, Stem cells.

[42]  B. Göttgens,et al.  Early dynamic fate changes in haemogenic endothelium characterized at the single-cell level , 2013, Nature Communications.

[43]  S. Horvath,et al.  Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing , 2013, Nature.

[44]  Ruiqiang Li,et al.  Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells , 2013, Nature Structural &Molecular Biology.

[45]  Julia Tischler,et al.  Investigating transcriptional states at single-cell-resolution. , 2013, Current opinion in biotechnology.

[46]  D. Dickel,et al.  Single site-specific integration targeting coupled with embryonic stem cell differentiation provides a high-throughput alternative to in vivo enhancer analyses , 2013, Biology Open.

[47]  Rastislav Bodík,et al.  Synthesis of biological models from mutation experiments , 2013, POPL.

[48]  Ioannis Xenarios,et al.  Hard-wired heterogeneity in blood stem cells revealed using a dynamic regulatory network model , 2013, Bioinform..

[49]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[50]  Fabian J Theis,et al.  Characterization of transcriptional networks in blood stem and progenitor cells using high-throughput single-cell gene expression analysis , 2013, Nature Cell Biology.

[51]  S. Orkin,et al.  Mapping cellular hierarchy by single-cell analysis of the cell surface repertoire. , 2013, Cell stem cell.

[52]  Ke Liu,et al.  Analysis of Dll4 regulation reveals a combinatorial role for Sox and Notch in arterial development , 2013, Proceedings of the National Academy of Sciences.

[53]  J. Fisher,et al.  Transcriptional hierarchies regulating early blood cell development. , 2013, Blood cells, molecules & diseases.

[54]  Yosuke Tanaka,et al.  PDGF Receptor Alpha+ Mesoderm Contributes to Endothelial and Hematopoietic Cells in Mice , 2013, Developmental dynamics : an official publication of the American Association of Anatomists.

[55]  Sean C. Bendall,et al.  viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia , 2013, Nature Biotechnology.

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

[57]  Åsa K. Björklund,et al.  Full-length RNA-seq from single cells using Smart-seq2 , 2014, Nature Protocols.

[58]  Avi Ma'ayan,et al.  Construction and Validation of a Regulatory Network for Pluripotency and Self-Renewal of Mouse Embryonic Stem Cells , 2014, PLoS Comput. Biol..

[59]  Patrick Lombard,et al.  CODEX: a next-generation sequencing experiment database for the haematopoietic and embryonic stem cell communities , 2014, Nucleic Acids Res..