title: Transcriptional regulation of Hhex in hematopoiesis and hematopoietic stem cell ontogeny.
abstract: Hematopoietic stem cells (HSCs) emerge during development via an endothelial-to-hematopoietic transition from hemogenic endothelium of the dorsal aorta (DA). Using in situ hybridization and analysis of a knock-in RedStar reporter, we show that the transcriptional regulator Hhex is expressed in endothelium of the dorsal aorta (DA) and in clusters of putative HSCs as they are specified during murine development. We exploited this observation, using the Hhex locus to define cis regulatory elements, enhancers and interacting transcription factors that are both necessary and sufficient to support gene expression in the emerging HSC. We identify an evolutionarily conserved non-coding region (ECR) in the Hhex locus with the capacity to bind the hematopoietic-affiliated transcriptional regulators Gata2, SCL, Fli1, Pu.1 and Ets1/2. This region is sufficient to drive the expression of a transgenic GFP reporter in the DA endothelium and intra-aortic hematopoietic clusters. GFP-positive AGM cells co-expressed HSC-associated markers c-Kit, CD34, VE-Cadherin, and CD45, and were capable of multipotential differentiation and long term engraftment when transplanted into myelo-ablated recipients. The Hhex ECR was also sufficient to drive expression at additional blood sites including the yolk sac blood islands, fetal liver, vitelline and umbilical arteries and the adult bone marrow, suggesting a common mechanism for Hhex regulation throughout ontogenesis of the blood system. To explore the physiological requirement for the Hhex ECR region during hematoendothelial development, we deleted the ECR element from the endogenous locus in the context of a targeted Hhex-RedStar reporter allele. Results indicate a specific requirement for the ECR in blood-associated Hhex expression during development and further demonstrate a requirement for this region in the adult HSC compartment. Taken together, our results identified the ECR region as an enhancer both necessary and sufficient for gene expression in HSC development and homeostasis. The Hhex ECR thus appears to be a core node for the convergence of the transcription factor network that governs the emergence of HSCs.
authors.affiliation: MRC Centre for Regenerative Medicine - Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, EH16 4UU Edinburgh, UK.
authors.forename: Rosa Portero
authors.initials: RP
authors.lastname: Migueles
authors.affiliation: MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK.
authors.forename: Louise
authors.initials: L
authors.lastname: Shaw
authors.affiliation: MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK; The European Cancer Stem Cell Research Institute, Cardiff University, School of Biosciences, Hadyn Ellis Building, Cardiff CF24 4HQ, UK.
authors.forename: Neil P
authors.initials: NP
authors.lastname: Rodrigues
authors.affiliation: MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK; Stem Cell Laboratory, UCL Cancer Institute, University College London, London WC1E 6DD, UK.
authors.forename: Gillian
authors.initials: G
authors.lastname: May
authors.affiliation: MRC Centre for Regenerative Medicine - Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, EH16 4UU Edinburgh, UK.
authors.forename: Korinna
authors.initials: K
authors.lastname: Henseleit
authors.affiliation: MRC Centre for Regenerative Medicine - Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, EH16 4UU Edinburgh, UK; The Danish Stem Cell Centre - DanStem, University of Copenhagen, 3B Blegdamsvej, DK-2200 Copenhagen, Denmark.
authors.forename: Kathryn G V
authors.initials: KG
authors.lastname: Anderson
authors.affiliation: Institute for Cancer Research, Chester Beatty Laboratories, London SW3 6JB, UK.
authors.forename: Hakan
authors.initials: H
authors.lastname: Goker
authors.affiliation: Institute for Cancer Research, Chester Beatty Laboratories, London SW3 6JB, UK.
authors.forename: C Michael
authors.initials: CM
authors.lastname: Jones
authors.affiliation: MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK.
authors.forename: Marella F T R
authors.initials: MF
authors.lastname: de Bruijn
authors.affiliation: MRC Centre for Regenerative Medicine - Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, EH16 4UU Edinburgh, UK; The Danish Stem Cell Centre - DanStem, University of Copenhagen, 3B Blegdamsvej, DK-2200 Copenhagen, Denmark. Electronic address: Joshua.brickman@sund.ku.dk.
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Brickman
authors.affiliation: MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK; Stem Cell Laboratory, UCL Cancer Institute, University College London, London WC1E 6DD, UK. Electronic address: T.enver@ucl.ac.uk.
authors.forename: Tariq
authors.initials: T
authors.lastname: Enver
mesh_terms: D000818:Animals; D002451:Cell Compartmentation; D019070:Cell Lineage / Q000235:genetics; D003114:Colony-Forming Units Assay; D017124:Conserved Sequence / Q000235:genetics; D004622:Embryo, Mammalian / Q000378:metabolism; D005786:Gene Expression Regulation; D056426:Genetic Loci; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006410:Hematopoiesis / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: C513080:Hhex protein, mouse; D018398:Homeodomain Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins
keywords: Blood; Development and differentiation; Enhancer; Transcription