title: Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification.
abstract: Vascular and haematopoietic cells organize into specialized tissues during early embryogenesis to supply essential nutrients to all organs and thus play critical roles in development and disease. At the top of the haemato-vascular specification cascade lies cloche, a gene that when mutated in zebrafish leads to the striking phenotype of loss of most endothelial and haematopoietic cells and a significant increase in cardiomyocyte numbers. Although this mutant has been analysed extensively to investigate mesoderm diversification and differentiation and continues to be broadly used as a unique avascular model, the isolation of the cloche gene has been challenging due to its telomeric location. Here we used a deletion allele of cloche to identify several new cloche candidate genes within this genomic region, and systematically genome-edited each candidate. Through this comprehensive interrogation, we succeeded in isolating the cloche gene and discovered that it encodes a PAS-domain-containing bHLH transcription factor, and that it is expressed in a highly specific spatiotemporal pattern starting during late gastrulation. Gain-of-function experiments show that it can potently induce endothelial gene expression. Epistasis experiments reveal that it functions upstream of etv2 and tal1, the earliest expressed endothelial and haematopoietic transcription factor genes identified to date. A mammalian cloche orthologue can also rescue blood vessel formation in zebrafish cloche mutants, indicating a highly conserved role in vertebrate vasculogenesis and haematopoiesis. The identification of this master regulator of endothelial and haematopoietic fate enhances our understanding of early mesoderm diversification and may lead to improved protocols for the generation of endothelial and haematopoietic cells in vivo and in vitro.
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA.|Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim 61231, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Reischauer
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA.|Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim 61231, Germany.
authors.forename: Oliver A
authors.initials: OA
authors.lastname: Stone
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA.|Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim 61231, Germany.
authors.forename: Alethia
authors.initials: A
authors.lastname: Villasenor
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA.|Department of Medicine, Division of Cardiology, Institute of Genomic Medicine, University of California San Diego, La Jolla, California 92037, USA.
authors.forename: Neil
authors.initials: N
authors.lastname: Chi
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA.
authors.forename: Suk-Won
authors.initials: SW
authors.lastname: Jin
authors.affiliation: Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Solna 17121, Sweden.
authors.forename: Marcel
authors.initials: M
authors.lastname: Martin
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Miler T
authors.initials: MT
authors.lastname: Lee
authors.affiliation: Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim 61231, Germany.
authors.forename: Nana
authors.initials: N
authors.lastname: Fukuda
authors.affiliation: Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim 61231, Germany.
authors.forename: Michele
authors.initials: M
authors.lastname: Marass
authors.affiliation: Department of Medicine, Division of Cardiology, Institute of Genomic Medicine, University of California San Diego, La Jolla, California 92037, USA.
authors.forename: Alec
authors.initials: A
authors.lastname: Witty
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA.
authors.forename: Ian
authors.initials: I
authors.lastname: Fiddes
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA.
authors.forename: Taiyi
authors.initials: T
authors.lastname: Kuo
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA.
authors.forename: Won-Suk
authors.initials: WS
authors.lastname: Chung
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA.
authors.forename: Sherveen
authors.initials: S
authors.lastname: Salek
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Lerrigo
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Alsiö
authors.affiliation: Illumina, San Diego, California 92122, USA.
authors.forename: Shujun
authors.initials: S
authors.lastname: Luo
authors.affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm 17177, Sweden.
authors.forename: Dominika
authors.initials: D
authors.lastname: Tworus
authors.affiliation: Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim 61231, Germany.
authors.forename: Sruthy M
authors.initials: SM
authors.lastname: Augustine
authors.affiliation: Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim 61231, Germany.
authors.forename: Sophie
authors.initials: S
authors.lastname: Mucenieks
authors.affiliation: Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, Uppsala 75124, Sweden.
authors.forename: Björn
authors.initials: B
authors.lastname: Nystedt
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Giraldez
authors.affiliation: Illumina, San Diego, California 92122, USA.
authors.forename: Gary P
authors.initials: GP
authors.lastname: Schroth
authors.affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm 17177, Sweden.
authors.forename: Olov
authors.initials: O
authors.lastname: Andersson
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA.|Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim 61231, Germany.
authors.forename: Didier Y R
authors.initials: DY
authors.lastname: Stainier
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001773:Blood Cells / Q000166:cytology* / Q000378:metabolism*; D001808:Blood Vessels / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics*; D017124:Conserved Sequence; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism*; D004843:Epistasis, Genetic; D017353:Gene Deletion; D018257:Helix-Loop-Helix Motifs; D006410:Hematopoiesis; D008648:Mesoderm / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D009154:Mutation; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000235:genetics; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D015027:Zebrafish / Q000196:embryology / Q000235:genetics; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D011518:Proto-Oncogene Proteins; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D029961:Zebrafish Proteins; C509408:Etv2 protein, zebrafish; C000609736:npas4l protein, zebrafish; C489367:tal1 protein, zebrafish