• A Corrigendum to this article was published on 21 March 2018

This article has been updated

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 cells1,2,3,4 and a significant increase in cardiomyocyte numbers5. Although this mutant has been analysed extensively to investigate mesoderm diversification and differentiation1,2,3,4,5,6,7 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.

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Change history

  • 21 March 2018

    Please see accompanying Corrigendum (http://doi.org/10.1038/nature25991). In the Author Information section, the accession for the PacBio whole-genome sequencing data was wrongly stated as LT571435 from the SRA, instead of PRJEB13442 from the European Nucleotide Archive (ENA). The original Letter has been corrected online.

Accessions

Primary accessions

European Nucleotide Archive

GenBank/EMBL/DDBJ

Gene Expression Omnibus

References

  1. 1.

    , , , & cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages. Development 121, 3141–3150 (1995)

  2. 2.

    et al. The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation. Development 124, 381–389 (1997)

  3. 3.

    et al. SCL/Tal-1 transcription factor acts downstream of cloche to specify hematopoietic and vascular progenitors in zebrafish. Genes Dev. 12, 621–626 (1998)

  4. 4.

    , , , & Hhex and Scl function in parallel to regulate early endothelial and blood differentiation in zebrafish. Development 127, 4303–4313 (2000)

  5. 5.

    , & Vessel and blood specification override cardiac potential in anterior mesoderm. Dev. Cell 13, 254–267 (2007)

  6. 6.

    , & Identification of novel vascular endothelial-specific genes by the microarray analysis of the zebrafish cloche mutants. Blood 106, 534–541 (2005)

  7. 7.

    , , & Fli1 acts at the top of the transcriptional network driving blood and endothelial development. Curr. Biol. 18, 1234–1240 (2008)

  8. 8.

    & Signaling pathways in vascular development. Annu. Rev. Cell Dev. Biol. 18, 541–573 (2002)

  9. 9.

    & Molecular regulation of angiogenesis and lymphangiogenesis. Nature Rev. Mol. Cell Biol. 8, 464–478 (2007)

  10. 10.

    & Molecular control of endothelial cell behaviour during blood vessel morphogenesis. Nat. Rev. Mol. Cell Biol. 12, 551–564 (2011)

  11. 11.

    Hemogenic endothelium during development and beyond. Blood 119, 4823–4827 (2012)

  12. 12.

    & Transcriptional control of endothelial cell development. Dev. Cell 16, 180–195 (2009)

  13. 13.

    et al. The gene SCL is expressed during early hematopoiesis and encodes a differentiation-related DNA-binding motif. Proc. Natl Acad. Sci. USA 86, 10128–10132 (1989)

  14. 14.

    , , & SCL/tal-1-dependent process determines a competence to select the definitive hematopoietic lineage prior to endothelial differentiation. EMBO J. 21, 6700–6708 (2002)

  15. 15.

    et al. Scl represses cardiomyogenesis in prospective hemogenic endothelium and endocardium. Cell 150, 590–605 (2012)

  16. 16.

    , , , & Regulation of hemangioblast development. Ann. NY Acad. Sci. 938, 96–108 (2001)

  17. 17.

    , , , & Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature 432, 625–630 (2004)

  18. 18.

    , , & A common progenitor for haematopoietic and endothelial lineages in the zebrafish gastrula. Nature 443, 337–339 (2006)

  19. 19.

    et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453, 524–528 (2008)

  20. 20.

    , , & An acyltransferase controls the generation of hematopoietic and endothelial lineages in zebrafish. Circ. Res. 102, 1057–1064 (2008)

  21. 21.

    , , , & Identification of a novel basic helix-loop-helix-PAS factor, NXF, reveals a Sim2 competitive, positive regulatory role in dendritic-cytoskeleton modulator Drebrin gene expression. Mol. Cell. Biol. 24, 608–616 (2004)

  22. 22.

    et al. Activity-dependent regulation of inhibitory synapse development by Npas4. Nature 455, 1198–1204 (2008)

  23. 23.

    et al. The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation. Blood 122, 376–385 (2013)

  24. 24.

    et al. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1α. Genes Dev. 12, 149–162 (1998)

  25. 25.

    , , & The transcription factor EPAS-1/hypoxia-inducible factor 2α plays an important role in vascular remodeling. Proc. Natl Acad. Sci. USA 97, 8386–8391 (2000)

  26. 26.

    , , & Formation of the digestive system in zebrafish. II. Pancreas morphogenesis. Dev. Biol. 261, 197–208 (2003)

  27. 27.

    et al. Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis. Genome Res. 22, 577–591 (2012)

  28. 28.

    & Cell-autonomous and non-autonomous requirements for the zebrafish gene cloche in hematopoiesis. Development 126, 2643–2651 (1999)

  29. 29.

    , , , & HIF-dependent hematopoietic factors regulate the development of the embryonic vasculature. Dev. Cell 11, 81–92 (2006)

  30. 30.

    , , & Hypoxia influences the vascular expansion and differentiation of embryonic stem cell cultures through the temporal expression of vascular endothelial growth factor receptors in an ARNT-dependent manner. Stem Cells 28, 799–809 (2010)

  31. 31.

    , , , & Cellular and molecular analyses of vascular tube and lumen formation in zebrafish. Development 132, 5199–5209 (2005)

  32. 32.

    et al. Duplex-specific nuclease efficiently removes rRNA for prokaryotic RNA-seq. Nucleic Acids Res. 39, e140 (2011)

  33. 33.

    et al. Comparison of RNA-seq by poly (A) capture, ribosomal RNA depletion, and DNA microarray for expression profiling. BMC Genomics 15, 419 (2014)

  34. 34.

    et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013)

  35. 35.

    & Fast gapped-read alignment with Bowtie 2. Nature Methods 9, 357–359 (2012)

  36. 36.

    , , & Identification of novel transcripts in annotated genomes using RNA-seq. Bioinformatics 27, 2325–2329 (2011)

  37. 37.

    & BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010)

  38. 38.

    et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011)

  39. 39.

    et al. In vivo genome editing using a high-efficiency TALEN system. Nature 491, 114–118 (2012)

  40. 40.

    , & Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc. Natl Acad. Sci. USA 110, 13904–13909 (2013)

  41. 41.

    Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at (2013)

  42. 42.

    & Haplotype-based variant detection from short-read sequencing. Preprint at (2012)

  43. 43.

    & High-resolution in situ hybridization to whole-mount zebrafish embryos. Nature Protocols 3, 59–69 (2008)

  44. 44.

    PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 20, 2471–2472 (2004)

  45. 45.

    et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013)

  46. 46.

    et al. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010)

  47. 47.

    et al. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36, W465–W469 (2008)

  48. 48.

    et al. Nine-amino-acid transactivation domain: establishment and prediction utilities. Genomics 89, 756–768 (2007)

  49. 49.

    , & Zebrafish scl functions independently in hematopoietic and endothelial development. Dev. Biol. 277, 522–536 (2005)

  50. 50.

    & Ets1-related protein is a key regulator of vasculogenesis in zebrafish. PLoS Biol. 4, e10 (2006)

  51. 51.

    et al. Chemically defined conditions for human iPSC derivation and culture. Nature Methods 8, 424–429 (2011)

  52. 52.

    et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8, 228–240 (2011)

  53. 53.

    et al. High-purity enrichment of functional cardiovascular cells from human iPS cells. Cardiovasc. Res. 95, 327–335 (2012)

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Acknowledgements

We thank all laboratory members who have worked on cloche over the years starting with W. Liao and H. Sawyer (UCSF), as well as J. Collins (Sanger Institute) for help in expanding the GRCz10 assembly, W. Coppieters (Liège), Z. Wang (JGI), X. Chen (BGI), H. Yuan (BGI) for their hard work in trying to resolve the cloche locus, C. Helker, M. Higuchi and C. Gerri for reagents, discussions and reading of the manuscript, A. Borchers (Marburg) for help with Xenopus experiments, and funding from the DFG (S.R.), AHA (S.R., S.-W.J., N.C.), NIH (N.C., A.J.G., D.Y.R.S.), the Ragnar Söderberg Foundation and Swedish Research Council (O.A.), and the Packard Foundation and Max Planck Society (D.Y.R.S.).

Author information

Author notes

    • Suk-Won Jin
    • , Miler T. Lee
    • , Ian Fiddes
    • , Taiyi Kuo
    • , Won-Suk Chung
    • , Sherveen Salek
    • , Robert Lerrigo
    • , Jessica Alsiö
    •  & Shujun Luo

    Present addresses: School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, 61005, South Korea and Yale Cardiovascular Research Center, Yale University School of Medicine, New Haven, Connecticut 06511, USA (S.-W.J.); Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA (M.T.L.); Genomics Institute, University of California Santa Cruz and Howard Hughes Medical Institute, Santa Cruz, California 95064, USA (I.F.); Department of Medicine and Berrie Diabetes Center, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA (T.K.); Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, South Korea (W.-S.C.); Johns Hopkins Hospital, Wilmer Eye Institute, Baltimore, Maryland 21224, USA (S.S.); Division of General Internal Medicine, University of Washington, Seattle, Washington 98104, USA (R.L.); Novartis, Basel 4056, Switzerland (J.A.); Personalis, Menlo Park, California 94025, USA (S.L.).

    • Sven Reischauer
    • , Oliver A. Stone
    •  & Alethia Villasenor

    These authors contributed equally to this work.

Affiliations

  1. Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA

    • Sven Reischauer
    • , Oliver A. Stone
    • , Alethia Villasenor
    • , Neil Chi
    • , Suk-Won Jin
    • , Ian Fiddes
    • , Taiyi Kuo
    • , Won-Suk Chung
    • , Sherveen Salek
    • , Robert Lerrigo
    • , Jessica Alsiö
    •  & Didier Y. R. Stainier
  2. Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim 61231, Germany

    • Sven Reischauer
    • , Oliver A. Stone
    • , Alethia Villasenor
    • , Nana Fukuda
    • , Michele Marass
    • , Sruthy M. Augustine
    • , Sophie Mucenieks
    •  & Didier Y. R. Stainier
  3. Department of Medicine, Division of Cardiology, Institute of Genomic Medicine, University of California San Diego, La Jolla, California 92037, USA

    • Neil Chi
    •  & Alec Witty
  4. Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Solna 17121, Sweden

    • Marcel Martin
  5. Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520, USA

    • Miler T. Lee
    •  & Antonio J. Giraldez
  6. Illumina, San Diego, California 92122, USA

    • Shujun Luo
    •  & Gary P. Schroth
  7. Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm 17177, Sweden

    • Dominika Tworus
    •  & Olov Andersson
  8. Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, Uppsala 75124, Sweden

    • Björn Nystedt

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Contributions

S.R., O.S., A.V. and D.Y.R.S. designed and performed experiments identifying cloche and its function. N.F., Mi.M, S.M.A. and S.M. performed reverse genetics and downstream analysis. N.C., S.-W.J., T.K., W.-S.C., S.S., R.L. and J.A. worked on the meiotic mapping. S.L., Ma.M., M.T.L., I.F. and D.T. contributed to sequencing and bioinformatics. Human embryonic stem cell assays were handled by A.W. Supervision by D.Y.R.S., N.C., O.A., G.P.S., A.J.G. and B.N. All authors contributed to data analysis and manuscript preparation.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Didier Y. R. Stainier.

Zebrafish npas4l sequences have been deposited to GenBank (KX066018, KX066019), RNA-seq data deposited to GEO (GSE76690), and PacBio whole-genome sequencing data to ENA (PRJEB13442).

Reviewer Information

Nature thanks K. Alitalo and K. Poss and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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https://doi.org/10.1038/nature18614

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