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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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.).
The authors declare no competing financial interests.
Nature thanks K. Alitalo and K. Poss and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Schematic representations show the wild-type protein (top) and the corresponding mutant protein (bottom) for each of the candidate genes in the cloche region. Functional protein domains are annotated, and the information about the molecular lesion(s) is indicated next to the protein’s name. Red numbers indicate the last native amino acid before the induced frameshift mutation. For the two genes that were targeted at more than one position, the extension of the longer mutant protein is indicated by transparency. Except for the npas4l mutation, none of the other 18 mutations causes an obvious morphological phenotype at 48 hpf. For proteins with incomplete sequence information (Inpp5a, Ptk7b, Srfb), primary structure was extrapolated from orthologues from other teleost species.
Extended Data Figure 2 5′ and 3′ RACE PCR for npas4l, and physical map of the cloche/npas4l locus in relation to klhdc3.
a, Primer design was based on a 513 bp fragment from GRCz10 that is deleted in clom39 mutants. b, c, Agarose gels showing the products of 5′ (b) and 3′ (c) RACE PCRs using cDNAs generated from 24 hpf or tailbud (TB) mRNAs. Multiple fragments were cloned for sequencing and bands that extended the npas4l sequence to the 5′ (b) and 3′ (c) are highlighted in blue boxes. d, Physical assembly of the cloche locus based on the extension of KN150101.1 by whole-genome sequencing. KN150101.1 (ex)/LT571435 is shown in reverse-complementary orientation with the blue region marking newly obtained sequence information. Alignment of the klhdc3 expressed sequence tag (EST) BC068372.1 establishes a physical connection between KN150101.1 (ex) and Chr. 13. The tightly linked SSLP marker z709 lies between klhdc3 and cloche/npas4l.
a–c, Individual clustering diagrams based on the amino acid sequence of the bHLH domain (a), the PAS-A domain (b), and the PAS-B domain (c) of mouse class I (NPAS1, NPAS3, NPAS4, HIF1A-HIF3A, AHR, SIM1/2) and class II (ARNT, ARNT2) bHLH-PAS proteins, together with the protein encoded by zebrafish cloche/npas4l. Analysis using the respective zebrafish proteins leads to similar results; the similarity between full-length zebrafish Npas4l and Npas4a is 39.5%, and between Npas4l and Npas4b it is 33%. d, GenBank accession numbers of proteins used in this analysis. e, Unrooted phylogenetic tree of Npas4 and Npas4l proteins across different phyla (using the sequence from the N terminus of the bHLH domain to the C terminus of the PAS-B domain). The cloche/npas4l gene is clearly present from lampreys to birds, but appears to be missing in mammals. f, Detailed information on the NPAS4 and NPAS4L protein sequences as well as synteny information of the corresponding genes. Predicted proteins were manually corrected when necessary using gene prediction methods (Genscan; AUGUSTUS) and BLAST tools.
a–f, Whole-mount in situ hybridization for kdrl (a, b), gata1a (c, d) and hbae3 (e, f) expression at the 15-somite stage in wild-type and npas4lbns59/bns59 embryos. g, h, Ventral views of Tg(kdrl:EGFP) sibling (g) and npas4lbns59 mutant (h) hearts at 48–hpf; a, atrium; v, ventricle. i, Quantification of the number of atrial endocardial cells in homozygous wild-type siblings (n = 9) and npas4lbns59 mutants (n = 9) at 60 hpf. Values represent means ± s.e.m. ***P < 0.001 by t-test. j, Penetrance of the vascular phenotype based on Tg(kdrl:EGFP) expression in 48 hpf npas4lbns59/bns59 embryos. k–v, Side-by-side comparison of endothelial and haematopoietic marker gene expression between different cloche alleles. Whole-mount in situ hybridization for etv2 (k–n), tal1 (o–r) and gata1a (s–v) expression at the 15-somite stage in wild-types and clom39, clos5 and npas4lbns59 mutants. x/y, number of embryos showing representative phenotype (x), number of embryos examined (y); m39 and s5 mutants were not confirmed by genotyping, hence the approximately 1/4 ratio in those panels. Scale bars, 200 μm (except for g and h, 100 μm).
Knockdown of Npas4l was achieved by co-injecting a translation blocking morpholino targeting the npas4l translational start site and a splice morpholino targeting the intron-exon splice site I2E3. a, Schematic representation of the npas4l genomic locus including the I2E3 splice morpholino (red) and the oligos used to test for splicing defects by RT–PCR (black). b, Analysis of npas4l mRNA splicing by RT–PCR shows a significant reduction in wild-type npas4l mRNA at the tailbud stage following injection of 6 ng of the I2E3 morpholino at the one-cell stage (3 different experiments, sMO1-sMO3, are shown). uc, uninjected control; sMO, splice morpholino injected; H2O, water control; wild-type product, 296 bp. c, d, Knockdown of Etv2 was achieved by injecting two previously published ATG morpholinos50. Fluorescence micrographs of Tg(kdrl:EGFP) control (ctrl) and etv2 MO1 + MO2 injected embryos. d–f, Knockdown of Tal1 was achieved by injecting a previously published splice morpholino49. d, e, Confocal projections of Tg(kdrl:EGFP) ctrl and tal1 MO injected embryos. g, Analysis of tal1 mRNA splicing by RT–PCR shows a significant reduction in wild-type tal1 mRNA at the two-somite stage after injecting 6.5 ng of tal1 MO at the one-cell stage. Three different experiments, sMO1–sMO3, are shown). uc, uninjected control; sMO, splice morpholino injected; H2O, water control; wild-type product, 401 bp. Scale bars, 200 μm.
a–f, Confocal projections of the anterior (a–c) and posterior (d–f) vasculature of 54 hpf Tg(kdrl:EGFP) sibling, clos5/s5 and clos5/s5 embryos injected with 25 pg npas4l mRNA at the one-cell stage. Scale bars, 200 μm.
a–c, RT–qPCR for npas4l, etv2 and tal1 expression; 20–40 embryos from single pair matings were pooled at each of the indicated stages; expression is normalized to the corresponding 24 hpf sample. Error bars represent s.e.m. from three biological replicates. d, Data extracted from ref. 27. npas4l expression clearly precedes etv2, tal1 and gata1a expression; it appears to be minimal until 6 hpf, peaks around the end of gastrulation/early somitogenesis (in 10 hpf sample from this data set), and is clearly down by 28 hpf, the stage when the other transcripts peak. Expression at each developmental stage represents a single RNA-seq experiment on 1,000 pooled embryos, and bars indicate the 95% confidence interval (Cufflinks). e, RT–qPCR for gata1a expression, performed and analysed as in a–c. Whole-mount in situ hybridization for gata1a expression in wild-type embryos at the 2-somite (f) and 18-somite (g) stages. Scale bars, 200 μm.
RT–qPCR analysis of etv2, fli1a, fli1b, lmo2, sox7, sox18 and tal1 expression in shield stage embryos injected with npas4l mRNA at the one-cell stage, normalized to uninjected controls. Error bars represent s.e.m. from three biological replicates of 20–40 pooled embryos per condition.
Extended Data Figure 9 NPAS4 expression during human embryonic stem cell derived vascular endothelial differentiation.
a, Scheme of the protocol used to differentiate human embryonic stem cells towards the vascular endothelial (VE) lineage highlighting the (1) mesoderm induction stage and (2) the VE lineage specification and differentiation stage. b, Representative flow cytometric analysis for day 5 KDR+ PDGFRA− VE progenitors and day 10 CD31+ KDR+ VE cells. c, RT–qPCR analysis of temporal expression of NPAS4 compared to OCT4, T, MESP1, ETV2, AHR, FLI1, CDH5 and PECAM1 over the course of human embryonic stem cell derived VE differentiation. Values are relative to the housekeeping gene TBP and depicted as a fold change in which average peak expression of the individual gene is set to 1. Raw data can be found in Figshare (http://dx.doi.org/10.6084/m9.figshare.3383572). Error bars represent s.e.m. from three biological replicates.
Extended Data Figure 10 AHR overexpression induces expression of early endothelial and haematopoietic markers in P19 mouse embryonic carcinoma cells.
a, RT–qPCR analysis of Etv2, Tal1, Lmo2 and Sox7 expression following overexpression of human AHR in P19 cells. Error bars represent s.e.m. from three biological replicates derived from independent transfections (*P < 0.05 and **P < 0.01 by t-test).
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Reischauer, S., Stone, O., Villasenor, A. et al. Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification. Nature 535, 294–298 (2016). https://doi.org/10.1038/nature18614
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