A right-handed signalling pathway drives heart looping in vertebrates

Abstract

Most animals show external bilateral symmetry, which hinders the observation of multiple internal left–right (L/R) asymmetries that are fundamental to organ packaging and function1,2. In vertebrates, left identity is mediated by the left-specific Nodal–Pitx2 axis that is repressed on the right-hand side by the epithelial–mesenchymal transition (EMT) inducer Snail1 (refs 3, 4). Despite some existing evidence3,5, it remains unclear whether an equivalent instructive pathway provides right-hand-specific information to the embryo. Here we show that, in zebrafish, BMP mediates the L/R asymmetric activation of another EMT inducer, Prrx1a, in the lateral plate mesoderm with higher levels on the right. Prrx1a drives L/R differential cell movements towards the midline, leading to a leftward displacement of the cardiac posterior pole through an actomyosin-dependent mechanism. Downregulation of Prrx1a prevents heart looping and leads to mesocardia. Two parallel and mutually repressed pathways, respectively driven by Nodal and BMP on the left and right lateral plate mesoderm, converge on the asymmetric activation of the transcription factors Pitx2 and Prrx1, which integrate left and right information to govern heart morphogenesis. This mechanism is conserved in the chicken embryo, and in the mouse SNAIL1 acts in a similar manner to Prrx1a in zebrafish and PRRX1 in the chick. Thus, a differential L/R EMT produces asymmetric cell movements and forces, more prominent from the right, that drive heart laterality in vertebrates.

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Figure 1: prrx1a L/R asymmetric expression in zebrafish.
Figure 2: Asymmetric L/R Prrx1a expression drives heart looping in zebrafish.
Figure 3: PRRX1 also drives heart looping in the chick embryo.
Figure 4: Two parallel and mutually exclusive pathways integrate left and right information.
Figure 5: A differential L/R EMT drives heart positioning in vertebrates.

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Acknowledgements

We thank members of M.A.N. laboratory for continuous and helpful discussions, J. M. Mingot (IN) for the purification of Cas9 protein, B. Alsina (Universitat Pompeu Fabra) for the myl12.1-mCherry transgenic line (originally from C. P. Heisenberg’s laboratory22), L. M. Gutiérrez (IN) for the LifeAct construct, J. Expósito (IN) for her technical advice with confocal microscopy, J. F. López Téllez (BIONAND) for the semithin histological sections and S. Ingham (IN) for his help with the graphics. This work was supported by the Spanish Ministry of Economy and Competitiveness (BFU2008-01042; BFU2014-53128-R co-financed by the European Regional Development Fund, ERDF), Generalitat Valenciana (PROMETEOII/2013/002) and European Research Council (ERC AdG 322694) to M.A.N., and BFU2014-52299-P, Instituto de Salud Carlos III-TERCEL network (RD12/0019-0022) and Junta de Andalucía (P11-CTS-07564) to R.M.-C. M.A.N. acknowledges financial support from the AEI, through the ‘Severo Ochoa’ Programme for Centres of Excellence in R&D (SEV-2013-0317) to Instituto de Neurociencias (IN).

Author information

O.H.O. performed the majority of the experiments, analysed the data and contributed to writing. H.C. performed histological and expression studies. C.M. generated the tbx5a zebrafish transgenic line, P.M. generated the anti-Prrx1 antibody in E.M.T.’s laboratory, and J.G. designed and performed the CRISPR-mediated prrx1a mutagenesis and the prrx1-promoter analysis. R.M.-C. carried out the morphological analysis and helped to interpret the data. M.A.N. conceived the project, interpreted the data and wrote the manuscript.

Correspondence to M. Angela Nieto.

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Competing interests

The authors declare no competing financial interests.

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Reviewer Information Nature thanks M. Bronner and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 prrx1a downregulation induces heart-looping defects.

a, Downregulation of prrx1a transcripts upon prrx1aMO1 microinjection. Ctrol, control. Data represent mean ± s.e.m.; ***P < 0.001; Student’s t-test; n = 3 biological replicates. b, Microinjection of a prrx1aMO2 morpholino located in the 5′ UTR also impairs normal heart looping at 50 hpf. Heart looping assessed by in situ hybridization with a myl7 probe in embryos microinjected with control or prrx1aMO2 morpholinos. The frequency of the phenotypes is indicated below the images. c, Scheme of the prrx1a locus indicating its exon organization. The coding sequence is depicted in blue and the location of the single-guide RNAs (sgRNA) is indicated. The location of primers used for screening is indicated by the two coloured arrows. d, Representative images of ‘crispants’ obtained by injection of the indicated sgRNA and Cas9. The frequency of the phenotypes is indicated below the images. e, Representative T7 endonuclease assays of four embryos per phenotype indicated in d and two wild-type (WT) embryos used as negative controls. f, T7 endonuclease assay of 8 crispants and one WT embryo after injection of the indicated sgRNA. g, The PCR products obtained from embryos 1, 2 and 5 in f were cloned and sequenced. Representative sequences of those embryos (embryo number indicated on the right), with the mutations indicated in red and the deletions with hyphens. The location of the sgRNA is indicated in green and the corresponding adjacent PAM is highlighted in yellow. Scale bars, 100 μm. Source data

Extended Data Figure 2 Cardiac phenotype of prrx1a-morphant embryos.

a, Bright-field images of whole-mount and haematoxylin–eosin staining of sagittal sections of control and prrx1a-morphant embryos (n = 5 embryos per condition). b, Snapshots of surface-rendering images of representative control and prrx1a-morphant embryos hybridized with the atrial marker amhc. The graph represents the area of amhc expression (μm2; n = 7 per condition). Data represent mean ± s.e.m.; ***P < 0.001; Student’s t-test. c, Parasagittal semithin sections of control and morphant embryos (48 hpf) obtained at the right (R), middle (M) and left (L) levels. The ventricle (V) is on the right and the sinus venosus (SV) is well-developed in the control. However, the morphant has the ventricle aligned with the atrium (A), and lacks a SV in the cardiac posterior pole (PP). OFT, outflow tract. d, Ventral view of a maximum-intensity projection and z-plane sections of confocal images (right) through the outflow tract showing the expression of the anterior SHF marker latent TGFβ binding protein-3 (ltbp3) in control and prrx1a-morphant zebrafish embryos. The cardiomyocytes are visualized by MF20 immunofluorescence. Scale bars, 50 μm. Source data

Extended Data Figure 3 Prrx1a expression in the early embryo.

a, Maximum-intensity projection of confocal images showing Prrx1 expression in Tg(tbx5a:eGFP) embryos with DAPI staining of cell nuclei (blue). Prrx1 and tbx5a colocalize in the LPM (left). The asymmetric L/R expression of Prrx1 in the right LPM is better seen in the single-channel view (middle) and the transverse sections at the level of the white dotted line (right). tbx5a is absent from the neural crest (NC) and the posterior LPM (PLPM). b, Prrx1 and tbx5 colocalization (yellow) identifies the Prrx1+ cardiac precursor population once the PHT has formed. Number of double-positive cells in the left and right halves of the LPM. Data represent mean ± s.e.m.; **P < 0.01; Student’s t-test; n = 6. c, Heat map of Prrx1-expression levels in the LPM cells identified in b. Colour code is shown on the right. Intensity was measured in individual cells of the selected regions in the left and right LPM and an average intensity value was obtained per side. The plot shows the average value obtained in the right relative to the left LPM. Data represent mean ± s.e.m.; *P < 0.05; Student’s paired t-test; n = 6. d, Dorsal view of zebrafish embryos to visualize the expression of prrx1a and the pan-neural crest marker crestin. Colocalization of prrx1a and crestin identifies the prrx1a+ neural crest cell population. tbx5 is excluded from the neural crest. NT, neural tube. Scale bars, 50 μm. Source data

Extended Data Figure 4 Prrx1 and Tbx5 co-localization in developing heart tissues.

ac, Ventral views or sagittal sections showing more Prrx1+tbx5a+ cells in the visceral pericardium (P) in contact with the heart tube (HT) on the right side (arrowheads in sections and enlarged images in a and c). Prrx1 is absent from the heart tube. Boxes in c indicate the areas highlighted on the right. d, Sagittal sections of a 48 hpf zebrafish embryo showing Prrx1 expression compared with the cardiomyocyte marker MF20, or the atrial marker S46. Scale bars, 50 μm. A, atrium; E, eye; PV, pericardial vesicle; SV, sinus venosus; V, ventricle.

Extended Data Figure 5 Cell movements during heart development in the zebrafish embryo.

a, Snapshots from time-lapse analysis of Tg(tbx5:eGFP) control and prrx1-morphant embryos at heart-looping stages. b, Randomization of cardiac jogging in prrx1a morphants. Representative images of control (Ctrol MO) and prrx1a-morphant (prrx1aMO1) embryos hybridized with a myosin light chain 7 (myl7) probe to assess heart tube jogging direction. Images are dorsal views with anterior to the top. Scale bars, 100 μm. c, Quantification of heart-jogging direction. A, atrium; PHT, primary heart tube; SV, sinus venosus; V, ventricle. Source data

Extended Data Figure 6 Photoablation of mesodermal cells in the LPM.

a, b, Photoablation was performed at 26–27 hpf on the left (a) or the right side (b). Ventral views with merged bright-field and z-plane confocal images, before and after unilateral photoablation (Pa) of tbx5:eGFP+ cells. The photoablated areas are located in the most anterior region of the LPM adjacent to the limit of the extra-embryonic tissue. Higher power images of the photoablated areas (right panels) correspond to the boxed areas. c, Analysis of heart location at 50 hpf. Hearts visualized after myl7 in situ hybridization in wild-type and left- or right-photoablated embryos. Mesocardia only develops in embryos photoablated on the right-hand side. d, Cartoon of a ventral view of a zebrafish embryo at 30–32 hpf with a boxed area that corresponds to that in the enlarged areas shown in Fig. 2f, g. e, Snapshots of actomyosin fibres, generated by the merged images extracted from time-lapse confocal microscopy in live control (left) and prrx1a-morphant embryos (right) of the Tg(tbx5a:eGFP) reporter line injected with LifeAct (n = 7 embryos per condition).Brackets indicate the region of the cable-like structure. f, Snapshots from time-lapse videos of Tg(tbx5a:eGFP) embryos treated with blebbistatin or with the vehicle alone. Scale bars, 50 μm.

Extended Data Figure 7 PRRX1 expression in chick embryos.

a, Whole-mount in situ hybridization in chick embryos showing the transient asymmetric expression of SNAIL1 in the LPM. b, Expression analysis by double fluorescence in situ hybridization of PRRX1 and NKX2.5 or TBX5 in whole-mount embryos. c, Transverse sections showing PRRX1 and NKX2.5 or TBX5 expression at the levels indicated by dotted lines in b. Boxes indicate the position of the corresponding enlarged images. The arrowhead indicates the centre of the posterior pole displaced to the left. d, Dual fluorescent in situ hybridization for PRRX1 and the atrial (AMHC) or ventricular (VMHC) myosins. High power images of transverse sections as indicated on the left showing the absence of myocardial PRRX1 expression. e, Downregulation of PRRX1 transcription after electroporation with siRNA. Data represent mean ± s.e.m.; ***P < 0.001; Student’s t-test; n = 3 biological replicates. fh, Quantification of heart location from experiments shown in Fig. 3c, d. RNAi, RNA interference. i, AMHC and VMHC in control embryos and in those electroporated with PRRX1 siRNA. j, Morphometric analysis of the atrium in chick embryos electroporated with control siRNA or PRRX1 siRNA. The graph represents the area of AMHC expression (μm2; n = 7 embryos per condition). Data represent mean ± s.e.m.; **P < 0.01; Student’s t-test. k, Expression of FGF8, required for outflow tract formation, is not altered by PRRX1 downregulation. l, PRRX1 and ISLET1 coexpression in the lateral right sinus venosus horn (RSH) (arrows). Scale bars, 500 μm (a), 10 μm (enlarged images in l) or 100 μm (all other panels). A, atrium; AIP; anterior intestinal portal; CF, cardiac folds; LPM, lateral plate mesoderm; LSH/RSH; left/right sinus venosus horns; OFT, outflow tract; SH, sinus venosus horns; V, ventricle. Source data

Extended Data Figure 8 PRRX1 is essential in the posterior pole of the heart.

a, Double fluorescent in situ hybridization for TBX18 and PRRX1 in chick embryos. b, TBX18 downregulation in embryos electroporated with PRRX1 siRNA. c, Maximum-intensity projection of dual fluorescent staining of PRRX1 and phalloidin (a mycotoxin that stains actin stress fibres). Stars indicate the area of asymmetry and colocalization. d, Heart looping assessed by MF20 immunofluorescence in embryos treated with vehicle or blebbistatin. e, Maximum-intensity projection showing the cells coexpressing PALLD (palladin) and PRRX1. f, PALLD is decreased upon PRRX1 downregulation. Data represent mean ± s.e.m. of four pools (five embryos each) of control and PRRX1-knockdown embryos; *P < 0.05, **P < 0.01; Student’s t-test. g, Visualization of electroporated areas in chick embryos shown in Fig. 4b. Embryos were co-electroporated on the right side with an eGFP-reporter construct plus an empty vector or a vector containing the NODAL-coding sequence. h, Heart location in embryos containing control BSA- or BMP4-loaded beads. i, Diagram of the PRRX1 promoter. Proximal (P) and distal (D) binding sites for SMAD1/SMAD5/SMAD8 and constructs used in j and k. j, ChIP with antibodies against SMAD5 or IgG (negative control). Data represent mean ± s.e.m.; **P < 0.01, ***P < 0.001; ns, not significant; Student’s t-test; n = 5 biological replicates. k, Luciferase activity of PRRX1 promoter. Both distal (D) and proximal (P) SMAD5-binding sites are functional. Fragments containing these sites can activate the promoter in primary chick fibroblasts upon BMP4 treatment. Mutations in these sites prevent the response to BMP4. Data represent mean ± s.e.m.; ***P < 0.001; ns, not significant; one-way ANOVA; n = 4 biological replicates. Dotted lines show the level of the sections. Scale bars, 100 μm or 10 μm (enlarged images and sections in a, b). A, atrium; AIP, anterior intestinal portal; CF, cardiac folds; LPM, lateral plate mesoderm; LSH/RSH, left/right sinus venosus horns; OFT, outflow tract; SV, sinus venosus; V, ventricle; VV, vitelline veins. Source data

Extended Data Figure 9 SNAIL1/snail1 and PRRX1 in heart looping in vertebrate embryos.

a, PRRX1 and SNAIL1 expression in whole-mount chick embryos and transverse sections of chick embryos. b, Maximum-intensity projection of confocal images showing heart location by triple immunofluorescence in embryos electroporated with control fluorescein-labelled morpholinos, with a mix of two fluorescein-morpholinos against SNAIL1 or a combination of SNAIL1 morpholinos plus PRRX1 RNA interference. An RFP construct was used as a reporter for electroporated cells. c, Schematic drawing and knockdown-efficiency assay of fluorescein-labelled morpholinos directed to SNAIL1 splicing sites. d, Quantitative analysis of heart location. e, snail1a is not expressed in the LPM in zebrafish embryos. f, snail1b expression in whole-mounted embryos and transverse sections at the levels of the dotted lines. g, Schematic drawing and knockdown efficiency assay of the snail1b splice site morpholino (snail1bMO). h, Quantification of heart location in control and snail1b morphants. i, snail1b is not expressed in the pericardium (P) in the zebrafish embryo. Scale bars, 500 μm (a, left panel), 50 μm (i) or 100 μm (all other panels). Source data

Extended Data Figure 10 Model for heart looping in vertebrates.

a, Cartoon depicting the comparative expression of Prrx1/PRRX1 and Snail1/SNAIL1 in zebrafish, chick and mouse embryos. b, Transverse sections showing the lack of PRRX2 expression in the sinus venosus horns of chick at HH9–10 and Prrx2 expression in mouse embryos at 8.5 dpc. Scale bars, 100 μm. c, Schematic representation of the proposed model for heart looping in vertebrates in relation to the expression of prrx1a/PRRX1/Prrx1 and snail1b/SNAIL1/Snail1. d, A prominent right-handed pathway drives differential L/R EMT and heart looping in vertebrates. In addition to the left-specific Nodal–Pitx2/PITX2 pathway that confers left identity and represses the right-handed pathway, BMP signalling activates EMT in the LPM in a L/R asymmetric manner to drive heart laterality and the repression of leftward information on the right through the repression of Pitx2/PITX2. This conserved cellular mechanism is implemented through the activation of different EMT inducers in different vertebrates. Nomenclature in the diagrams is unified for all species for simplicity. Correct names are as in the legend. A, atrium; AIP, anterior intestinal portal; C, coelom; DSpLM, dorsal splanchnic lateral mesoderm; Ec, ectoderm; FG, foregut; LA, left atrium; LSH, left sinus venosus horn; LV, left ventricle; LVV, left vitelline vein; NC, neural crest; N, notochord; NP, neural plate; NT, neural tube; OFT, outflow tract; PV, pericardial vesicle; RA, right atrium; RSH, right sinus venosus horn; RV, right ventricle; RVV, right vitelline vein; SoLM, somatic lateral mesoderm; S, somite; SV, sinus venosus; V, ventricle; VSpLM, ventral splanchnic lateral mesoderm; YS, yolk sac.

Supplementary information

Supplementary Information (PDF 200 kb)

Asymmetric cell movements during heart looping in zebrafish

The Tg(tbx5a:eGFP) line shows both the heart tube and late cardiac progenitors contributing to the posterior pole. Embryos were microinjected with Control MO at 1 cell stage and the time-lapse 4D videos were recorded from 28 hpf to 44 hpf taking one picture every 10 minutes. The video shows a ventral view (right side on the left) of surface rendering of a maximum intensity projection. Note the bilaterally asymmetric cell contribution, with more cells from the right side. (MOV 6879 kb)

prrx1a knockdown prevents heart looping in zebrafish

Tg(tbx5a:eGFP) embryos were microinjected with prrx1aMO1 at 1 cell stage and the time lapse 4D videos were recorded from 28 hpf to 44 hpf stage taking one picture every 10 minutes. The video shows images similar to those described in legend to Video S1. Note the decrease in the Tbx5a+ progenitor cells and the absence of heart looping. (MOV 4406 kb)

Down regulation of prrx1a produces randomized heart jogging

Tg(tbx5a:eGFP) embryos were microinjected with prrx1aMO1 at 1 cell stage and the time lapse 4D videos were recorded from 27hpf to 48hpf taking one picture every 10 minutes. The video shows a maximum intensity projection of confocal images. While the jogging is to the left side as in control embryos the heart fails to loop and remains as a straight tube in the midline.Tg(tbx5a:eGFP) embryos were microinjected with prrx1aMO1 at 1 cell stage and the time lapse 4D videos were recorded from 27hpf to 48hpf taking one picture every 10 minutes. The video shows a maximum intensity projection of confocal images. While the jogging is to the left side as in control embryos the heart fails to loop and remains as a straight tube in the midline. (MOV 4837 kb)

Down regulation of prrx1a produces randomized heart jogging

Tg(tbx5a:eGFP) embryos were microinjected with prrx1aMO1 at 1 cell stage and the time lapse 4D videos were recorded from 27hpf to 48hpf taking one picture every 10 minutes. The video shows a maximum intensity projection of confocal images. While the jogging is to the right side the heart fails to loop and remains as an straight tube in the midline. (MOV 4727 kb)

Asymmetric actomyosin forces during heart looping in zebrafish

Tg(tbx5a:eGFP) embryos were microinjected with Control MO plus LifeAct:RFP mRNA at 1 cell-stage and the time-lapse 4D videos were recorded from 28hpf to 38hpf stage taking one picture every 10 minutes. The video shows ventral views (right side on the left) of a maximum intensity projection and was generated with a colocalization channel. Note how actomyosin fibres make contact at the posterior pole of the heart on the right side. (MOV 1640 kb)

Prrx1a drives the asymmetric actomyosin forces necessary for the heart looping in zebrafish

Tg(tbx5a:eGFP) embryos were microinjected with prrx1aMO1 plus LifeAct:RFP mRNA at 1 cell-stage and the time-lapse 4D videos were recorded and presented as described in Video S5. Note the disruption of actomyosin fibres and the absence of the cable-like structure upon prrx1a downregulation. (MOV 813 kb)

prrx1a regulates cell shape and actomyosin cytoskeleton-Control

3D reconstruction at 30-32 hpf in a Tg(actb2:myl12.1-mCherry) zebrafish embryo injected with LifeAct:GFP mRNA and control morpholino (Ctrol MO) at 1 cell-stage embryo to visualize F-actin (lifeAct:GFP, green), myosin II (myl12.1 mCherry, red) and Prrx1 expression (anti-Prrx1, white). (MOV 4822 kb)

prrx1a regulates cell shape and actomyosin cytoskeleton-prrx1aMO1.

3D reconstruction at 30-32 hpf in a Tg(actb2:myl12.1-mCherry) zebrafish embryo injected with LifeAct:GFP mRNA and prrx1a morpholino (prrx1aMO1) at 1 cell-stage embryo to visualize, F-actin (lifeAct:GFP, green), myosin II (myl12.1-mCherry, red) and Prrx1 expression (anti-Prrx1, white). (MOV 4810 kb)

Heart looping in zebrafish-control

Tg(tbx5a:eGFP) embryos were treated with vehicle from 26hpf and the time lapse 4D videos were recorded from 26hpf to 44hpf stage taking one picture every 10 minutes. The video shows a maximum intensity projection. Note the proper dextral looping of the heart. (MOV 4435 kb)

Actomyosin inhibition prevents heart looping in zebrafish

Tg(tbx5a:eGFP) embryos were treated with blebbistatin from 26hpf and the time lapse 4D videos were recorded and presented as described for Video S9. Note how upon blebbistatin treatment the dextral looping of the heart is impeded and the heart remains as a straight tube. (MOV 5648 kb)

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Ocaña, O., Coskun, H., Minguillón, C. et al. A right-handed signalling pathway drives heart looping in vertebrates. Nature 549, 86–90 (2017). https://doi.org/10.1038/nature23454

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