A predictive model of asymmetric morphogenesis from 3D reconstructions of mouse heart looping dynamics

  1. Jean-François Le Garrec
  2. Jorge N Domínguez
  3. Audrey Desgrange
  4. Kenzo D Ivanovitch
  5. Etienne Raphaël
  6. J Andrew Bangham
  7. Miguel Torres
  8. Enrico Coen
  9. Timothy J Mohun
  10. Sigolène M Meilhac  Is a corresponding author
  1. Laboratory of Heart Morphogenesis, France
  2. Université Paris Descartes, France
  3. University of Jaén, CU Las Lagunillas, Spain
  4. Centro Nacional de Investigaciones Cardiovasculares, CNIC, Spain
  5. University of East Anglia, United Kingdom
  6. Norwich Research Park, United Kingdom
  7. The Francis Crick Institute, United Kingdom
10 figures, 4 videos, 1 table and 6 additional files

Figures

Stages depicting the progression of heart looping in the mouse.

(A) Schematic representation of shape changes during the formation and looping of the heart tube (orange) in the E8.5 mouse embryo. Until E8.5f, the mouse embryo appears bilaterally symmetrical, and …

https://doi.org/10.7554/eLife.28951.003
Figure 1—source data 1

3D reconstructions of heart stages during the looping process, in a 3D pdf format.

3D visualisation of the heart, as reconstructed from HREM images, at the six stages shown in Figure 1. This file should be opened with Adobe Acrobat Reader. Click on the image to activate the manual rotation of the reconstruction. Each image may be rotated at will with the mouse (hold left click). Zoom in and out with the mouse wheel. Shortcuts at the top align all images in a ventral or dorsal view, with the notochord vertical.

https://doi.org/10.7554/eLife.28951.004
The heart tube elongates and loops between fixed poles.

(A) HREM image of an embryo section at E8.5f, with the notochord (green dot) and the centroid (red dot) of the myocardial tube (pale red) outlined. (B) Ventral view of a 3D reconstruction of the …

https://doi.org/10.7554/eLife.28951.005
Progression of dorsal mesocardium breakdown during heart looping.

(A) Transverse section from an HREM image of an embryo at E8.5f showing the attachment of the heart tube to the body via the dorsal mesocardium (lateral thickness between red arrowheads). The …

https://doi.org/10.7554/eLife.28951.007
Figure 4 with 1 supplement
Rotation of the arterial pole at E8.5f.

(A) Transverse section from an HREM image of an embryo at E8.5f, in which the angles (right and left) between the heart tube and the dorsal pericardial wall, at the dorsal mesocardium, are shown. …

https://doi.org/10.7554/eLife.28951.008
Figure 4—figure supplement 1
Asymmetry of the dorsal pericardial wall.

(A) Transverse embryo section at E8.5g, in which the width of the dorsal pericardial wall on either sides of the dorsal mesocardium is shown. The position of the section is indicated in brackets, as …

https://doi.org/10.7554/eLife.28951.009
Asymmetric cell ingression at the venous pole at E8.5g.

(A) 3D reconstruction of the heart tube at E8.5i, aligned with the notochord vertical (green), showing the axis of the myocardial tube (red) and the last tube section before the bifurcation (dotted …

https://doi.org/10.7554/eLife.28951.010
Figure 6 with 1 supplement
Computer simulations of heart looping integrating mechanical constraints and left-right asymmetries.

(A) Timeline of the simulations, with the successive events reflecting experimental observations. (B) Simulation of heart shape changes in 3D with a Finite Element model of a straight tube, seen …

https://doi.org/10.7554/eLife.28951.011
Figure 6—figure supplement 1
Parameters of the computer model.

(A) Hollow-cylinder model of the heart tube, made of 1800 finite elements. (B) Mechanical constraints due to the attachment of the heart tube to the rest of the body: the arterial pole extremities …

https://doi.org/10.7554/eLife.28951.012
Variations in left-right asymmetries at the poles impact heart shape.

In the computer simulation, parameters of pole asymmetries were explored to analyse the variety of output shapes of the heart tube. Simulated shapes are shown at step 80 with the ventral line in …

https://doi.org/10.7554/eLife.28951.014
Figure 8 with 1 supplement
Model prediction and experimental validation of the rotation of the ventral line.

(A–B) Computer simulations of heart looping with the fate of the initial ventral line shown in blue. The simulated shape is shown at step 90 in ventral (left) and right-lateral (right) views. When …

https://doi.org/10.7554/eLife.28951.015
Figure 8—figure supplement 1
Live-imaging of the rotation of the arterial pole.

Polr2aCreERT2/+; R26Rtdtomato/YFP embryos, are shown, after injection of a low dose of tamoxifen generating fluorescent cell mosaicism. (A–B) Initial and final images of a time-lapse movie between …

https://doi.org/10.7554/eLife.28951.016
Figure 9 with 3 supplements
Model prediction and experimental validation, in the case of persistent dorsal mesocardium.

(A) Computer simulations of heart looping in control conditions (left), as in Figure 6, and when the dorsal mesocardium is persistent (right, simulated mutant). Simulated shapes are shown at step 90 …

https://doi.org/10.7554/eLife.28951.018
Figure 9—source data 1

3D reconstructions of all hearts of Shh-/- mutants and control littermates analysed at E8.5.

Ventral (left) and dorsal (right) views are shown, aligned with the notochord vertical (green). The myocardial layer (yellow) is made transparent, revealing the tube axis (red). Samples are identified with a number (S). The length of the heart tube is indicated, as well as the somite number (So) of the corresponding embryo. The genotype of control samples is Shh+/+ (S11, S17, S20) or Shh+/- (S8, S14). Scale bar: 100 µm.

https://doi.org/10.7554/eLife.28951.022
Figure 9—figure supplement 1
Structure of the dorsal mesocardium in Shh-/- mutants and control littermates at E8.5.

(A, C) Lateral thickness of the dorsal mesocardium, measured as in Figure 3D, in five controls (A) and 5 Shh-/- mutant hearts (C) at E8.5. Samples are identified with a number (S), and the somite …

https://doi.org/10.7554/eLife.28951.019
Figure 9—figure supplement 2
Shh-/- mutants and control littermates at E9.5.

(A) 3D visualisation of HREM images of a control (left) and Shh-/- mutant (right) embryos, seen in a ventral view. The presence of the pericardium (S55) provides a rough appearance to the heart. (B, …

https://doi.org/10.7554/eLife.28951.020
Figure 9—figure supplement 3
Alternative simulations and comparison with Shh-/- mutant hearts.

Comparison between quantitative predictions for four geometrical parameters raised by two types of computer simulations and biological measures in Shh-/-mutant hearts at E8.5. Simulation with 50% …

https://doi.org/10.7554/eLife.28951.021
Matrix metalloproteases are required for dorsal mesocardium breakdown and heart looping.

(A) Immunostaining of Mmp2, showing expression in the foregut (fg) at E8.5g, and vesicular localisations in the cardiac region (arrowheads in A1), but not in the neural tube (nt, A2). Maximum …

https://doi.org/10.7554/eLife.28951.024

Videos

Video 1
Example of a 3D stack of HREM images acquired from an embryo at E8.5h.

Video showing the successive sections of an E8.5h embryo, at the level of the heart. Images were acquired by HREM every 2 µm.

https://doi.org/10.7554/eLife.28951.006
Video 2
Computer simulation of heart looping in the control situation.
https://doi.org/10.7554/eLife.28951.013
Video 3
Example of live-imaging of the arterial pole rotation.
https://doi.org/10.7554/eLife.28951.017
Video 4
Computer simulation of heart looping in the case of persistent dorsal mesocardium.
https://doi.org/10.7554/eLife.28951.023

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)wild-type, Swiss backgroundJanvier
Strain, strain background (Mus musculus)wild-type, C57Bl6 backgroundJanvier
Strain, strain background (Mus musculus)T4nLacZ, Swiss backgroundBiben et al. (1996) doi:10.1006/dbio.1996.0017PMID 8575622
strain, strain background (Mus musculus)Shh+/-, C57Bl6J backgroundGonzalez-Reyes et al. (2012) doi:10.1016/j.neuron.2012.05.018MGI:5440762
Strain, strain background (Mus musculus)Polr2aCreERT2/+, C57Bl6 backgroundGuerra et al. (2003) PMID:12957286MGI:3772332
Strain, strain background (Mus musculus)R26YFP/+, C57Bl6 backgroundSrinivas et al. (2001) PMID:11299042MGI:2449038
Strain, strain background (Mus musculus)R26Rtdtomato/+ (Ai14), C57Bl6 backgroundMadisen et al. (2010) doi:10.1038/nn.2467MGI:3809524
Antibodyanti-PH3 (rabbit monoclonal)AbcamAbcam: ab32107(1:100)
Antibodyanti-Isl1 (mouse monoclonal)Developmental Studies Hybridoma BankDSHB: 39.4D5(1:50)
Antibodyanti-MMP2 (mouse monoclonal)Santa CruzSanta Cuz: sc13594(1:50)
Antibodygoat anti-rabbit IgG Alexa Fluor 546InvitrogenInvitrogen: A11035(1:500)
Antibodygoat anti-mouse IgG2b Alexa Fluor 488InvitrogenInvitrogen: A21141(1:500)
Antibodygoat anti-mouse IgG1 Alexa Fluor 488InvitrogenInvitrogen: A21121(1:500)
Commercial assay or kitJB-4 embedding kitPolysciencesPolysciences: 00226–1
Chemical compound, drugGM6001 (Ilomast)MilliporeMillipore: CC100010 µM
Software, algorithmGftboxKennaway et al. (2011) doi:10.1371/journal.pcbi.1002071Matlab Finite Element Analysis package simulating biological growth
SoftwareICYde Chaumont et al. 2012 doi:10.1038/nmeth.2075Open platform for bioimage informatics

Additional files

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