Pseudo-dynamic analysis of heart tube formation in the mouse reveals strong regional variability and early left-right asymmetry

Background:

Heart morphogenesis is a complex process that involves a series of changes in cell behaviour and tissue patterning. During gastrulation in mouse embryos, the lateral plate mesoderm splits into two layers, the splanchnic mesoderm located dorsally to the endoderm; and the somatic mesoderm underneath the ectoderm. The primitive heart tube is derived from the splanchnic mesoderm. At the onset of embryonic day 7.5 (E7.5), the cardiogenic mesoderm forms a bilateral field at the anterior region of the primitive streak. The bilateral field merges anteriorly and creates the so-called “cardiac crescent”. The cardiac crescent includes two progenitor pools contributing to the different structures during heart development, including the first heart field (FHF) and the secondary heart field (SHF). From E7.5 to E8.5, cardiac progenitors from two sides migrate toward the midline and coalesce medially to form a single primitive heart tube. From E8.5 onward, the primitive ventricle starts bulging while the anterior and venous poles are elongating. At the same time, the dorsal mesocardium detaches from the dorsal pericardial wall. The primitive ventricle becomes suspended within the pericardial cavity with two poles connecting to the embryo body by the end of these processes. The primitive ventricle gradually loops from left to right, orienting the chamber toward the correct position [1].

Morphometric analysis of tissues is critical for gaining a deeper understand of the molecular mechanism driving organogenesis. However, the morphological changes from cardiac crescent to heart looping occur rapidly and in a brief time window, representing a technical challenge for capturing the spatiotemporal changes of the heart’s structure. Previous methods using serial tissue sections to reconstruct the 3D model produced low-resolution images and were mainly suitable for larger tissue sizes [2]. Here, Esteban et al. study employed a whole-mount imaging technique to scan an entire cardiac region at different developmental stages, establishing new geometrical parameters and generating a high-resolution 3D+t digital model of heart morphogenesis.

Key findings:

 1. Image acquisition and segmentation methods:

Mesoderm posterior 1 (Mesp1) is expressed in the nascent mesoderm at the onset of gastrulation and is one of the earliest markers of cardiac progenitors. To visualize tissues derived from the mesoderm lineage, embryos carrying Mesp1-Cre driving the expression of two reporter alleles, R26mTmG and R26Tomato, were used for whole-mount imaging. In these embryos, the mesoderm derived cells were labelled with the green membrane (mGFP) and red cytoplasm (tdTomato); and the remaining cells were distinguished by the red membrane (mTomato). Fifty-two embryos from E7.75 to E8.25 were used to build the tissue atlas, approximately equivalent to one embryo every 20 minutes. The embryos samples were cleared, embedded in 1% agarose gel, and scanned for a whole cardiac region. Depending on the tissue structure, the segmentation proceeded in either automatic or semi-automatic manners. For example, the foregut endoderm was fully automatically segmented, while the myocardium required additional morphological and topological criteria to differentiate from the rest of the splanchnic mesoderm. The segmentation was firstly processed in 2D images, later combined, and then rendered in 3D. The images were subjected to post – segmentation and idealized surface processing to remove noisy voxels and smoothen objects’ surfaces. This methodology successfully reconstructed 3D+t models of the myocardium from cardiac crescent until heart looping, and all mesoderm related tissues including splanchnic, somatic, and paraxial mesoderm and other origin derived tissues such as foregut endoderm and circulatory system.

Figure 1: The morphology of the myocardium together with other tissue across stages. FGE, (Foregut endoderm); AP, arterial poles; VP, venous poles; RSH, right sinus horn; LSH, left sinus horn; EL, endocardium lumen; AOL, aortic lumen. Images were obtained from Figure 2 (C-D) in the Esteban I. et al. preprint. DOI: https://doi.org/10.1101/2021.10.07.463475

2. A new morphometric staging system for early heart development:

From E7.75 to E8.25, mouse embryos undergo drastically morphological changes in the structure of the heart tube, foregut socket, head folds and embryo shape. Conventional embryo staging methods relying on the day post coitum (dpc), numbers of somites or head fold shape are therefore not accurate and introduce potential variability in the analysis of heart development. Thus, with 3D reconstructed images, the authors aimed to establish novel morphometric parameters to describe the temporal evolution change of the heart shape more precisely, providing an alternative method for staging embryos.

Based on the anatomical landmarks of the myocardium, mesodermal layers, and foregut, six different measurements were calculated. The first one is the d1/d2 ratio, in which d1 is the length of the border between the myocardium and juxta-cardiac field (JCF) – an anterior tissue above the cardiac crescent, and d2 is the length of the border between the myocardium and secondary heart field (SHF). The second parameter is the proportion between myocardium height to myocardium width (h:w). The third parameter is the ratio between d1 and JCF height (d1:jh). The fourth and fifth are the division of d1 to the foregut length (d1:fl) and the foregut width (d1:fw), respectively. Lastly, the sixth is the ratio between d2 and the height of the somatic mesoderm (d2:sh). To determine the most suitable parameter for staging embryos and the number of embryo groups (k-mean classifier), a k-mean clustering algorithm was applied to each parameter of every embryo. The accuracy of each clustering method was evaluated using average silhouette coefficient (s). Among six parameters, d1/d2 ratio has the best s value and is the most consistent parameter for grouping embryos. Additionally, d1/d2 ratio is well correlated with the heigh-to-width ratio of the heart tube. Based on d1/d2 ratio, embryos could be staged into ten groups in which group 1 – 4 can be assigned to cardiac crescent, group 5 – 8 to heart tube, and group 9 – 10 to heart looping.

3. Standardizing the embryonic geometries of the heart and mapping local shape variability

Given that the heart shapes are highly variable even within the assigned groups, the next aim was to standardize a consensus geometry of the heart tube and the associated tissues in each group and define the local shape variability. The team developed a new quantitative methodology to compute a surface map of each heart sample using equivalent landmark points and curves across stages. Each surface map contains a vertices index for each sample. Then, a vertex-to-vertex correspondence between samples was used to align all heart shapes to a reference shape of the group. All measurements were averaged to create a Mean Shape for each group (MSG). Besides, all shapes in one group were uniformly scaled. Each group selected a medoid shape with the least minimal dissimilarity to be Stage Group Representative (SGR). The concrete set of MSG and SGR is a valuable atlas of the heart structure during heart tube formation and looping. By using MSG to identify the hotspot of natural variability of the cardiac crescent and heart tube, the authors showed that the first morphological variability was found in the bilateral bulges of the cardiac crescent and later at the medial region of the primitive ventricle. At stage 9, when the looping begins, the high morphological variability was also seen in the areas of the out and inflow tracts.

4. Detection of the earliest left-right asymmetry, and 3D + t model of the heart tube formation

Although the left-right asymmetry of the heart is visible at stages 9 and 10 when the heart tube starts to turn rightward, there could be less obvious signs of heart asymmetry happening before the heart looping. Indeed, analysis of 3D quantitative maps of MSG suggested that the angle of insertion of the left and right inflow is not identical. Two measurements were implemented to assess the sinus venous asymmetric structure. One is the φ angle between the direction of inflow insertion and the z-axis, and the other is the θ angle between the orthogonal projection of the direction of the inflow insertion on the XY plane and the x-axis. Then, the changes of these angles were examined relative to the d1/d2 ratio across stages. Interestingly, there was a significant deviation of the θ angle from symmetry initiating at the stages between groups 5 and 6; in contrast, the φ angle on average was unchanged at any stage. Of note, there is an acute angle in the insertion of the right inflow, which is opposite to the smooth transition in the left influx. These results suggested that the left-right asymmetry occurs at a certain angle of the inflow regions.

Finally, the authors used the collection of MSG and SGR to build a temporal dynamic transformation of the myocardium and the associated tissue across ten stages. Tissue meshes from the first shape to the final shape were connected and aligned using vertex coordination. Thirty interpolated shapes were additionally inserted between each stage to smoothen the transition. This approach produces a 3D + t model of heart tube formation, presenting comprehensively morphological changes from heart crescent to heart tube formation and looping.

What I liked about this preprint:

With new embryo imaging techniques and quantitative methodologies, this paper has built an intricate and high-resolution atlas of heart morphology from cardiac crescent to heart tube formation and looping, providing a new set of geometrical parameters for myocardium and the associated tissues during heart development. This atlas not only allows the embryologist to stage embryonic hearts more precisely and minimize the natural variability within embryo samples but also contributes significantly to the studying of congenital heart disease in the aspect of providing morphometric criteria to assess the atypical structure of the different genetically mutated embryonic heart. Finally, the 3D reconstruction method, quantitative analysis, and statistical framework developed in this paper could be applicable to examine the morphogenesis of other organs in mouse embryos or the heart tissue in other species.

Question to the authors

In this preprint, the surface macro-morphology of the myocardium was carefully addressed and reconstructed into a 3D model. It has been known that the myocardial wall of the early embryonic heart tube is also complex that composed of multi-layers with the outer continuous layer and the luminal laminated layer. I wonder if the authors have a plan to build a similar 3D atlas with an exclusive focus on the cross-sectional morphology of the myocardium and whether the same methodology could be applicable to study its structure?

Acknowledgement

I would like to thank Dr Osvaldo Conteras (Victor Chang Cardiac Institute, Australia) and Dr Helen Robertson (preLights Community Manager) for proofreading the highlight.

References

1. Kelly RG et al., Heart fields and cardiac morphogenesis. Cold Spring Harb Perspect Med. 2014
2. de Boer BA et al., Growth of the developing mouse heart: an interactive qualitative and quantitative 3D atlas. Dev Biol. 2012.

Tags: 3d image, embryonic heart atlas, heart development

doi: https://doi.org/10.1242/prelights.31061

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