The developing heart proceeds through a series of complex formative events (morphogenetic events), involving numerous cell types to form an organ that begins its vital function well before reaching its final intricate shape1. That this process is error-prone is highlighted by the high incidence of congenital defects in the heart and vasculature, associated with morbidity and adult onset disease in humans2. New technologies, including high-resolution imaging and computational modelling offer a real-time overview to the process, allowing comprehensive narrative of the normal heart’s development as a precondition to understand the aetiology of congenital defects1,3.
Early in embryogenesis, the heart starts beating and pumping blood, when still a linear tube with no valves and chambers, to ensure survival throughout embryonic fetal life1. Under conditions of blood flow, key morphogenetic changes transform the heart into a complex, multi-chambered structure resembling the adult heart (Fig 1).
Cardiovascular development is highly conserved among vertebrate species with a variety of animal models employed to study heart development, in particular, chick and mouse embryo models are used to assess genetic/hemodynamic complications linked to cardiac defects (Table 1)1,4.
Advanced imaging techniques have enabled dynamic, subject-specific models of the heart, with potential to revolutionize the field of heart development and congenital heart disease investigation4. For instance, the recent use of a technique known as multi-photon live-imaging to visualize cardiogenesis, has enabled tissue-level characterization of heart tube (HT) formation in a growing mouse embryo, within a culture system, with high-resolution imaging to generate progressive, 4 dimensional (4D) time-lapse movies5.
BACKGROUND: CLASSICAL STUDIES AND EARLY IMAGING TECHNIQUES
In the early 1900’s, an official assortment of human embryos for research purposes known as the Carnegie collection was launched. Appearance of the developing human embryonic heart was illustrated by Carl L. Davis in 1923 with embryo #3709 at stage 9 using fixed and sectioned samples from the collection - to illuminate vertebrate heart compartmentalization, the same embryo is now digitally reconstructed in 3D. Classical studies of embryogenesis were also performed in continuous, uninterrupted sequence for 24-hours with chicken embryo, under microscopic observations through thin tissue sections in 1920, to create several monographs of the subject6.
In recent years, studies with embryonic chick hearts shed light on the straight embryonic vertebrate heart tube’s transformation into a helically wound loop known as cardiac looping, using scanning electron microscopy (SEM)7,8. Although descriptive studies enabled visualization of the developing heart’s anatomy, they were conducted ex vivo i.e. in a fixed medium, removed from normal development and were thus unable to quantify simultaneous biomechanical events of cardiogenesis.
IN VIVO EMBRYONIC HEART IMAGING TECHNIQUES
Embryonic imaging in the natural environment i.e. in vivo, offers qualitative assessment of early heart formation, with a variety of new techniques2, including stereomicroscopy, confocal and video microscopy, to detail biomechanical landscapes of heart development2,9. However, due to limited depth of view, the techniques were confined to imaging tissue surfaces and used only in early stage embryos or with smaller animal models9.
Optical coherence tomography (OCT) is increasingly used for live-imaging chick embryo, due to non-invasive high resolution imaging, although limited by light scattering in embryonic tissue and a depth of view ranging from 1-2 mm10. Long-term imaging of live chick embryos can be done in lab over a time-lapse of hours/days with a single embryo, by constructing a chamber with conditions similar to a biological environment (Fig 2), although maintaining the live-embryo in an exposed, stationary state to enable continuous imaging is a challenge11. Despite advanced imaging techniques that enabled in vivo and live embryo imaging in lab, quantitative analysis of the biomechanics of cardiovascular transformation remain lacking2.
The technique of multi-photon microscopy (MPM) combined with long distance stereomicroscopy offers greater imaging depth, while providing 3D microstructural images over time2. Elastin in the artery, for instance, can be imaged in detail without external stains due to intrinsic fluorescence using two-photon excitation (2-PE) microscopy12.
TRACKING EARLY CARDIAC DEVELOPMENT IN REAL TIME: MULTI-PHOTON LIVE IMAGING AND 4-D CELL TRACKING IN MOUSE EMBRYO
A novel study protocol for whole embryo live-imaging based on multi-photon and confocal microscopy, tracks deeper complexities of cardiogenesis with cellular resolution, given that early cell movements during heart tube formation, have not been captured in real time so far. In a complex, transformative process that occurs early in cardiogenesis (at embryonic stage 7.5-8.25 for mouse), a bilateral structure known as the cardiac crescent transforms into an early heart tube (HT), initially open dorsally to undergo closure with development (Fig 3). At the tissue-level, this process involves precise and timely integration of different cell populations5,13.
During vertebrate heart development, two progenitor cell populations; first and second heart fields (FHF and SHF), sequentially contribute to heart tube formation and subsequently form the mature heart5,13. In view of that, cells in the developing mouse embryo were fluorescently labelled with genetic tracing tools, for 4D cell tracking and live-imaging cardiac differentiation over time with cellular resolution, for the first time. A culture system14 was reproduced, to allow embryonic growth externally, while immobilized for live imaging, to ensure the developing whole mouse embryo was stable throughout the process of live-imaging early cardiogenesis (Fig 4A)5. The system, coupled to live-confocal and -multiphoton microscopy, enabled high-resolution images (Fig 4B) and 4-D movies via time-lapse acquisition (Fig 5-7)5.
Live-imaging and 4-D cell tracking experiments sequentially captured dynamics of cell differentiation and cell movement during early heart tube formation to uncover cell population-based co-ordination of heart tube morphogenesis5. Three distinct stages of heart tube formation were observed with time to reveal 1) an early phase where cellular precursors differentiated to form a cardiac crescent, 2) followed by a 2nd phase to form a dorsally open heart tube and 3) a final phase of its dorsal closure5. Representative figures outline key features of the study (Fig 5-7)5.
The study further enabled quantification of cardiac cell differentiation during heart tube formation, using the 4D microscopy time-lapse movies and Imaris software, where each object (cell of interest) was identified according to pixel intensity (Fig 7). Study limitations included inability to image all cells located inside live tissue at the final stages recorded with multiphoton-imaging, due to limited depth of penetration, overcome by fixing embryos post-live-imaging for confocal microscopy analysis5. Further studies will also be required to verify key observations detailed.
The experiments collectively illustrated essential properties of heart development at cellular resolution, for the first time, with mouse whole embryo live-analysis, and are projected to similarly uncover unexpected/novel mechanisms of organogenesis in the future. As recent investigations of congenital disease have increasingly relied on experimental models of vertebrate embryo15,16, the evolving imaging techniques can revolutionize the field of heart development and early investigations of disease.
Poster Image: Confocal images of immunostained embryo representing early heart development (Figure 3A, reference 5).
- Goenezen, S., Rennie, M. Y. & Rugonyi, S. Biomechanics of early cardiac development. Biomech Model Mechanobiol 11, 1187-1204, (2012).
- Kowalski, W. J., Pekkan, K., Tinney, J. P. & Keller, B. B. Investigating developmental cardiovascular biomechanics and the origins of congenital heart defects. Frontiers in Physiology 5, 408, (2014).
- Rugonyi, S., Shaut, C., Liu, A., Thornburg, K. & Wang, R. K. Changes in wall motion and blood flow in the outflow tract of chick embryonic hearts observed with optical coherence tomography after outflow tract banding and vitelline-vein ligation. Phys Med Biol 53, 5077-5091, (2008).
- Jenkins, M. W., Watanabe, M. & Rollins, A. M. Longitudinal Imaging of Heart Development With Optical Coherence Tomography. IEEE journal of selected topics in quantum electronics : a publication of the IEEE Lasers and Electro-optics Society 18, 1166-1175, (2012).
- Ivanovitch, K., Temino, S. & Torres, M. Live imaging of heart tube development in mouse reveals alternating phases of cardiac differentiation and morphogenesis. bioRxiv, (2017).
- Patten, B. M. The early embryology of the chick, by Bradley M. Patten ... with 55 illustrations containing 182 figures. (P. Blakiston's Son & Co., 1920).
- van den Berg, G. & Moorman, A. F. M. Development of the Pulmonary Vein and the Systemic Venous Sinus: An Interactive 3D Overview. PLOS ONE 6, e22055, (2011).
- Manner, J. The anatomy of cardiac looping: a step towards the understanding of the morphogenesis of several forms of congenital cardiac malformations. Clinical anatomy (New York, N.Y.) 22, 21-35, (2009).
- Al Naieb, S., Happel, C. M. & Yelbuz, T. M. A detailed atlas of chick heart development in vivo. Annals of Anatomy - Anatomischer Anzeiger 195, 324-341, (2013).
- Happel, C. M., Thrane, L., Thommes, J., Manner, J. & Yelbuz, T. M. Integration of an optical coherence tomography (OCT) system into an examination incubator to facilitate in vivo imaging of cardiovascular development in higher vertebrate embryos under stable physiological conditions. Annals of anatomy = Anatomischer Anzeiger : official organ of the Anatomische Gesellschaft 193, 425-435, (2011).
- El-Ghali, N., Rabadi, M., Ezin, A. M. & De Bellard, M. E. New methods for chicken embryo manipulations. Microscopy research and technique 73, 58-66, (2010).
- Robertson, A. M., Hill, M. R. & Li, D. in Modeling of Physiological Flows (eds Davide Ambrosi, Alfio Quarteroni, & Gianluigi Rozza) 143-185 (Springer Milan, 2012).
- Buckingham, M. in Congenital Heart Diseases: The Broken Heart: Clinical Features, Human Genetics and Molecular Pathways (eds Silke Rickert-Sperling, Robert G. Kelly, & David J. Driscoll) 25-40 (Springer Vienna, 2016).
- Nonaka, S., Shiratori, H., Saijoh, Y. & Hamada, H. Determination of left-right patterning of the mouse embryo by artificial nodal flow. Nature 418, 96-99, (2002).
- Shi, H. et al. NAD Deficiency, Congenital Malformations, and Niacin Supplementation. New England Journal of Medicine 377, 544-552, (2017).
- Ma, H. et al. Correction of a pathogenic gene mutation in human embryos. Nature, (2017).