The traditional description of cardiac development involves progression from a cardiac crescent to a linear heart tube, which in the phase of transformation into a mature heart forms a cardiac loop and is divided with the septa into individual cavities. Cardiac morphogenesis involves numerous types of cells originating outside the initial cardiac crescent, including neural crest cells, cells of the second heart field origin, and epicardial progenitor cells. The development of the fetal heart and circulatory system is subject to regulatation by both genetic and environmental processes. The etiology for cases with congenital heart defects (CHDs) is largely unknown, but several genetic anomalies, some maternal illnesses, and prenatal exposures to specific therapeutic and non-therapeutic drugs are generally accepted as risk factors. New techniques for studying heart development have revealed many aspects of cardiac morphogenesis that are important in the development of CHDs, in particular transposition of the great arteries.

Despite their burden, most congenital defects remain poorly understood, due to lack of knowledge of embryological mechanisms. Here, we identify Greb1l mutants as a mouse model of crisscross heart. Based on 3D quantifications of shape changes, we demonstrate that torsion of the atrioventricular canal occurs together with supero-inferior ventricles at E10.5, after heart looping. Mutants phenocopy partial deficiency in retinoic acid signaling, which reflect overlapping pathways in cardiac precursors. Spatiotemporal gene mapping and cross-correlated transcriptomic analyses further reveal the role of Greb1l in maintaining a pool of dorsal pericardial wall precursor cells during heart tube elongation, likely by controlling ribosome biogenesis and cell differentiation. Consequently, we observe growth arrest and malposition of the outflow tract, which are predictive of abnormal tube remodeling in mutants. Our work on a rare cardiac malformation opens novel perspectives on the origin of a broader spectrum of congenital defects associated with GREB1L in humans.

Heart morphogenesis is a complex and dynamic process that has captivated researchers for almost a century. This process involves three main stages, during which the heart undergoes growth and folding on itself to form its common chambered shape. However, imaging heart development presents significant challenges due to the rapid and dynamic changes in heart morphology. Researchers have used different model organisms and developed various imaging techniques to obtain high-resolution images of heart development. Advanced imaging techniques have allowed the integration of multiscale live imaging approaches with genetic labeling, enabling the quantitative analysis of cardiac morphogenesis. Here, we discuss the various imaging techniques used to obtain high-resolution images of whole-heart development. We also review the mathematical approaches used to quantify cardiac morphogenesis from 3D and 3D+time images and to model its dynamics at the tissue and cellular levels.

Despite the extensive information available on the different genetic, epigenetic, and molecular features of cardiogenesis, the origin of congenital heart defects remains unknown. Most genetic and molecular studies have been conducted outside the context of the progressive anatomical and histological changes in the embryonic heart, which is one of the reasons for the limited knowledge of the origins of congenital heart diseases. We integrated the findings of descriptive studies on human embryos and experimental studies on chick, rat, and mouse embryos. This research is based on the new dynamic concept of heart development and the existence of two heart fields. The first field corresponds to the straight heart tube, into which splanchnic mesodermal cells from the second heart field are gradually recruited. The overall aim was to create a new vision for the analysis, diagnosis, and regionalized classification of congenital defects of the heart and great arteries. In addition to highlighting the importance of genetic factors in the development of congenital heart disease, this study provides new insights into the composition of the straight heart tube, the processes of twisting and folding, and the fate of the conus in the development of the right ventricle and its outflow tract. The new vision, based on in vivo labeling and cell tracking and enhanced by models such as gastruloids and organoids, has contributed to a better understanding of important errors in cardiac morphogenesis, which may lead to several congenital heart diseases.

Structural and Doppler velocity data collected from optical coherence tomography have already provided crucial insights into cardiac morphogenesis. X-ray microtomography and other ex vivo methods have elucidated structural details of developing hearts. However, by itself, no single imaging modality can provide comprehensive information allowing to fully decipher the inner workings of an entire developing organ. Hence, we introduce a specimen-specific correlative multimodal imaging workflow combining OCT and micro-CT imaging which is applicable for modeling of early chick heart development-a valuable model organism in cardiovascular development research. The image acquisition and processing employ common reagents, lab-based micro-CT imaging, and software that is free for academic use. Our goal is to provide a step-by-step guide on how to implement this workflow and to demonstrate why those two modalities together have the potential to provide new insight into normal cardiac development and heart malformations leading to congenital heart disease.

Congenital heart disease is the most frequent birth defect and the leading cause of death for the fetus and in the first year of life. The wide phenotypic diversity of congenital heart defects requires expert diagnosis and sophisticated repair surgery. Although these defects have been described since the seventeenth century, it was only in 2005 that a consensus international nomenclature was adopted, followed by an international classification in 2017 to help provide better management of patients. Advances in genetic engineering, imaging, and omics analyses have uncovered mechanisms of heart formation and malformation in animal models, but approximately 80% of congenital heart defects have an unknown genetic origin. Here, we summarize current knowledge of congenital structural heart defects, intertwining clinical and fundamental research perspectives, with the aim to foster interdisciplinary collaborations at the cutting edge of each field. We also discuss remaining challenges in better understanding congenital heart defects and providing benefits to patients.

Vertebrate heart development requires the complex morphogenesis of a linear tube to form the mature organ, a process essential for correct cardiac form and function, requiring coordination of embryonic laterality, cardiac growth, and regionalized cellular changes. While previous studies have demonstrated broad requirements for extracellular matrix (ECM) components in cardiac morphogenesis, we hypothesized that ECM regionalization may fine tune cardiac shape during heart development.
Using live in vivo light sheet imaging of zebrafish embryos, we describe a left-sided expansion of the ECM between the myocardium and endocardium prior to the onset of heart looping and chamber ballooning. Analysis using an ECM sensor revealed the cardiac ECM is further regionalized along the atrioventricular axis. Spatial transcriptomic analysis of gene expression in the heart tube identified candidate genes that may drive ECM expansion. This approach identified regionalized expression of hapln1a, encoding an ECM cross-linking protein. Validation of transcriptomic data by in situ hybridization confirmed regionalized hapln1a expression in the heart, with highest levels of expression in the future atrium and on the left side of the tube, overlapping with the observed ECM expansion. Analysis of CRISPR-Cas9-generated hapln1a mutants revealed a reduction in atrial size and reduced chamber ballooning. Loss-of-function analysis demonstrated that ECM expansion is dependent upon Hapln1a, together supporting a role for Hapln1a in regionalized ECM modulation and cardiac morphogenesis. Analysis of hapln1a expression in zebrafish mutants with randomized or absent embryonic left-right asymmetry revealed that laterality cues position hapln1a-expressing cells asymmetrically in the left side of the heart tube.
We identify a regionalized ECM expansion in the heart tube which promotes correct heart development, and propose a novel model whereby embryonic laterality cues orient the axis of ECM asymmetry in the heart, suggesting these two pathways interact to promote robust cardiac morphogenesis.

The secreted factor Nodal, known as a major left determinant, is associated with severe heart defects. Yet, it has been unclear how it regulates asymmetric morphogenesis such as heart looping, which align cardiac chambers to establish the double blood circulation. Here, we report that Nodal is transiently active in precursors of the mouse heart tube poles, before looping. In conditional mutants, we show that Nodal is not required to initiate asymmetric morphogenesis. We provide evidence of a heart-specific random generator of asymmetry that is independent of Nodal. Using 3D quantifications and simulations, we demonstrate that Nodal functions as a bias of this mechanism: it is required to amplify and coordinate opposed left-right asymmetries at the heart tube poles, thus generating a robust helical shape. We identify downstream effectors of Nodal signaling, regulating asymmetries in cell proliferation, differentiation, and extracellular matrix composition. Our study uncovers how Nodal regulates asymmetric organogenesis.

Cohesin mediates sister chromatid cohesion and 3D genome folding. Two versions of the complex carrying STAG1 or STAG2 coexist in somatic vertebrate cells. STAG2 is commonly mutated in cancer, and germline mutations have been identified in cohesinopathy patients. To better understand the underlying pathogenic mechanisms, we report the consequences of Stag2 ablation in mice. STAG2 is largely dispensable in adults, and its tissue-wide inactivation does not lead to tumors but reduces fitness and affects both hematopoiesis and intestinal homeostasis. STAG2 is also dispensable for murine embryonic fibroblasts in vitro. In contrast, Stag2-null embryos die by mid-gestation and show global developmental delay and defective heart morphogenesis, most prominently in structures derived from secondary heart field progenitors. Both decreased proliferation and altered transcription of tissue-specific genes contribute to these defects. Our results provide compelling evidence on cell- and tissue-specific roles of different cohesin complexes and how their dysfunction contributes to disease.

Dynamic imaging is a powerful approach to assess the function of a developing organ system. The heart is a dynamic organ that undergoes quick morphological and mechanical changes through early embryonic development. Defining the embyonic mouse heart\'s normal function is important for our own understanding of human heart development and will inform us on treatments and prevention of congenital heart defects (CHD). Traditional methods such as ultrasound or fluorescence-based microscopy are suitable for live dynamic imaging, are excellent to visualize structure and connect gene expression to phenotypes, but can be of low quality in resolving fine features and lack imaging depth and scale to fully appreciate organ morphogenesis. Additionally, previous methods can be limited in accommodating a live imaging apparatus capable of sustaining whole embryo development for extended periods time. Optical coherence tomography (OCT) is unique in this circumstance because acquisition of three-dimensional images without contrast reagents, at single cell resolution make it a suitable modality to visualize fine structures in the developing embryo. OCT setups are highly customizable for live imaging because of the tethered imaging arm, due to its setup as a fiber-based interferometer. OCT allows for 4D (3D + time) functional imaging of living mouse embryos and can provide functional and mechanical information to ascertain how the heart\'s pump function changes through development. In this chapter, we will focus on how we use OCT to visualize live heart dynamics at different stages of development and provide mechanical information to reveal functional properties of the developing heart.