Mechanical forces in avian embryo development.

Research using avian embryos has led to major conceptual advances in developmental biology, virology, immunology, genetics and cell biology. The avian embryo has several significant advantages, including ready availability and ease of accessibility, rapid development with marked similarities to mammals and a high amenability to manipulation. As mechanical forces are increasingly recognised as key drivers of morphogenesis, this powerful model system is shedding new light on the mechanobiology of embryonic development. Here, we highlight progress in understanding how mechanical forces direct key morphogenetic processes in the early avian embryo. Recent advances in quantitative live imaging and modelling are elaborating upon traditional work using physical models and embryo manipulations to reveal cell dynamics and tissue forces in ever greater detail. The recent application of transgenic technologies further increases the strength of the avian model and is providing important insights about previously intractable developmental processes.

Morphogenetic control of zebrafish cardiac looping by Bmp signaling.

Cardiac looping is an essential and highly conserved morphogenetic process that places the different regions of the developing vertebrate heart tube into proximity of their final topographical positions. High-resolution 4D live imaging of mosaically labelled cardiomyocytes reveals distinct cardiomyocyte behaviors that contribute to the deformation of the entire heart tube. Cardiomyocytes acquire a conical cell shape, which is most pronounced at the superior wall of the atrioventricular canal and contributes to S-shaped bending. Torsional deformation close to the outflow tract contributes to a torque-like winding of the entire heart tube between its two poles. Anisotropic growth of cardiomyocytes based on their positions reinforces S-shaping of the heart. During cardiac looping, bone morphogenetic protein pathway signaling is strongest at the future superior wall of the atrioventricular canal. Upon pharmacological or genetic inhibition of bone morphogenetic protein signaling, myocardial cells at the superior wall of the atrioventricular canal maintain cuboidal cell shapes and S-shaped bending is impaired. This description of cellular rearrangements and cardiac looping regulation may also be relevant for understanding the etiology of human congenital heart defects.

Intrinsic cellular chirality regulates left-right symmetry breaking during cardiac looping.

The vertebrate body plan is overall symmetrical but left-right (LR) asymmetric in the shape and positioning of internal organs. Although several theories have been proposed, the biophysical mechanisms underlying LR asymmetry are still unclear, especially the role of cell chirality, the LR asymmetry at the cellular level, on organ asymmetry. Here with developing chicken embryos, we examine whether intrinsic cell chirality or handedness regulates cardiac C looping. Using a recently established biomaterial-based 3D culture platform, we demonstrate that chick cardiac cells before and during C looping are intrinsically chiral and exhibit dominant clockwise rotation in vitro. We further show that cells in the developing myocardium are chiral as evident by a rightward bias of cell alignment and a rightward polarization of the Golgi complex, correlating with the direction of cardiac tube rotation. In addition, there is an LR polarized distribution of N-cadherin and myosin II in the myocardium before the onset of cardiac looping. More interestingly, the reversal of cell chirality via activation of the protein kinase C signaling pathway reverses the directionality of cardiac looping, accompanied by a reversal in cellular biases on the cardiac tube. Our results suggest that myocardial cell chirality regulates cellular LR symmetry breaking in the heart tube and the resultant directionality of cardiac looping. Our study provides evidence of an intrinsic cellular chiral bias leading to LR symmetry breaking during directional tissue rotation in vertebrate development.

On the Biomechanics of Cardiac S-Looping in the Chick: Insights From Modeling and Perturbation Studies.

Cardiac looping is an important embryonic developmental stage where the primitive heart tube (HT) twists into a configuration that more closely resembles the mature heart. Improper looping leads to congenital defects. Using the chick embryo as the experimental model, we study cardiac s-looping wherein the primitive ventricle, which lay superior to the atrium, now assumes its definitive position inferior to it. This process results in a heart loop that is no longer planar with the inflow and outflow tracts now lying in adjacent planes. We investigate the biomechanics of s-looping and use modeling to understand the nonlinear and time-variant morphogenetic shape changes. We developed physical and finite element models and validated the models using perturbation studies. The results from experiments and models show how force actuators such as bending of the embryonic dorsal wall (cervical flexure), rotation around the body axis (embryo torsion), and HT growth interact to produce the heart loop. Using model-based and experimental data, we present an improved hypothesis for early cardiac s-looping.

Mespaa can potently induce cardiac fates in zebrafish.

The Mesp family of transcription factors have been implicated in the early formation and migration of the cardiac lineage, although the precise molecular mechanisms underlying this process remain unknown. In this study we examine the function of Mesp family members in zebrafish cardiac development and find that Mespaa is remarkably efficient at promoting cardiac fates in normally non-cardiogenic cells. However, Mespaa is dispensable for normal cardiac formation. Despite no overt defects in cardiovascular specification, we find a consistent defect in cardiac laterality in mespaa null embryos. This is further exacerbated by the depletion of other mesp paralogues, highlighting a conserved role for the mesp family in left-right asymmetry, distinct from a function in cardiac specification. Despite an early requirement for mespaa to promote cardiogenesis, cells over-expressing mespaa are found to both exhibit unique cellular behaviors and activate the transcription of gata5 only after the completion of gastrulation. We propose that while mespaa remains capable of driving cardiac progenitor formation in zebrafish, it may not play an essential role in the cardiac regulatory network. Furthermore, the late activation of migration and cardiac gene transcription in mespaa over-expressing cells challenges previous studies on the timing of these events and provides intriguing questions for future study.

Cooperation between Nodal and FGF signals regulates zebrafish cardiac cell migration and heart morphogenesis.

Asymmetric vertebrate heart development is driven by an intricate sequence of morphogenetic cell movements, the coordination of which requires precise interpretation of signaling cues by heart primordia. Here we show that Nodal functions cooperatively with FGF during heart tube formation and asymmetric placement. Both pathways act as migratory stimuli for cardiac progenitor cells (CPCs), but FGF is dispensable for directing heart tube asymmetry, which is governed by Nodal. We further find that Nodal controls CPC migration by inducing left-right asymmetries in the formation of actin-based protrusions in CPCs. Additionally, we define a developmental window in which FGF signals are required for proper heart looping and show cooperativity between FGF and Nodal in this process. We present evidence FGF may promote heart looping through addition of the secondary heart field. Finally, we demonstrate that loss of FGF signaling affects proper development of the atrioventricular canal (AVC), which likely contributes to abnormal chamber morphologies in FGF-deficient hearts. Together, our data shed insight into how the spatiotemporal dynamics of signaling cues regulate the cellular behaviors underlying organ morphogenesis.

The Functional Significance of Cardiac Looping: Comparative Embryology, Anatomy, and Physiology of the Looped Design of Vertebrate Hearts.

The flow path of vertebrate hearts has a looped configuration characterized by curved (sigmoid) and twisted (chiral) components. The looped heart design is phylogenetically conserved among vertebrates and is thought to represent a significant determinant of cardiac pumping function. It evolves during the embryonic period of development by a process called "cardiac looping". During the past decades, remarkable progress has been made in the uncovering of genetic, molecular, and biophysical factors contributing to cardiac looping. Our present knowledge of the functional consequences of cardiac looping lags behind this impressive progress. This article provides an overview and discussion of the currently available information on looped heart design and its implications for the pumping function. It is emphasized that: (1) looping seems to improve the pumping efficiency of the valveless embryonic heart. (2) bilaterally asymmetric (chiral) looping plays a central role in determining the alignment and separation of the pulmonary and systemic flow paths in the multi-chambered heart of tetrapods. (3) chiral looping is not needed for efficient pumping of the two-chambered hearts of fish. (4) it is the sigmoid curving of the flow path that may improve the pumping efficiency of lower as well as higher vertebrate hearts.

Timeline of Developmental Defects Generated upon Genetic Inhibition of the Retinoic Acid Receptor Signaling Pathway.

It has been established for almost 30 years that the retinoic acid receptor (RAR) signalling pathway plays essential roles in the morphogenesis of a large variety of organs and systems. Here, we used a temporally controlled genetic ablation procedure to precisely determine the time windows requiring RAR functions. Our results indicate that from E8.5 to E9.5, RAR functions are critical for the axial rotation of the embryo, the appearance of the sinus venosus, the modelling of blood vessels, and the formation of forelimb buds, lung buds, dorsal pancreatic bud, lens, and otocyst. They also reveal that E9.5 to E10.5 spans a critical developmental period during which the RARs are required for trachea formation, lung branching morphogenesis, patterning of great arteries derived from aortic arches, closure of the optic fissure, and growth of inner ear structures and of facial processes. Comparing the phenotypes of mutants lacking the 3 RARs with that of mutants deprived of all-trans retinoic acid (ATRA) synthesising enzymes establishes that cardiac looping is the earliest known morphogenetic event requiring a functional ATRA-activated RAR signalling pathway.

An integrative multiscale view of early cardiac looping.

The heart is the first organ to form and function during the development of an embryo. Heart development consists of a series of events believed to be highly conserved in vertebrates. Development of heart begins with the formation of the cardiac fields followed by a linear heart tube formation. The straight heart tube then undergoes a ventral bending prior to further bending and helical torsion to form a looped heart. The looping phase is then followed by ballooning, septation, and valve formation giving rise to a four-chambered heart in avians and mammals. The looping phase plays a central role in heart development. Successful looping is essential for proper alignment of the future cardiac chambers and tracts. As aberrant looping results in various congenital heart diseases, the mechanisms of cardiac looping have been studied for several decades by various disciplines. Many groups have studied anatomy, biology, genetics, and mechanical processes during heart looping, and have proposed multiple mechanisms. Computational modeling approaches have been utilized to examine the proposed mechanisms of the looping process. Still, the exact underlying mechanism(s) controlling the looping phase remain poorly understood. Although further experimental measurements are obviously still required, the need for more integrative computational modeling approaches is also apparent in order to make sense of the vast amount of experimental data and the complexity of multiscale developmental systems. Indeed, there needs to be an iterative interaction between experimentation and modeling in order to properly find the gap in the existing data and to validate proposed hypotheses. This article is categorized under: Cardiovascular Diseases > Genetics/Genomics/Epigenetics Cardiovascular Diseases > Computational Models Cardiovascular Diseases > Molecular and Cellular Physiology.

Pyridaben induced cardiotoxicity during the looping stages of zebrafish (Danio rerio) embryos.

Pyridaben is a widely used acaricide in agriculture and reaches a high concentration (97 µg/L) in paddy water for a short time when pyridaben was applied to rice. However, its toxicity to aquatic organisms is still poorly understood. Therefore, we assessed the pyridaben cardiotoxicity to aquatic organisms using the zebrafish (Danio rerio) model. We found that pyridaben is highly toxic to aquatic organisms, and LC50 of pyridaben for zebrafish at 72 hpf was 100.6 µg/L. Pyridaben caused severe cardiac malformations and functional abnormalities. Morphologic abnormity included severe pericardial edema, cardiomegaly, decreased cardiomyocytes, thinning of the myocardial layer, linear heart, and increased the distance between sinus venous and bulbus arteriosus (SV-BA). Functional failure included arrhythmia, heart failure, and reduced pumping efficiency. The genes involved in heart development, WNT signaling, BMP signaling, ATPase, and cardiac troponin C were abnormally expressed in the pyridaben treatment group. Exposure to pyridaben increased oxidative stress and induced cell apoptosis. The above causes may lead to cardiac toxicity. The results suggest that pyridaben exposure induced elevated oxidative stress through the WNT signaling pathway, which in turn led to apoptosis in the heart and cardiotoxicity. Besides, pyridaben exposure at the critical stage of cardiac looping (24-36 hpf) resulted in the greatest cardiotoxicity. The chorion reduced the entry of pyridaben and protected zebrafish embryos, resulting in cardiotoxicity second only to the stage of cardiac looping. The study should provide valuable information that pyridaben exposure causes cardiotoxicity in zebrafish embryos and have potential health risks for other aquatic organisms and humans.

Bending and twisting the embryonic heart: a computational model for c-looping based on realistic geometry.

The morphogenetic process of cardiac looping transforms the straight heart tube into a curved tube that resembles the shape of the future four-chambered heart. Although great progress has been made in identifying the molecular and genetic factors involved in looping, the physical mechanisms that drive this process have remained poorly understood. Recent work, however, has shed new light on this complicated problem. After briefly reviewing the current state of knowledge, we propose a relatively comprehensive hypothesis for the mechanics of the first phase of looping, termed c-looping, as the straight heart tube deforms into a c-shaped tube. According to this hypothesis, differential hypertrophic growth in the myocardium supplies the main forces that cause the heart tube to bend ventrally, while regional growth and cytoskeletal contraction in the omphalomesenteric veins (primitive atria) and compressive loads exerted by the splanchnopleuric membrane drive rightward torsion. A computational model based on realistic embryonic heart geometry is used to test the physical plausibility of this hypothesis. The behavior of the model is in reasonable agreement with available experimental data from control and perturbed embryos, offering support for our hypothesis. The results also suggest, however, that several other mechanisms contribute secondarily to normal looping, and we speculate that these mechanisms play backup roles when looping is perturbed. Finally, some outstanding questions are discussed for future study.

The Transitional Cardiac Pumping Mechanics in the Embryonic Heart.

Several studies have linked abnormal blood flow dynamics to the formation of congenital heart defects during the early stages of development. The objective of this study is to document the transition of pumping mechanics from the early tube stage to the late looping stage of the embryonic heart. The optically transparent zebrafish embryonic heart was utilized as the in vivo model and was studied using standard bright field microscopy at three relevant stages within the transitional period: (1) tube stage at 30 hours post-fertilization (hpf); (2) early cardiac looping stage at 36 hpf; and (3) late cardiac looping stage at 48 hpf. High-speed videos were collected at 1000 fps at a spatial resolution of 1.1 µm/pixel at each of these stages and were post-processed to yield blood velocity patterns as well as wall kinematics. Results show that several relevant trends exist. Morphological trends from tube through late looping include: (a) ballooning of the chambers, (b) increasing constriction at the atrioventricular junction (AVJ), and (c) repositioning of the ventricle toward the side of the atrium. Blood flow trends include: (a) higher blood velocities, (b) increased AVJ regurgitation, and (c) larger percentages of blood from the upper atrium expelled backward toward the atrial inlet. Pumping mechanics trends include: (a) increasing contraction wave delay at the AVJ, (b) the AVJ begins acting as a rudimentary valve, (c) decreasing chamber constriction during maximum contraction, and (d) a transition in ventricular kinematics from a pronounced propagating wave to an independent, full-chamber contraction. The above results provide new insight into the transitional pumping mechanics from peristalsis-like pumping to a displacement pumping mechanism.