Cardiac forces regulate zebrafish heart valve delamination by modulating Nfat signaling.

In the clinic, most cases of congenital heart valve defects are thought to arise through errors that occur after the endothelial-mesenchymal transition (EndoMT) stage of valve development. Although mechanical forces caused by heartbeat are essential modulators of cardiovascular development, their role in these later developmental events is poorly understood. To address this question, we used the zebrafish superior atrioventricular valve (AV) as a model. We found that cellularized cushions of the superior atrioventricular canal (AVC) morph into valve leaflets via mesenchymal-endothelial transition (MEndoT) and tissue sheet delamination. Defects in delamination result in thickened, hyperplastic valves, and reduced heart function. Mechanical, chemical, and genetic perturbation of cardiac forces showed that mechanical stimuli are important regulators of valve delamination. Mechanistically, we show that forces modulate Nfatc activity to control delamination. Together, our results establish the cellular and molecular signature of cardiac valve delamination in vivo and demonstrate the continuous regulatory role of mechanical forces and blood flow during valve formation.

Mechanical control of tissue shape: Cell-extrinsic and -intrinsic mechanisms join forces to regulate morphogenesis.

During vertebrate development, cells must proliferate, move, and differentiate to form complex shapes. Elucidating the mechanisms underlying the molecular and cellular processes involved in tissue morphogenesis is essential to understanding developmental programmes. Mechanical stimuli act as a major contributor of morphogenetic processes and impact on cell behaviours to regulate tissue shape and size. Specifically, cell extrinsic physical forces are translated into biochemical signals within cells, through the process of mechanotransduction, activating multiple mechanosensitive pathways and defining cell behaviours. Physical forces generated by tissue mechanics and the extracellular matrix are crucial to orchestrate tissue patterning and cell fate specification. At the cell scale, the actomyosin network generates the cellular tension behind the tissue mechanics involved in building tissue. Thus, understanding the role of physical forces during morphogenetic processes requires the consideration of the contribution of cell intrinsic and cell extrinsic influences. The recent development of multidisciplinary approaches, as well as major advances in genetics, microscopy, and force-probing tools, have been key to push this field forward. With this review, we aim to discuss recent work on how tissue shape can be controlled by mechanical forces by focusing specifically on vertebrate organogenesis. We consider the influences of mechanical forces by discussing the cell-intrinsic forces (such as cell tension and proliferation) and cell-extrinsic forces (such as substrate stiffness and flow forces). We review recently described processes supporting the role of intratissue force generation and propagation in the context of shape emergence. Lastly, we discuss the emerging role of tissue-scale changes in tissue material properties, extrinsic forces, and shear stress on shape establishment.

Role of tissue biomechanics in the formation and function of myocardial trabeculae in zebrafish embryos.

Cardiac trabeculae are uneven ventricular muscular structures that develop during early embryonic heart development at the outer curvature of the ventricle. Their biomechanical function is not completely understood, and while their formation is known to be mechanosensitive, it is unclear whether ventricular tissue internal stresses play an important role in their formation. Here, we performed imaging and image-based cardiac biomechanics simulations on zebrafish embryonic ventricles to investigate these issues. Microscopy-based ventricular strain measurements show that the appearance of trabeculae coincided with enhanced deformability of the ventricular wall. Image-based biomechanical simulations reveal that the presence of trabeculae reduces ventricular tissue internal stresses, likely acting as structural support in response to the geometry of the ventricle. Passive ventricular pressure-loading experiments further reveal that the formation of trabeculae is associated with a spatial homogenization of ventricular tissue stiffnesses in healthy hearts, but gata1 morphants with a disrupted trabeculation process retain a spatial stiffness heterogeneity. Our findings thus suggest that modulating ventricular wall deformability, stresses, and stiffness are among the biomechanical functions of trabeculae. Further, experiments with gata1 morphants reveal that a reduction in fluid pressures and consequently ventricular tissue internal stresses can disrupt trabeculation, but a subsequent restoration of ventricular tissue internal stresses via vasopressin rescues trabeculation, demonstrating that tissue stresses are important to trabeculae formation. Overall, we find that tissue biomechanics is important to the formation and function of embryonic heart trabeculation. KEY POINTS: Trabeculations are fascinating and important cardiac structures and their abnormalities are linked to embryonic demise. However, their function in the heart and their mechanobiological formation processes are not completely understood. Our imaging and modelling show that tissue biomechanics is the key here. We find that trabeculations enhance cardiac wall deformability, reduce fluid pressure stresses, homogenize wall stiffness, and have alignments that are optimal for providing load-bearing structural support for the heart. We further discover that high ventricular tissue internal stresses consequent to high fluid pressures are needed for trabeculation formation through a rescue experiment, demonstrating that myocardial tissue stresses are as important as fluid flow wall shear stresses for trabeculation formation.

Fluid mechanics of the zebrafish embryonic heart trabeculation.

Embryonic heart development is a mechanosensitive process, where specific fluid forces are needed for the correct development, and abnormal mechanical stimuli can lead to malformations. It is thus important to understand the nature of embryonic heart fluid forces. However, the fluid dynamical behaviour close to the embryonic endocardial surface is very sensitive to the geometry and motion dynamics of fine-scale cardiac trabecular surface structures. Here, we conducted image-based computational fluid dynamics (CFD) simulations to quantify the fluid mechanics associated with the zebrafish embryonic heart trabeculae. To capture trabecular geometric and motion details, we used a fish line that expresses fluorescence at the endocardial cell membrane, and high resolution 3D confocal microscopy. Our endocardial wall shear stress (WSS) results were found to exceed those reported in existing literature, which were estimated using myocardial rather than endocardial boundaries. By conducting simulations of single intra-trabecular spaces under varied scenarios, where the translational or deformational motions (caused by contraction) were removed, we found that a squeeze flow effect was responsible for most of the WSS magnitude in the intra-trabecular spaces, rather than the shear interaction with the flow in the main ventricular chamber. We found that trabecular structures were responsible for the high spatial variability of the magnitude and oscillatory nature of WSS, and for reducing the endocardial deformational burden. We further found cells attached to the endocardium within the intra-trabecular spaces, which were likely embryonic hemogenic cells, whose presence increased endocardial WSS. Overall, our results suggested that a complex multi-component consideration of both anatomic features and motion dynamics were needed to quantify the trabeculated embryonic heart fluid mechanics.

Actin dynamics and the Bmp pathway drive apical extrusion of proepicardial cells.

The epicardium, the outer mesothelial layer enclosing the myocardium, plays key roles in heart development and regeneration. During embryogenesis, the epicardium arises from the proepicardium (PE), a cell cluster that appears in the dorsal pericardium (DP) close to the venous pole of the heart. Little is known about how the PE emerges from the pericardial mesothelium. Using a zebrafish model and a combination of genetic tools, pharmacological agents and quantitative in vivo imaging, we reveal that a coordinated collective movement of DP cells drives PE formation. We found that Bmp signaling and the actomyosin cytoskeleton promote constriction of the DP, which enables PE cells to extrude apically. We provide evidence that cell extrusion, which has been described in the elimination of unfit cells from epithelia and the emergence of hematopoietic stem cells, is also a mechanism for PE cells to exit an organized mesothelium and fulfil their developmental fate to form a new tissue layer, the epicardium.

The cilium as a force sensor-myth versus reality.

Cells need to sense their mechanical environment during the growth of developing tissues and maintenance of adult tissues. The concept of force-sensing mechanisms that act through cell-cell and cell-matrix adhesions is now well established and accepted. Additionally, it is widely believed that force sensing can be mediated through cilia. Yet, this hypothesis is still debated. By using primary cilia sensing as a paradigm, we describe the physical requirements for cilium-mediated mechanical sensing and discuss the different hypotheses of how this could work. We review the different mechanosensitive channels within the cilium, their potential mode of action and their biological implications. In addition, we describe the biological contexts in which cilia are acting - in particular, the left-right organizer - and discuss the challenges to discriminate between cilium-mediated chemosensitivity and mechanosensitivity. Throughout, we provide perspectives on how quantitative analysis and physics-based arguments might help to better understand the biological mechanisms by which cells use cilia to probe their mechanical environment.

Notch and Bmp signaling pathways act coordinately during the formation of the proepicardium.

BACKGROUND: The epicardium is the outer mesothelial layer of the heart. It encloses the myocardium and plays key roles in heart development and regeneration. It derives from the proepicardium (PE), cell clusters that appear in the dorsal pericardium (DP) close to the atrioventricular canal and the venous pole of the heart, and are released into the pericardial cavity. PE cells are advected around the beating heart until they attach to the myocardium. Bmp and Notch signaling influence PE formation, but it is unclear how both signaling pathways interact during this process in the zebrafish. RESULTS: Here, we show that the developing PE is influenced by Notch signaling derived from the endothelium. Overexpression of the intracellular receptor of notch in the endothelium enhances bmp expression, increases the number of pSmad1/5 positive cells in the DP and PE, and enhances PE formation. On the contrary, pharmacological inhibition of Notch1 impairs PE formation. bmp2b overexpression can rescue loss of PE formation in the presence of a Notch1 inhibitor, but Notch gain-of-function could not recover PE formation in the absence of Bmp signaling. CONCLUSIONS: Endothelial Notch signaling activates bmp expression in the heart tube, which in turn induces PE cluster formation from the DP layer.

Anisotropic shear stress patterns predict the orientation of convergent tissue movements in the embryonic heart.

Myocardial contractility and blood flow provide essential mechanical cues for the morphogenesis of the heart. In general, endothelial cells change their migratory behavior in response to shear stress patterns, according to flow directionality. Here, we assessed the impact of shear stress patterns and flow directionality on the behavior of endocardial cells, the specialized endothelial cells of the heart. At the early stages of zebrafish heart valve formation, we show that endocardial cells are converging to the valve-forming area and that this behavior depends upon mechanical forces. Quantitative live imaging and mathematical modeling allow us to correlate this tissue convergence with the underlying flow forces. We predict that tissue convergence is associated with the direction of the mean wall shear stress and of the gradient of harmonic phase-averaged shear stresses, which surprisingly do not match the overall direction of the flow. This contrasts with the usual role of flow directionality in vascular development and suggests that the full spatial and temporal complexity of the wall shear stress should be taken into account when studying endothelial cell responses to flow in vivo.

Hemodynamics driven cardiac valve morphogenesis.

Mechanical forces are instrumental to cardiovascular development and physiology. The heart beats approximately 2.6 billion times in a human lifetime and heart valves ensure that these contractions result in an efficient, unidirectional flow of the blood. Composed of endocardial cells (EdCs) and extracellular matrix (ECM), cardiac valves are among the most mechanically challenged structures of the body both during and after their development. Understanding how hemodynamic forces modulate cardiovascular function and morphogenesis is key to unraveling the relationship between normal and pathological cardiovascular development and physiology. Most valve diseases have their origins in embryogenesis, either as signs of abnormal developmental processes or the aberrant re-expression of fetal gene programs normally quiescent in adulthood. Here we review recent discoveries in the mechanobiology of cardiac valve development and introduce the latest technologies being developed in the zebrafish, including live cell imaging and optical technologies, as well as modeling approaches that are currently transforming this field. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Live imaging and modeling for shear stress quantification in the embryonic zebrafish heart.

Hemodynamic shear stress is sensed by the endocardial cells composing the inner cell layer of the heart, and plays a major role in cardiac morphogenesis. Yet, the underlying hemodynamics and the associated mechanical stimuli experienced by endocardial cells remains poorly understood. Progress in the field has been hampered by the need for high temporal resolution imaging allowing the flow profiles generated in the beating heart to be resolved. To fill this gap, we propose a method to analyze the wall dynamics, the flow field, and the wall shear stress of the developing zebrafish heart. This method combines live confocal imaging and computational fluid dynamics to overcome difficulties related to live imaging of blood flow in the developing heart. To provide an example of the applicability of the method, we discuss the hemodynamic frequency content sensed by endocardial cells at the onset of valve formation, and how the fundamental frequency of the wall shear stress represents a unique mechanical cue to endocardial, heart-valve precursors.

Pulse propagation by a capacitive mechanism drives embryonic blood flow.

Pulsatile flow is a universal feature of the blood circulatory system in vertebrates and can lead to diseases when abnormal. In the embryo, blood flow forces stimulate vessel remodeling and stem cell proliferation. At these early stages, when vessels lack muscle cells, the heart is valveless and the Reynolds number (Re) is low, few details are available regarding the mechanisms controlling pulses propagation in the developing vascular network. Making use of the recent advances in optical-tweezing flow probing approaches, fast imaging and elastic-network viscous flow modeling, we investigated the blood-flow mechanics in the zebrafish main artery and show how it modifies the heart pumping input to the network. The movement of blood cells in the embryonic artery suggests that elasticity of the network is an essential factor mediating the flow. Based on these observations, we propose a model for embryonic blood flow where arteries act like a capacitor in a way that reduces heart effort. These results demonstrate that biomechanics is key in controlling early flow propagation and argue that intravascular elasticity has a role in determining embryonic vascular function.

Blood flow mechanics in cardiovascular development.

Hemodynamic forces are fundamental to development. Indeed, much of cardiovascular morphogenesis reflects a two-way interaction between mechanical forces and the gene network activated in endothelial cells via mechanotransduction feedback loops. As these interactions are becoming better understood in different model organisms, it is possible to identify common mechanogenetic rules, which are strikingly conserved and shared in many tissues and species. Here, we discuss recent findings showing how hemodynamic forces potentially modulate cardiovascular development as well as the underlying fluid and tissue mechanics, with special attention given to the flow characteristics that are unique to the small scales of embryos.

Fluid flows and forces in development: functions, features and biophysical principles.

Throughout morphogenesis, cells experience intracellular tensile and contractile forces on microscopic scales. Cells also experience extracellular forces, such as static forces mediated by the extracellular matrix and forces resulting from microscopic fluid flow. Although the biological ramifications of static forces have received much attention, little is known about the roles of fluid flows and forces during embryogenesis. Here, we focus on the microfluidic forces generated by cilia-driven fluid flow and heart-driven hemodynamics, as well as on the signaling pathways involved in flow sensing. We discuss recent studies that describe the functions and the biomechanical features of these fluid flows. These insights suggest that biological flow determines many aspects of cell behavior and identity through a specific set of physical stimuli and signaling pathways.

Desmin in muscle and associated diseases: beyond the structural function.

Desmin is a muscle-specific type III intermediate filament essential for proper muscular structure and function. In human, mutations affecting desmin expression or promoting its aggregation lead to skeletal (desmin-related myopathies), or cardiac (desmin-related cardiomyopathy) phenotypes, or both. Patient muscles display intracellular accumulations of misfolded proteins and desmin-positive insoluble granulofilamentous aggregates, leading to a large spectrum of molecular alterations. Increasing evidence shows that desmin function is not limited to the structural and mechanical integrity of cells. This novel perception is strongly supported by the finding that diseases featuring desmin aggregates cannot be easily associated with mechanical defects, but rather involve desmin filaments in a broader spectrum of functions, such as in organelle positioning and integrity and in signaling. Here, we review desmin functions and related diseases affecting striated muscles. We detail emergent cellular functions of desmin based on reported phenotypes in patients and animal models. We discuss known desmin protein partners and propose an overview of the way that this molecular network could serve as a signal transduction platform necessary for proper muscle function.

The dynein regulatory complex is required for ciliary motility and otolith biogenesis in the inner ear.

In teleosts, proper balance and hearing depend on mechanical sensors in the inner ear. These sensors include actin-based microvilli and microtubule-based cilia that extend from the surface of sensory hair cells and attach to biomineralized 'ear stones' (or otoliths). Otolith number, size and placement are under strict developmental control, but the mechanisms that ensure otolith assembly atop specific cells of the sensory epithelium are unclear. Here we demonstrate that cilia motility is required for normal otolith assembly and localization. Using in vivo video microscopy, we show that motile tether cilia at opposite poles of the otic vesicle create fluid vortices that attract otolith precursor particles, thereby biasing an otherwise random distribution to direct localized otolith seeding on tether cilia. Independent knockdown of subunits for the dynein regulatory complex and outer-arm dynein disrupt cilia motility, leading to defective otolith biogenesis. These results demonstrate a requirement for the dynein regulatory complex in vertebrates and show that cilia-driven flow is a key epigenetic factor in controlling otolith biomineralization.

The wall-stress footprint of blood cells flowing in microvessels.

It is well known that mechanotransduction of hemodynamic forces mediates cellular processes, particularly those that lead to vascular development and maintenance. Both the strength and space-time character of these forces have been shown to affect remodeling and morphogenesis. However, the role of blood cells in the process remains unclear. We investigate the possibility that in the smallest vessels blood's cellular character of itself will lead to forces fundamentally different than the time-averaged forces usually considered, with fluctuations that may significantly exceed their mean values. This is quantitated through the use of a detailed simulation model of microvessel flow in two principal configurations: a diameter D=6.5 µm tube-a model for small capillaries through which red blood cells flow in single-file-and a D=12 µm tube-a model for a nascent vein or artery through which the cells flow in a confined yet chaotic fashion. Results in both cases show strong sensitivity to the mean flow speed U. Peak stresses exceed their means by greater than a factor of 10 when U/D≲10 s(-1), which corresponds to the inverse relaxation time of a healthy red blood cell. This effect is more significant for smaller D cases. At faster flow rates, including those more commonly observed under normal, nominally static physiological conditions, the peak fluctuations are more comparable with the mean shear stress. Implications for mechanotransduction of hemodynamic forces are discussed.

Modeling new conceptual interpretations of development.

In April 2011, researchers from diverse background met at the Gulbenkian Institute (Oeiras, Portugal) to discuss the emerging input of biophysics into the field of developmental biology. The scope of the workshop was to bring together scientists working in different model systems and to discuss some of the most recent advances towards understanding how physical forces affect embryonic development. Discussions and talks highlighted two main trends: that many aspects of embryogenesis can be accurately quantified and translated into a limited number of physical forces and biochemical parameters; and that simulations and modeling provide new conceptual interpretations of classical developmental questions.

Calcium Signal Analysis in the Zebrafish Heart via Phase Matching of the Cardiac Cycle.

Calcium signalling in the endocardium is critical for heart valve development. Calcium ion pulses in the endocardium are generated in response to mechanical forces due to blood flow and can be visualised in the beating zebrafish heart using a genetically encoded calcium indicator such as GCaMP7a. Analysing these pulses is challenging because of the rapid movement of the heart during heartbeat. This protocol outlines an imaging analysis method used to phase-match the cardiac cycle in single z-slice movies of the beating heart, allowing easy measurement of the calcium signal. Key features • Software to synchronise and analyse frames from movies of the beating heart corresponding to a user-defined phase of the cardiac cycle. • Software to measure the fluorescence intensity of the beating heart corresponding to a user-defined region of interest.

Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development.

The transcription factor neurogenin 3 (Neurog3 or Ngn3) controls islet cell fate specification in multipotent pancreatic progenitor cells in the mouse embryo. However, our knowledge of the genetic programs implemented by Ngn3, which control generic and islet subtype-specific properties, is still fragmentary. Gene expression profiling in isolated Ngn3-positive progenitor cells resulted in the identification of the uncharacterized winged helix transcription factor Rfx6. Rfx6 is initially expressed broadly in the gut endoderm, notably in Pdx1-positive cells in the developing pancreatic buds, and then becomes progressively restricted to the endocrine lineage, suggesting a dual function in both endoderm development and islet cell differentiation. Rfx6 is found in postmitotic islet progenitor cells in the embryo and is maintained in all developing and adult islet cell types. Rfx6 is dependent on Ngn3 and acts upstream of or in parallel with NeuroD, Pax4 and Arx transcription factors during islet cell differentiation. In zebrafish, the Rfx6 ortholog is similarly found in progenitors and hormone expressing cells of the islet lineage. Loss-of-function studies in zebrafish revealed that rfx6 is required for the differentiation of glucagon-, ghrelin- and somatostatin-expressing cells, which, in the absence of rfx6, are blocked at the progenitor stage. By contrast, beta cells, whose number is only slightly reduced, were no longer clustered in a compact islet. These data unveil Rfx6 as a novel regulator of islet cell development.

TissUExM protocol for ultrastructure expansion microscopy of zebrafish larvae and mouse embryos.

Expansion microscopy of millimeter-large mechanically heterogeneous tissues, such as whole vertebrate embryos, has been limited, particularly when combined with post-expansion immunofluorescence. Here, we present a protocol to perform ultrastructure expansion microscopy of whole vertebrate embryos, optimized to perform post-expansion labeling. We describe steps for embedding and denaturing zebrafish larvae or mouse embryos. We then detail procedures for hydrogel handling and mounting. This protocol is particularly well suited for super-resolution imaging of macromolecular protein complexes in situ but does not preserve lipids. For complete details on the use and execution of this protocol, please refer to Steib et al.1.