Francesco Boselli

Endothelial Cilia Mediate Low Flow Sensing during Zebrafish Vascular Development

VIDEO ABSTRACT The pattern of blood flow has long been thought to play a significant role in vascular morphogenesis, yet the flow-sensing mechanism that is involved at early embryonic stages, when flow forces are low, remains unclear. It has been proposed that endothelial cells use primary cilia to sense flow, but this has never been tested in vivo. Here we show, by noninvasive, high-resolution imaging of live zebrafish embryos, that endothelial cilia progressively deflect at the onset of blood flow and that the deflection angle correlates with calcium levels in endothelial cells. We demonstrate that alterations in shear stress, ciliogenesis, or expression of the calcium channel PKD2 impair the endothelial calcium level and both increase and perturb vascular morphogenesis. Altogether, these results demonstrate that endothelial cilia constitute a highly sensitive structure that permits the detection of low shear forces during vascular morphogenesis.

Oscillatory Flow Modulates Mechanosensitive klf2a Expression through trpv4 and trpp2 during Heart Valve Development

In vertebrates, heart pumping is required for cardiac morphogenesis and altering myocardial contractility leads to abnormal intracardiac flow forces and valve defects. Among the different mechanical cues generated in the developing heart, oscillatory flow has been proposed to be an essential factor in instructing endocardial cell fate toward valvulogenesis and leads to the expression of klf2a, a known atheroprotective transcription factor. To date, the mechanism by which flow forces are sensed by endocardial cells is not well understood. At the onset of valve formation, oscillatory flows alter the spectrum of the generated wall shear stress (WSS), a key mechanical input sensed by endothelial cells. Here, we establish that mechanosensitive channels are activated in response to oscillatory flow and directly affect valvulogenesis by modulating the endocardial cell response. By combining live imaging and mathematical modeling, we quantify the oscillatory content of the WSS during valve development and demonstrate it sets the endocardial cell response to flow. Furthermore, we show that an endocardial calcium response and the flow-responsive klf2a promoter are modulated by the oscillatory flow through Trpv4, a mechanosensitive ion channel specifically expressed in the endocardium during heart valve development. We made similar observations for Trpp2, a known Trpv4 partner, and show that both the absence of Trpv4 or Trpp2 leads to valve defects. This work identifies a major mechanotransduction pathway involved during valve formation in vertebrates.

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.

Developmental Alterations in Heart Biomechanics and Skeletal Muscle Function in Desmin Mutants Suggest an Early Pathological Root for Desminopathies

Desminopathies belong to a family of muscle disorders called myofibrillar myopathies that are caused by Desmin mutations and lead to protein aggregates in muscle fibers. To date, the initial pathological steps of desminopathies and the impact of desmin aggregates in the genesis of the disease are unclear. Using live, high-resolution microscopy, we show that Desmin loss of function and Desmin aggregates promote skeletal muscle defects and alter heart biomechanics. In addition, we show that the calcium dynamics associated with heart contraction are impaired and are associated with sarcoplasmic reticulum dilatation as well as abnormal subcellular distribution of Ryanodine receptors. Our results demonstrate that desminopathies are associated with perturbed excitation-contraction coupling machinery and that aggregates are more detrimental than Desmin loss of function. Additionally, we show that pharmacological inhibition of aggregate formation and Desmin knockdown revert these phenotypes. Our data suggest alternative therapeutic approaches and further our understanding of the molecular determinants modulating Desmin aggregate formation.

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.

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.

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. Summary: Blood flow modeling shows that dynamic shear stress patterns, rather than mean flow direction, predict the stereotypical behavior of endocardial cells during the early steps of heart valve formation.

A quantitative approach to study endothelial cilia bending stiffness during blood flow mechanodetection in vivo.

Primary cilia are necessary for shear stress sensing in different developing organs such as the kidneys and blood vessels. In endothelial cells (ECs), primary cilia bend in response to blood flow forces and are necessary for flow sensing as well as for the control of angiogenesis. The different parameters guiding cilia bending reflect the forces generated at the surface of the ECs and the mechanical properties of the endothelial cilia. Here, we present an approach allowing the calculation of the bending rigidity of endothelial cilia based on live imaging. The method relies on segmentation and mathematical modeling to extract the critical parameters needed for the calculation.

Three-dimensional microscopy and image analysis methodology for mapping and quantification of nuclear positions in tissues with approximate cylindrical geometry

Organogenesis involves extensive and dynamic changes of tissue shape during development. It is associated with complex morphogenetic events that require enormous tissue plasticity and generate a large variety of transient three-dimensional geometries that are achieved by global tissue responses. Nevertheless, such global responses are driven by tight spatio-temporal regulation of the behaviours of individual cells composing these tissues. Therefore, the development of image analysis tools that allow for extraction of quantitative data concerning individual cell behaviours is central to study tissue morphogenesis. There are many image analysis tools available that permit extraction of cell parameters. Unfortunately, the majority are developed for tissues with relatively simple geometries such as flat epithelia. Problems arise when the tissue of interest assumes a more complex three-dimensional geometry. Here, we use the endothelium of the developing zebrafish dorsal aorta as an example of a tissue with cylindrical geometry and describe the image analysis routines developed to extract quantitative data on individual cells in such tissues, as well as the image acquisition and sample preparation methodology. This article is part of the Theo Murphy meeting issue ‘Mechanics of development’.

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.