Final Report Summary - FLOWBUILT (Biological flows and embryonic development) HypothesisThe cardiovascular system is vital to every vertebrate and is the first functional organ in the developing embryo. As a consequence, the heartbeats generate hemodynamic forces that physically stimulate growing arteries and veins at the earliest steps of their development. The mechanisms involved in vascular morphogenesis are poorly understood. Primarily, our goal is to address the roles of blood flow patterns in specific regions of the cardiovascular system at different developmental stages. Secondarily, these experiments will be combined with molecular biology techniques to identify the genes activated by flows and conventional imaging to characterize the effects of flow alterations. In particular, we will develop live flow reporters to correlate the flow response with gene expression in vivo. Danio rerio, is a powerful animal model to study vessel formation during embryogenesis. Accessibility to large-scale genetic and experimental access together with its small size, optical transparency and external development of its embryos strongly facilitates in vivo analysis of both blood and lymphatic vessels development. Zebrafish imaging using fast confocal imaging have proven that the characterization of blood cell motion during cardiogenesis is possible (Vermot et al., 2009). Furthermore, the vascular system of the zebrafish develops in many ways in a similar fashion to others vertebrates allowing insightful comparison across species. Most importantly, embryo does not need heart function during the first 5 days of development (which corresponds to the end of organogenesis stage in fish) because oxygen in water diffuses within the embryo to sustain proper oxygenation. As a consequence, zebrafish is an excellent model to address the roles of flow forces during angiogenesis.Our objectives are to:1. Identify the roles of blood flow during vascular development 2. Address the cellular mechanisms controlling valve development in response to blood flow3. Address the mechanisms of flow function at the cellular and molecular level in the endothelium and in the pericardial cavity4. Developing models of cardiomyopathies suitable for drug screeningThe insights gained from these in vivo studies will significantly increase our knowledge on how genetic networks interact with their physical environment during vascular development. Ultimately, this work should lead to relevant findings for a better understanding of congenital cardiovascular diseases.Tasks achieved during the reporting period1. Identify the roles of blood flow during vascular development a. Question: The vascular system biomechanics in response to hemodynamic forcesThe development of the cardiovascular system is finely regulated to enable adequate perfusion of adult tissues throughout life. Unidirectional laminar, pulsatile or bidirectional flows are the most common type of flows found in normal vasculature (Hahn and Schwartz, 2009). Disturbances of fluid flow in specific regions or the cardiovascular system, in concert with major risk factors, is a key feature in atherosclerosis (Hahn and Schwartz, 2009). In vivo and in vitro studies have shown that endothelial cells align their cytoskeleton along a laminar flow, repress expression of inflammatory mediators and favor expression of a series of atheroprotective genes, many of them depending on increased levels of the transcription factor Krüppel-like factor 2 (Klf2)(Parmar et al., 2006). However, many of these in vitro observations remain to be validated in vivo because blood vessel biomechanics quantification is extremely challenging in whole embryos. Here we addressed the blood vessel biomechanics and the endothelial genes profile activated by blood flow directly in the embryo using zebrafish as a model organism.b. Approach 1: Characterizing blood vessel biomechanics in response to flow forces during angiogenesis. (work published (Anton et al., 2013))We developed a method to address the biomechanics of flow propagation during angiogenesis using optical tweezers. This new approach to flow probe allowed us to directly address the mechanism by which flow changes its shape along the vascular network. We found that the flow profile generated at artery/vessel branches is unique, notably because of the presence of a global deformation observed along the whole antero-posterior axis of the aorta. Using fast live imaging and optical tweezing, we demonstrated that this deformability is critical for generating an attracting flow at the entry of the connecting vessels and for creating an additional pressure gradient, which is distinct from the main pressure gradient dictated by the heart. Optical tweezing was developed with Sébastien Harlepp (IPCMS Strasbourg), a specialist in the field. We show the deformation permits the flow rectification observed between the arterial and venous network. The data explain flow rectification in vivo and show that heart effort benefits from such a pumping mechanism. It also help to clarify the origin of blood flow defects from a biophysical perspective. Finally, it shows that even though the physical laws that drive flow forces in adults (where inertia influence is prominent) and in embryos (where viscosity is taking over) are completely different, they share unexpected similarities in using vascular wall elasticity to bias flow profiles. The technical advances and conclusions presented in this study will be very useful for the community of cell biologists, developmental biologists and biophysicists as well as to clinicians interested in vascular diseases associated with abnormal hemodynamics in humans. c. Approach2: Blood flow modeling (work published (Anton et al., 2013) and (Freund and Vermot, 2014))Our work is based on the fact that all predictive models—stepping beyond purely phenomenological models—for gene expression or other cellular processes leading to angiogenesis will require quantitative inputs of hemodynamic stress at the cell and tissue levels. This requires a mathematically advanced simulation model to characterize the physical forces controlling blood vessel genesis and to identify the biomechanical mechanism involved in sensing mechanical stimuli generated by circulating blood during this process. So far, we have developed a 2D model of the entire zebrafish vascular network based on a lumped model that mimics very well the vascular flow observed in vivo (Anton et al., 2013) as well as a 3D model (Freund and Vermot, 2014). The results show that stress at narrow junctions, such as those between newly connected vessels, can increase by factors of 10 due to the presence of red cells. Our current work is now focused on understanding this influence at the molecular level and through more complete models.d. Approach3: Gene profiling of the endothelial flow response We used high throughput RNA sequencing analysis to examine the effect of interfering with blood flow on the endothelial transcriptome in zebrafish embryo. We used a microdissection approach where endothelial cells mRNA of the trunk were extracted along with muscular and blood cells. mRNA were extracted from dissected trunk of control and embryos that did not experienced flow (using a silent heart mutant that have no blood flow). Preliminary analysis identified differentially expressed gene potentially involved in the cellular functions regulated by the flow during endothelium maturation. These data should also be very informative with respect to the identification of responsive pathways, which are associated in several developmental systems and in diseases. For comparisons purpose, we used an approach to specifically isolate mRNA of endothelial cells by cell sorting through FACS in embryos expressing the eGFP in the endothelial cells (flk1:eGFP) as done in (Covassin et al., 2006). Endothelial cells mRNA that have not been in contact with flow using silent heart mutants have been compared to the control mRNA. A manuscript is now in preparation.e. Expected resultsSince flow responsive genes are frequently associated in development and disease, we expect our results to have a significant impact on the understanding of the cellular functions and the cross talk of key developmental pathways involved during cardiogenesis. 2. Cellular mechanisms controlling valve development in response to blood flowa. QuestionThis project is a direct follow up of the study published in 2009 about the roles of klf2a during valvulogenesis. Our goal was to characterize the mechanism activating the response to flow during valve development and the process of valve morphogenesis in response to flow.b. Approach: Characterization of the cellular behavior activated in response to flow during valve development and identification of two genes coding for proteins known to act as mechanodetectors.We studied several zebrafish valve mutants (provided by the Sanger Institute, Cambridge and Dimitris Beis, Academy of Athens), that are either affected in the intracardiac flow patterns (cmlc1, cmlc2, weak atrium). These animals have been studied from the cellular and biomechanical angle using intravital imaging of transgenic lines expressing fluorescent markers in specific parts of the endocardium. These studies allow us to understand the contribution of flow in the cellular rearrangements occurring during valve formation as well as the mechanotransduction mechanism operating in the endocardium. A manuscript is now in preparation.c. Expected resultsThis approach will permit to identify the genes activated or repressed by blood flow and their roles during valve morphogenesis, which is key for understanding the environmental factors involved in valvular congenital diseases.Contributors: Francesco Boselli/Emily Steed/Emilie Heckel, post docs3. Access the dynamics of mechanotransduction at the cellular and molecular level in the endothelium and in the pericardial cavity a. QuestionFlow sensing has been shown to control morphogenesis of the heart valve, of the vascular system, and during left-right patterning(Freund et al., 2012). Furthermore it has been proposed primary cilia constitute a key structure involved in flow sensing(Freund et al., 2012). Thus we hypothesise that at the time of angiogenesis and left-right patterning, cilia mediated mechanotransduction permits cells (EC) to modulate the shape of the vascular tree and cilia positioning according to the flow forces they experience during development.b. Approach 1: Access the dynamics of mechanotransduction at the cellular and molecular level in the endothelium. Ongoing work and published (Goetz et al., in press) and (Goetz et al., 2014).Here, we tested the biomechanical input of hemodynamics to vascular development and dissected the mechano-detection mechanisms at play in the developing endothelium. We hypothetized that primary cilia could be the mechanodetector operating at these early stages because cilia can protrude as flexible and deflectable antennaes in the lumen of multiple flow-filled biological tubes and are biologically suited for mechano-sensing. We observed that primary cilia line up the developing vessels and are progressively deflected by flow at the onset of heart contraction. Using a CLEM approach (Correlative and Light Electron Microscopy) coupled to electron tomography, we deciphered the architecture of the endothelial cilia. We published a detailed protocol of this unique technique (Goetz et al., in press). Our results suggest that early hemodynamic forces are detected by primary cilia whose deflection allows the gating of a calcium channel and the control of pro-angiogenic signaling. We used similar methods to characterize mechanodetection in the heart endocardium and found many similarities with the vascular endothelium. Here we identified the mechanodetector as TRPV4, a TRP channel involved in cilia mediated mechanotransduction(Kottgen et al., 2008; Thodeti et al., 2009). We found that the endocardium is ciliated and that contraction is followed by cyclical activation of calcium fluxes. Altogether, these data opens the possibility that valvulogenesis and ciliopathies could be associated. c. Approach2: Mapping the flow forces and function during pericardial development. Ongoing work and published (Peralta et al., 2013). In collaboration with the Mercader group (CNIC, Madrid) we addressed the role of low forces during epicardium development. The epicardium, the cell layer covering the myocardium, plays an essential role in heart maturation. It derives from the proepicardium (PE), a cluster of cells emerging from the inner lining of the pericardial wall, in which the heart is located. During development, PE cells need to be transferred to the surface of the myocardium, in order to form the epicardial layer. Using fast imaging and optical tweezers, we have shown that adhesion is ordered and depends on the pericardial flow. We also showed that adhesion was possible wherever the cells were trapped. Using optical tweezing, we quantified the time dependence when a cell adheres to the ventricle. The time dependence can be obtained by trapping cells at different distances from the heart. Thus, two hydrodynamic forces couple cardiac development and function: first, blood flow inside the heart, and second, the pericardial fluid advections outside the heart identified here. This pericardiac fluid flow is essential for epicardium formation and represents the first example of hydrodynamic flow forces controlling organogenesis through an action on mesothelial cells. d. Expected resultsThese approaches will permit to identify the roles of flow forces in broader contexts as well as to understand the environmental factors involved in other congenital diseases affecting heart development and cilia functions.Contributors: Dave Wu/Francesco Boselli/Emily Steed/Emilie Heckel/Jacky Goetz, post docs4. Developing models of cardiomyopathies suitable for drug screening. Ongoing work.a. HypothesisIn vertebrates, biomechanics is essential for many developmental processes and several mutations affecting molecular motors have been shown to perturb embryonic development. For example, contractions generated by beating heart are essential for embryonic heart patterning as well as morphogenesis, and aberrant contractile activity is often associated with lethal diseases and malformation in humans (Thierfelder et al., 1994). Furthermore, mechanical activities of the contractions could be related to human diseases such as hypertrophic cardiomyopathies or dilated cardiomyopathies (Seidman and Seidman, 2001). Nevertheless, heart contractions roles during embryonic development and the mechanisms by which contractions affects cardiomyopathies are still poorly understood. Desmin Related Myopathy (DRM or Desminopathy) is a subgroup of the myofibrillar myopathy diseases that leads to progressive muscular weakness, neuropathy and cardiomyopathy. Several genes have been identified for DRM, including desmin, but their function during normal and pathological muscles are still unclear. In particular, DRM can be the result of a mutation in the gene that codes for desmin inducing the formation of aggregates of desmin and other proteins throughout the cell. The roles of desmin aggregates during the genesis of myopathies, cardiomyopathy and cardiogenesis are unexplored. We address this point in vivo, we characterized an animal model of the desminopathy allowing to perform imaging of aggregates formation and amenable for drug screening to identify compounds able to correct the muscular activity seen in this neuromuscular disease. b. Approach: Characterization of engineered desmin1a linesThis project necessitated the generation of a desmin1a mutant through a tilling approach (provided by the Sanger institute), the characterization of a knock in desmin1a line promoting desmin aggregation (provided by Le trihn, Caltech) and the generation of a ‘humanized’ zebrafish model expressing the human desmin found in human patient. This project will concentrated on addressing the function of desmin1a during heart development and tail myogenesis and lead to the development of an animal model of the DRM using zebrafish. We also screened for chemical coumpounds inhibiting the formation of DESMIN aggregates and restoring normal heart activity. The short terms goals of this project was to validate that zebrafish is a good animal model to study mutated forms of the human Desmin involved in the desminopathy during muscle development of zebrafish. Our results show that desmin impacts on (i) the myocardial maturation and function during heart and vessel development in the embryo, (ii) myofibril assembly in skeletal muscles. A manuscript is now in preparation.c. Expected resultsThis project leads to the generation of genetic tools that will have an important impact for understanding cardiomyopathies: (i) we will generate animals allowing for high throughput screening of of myocardial contraction and muscle formation after desmin knock out or overexpression through transgenesis, (ii) we will develop fusion protein using photoactivable fluorescent proteins allowing to follow the molecular dynamics of myofibril assembly within the cells during myocardial contractions in normal and transgenic animals suitable with high throughput imaging and drug screening. REFERENCESAnton, H., Harlepp, S., Ramspacher, C., Wu, D., Monduc, F., Bhat, S., Liebling, M., Paoletti, C., Charvin, G., Freund, J.B. et al. (2013). Pulse propagation by a capacitive mechanism drives embryonic blood flow. Development 140, 4426-4434.2. Covassin, L., Amigo, J.D. Suzuki, K., Teplyuk, V., Straubhaar, J., and Lawson, N.D. (2006). Global analysis of hematopoietic and vascular endothelial gene expression by tissue specific microarray profiling in zebrafish. Dev Biol 299, 551-562.3. Freund, J.B. Goetz, J.G. Hill, K.L. and Vermot, J. (2012). Fluid flows and forces in development: functions, features and biophysical principles. Development 139, 1229-1245.4. Freund, J.B. and Vermot, J. (2014). The Wall-stress Footprint of Blood Cells Flowing in Microvessels. Biophys J 106, 752-762.5. Goetz, J.G. Monduc, F., Schwab, Y., and Vermot, J. (in press). Using correlative light and electron microscopy6. to study zebrafish vascular morphogenesis. Methods in Molecular Biology.7. Goetz, J.G. Steed, E., Ferreira, R.R. Roth, S., Ramspacher, C., Boselli, F., Charvin, G., Liebling, M., Wyart, C., Schwab, Y., et al. (2014). Endothelial Cilia Mediate Low Flow Sensing during Zebrafish Vascular Development. Cell reports.8. Hahn, C., and Schwartz, M.A. (2009). Mechanotransduction in vascular physiology and atherogenesis. Nature reviews 10, 53-62.9. Parmar, K.M. Larman, H.B. Dai, G., Zhang, Y., Wang, E.T. Moorthy, S.N. Kratz, J.R. Lin, Z., Jain, M.K. Gimbrone, M.A. Jr., et al. (2006). Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2. J Clin Invest 116, 49-58.10. Peralta, M., Steed, E., Harlepp, S., Gonzalez-Rosa, J.M. Monduc, F., Ariza-Cosano, A., Cortes, A., Rayon, T., Gomez-Skarmeta, J.L. Zapata, A., et al. (2013). Heartbeat-driven pericardiac fluid forces contribute to epicardium morphogenesis. Curr Biol 23, 1726-1735.11. Seidman, J.G. and Seidman, C. (2001). The genetic basis for cardiomyopathy: from mutation identification to mechanistic paradigms. Cell 104, 557-567.12. Thierfelder, L., Watkins, H., MacRae, C., Lamas, R., McKenna, W., Vosberg, H.P. Seidman, J.G. and Seidman, C.E. (1994). Alpha-tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell 77, 701-712.13. Vermot, J., Forouhar, A.S. Liebling, M., Wu, D., Plummer, D., Gharib, M., and Fraser, S.E. (2009). Reversing blood flows act through klf2a to ensure normal valvulogenesis in the developing heart. PLoS Biol 7, e1000246.