Final Report Summary - VASCULOFLOW (Understanding the dynamics of vasculogenesis from a biomechanical perspective)
The 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 cardiovascular development. The mechanisms involved in vascular morphogenesis are poorly understood. One of the fundamental questions is whether vascular morphogenesis depends solely on genetic pre-programming or whether additional environmental cues provided by blood flow are needed. During the time-course of that fellowship, we first dissected early embryonic hemodynamics at blood flow onset and investigated the mechano-detection mechanisms at play in the developing endothelium. We show that the first heartbeats progressively increase blood flows velocities in the developing vasculature. At that stage, the endothelium is composed of protruding cilia, which display a non-conventional inner architecture. Cilia are progressively deflected by flow at the onset of blood circulation and regulate the calcium influx through the cilia-linked calcium-permeable channel pkd2. Alteration of either blood flow profiles or pkd2 expression impairs endothelial calcium influx and perturbs the subsequent angiogenesis. These results demonstrate that blood flow onset is mechanically sensed by cilia whose deflection finely tunes the resulting calcium influx and regulate endothelial homeostasis.
Characterisation of the early embryonic cardiac and vascular hemodynamics
Although the role of hemodynamic forces is thus much appreciated, we know very little about the forces generated in the developing embryo at the onset of blood flow. Moreover, it remains unknown whether and how the hemodynamic forces imposed on the endothelial wall at this developmental stage are detected and transduced in the developing endothelium. Here, we took advantages of the optical clarity and external development of the zebrafish (Danio Rerio) embryo and challenged early cardiac morphogenic events and hemodynamics quantification using high temporal resolution three-dimensional (3D) imaging and sophisticated image processing. Our observations show that the first heartbeats progressively increase blood flow velocities in the developing vasculature. Using the central region of the dorsal aorta (DA) as a reference region of the zebrafish developing vasculature, we observed that very low flow velocities accompany the onset of heart contractions (24 hours post-fertilisation (hpf)) (about 50 - 100 µm*s-1). Progressively, from 26 to 28 hpf, heart contraction gain in efficacy and generate increasing blood flow velocities in the DA ranging from about 300 to 600 µm*s-1
Early embryonic vessels are ciliated
We next investigated whether early embryonic vasculature had developed mechanodetection mechanisms allowing flow sensing during this critical transition from a very low flow situation to (high shear stress values). Because primary 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 hypothesised that they could be the mechanodetector operating at blood flow onset. We thus screened early embryonic vasculature for primary cilia and observed that most of the endothelial cells displayed a protruding and deflected cilia in the vessel lumen. Using correlative light and electron microscopy (CLEM) allowing us to simultaneously image a single cilia at high temporal and spatial resolution and, thereafter, to study the same cilia at the ultrastructural level, we were able to track several cilia of interest in a 28-hpf old embryo. We observed that endothelial cilia in early embryos display non-conventional microtubule architecture in the axonemal region, whereas their basal body had the conventional 9-fold symmetry composition of microtubule triplets. While pronephric motile cilia displayed the conventional 9+2 microtubule doublets in their axoneme, this non-conventional microtubule content was observed in both venous and arterial endothelial cilia.
Blood flow onset is mechanically detected and transduced by primary cilia
Using a combination of in vivo high temporal resolution imaging and image processing of single cilia double-transgenic embryos (Tg (actin:Arl13b-egfp) Tg(flk1:mCherry)), we next investigated whether endothelial cilia sense blood flow in the developing vasculature. We observed that pulsatile flow correlated with oscillatory cilia deflection in the main artery of the embryo and that the highest cilia deflection correlated with the highest flow velocity measured, suggesting that endothelial cilia behave as fine flow sensors. At stages where blood flow is not sufficient to deflect cilia, the latter remain perpendicular to the vessel wall. Because cilia deflection had been shown to increase calcium signalling in vitro in kidney epithelial cells and endothelial cells, we next investigated whether endothelial cilia deflection lead to increased calcium signalling during blood flow onset using in vivo imaging. Using the endothelial-specific expression of a genetically encoded calcium probe (Gcamp 3.0) we observed a progressive increase in calcium content in the DA from 24 to 28 hpf, reliable to the progressive flow-mediated cilia deflection. Altogether, this result suggests that endothelial cilia are potent flow sensors capable of transducing the flow velocity into calcium signalling in the developing vasculature.
Primary cilia sense subtle blood flow perturbation and control embryonic angiogenesis
We next reasoned that if endothelial cilia are capable of detecting subtle flow variations at the onset of blood flow, blocking or impairing blood flow should be sensed by cilia and lead to impaired endothelial homeostasis. In mutant embryos that lack heart contraction but are able to survive from oxygen diffusion (Silent-heart) endothelial cilia are not deflected and display a passive and stochastic movement. Consequently, we observed that this 'no flow' situation results in decreased calcium content in the developing endothelium, which leads to impaired angiogenesis. Cilia-mediated calcium influx had been linked to the gating of the calcium-permeable channel pkd2/PC2/TRPP2 in several ciliated structures such as the left-right organiser, kidney epithelial cells and endothelial cells. We found that depletion of pkd2 impaired calcium influx without affecting cilia deflection and drastically hampered angiogenesis, phenocopying silent-heart embryos.
In the present study, we provide for the first time in vivo evidence that mild but increasing hemodynamic forces generated at blood flow onset are detected and transduced to the endothelium by protruding primary cilia. This confirms that the vasculature has developed highly sensitive mechanodetection mechanisms which allow a fine sensing of forces during embryonic development. Consistently, hair-like glycocalyx was recently shown to be present in the early vasculature of the quail embryo and capable of detecting forces carried by bloow flow at its onset13. Altogether, these data show that:
(1) the early embryonic vasculature of the zebrafish is ciliated;
(2) endothelial cilia of zebrafish display a non-conventional architecture characterised by fewer microtubules;
(3) blood flow onset progressively deflect endothelial cilia;
(4) cilia are fine mechanosensors able to detect subtle variations in flow profiles;
(5) flow-mediated cilia deflection allows a pkd2-dependent calcium influx in the endothelium and concomitant normal vessel homeostasis.
Characterisation of the early embryonic cardiac and vascular hemodynamics
Although the role of hemodynamic forces is thus much appreciated, we know very little about the forces generated in the developing embryo at the onset of blood flow. Moreover, it remains unknown whether and how the hemodynamic forces imposed on the endothelial wall at this developmental stage are detected and transduced in the developing endothelium. Here, we took advantages of the optical clarity and external development of the zebrafish (Danio Rerio) embryo and challenged early cardiac morphogenic events and hemodynamics quantification using high temporal resolution three-dimensional (3D) imaging and sophisticated image processing. Our observations show that the first heartbeats progressively increase blood flow velocities in the developing vasculature. Using the central region of the dorsal aorta (DA) as a reference region of the zebrafish developing vasculature, we observed that very low flow velocities accompany the onset of heart contractions (24 hours post-fertilisation (hpf)) (about 50 - 100 µm*s-1). Progressively, from 26 to 28 hpf, heart contraction gain in efficacy and generate increasing blood flow velocities in the DA ranging from about 300 to 600 µm*s-1
Early embryonic vessels are ciliated
We next investigated whether early embryonic vasculature had developed mechanodetection mechanisms allowing flow sensing during this critical transition from a very low flow situation to (high shear stress values). Because primary 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 hypothesised that they could be the mechanodetector operating at blood flow onset. We thus screened early embryonic vasculature for primary cilia and observed that most of the endothelial cells displayed a protruding and deflected cilia in the vessel lumen. Using correlative light and electron microscopy (CLEM) allowing us to simultaneously image a single cilia at high temporal and spatial resolution and, thereafter, to study the same cilia at the ultrastructural level, we were able to track several cilia of interest in a 28-hpf old embryo. We observed that endothelial cilia in early embryos display non-conventional microtubule architecture in the axonemal region, whereas their basal body had the conventional 9-fold symmetry composition of microtubule triplets. While pronephric motile cilia displayed the conventional 9+2 microtubule doublets in their axoneme, this non-conventional microtubule content was observed in both venous and arterial endothelial cilia.
Blood flow onset is mechanically detected and transduced by primary cilia
Using a combination of in vivo high temporal resolution imaging and image processing of single cilia double-transgenic embryos (Tg (actin:Arl13b-egfp) Tg(flk1:mCherry)), we next investigated whether endothelial cilia sense blood flow in the developing vasculature. We observed that pulsatile flow correlated with oscillatory cilia deflection in the main artery of the embryo and that the highest cilia deflection correlated with the highest flow velocity measured, suggesting that endothelial cilia behave as fine flow sensors. At stages where blood flow is not sufficient to deflect cilia, the latter remain perpendicular to the vessel wall. Because cilia deflection had been shown to increase calcium signalling in vitro in kidney epithelial cells and endothelial cells, we next investigated whether endothelial cilia deflection lead to increased calcium signalling during blood flow onset using in vivo imaging. Using the endothelial-specific expression of a genetically encoded calcium probe (Gcamp 3.0) we observed a progressive increase in calcium content in the DA from 24 to 28 hpf, reliable to the progressive flow-mediated cilia deflection. Altogether, this result suggests that endothelial cilia are potent flow sensors capable of transducing the flow velocity into calcium signalling in the developing vasculature.
Primary cilia sense subtle blood flow perturbation and control embryonic angiogenesis
We next reasoned that if endothelial cilia are capable of detecting subtle flow variations at the onset of blood flow, blocking or impairing blood flow should be sensed by cilia and lead to impaired endothelial homeostasis. In mutant embryos that lack heart contraction but are able to survive from oxygen diffusion (Silent-heart) endothelial cilia are not deflected and display a passive and stochastic movement. Consequently, we observed that this 'no flow' situation results in decreased calcium content in the developing endothelium, which leads to impaired angiogenesis. Cilia-mediated calcium influx had been linked to the gating of the calcium-permeable channel pkd2/PC2/TRPP2 in several ciliated structures such as the left-right organiser, kidney epithelial cells and endothelial cells. We found that depletion of pkd2 impaired calcium influx without affecting cilia deflection and drastically hampered angiogenesis, phenocopying silent-heart embryos.
In the present study, we provide for the first time in vivo evidence that mild but increasing hemodynamic forces generated at blood flow onset are detected and transduced to the endothelium by protruding primary cilia. This confirms that the vasculature has developed highly sensitive mechanodetection mechanisms which allow a fine sensing of forces during embryonic development. Consistently, hair-like glycocalyx was recently shown to be present in the early vasculature of the quail embryo and capable of detecting forces carried by bloow flow at its onset13. Altogether, these data show that:
(1) the early embryonic vasculature of the zebrafish is ciliated;
(2) endothelial cilia of zebrafish display a non-conventional architecture characterised by fewer microtubules;
(3) blood flow onset progressively deflect endothelial cilia;
(4) cilia are fine mechanosensors able to detect subtle variations in flow profiles;
(5) flow-mediated cilia deflection allows a pkd2-dependent calcium influx in the endothelium and concomitant normal vessel homeostasis.