Jiahong Raymond Zhang, Julien Vermot, Riccardo Gherardi, Hajime Fukui and Wei-Yan Renee Chow : Calcium Signal Analysis in the Zebrafish Heart via Phase Matching of the Cardiac Cycle., Bio-protocol, Vol.14, No.10, 2024.
(Summary)
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.
Hiroyuki Nakajima, Hiroyuki Ishikawa, Takuya Yamamoto, Ayano Chiba, Hajime Fukui, Keisuke Sako, Moe Fukumoto, Kenny Mattonet, Hyouk-Bum Kwon, P Subhra Hui, D Gergana Dobreva, Kazu Kikuchi, M Christian S Helker, R Didier Y Stainier and Naoki Mochizuki : Endoderm-derived islet1-expressing cells differentiate into endothelial cells to function as the vascular HSPC niche in zebrafish., Developmental Cell, Vol.58, No.3, 224-238.e7, 2023.
(Summary)
Endothelial cells (ECs) line blood vessels and serve as a niche for hematopoietic stem and progenitor cells (HSPCs). Recent data point to tissue-specific EC specialization as well as heterogeneity; however, it remains unclear how ECs acquire these properties. Here, by combining live-imaging-based lineage-tracing and single-cell transcriptomics in zebrafish embryos, we identify an unexpected origin for part of the vascular HSPC niche. We find that islet1 (isl1)-expressing cells are the progenitors of the venous ECs that constitute the majority of the HSPC niche. These isl1-expressing cells surprisingly originate from the endoderm and differentiate into ECs in a process dependent on Bmp-Smad signaling and subsequently requiring npas4l (cloche) function. Single-cell RNA sequencing analyses show that isl1-derived ECs express a set of genes that reflect their distinct origin. This study demonstrates that endothelial specialization in the HSPC niche is determined at least in part by the origin of the ECs.
Hélène Vignes, Christina Vagena-Pantoula, Mangal Prakash, Hajime Fukui, Caren Norden, Naoki Mochizuki, Florian Jug and Julien Vermot : Extracellular mechanical forces drive endocardial cell volume decrease during zebrafish cardiac valve morphogenesis., Developmental Cell, Vol.57, No.5, 598-609.e5, 2022.
(Summary)
Organ morphogenesis involves dynamic changes of tissue properties while cells adapt to their mechanical environment through mechanosensitive pathways. How mechanical cues influence cell behaviors during morphogenesis remains unclear. Here, we studied the formation of the zebrafish atrioventricular canal (AVC) where cardiac valves develop. We show that the AVC forms within a zone of tissue convergence associated with the increased activation of the actomyosin meshwork and cell-orientation changes. We demonstrate that tissue convergence occurs with a reduction of cell volume triggered by mechanical forces and the mechanosensitive channel TRPP2/TRPV4. Finally, we show that the extracellular matrix component hyaluronic acid controls cell volume changes. Together, our data suggest that multiple force-sensitive signaling pathways converge to modulate cell volume. We conclude that cell volume reduction is a key cellular feature activated by mechanotransduction during cardiovascular morphogenesis. This work further identifies how mechanical forces and extracellular matrix influence tissue remodeling in developing organs.
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.
Hajime Fukui, Wei-Yan Renee Chow, Jing Xie, Yin Yoke Foo, Hwai Choon Yap, Nicolas Minc, Naoki Mochizuki and Julien Vermot : Bioelectric signaling and the control of cardiac cell identity in response to mechanical forces., Science, Vol.374, No.6565, 351-354, 2021.
(Summary)
) pulses and nuclear factor of activated T cells 1 (Nfatc1) activation. Thus, mechanical forces are converted into discrete bioelectric signals by an ATP-Ca
R Rita Ferreira, Guillaume Pakula, Lhéanna Klaeyle, Hajime Fukui, Andrej Vilfan, Willy Supatto and Julien Vermot : Chiral Cilia Orientation in the Left-Right Organizer., Cell Reports, Vol.25, No.8, 2008-2016.e4, 2018.
(Summary)
Chirality is a property of asymmetry between an object and its mirror image. Most biomolecules and many cell types are chiral. In the left-right organizer (LRO), cilia-driven flows transfer such chirality to the body scale. However, the existence of cellular chirality within tissues remains unknown. Here, we investigate this question in Kupffer's vesicle (KV), the zebrafish LRO. Quantitative live imaging reveals that cilia populating the KV display asymmetric orientation between the right and left sides, resulting in a chiral structure, which is different from the chiral cilia rotation. This KV chirality establishment is dynamic and depends on planar cell polarity. While its impact on left-right (LR) symmetry breaking remains unclear, we show that this asymmetry does not depend on the LR signaling pathway or flow. This work identifies a different type of tissue asymmetry and sheds light on chirality genesis in developing tissues.
Hajime Fukui, Takahiro Miyazaki, Wei-Yan Renee Chow, Hiroyuki Ishikawa, Hiroyuki Nakajima, Julien Vermot and Naoki Mochizuki : Hippo signaling determines the number of venous pole cells that originate from the anterior lateral plate mesoderm in zebrafish., eLife, Vol.7, 2018.
(Summary)
, a key transcription factor that is involved in the differentiation of atrial cardiomyocytes. Collectively, these results demonstrate that Hippo signaling defines venous pole cardiomyocyte number by modulating both the number and the identity of the ALPM cells that will populate the atrium of the heart.
Koji Ando, Shigetomo Fukuhara, Nanae Izumi, Hiroyuki Nakajima, Hajime Fukui, N Robert Kelsh and Naoki Mochizuki : Clarification of mural cell coverage of vascular endothelial cells by live imaging of zebrafish., Development, Vol.143, No.8, 1328-1339, 2016.
(Summary)
Mural cells (MCs) consisting of vascular smooth muscle cells and pericytes cover the endothelial cells (ECs) to regulate vascular stability and homeostasis. Here, we clarified the mechanism by which MCs develop and cover ECs by generating transgenic zebrafish lines that allow live imaging of MCs and by lineage tracing in vivo To cover cranial vessels, MCs derived from either neural crest cells or mesoderm emerged around the preformed EC tubes, proliferated and migrated along EC tubes. During their migration, the MCs moved forward by extending their processes along the inter-EC junctions, suggesting a role for inter-EC junctions as a scaffold for MC migration. In the trunk vasculature, MCs derived from mesoderm covered the ventral side of the dorsal aorta (DA), but not the posterior cardinal vein. Furthermore, the MCs migrating from the DA or emerging around intersegmental vessels (ISVs) preferentially covered arterial ISVs rather than venous ISVs, indicating that MCs mostly cover arteries during vascular development. Thus, live imaging and lineage tracing enabled us to clarify precisely how MCs cover the EC tubes and to identify the origins of MCs.
Jun-Dal Kim, Kyung-Eui Park, Junji Ishida, Koichiro Kako, Juri Hamada, Shuichi Kani, Miki Takeuchi, Kana Namiki, Hajime Fukui, Shigetomo Fukuhara, Masahiko Hibi, Makoto Kobayashi, Yasunori Kanaho, Yoshitoshi Kasuya, Naoki Mochizuki and Akiyoshi Fukamizu : PRMT8 as a phospholipase regulates Purkinje cell dendritic arborization and motor coordination., Science Advances, Vol.1, No.11, 2015.
(Summary)
The development of vertebrate neurons requires a change in membrane phosphatidylcholine (PC) metabolism. Although PC hydrolysis is essential for enhanced axonal outgrowth mediated by phospholipase D (PLD), less is known about the determinants of PC metabolism on dendritic arborization. We show that protein arginine methyltransferase 8 (PRMT8) acts as a phospholipase that directly hydrolyzes PC, generating choline and phosphatidic acid. We found that PRMT8 knockout mice (prmt8 (-/-)) displayed abnormal motor behaviors, including hindlimb clasping and hyperactivity. Moreover, prmt8 (-/-) mice and TALEN-induced zebrafish prmt8 mutants and morphants showed abnormal phenotypes, including the development of dendritic trees in Purkinje cells and altered cerebellar structure. Choline and acetylcholine levels were significantly decreased, whereas PC levels were increased, in the cerebellum of prmt8 (-/-) mice. Our findings suggest that PRMT8 acts both as an arginine methyltransferase and as a PC-hydrolyzing PLD that is essential for proper neurological functions.
Hajime Fukui, Dai Shiba, Kazuhide Asakawa, Koichi Kawakami and Takahiko Yokoyama : The ciliary protein Nek8/Nphp9 acts downstream of Inv/Nphp2 during pronephros morphogenesis and left-right establishment in zebrafish., FEBS Letters, Vol.586, No.16, 2273-2279, 2012.
(Summary)
Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease. Among 12 reported Nphp gene products, Inv/Nphp2, Nphp3 and Nek8/Nphp9 are localized to the proximal segment in the primary cilium. However, the functional relationships are unknown. This study focused on phenotype analysis of nek8 knockdown embryos and the genetic relationship between nek8 and inv in zebrafish. Knockdown of nek8 produced both pronephric cysts and abnormal cardiac looping. Simultaneous knockdown of nek8 and inv synergistically increased the incidence of these defects. Interestingly, nek8 mRNA rescued inv morphant phenotypes, although inv mRNA could not rescue nek8 morphant phenotypes. These results suggest that Nek8 acts downstream of Inv function.
Hajime Fukui, Ryuki Hanaoka and Atsuo Kawahara : Noncanonical activity of seryl-tRNA synthetase is involved in vascular development., Circulation Research, Vol.104, No.11, 1253-1259, 2009.
(Summary)
Vascular endothelial growth factor (Vegf) plays central roles in the establishment of stereotypic vascular patterning in vertebrates. However, it is not fully understood how the network of blood vessels is established and maintained during vascular development. A zebrafish ko095 mutant presented the disorganized vessels with abnormal branching of the established intersegmental vessels (ISVs) after 60 hours postfertilization. The gene responsible for ko095 encodes seryl-tRNA synthetase (Sars) with a nonsense mutation. The abnormal branching of ISVs in ko095 mutant was suppressed by the introduction of either wild-type Sars or a mutant Sars (T429A) lacking the enzymatic activity that catalyzes aminoacylation of transfer RNA for serine (canonical activity), suggesting that the abnormal branching is attributable to the loss of function of Sars besides its canonical activity. We further found the increased expression of vegfa in ko095 mutant at 72 hours postfertilization, which was also reversed by the introduction of Sars (T429A). Furthermore, the abnormal branching of ISVs in the mutant was suppressed by knockdown of vegfa or vegfr2 (kdra and kdrb). Knockdown of vegfc or vegfr3 rescued the abnormal ISV branching in ko095 mutant. These results suggest that the abnormal ISV branching in ko095 mutant is caused by the activated Vegfa-Vegfr2 signal and requires the Vegfc-Vegfr3 signal, because the latter is needed for general angiogenesis. Hence, we conclude that noncanonical activity of Sars is involved in vascular development presumably by modulating the expression of vegfa.
Atsuo Kawahara, Tsuyoshi Nishi, Yu Hisano, Hajime Fukui, Akihito Yamaguchi and Naoki Mochizuki : The sphingolipid transporter spns2 functions in migration of zebrafish myocardial precursors., Science, Vol.323, No.5913, 524-527, 2008.
(Summary)
Sphingosine-1-phosphate (S1P) is a secreted lipid mediator that functions in vascular development; however, it remains unclear how S1P secretion is regulated during embryogenesis. We identified a zebrafish mutant, ko157, that displays cardia bifida (two hearts) resembling that in the S1P receptor-2 mutant. A migration defect of myocardial precursors in the ko157 mutant is due to a mutation in a multipass transmembrane protein, Spns2, and can be rescued by S1P injection. We show that the export of S1P from cells requires Spns2. spns2 is expressed in the extraembryonic tissue yolk syncytial layer (YSL), and the introduction of spns2 mRNA in the YSL restored the cardiac defect in the ko157 mutant. Thus, Spns2 in the YSL functions as a S1P transporter in S1P secretion, thereby regulating myocardial precursor migration.
Rita Ferreira R, Hajime Fukui, Renee Chow, Andrej Vilfan and Julien Vermot : The cilium as a force sensor-myth versus reality., Journal of Cell Science, Vol.132, No.14, Jul. 2019.
(Summary)
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.
Hajime Fukui, Ayano Chiba, Takahiro Miyazaki, Haruko Takano, Hiroyuki Ishikawa, Toyonori Omori and Naoki Mochiuzki : Spatial Allocation and Specification of Cardiomyocytes during Zebrafish Embryogenesis., Korean Circulation Journal, Vol.47, No.2, 160-167, Mar. 2017.
(Summary)
Incomplete development and severe malformation of the heart result in miscarriage of embryos because of its malfunction as a pump for circulation. During cardiogenesis, development of the heart is precisely coordinated by the genetically-primed program that is revealed by the sequential expression of transcription factors. It is important to investigate how spatial allocation of the heart containing cardiomyocytes and other mesoderm-derived cells is determined. In addition, the molecular mechanism underlying cardiomyocyte differentiation still remains elusive. The location of ectoderm-, mesoderm-, and endoderm-derived organs is determined by their initial allocation and subsequent mutual cell-cell interactions or paracrine-based regulation. In the present work, we provide an overview of cardiac development controlled by the germ layers and discuss the points that should be uncovered in future for understanding cardiogenesis.
Shigetomo Fukuhara, Hajime Fukui, Yuki Wakayama, Koji Ando, Hiroyuki Nakajima and Naoki Mochizuki : Looking back and moving forward: recent advances in understanding of cardiovascular development by imaging of zebrafish., Development Growth & Differentiation, Vol.57, No.4, 333-340, Apr. 2015.
(Summary)
Development requires cell proliferation, migration, differentiation, apoptosis, and many kinds of cell responses. Cells prepare intracellular conditions to respond to extracellular cues from neighboring cells. We have studied the development of the cardiovascular system (CVS) by visualizing morphology and signaling simultaneously using zebrafish, which express probes for both. Endodermal sheet is required for the bilateral cardiac precursor cell (CPC) migration toward the midline. Endothelial cells (ECs) proliferate specifically in the certain regions of blood vessels. Bone morphogenetic proteins (BMP) induce the remodeling of the caudal vein plexus (CVP) to form the caudal vein (CV). Our findings point to the pre-existing neighboring cells as the cells exhibiting certain responses during the development of CVS. In this review, we introduce recent results of our research on angiogenesis and cardiogenesis by spotlighting the mechanism by which ECs and CPCs are regulated by the cells next to themselves. In addition, we discuss the unanswered questions that should be clarified in the future in the field of CVS development.
(Keyword)
Animals / Cardiovascular System / Cell Movement / Cell Proliferation / Transcriptional Activation / Zebrafish