Kota Miyasaka

Haemodynamically dependent valvulogenesis of zebrafish heart is mediated by flow-dependent expression of miR-21

Heartbeat is required for normal development of the heart, and perturbation of intracardiac flow leads to morphological defects resembling congenital heart diseases. These observations implicate intracardiac haemodynamics in cardiogenesis, but the signalling cascades connecting physical forces, gene expression and morphogenesis are largely unknown. Here we use a zebrafish model to show that the microRNA, miR-21, is crucial for regulation of heart valve formation. Expression of miR-21 is rapidly switched on and off by blood flow. Vasoconstriction and increasing shear stress induce ectopic expression of miR-21 in the head vasculature and heart. Flow-dependent expression of mir-21 governs valvulogenesis by regulating the expression of the same targets as mouse/human miR-21 (sprouty, pdcd4, ptenb) and induces cell proliferation in the valve-forming endocardium at constrictions in the heart tube where shear stress is highest. We conclude that miR-21 is a central component of a flow-controlled mechanotransduction system in a physicogenetic regulatory loop.

Daam1 regulates the endocytosis of EphB during the convergent extension of the zebrafish notochord

Convergent extension (CE) movement of cells is one of the fundamental processes that control the organized morphogenesis of tissues and organs. The molecular events connecting the noncanonical Wnt pathway and CE movement, however, are not well understood. We show that subcellular localization of Daam1, an essential component of noncanonical Wnt signaling, changes dynamically during notochord formation. In the early phases, Daam1 complexes with EphB receptors and Disheveled 2. This complex is incorporated into endocytic vesicles in a dynamin-dependent manner, thereby resulting in the removal of EphB from the cell surface with subsequent switching of cell adhesiveness. In the next step, Daam1 colocalizes with the actin cytoskeleton to induce morphological extension of cells. We elucidate the molecular mechanism underlying the CE movement of notochord cells with Daam1 as a dynamic coordinator of endocytosis and cytoskeletal remodeling.

Csrp1 regulates dynamic cell movements of the mesendoderm and cardiac mesoderm through interactions with Dishevelled and Diversin

Zebrafish Csrp1 is a member of the cysteine- and glycine-rich protein (CSRP) family and is expressed in the mesendoderm and its derivatives. Csrp1 interacts with Dishevelled 2 (Dvl2) and Diversin (Div), which control cell morphology and other dynamic cell behaviors via the noncanonical Wnt and JNK pathways. When csrp1 message is knocked down, abnormal convergent extension cell movement is induced, resulting in severe deformities in midline structures. In addition, cardiac bifida is induced as a consequence of defects in cardiac mesoderm cell migration. Our data highlight Csrp1 as a key molecule of the noncanonical Wnt pathway, which orchestrates cell behaviors during dynamic morphogenetic movements of tissues and organs.

Heartbeat regulates cardiogenesis by suppressing retinoic acid signaling via expression of miR-143.

Cardiogenesis proceeds with concomitant changes in hemodynamics to accommodate the circulatory demands of developing organs and tissues. In adults, circulatory adaptation is critical for the homeostatic regulation of blood circulation. In these hemodynamics-dependent processes of morphogenesis and adaptation, a mechanotransduction pathway, which converts mechanical stimuli into biological outputs, plays an essential role, although its molecular nature is largely unknown. Here, we report that expression of zebrafish miR-143 is dependent on heartbeat. Knocking-down miR-143 results in de-repression of retinoic acid signaling, and produces abnormalities in the outflow tracts and ventricles. Our data uncover a novel epigenetic link between heartbeat and cardiac development, with miR-143 as an essential component of the mechanotransduction cascade.

Notch and Hippo signaling converge on Strawberry Notch 1 (Sbno1) to synergistically activate Cdx2 during specification of the trophectoderm

The first binary cell fate decision occurs at the morula stage and gives rise to two distinct types of cells that constitute the trophectoderm (TE) and inner cell mass (ICM). The cell fate determinant, Cdx2, is induced in TE cells and plays an essential role in their differentiation and maintenance. Notch and Hippo signaling cascades are assumed to converge onto regulatory elements of Cdx2, however, the underlying molecular mechanisms are largely unknown. Here, we show involvement of Strawberry Notch1 (Sbno1), a novel chromatin factor of the helicase superfamily 2, during preimplantation development. Sbno1 knockout embryos die at the preimplantation stage without forming a blastocoel, and Cdx2 is not turned on even though both Yap and Tead4 reside normally in nuclei. Accordingly, Sbno1 acts on the trophectoderm-enhancer (TEE) of Cdx2, ensuring its robust and synergistic activation by the Yap/Tead4 and NICD/Rbpj complexes. Interestingly, this synergism is enhanced when cells are mechanically stretched, which might reflect that TE cells are continuously stretched by the expanding ICM and blastocoel cavity. In addition, the histone chaperone, FACT (FAcilitates Chromatin Transcription) physically interacts with Sbno1. Our data provide new evidence on TE specification, highlighting unexpected but essential functions of the highly conserved chromatin factor, Sbno1.

Organ-explanted bionic simulator (OBiS): Concurrent microcardiovascular anastomosis of chick embryo

In this paper, we newly propose an organ-explanted bionic simulator (OBiS) using an isolated organ tissue. We use a heart isolated from the chick embryo for proposed simulator. To achieve the OBiS, we perform the concurrent microvascular anastomosis by using suction-induced vascular fixation (SVF) method for easily connection to artificial tubes with blood vessels led to the explanted chicks heart. Through the experiment with a simulated cardiovascular, it is confirmed that vessels can be aligned by using the proposed method. In addition, we have succeeded in connecting the Alg tubes with the blood vessels led to the heart of chick embryo. OBiS is available for the evaluation of new drugs immune response not only without using large animals but also as a response of organs beyond cellular level.

ALL-in-one microfluidic device for microvascular connection

We newly propose a microfluidic device with multiple functions for simple microvascular connection. By using this microfluidic device with suction mechanism, we can operate the blood vessels in a single device for assisting the microvascular connection procedure such as fixing the position of the blood vessels, expanding the diameter of the blood vessels, and pouring the adhesion and drugs. We have succeeded in connecting the microcardiovascular of the extracted chicken embryonic heart to artificial mictotubes in a shorter time than the manual operation such as suturing. We have also succeeded in constructing the biological simulator that can observe a living organ continuously while circulating the culture medium through the inside of the heart. Finally, we showed the prototype platform of the evaluating the migration of the cells at the connection part between the extracted tissue from living organism and the assembled tissue structure.

Aligning collagen fibers by cyclic mechanical stretch for efficiently muscle cell actuator

Muscle cell actuators are composed of skeletal muscle cells and collagen intercellular matrix. Muscle cells adhere to collagen fibers and contraction along with the direction of collagen fiber. Collagen fibers in a muscle cell actuator transmit contraction force of each muscle cells along longitudinal direction. In order to improve contraction force of muscle cell actuators, it is very important to align collagen fibers. In this study, we aim to achieve alignment of collagen fibers in the actuator. We developed the automatic culture system which apply cyclic stretch to muscle cell actuators. We estimated the structure the actuator by using a scanning electron microscope and fluorescent observation. And we confirmed that cyclic stretch induced alignment of collagen fibers in the muscle cell actuators.

Remodeling Muscle Cells by Inducing Mechanical Stimulus

A muscle cell actuator has been attracting a lot of attention since it is a key technology for realizing bio-machine hybrid systems. This study especially intend to deal with micro robot driven by real muscle cell actuators. To fabricate the actuator, we should study how to control cell aggregation for efficient power generation. This paper proposes a method for remodeling muscle cells by exploiting mechanical stimulus so that we can get an appropriate structure of the actuator. The experimental results demonstrate that the three factors, cell density, cell-matrix adhesion, and mechanical stimulation period, largely contribute to the remodeling of the muscle cells.

Simple and rapid connection of chicken embryonic cardiovascular system

We propose a simple and rapid microcardiovascular connection method called suction-induced vascular fixation (SVF) method for the achievement of bionic simulator with using chicken embryonic heart. The advantages of proposed method with using a microfluidic device are as follows: (1) operation of flexible objects (blood vessels), (2) alignment the blood vessels concurrently, and (3) reduction the DOF of the blood vessels. From the experimental results, we confirmed that four cardiovascular can be induced into the fabricated device concurrently. Furthermore, we have succeeded in connecting to a heart of chick embryo with microtubes. We have also succeeded in construction of hybrid circulatory system between artificial tubes and blood vessels led to the heart of chick embryo.