Katsumi Ando

Development of Proximal Coronary Arteries in Quail Embryonic Heart. Multiple Capillaries Penetrating the Aortic Sinus Fuse to Form Main Coronary Trunk

Abstract— Studies have shown that the proximal coronary artery (PCA) develops via endothelial ingrowth from the peritruncal ring (PR) of the coronary vasculature. However, the details of PCA formation remain unclear. We examined the development of PCAs in quail embryonic hearts from 5 to 9 days of incubation (embryonic day [ED]) using double-immunostaining for QH1 (quail endothelial marker) and smooth muscle &agr;-actin. At 6 to 7 ED, several QH1-positive endothelial strands from the PR penetrated the facing sinuses, and in some embryos, several endothelial strands penetrated the posterior (noncoronary) sinus. At 7 to 8 ED, the endothelial strands penetrating the facing sinuses seemed to fuse, forming a proximal coronary stem that was demarcated from the aortic wall by the nascent smooth muscle layer of the coronary artery. By 9 ED, two coronary stems were completely formed, and the endothelial strands previously penetrating the noncoronary sinus had disappeared. Confocal microscopy at 6 ED revealed discontinuous QH1-positive endothelial progenitors in the aortic wall at sites where the endothelial strands would later develop. Observations demonstrate that during the formation of the PCA, endothelial strands from the PR penetrate the facing sinuses and then fuse, whereas those strands penetrating the noncoronary sinus disappear. Thereafter, the coronary artery tunica media demarcates the definitive PCA from the aortic media.

Understanding heart development and congenital heart defects through developmental biology: A segmental approach

ABSTRACT  The heart is the first organ to form and function during development. In the pregastrula chick embryo, cells contributing to the heart are found in the postero‐lateral epiblast. During the pregastrula stages, interaction between the posterior epiblast and hypoblast is required for the anterior lateral plate mesoderm (ALM) to form, from which the heart will later develop. This tissue interaction is replaced by an Activin‐like signal in culture. During gastrulation, the ALM is committed to the heart lineage by endoderm‐secreted BMP and subsequently differentiates into cardiomyocyte. The right and left precardiac mesoderms migrate toward the ventral midline to form the beating primitive heart tube. Then, the heart tube generates a right‐side bend, and the d‐loop and presumptive heart segments begin to appear segmentally: outflow tract (OT), right ventricle, left ventricle, atrioventricular (AV) canal, atrium and sinus venosus. T‐box transcription factors are involved in the formation of the heart segments: Tbx5 identifies the left ventricle and Tbx20 the right ventricle. After the formation of the heart segments, endothelial cells in the OT and AV regions transform into mesenchyme and generate valvuloseptal endocardial cushion tissue. This phenomenon is called endocardial EMT (epithelial‐mesenchymal transformation) and is regulated mainly by BMP and TGFβ. Finally, heart septa that have developed in the OT, ventricle, AV canal and atrium come into alignment and fuse, resulting in the completion of the four‐chambered heart. Altered development seen in the cardiogenetic process is involved in the pathogenesis of congenital heart defects. Therefore, understanding the molecular nature regulating the ‘nodal point’ during heart development is important in order to understand the etiology of congenital heart defects, as well as normal heart development.

Expression of tbx20 RNA during chick heart development

The T‐box gene family encodes a set of transcription factors that are involved in various developmental processes. We isolated tbx20 gene from chick embryos and examined in detail its expression patterns during heart development. In situ hybridization showed that tbx20 was expressed in the lateral plate mesoderm and subsequently in the primitive heart tube. At stages of looped heart, tbx20 was localized in the outflow tract (OT) and atrioventricular (AV) canal, in which valvuloseptal endocardial cushion develops. At later stages, although tbx20 was expressed predominantly in the nascent right ventricle, transcripts of tbx20 were down‐regulated in the left ventricle. These results suggest that tbx20 may play important roles in a variety of developmental processes in cardiogenesis, such as chamber‐specification and septation. Developmental Dynamics 230:576–580, 2004.

Roles of TGFβ and BMP during valvulo–septal endocardial cushion formation

The primordia of valves and the atrioventricular septum arise from endocardial cushion tissue that is formed in the outflow tract (OFT) and in the atrioventricular (AV) regions during cardiogenesis. Abnormal development of the endocardial cushion results in various congenital heart diseases. Endocardial epithelial–mesenchymal transformation (EMT) is a critical process in cushion tissue formation and is regulated by many factors, such as growth factors, intercellular signaling molecules, transcription factors, and extracellular matrices. A signal that is produced by the myocardium of the AV and OFT regions and transferred to the adjacent endocardium across the extracellular matrix mediates EMT. Studies in vitro and genetic analyses have shown that transforming growth factor β and bone morphogenetic protein play central roles in the regulation of EMT during cushion tissue formation.

Expression of bone morphogenetic protein-5 gene during chick heart development: possible roles in valvuloseptal endocardial cushion formation.

The bone morphogenetic protein (BMP) family, comprising multifunctional peptide growth factors, regulates many developmental processes in a variety of tissues. We examined the spatiotemporal expression of BMP5 by in situ hybridization in chick embryonic hearts from stages 5 to 33. The BMP5 gene was first expressed in the endoderm underlying the precardiac mesoderm at stages 5 to 8. Thereafter, BMP5 expression was restricted to the myocardium of the atrioventricular (AV) canal and outflow tract (OT) regions, where the valvuloseptal endocardial cushion tissue is induced. These results suggest that BMP5 may play important roles not only in myocardial differentiation, but also in the formation and maintenance of endocardial cushion tissue. Anat Rec 264:313–316, 2001.

Tenascin C may regulate the recruitment of smooth muscle cells during coronary artery development

Tenascin C (TNC) is an extracellular glycoprotein that is thought to be involved in tissue remodeling during organogenesis and regeneration. Using avian embryonic hearts, we investigated the spatiotemporal expression patterns of TNC during the formation of the proximal coronary artery. Immunohistochemistry showed that TNC was deposited around the developing coronary stem and that TNC colocalized with vascular smooth muscle α-actin. A quail-chick chimera, in which a quail proepicardial organ (PEO) had been transplanted, showed that quail tissue-derived cells contributed to the establishment of the endothelial and mural cells of the proximal coronary artery, and the quail tissue-derived mural cells displayed TNC. Proepicardial cells cultured in TNC showed the myofibroblast/smooth muscle cell phenotype and neutralizing anti-TNC antibody suppressed the expression of smooth muscle markers. These observations suggest that TNC plays a role in the mural smooth muscle development of the nascent proximal coronary artery.

Expression of the Tgfβ2 Gene During Chick Embryogenesis

We performed a comprehensive analysis of the expression of transforming growth factor (TGF) β2 during chick embryogenesis from stage 6 to 30 (Hamburger and Hamilton, J Morphol 1951;88:49–92) using in situ hybridization. During cardiogenesis, Tgfβ2 was expressed in the endothelial/mesenchymal cells of the valvulo‐septal endocardial cushion tissue and in the epicardium until the end of embryogenesis. During the formation of major arteries, Tgfβ2 was localized in smooth muscle progenitors but not in the vascular endothelium. During limb development, Tgfβ2 was expressed in the mesenchymal cells in the presumptive limb regions at stage 16, and thereafter it was localized in the skeletal muscle progenitors. In addition, strong Tgfβ2 expression was seen in the mesenchymal cells in the pharyngeal arches. Tgfβ2 mRNA was also detected in other mesoderm‐derived tissues, such as the developing bone and pleura. During ectoderm development, Tgfβ2 was expressed in the floor plate of the neural tube, lens, optic nerve, and otic vesicle. In addition, Tgfβ2 was expressed in the developing gut epithelium. Our results suggest that TGFβ2 plays an important role not only in epithelial‐mesenchymal interactions but also in cell differentiation and migration and cell death during chick embryogenesis. We also found that chick and mouse Tgfβ2 RNA show very similar patterns of expression during embryogenesis. Chick embryos can serve as a useful model to increase our understanding in the roles of TGFβ2 in cell–cell interactions, cell differentiation, and proliferation during organogenesis. Anat Rec, 2012.

Heart Myofibrillogenesis Occurs in Isolated Chick Posterior Blastoderm: A Culture Model

Early cardiogenesis including myofibrillogenesis is a critical event during development. Recently we showed that prospective cardiomyocytes reside in the posterior lateral blastoderm in the chick embryo. Here we cultured the posterior region of the chick blastoderm in serum-free medium and observed the process of myofibrillogenesis by immunohistochemistry. After 48 hours, explants expressed sarcomeric proteins (sarcomeric α-actinin, 61%; smooth muscle α-actin, 95%; Z-line titin, 56%; sarcomeric myosin, 48%); however, they did not yet show a mature striation. After 72 hours, more than 92% of explants expressed I-Z-I proteins, which were incorporated into the striation in 75% of explants or more (sarcomeric α-actinin, 75%; smooth muscle α-actin, 81%; Z-line titin, 83%). Sarcomeric myosin was expressed in 63% of explants and incorporated into A-bands in 37%. The percentage incidence of expression or striation of I-Z-I proteins was significantly higher than that of sarcomeric myosin. Results suggested that the nascent I-Z-I components appeared to be generated independently of A-bands in the cultured posterior blastoderm, and that the process of myofibrillogenesis observed in our culture model faithfully reflected that in vivo. Our blastoderm culture model appeared to be useful to investigate the mechanisms regulating the early cardiogenesis.