Yuji Nakajima

Mechanisms involved in valvuloseptal endocardial cushion formation in early cardiogenesis: Roles of transforming growth factor (TGF)-β and bone morphogenetic protein (BMP)

Endothelial‐mesenchymal transformation (EMT) is a critical event in the generation of the endocardial cushion, the primordia of the valves and septa of the adult heart. This embryonic phenomenon occurs in the outflow tract (OT) and atrioventricular (AV) canal of the embryonic heart in a spatiotemporally restricted manner, and is initiated by putative myocardially derived inductive signals (adherons) which are transferred to the endocardium across the cardiac jelly. Abnormal development of endocardial cushion tissue is linked to many congenital heart diseases. At the onset of EMT in chick cardiogenesis, transforming growth factor (TGFβ)‐3 is expressed in transforming endothelial and invading mesenchymal cells, while bone morphogenetic protein (BMP)‐2 is expressed in the subjacent myocardium. Three‐dimensional collagen gel culture experiments of the AV endocardium show that 1) myocardially derived inductive signals upregulate the expression of AV endothelial TGFβ3 at the onset of EMT, 2) TGFβ3 needs to be expressed by these endothelial cells to trigger the initial phenotypic changes of EMT, and 3) myocardial BMP2 acts synergistically with TGFβ3 in the initiation of EMT. Anat Rec 258:119–127, 2000.

Bone morphogenetic protein-2 acts synergistically with transforming growth factor-β3 during endothelial-mesenchymal transformation in the developing chick heart

In the early embryonic heart, endothelial cells in atrioventricular (AV) and outflow tract (OT) regions are transformed into the invasive mesenchymal cells that form endocardial cushion tissue (endothelial‐mesenchymal transformation). It has been reported that bone morphogenetic proteins (BMPs) are transcribed in the AV and OT regions of the embryonic mouse heart. We previously reported that transforming growth factor beta 3 (TGFβ3) triggers the initial phenotypic changes seen in endothelial‐mesenchymal transformation. We cloned BMP2 from embryonic chick hearts and examined its functional role during endocardial cushion tissue formation. In situ hybridization showed BMP2 transcripts in the myocardium of the AV and OT regions, but not in endothelial/mesenchymal cells. Antisense oligodeoxynucleotides to BMP2 inhibited mesenchyme formation in AV endocardium cocultured with associated myocardium. This inhibitory effect was reversed by the addition of recombinant BMP2. In cultured AV endothelial monolayers, recombinant BMP2 did not induce any cellular phenotypic changes characteristic of endothelial‐mesenchymal transformation. However, BMP2 enhanced the TGFβ‐induced initial phenotypic changes associated with endothelial‐mesenchymal transformation. These results suggest that BMP2 1) plays an important role in the formation of endocardial cushion tissue and 2) acts synergistically with TGFβ3 in the regulation of this developmental event. J. Cell. Physiol. 180:35–45, 1999.

Expression of TGFβ3 RNA during chick embryogenesis: a possible important role in cardiovascular development

Abstract. We examined the temporal and spatial expression pattern of transforming growth factor (TGF)-β3 RNA during chick embryogenesis from stagexa06 to stagexa033 (Hamburger and Hamilton scale) by using in situ hybridization. During cardiogenesis, TGFβ3 mRNA was first expressed in the premyocardium at stagexa08 and thereafter it was localized in endocardial cushion tissue and the ventricular myocardium until the end of embryogenesis. During the formation of the major arteries, mRNA for TGFβ3 was found in smooth muscle progenitor cells, but not in endothelium. In addition, TGFβ3 mRNA was detectable in various mesoderm-derived tissues, such as the notochord, myotome, pleura, peritoneum, mesenchymal cells in the limb, and developing bone. These results suggest that TGFβ3 plays an important role in the development of the cardiovascular system and of other mesodermal derivatives during chicken embryogenesis.

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

ABSTRACTu2003 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.

Rho kinases regulate endothelial invasion and migration during valvuloseptal endocardial cushion tissue formation

Rho‐associated kinase (ROCK) is a downstream effector of small Rho‐GTPases, and phosphorylates several substrates to regulate cell functions, including actin cytoskeletal reorganization and cellular motility. Endothelial–mesenchymal transformation (EMT) is a critical event in the formation of valves and septa during cardiogenesis. It has been reported that ROCK plays an important role in the regulation of endocardial cell differentiation and migration during mouse cardiogenesis (Zhao and Rivkees [ 2004 ] Dev. Biol. 275:183–191). Immunohistochemistry showed that, during chick cardiogenesis, ROCK1 and ‐2 were expressed in the transforming and migrating endothelial/mesenchymal cells in the outflow tract (OT) and atrioventricular (AV) canal regions from which valvuloseptal endocardial cushion tissue would later develop. Treatment with Y27632, a specific ROCK inhibitor, of cultured AV explants or AV endothelial monolayers of stage 14‐minus heart (preactivated stage for EMT) on three‐dimensional collagen gel perturbed the seeding of mesenchymal cells into the gel lattice. In these experiments, Y27632 did not suppress the expression of an early transformation marker, smooth muscle α‐actin. Moreover, Y27632 inhibited the mesenchymal invasion in stage 14–18 AV explants, in which endothelial cells had committed to undergo EMT. ML‐9, a myosin light chain kinase inhibitor, also inhibited the mesenchymal invasion in cultured AV explants. These results suggest that ROCKs have a critical role in the mesenchymal cell invasion/migration that occurs at the late onset of EMT. Developmental Dynamics 235:94–104, 2006.

Hypoplasia of Cushion Ridges in the Proximal Outflow Tract Elicits Formation of a Right Ventricle-to-Aortic Route in Retinoic Acid-Induced Complete Transposition of the Great Arteries in the Mouse: Scanning Electron Microscopic Observations of Corrosion Cast Models

The major morphologic change associated with retinoic acid (RA)‐induced complete transposition of the great arteries (TGA), a congenital malformation of the heart, was investigated in a mouse model in which TGA was found in 80% of surviving fetuses.

Distribution of fibronectin, type I collagen, type IV collagen, and laminin in the cardiac jelly of the mouse embryonic heart with retinoic acid-induced complete transposition of the great arteries.

In the mouse model of complete transposition of the great arteries (TGA) produced by all‐trans retinoic acid (RA), parietal and septal ridges in the outflow tract (OT) are hypoplastic. At first, these ridges are generated by an expanded cardiac jelly (mainly myocardial basement membrane). Thereafter, endothelial cells delaminate and invade into the adjacent cardiac jelly to form endocardial cushion tissue (formation of cushion ridge). During cushion tissue formation, basement membrane antigens play an important role in the regulation of this endothelial‐mesenchymal transformation.

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.

Antisense oligodeoxynucleotide complementary to smooth muscle ?-actin inhibits endothelial-mesenchymal transformation during chick cardiogenesis

α‐Smooth‐muscle actin (SMA) is the major isoform of adult vascular tissues. During early development, SMA is expressed in various mesodermally derived tissues in a spatiotemporally restricted manner; however, its exact role remains unknown. We examined its role in the formation of chicken atrioventricular (AV) endocardial cushion tissue. This developmental process possesses the characteristics of endothelial–mesenchymal transformation and is partly TGFβ‐dependent. Immunohistochemistry showed that SMA was (1) expressed homogeneously in the newly formed appendages of transforming endothelial/mesenchymal cells, and (2) distributed in a punctate manner in the lamellipodia/filopodia of invading mesenchymal cells. Antisense oligodeoxynucleotide (ODNs) specific for SMA reduced both SMA expression and mesenchymal formation in AV endothelial cells cultured with myocardium on a collagen gel lattice. Perturbation of SMA by antisense ODN also inhibited TGFβ‐inducible migratory appendage formation in a cultured AV endothelial monolayer. However, it did not inhibit cell:cell separation or cellular hypertrophy. These results suggest that the expression of SMA is necessary for migratory appendage formation during the TGFβ‐dependent initial phenotypic changes that occur in endothelial–mesenchymal transformation. Dev Dyn 1999;216:489–498. ©1999 Wiley‐Liss, Inc.

Immunolocalization of latent transforming growth factor-ß binding protein-1 (LTBP1) during mouse development: possible roles in epithelial and mesenchymal cytodifferentiation

Abstract Latent transforming growth factor-β binding protein-1 (LTBP1) is a member of the fibrillin family; it is a glycoprotein of more than 190 kDa that is characterized by its possession of 16–18 epidermal growth factor-like motifs and 8 cysteine residues. The secretion of transforming growth factor-β involves its release from cells in a large latent complex containing LTBP1, a latency-associated peptide, and the mature region of the growth factor. Using a polyclonal antibody specific for LTBP1 (Ab39), we examined the immunohistochemical localization of this molecule during mouse embryogenesis between 8.5 and 13.5 embryonic days. An extracellular fibrillar structure containing LTBP1 was found in both the basement membrane of epithelia and mesenchymal tissue in which extensive tissue remodeling is carried out. Immunoelectron microscopy revealed Ab39 immunoreactivity on a 5- to 10-nm microfibrillar component of these basement membranes as well as in mesenchymal tissue. These results suggest that LTBP1 is one of the extracellular microfibrillar components of the basement membrane and of mesenchymal tissue, and that it may play an important role in the regulation of developmental phenomena involved in epithelial-mesenchymal interaction and epithelial differentiation, processes in which transforming growth factor-β is required for the control of cellular differentiation.