Masahide Sakabe

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.

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.

Heart development before beating

During heart development at the pregastrula stage, prospective heart cells reside in the posterior lateral region of the epiblast layer. Interaction of tissues between the posterior epiblast and hypoblast is necessary to generate the future heart mesoderm. Signaling regulating the interaction involves fibroblast growth factor (FGF)-8, Nodal, bone morphogenetic protein (BMP)-antagonist, and canonical Wnt and acts on the posterior epiblast to induce the expression of genes specific for the anterior lateral mesoderm. At the early gastrula stage, prospective heart cells accumulate at the posterior midline and migrate to the anterior region of the primitive streak. During gastrulation, future heart cells leave the primitive streak and migrate anterolaterally to form the left and right anterior lateral plate mesoderm including the precardiac mesoderm. At this stage, prospective heart cells receive endoderm-derived signals, including BMP, FGF, and Wnt-antagonist, and thereby become committed to the heart lineage. At the neurula stage, the left and right precardiac mesoderm move to the ventral midline and fuse, resulting in the formation of a single primitive heart tube. Therefore, a two-step signaling cascade, which includes tissue interaction between epiblast and hypoblast at the blastula stage and endoderm-derived signals during gastrulation, is required to generate a beating heart.

Ectopic retinoic acid signaling affects outflow tract cushion development through suppression of the myocardial Tbx2-Tgfβ2 pathway.

The progress of molecular genetics has enabled us to identify the genes responsible for congenital heart malformations. However, recent studies suggest that congenital heart diseases are induced not only by mutations in certain genes, but also by abnormal maternal factors. A high concentration of maternal retinoic acid (RA), the active derivative of vitamin A, is well known as a teratogenic agent that can cause developmental defects. Our previous studies have shown that the maternal administration of RA to mice within a narrow developmental window induces outflow tract (OFT) septum defects, a condition that closely resembles human transposition of the great arteries (TGA), although the responsible factors and pathogenic mechanisms of the TGA induced by RA remain unknown. We herein demonstrate that the expression of Tbx2 in the OFT myocardium is responsive to RA, and its downregulation is associated with abnormal OFT development. We found that RA could directly downregulate the Tbx2 expression through a functional retinoic acid response element (RARE) in the Tbx2 promoter region, which is also required for the initiation of Tbx2 transcription during OFT development. Tgfb2 expression was also downregulated in the RA-treated OFT region and was upregulated by Tbx2 in a culture system. Moreover, defective epithelial-mesenchymal transition caused by the excess RA was rescued by the addition of Tgfβ2 in an organ culture system. These data suggest that RA signaling participates in the Tbx2 transcriptional mechanism during OFT development and that the Tbx2-Tgfβ2 cascade is one of the key pathways involved in inducing the TGA phenotype.

Induction of initial cardiomyocyte α-actin—smooth muscle α-actin—in cultured avian pregastrula epiblast: A role for nodal and BMP antagonist

During early cardiogenesis, endoderm‐derived bone morphogenetic protein (BMP) induces the expression of both heart‐specific transcription factors and sarcomeric proteins. However, BMP antagonists do not inhibit the expression of the “initial heart α‐actin”—smooth muscle α‐actin (SMA)—which is first expressed in the anterior lateral mesoderm and then recruited into the initial myofibrils (Nakajima et al. [2002] Dev. Biol. 245:291–303). Therefore, mechanisms that regulate the expression of SMA in the heart‐forming mesoderm are not well‐understood. Regional explantation experiments using chick blastoderm showed that the posterolateral region of the epiblast differentiated into cardiomyocytes. Posterior epiblast cultured with or without the associated hypoblast showed that interaction between the tissues of these two germ layers at the early pregastrula stage (stages X–XI) was a prerequisite for the expression of SMA. Posterior epiblast that is cultured without hypoblast could also be induced to express SMA if TGF‐β or activin was added to the culture medium. However, neither neutralizing antibodies against TGF‐βs nor follistatin perturbed the expression of SMA in cultured blastoderm. Adding BMP to the cultured blastoderm inhibited the expression of SMA, whereas BMP antagonists, such as chordin, were able to induce the expression of SMA in cultured posterior epiblast. Furthermore, adding lefty‐1, a nodal antagonist, to the blastoderm inhibited the expression of SMA, and nodal plus BMP antagonist up‐regulated the expression of SMA in cultured posterior epiblast. Results indicate that the interaction between the tissues of the posterior epiblast and hypoblast is necessary to initiate the expression of SMA during early cardiogenesis and that nodal and BMP antagonist may play an important role in the regulation of SMA expression. Developmental Dynamics 233:1419–1429, 2005.

Rho kinase inhibitor Y27632 affects initial heart myofibrillogenesis in cultured chick blastoderm

During early vertebrate development, Rho‐associated kinases (ROCKs) are involved in various developmental processes. Here, we investigated spatiotemporal expression patterns of ROCK1 protein and examined the role of ROCK during initial heart myofibrillogenesis in cultured chick blastoderm. Immunohistochemistry showed that ROCK1 protein was distributed in migrating mesendoderm cells, visceral mesoderm of the pericardial coelom (from which cardiomyocytes will later develop), and cardiomyocytes of the primitive heart tube. Pharmacological inhibition of ROCK by Y27632 did not alter the myocardial specification process in cultured posterior blastoderm. However, Y27632 disturbed the formation of striated heart myofibrils in cultured posterior blastoderm. Furthermore, Y27632 affected the formation of costamere, a vinculin/integrin‐based rib‐like cell adhesion site. In such cardiomyocytes, cell–cell adhesion was disrupted and N‐cadherin was distributed in the perinuclear region. Pharmacological inactivation of myosin light chain kinase, a downstream of ROCK, by ML‐9 perturbed the formation of striated myofibrils as well as costameres, but not cell–cell adhesion. These results suggest that ROCK plays a role in the formation of initial heart myofibrillogenesis by means of actin–myosin assembly, and focal adhesion/costamere and cell–cell adhesion. Developmental Dynamics 236:461–472, 2007.

ROCK1 Expression is Regulated by TGFβ3 and ALK2 During Valvuloseptal Endocardial Cushion Formation

During early heart development at the looped heart stage, endothelial cells in the outflow tract and atrioventricular (AV) regions transform into mesenchyme to generate endocardial cushion tissue. This endocardial epithelial–mesenchymal transition (EMT) is regulated by several regulatory pathways, including the transforming growth factor‐beta (TGFβ), bone morphogenetic protein (BMP), and Rho‐ROCK pathways. Here, we investigated the spatiotemporal expression pattern of ROCK1 mRNA during EMT in chick and examined whether TGFβ or BMP could induce the expression of ROCK1. At the onset of EMT, ROCK1 expression was up‐regulated in endothelial/mesenchymal cells. A three‐dimensional collagen gel assay was used to examine the mechanisms regulating the expression of ROCK1. In AV endocardium co‐cultured with associated myocardium, ROCK1 expression was inhibited by either anti‐TGFβ3 antibody, anti‐ALK2 antibody or noggin, but not SB431542 (ALK5 inhibitor). In cultured preactivated AV endocardium, TGFβ3 protein induced the expression of ROCK1, but BMP did not. AV endothelial cells that were cultured in medium supplemented with TGFβ3 plus anti‐ALK2 antibody failed to express ROCK1. These results suggest that the expression of ROCK1 is up‐regulated at the onset of EMT and that signaling mediated by TGFβ3/ALK2 together with BMP is involved in the expression of ROCK1. Anat Rec, 291:845‐857, 2008.

Induction of initial heart α-actin, smooth muscle α-actin, in chick pregastrula epiblast : The role of hypoblast and fibroblast growth factor-8

During heart development at the gastrula stage, inhibition of bone morphogenetic protein (BMP) activity affects the heart specification but does not impair the expression of smooth muscle α‐actin (SMA), which is first expressed in the heart mesoderm and recruited into initial heart myofibrils. Interaction of tissues between posterior epiblast and hypoblast at the early blastula stage is necessary to induce the expression of SMA, in which Nodal and Chordin are thought to be involved. Here we investigated the role of fibroblast growth factor‐8 (FGF8) in the expression of SMA. In situ hybridization and reverse transcription–polymerase chain reaction showed that Fgf8b is expressed predominantly in the nascent hypoblast. Anti‐FGF8b antibody inhibited the expression of SMA, cTNT, and Tbx5, which are BMP‐independent heart mesoderm/early cardiomyocyte genes, but not Brachyury in cultured posterior blastoderm, and combined FGF8b and Nodal, but neither factor alone induced the expression of SMA in association with heart specific markers in cultured epiblast. Although FGF8b did not induce the upregulation of phospho‐Smad2, anti‐FGF8b properties suppressed phospho‐Smad2 in cultured blastoderm. FGF8b was able to reverse the BMP‐induced inhibition of cardiomyogenesis. The results suggest that FGF8b acts on the epiblast synergistically with Nodal at the pregastrula stage and may play a role in the expression of SMA during early cardiogenesis.

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.

Cover Image: TMEM100 for EndMT During Cardiac Development

Background: Endothelial‐mesenchymal transformation (EndMT) is essential for endocardial cushion formation during cardiac morphogenesis. We recently identified Tmem100 as an endothelial gene indispensable for vascular development. In this study, we further investigated its roles for EndMT during atrioventricular canal (AVC) cushion formation. Results: Tmem100 was expressed in AVC endocardial cells, and Tmem100 null embryos showed severe EndMT defect in the AVC cushions. While calcineurin‐dependent suppression of vascular endothelial growth factor (VEGF) expression in the AVC myocardium is important for EndMT, significant up‐regulation of Vegfa expression was observed in Tmem100 null heart. EndMT impaired in Tmem100 null AVC explants was partially but significantly restored by the expression of constitutively‐active calcineurin A, suggesting dysregulation of myocardial calcineurin‐VEGF signaling in Tmem100 null heart. Moreover, Tmem100 null endocardial cells in explant culture did not show EndMT in response to the treatment with myocardium‐derived growth factors, transforming growth factor β2 and bone morphogenetic protein 2, indicating involvement of an additional endocardial‐specific abnormality in the mechanism of EndMT defect. The lack of NFATc1 nuclear translocation in endocardial cells of Tmem100 null embryos suggests impairment of endocardial calcium signaling. Conclusions: The Tmem100 deficiency causes EndMT defect during AVC cushion formation possibly via disturbance of multiple calcium‐related signaling events. Developmental Dynamics 244:31–42, 2015.