Although de la Cruz and colleagues showed as early as 1977 that the outflow tract was added after the heart tube formed, the source of these secondarily added cells was not identified for nearly 25 years. In 2001, three pivotal publications described a secondary or anterior heart field that contributed to the developing outflow tract. This review details the history of the heart field, the discovery and continuing elucidation of the secondarily adding myocardial cells, and how the different populations identified in 2001 are related to the more recent lineage tracing studies that defined the first and second myocardial heart field/lineages. Much recent work has focused on secondary heart field progenitors that give rise to the myocardium and smooth muscle at the definitive arterial pole. These progenitors are the last to be added to the arterial pole and are particularly susceptible to abnormal development, leading to conotruncal malformations in children. The major signaling pathways (Wnt, BMP, FGF8, Notch, and Shh) that control various aspects of secondary heart field progenitor behavior are discussed.

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2–DNAAF4–HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins.

The Sonic hedgehog (Shh)-null mouse was initially described as a phenotypic mimic of Tetralogy of Fallot with pulmonary atresia (Washington-Smoak et al, 2005); however, subsequent reports describe only a single outflow tract, leaving the phenotype and its developmental mechanism unclear. We hypothesized that the phenotype that occurs in response to Shh knockdown is pulmonary atresia and is directly related to the abnormal development of the secondary heart field. We found that Shh was expressed by the pharyngeal endoderm adjacent to the secondary heart field and that its receptor Ptc2 was expressed in a gradient in the secondary heart field, with the most robust expression in the caudal secondary heart field, closest to the Shh expression. In vitro culture of secondary heart field with the hedgehog inhibitor cyclopamine significantly reduced proliferation. In ovo, cyclopamine treatment before the secondary heart field adds to the outflow tract reduced proliferation only in the caudal secondary heart field, which coincided with the region of high Ptc2 expression. After outflow tract septation should occur, embryos treated with cyclopamine exhibited pulmonary atresia, pulmonary stenosis, and persistent truncus arteriosus. Cardiac neural crest-derived cells, which form the outflow tract septum, migrated into the outflow tract and formed a septum. However, this septum divided the outflow tract into two unequal sized vessels and effectively closed off the pulmonary outlet. These experiments show that Shh is necessary for secondary heart field proliferation, which is required for normal pulmonary trunk formation, and that embryos with pulmonary atresia have an outflow tract septum.

The bone morphogenetic protein (BMP) family of proteins has a multitude of roles throughout the body. In embryonic development, BMPs promote endothelial specification and subsequent venous differentiation. The BMP pathway also plays important roles in the adult vascular endothelium, promoting angiogenesis and mediating shear and oxidative stress. The canonical BMP pathway functions through the Smad transcription factors; however, other intracellular signaling cascades can be activated, and receptor complexes beyond the traditional type I and type II receptors add additional layers of regulation. Dysregulated BMP signaling has been linked to vascular diseases, including pulmonary hypertension and atherosclerosis. This review addresses recent advances in the roles of BMP signaling in the endothelium and how BMPs affect endothelial dysfunction and human disease.

Rationale Among the extracellular modulators of Bmp (bone morphogenetic protein) signaling, Bmper (Bmp endothelial cell precursor-derived regulator) both enhances and inhibits Bmp signaling. Recently we found that Bmper modulates Bmp4 activity via a concentration-dependent, endocytic trap-and-sink mechanism. Objective To investigate the molecular mechanisms required for endocytosis of the Bmper/Bmp4 and signaling complex and determine the mechanism of Bmper’s differential effects on Bmp4 signaling. Methods and Results Using an array of biochemical and cell biology techniques, we report that LRP1 (Low density lipoprotein receptor-related protein 1), a member of the LDL receptor family, acts as an endocytic receptor for Bmper and a co-receptor of Bmp4 to mediate the endocytosis of the Bmper/Bmp4 signaling complex. Furthermore, we demonstrate that LRP1-dependent Bmper/Bmp4 endocytosis is essential for Bmp4 signaling, as evidenced by the phenotype of lrp1-deficient zebrafish, which have abnormal cardiovascular development and decreased Smad1/5/8 activity in key vasculogenic structures. Conclusions Together, these data reveal a novel role for LRP1 in the regulation of Bmp4 signaling by regulating receptor complex endocytosis. In addition, these data introduce LRP1 as a critical regulator of vascular development. These observations demonstrate Bmper’s ability to fine-tune Bmp4 signaling at the single-cell level, unlike the spatial regulatory mechanisms applied by other Bmp modulators.

Objective— Bone morphogenetic proteins (Bmps) are important mediators of inflammation and atherosclerosis, though their mechanism of action is not fully understood. To better understand the contribution of the Bmp signaling pathway in vascular inflammation, we investigated the role of Bmper (Bmp endothelial cell precursor–derived regulator), an extracellular Bmp modulator, in an induced in vivo model of inflammation and atherosclerosis. Methods and Results— We crossed apolipoprotein E–deficient (ApoE −/− ) mice with mice missing 1 allele of Bmper (Bmper +/− mice used in the place of Bmper −/− mice that die at birth) and measured the development of atherosclerosis in mice fed a high-fat diet. Bmper haploinsufficiency in ApoE −/− mice (Bmper +/− ;ApoE −/− mice) led to a more severe phenotype compared with Bmper +/+ ;ApoE −/− mice. Bmper +/− ;ApoE −/− mice also exhibited increased Bmp activity in the endothelial cells in both the greater and lesser curvatures of the aortic arch, suggesting a role for Bmper in regulating Bmp-mediated inflammation associated with laminar and oscillatory shear stress. Small interfering RNA knockdown of Bmper in human umbilical vein endothelial cells caused a dramatic increase in the inflammatory markers intracellular adhesion molecule 1 and vascular cell adhesion molecule 1 at rest and after exposure to oscillatory and laminar shear stress. Conclusion— We conclude that Bmper is a critical regulator of Bmp-mediated vascular inflammation and that the fine-tuning of Bmp and Bmper levels is essential in the maintenance of normal vascular homeostasis.

The endothelium is composed of specialized epithelial cells that line the vasculature, the lymph vessels, and the heart. These endothelial cells are characterized by their stratification and are connected via intercellular junctions that confer specific permeability. Although all endothelium acts as a barrier, considerable heterogeneity exists among different organs and even within vessels. During development, the endothelial cells are specified before they migrate to their final destination, and then they commit to an arterial or venous fate. From the venous endothelial cell population, a subset of cells is further specified as lymphatic endothelium. The endothelium can be highly permeable, as in the lymph vessels, or impenetrable, as in the blood-brain barrier. These differences arise during development and are orchestrated through a series of signaling pathways. This review details how endothelial cells arise and are directed to their specific fate, specifically targeting what differentiates endothelial populations.

Summary The connection of the coronary vasculature to the aorta is one of the last essential steps of cardiac development. However, little is known about the signaling events that promote normal coronary artery formation. The bone morphogenetic protein (BMP) signaling pathway regulates multiple aspects of endothelial cell biology but has not been specifically implicated in coronary vascular development. BMP signaling is tightly regulated by numerous factors, including BMP-binding endothelial cell precursor-derived regulator (BMPER), which can both promote and repress BMP signaling activity. In the embryonic heart, BMPER expression is limited to the endothelial cells and the endothelial-derived cushions, suggesting that BMPER may play a role in coronary vascular development. Histological analysis of BMPER−/− embryos at early embryonic stages demonstrates that commencement of coronary plexus differentiation is normal and that endothelial apoptosis and cell proliferation are unaffected in BMPER−/− embryos compared with wild-type embryos. However, analysis between embryonic days 15.5-17.5 reveals that, in BMPER−/− embryos, coronary arteries are either atretic or connected distal to the semilunar valves. In vitro tubulogenesis assays indicate that isolated BMPER−/− endothelial cells have impaired tube formation and migratory ability compared with wild-type endothelial cells, suggesting that these defects may lead to the observed coronary artery anomalies seen in BMPER−/− embryos. Additionally, recombinant BMPER promotes wild-type ventricular endothelial migration in a dose-dependent manner, with a low concentration promoting and high concentrations inhibiting migration. Together, these results indicate that BMPER-regulated BMP signaling is critical for coronary plexus remodeling and normal coronary artery development.