Mechanisms of heart valve development and disease.

The valves of the heart are crucial for ensuring that blood flows in one direction from the heart, through the lungs and back to the rest of the body. Heart valve development is regulated by complex interactions between different cardiac cell types and is subject to blood flow-driven forces. Recent work has begun to elucidate the important roles of developmental pathways, valve cell heterogeneity and hemodynamics in determining the structure and function of developing valves. Furthermore, this work has revealed that many key genetic pathways involved in cardiac valve development are also implicated in diseased valves. Here, we review recent discoveries that have furthered our understanding of the molecular, cellular and mechanosensitive mechanisms of valve development, and highlight new insights into congenital and acquired valve disease.

Krox20 defines a subpopulation of cardiac neural crest cells contributing to arterial valves and bicuspid aortic valve.

Although cardiac neural crest cells are required at early stages of arterial valve development, their contribution during valvular leaflet maturation remains poorly understood. Here, we show in mouse that neural crest cells from pre-otic and post-otic regions make distinct contributions to the arterial valve leaflets. Genetic fate-mapping analysis of Krox20-expressing neural crest cells shows a large contribution to the borders and the interleaflet triangles of the arterial valves. Loss of Krox20 function results in hyperplastic aortic valve and partially penetrant bicuspid aortic valve formation. Similar defects are observed in neural crest Krox20-deficient embryos. Genetic lineage tracing in Krox20-/- mutant mice shows that endothelial-derived cells are normal, whereas neural crest-derived cells are abnormally increased in number and misplaced in the valve leaflets. In contrast, genetic ablation of Krox20-expressing cells is not sufficient to cause an aortic valve defect, suggesting that adjacent cells can compensate this depletion. Our findings demonstrate a crucial role for Krox20 in arterial valve development and reveal that an excess of neural crest cells may be associated with bicuspid aortic valve.

New imaging techniques project the cellular and molecular alterations underlying bicuspid aortic valve development.

Bicuspid aortic valve (BAV) disease is the most common congenital cardiac malformation associated with an increased lifetime risk and a high rate of surgically-relevant valve deterioration and aortic dilatation. Genomic data revealed that different genes are associated with BAV. A dominant genetic factor for the recent past was the basis to the recommendation for a more extensive aortic intervention. However very recent evidence that hemodynamic stressors and alterations of wall shear stress play an important role independent from the genetic trait led to more conservative treatment recommendations. Therefore, there is a current need to improve the ability to risk stratify BAV patients in order to obtain an early detection of valvulopathy and aortopathy while also to predict valve dysfunction and/or aortic disease development. Imaging studies based on new cutting-edge technologies, such us 4-dimensional (4D) flow magnetic resonance imaging (MRI), two-dimensional (2D) or three-dimensional (3D) speckle-tracking imaging (STI) and computation fluid dynamics, combined with studies demonstrating new gene mutations, specific signal pathways alterations, hemodynamic influences, circulating biomarkers modifications, endothelial progenitor cell impairment and immune/inflammatory response, all detected BAV valvulopathy progression and aortic wall abnormality. Overall, the main purpose of this review article is to merge the evidences of imaging and basic science studies in a coherent hypothesis that underlies and thus projects the development of both BAV during embryogenesis and BAV-associated aortopathy and its complications in the adult life, with the final goal to identifying aneurysm formation/rupture susceptibility to improve diagnosis and management of patients with BAV-related aortopathy.

Hemodynamics driven cardiac valve morphogenesis.

Mechanical forces are instrumental to cardiovascular development and physiology. The heart beats approximately 2.6 billion times in a human lifetime and heart valves ensure that these contractions result in an efficient, unidirectional flow of the blood. Composed of endocardial cells (EdCs) and extracellular matrix (ECM), cardiac valves are among the most mechanically challenged structures of the body both during and after their development. Understanding how hemodynamic forces modulate cardiovascular function and morphogenesis is key to unraveling the relationship between normal and pathological cardiovascular development and physiology. Most valve diseases have their origins in embryogenesis, either as signs of abnormal developmental processes or the aberrant re-expression of fetal gene programs normally quiescent in adulthood. Here we review recent discoveries in the mechanobiology of cardiac valve development and introduce the latest technologies being developed in the zebrafish, including live cell imaging and optical technologies, as well as modeling approaches that are currently transforming this field. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Bicuspid Aortic Valve: A Review of its Genetics and Clinical Significance.

The aim of this review was to describe recent advancements in the understanding of bicuspid aortic valve (BAV). BAV is the most common congenital cardiac anomaly, and affects between 0.46% and 1.37% of the population. There is a male predominance of approximately 3:1.While isolated BAV is found in certain patients, it is often associated with other congenital cardiac lesions, including dilatation of the thoracic aorta, coarctation of the aorta and abnormalities of the coronary anatomy. In most cases, it remains undetected until the patient contracts infective endocarditis, or calcification occurs. Alternatively, the BAV may remain functional for the entirety of the subjects' life, or it may develop progressive calcification, stenosis and regurgitation, with or without infection. Additionally, BAV is associated with aortic aneurysm formation and aortic dissection. Because BAV is a disease of both the valve and the aorta, surgical decision-making is complicated and remains an important challenge to the surgeon. Although recent reports have improved the current knowledge of the disease, many questions remain unresolved. The present review summarizes the current knowledge regarding the genetic basis of BAV and highlights some of the recent findings that have shed a light on the complications of this disease.

Evaluation of fetal cardiac valve anomalies by four-dimensional echocardiography with spatiotemporal image correlation (4DSTIC).

BACKGROUND: Prenatal diagnosis of cardiac valve anomalies challenged most screening sonographers. The purpose of the study was to evaluate the use of four-dimensional echocardiography with spatiotemporal image correlation (4DSTIC) in detecting normal and abnormal fetal cardiac valves. METHODS: Forty-three cases of confirmed cardiac valve anomalies identified by two-dimensional echocardiography (2DE) were retrospectively reviewed in this study. Additional 121 confirmed normal fetuses were included as controls. Four-dimensional volumes were acquired from each fetus using a transverse sweep. Four-dimensional rendered images were retrieved from the volumes for each of the cardiac valves for the normal fetuses and for the intended valves for fetuses with valve malformations. RESULTS: The visualization rates of cardiac valves retrieved from 4D volumes in the normal fetuses ranged from 72.5% to 97.5% before 33 gestational weeks and from 46.3% to 80.5% in late pregnancy. Furthermore, 4D rendered images were successfully obtained in 38 of 43 (88.4%) fetuses with cardiac valve lesions. CONCLUSIONS: The 4D images and cine loops displayed the valves anatomy vividly in both normal and abnormal fetuses, including some subtle malformations which were not identified by traditional 2DE. The standardized protocol we propose herein was important in obtaining the 4D images from the volumes. The 4D modality allows a better visualization of fetal cardiac valves and should be considered a valuable addition to traditional 2DE imaging.

Quadricuspid aortic valves in Syrian hamsters and their formation according to current knowledge on valvulogenesis.

Occurrence of quadricuspid aortic valves has been reported in humans, in nine dogs and in a greater white-toothed shrew. Moreover, two cases of developing aortic valves with four anticipated leaflets have been described in Syrian hamster embryos. Currently, however, no case of quadricuspid aortic valve in adult hamsters has been recorded. The aim here is to present four adults of this rodent species, two of them with unequivocally quadricuspid aortic valves and the other two with quadricuspid-like aortic valves. The four anomalous aortic valves were detected among 4,190 Syrian hamsters examined in our laboratory, representing an incidence of 0.09%. None of the affected hamsters showed apparent signs of disease. The present findings are considered on the light of current empirical knowledge about the morphogenesis of quadricuspid and bicuspid aortic and pulmonary valves. Quadricuspid aortic valves result from the partition of one of the normal mesenchymal cushions which normally give rise to normal (tricuspid) valves, while quadricuspid-like valves might be the product of a combined mechanism of fusion and partition of the cushions at the onset of the valvulogenesis. The presence of aortic valves with four leaflets in ancient mammalian lineages such as insectivors and rodents suggest that quadricuspid aortic valves, although showing almost certainly a low incidence, may be widespread among the different groups of mammals, including domestic animals.

Etiology of valvular heart disease-genetic and developmental origins.

Valvular heart disease occurs as either a congenital or acquired condition and advances in medical care have resulted in valve disease becoming increasingly prevalent. Unfortunately, treatments remain inadequate because of our limited understanding of the genetic and molecular etiology of diseases affecting the heart valves. Therefore, surgical repair or replacement remains the most effective option, which comes with additional complications and no guarantee of life-long success. Over the past decade, there have been significant advances in our understanding of cardiac valve development and, not surprisingly, mutations in these developmental genes have been identified in humans with congenital valve malformations. Concurrently, there has been a greater realization that acquired valve disease is not simply a degenerative process. Molecular investigation of acquired valve disease has identified that numerous signaling pathways critical for normal valve development are re-expressed in diseased valves. This review will discuss recent advances in our understanding of the development of the heart valves, as well as the implications of these findings on the genetics of congenital and acquired valvular heart disease.

Maternal aspects of fetal cardiac intervention.

OBJECTIVES: Fetal cardiac interventions have the potential to alter natural disease progression and reduce morbidity and mortality in children. Although there are already encouraging data on fetal outcome, information on maternal morbidity and mortality after intervention is scarce. The aim of the present study was to assess maternal aspects, pregnancy-associated risks and adverse events in 53 intrauterine cardiac interventions. METHODS: Between October 2000 and December 2012, 53 fetal cardiac interventions were performed in 47 patients (43 aortic valve dilations in 39 patients, seven pulmonary valve dilations in six patients and three balloon atrioseptostomies in two patients). Median gestational age was 26 + 4 (range, 20 + 3 to 33 + 1) weeks. Interventions were performed by an ultrasound-guided percutaneous approach under general anesthesia. All medical records and patient charts were analyzed retrospectively. RESULTS: All women were considered to be healthy in the preoperative assessment; 39 (83%) patients continued pregnancy until term and eight of 47 patients had an intrauterine fetal death (IUFD) and were induced. Postoperative nausea was reported in 29.8% of patients and abdominal pain in 36.2% of patients on the day of surgery. Preterm contractions were observed in two patients; no preterm prelabor rupture of membranes occurred. One severe postpartum hemorrhage was observed in a patient with IUFD and subsequent induction; however, this was unrelated to the balloon valvuloplasty. No intensive care unit admission and no major anesthesia-associated complications (aspiration, anaphylactic reaction, cardiovascular collapse, damage to teeth, laryngeal damage, awareness or hypoxic brain damage) were observed. Maternal mortality was zero. A significant learning curve was observed in terms of duration of intervention. CONCLUSION: In our experience, percutaneous needle-guided fetal cardiac intervention seems to be a safe procedure for the mother. In 53 procedures no major maternal complication directly related to the intervention was observed.

Assessment of fetal cardiomyopathy in early-stage twin-twin transfusion syndrome: comparison between commonly reported cardiovascular assessment scores.

OBJECTIVES: To assess the relationship between commonly reported fetal cardiomyopathy scoring systems in early-stage twin-twin transfusion syndrome (TTTS). METHODS: We reviewed retrospectively 100 cases of Quintero Stages I and II TTTS referred to our center for evaluation from 2008 to 2010. The cases were divided into groups of 25, representing each of four grades of TTTS cardiomyopathy as assessed by Cincinnati stage: no cardiomyopathy, Stage IIIa, Stage IIIb and Stage IIIc. Spearman correlation (rs ) was calculated between the Children's Hospital of Philadelphia (CHOP) score, cardiovascular profile score (CVPS), Cincinnati stage and myocardial performance index (MPI). RESULTS: There was a weak correlation between the Cincinnati stage and the CHOP score (rs = 0.36) and CVPS (rs = -0.39), while correlation was strong between the CHOP score and CVPS (rs = -0.72). MPI elevation was concordant with Cincinnati stage more frequently (82% of cases) than were ventricular hypertrophy (43%) or atrioventricular valve regurgitation (28%). 51% of fetuses with minimally elevated CHOP score (0-1) and 48% of fetuses with minimally depressed CVPS (9-10) had significant elevation (Z-score ≥ +3) in right ventricular or left ventricular MPI. CONCLUSIONS: MPI has a strong influence on grading the severity of fetal cardiomyopathy using the Cincinnati stage among fetuses with mild TTTS. Furthermore, significant elevation of the MPI is common among fetuses with mild disease as assessed by the CHOP score and CVPS. These differences should be understood when assessing and grading cardiomyopathy in TTTS, particularly in early (Quintero Stages I and II) disease.

Bicuspid aortic valve morphology and associated cardiovascular abnormalities in fetal Turner syndrome: a pathomorphological study.

INTRODUCTION: Bicuspid aortic valve (BAV) is common in Turner syndrome (TS). In adult TS, 82-95% of BAVs have fusion of the right and left coronary leaflets. Data in fetal stages are scarce. The purpose of this study was to gain insight into aortic valve morphology and associated cardiovascular abnormalities in a fetal TS cohort with adverse outcome early in development. MATERIAL AND METHODS: We studied post-mortem heart specimens of 36 TS fetuses and 1 TS newborn. RESULTS: BAV was present in 28 (76%) hearts. BAVs showed fusion of the right and left coronary leaflet (type 1 BAV) in 61%, and fusion of the right coronary and non-coronary leaflet (type 2 BAV) in 39%. There were no significant differences in occurrence of additional cardiovascular abnormalities between type 1 and type 2 BAV. However, all type 2 BAV hearts showed ascending aorta hypoplasia and tubular hypoplasia of the B segment, as opposed to only 55 and 64% of type 1 BAV hearts, respectively. DISCUSSION: The proportion of type 2 BAV seems higher in TS fetuses than in adults. Fetal type 2 BAV hearts all had severe aortic pathology, possibly contributing to a worse prognosis of type 2 than type 1 BAV in TS.

Insufficient versican cleavage and Smad2 phosphorylation results in bicuspid aortic and pulmonary valves.

Bicuspid or bifoliate aortic valve (BAV) results in two rather than three cusps and occurs in 1-2% of the population placing them at higher risk of developing progressive aortic valve disease. Only NOTCH-1 has been linked to human BAV, and genetically modified mouse models of BAV are limited by low penetrance and additional malformations. Here we report that in the Adamts5(-/-) valves, collagen I, collagen III, and elastin were disrupted in the malformed hinge region that anchors the mature semilunar cusps and where the ADAMTS5 proteoglycan substrate versican, accumulates. ADAMTS5 deficient prevalvular mesenchyme also exhibited a reduction of α-smooth muscle actin and filamin A suggesting versican cleavage may be involved in TGFß signaling. Subsequent evaluation showed a significant decrease of pSmad2 in regions of prevalvular mesenchyme in Adamts5(-/-) valves. To test the hypothesis that ADAMTS5 versican cleavage is required, in part, to elicit Smad2 phosphorylation we further reduced Smad2 in Adamts5(-/-) mice through intergenetic cross. The Adamts5(-/-);Smad2(+/-) mice had highly penetrant BAV and bicuspid pulmonary valve (BPV) malformations as well as increased cusp and hinge size compared to the Adamts5(-/-) and control littermates. These studies demonstrate that semilunar cusp malformations (BAV and BPV) can arise from a failure to remodel the proteoglycan-rich provisional ECM. Specifically, faulty versican clearance due to ADAMTS5 deficiency blocks the initiation of pSmad2 signaling, which is required for excavation of endocardial cushions during aortic and pulmonary valve development. Further studies using the Adamts5(-/-); Smad2(+/-) mice with highly penetrant and isolated BAV, may lead to new pharmacological treatments for valve disease.

UDP-glucose dehydrogenase polymorphisms from patients with congenital heart valve defects disrupt enzyme stability and quaternary assembly.

Cardiac valve defects are a common congenital heart malformation and a significant clinical problem. Defining molecular factors in cardiac valve development has facilitated identification of underlying causes of valve malformation. Gene disruption in zebrafish revealed a critical role for UDP-glucose dehydrogenase (UGDH) in valve development, so this gene was screened for polymorphisms in a patient population suffering from cardiac valve defects. Two genetic substitutions were identified and predicted to encode missense mutations of arginine 141 to cysteine and glutamate 416 to aspartate, respectively. Using a zebrafish model of defective heart valve formation caused by morpholino oligonucleotide knockdown of UGDH, transcripts encoding the UGDH R141C or E416D mutant enzymes were unable to restore cardiac valve formation and could only partially rescue cardiac edema. Characterization of the mutant recombinant enzymes purified from Escherichia coli revealed modest alterations in the enzymatic activity of the mutants and a significant reduction in the half-life of enzyme activity at 37 °C. This reduction in activity could be propagated to the wild-type enzyme in a 1:1 mixed reaction. Furthermore, the quaternary structure of both mutants, normally hexameric, was destabilized to favor the dimeric species, and the intrinsic thermal stability of the R141C mutant was highly compromised. The results are consistent with the reduced function of both missense mutations significantly reducing the ability of UGDH to provide precursors for cardiac cushion formation, which is essential to subsequent valve formation. The identification of these polymorphisms in patient populations will help identify families genetically at risk for valve defects.

Atrioventricular valve development: new perspectives on an old theme.

Atrioventricular valve development commences with an EMT event whereby endocardial cells transform into mesenchyme. The molecular events that induce this phenotypic change are well understood and include many growth factors, signaling components, and transcription factors. Besides their clear importance in valve development, the role of these transformed mesenchyme and the function they serve in the developing prevalve leaflets is less understood. Indeed, we know that these cells migrate, but how and why do they migrate? We also know that they undergo a transition to a mature, committed cell, largely defined as an interstitial fibroblast due to their ability to secrete various matrix components including collagen type I. However, we have yet to uncover mechanisms by which the matrix is synthesized, how it is secreted, and how it is organized. As valve disease is largely characterized by altered cell number, cell activation, and matrix disorganization, answering questions of how the valves are built will likely provide us with information of real clinical relevance. Although expression profiling and descriptive or correlative analyses are insightful, to advance the field, we must now move past the simplicity of these assays and ask fundamental, mechanistic based questions aimed at understanding how valves are "built". Herein we review current understandings of atrioventricular valve development and present what is known and what isn't known. In most cases, basic, biological questions and hypotheses that were presented decades ago on valve development still are yet to be answered but likely hold keys to uncovering new discoveries with relevance to both embryonic development and the developmental basis of adult heart valve diseases. Thus, the goal of this review is to remind us of these questions and provide new perspectives on an old theme of valve development.

The inter-relationship of periostin, TGF beta, and BMP in heart valve development and valvular heart diseases.

Recent studies have suggested an important role for periostin and transforming growth factor beta (TGF beta) and bone morphogenetic protein (BMP) ligands in heart valve formation and valvular heart diseases. The function of these molecules in cardiovascular development has previously been individually reviewed, but their association has not been thoroughly examined. Here, we summarize the current understanding of the association between periostin and TGF beta and BMP ligands, and discuss the implications of this association in the context of the role of these molecules in heart valve development and valvular homeostasis. Information about hierarchal connections between periostin and TGF beta and BMP ligands in valvulogenesis will increase our understanding of the pathogenesis, progression, and medical treatment of human valve diseases.

Scleraxis is required for cell lineage differentiation and extracellular matrix remodeling during murine heart valve formation in vivo.

Heart valve structures, derived from mesenchyme precursor cells, are composed of differentiated cell types and extracellular matrix arranged to facilitate valve function. Scleraxis (scx) is a transcription factor required for tendon cell differentiation and matrix organization. This study identified high levels of scx expression in remodeling heart valve structures at embryonic day 15.5 through postnatal stages using scx-GFP reporter mice and determined the in vivo function using mice null for scx. Scx(-/-) mice display significantly thickened heart valve structures from embryonic day 17.5, and valves from mutant mice show alterations in valve precursor cell differentiation and matrix organization. This is indicated by decreased expression of the tendon-related collagen type XIV, increased expression of cartilage-associated genes including sox9, as well as persistent expression of mesenchyme cell markers including msx1 and snai1. In addition, ultrastructure analysis reveals disarray of extracellular matrix and collagen fiber organization within the valve leaflet. Thickened valve structures and increased expression of matrix remodeling genes characteristic of human heart valve disease are observed in juvenile scx(-/-) mice. In addition, excessive collagen deposition in annular structures within the atrioventricular junction is observed. Collectively, our studies have identified an in vivo requirement for scx during valvulogenesis and demonstrate its role in cell lineage differentiation and matrix distribution in remodeling valve structures.

Embryonic development of bicuspid aortic valves.

Bicuspid aortic valve (BAV) is the most common congenital cardiac malformation, frequently associated with aortopathies and valvulopathies. The congenital origin of BAV is suspected to impact the development of the disease in the adult life. During the last decade, a number of studies dealing with the embryonic development of congenital heart disease have significantly improved our knowledge on BAV etiology. They describe the developmental defects, at the molecular, cellular and morphological levels, leading to congenital cardiac malformations, including BAV, in animal models. These models consist of a spontaneous hamster and several mouse models with different genetic manipulations in genes belonging to a variety of pathways. In this review paper, we aim to gather information on the developmental defects leading to BAV formation in these animal models, in order to tentatively explain the morphogenetic origin of the spectrum of valve morphologies that characterizes human BAV. BAV may be the only defect resulting from gene manipulation in mice, but usually it appears as the less severe defect of a spectrum of malformations, most frequently affecting the cardiac outflow tract. The genes whose alterations cause BAV belong to different genetic pathways, but many of them are direct or indirectly associated with the NOTCH pathway. These molecular alterations affect three basic cellular mechanisms during heart development, i.e., endocardial-to-mesenchymal transformation, cardiac neural crest (CNC) cell behavior and valve cushion mesenchymal cell differentiation. The defective cellular functions affect three possible morphogenetic mechanisms, i.e., outflow tract endocardial cushion formation, outflow tract septation and valve cushion excavation. While endocardial cushion abnormalities usually lead to latero-lateral BAVs and septation defects to antero-posterior BAVs, alterations in cushion excavation may give rise to both BAV types. The severity of the original defect most probably determines the specific aortic valve phenotype, which includes commissural fusions and raphes. Based on current knowledge on the developmental mechanisms of the cardiac outflow tract, we propose a unified hypothesis of BAV formation, based on the inductive role of CNC cells in the three mechanisms of BAV development. Alterations of CNC cell behavior in three possible alternative key valvulogenic processes may lead to the whole spectrum of BAV.

Reactivation of Notch signaling is required for cardiac valve regeneration.

Cardiac Valve Disease is one of the most common heart disorders with an emerging epidemic of cardiac valve degeneration due to aging. Zebrafish can regenerate most of their organs, including their heart. We aimed to explore the regenerative potential of cardiac valves and the underlying molecular mechanisms involved. We used an inducible, tissue-specific system of chemogenetic ablation and showed that zebrafish can also regenerate their cardiac valves. Upon valvular damage at larval stages, the intracardiac flow pattern becomes reminiscent of the early embryonic stages, exhibiting an increase in the retrograde flow fraction through the atrioventricular canal. As a result of the altered hemodynamics, notch1b and klf2a expression are ectopically upregulated, adopting the expression pattern of earlier developmental stages. We find that Notch signaling is re-activated upon valvular damage both at larval and adult stages and that it is required during the initial regeneration phase of cardiac valves. Our results introduce an animal model of cardiac valve specific ablation and regeneration.

Bicuspid aortic valve formation: Nos3 mutation leads to abnormal lineage patterning of neural crest cells and the second heart field.

The bicuspid aortic valve (BAV), a valve with two instead of three aortic leaflets, belongs to the most prevalent congenital heart diseases in the world, occurring in 0.5-2% of the general population. We aimed to understand how changes in early cellular contributions result in BAV formation and impact cardiovascular outflow tract development. Detailed 3D reconstructions, immunohistochemistry and morphometrics determined that, during valvulogenesis, the non-coronary leaflet separates from the parietal outflow tract cushion instead of originating from an intercalated cushion. Nos3-/- mice develop a BAV without a raphe as a result of incomplete separation of the parietal outflow tract cushion into the right and non-coronary leaflet. Genetic lineage tracing of endothelial, second heart field and neural crest cells revealed altered deposition of neural crest cells and second heart field cells within the parietal outflow tract cushion of Nos3-/- embryos. The abnormal cell lineage distributions also affected the positioning of the aortic and pulmonary valves at the orifice level. The results demonstrate that the development of the right and non-coronary leaflets are closely related. A small deviation in the distribution of neural crest and second heart field populations affects normal valve formation and results in the predominant right-non-type BAV in Nos3-/- mice.

Variations in structure of the outflow tract of the human embryonic heart: A new hypothesis for generating bicuspid aortic semilunar valves.

Outflow tract development of the heart is complex. The presence, differential growth and interactions of the various tissues through space and time contribute to the final development of the tract. This paper presents a novel interpretation of observations of outflow tract development, in particular of the aortic and pulmonary semilunar valves in embryos from the Shaner Collection at the University of Alberta. Three-dimensional reconstructions assist in the visualization of the spatial relationships of the developing valve tissues. In some embryos the aortic intercalated valve swelling is displaced proximally, giving rise to a bicuspid aortic semilunar valve more distally. In addition, the developing valve tissue first appears external to the myocardial cuff. The pulmonary semilunar valve regions appear to be more normal. This paper thus proposes a novel mechanism for generating a bicuspid aortic valve and also supports the idea that there is some independence of the aortic and pulmonary regions from each other during development.