The anatomical components of the cardiac outflow tract of the gray bichir,Polypterus senegalus: their evolutionary significance

It has been reported that in chondrichthyans the cardiac outflow tract is composed of the myocardial conus arteriosus, while in most teleosteans it consists of the nonmyocardial bulbus arteriosus. Classical studies already indicated that a conus and a bulbus coexist in several ancient actinopterygian and teleost groups. Recent work has shown that a cardiac outflow tract consisting of a conus and a bulbus is common to both cartilaginous and bony fishes. Nonetheless and despite their position at the base of the actinopterygian phylogenetic lineage, the anatomical arrangement of the cardiac outflow tract of the Polypteriformes remained uncertain. The present study of hearts from gray bichirs was intended to fill this gap. The cardiac outflow tract of the bichir consists of two main components, namely a very long conus arteriosus, furnished with valves, and a short, intrapericardial, arterial-like bulbus arteriosus, which differs from the ventral aorta because it is covered by epicardium, shows a slightly different spatial arrangement of the histological elements and is crossed by coronary arteries. Histomorphologically, the outflow tract consists of three longitudinal regions, distal, middle and proximal, an arrangement which has been suggested to be common to all vertebrates. The distal region corresponds to the bulbus, while the conus comprises the middle and proximal regions. The present findings reinforce the notion that the bulbus arteriosus of fish has played an essential role in vertebrate heart evolution as it is the precursor of the intrapericardial trunks of the aorta and pulmonary artery of birds and mammals.     

The anatomical components of the cardiac outflow tract of chondrichthyans and actinopterygians

The outflow tract of the fish heart is the segment interposed between the ventricle and the ventral aorta. It holds the valves that prevent blood backflow from the gill vasculature to the ventricle. The anatomical composition, histological structure and evolutionary changes in the fish cardiac outflow tract have been under discussion for nearly two centuries and are still subject to debate. This paper offers a brief historical review of the main conceptions about the cardiac outflow tract components of chondrichthyans (cartilaginous fish) and actinopterygians (ray‐finned fish) which have been put forward since the beginning of the nineteenth century up to the current day. We focus on the evolutionary origin of the outflow tract components and the changes to which they have been subject in the major extant groups of chondrichthyans and actinopterygians. In addition, an attempt is made to infer the primitive anatomical design of the heart of the gnathostomes (jawed vertebrates). Finally, several areas of further investigation are suggested. Recent work on fish heart morphology has shown that the cardiac outflow tract of chondrichthyans does not consist exclusively of the myocardial conus arteriosus as classically thought. A conus arteriosus and a bulbus arteriosus, devoid of myocardium and mainly composed of elastin and smooth muscle, are usually present in cartilaginous and ray‐finned fish. This is consistent with the suggestion that both components coexisted from the onset of the gnathostome radiation. There is evidence that the conus arteriosus appeared in the agnathans. By contrast, the evolutionary origin of the bulbus is still unclear. It is almost certain that in all fish, both the conus and bulbus develop from the embryonic second heart field. We suggest herein that the primitive anatomical heart of the jawed vertebrates consisted of a sinus venosus containing the pacemaker tissue, an atrium possessing trabeculated myocardium, an atrioventricular region with compact myocardium which supported the atrioventricular valves, a ventricle composed of mixed myocardium, and an outflow tract consisting of a conus arteriosus, with compact myocardium in its wall and valves at its luminal side, and a non‐myocardial bulbus arteriosus that connected the conus with the ventral aorta. Chondrichthyans have retained this basic anatomical design of the heart. In actinopterygians, the heart has been subject to notable changes during evolution. Among them, the following two should be highlighted: (i) a decrease in size of the conus in combination with a remarkable development of the bulbus, especially in teleosts; and (ii) loss of the myocardial compact layer of the ventricle in many teleost species.     

Differentiation of the cardiac outflow tract components in alevins of the sturgeon Acipenser naccarii (Osteichthyes, Acipenseriformes): implications for heart evolution

Previous work showed that in the adult sturgeon an intrapericardial, nonmyocardial segment is interposed between the conus arteriosus of the heart and the ventral aorta. The present report illustrates the ontogeny of this intermediate segment in Acipenser naccarii. The sample studied consisted of 178 alevins between 1 and 24 days posthatching. They were examined using light and electron microscopy. Our observations indicate that the entire cardiac outflow tract displays a myocardial character during early development. Between the fourth and sixth days posthatching, the distal portion of the cardiac outflow tract undergoes a phenotypical transition, from a myocardial to a smooth muscle-like phenotype. The length of this region with regard to the whole outflow tract increases only moderately during subsequent developmental stages, becoming more and more cellularized. The cells soon organize into a pattern that resembles that of the arterial wall. Elastin appears at this site by the seventh day posthatching. Therefore, two distinct components, proximal and distal, can be recognized from the fourth day posthatching in the cardiac outflow tract of A. naccarii. The proximal component is the conus arteriosus, characterized by its myocardial nature and the presence of endocardial cushions. The distal component transforms into the intrapericardial, nonmyocardial segment mentioned above, which is unequivocally of cardiac origin. We propose to designate this segment the "bulbus arteriosus" because it is morphogenetically equivalent to the bulbus arteriosus of teleosts. The present findings, together with data from the literature, point to the possibility that cells from the cardiac neural crest are involved in the phenotypical transition that takes place at the distal portion of the cardiac outflow tract, resulting in the appearance of the bulbus arteriosus. Moreover, they suggest that the cardiac outflow tract came to be formed by a bulbus arteriosus and a conus arteriosus from an early period of the vertebrate evolutionary story. Finally, we hypothesize that the embryonic truncus of birds and mammals is homologous to the bulbus arteriosus of fish.     

Cardiac neural crest is dispensable for outflow tract septation in Xenopus

In vertebrate embryos, cardiac precursor cells of the primary heart field are specified in the lateral mesoderm. These cells converge at the ventral midline to form the linear heart tube, and give rise to the atria and the left ventricle. The right ventricle and the outflow tract are derived from an adjacent population of precursors known as the second heart field. In addition, the cardiac neural crest contributes cells to the septum of the outflow tract to separate the systemic and the pulmonary circulations. The amphibian heart has a single ventricle and an outflow tract with an incomplete spiral septum; however, it is unknown whether the cardiac neural crest is also involved in outflow tract septation, as in amniotes. Using a combination of tissue transplantations and molecular analyses in Xenopus we show that the amphibian outflow tract is derived from a second heart field equivalent to that described in birds and mammals. However, in contrast to what we see in amniotes, it is the second heart field and not the cardiac neural crest that forms the septum of the amphibian outflow tract. In Xenopus, cardiac neural crest cells remain confined to the aortic sac and arch arteries and never populate the outflow tract cushions. This significant difference suggests that cardiac neural crest cell migration into the cardiac cushions is an amniote-specific characteristic, presumably acquired to increase the mass of the outflow tract septum with the evolutionary need for a fully divided circulation.     

Evolutionary and developmental origins of the cardiac neural crest: Building a divided outflow tract

The cardiac neural crest cells (CNCCs) have played an important role in the evolution and development of the vertebrate cardiovascular system: from reinforcement of the developing aortic arch arteries early in vertebrate evolution, to later orchestration of aortic arch artery remodeling into the great arteries of the heart, and finally outflow tract septation in amniotes. A critical element necessary for the evolutionary advent of outflow tract septation was the co‐evolution of the cardiac neural crest cells with the second heart field. This review highlights the major transitions in vertebrate circulatory evolution, explores the evolutionary developmental origins of the CNCCs from the third stream cranial neural crest, and explores candidate signaling pathways in CNCC and outflow tract evolution drawn from our knowledge of DiGeorge Syndrome. Birth Defects Research (Part C) 102:309–323, 2014. © 2014 Wiley Periodicals, Inc.     

Ventricle and outflow tract of the African lungfish Protopterus dolloi

We report a morphologic study of the heart ventricle and outflow tract of the African lungfish Protopterus dolloi. The ventricle is saccular and appears attached to the anterior pericardial wall by a thick tendon. An incomplete septum divides the ventricle into two chambers. Both the free ventricular wall and the incomplete ventricular septum are entirely trabeculated. Only a thin rim of myocardium separates the trabecular system from the subepicardial space. The outflow tract consists of proximal, middle, and distal portions, separated by two flexures, proximal and distal. The proximal outflow tract portion is endowed with a layer of compact, well-vascularized myocardium. This portion is homologous to the conus arteriosus observed in the heart of most vertebrates. The middle and distal outflow tract portions are arterial-like, thus being homologous to the bulbus arteriosus. However, the separation between the muscular and arterial portions of the outflow tract is not complete in the lungfish. A thin layer of myocardium covers the arterial tissue, and a thin layer of elastic tissue underlies the conus myocardium. Two unequal ridges composed of loose connective tissue, the spiral and bulbar folds, run the length of the outflow tract. They form an incomplete division of the outflow tract, but fuse at the distal end. The two folds are covered by endocardium and contain collagen, elastin, and fibroblast-like cells. They appear to be homologous to the dextro-dorsal and sinistro-ventral ridges observed during the development of the avian and mammalian heart. Two to three rows of vestigial arterial-like valves appear in the dorsal and ventral aspects of the conus. These valves are unlikely to have a functional role. The possible functional significance of the "gubernaculum cordis," the thick tendon extending between the anterior ventricular surface and the pericardium, is discussed.     

Cardiac neural crest contributes to cardiomyogenesis in zebrafish

In birds and mammals, cardiac neural crest is essential for heart development and contributes to conotruncal cushion formation and outflow tract septation. The zebrafish prototypical heart lacks outflow tract septation, raising the question of whether cardiac neural crest exists in zebrafish. Here, results from three distinct lineage-labeling approaches identify zebrafish cardiac neural crest cells and indicate that these cells have the ability to generate MF20-positive muscle cells in the myocardium of the major chambers during development. Fate-mapping demonstrates that cardiac neural crest cells originate both from neural tube regions analogous to those found in birds, as well as from a novel region rostral to the otic vesicle. In contrast to other vertebrates, cardiac neural crest invades the myocardium in all segments of the heart, including outflow tract, atrium, atrioventricular junction, and ventricle in zebrafish. Three distinct groups of premigratory neural crest along the rostrocaudal axis have different propensities to contribute to different segments in the heart and are correspondingly marked by unique combinations of gene expression patterns. Zebrafish will serve as a model for understanding interactions between cardiac neural crest and cardiovascular development.     

Model systems for the study of heart development and disease. Cardiac neural crest and conotruncal malformations

Neural crest cells are multipotential cells that delaminate from the dorsal neural tube and migrate widely throughout the body. A subregion of the cranial neural crest originating between the otocyst and somite 3 has been called "cardiac neural crest" because of the importance of these cells in heart development. Much of what we know about the contribution and function of the cardiac neural crest in cardiovascular development has been learned in the chick embryo using quail-chick chimeras to study neural crest migration and derivatives as well as using ablation of premigratory neural crest cells to study their function. These studies show that cardiac neural crest cells are absolutely required to form the aorticopulmonary septum dividing the cardiac arterial pole into systemic and pulmonary circulations. They support the normal development and patterning of derivatives of the caudal pharyngeal arches and pouches, including the great arteries and the thymus, thyroid and parathyroids. Recently, cardiac neural crest cells have been shown to modulate signaling in the pharynx during the lengthening of the outflow tract by the secondary heart field. Most of the genes associated with cardiac neural crest function have been identified using mouse models. These studies show that the neural crest cells may not be the direct cause of abnormal cardiovascular development but they are a major component in the complex tissue interactions in the caudal pharynx and outflow tract. Since, cardiac neural crest cells span from the caudal pharynx into the outflow tract, they are especially susceptible to any perturbation in or by other cells in these regions. Thus, understanding congenital cardiac outflow malformations in human sequences of malformations as represented by the DiGeorge syndrome will necessarily require understanding development of the cardiac neural crest.     

Expression of cardiac neural crest and heart genes isolated by modified differential display

The invasion of the cardiac neural crest (CNC) into the outflow tract (OFT) and subsequent outflow tract septation are critical events during vertebrate heart development. We have performed four modified differential display screens in the chick embryo to identify genes that may be involved in CNC, OFT, secondary heart field, and heart development. The screens included differential display of RNA isolated from three different axial segments containing premigratory cranial neural crest cells; of RNA from distal outflow tract, proximal outflow tract, and atrioventricular tissue of embryonic chick hearts; and of RNA isolated from left and right cranial tissues, including the early heart fields. These screens have resulted in the identification of the five cDNA clones presented here, which are expressed in the cardiac neural crest, outflow tract and developing heart in patterns that are unique in heart development.     

Cardiac neural crest ablation alters Id2 gene expression in the developing heart

Id proteins are negative regulators of basic helix-loop-helix gene products and participate in many developmental processes. We have evaluated the expression of Id2 in the developing chick heart and found expression in the cardiac neural crest, secondary heart field, outflow tract, inflow tract, and anterior parasympathetic plexus. Cardiac neural crest ablation in the chick embryo, which causes structural defects of the cardiac outflow tract, results in a significant loss of Id2 expression in the outflow tract. Id2 is also expressed in Xenopus neural folds, branchial arches, cardiac outflow tract, inflow tract, and splanchnic mesoderm. Ablation of the premigratory neural crest in Xenopus embryos results in abnormal formation of the heart and a loss of Id2 expression in the heart and splanchnic mesoderm. This data suggests that the presence of neural crest is required for normal Id2 expression in both chick and Xenopus heart development and provides evidence that neural crest is involved in heart development in Xenopus embryos.     

Sonic hedgehog maintains proliferation in secondary heart field progenitors and is required for normal arterial pole formation

The Sonic hedgehog (Shh)-null mouse was initially described as a phenotypic mimic of Tetralogy of Fallot with pulmonary atresia (Washington Smoak, I., Byrd, N.A., Abu-Issa, R., Goddeeris, M.M., Anderson, R., Morris, J., Yamamura, K., Klingensmith, J., and Meyers, E.N. 2005. Sonic hedgehog is required for cardiac outflow tract and neural crest cell development. Dev. Biol. 283, 357–372.); 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. In hearts with pulmonary atresia, 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.     

基于Rag1基因序列变异的鲤形目鱼类系统发育重建:采样策略对生命之树的影响

The phylogenetic relationships of species are fundamental to any biological investigation, including all evolutionary studies. Accurate inferences of sister group relationships provide the researcher with an historical framework within which the attributes or geographic origin of species (or supraspecific groups) evolved. Taken out of this phylogenetic context, interpretations of evolutionary processes or origins, geographic distributions, or speciation rates and mechanisms, are subject to nothing less than a biological experiment without controls. Cypriniformes is the most diverse clade of freshwater fishes with estimates of diversity of nearly 3,500 species. These fishes display an amazing array of morphological, ecological, behavioral, and geographic diversity and offer a tremendous opportunity to enhance our understanding of the biotic and abiotic factors associated with diversification and adaptation to environments. Given the nearly global distribution of these fishes, they serve as an important model group for a plethora of biological investigations, including indicator species for future climatic changes. The occurrence of the zebrafish, Danio rerio, in this order makes this clade a critical component in understanding and predicting the relationship between mutagenesis and phenotypic expressions in vertebrates, including humans. With the tremendous diversity in Cypriniformes, our understanding of their phylogenetic relationships has not proceeded at an acceptable rate, despite a plethora of morphological and more recent molecular studies. Most studies are pre-Hennigian in origin or include relatively small numbers of taxa. Given that analyses of small numbers of taxa for molecular characters can be compromised by peculiarities of long-branch attraction and nodal-density effect, it is critical that significant progress in our understanding of the relationships of these important fishes occurs with increasing sampling of species to mitigate these potential problems. The recent Cypriniformes Tree of Life initiative is an effort to achieve this goal with morphological and molecular (mitochondrial and nuclear) data. In this early synthesis of our understanding of the phylogenetic relationships of these fishes, all types of data have contributed historically to improving our understanding, but not all analyses are complementary in taxon sampling, thus precluding direct understanding of the impact of taxon sampling on achieving accurate phylogenetic inferences. However, recent molecular studies do provide some insight and in some instances taxon sampling can be implicated as a variable that can influence sister group relationships. Other instances may also exist but without inclusion of more taxa for both mitochondrial and nuclear genes, one cannot distinguish between inferences being dictated by taxon sampling or the origins of the molecular data.     

Mesoderm progenitor cells of common origin contribute to the head musculature and the cardiac outflow tract

During early embryogenesis, heart and skeletal muscle progenitor cells are thought to derive from distinct regions of the mesoderm (i.e. the lateral plate mesoderm and paraxial mesoderm, respectively). In the present study, we have employed both in vitro and in vivo experimental systems in the avian embryo to explore how mesoderm progenitors in the head differentiate into both heart and skeletal muscles. Using fate-mapping studies, gene expression analyses, and manipulation of signaling pathways in the chick embryo, we demonstrate that cells from the cranial paraxial mesoderm contribute to both myocardial and endocardial cell populations within the cardiac outflow tract. We further show that Bmp signaling affects the specification of mesoderm cells in the head: application of Bmp4, both in vitro and in vivo, induces cardiac differentiation in the cranial paraxial mesoderm and blocks the differentiation of skeletal muscle precursors in these cells. Our results demonstrate that cells within the cranial paraxial mesoderm play a vital role in cardiogenesis, as a new source of cardiac progenitors that populate the cardiac outflow tract in vivo. A deeper understanding of mesodermal lineage specification in the vertebrate head is expected to provide insights into the normal, as well as pathological, aspects of heart and craniofacial development.     

Vangl2-Regulated Polarisation of Second Heart Field-Derived Cells Is Required for Outflow Tract Lengthening during Cardiac Development

Planar cell polarity (PCP) is the mechanism by which cells orient themselves in the plane of an epithelium or during directed cell migration, and is regulated by a highly conserved signalling pathway. Mutations in the PCP gene Vangl2, as well as in other key components of the pathway, cause a spectrum of cardiac outflow tract defects. However, it is unclear why cells within the mesodermal heart tissue require PCP signalling. Using a new conditionally floxed allele we show that Vangl2 is required solely within the second heart field (SHF) to direct normal outflow tract lengthening, a process that is required for septation and normal alignment of the aorta and pulmonary trunk with the ventricular chambers. Analysis of a range of markers of polarised epithelial tissues showed that in the normal heart, undifferentiated SHF cells move from the dorsal pericardial wall into the distal outflow tract where they acquire an epithelial phenotype, before moving proximally where they differentiate into cardiomyocytes. Thus there is a transition zone in the distal outflow tract where SHF cells become more polarised, turn off progenitor markers and start to differentiate to cardiomyocytes. Membrane-bound Vangl2 marks the proximal extent of this transition zone and in the absence of Vangl2, the SHF-derived cells are abnormally polarised and disorganised. The consequent thickening, rather than lengthening, of the outflow wall leads to a shortened outflow tract. Premature down regulation of the SHF-progenitor marker Isl1 in the mutants, and accompanied premature differentiation to cardiomyocytes, suggests that the organisation of the cells within the transition zone is important for maintaining the undifferentiated phenotype. Thus, Vangl2-regulated polarisation and subsequent acquisition of an epithelial phenotype is essential to lengthen the tubular outflow vessel, a process that is essential for on-going cardiac morphogenesis.     

Phylogenetic perspectives in the evolution of parental care in ray-finned fishes

Among major vertebrate groups, ray-finned fishes (Actinopterygii) collectively display a nearly unrivaled diversity of parental care activities. This fact, coupled with a growing body of phylogenetic data for Actinopterygii, makes these fishes a logical model system for analyzing the evolutionary histories of alternative parental care modes and associated reproductive behaviors. From an extensive literature review, we constructed a supertree for ray-finned fishes and used its phylogenetic topology to investigate the evolution of several key reproductive states including type of parental care (maternal, paternal, or biparental), internal versus external fertilization, internal versus external gestation, nest construction behavior, and presence versus absence of sexual dichromatism (as an indicator of sexual selection). Using a comparative phylogenetic approach, we critically evaluate several hypotheses regarding evolutionary pathways toward parental care. Results from maximum parsimony reconstructions indicate that all forms of parental care, including paternal, biparental, and maternal (both external and internal to the female reproductive tract) have arisen repeatedly and independently during ray-finned fish evolution. The most common evolutionary transitions were from external fertilization directly to paternal care and from external fertilization to maternal care via the intermediate step of internal fertilization. We also used maximum likelihood phylogenetic methods to test for statistical correlations and contingencies in the evolution of pairs of reproductive traits. Sexual dichromatism and nest construction proved to be positively correlated with the evolution of male parental care in species with external fertilization. Sexual dichromatism was also positively correlated with female-internal fertilization and gestation. No clear indication emerged that female-only care or biparental care were evolutionary outgrowths of male-only care, or that biparental care has been a common evolutionary stepping stone between paternal and maternal care. Results are discussed in the context of prior thought about the evolution of alternative parental care modes in vertebrates.     

Cardiac outflow tract defects in mice lacking ALK2 in neural crest cells

Cardiac neural crest cells are multipotent migratory cells that contribute to the formation of the cardiac outflow tract and pharyngeal arch arteries. Neural crest-related developmental defects account for a large proportion of congenital heart disorders. Recently, the genetic bases for some of these disorders have been elucidated, and signaling pathways required for induction, migration and differentiation of cardiac neural crest have emerged. Bone morphogenetic proteins comprise a family of secreted ligands implicated in numerous aspects of organogenesis, including heart and neural crest development. However, it has remained generally unclear whether BMP ligands act directly on neural crest or cardiac myocytes during cardiac morphogenesis, or function indirectly by activating other cell types. Studies on BMP receptor signaling during organogenesis have been hampered by the fact that receptor knockouts often lead to early embryonic lethality. We have used a Cre/loxP system for neural crest-specific deletion of the type I receptor, ALK2, in mouse embryos. Mutant mice display cardiovascular defects, including persistent truncus arteriosus, and abnormal maturation of the aortic arch reminiscent of common forms of human congenital heart disease. Migration of mutant neural crest cells to the outflow tract is impaired, and differentiation to smooth muscle around aortic arch arteries is deficient. Moreover, in Alk2 mutants, the distal outflow tract fails to express Msx1, one of the major effectors of BMP signaling. Thus, the type I BMP receptor ALK2 plays an essential cell-autonomous role in the development of the cardiac outflow tract and aortic arch derivatives.