Impairment of gastric acid secretion and increase of embryonic lethality in Foxq1-deficient mice.

The mouse Foxq1 gene, also known as Hfh1, encodes a winged helix/forkhead transcription factor. In adult mice, Foxq1 is highly expressed in kidney and stomach. Here, we report that Foxq1 is expressed during prenatal and postnatal stomach development and the transcripts are restricted to acid secreting parietal cells. Mice homozygous for a deletion of the Foxq1 locus on a 129/Sv x C57BL/6J hybrid genetic background display variable phenotypes consistent with requirement of the gene during embryogenesis. Approximately 50% of Foxq1-/- embryos die in utero. Surviving homozygous mutants are normal and fertile, and have a silky shiny coat. Although the parietal cell development is not affected in the absence of Foxq1, there is a lack of gastric acid secretion in response to various secretagogue stimuli. Ultrastructural analysis suggests that the gastric acid secretion defect in Foxq1-deficient mice might be due to impairment in the fusion of cytoplasmic tubulovesicles to the apical membrane of secretory canaliculi.

Does the stage 16 embryo in Hamburger-Hamilton's "Series of normal stages in the development of the chick embryo" have a potential "conotruncal" heart defect?

The classical Hamburger and Hamilton (HH) paper demonstrates the normal stages of development of the chick embryo that have been extensively used as the basis of understanding normal and abnormal development of the chick embryo heart. Careful examination of the series of images published in this seminal paper indicates that the cardiac images of stage 16 embryo shown in this article may reflect an abnormally developed heart. In this article, the argument is presented that the embryo depicted in the HH paper is not normal, but instead inflicted with a conotruncal heart defect.

Prenatal diagnosis of a large de novo terminal deletion of chromosome 11q.

OBJECTIVE: To describe the prenatal phenotype of the 11q deletion syndrome (Jacobsen syndrome) and present the molecular characterization of the deletion in the case presented. CASE: Ultrasound at 18 and 20 weeks of gestation, on a 34-year-old woman who presented for amniocentesis, revealed slow movements, oligohydramnios and dilatation of the cerebral ventricles in the fetus. Maternal and paternal ages were 34 and 38 years, respectively. RESULTS: Prenatal karyotyping of cultured amniotic fluid cells revealed an 11q terminal deletion, 46,XX,del(11)(q23) (Jacobsen syndrome). Real-time quantitative PCR analysis was used to identify and map the breakpoint physically to a 45-kb region located 14.5 Mb from the 11q telomere. Polymorphic DNA marker analysis showed that DNA sequences on the paternally derived chromosome are deleted. At autopsy, facial dysmorphism without major malformations was recorded. Examination of the internal organs disclosed the following abnormalities: a Meckels' diverticulum of 4-mm length, adhesion between the gall bladder and the transverse colon, and bilaterally bilobed lungs without further situs anomalies. CONCLUSION: Our case demonstrates significant phenotypic variability of Jacobsen syndrome at midtrimester pregnancy; the syndrome may be manifested at this stage only by mild to moderate ventriculomegaly of the brain.

Does an equivalent of the "ventral node" exist in chick embryos? A scanning electron microscopic study.

The internal organs of vertebrates show species-specific left-right (L-R) asymmetries. Questions on the embryonic origin of these asymmetries have been fascinating embryologists since the 19th century. During the past years, remarkable progress has been made in answering these questions. Evolutionary highly conserved molecular signaling cascades have been identified that start from Hensen's node and transfer side-specific identity to the embryonic left and right halves. However, the question of what initiates these signaling cascades has remained unanswered. Studies on mouse embryos have shown that the ventral surface of Hensen's node consists of a ciliated epithelium called the ventral node. Recent findings suggest that the monocilia of ventral nodel cells generate a leftward flow of extracellular fluid possibly leading to the accumulation of an unknown morphogen at the left of the node, which then might start the signaling cascades. This hypothesis might explain the fact that gene defects causing ciliary dyskinesia are frequently associated with situs anomalies. Studies on chick embryos led to the discovery of the L-R signaling cascades. However, whether an equivalent of the ventral node exists in avian embryos remained unknown. Therefore, I examined the endoderm and epiblast of early chick embryos for the presence of monociliated cells. In the endoderm, a population of monociliated cells indeed was present. These cells, however, were neither confined to the area of Hensen's node nor did they form the predominant cell population at this location. In the epiblast, monociliated cells formed the predominant cell population at the periphery of the blastodisc but only a relatively small subpopulation of epiblast cells at Hensen's node. These findings suggest that, in the early chick embryo, an equivalent of the ventral node of mouse embryos neither exists on the ventral nor the dorsal surface of Hensen's node. It is unlikely that nodal cilia are required for initiating the L-R patterning in chick embryos.

The origin, formation and developmental significance of the epicardium: a review.

Questions on the embryonic origin and developmental significance of the epicardium did not receive much recognition for more than a century. It was generally thought that the epicardium was derived from the outermost layer of the primitive myocardium of the early embryonic heart tube. During the past few years, however, there has been an increasing interest in the development of the epicardium. This was caused by a series of new embryological data. The first data showed that the epicardium did not derive from the primitive myocardium but from a primarily extracardiac primordium, called the proepicardial serosa. Subsequent data then suggested that the proepicardial serosa and the newly formed epicardium provided nearly all cellular elements of the subepicardial and intermyocardial connective tissue, and of the coronary vasculature. Recent data even suggest important modulatory roles of the epicardium and of other proepicardium-derived cells in the differentiation of the embryonic myocardium and cardiac conduction system. The present paper reviews our current knowledge on the origin and embryonic development of the epicardium.

Horseshoe lung: report on a new variant--"inverted" horseshoe lung--with embryological reflections on the formal pathogenesis of horseshoe lungs.

The term "horseshoe lung" is used to describe a rare congenital anomaly of the lungs that is characterized by the presence of a midline isthmus of pulmonary parenchyma connecting the posterobasal regions of the right and left lungs. Since the introduction of the term horseshoe lung in the 1960's, almost 40 cases have been reported in the literature. In all these cases, the right and left lungs were joined in their posterobasal regions, the situation resembling that found in horseshoe kidneys. Here we present a case of connection between the right and left lungs found during necropsy of a human fetus. In this case, a midline isthmus of pulmonary parenchyma covered by visceral pleura joined the apical regions of the right and left lungs behind the trachea and esophagus. This connection resulted in a "horseshoe"-shaped lung in which the "horseshoe" was turned by 180 degrees compared to classical cases of horseshoe lung. To the best of our knowledge, this is the first report on an "inverted" horseshoe lung. Embryological reflections on the formal pathogenesis of inverted and classical horseshoe lungs are presented.

Cardiac looping in the chick embryo: a morphological review with special reference to terminological and biomechanical aspects of the looping process.

Understanding early cardiac morphogenesis, especially the process of cardiac looping, is of fundamental interest for diverse biomedical disciplines. During the past few years, remarkable progress has been made in identifying molecular signaling cascades involved in the control of cardiac looping. Given the rapid accumulation of new data on genetic, molecular, and cellular aspects of early cardiac morphogenesis, and given the widespread interest in cardiac looping, it seems worth reviewing those aspects of the looping process that have received less attention during the past few years. These are terminological problems, the "gross" morphological aspects, and the biomechanical concepts of cardiac looping. With respect to terminology, emphasis is given to the unperceived fact that different viewpoints exist as to which part of the normal sequence of morphogenetic events should be called cardiac looping. In a short-term version, which is preferred by developmental biologists, cardiac looping is also called dextral- or rightward-looping. Dextral-looping comprises only those morphogenetic events leading to the transformation of the originally straight heart tube into a c-shaped loop, whose convexity is normally directed toward the right of the body. Cardioembryologists, however, regard cardiac looping merely as a long-term process that may continue until the subdivisions of the heart tube and vessel primordia have approximately reached their definitive topographical relationship to each other. Among cardioembryologists, therefore, three other definitions are used. Taking into account the existence of four different definitions of the term cardiac looping will prevent some confusion in communications on early cardiac morphogenesis. With respect to the gross morphological aspects, emphasis is given to the following points. First, the straight heart tube does not consist of all future regions of the mature heart but only of the primordia of the apical trabeculated regions of the future right and left ventricles, and possibly a part of the primitive conus (outflow tract). The remaining part of the primitive conus and the primordia of the great arteries (truncus arteriosus), the inflow of both ventricles, the primitive atria, and the sinus venosus only appear during looping at the arterial (truncus arteriosus) and venous pole (other primordia). Second, dextral-looping is not simply a bending of the straight heart tube toward the right of the body, as it has frequently been misinterpreted. It results from three different morphogenetic events: (a) bending of the primitive ventricular region of the straight heart tube toward its original ventral side; (b) rotation or torsion of the bending ventricular region around a craniocaudal axis to the right of the body, so that the original ventral side of the heart tube finally forms the right convex curvature and the original dorsal side forms the left concave curvature of the c-shaped heart loop; (c) displacement of the primitive conus to the right of the body by kinking with respect to the arterial pole. Third, dextral-looping does not bring the subdivisions of the heart tube and vessel primordia approximately into their definitive topographical relationship to each other. This is achieved by the morphogenetic events following dextral-looping. This review seeks to bring together data from the diverse disciplines working on the developing heart.

Embryology of congenital ventriculo-coronary communications: a study on quail-chick chimeras.

Ventriculo-coronary arterial communications are rare congenital heart defects which have been explained traditionally on the basis of abnormal persistence of such communications found in the normal developing heart. Recent studies, however, have suggested that these embryonic communications might be an incidental finding rather than a normal feature. Thus, it has been suggested that congenital ventriculo-coronary communications do not represent remnants of normal embryonic vessels, but rather represent acquired lesions. In the present study, hearts were constructed in embryonic chicks in which the coronary vasculature was almost completely derived from a quail-donor. After immunohistochemical staining of the quail-derived coronary endothelium, chimeric hearts were analysed with respect to the presence of embryonic ventriculo-coronary communications, and with respect to the origin of these structures from either coronary arteries or endocardium. The results demonstrate the normal presence of ventriculo-coronary communications in avian embryonic hearts. They show, furthermore, that these structures are of coronary endothelial origin. The findings are in accord with the traditional view on the pathogenesis of congenital ventriculo-coronary communications. The roles of elevated ventricular pressure, abnormal remodelling of the developing myocardium, and of abnormal growth of the coronary vasculature are discussed relative to the pathogenesis of congenital ventriculo-coronary communications.

Does the subepicardial mesenchyme contribute myocardioblasts to the myocardium of the chick embryo heart? A quail-chick chimera study tracing the fate of the epicardial primordium.

Morris (J. Anat., 1976;121:47-64) proposed that the subepicardial mesenchyme might represent a continuing source of myocardioblasts during embryonic and fetal development. Recent studies have shown that the epicardium and subepicardial mesenchyme, and the coronary vasculature are all derived from a region of the pericardial wall, called the proepicardial serosa. In avian embryos, the cells from the proepicardial serosa colonize the heart via a secondary tissue bridge formed by attachment of proepicardial villi to the heart. In the present study, Morris's hypothesis was tested by tracing the fate of the proepicardial serosa. This was achieved by constructing quail-chick chimeras. The proepicardial serosa was transplanted from HH16/17 quail embryos to HH16/17 chick embryos (ED3). A new transplantation technique facilitated an orthotopic attachment of the quail proepicardial villi to the chicken heart, and prevented the attachment of the chicken proepicardial villi to the heart. The fate of the grafted quail cells was traced in chimeras from ED4 to ED18 with immunohistochemistry, using quail-specific antibodies (QCPN, QH-1). From ED4 onward, the transplant was connected to the dorsal heart wall via its proepicardial villi. Starting from the point of attachment of the quail proepicardial villi to the heart, the originally naked myocardium became almost completely covered by quail-derived epicardium, and quail mesenchymal cells populated the subepicardial, myocardial, and subendocardial layers including the av-endocardial cushions. Quail cells formed the endothelial and smooth muscles cells of the coronary vessels, and the perivascular and intramyocardial fibroblasts. Quail myocardial cells were never found in the subepicardial, myocardial, and subendocardial layers. This suggests that the subepicardial mesenchyme normally does not contribute a substantial number of myocardioblasts to the developing avian heart. The new transplantation technique presented facilitates the production of chimeric hearts in which the derivatives of the proepicardial serosa are almost completely of donor origin. This technique might be useful for future studies analyzing the role of certain genes in cardiac development by the creation of somatic transgenics.

Complete transposition in a chick embryo demonstrated by scanning electron microscopy.

Chick embryos are frequently used as animal models when researching the developing heart. In the past, every attempt to induce complete transposition (the combination of concordant atrioventricular and discordant ventriculo-arterial connections) failed in chicks, suggesting that it might be impossible to develop a chicken model for this malformation. We demonstrate, to the best of our knowledge, the first well-documented case of complete transposition occurring in the chick.

Embryological observations on the formal pathogenesis of double-outlet left ventricle with a right-ventricular infundibulum.

The term 'double-outlet left ventricle' (DOLV) denotes congenital heart malformations in which the aorta and the pulmonary trunk both arise entirely or predominantly above the morphologically left ventricle. In the past, the formal pathogenesis of DOLV was explained by an abnormal topogenesis of the origin of the great arteries, which could be caused by an excessive leftward shift of the embryonic conotruncus, or by errors in differential conal growth or absorption. However, modern embryological and pathological research is casting doubt on the validity of these concepts. In the present paper we demonstrate a case of DOLV found in a chick fetus. In this heart the main derivatives of the embryonic conotruncus (right-ventricular infundibulum and proximal portions of the great arteries) principally are in the normal position and of normal dimensions. The anomaly leading to DOLV under these conditions is a misalignment of the ventricular septum. The subarterial portion of the ventricular septum above the crista supraventricularis is not oriented in the normal oblique plane between the pulmonary and aortic valve, but is oriented in a frontal plane anterior to the origin of both great arteries. The consequences of this anomaly are the separation of the right-ventricular infundibulum from the origin of both great vessels (DOLV) and a lack of continuity between the malpositioned portion of the ventricular septum (posterior wall of the right-ventricular infundibulum) and a septum dividing the semilunar valve level. The infundibulum of the right ventricle is derived from the upstream portion of the embryonic conotruncus (conus) whereas the semilunar valves and great arteries are derived from its downstream protion (truncus arteriosus) and the aortic sac. Therefore, our findings suggest that the division of the conotruncus is performed by at least two different septa, one dividing the conus and another dividing the truncus arteriosus and aortic sac. The misalignment of the ventricular septum leading to the presented type of DOLV could result from a misalignment of the septal anlagen of the embryonic conus, the conus ridges. Our findings are discussed with respect to human cases of DOLV and with respect to a recent concept on the septation of the embryonic conotruncus.

The formal pathogenesis of isolated common carotid or innominate arteries: the concept of malseptation of the aortic sac.

Deficient connections (= isolation) of the innominate artery or the common carotid artery to the aorta are rare congenital anomalies of the human aortic arch complex that are usually associated with a patent vascular connection between the isolated artery and a pulmonary artery. In the present study we demonstrate chick fetuses with a corresponding anomaly, the isolation of the brachiocephalic artery. In our chick fetuses the left brachiocephalic artery did not arise from the aortic arch, but was connected to the pulmonary trunk proximal (upstream) to the patent left and right ductus arteriosus. These cases are of interest because the presence of a congenital pulmonary-systemic arterial connection proximal (upstream) to the ductus arteriosus cannot be explained by the traditional concept of the morphogenesis of the aortic arch complex. The development of the normal and abnormal branching patterns of the aortic arch arteries is traditionally explained by transformation of the primitive embryonic pharyngeal arch arterial system due to obliteration of some of its vascular segments. Based on this concept, the isolation of an aortic arch artery can be explained by obliteration of vascular segments proximal and distal to this artery, whereas its connection to a pulmonary artery can be explained only by deficient obliteration (persistence) of the distal portion of the right or left sixth pharyngeal arch artery. The connecting "vascular segment" between an isolated aortic arch artery and the pulmonary circulation, therefore, is traditionally interpreted as a patent ductus arteriosus. The formal pathogenesis of congenital pulmonary-systemic arterial connections proximal (upstream) to the ductus arteriosus is discussed. The presented cases of isolation of the brachiocephalic artery are explained by disturbances in the partition of the embryonic aortic sac, possibly due to abnormal development of the "cardiac" neural crest.

Experimental study on the significance of abnormal cardiac looping for the development of cardiovascular anomalies in neural crest-ablated chick embryos.

During normal development, ectomesenchyme from the cardiac neural crest migrates to pharyngeal arches 3, 4, 6 and the developing heart. It participates in the formation of the aorticopulmonary septum and the wall of the great arteries. Removal of the cardiac neural crest resulted in anomalies of the great arteries and in two categories of severe heart defects: (1) outflow septation defects of the persistent truncus arteriosus (PTA) type, (2) alignment defects. It has been hypothesized that PTA occurs if the number of cardiac neural crest cells is reduced below a level critical for complete formation of the aorticopulmonary septum. Alignment defects would be indirect consequences of neural crest defects, possibly caused by altered blood flow in the pharyngeal arch region. We found that these concepts were not in agreement with some experimental facts reported previously, so we considered whether there could be other mechanisms responsible for the heart defects described. To investigate whether mechanical interference with cardiac looping could possibly contribute to the pathogenesis of these anomalies, we removed the entire cardiac neural crest in chick embryos with micro-needles. Postoperative development was checked during cardiac looping and after normal completion of cardiac septation. Our data suggested that abnormal cardiac looping did not contribute to the pathogenesis of the aortic arch artery anomalies and PTA. With respect to the alignment heart defects, we could not elucidate the role of looping anomalies because we did not observe such heart defects. Moreover, PTA occurred only in 28% of survivors. This finding conflicts with previous studies where extensive ablation of the cardiac neural crest has led to a high incidence of PTA (73-100% of survivors). The possible reasons for this discrepancy are discussed. It is shown that the use of different microsurgical techniques (mechanical cutting/microcautery) may be responsible for the different incidence of PTA. We speculate that microcautery hampers a normal complete repair of neural crest defects, possibly by release of abnormally high levels of growth factors.

Embryological observations on the morphogenesis of double-outlet right ventricle with subaortic ventricular septal defect and normal arrangement of the great arteries.

Double-outlet ventricle (DORV) is generally regarded as a congenital heart defect resulting from impaired morphogenesis of either the outflow portion (conotruncus) or the conoventricular flange (crista prima) of the embryonic heart. However, we demonstrate in this study chicken fetal hearts with DORV in which the conotruncal derivatives (great arteries and subpulmonary part of the ventricular septum = conus septum) and the region of the crista prima are normally developed. The anomalies leading to DORV under these conditions are found at the atrioventricular region. The posterior-anterior axis of the tricuspid orifice is not directed to the right anterior but to the left anterior side of the heart. Thereby the posterior connection line between the muscular ventricular septum and conus septum, which usually follows the left margin of the tricuspid orifice, is not connected to the right portion of the conus septum but instead is directed towards the left portion of the conus septum. In consequence of the abnormal connection between the muscular ventricular septum and the conus septum, the interventricular foramen is formed at the left side of the subaortic flow path. The subaortic flow path arises from the right ventricle. These findings show that DORV can result not only from impaired development of the conotruncus or conoventricular flange, but also from abnormal development of the atrioventricular region. We suggest distinguishing between conotruncal, conoventricular (crista prima), and atrioventricular types of DORV.

The role of extracardiac factors in normal and abnormal development of the chick embryo heart: cranial flexure and ventral thoracic wall.

The present study was designed to investigate whether the formation of the cranial flexure is involved in the normal positional changes of the embryonic heart tube that occur during its transformation from the c- to s-shaped loop. For this purpose, the formation of the cranial flexure was locally suppressed in chick embryos by introducing a straight hair into the neural canal. In the experimental embryos, prevention of cranial flexure did not suppress the normal positional changes of the heart tube. However, other anomalies in the looping of the heart tube were frequently observed. These anomalies were caused by alterations in the formation of the ventral thoracic wall, which in turn seemed to be related not to the prevention of the cranial flexure but rather to accidental injuries during the implantation of the hair. In the embryos with abnormal looping of the heart tube, the incidence of delayed/defective septation of the heart was significantly higher than in embryos with normal looping. These results show that in the chick embryo: (1) cranial flexure is not involved in normal positional changes of the heart loop; (2) manipulations at the head region of the embryo can unintentionally result in developmental disorders of the ventral thoracic wall; (3) such disorders can result in congenital heart defects through mechanical interference with normal looping of the embryonic heart. The possible significance of these findings for the evaluation of experimental studies of chick embryos is discussed in the context of anomalies observed after surgical ablation of the premigratory cranial neural crest.

Formation of the cervical flexure: an experimental study on chick embryos.

It has been proposed that the cervical flexure of vertebrate embryos arises from the normal morphogenesis of the heart. This hypothesis is based on experiments in which the heart tube is removed or disrupted in early chick embryos. It has been reported that, in normal atmosphere, these embryos continued normal morphogenesis except for cervical flexure formation. In the present study, we performed similar experiments. In contrast to previous work, however, only one set of our heart-deprived chick embryos was reincubated in normal air. The other sets were reincubated in oxygen-enriched air. Under normoxia, heart removal resulted not only in prevention of the cervical flexure, but also in mesenchymal defects, and in a remarkable hypoplasia of the craniocervical region. Under hyperoxia, heart-deprived embryos developed no severe mesenchymal defects and the growth of the upper body portion was more normal, with the hypoplasia confined to the cranial region. The formation of the cervical flexure was now normalized. These results show that cervical flexure formation is not directly dependent on normal morphogenesis of the heart, but does depend on a sufficient oxygen supply to the cervical region. During early development, the cranio-cervical region of a chick embryo is more sensitive to circulatory failure than the trunk.