Showing papers in "Anatomical Record-advances in Integrative Anatomy and Evolutionary Biology in 2000"

Developmental patterning of the myocardium.

Abstract: The heart in higher vertebrates develops from a simple tube into a complex organ with four chambers specialized for efficient pumping at pressure. During this period, there is a concomitant change in the level of myocardial organization. One important event is the emergence of trabeculations in the luminal layers of the ventricles, a feature which enables the myocardium to increase its mass in the absence of any discrete coronary circulation. In subsequent development, this trabecular layer becomes solidified in its deeper part, thus increasing the compact component of the ventricular myocardium. The remaining layer adjacent to the ventricular lumen retains its trabeculations, with patterns which are both ventricle- and species-specific. During ontogenesis, the compact layer is initially only a few cells thick, but gradually develops a multilayered spiral architecture. A similar process can be charted in the atrial myocardium, where the luminal trabeculations become the pectinate muscles. Their extent then provides the best guide for distinguishing intrinsically the morphologically right from the left atrium. We review the variations of these processes during the development of the human heart and hearts from commonly used laboratory species (chick, mouse, and rat). Comparison with hearts from lower vertebrates is also provided. Despite some variations, such as the final pattern of papillary or pectinate muscles, the hearts observe the same biomechanical rules, and thus share many common points. The functional importance of myocardial organization is demonstrated by lethality of mouse mutants with perturbed myocardial architecture. We conclude that experimental studies uncovering the rules of myocardial assembly are relevant for the full understanding of development of the human heart.

Morphology, distribution, and variability of the epicardiac neural ganglionated subplexuses in the human heart

Abstract: Concomitant with the development of surgical treatment of cardiac arrythmias and management of myocardial ischemia, there is renewed interest in morphology of the intrinsic cardiac nervous system. In this study, we analyze the topography and structure of the human epicardiac neural plexus (ENP) as a system of seven ganglionated subplexuses. The morphology of the ENP was revealed by a histochemical method for acetylcholinesterase in whole hearts of 21 humans and examined by stereoscopic, contact, and bright-field microscopy. According to criteria established to distinguish ganglionated subplexuses, they are epicardiac extensions of mediastinal nerves entering the heart through discrete sites of the heart hilum and proceeding separately into regions of innervation by seven pathways, on the courses of which epicardiac ganglia, as wide ganglionated fields, are plentifully located. It was established that topography of epicardiac subplexuses was consistent from heart to heart. In general, the human right atrium was innervated by two subplexuses, the left atrium by three, the right ventricle by one, and the left ventricle by three subplexuses. The highest density of epicardiac ganglia was identified near the heart hilum, especially on the dorsal and dorsolateral surfaces of the left atrium, where up to 50% of all cardiac ganglia were located. The number of epicardiac ganglia identified for the human hearts in this study ranged from 706 up to 1,560 and was not correlated with age in most heart regions. The human heart contained on average 836 +/- 76 epicardiac ganglia. The structural organization of ganglia and nerves within subplexuses was observed to vary considerably from heart to heart and in relation to age. The number of neurons identified for any epicardiac ganglion was significantly fewer in aged human compared with infants. By estimating the number of neurons within epicardiac ganglia and relating this to the number of ganglia in the human epicardium, it was calculated that approximately 43,000 intrinsic neurons might be present in the ENP in adult hearts and 94,000 neurons in young hearts (fetuses, neonates, and children). In conclusion, this study demonstrates the total ENP in humans using staining for acetylcholinesterase, and provides a morphological framework for an understanding of how intrinsic ganglia and nerves are structurally organized within the human heart.

Mechanisms involved in valvuloseptal endocardial cushion formation in early cardiogenesis: Roles of transforming growth factor (TGF)‐β and bone morphogenetic protein (BMP)

Abstract: Endothelial-mesenchymal transformation (EMT) is a critical event in the generation of the endocardial cushion, the primordia of the valves and septa of the adult heart. This embryonic phenomenon occurs in the outflow tract (OT) and atrioventricular (AV) canal of the embryonic heart in a spatiotemporally restricted manner, and is initiated by putative myocardially derived inductive signals (adherons) which are transferred to the endocardium across the cardiac jelly. Abnormal development of endocardial cushion tissue is linked to many congenital heart diseases. At the onset of EMT in chick cardiogenesis, transforming growth factor (TGFbeta)-3 is expressed in transforming endothelial and invading mesenchymal cells, while bone morphogenetic protein (BMP)-2 is expressed in the subjacent myocardium. Three-dimensional collagen gel culture experiments of the AV endocardium show that 1) myocardially derived inductive signals upregulate the expression of AV endothelial TGFbeta3 at the onset of EMT, 2) TGFbeta3 needs to be expressed by these endothelial cells to trigger the initial phenotypic changes of EMT, and 3) myocardial BMP2 acts synergistically with TGFbeta3 in the initiation of EMT.

Extracellular matrix of the human aortic media: an ultrastructural histochemical and immunohistochemical study of the adult aortic media.

Abstract: Aortic distensability is the key to normal aortic function and relates to the lamellar unit in the media. However, the organization of the extracellular matrix components in these lamellar units, which are largely responsible for the distensability, is insufficiently known, especially in the human. We therefore performed a detailed ultrastructural analysis of these components. Thoracic aortas of 56 individuals (age 45-74 years), none of whom suffered from aortic disease, were studied by immunoelectron microscopy of elastin, collagen types I, III, IV, V, and VI, fibronectin, and fibrillin-1, and by ultrastructural histochemistry of proteoglycans, which were further characterized by enzymatic digestion. The elastic lamellae were closely associated with thick collagen fibers containing types I, III, and V collagen. Between these collagen fibers, numerous complex, circumferentially oriented streaks of elastin protruded from the lamellae. In contrast to what is usually reported in the aortas of experimental animals, the smooth muscle cells preferentially adhered to these ill-defined streaks rather than directly to the solid lamellae. Fibrillin-1- and type VI collagen-containing bundles of microfibrils (oxytalan fibers) were also involved in the smooth muscle cell-elastin contact. The smooth muscle cells were invested by basal lamina-like layers connecting them to each other as well as to the oxytalan fibers. Unexpectedly, these layers were abundantly labeled by anti-fibronectin, whereas type IV collagen, a specific basement membrane component, was mainly found in larger, flocculent deposits. The proteoglycans present were collagen-associated dermatan sulfate proteoglycan, cell-associated heparan sulfate proteoglycan, and interstitial chondroitin sulfate proteoglycan. Our observations demonstrate that the extracellular matrix in the human aorta is extremely complex and therefore differs from most descriptions based on experimental animals. They serve as reference for future studies on aortic diseases, such as aneurysmas and dissections.

Structure and function of the developing zebrafish heart.

Abstract: The combination of optical clarity and large scale of mutants makes the zebrafish vital for developmental biologists. However, there is no comprehensive reference of morphology and function for this animal. Since study of gene expression must be integrated with structure and function, we undertook a longitudinal study to define the cardiac morphology and physiology of the developing zebrafish. Our studies included 48-hr, 5-day, 2-week, 4-week, and 3-month post-fertilization zebrafish. We measured ventricular and body wet weights, and performed morphologic analysis on the heart with H&E and MF-20 antibody sections. Ventricular and dorsal aortic pressures were measured with a servonull system. Ventricular and body weight increased geometrically with development, but at different rates. Ventricle-to-body ratio decreased from 0.11 at 48-hr to 0.02 in adult. The heart is partitioned into sinus venosus, atrium, ventricle, and bulbus arteriosus as identified by the constriction between the segments at 48-hr. Valves were formed at 5-day post-fertilization. Until maturity, the atrium showed extensive pectinate muscles, and the atrial wall increased to two to three cell layers. The ventricular wall and the compact layer increased to three to four cell layers, while the extent and complexity in trabeculation continued. Further thickening of the heart wall was mainly by increase in cell size. The bulbus arteriosus had similar characteristics to the myocardium in early stages, but lost the MF-20 positive staining, and transitioned to smooth muscle layer. All pressures increased geometrically with development, and were linearly related to stage-specific values for body weight (P < 0.05). These data define the parameters of normal cardiac morphology and ventricular function in the developing zebrafish.

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

Abstract: 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. Anat Rec 259:248–262, 2000. © 2000 Wiley-Liss, Inc.

Cell death in denervated skeletal muscle is distinct from classical apoptosis

Abstract: Denervation of skeletal muscle is followed by the progressive loss of tissue mass and impairment of its functional properties. The purpose of the present study was to investigate the occurrence of cell death and its mechanism in rat skeletal muscle undergoing post-denervation atrophy. We studied the expression of specific markers of apoptosis and necrosis in experimentally denervated tibialis anterior, extensor digitorum longus and soleus muscles of adult rats. Fluorescent staining of nuclear DNA with propidium iodide revealed the presence of nuclei with hypercondensed chromatin and fragmented nuclei typical of apoptotic cells in the muscle tissue 2, 4 and to a lesser extent 7 months after denervation. This finding was supported by electron microscopy of the denervated muscle. We found clear morphological manifestations of muscle cell death, with ultrastructural characteristics very similar if not identical to those considered as nuclear and cytoplasmic markers of apoptosis. With increasing time of denervation, progressive destabilization of the differentiated phenotype of muscle cells was observed. It included disalignment and spatial disorganization of myofibrils as well as their resorption and formation of myofibril-free zones. These changes initially appeared in subsarcolemmal areas around myonuclei, and by 4 months following nerve transection they were spread throughout the sarcoplasm. Despite an increased number of residual bodies and secondary lysosomes in denervated muscle, we did not find any evidence of involvement of autophagocytosis in the resorption of the contractile system. Dead muscle fibers were usually surrounded by a folded intact basal lamina; they had an intact sarcolemma and highly condensed chromatin and sarcoplasm. Folds of the basal lamina around the dead cells resulted from significant shrinkage of cell volume. Macrophages were occasionally found in close proximity to dead myocytes. We detected no manifestations of inflammation in the denervated tissue. Single myocytes expressing traits of the necrotic phenotype were very rare. A search for another marker of apoptosis, nuclear DNA fragmentation, using terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (the TUNEL method) in situ, revealed the presence of multiple DNA fragments in cell nuclei in only a very small number of cell nuclei in 2 and 4 month denervated muscle and to less extent in 7 month denervated muscle. Virtually no TUNEL reactivity was found in normal muscle. Double labeling of tissue denervated for 2 and 4 months for genome fragmentation with the TUNEL method and for total nuclear DNA with propidium iodide demonstrated co-localization of the TUNEL-positive fragmented DNA in some of the nuclei containing condensed chromatin and in fragmented nuclei. However, the numbers of nuclei of abnormal morphology containing condensed and/or irregular patterns of chromatin distribution, as revealed by DNA staining and electron microscopy, exceeded by 33-38 times the numbers of nuclei positive for the TUNEL reaction. Thus, we found a discrepancy between the frequences of expression of morphological markers of apoptosis and DNA fragmentation in denervated muscle. This provides evidence that fragmentation of the genomic DNA is not an obligatory event during atrophy and death of muscle cells, or, alternatively, it may occur only for a short period of time during this process. Unlike classical apoptosis described in mammalian thymocytes and lymphoid cells, non-inflammatory death of muscle fibers in denervated muscle occurs a long time after the removal of myotrophic influence of the nerve and is preceded by the progressive imbalance of the state of terminal differentiation. Our results indicate that apoptosis appears to be represented by a number of distinct isotypes in animals belonging to different taxonomic groups and in different cell lineages of the same organism.

Neuronal density in the human retinal ganglion cell layer from 16–77 years

Abstract: Literature assessing whether or not neurons (retinal ganglion cells and displaced amacrine cells) are lost from the retinal ganglion cell layer in mammals with age is still controversial, some studies finding a decrease in cell density and others not. To date there have been no studies estimating the total number of neurons in the retinal ganglion cell layer of humans throughout life. Recent studies have concentrated on the macular region and examined cell densities, which are reported to decrease during aging. In a study of the human retinal pigment epithelium (RPE), we showed that, while RPE cell number does not change, cell density increases significantly in central temporal retina (macular region) as the retina ages. We speculated that the increase in density represents a "drawing together" of the retinal sheet to maintain high cell densities, in this region of the neural retina, in the face of presumed cell loss from the ganglion cell layer due to aging. Here, therefore, we have sampled the entire ganglion cell layer of the human retina and estimated total neuron numbers in 12 retinae aged from 16 to 77 years. Human retinae, fixed in formalin, were obtained from the Queensland Eye Bank and whole-mounted, ganglion cell layer uppermost. The total number of neurons was lower in the older than younger retinae and neuronal density was lower in most retinal regions in older retinae. Retinal area increased with age and neuronal density fell throughout the retina with a mean reduction of 0.53% per year. However, the percentage reduction in density was much lower for the macular region, with a value of 0.29% per year. It is possible that this lesser reduction in cell density in the macula is a result of the drawing together of the retinal sheet in this region as we speculated from RPE data.

Angiogenesis: new insights and therapeutic potential.

Abstract: Angiogenesis, the formation of vessels from pre-existing vessels, is of critical importance not only during normal growth, but also in pathological situations. In the latter, some diseases are enhanced by excessive vascular growth (e.g., tumors), whereas in others inadequate vascular growth contributes to morbidity and mortality (e.g., ischemic heart disease). Our current state of knowledge makes it clear that the cascade of angiogenic events depends on complex processes that include cell-cell interactions, various intracellular signaling pathways, and the appropriate extracellular microenvironment. The literature regarding angiogenesis has increased exponentially during the last decade. Progress in this area is largely a consequence of advances in our understanding of angiogenic growth factor and cytokine function, in part due to the determination of their complete amino acid sequences and cloning of their genes. Other factors also play key roles in angiogenesis, including the extracellular matrix, adhesion molecules and their inhibitors, and metabolic and mechanical factors. The potential for developing therapeutic protocols has been enhanced by data from both in vitro and in vivo studies and has provided the rationale for clinic trials. Angiogenic therapy strategies include inhibition of aberrant angiogenesis, as seen in tumors or diabetes, as well as stimulation of angiogenesis in conditions of ischemia, such as ischemic heart or peripheral vascular disease. Anat Rec (New Anat) 261:126–135, 2000. © 2000 Wiley-Liss, Inc.

Skeletal muscle satellite cell populations in healthy young and older men and women.

Abstract: The purpose of the present investigation was to assess satellite cell populations and morphology in m. vastus lateralis biopsies obtained from young (20-30 years) and older (65-75 years) healthy, sedentary men and women. Multiple muscle biopsies were obtained from 14 young individuals (men, n = 7; women, n = 7) and 15 older individuals (men, n = 8; women, n = 7). Muscle fibers were viewed longitudinally using a Zeiss EM 10 CA electron microscope. All myonuclei and satellite cells were counted and satellite cells were photographed for morphological analysis. The proportion of satellite cells [satellite cells/(myonuclei + satellite cells)] did not differ among the four subject groups (1.7-2.8%), nor did proportions differ when subject groups were combined for age and gender comparisons. Few morphological differences were noted between groups; however, lipofuscin granules were more prominent in satellite cells from older subjects and women demonstrated significantly larger satellite cell and satellite cell nucleus areas than men. Mitochondria from satellite cells (regardless of group) were more pallid and exhibited fewer cristae than mitochondria located in the adjacent muscle fiber. The results of the current investigation suggest that, despite findings in animal models, satellite cell populations are not significantly lower in healthy, sedentary older compared to young adult men and women.

Remodeling of the vascular bed and progressive loss of capillaries in denervated skeletal muscle.

Abstract: Very little is known regarding structural and functional responses of the vascular bed of skeletal muscle to denervation and about the role of microcirculatory changes in the pathogenesis of post-denervation muscle atrophy The purpose of the present study was to investigate the changes of the anatomical pattern of vascularization of the extensor digitorum longus muscle in WI/HicksCar rats 1, 2, 4, 7, 12, and 18 months following denervation of the limb We found that the number of capillaries related to the number of muscle fibers, ie the capillary-to-fiber ratio (CFR), decreased by 88%, from 155 +/- 035 to 019 +/- 004, during the first 7 months after denervation and then slightly declined at a much lower rate during the next 11 months of observation to 10% of the CFR in normal muscle Between months 2 and 4 after denervation, the CRF decreased by 24 times, from 58% to 24% of the control value The loss of capillaries during the first 4 months following nerve transection was nearly linear and progressed with an average decrement of 416% per week Electron microscopy demonstrated progressive degeneration of capillaries following nerve transection In muscle cells close to degenerating capillaries, the loss of subsarcolemmal and intermyofibrillar mitochondria, local disassembly of myofibrils and other manifestations of progressive atrophy were frequently observed The levels of devascularization and the degree of degenerative changes varied greatly within different topographical areas, resulting in significant heterogeneity of intercapillary distances and local capillary densities within each sample of denervated muscle Perivascular and interstitial fibrosis that rapidly developed after denervation resulted in the spatial separation of blood vessels from muscle cells and their embedment in a dense lattice of collagen As a result of this process, diffusion distances between capillaries and the surfaces of muscle fibers increased 10-400 times Eighteen months after denervation most of the capillaries were heavily cushioned with collagen, and on the average 40% of the muscle cells were completely avascular Devascularization of the tissue was accompanied by degeneration and death of muscle cells that had become embedded in a dense lattice of collagen Immunofluorescent staining for the vascular isoform of alpha-actin revealed preservation of major blood vessels and a greater variability in thickness of their medial layer Hyperplastic growth of the medial layer in some blood vessels resulted in narrowing of their lumens By the end of month 7 after denervation, large deposits of collagen around arterioles often exceeded their diameters Identification of oxidative muscle fibers after immunostaining for slow-twitch myosin, as well as using ultrastructural criteria, has shown that after 2 months of denervation oxidative muscle fibers were less susceptible to atrophy than glycolytic fibers The lower rate of atrophy of type I muscle fibers at early stages of denervation may be explained by their initially better vascularization in normal muscle and their higher capacity to retain capillaries shortly after denervation Thus, degeneration and loss of capillaries after denervation occurs more rapidly than the loss of muscle fibers, which results in progressive decrease of the CFR in denervated muscle The change of capillary number in denervated muscle is biphasic: the phase of a rapid decrease of the CFR during the first 7 months after nerve transection is followed by the phase of stabilization The presence of areas completely devoid of capillaries in denervated muscle and the virtual absence of such areas in normal muscle indicate the development of foci of regional hypoxia during long-term denervation The anatomical pattern of muscle microvascularization changes dramatically after nerve transection Each muscle fiber in normal muscle directly contacts on average 3-5 capillaries (ABSTRACT TRUNCATED)

Reproliferation and relocation of mouse male germ cells (gonocytes) during prespermatogenesis

Abstract: In the prespermatogenesis period, male germ cells (gonocytes) begin to reproliferate and move to the basement membrane of the seminiferous tubule. Although these two events-reproliferation and relocation-are important for establishment of spermatogenesis, they have not been greatly analyzed both in a mechanical and in an endocrine or paracrine aspect. In this study, the relationship between reproliferation and relocation of gonocytes was examined, using the thymidine analog bromodeoxyuridine (BrdU) labeling method and transmission electron microscopy (TEM). BrdU was injected into the fetuses [day 13.5 post coitus (dpc) to 18.5 dpc] and pups [day 0. 5 post partum (dpp) to 6.5 dpp] of C57BL/6J mice. Two hours later, BrdU positive gonocytes were examined immunohistochemically and these data were analyzed. TEM and LM observation was carried out as well. Gonocytes began to relocate on the basement membrane from 18.5 dpc (1.4%) while BrdU-labeled gonocytes were first detected on 1.5 dpp (13.6%). Relocated BrdU-negative gonocytes were recognized from 18.5 dpc (1.4%), and relocated BrdU-labeled gonocytes were recognized from 1.5 dpp (8.4%). On the other hand, non-relocated BrdU-labeled gonocytes were detected from 1.5 dpp (5.2%). Gonocyte relocation began 2 days earlier than reproliferation during the late fetal period. After birth, the two events occurred at random. These results indicate that the reproliferation of the gonocyte does not correlate with relocation. The two events may be regulated by different mechanisms.

Mist1 expression is a common link among serous exocrine cells exhibiting regulated exocytosis

Abstract: Mist1 is a basic helix-loop-helix transcription factor that represses E-box-mediated transcription. Previous studies have suggested that the Mist1 gene is expressed in a wide range of tissues, although a complete characterization of Mist1 protein accumulation in the adult organism has not been described. In an effort to identify specific cell types that contain the Mist1 protein, antibodies specific for Mist1 were generated and used in Western blot and immunohistochemical assays. Our studies show that the Mist1 protein is present in many different tissues but that it is restricted to cell types that are exclusively secretory in nature. Pancreatic acinar cells, serous or seromucous cells of the salivary glands, chief cells of the stomach, and secretory cells of the prostate and seminal vesicle show high levels of Mist1 protein, whereas nonserous exocrine cells, including the mucus-producing cells of the salivary glands, remain Mist1 negative. These results identify Mist1 as the first transcription factor that exhibits this unique serous-specific expression pattern and suggest that Mist1 may have a key role in establishing and maintaining a pathway responsible for the exocytosis of serous secretions.

Atrial development in the human heart: An immunohistochemical study with emphasis on the role of mesenchymal tissues

Abstract: The development of the atrial chambers in the human heart was investigated immunohistochemically using a set of previously described antibodies. This set included the monoclonal antibody 249-9G9, which enabled us to discriminate the endocardial cushion-derived mesenchymal tissues from those derived from extracardiac splanchnic mesoderm, and a monoclonal antibody recognizing the B isoform of creatine kinase, which allowed us to distinguish the right atrial myocardium from the left. The expression patterns obtained with these antibodies, combined with additional histological information derived from the serial sections, permitted us to describe in detail the morphogenetic events involved in the development of the primary atrial septum (septum primum) and the pulmonary vein in human embryos from Carnegie stage 14 onward. The level of expression of creatine kinase B (CK-B) was found to be consistently higher in the left atrial myocardium than in the right, with a sharp boundary between high and low expression located between the primary septum and the left venous valve indicating that the primary septum is part of the left atrial gene-expression domain. This expression pattern of CK-B is reminiscent of that of the homeobox gene Pitx2, which has recently been shown to be important for atrial septation in the mouse. This study also demonstrates a poorly appreciated role of the dorsal mesocardium in cardiac development. From the earliest stage investigated onward, the mesenchyme of the dorsal mesocardium protrudes into the dorsal wall of the primary atrial segment. This dorsal mesenchymal protrusion is continuous with a mesenchymal cap on the leading edge of the primary atrial septum. Neither the mesenchymal tissues of the dorsal protrusion nor the mesenchymal cap on the edge of the primary septum expressed the endocardial tissue antigen recognized by 249-9G9 at any of the stages investigated. The developing pulmonary vein uses the dorsal mesocardium as a conduit to reach the primary atrial segment. Initially, the pulmonary pit, which will becomes the portal of entry for the pulmonary vein, is located along the midline, flanked by two myocardial ridges. As development progresses, tissue remodeling results in the incorporation of the portal of entry of the pulmonary vein in left atrial myocardium, which is recognized because of its high level of creatine. Closure of the primary atrial foramen by the primary atrial septum occurs as a consequence of the fusion of these mesenchymal structures. Copyright 2000 Wiley-Liss, Inc.

Anatomy of the human atrioventricular junctions revisited.

Abstract: There have been suggestions made recently that our understanding of the atrioventricular junctions of the heart is less than adequate, with claims for several new findings concerning the arrangement of the ordinary working myocardium and the specialised pathways for atrioventricular conduction. In reality, these claims are grossly exaggerated. The structure and architecture of the pathways for conduction between the atrial and ventricular myocardium are exactly as described by Tawara nearly 100 years ago. The recent claims stem from a failure to assess histological findings in the light of criterions established by Monckeberg and Aschoff following a similar controversy in 1910. The atrioventricular junctions are the areas where the atrial myocardium inserts into, and is separated from, the base of the ventricular mass, apart from at the site of penetration of the specialised axis for atrioventricular conduction. There are two such junctions in the normal heart, surrounding the orifices of the mitral and tricuspid valves. The true septal area between the junctions is of very limited extent, being formed by the membranous septum. Posterior and inferior to this septal area, the atrial myocardium overlies the crest of the ventricular septum, with the atrial component being demarcated by the landmarks of the triangle of Koch. The adjacent structures, and in particular the so-called inferior pyramidal space, were accurately described by McAlpine (Heart and Coronary Arteries, 1975). Thus, again there is no need for revision of our understanding. The key to unravelling much of the alleged controversy is the recognition that, as indicated by Tawara, the atrioventricular node becomes the atrioventricular bundle at the point where the overall axis for conduction penetrates into the central fibrous body. There are also marked differences in arrangement, also described by Tawara, between the disposition of the conduction axis in man as compared to the dog.

Functional anatomy of the hypoglossal innervated muscles of the rat tongue: a model for elongation and protrusion of the mammalian tongue.

Abstract: This anatomical investigation in the rat was designed to illustrate the detailed organization of the tongue's muscles and their innervation in order to elucidate the actions of the muscles of the higher mammalian tongue and thereby clarify the protrusor subdivision of the hypoglossal-tongue complex. The hypoglossal innervated, extrinsic styloglossus, hyoglossus, and genioglossus and the intrinsic transversus, verticalis and longitudinalis linguae muscles were observed by microdissection and analysis of serial transverse-sections of the tongue. Sihler's staining technique was applied to whole rat tongues to demonstrate the hypoglossal nerve branching patterns. Dissections of the tongue demonstrate the angles at which the extrinsic muscles act on the base of the tongue. The Sihler stained hypoglossal nerves demonstrate branches to the styloglossus and hyoglossus emanating from its lateral division while branches to the genioglossus muscle exit from its medial division. The largest portions of both XIIth nerve divisions can be seen to enter the body of the tongue to innervate the intrinsic muscles. Transverse sections of the tongue demonstrate the organization of the intrinsic muscle fibers of the tongue. Longitudinal muscle fibers run along the entire circumference of the tongue. Alternating sheets of transverse lingual and vertical lingual muscles can be observed to insert into the circumference of the tongue. Most importantly in clarifying tongue protrusion, we demonstrate the transversus muscle fibers enveloping the most superior and inferior portions of the longitudinalis muscles. Longitudinal muscle fascicles are completely encircled and thus are likely to be compressed by transverse muscle fascicles resulting in elongation of the tongue. We discuss our findings in relation to biomechanical studies, that describe the tongue as a muscular hydrostat and thereby define the "elongation-protrusion apparatus" of the mammalian tongue. In so doing, we clarify the functional organization of the hypoglossal-tongue complex.

Study of the functional anatomy of bovine oviductal mucosa.

Abstract: The oviducts of 31 cyclic cows were examined to study the structure and nature of the oviductal mucosa. The general distribution of spermatozoa within the oviductal mucosa was studied in five additional cows. The oviductal infundibulum is an asymmetric funnel-shaped structure surrounding the ostium. It is divided along the free boarder of the mesosalpinx and presents one wide and one narrow side. The mucosa of the wide side possesses a system of low interconnected cords that converge distally forming primary folds. The folds on the narrow side start sharply from the free margin and fuse toward the ostium abdominale. Areas between folds throughout the lumen of the oviduct show a high degree of complex organization. Interfold spaces are occupied by secondary and small interconnected folds which join to form a system of cul-de-sacs. In the infundibulum, these cul-de-sacs open toward the ovary, while cul-de-sacs present in the caudal isthmus and in the UTJ open toward the uterus. Marked variations were observed in the oviductal epithelium depending on the oviductal segment, basal or apical areas of the folds, and phase of the oestrous cycle. Near to the time of ovulation, numerous spermatozoa were found in the periphery of the caudal isthmus within pockets of basal interfold areas, as well as within pockets and cul-de-sacs of the tubo-uterine junction. Individual spermatozoa were also observed in peripheral areas of the ampullary-isthmic junction and ampulla. The topography of the oviduct provides a complex system of regulation which may influence not only the passage of gametes and/or embryos, but also movement of fluid within the oviductal canal. Anat Rec 260:268–278, 2000. © 2000 Wiley-Liss, Inc.

Sensory nerve supply of the human oro- and laryngopharynx: A preliminary study

Abstract: To date, the details of human sensory innervation to the pharynx and upper airway have not been demonstrated. In this study, a single human oro- and laryngopharynx obtained from autopsy was processed with a whole-mount nerve staining technique, Sihler's stain, to determine its entire sensory nerve supply. The Sihler's stain rendered all mucosa and soft tissue translucent while counterstaining nerves. The stained specimen was then dissected and the nerves were traced from their origins to the terminal branches. It was found that the sensory innervation of the human pharynx is organized into discrete primary branches that innervate specific areas, although these areas are often connected by small neural anastomoses. The density of innervation varied, with some areas receiving almost no identifiable nerve supply (e.g., posterior wall of the hypopharynx) and certain areas contained much higher density of sensory nerves: the posterior tonsillar pillars; the laryngeal surface of the epiglottis; and the postcricoid and arytenoid regions. The posterior tonsillar pillar was innervated by a dense plexus formed by the pharyngeal branches of the IX and X nerves. The epiglottis was densely innervated by the internal superior laryngeal nerve (ISLN) and IX nerve. Finally, the arytenoid and postcricoid regions were innervated by the ISLN. The postcricoid region had higher density of innervation than the arytenoid area. The use of the Sihler's stain allowed the entire sensory nerve supply of the pharyngeal areas in a human to be demonstrated for the first time. The areas of dense sensory innervation are the same areas that are known to be the most sensitive for triggering reflex swallowing or glottic protection. The data would be useful for further understanding swallowing reflex and guiding sensory reinnervation of the pharynx to treat neurogenic dysphagia and aspiration disorders.

Osteogenic protein‐1, a bone morphogenetic protein, induces angiogenesis in the chick chorioallantoic membrane and synergizes with basic fibroblast growth factor and transforming growth factor‐β1

Abstract: Capillary invasion is a vital regulatory signal during bone morphogenesis that is influenced by angiogenic molecules such as fibroblast growth factor (FGF) and some members of the transforming growth factor-β (TGF-β) superfamily, including TGF-βs themselves. Bone morphogenetic proteins (BMPs), which are members of the TGF-β superfamily, have previously not been shown to possess direct angiogenic properties. Osteogenic protein-1 (OP-1; BMP-7) is a potent regulator of cartilage and bone differentiation in vivo. The osteogenic and angiogenic properties of OP-1 at both ortho- and heterotopic sites in adult chacma baboons (Papio ursinus) are enhanced synergistically by the simultaneous application of relatively low doses of TGF-β1. The single application of relatively high doses of TGF-β1 (20 ng), and bFGF (500 ng) or relatively low (100 ng) and high (1,000 ng) doses of OP-1 in the chick chorioallantoic membrane (CAM) assay elicited a prominent and (for OP-1) dose-dependent angiogenic response. The binary application of a relatively low dose of OP-1 (100 ng) with a relatively low dose of bFGF (100 ng) or with a relatively low (5 ng) or high (20 ng) dose of TGF-β1 resulted in a synergistic enhancement of the angiogenic response. The angiogenic effect of the relatively low doses of the combined morphogens was distinctly more pronounced than that of the single application of the relatively high doses of the respective factors. The present findings suggest that these morphogens may be deployed in binary combination in order to accentuate experimental angiogenesis. The cooperative interaction of the different morphogens in the CAM assay may provide important biological clues towards the control of clinical angiogenesis. Anat Rec 259:97-107, 2000. © 2000 Wiley-Liss, Inc.

Islet‐1 marks the early heart rudiments and is asymmetrically expressed during early rotation of the foregut in the chick embryo

Abstract: Islet-1 (Isl-1), the LIM domain homeobox gene, is a well-known early marker of neuronal specification. Here, we show its spatial and temporal patterns of expression during early heart and gut development in the chick embryo. Isl-1 transcripts are first detected in the early cardiac progenitors and underlying endoderm at late stage 4. By stages 5-6, it is also expressed in the prechordal plate. From stage 6 onward, transcripts are also detected in the endoderm forming the anterior intestinal portal and floor of the caudal foregut. With progressive rostrocaudal fusion of the paired cardiac rudiments, Isl-1 expression is maintained in the cardiac mesoderm and associated endoderm. By the onset of heart beating, transcripts become restricted to the dorsal mesocardium and more caudal medial splanchnic mesoderm flanking the open gut. Within the foregut, Isl-1 is expressed in the endoderm of the oral membrane, thyroid rudiment, and second pharyngeal pouches, as well as within the second branchial grooves adjacent to the secondary pouches. Interestingly, with the onset of gut rotation, Isl-1 expression is detected unilaterally in the splanchnic mesodermal wall of the future greater curvature of the caudal stomach/rostral duodenum. Thus, Isl-1 is a novel and useful marker of the early cardiac rudiments and of the original left side of the rotating foregut. During early organogenesis, Isl-1 is also expressed in several other discrete domains as reported previously. Additionally, it is expressed at the interface between the hind limbs and trunk.

Going virtual with quicktime VR: New methods and standardized tools for interactive dynamic visualization of anatomical structures

Abstract: Continuing evolution of computer-based multimedia technologies has produced QuickTime, a multiplatform digital media standard that is supported by stand-alone commercial programs and World Wide Web browsers. While its core functions might be most commonly employed for production and delivery of conventional video programs (e.g., lecture videos), additional QuickTime VR "virtual reality" features can be used to produce photorealistic, interactive "non-linear movies" of anatomical structures ranging in size from microscopic through gross anatomic. But what is really included in QuickTime VR and how can it be easily used to produce novel and innovative visualizations for education and research? This tutorial introduces the QuickTime multimedia environment, its QuickTime VR extensions, basic linear and non-linear digital video technologies, image acquisition, and other specialized QuickTime VR production methods. Four separate practical applications are presented for light and electron microscopy, dissectable preserved specimens, and explorable functional anatomy in magnetic resonance cinegrams.

Extracellular matrix environment influences chondrogenic pattern formation in limb bud micromass culture: experimental verification of theoretical models.

Abstract: Various theoretical models have been proposed to explain the periodicity in the pattern of limb chondrogenesis, but experimental comparison of these models have seldom been performed properly. In the present study, micromass culture of limb bud mesenchyme cells was undertaken to test the validity of three theoretical models: the reaction-diffusion model, the cell sorting model, and the mechanochemical model. Computer simulations were undertaken to predict the factors that can affect the coarseness of the chondrogenic pattern. According to the predictions, we performed micromass culture of limb mesenchyme in collagen and agarose gel. Then we carried out time-lapse observation to analyze the cell movement during pattern formation. From computer simulations it was theoretically predicted that changes in the surrounding extracellular matrix should alter the periodicity of the chondrogenic pattern in vitro, and we found that pattern changes actually occurred under different culture conditions. When compared with the culture in a liquid medium, the chondrogenic pattern became less coarse when the cells were cultured in collagen or agarose gel, and the pattern change appeared to be independent of the cell differentiation. Time-lapse observation revealed a decrease in cell motility when the cells were cultured in gel. It was found that both the reaction-diffusion and cell sorting models fit the pattern change produced and that the mechanochemical model is not primarily important in the chondrogenic pattern formation in vitro. Anat Rec 258:100–107, 2000. © 2000 Wiley-Liss, Inc.

Animated PowerPoint as a tool to teach anatomy

Abstract: Anatomy is a visual science. For centuries, anatomic information has been conveyed through drawings that have been presented to students through every available medium. The projection of animated images from a computer is a medium that offers great promise in effecting improved communication of anatomic information. Using Microsoft PowerPoint software, we have developed animated presentations for all of our lectures in Gross and Developmental Anatomy. As a starting point, we scan pen-and-ink drawings to create a digital image. The image may be edited and manipulated in an image processing program. Next, the image is imported into a PowerPoint slide where it is labeled and otherwise enhanced (arteries overlaid with red color, veins in blue, etc.) and the enhancements are animated, as we describe here step by step. For the lecture, the file is loaded on a server that is accessible through a network from a computer in the lecture hall. The output is directed to a video projector and the PowerPoint presentation is projected in the "Slide Show" mode. We use a wireless mouse that allows us to control the presentation from anywhere in the room. Before the lecture, students are provided with the same unlabeled drawings as handouts, and during the lecture the students are actively engaged in labeling the drawings and making related notes. After the lecture, the file is saved in HTML format and posted on our course web site where students can access the slides. Evaluation by the students at the end of the course demonstrated that this style of presentation was very favorably received.

Emotional reactions of medical students to dissecting human bodies: a conceptual approach and its evaluation.

Abstract: In recent years, the medical curriculum has been changed in many institutions worldwide. New concepts such as problem-based or evidence-based learning have consequences for teaching anatomy (Drake, 1998). Various papers have been published, for example, on the effects of self-directed learning on teaching anatomy (Peplow, 1990), small group case-based learning programs (Hansen and Krackov, 1994; Peplow, 1992), multiple choice testing in anatomy (Nnodim, 1992), or using computerized representations of anatomy (Rosse, 1995; Walsh and Bohn, 1990). Dissection has also been compared with prosection (Nnodim, 1990). In other articles, the hours available for teaching anatomy have been compared between medical schools in Great Britain, and aspects of teacher training for anatomists have been discussed (Fitzgerald, 1992). One aspect, however, has seldom been discussed (Putz, 1999) and has rarely been the subject of scientific interest. How do we prepare our young medical students to touch and dissect a human body? Irrespective of whether it is the classical dissection course or problem-based learning using prosected material, it will probably be the first time that students will come into close contact with a dead body and thus be confronted with issues of dying and death. Articles on this topic have been published in journals but not often read by anatomists (Bertman and Marks, 1985; Horne et al., 1990). In our experience, the vast majority of first year medical students have seldom had contact with dying relatives or friends, nor have they actually seen a dead person.

Intimal thickening involves transdifferentiation of embryonic endothelial cells.

Abstract: Morphological studies have hypothesized different origins for the precursors of the vascular smooth muscle cells (SMCs). The intriguing possibility that intimal SMCs may arise from the endothelium has newly emerged. As a first step towards understanding of the possible mechanisms involved in the transdifferentiation of endothelium into smooth muscle cells, we characterized the in vivo phenotype of the cells located in the aortic wall (distal to the aortic arches). This was accomplished using advanced stages of chicken embryo development. Furthermore, we investigated whether the cells present at the intimal thickening derive from the endothelial cell transdifferentiation. Immunolabeling of serial cryosections suggested that mesenchymal cells observed in the intimal thickening may arise from the endothelium. These cells may persist either as non-muscle throughout the development or possibly convert to cells expressing smooth muscle alpha-actin (SM alpha-actin). To determine whether endothelial cells may actually transdifferentiate into mesenchymal cells, aortic explants from 14-day-old chicken embryos (stage 40) were used. We found that explanted endothelial cells lose their cobblestone-appearance and migrate toward cell-free area. Some of these cells maintain the vWf immunoreactivity, whereas other cells coordinately lose vWf and gain SM alpha-actin expression (transitional cells). Taken together these findings strongly support the possibility that embryonic aortic endothelial transdifferentiate into mesenchymal cells, some of which express SM alpha-actin. Since TGFbeta-3 is considered an essential factor during epithelial to mesenchymal transitions in earlier chicken heart development, we also investigated the distribution of this growth factor at day 14. Our observations indicated that the immunoreactivity for TGFbeta-3 in this stage may be associated with migrating mesenchymal cells and that this immunoreactivity appears to decrease as cell differentiation advances. Therefore, the present study provides evidence that could help to explain 1) the presence of cells displaying a phenotype reminiscent of fetal-like cells in the normal chicken aorta and in the intimal region of the human aorta; 2) the SM lineage diversity in the chicken embryo reported by others; 3) a subpopulation of immature cells in the subendothelial region of the main pulmonary arteries of fetal, neonatal and adult bovines; and 4) the presence of intimal cushions, intimal pads, eccentric and diffuse intimal thickening that are observed in mammalian and avian vessels at birth.

Blood cells of sea bass (Dicentrarchus labrax l.). Flow cytometric and microscopic studies

Abstract: Studies of fish blood cells made to date presented numerous problems derived from both the nomenclature and the techniques used. A combination of quantitative and morphological methods is needed if the classification of fish blood cells is to advance from it present provisional state. The aim of the present paper was first to isolate sea bass blood cell populations by flow cytometry and second to characterize then microscopically. Blood cell populations from sea bass (Dicentrarchus labrax L.) were isolated according to their FSC (size) and SSC (granularity) properties by flow cytometry. The isolated populations were then processed for light and transmission and scanning electron microscopic characterization. Sea bass blood leukocytes isolated by flow cytometry consisted of two main cell subpopulations. Subsequent microscopic study of these cells revealed that the first subpopulation was composed of small cells (3-5 microm) of low granularity and consisted of thrombocytes and lymphocytes whereas, the second subpopulation was formed of 6-9 microm sized cells of high granularity consisting of granulocytes and monocyte/macrophages. The combined use of flow cytometry and electron microscopy makes it possible to characterize the different cell types present in sea bass peripheral blood with a high degree of certainty. Although sea bass basically follows the common vertebrate hematological pattern, significant modifications such as the presence of circulating immature erythrocytes, plasma cells and monocyte/macrophages and different forms of thrombocytes can be established with respect to this pattern.

New ideas about binocular coordination of eye movements: is there a chameleon in the primate family tree?

Abstract: Many animals with laterally placed eyes, such as chameleons, move their eyes independently of one another In contrast, primates with frontally placed eyes and binocular vision must move them together so that both eyes are aimed at the same point in visual space Is binocular coordination an innate feature of how our brains are wired, or have we simply learned to move our eyes together? This question sparked a controversy in the 19th century between two eminent German scientists, Ewald Hering and Hermann von Helmholtz Hering took the position that binocular coordination was innate and vigorously challenged von Helmholtz's view that it was learned Hering won the argument and his hypothesis, known as Hering's Law of Equal Innervation, became generally accepted New evidence suggests, however, that similar to chameleons, primates may program movements of each eye independently Binocular coordination is achieved by a neural network at the motor periphery comprised of motoneurons and specialized interneurons located near or in the cranial nerve nuclei that innervate the extraocular muscles It is assumed that this network must be trained and calibrated during infancy and probably throughout life in order to maintain the precise binocular coordination characteristic of primate eye movements despite growth, aging effects, and injuries to the eye movement neuromuscular system Malfunction of this network or its ability to adaptively learn may be a contributing cause of strabismus Anat Rec (New Anat) 261:153–161, 2000 © 2000 Wiley-Liss, Inc

MMP-2 expression during early avian cardiac and neural crest morphogenesis.

Abstract: Matrix metalloproteinase-type 2 (MMP-2) degrades extracellular matrix, mediates cell migration and tissue remodeling, and is implicated in mediating neural crest (NC) and cardiac development. However, there is little information regarding the expression and distribution of MMP-2 during cardiogenesis and NC morphogenesis. To elucidate the role of MMP-2, we performed a comprehensive study on the temporal and spatial distribution of MMP-2 mRNA and protein during critical stages of early avian NC and cardiac development. We found that ectodermally derived NC cells did not express MMP-2 mRNA during their initial formation and early emigration but encountered MMP-2 protein in basement membranes deposited by mesodermal cells. While NC cells did not synthesize MMP-2 mRNA early in migration, MMP-2 expression was seen in NC cells within the cranial paraxial and pharyngeal arch mesenchyme at later stages but was never detected in NC-derived neural structures. This suggested NC MMP-2 expression was temporally and spatially dependent on tissue interactions or differed within the various NC subpopulations. MMP-2 was first expressed within cardiogenic splanchnic mesoderm before and during the formation of the early heart tube, at sites of active pharyngeal arch and cardiac remodeling, and during cardiac cushion cell migration. Collectively, these results support the postulate that MMP-2 has an important functional role in early cardiogenesis, NC cell and cardiac cushion migration, and remodeling of the pharyngeal arches and cardiac heart tube. Anat Rec 259:168–179, 2000. © 2000 Wiley-Liss, Inc.

Comparative respiratory morphology: themes and principles in the design and construction of the gas exchangers.

Abstract: Along the evolutionary continuum, a kaleidoscope of gas exchangers has evolved from the simple cell membrane of the primeval unicells. The most momentous events in this process were: the intensification of molecular oxygen in the biosphere and its appropriation into aerobic metabolism, the rise of multicellular organisms, the development of a circulatory system and carrier pigments in blood, the advocacy of air breathing, adoption of suctional breathing, and the shift to endothermy. To satisfy species-specific needs for oxygen, some constraints were overcome through transactions that obliged certain compromises and trade-offs. Optimal designs of the gas exchangers for particular phylogenetic levels of development, habitat, and lifestyle have developed only so far as to satisfy prescribed needs. The efficiency of the human lung, for example, falls well below those of certain taxa that are considered to be relatively “less advanced.” Utilizing different resources and strategies, in fascinating processes of conformity, different groups of animals have developed similar respiratory structures. In most cases, the analogy reflects evolutionary convergence in response to corresponding selective pressures rather than common ancestry. Anat Rec (New Anat) 261:25‐ 44, 2000. © 2000 Wiley-Liss, Inc.

Conotruncal anomalies in the trisomy 16 mouse: an immunohistochemical analysis with emphasis on the involvement of the neural crest.

Abstract: The trisomy 16 (Ts16) mouse is generally considered a model for human Down's syndrome (trisomy 21). However, many of the cardiac defects in the Ts16 mouse do not reflect the heart malformations seen in patients suffering from this chromosomal disorder. In this study we describe the conotruncal malformations in mice with trisomy 16. The development of the outflow tract was immunohistochemically studied in serially sectioned hearts from 34 normal and 26 Ts16 mouse embryos ranging from 8.5 to 14.5 embryonic days. Conotruncal malformations observed in the Ts 16 embryos included double outlet right ventricle, persistent truncus arteriosus, Tetralogy of Fallot, and right-sided aortic arch. This spectrum of malformations is remarkably similar to that seen in humans suffering from DiGeorge syndrome (DGS). As perturbation of neural crest development has been proposed in the pathogenesis of DGS we specifically focussed on the fate of neural crest derived cells during outflow tract development of the Ts16 mouse using an antibody that enabled us to trace these cells during development. Severe perturbation of the neural crest-derived cell population was observed in each trisomic specimen. The abnormalities pertained to: 1) the size of the columns of neural crest-derived cells (or prongs); 2) the spatial orientation of these prongs within the mesenchymal tissues of the outflow tract; and 3) the location in which the neural crest cells interact with the myocardium. The latter abnormality appeared to be responsible for ectopic myocardialization found in trisomic embryos. Our observations strongly suggest that abnormal neural crest cell behavior is involved in the pathogenesis of the conotruncal malformations in the Ts16 mouse.