An in ovo investigation into the hepatotoxicity of cadmium and chromium evaluated with light- and transmission electron microscopy and electron energy-loss spectroscopy.

Excessive agriculture, transport and mining often lead to the contamination of valuable water resources. Communities using this water for drinking, washing, bathing and the irrigation of crops are continuously being exposed to these heavy metals. The most vulnerable is the developing fetus. Cadmium (Cd) and chrome (Cr) were identified as two of the most prevalent heavy metal water contaminants in South Africa. In this study, chicken embryos at the stage of early organogenesis were exposed to a single dosage of 0.430 µM physiological dosage (PD) and 430 µM (×1000 PD) CdCl2, as well as 0.476 µM (PD) and 746 µM (×1000 PD) K2Cr2O7. At day 14, when all organ systems were completely developed, the embryos were terminated and the effect of these metals on liver tissue and cellular morphology was determined with light- and transmission electron microscopy (TEM). The intracellular localization of these metals was determined using electron energy-loss spectroscopy (EELS). With light microscopy, the PD of both Cd and Cr had no effect on liver tissue or cellular morphology. At ×1000 PD both Cd and Cr caused sinusoid dilation and tissue necrosis. With TEM analysis, Cd exposed hepatocytes presented with irregular chromatin condensation, ruptured cellular membranes and damaged or absent organelles. In contrast Cr caused only slight mitochondrial damage. EELS revealed the bio-accumulation of Cd and Cr along the cristae of the mitochondria and chromatin of the nuclei.

A technique to increase accessibility to late-stage chick embryos for in ovo manipulations.

BACKGROUND: During early development, avian embryos are easily accessible in ovo for transplantations and experimental perturbations. However, these qualities of the avian embryonic model rapidly wane shortly after embryonic day (E)4 when the embryo is obscured by extraembryonic membranes, making it difficult to study developmental events that occur at later stages in vivo. RESULTS: In this study, we describe a multistep method that involves initially windowing eggs at E3, followed by dissecting away extraembryonic membranes at E5 to facilitate embryo accessibility in ovo until later stages of development. The majority of the embryos subjected to this technique remain exposed between E5 and E8, then become gradually displaced by the growing allantois from posterior to anterior regions. CONCLUSIONS: Exposed embryos are viable and compatible with embryological and modern developmental biology techniques including tissue grafting and ablation, gene manipulation by electroporation, and protein expression. This technique opens up new avenues for studying complex cellular interactions during organogenesis and can be further extrapolated to regeneration and stem cell studies.

Regional differences in actomyosin contraction shape the primary vesicles in the embryonic chicken brain.

In the early embryo, the brain initially forms as a relatively straight, cylindrical epithelial tube composed of neural stem cells. The brain tube then divides into three primary vesicles (forebrain, midbrain, hindbrain), as well as a series of bulges (rhombomeres) in the hindbrain. The boundaries between these subdivisions have been well studied as regions of differential gene expression, but the morphogenetic mechanisms that generate these constrictions are not well understood. Here, we show that regional variations in actomyosin-based contractility play a major role in vesicle formation in the embryonic chicken brain. In particular, boundaries did not form in brains exposed to the nonmuscle myosin II inhibitor blebbistatin, whereas increasing contractile force using calyculin or ATP deepened boundaries considerably. Tissue staining showed that contraction likely occurs at the inner part of the wall, as F-actin and phosphorylated myosin are concentrated at the apical side. However, relatively little actin and myosin was found in rhombomere boundaries. To determine the specific physical mechanisms that drive vesicle formation, we developed a finite-element model for the brain tube. Regional apical contraction was simulated in the model, with contractile anisotropy and strength estimated from contractile protein distributions and measurements of cell shapes. The model shows that a combination of circumferential contraction in the boundary regions and relatively isotropic contraction between boundaries can generate realistic morphologies for the primary vesicles. In contrast, rhombomere formation likely involves longitudinal contraction between boundaries. Further simulations suggest that these different mechanisms are dictated by regional differences in initial morphology and the need to withstand cerebrospinal fluid pressure. This study provides a new understanding of early brain morphogenesis.

Multiscale cardiac imaging spanning the whole heart and its internal cellular architecture in a small animal model.

Cardiac pumping depends on the morphological structure of the heart, but also on its subcellular (ultrastructural) architecture, which enables cardiac contraction. In cases of congenital heart defects, localized ultrastructural disruptions that increase the risk of heart failure are only starting to be discovered. This is in part due to a lack of technologies that can image the three-dimensional (3D) heart structure, to assess malformations; and its ultrastructure, to assess organelle disruptions. We present here a multiscale, correlative imaging procedure that achieves high-resolution images of the whole heart, using 3D micro-computed tomography (micro-CT); and its ultrastructure, using 3D scanning electron microscopy (SEM). In a small animal model (chicken embryo), we achieved uniform fixation and staining of the whole heart, without losing ultrastructural preservation on the same sample, enabling correlative multiscale imaging. Our approach enables multiscale studies in models of congenital heart disease and beyond.

The heart is our hardest-working organ and beats around 100,000 times a day, pumping blood through a vast system of vessels to all areas of the body. Specialized heart cells make the heart contract rhythmically, enabling it to work efficiently. Contractile molecules inside these cells, called myofibrils, align within the heart cells, and heart cells align to each other, so that the heart tissue contracts effectively. However, when the heart has defects or is diseased this organization can be lost, and the heart may no longer pump blood efficiently, leading to sometimes life-threatening complications. For example, around one in a hundred newborn babies suffer from congenital heart defects, and despite medical advances, these conditions remain the main cause of non-infectious mortality in children. Many cases of congenital heart disease are diagnosed before a baby is born during an ultrasound scan. However, these scans, as well as subsequent diagnostic tools, lack the precision to detect problems within the heart cells. Now, Rykiel et al. used two complementary imaging techniques known as micro-computed tomography and scanning electron microscopy to acquire pictures of the whole heart as well as of the organization inside the heart cells. This made it possible to capture the structure of the heart tissue at both micrometer (the whole heart) and nanometer resolution (the inside of the cells), and to study what happens within the heart and its cells when the heart has a defect. Rykiel et al. tested the imaging technology on the hearts of chicken embryos, at stages equivalent to a five to six-month-old human fetus, and compared a healthy heart with a heart with a defect called tetralogy of Fallot. They found that the tissues in the heart with a defect had a sponge-like appearance, with increased space in between cells. Moreover, the myofibrils of the heart with a defect were aligned differently compared to those in the normal heart. More research is needed to fully understand what happens when the heart has a defect. However, the imaging technology used in this study offers the possibility of examining the heart at an unprecedented level of detail. This will deepen our understanding of how structural heart defects arise and how they affect the pumping of the heart, and will give us clues to design better treatments for patients with heart defects and other heart anomalies.

Ultrastructural evidence of a vesicle-mediated mode of cell degranulation in chicken chromaffin cells during the late phase of embryonic development.

In the present investigation, we attempted to determine whether ultrastructural features indicative of a vesicle-mediated mode of cell secretion were detectable in chick chromaffin cells during embryo development. The adrenal anlagen of domestic fowls were examined at embryonic days (E) 12, 15, 19 and 21 by electron microscopy quantitative analysis. Morphometric evaluation revealed a series of granule and cytoplasmic changes highly specific for piecemeal degranulation (PMD), a secretory process based on vesicular transport of cargoes from within granules for extracellular release. At E19 and E21 we found a significant peak in the percentage of granules exhibiting changes indicative of progressive release of secretory materials, i.e. granules with lucent areas in their cores, reduced electron density, disassembled matrices, residual cores and membrane empty containers. A dramatic raise in the density of 30-80-nm-diameter, membrane-bound, electron-dense and electron-lucent vesicles--which were located either next to granules or close to the plasma membrane--was recognizable at E19, that is, during the prehatching phase. The cytoplasmic burst of dense and clear vesicles was paralleled by the appearance of chromaffin granules showing outpouches or protrusions of their profiles ('budding features'). These ultrastructural data are indicative of an augmented vesicle-mediated transport of chromaffin granule products for extracellular release in chick embryo chromaffin cells during the prehatching stage. In conclusion, this study provides new data on the fine structure of chromaffin cell organelles during organ development and suggests that PMD may be part of an adrenomedullary secretory response that occurs towards the end of chicken embryogenesis. From an evolutionary point of view, this study lends support to the concept that PMD is a secretory mechanism highly conserved throughout vertebrate classes.

An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.

Scanning confocal microscopes offer improved rejection of out-of-focus noise and greater resolution than conventional imaging. In such a microscope, the imaging and condenser lenses are identical and confocal. These two lenses are replaced by a single lens when epi-illumination is used, making confocal imaging particularly applicable to incident light microscopy. We describe the results we have obtained with a confocal system in which scanning is performed by moving the light beam, rather than the stage. This system is considerably faster than the scanned stage microscope and is easy to use. We have found that confocal imaging gives greatly enhanced images of biological structures viewed with epifluorescence. The improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections.

Isolation and distribution of West Nile virus in embryonated chicken eggs.

West Nile virus (WNV) was identified from domestic psittacine birds by inoculating embryonated chicken eggs. Most of the embryos died 5 days postinoculation; flavivirus was detected in some by negative-staining electron microscopy. Immunohistochemistry performed on the embryos and their supporting structures detected the WNV antigen mainly in the chorioallantoic membrane, regardless of the inoculation route or passage number. WNV antigen was also found in the embryonic muscle (both skeletal and smooth muscles) and in multifocal areas of the skin. WNV was not detected in the viscera of the embryo or yolk sac. This study provides evidence of isolation and identification of WNV via embryonated chicken eggs.

Transcriptional profiles in the chicken ductus arteriosus during hatching.

The ductus arteriosus, an essential embryonic blood vessel between the pulmonary artery and the descending aorta, constricts after birth or hatching and eventually closes to terminate embryonic circulation. Chicken embryos have two long ductus arteriosi, which anatomically differ from mammal ductus arteriosus. Each long ductus arteriosus is divided into two parts: the pulmonary artery-sided and descending aorta-sided ductus arteriosi. Although the pulmonary artery-sided and descending aorta-sided ductus arteriosi have distinct functional characteristics, such as oxygen responsiveness, the difference in their transcriptional profiles has not been investigated. We performed a DNA microarray analysis (GSE 120116 at NCBI GEO) with pooled tissues from the chicken pulmonary artery-sided ductus arteriosus, descending aorta-sided ductus arteriosus, and aorta at the internal pipping stage. Although several known ductus arteriosus-dominant genes such as tfap2b were highly expressed in the pulmonary artery-sided ductus arteriosus, we newly found genes that were dominantly expressed in the chicken pulmonary artery-sided ductus arteriosus. Interestingly, cluster analysis showed that the expression pattern of the pulmonary artery-sided ductus arteriosus was closer to that of the descending aorta-sided ductus arteriosus than that of the aorta, whereas the morphology of the descending aorta-sided ductus arteriosus was closer to that of the aorta than that of the pulmonary artery-sided ductus arteriosus. Subsequent pathway analysis with DAVID bioinformatics resources revealed that the pulmonary artery-sided ductus arteriosus showed enhanced expression of the genes involved in melanogenesis and tyrosine metabolism compared with the descending aorta-sided ductus arteriosus, suggesting that tyrosinase and the related genes play an important role in the proper differentiation of neural crest-derived cells during vascular remodeling in the ductus arteriosus. In conclusion, the transcription profiles of the chicken ductus arteriosus provide new insights for investigating the mechanism of ductus arteriosus closure.

Chick Barx2b, a marker for myogenic cells also expressed in branchial arches and neural structures.

We have isolated a new chicken gene, cBarx2b, which is related to mBarx2 in sequence, although the expression patterns of the two genes are quite different from one another. The cBarx2b gene is expressed in craniofacial structures, regions of the neural tube, and muscle groups in the limb, neck and cloaca. Perturbation of anterior muscle pattern by application of Sonic Hedgehog protein results in a posteriorization of cBarx2b expression.

Expression of the galactose-binding lectins during the formation of organ primordia in the chick embryo.

Early chick embryos contain two beta-galactoside-binding lectins of 16 kDa and 14 kDa. using several antisera to these proteins, we have studied lectin expression at embryonic stages when the segregation and early differentiation of organ primordia are taking place. With antisera to the 16 kDa lectin that display similar immunoreactivity in immunoblot analysis, we show that these antisera exhibit varying immunoreactivity in embryo sections. One antiserum reacts preferentially with a matrix form of lectin while another detects mainly a cellular form of this protein. During early development, galactoside-binding lectins of the matrix type are expressed in the vitelline membrane, the outer and inner limiting membranes of the neural tube, the surface of the notochord and the coelomic surface of the cardiac rudiments. The cellular form of the lectin occurs in the intracellular yolk of early embryos, in the primordial germ cells, the myocardium, in the early myotome, and in a cohort of cells which are presumed to belong to the neural crest. Our results indicate that, although all of the antisera recognize the intracellular lectin of the extraembryonic endoderm, some antisera to the 16 kDa lectin exhibit preferential reactivity with different lectin isoforms. The extracellular matrix form of lectin is transiently expressed during early development at the stages when the segregation of organ primordia is occurring. It's expression could be related to the acquisition of polarity in developing epithelia. Results also suggest that various versions of the same protein may perform distinct developmental roles in the embryo.

Organelle distribution in chick neuroepithelial cells: effects of colchicine and cytochalasin B.

The intracellular distribution of mitochondria, cytoplasmic inclusions and rough endoplasmic reticulum cisternae of chick neuroepithelial cells was investigated at neurulation stages 6, 8, 10 and 12. These neuroepithelial cells were subdivided into three zones: apical, median and basal and the distribution percentages of distribution of these organelles were obtained. Mitochondrial distribution was related to the energy supply that mitochondria provide for apical microfilament contraction. Cytoplasmic inclusions were distributed preferentially in the apical zone of the neuroepithelial cells during the four stages. Rough endoplasmic reticulum cisternae were homogeneously distributed in the three zones at stages 10 and 12, but at stages 6 and 8 there are more elevated percentages of rough endoplasmic reticulum in the apical zones than in the other zones. Experimental treatments with colchicine and cytochalasin B does not modify the patterns of mitochondria and rough endoplasmic reticulum cisternae but alters the distribution of cytoplasmic inclusions. Finally, there is a correlation in the normal neurulating neuroepithelial cells between the distributions of mitochondria and rough endoplasmic reticulum distribution and between the distributions of mitochondria and cytoplasmic inclusions distribution. This relationship is retained in the treated neuroepithelial cells.

Retention and structure of extracellular matrix in early chick embryos after quick-freezing and freeze-substitution.

Early chick embryos, stages 11 to 14, were isolated, quick-frozen by immersion in isopentane/propane cryogen (-185 degrees C) and freeze-substituted for study by scanning electron microscopy. Emphasis was placed on the extracellular matrix (ECM) in the axial region of the segmental plate and developing somites. Ultrarapid freezing, followed by delicate freeze-substitution, immobilizes and retains much more ECM than chemical fixatives that include tannic acid (TA). The matrix on the dorsal surface of the neural tube is preserved as delicate filaments which are expressed bilaterally over the tube in a dorso-ventral orientation. These parallel primary ridges of ECM have a spacing of 1 to 3 micron, forming grooves on the wall of the neural tube. Interrupting this pattern are funnel-shaped ridges about 80 to 100 micron apart along the neural tube. The ridges become decorated with cross-bridges creating a dense lattice in the region of somite development, to the extent that a basal lamina composed of dense fibrillar network and amorphous mats of matrix accumulates on the lateral wall of the neural tube. Heavy strands and fenestrated lamellae of ECM interconnect the neural tube, notochord and somites, and attach the overlying epithelium to the upper surface of the somites. The pattern of ECM is complimentary to the migratory pathways of ventrally migrating neural crest cells and is the basis for suggesting that a physical substratum influencing the direction of neural crest cell migration is an idea that should be revived.

Electron microscopic study on the development of the carotid body and glomus cell groups distributed in the wall of the common carotid artery and its branches in the chicken.

Development of the carotid body and the glomus cell groups in the wall of the common carotid artery and its branches was studied in chickens at various developmental stages by electron microscopy. At 8 days of incubation, the carotid body anlage consisted of mesenchyme-like cells, whereas the clusters of epithelial cells, which occasionally contained a few dense-cored vesicles and were accompanied by unmyelinated nerve fibers, were located in the region surrounding the carotid body anlage and in the wall of the common carotid artery. Subsequently, the granule-containing cells together with nerve fibers were detected in the periphery of the carotid body anlage. At 12 days of incubation, a large number of granule-containing cells (glomus cells) were dispersed throughout the carotid body parenchyma and were also widely distributed along the common carotid artery and its branches. The cells frequently extended long cytoplasmic processes that made contact with other glomus cells and nerve fibers. Synaptic junctions which showed desmosome-like thickening of pre- and postsynaptic membranes and accumulations of small clear vesicles (around 50 nm in diameter) were first detected along the contact between the long axons and glomus cells at 12 days of incubation. In the wall of the common carotid artery, interdigitations between the cytoplasmic processes of glomus cells and smooth muscle cells began to form. Sustentacular cells investing partly the glomus cells were also discerned both in the carotid body and around the arteries at this stage. At 14 days of incubation, the glomus cells expressed most of the characteristics of the mature cells, and the synaptic junctions displaying afferent morphology appeared; the secretory granules of glomus cells were accumulated near and attached to the desmosome-like thickening of apposed membranes.

Disruption of the pial basal lamina during early avian embryonic development inhibits histogenesis and axonal pathfinding in the optic tectum.

Bacterial collagenase was injected into the ventricular cavity of the optic tectum of chick and quail embryos. Histological examination up to 6 days after enzyme injection revealed that the collagenase disrupted the pial basal lamina, which was evident by the fragmented distribution of basal lamina proteins at the pial surface of the midbrain and the brainstem. Although the disrupted basal lamina was not reestablished at later stages of development, the pial basal lamina of the newly developing neuroepithelium in the caudal part of the tectum was continuous and intact. Western blot analysis showed that the collagenase digested collagens but spared noncollagenous proteins. The disruption of the pial basal lamina caused the neuroepithelial cells to retract their pial end feet and caused tectal axons to exit the brain tissue into the adjacent mesenchyme. The vertical migration of neuroblasts to the pial layers of the tectum was inhibited, leading to a disruption of the tectal histogenesis. In the developing optic pathways, retinal axons were misguided at the optic chiasma and terminated in the head mesenchyme instead of the tectum. None of the abnormalities in histogenesis and axonal pathways were observed when the basal lamina was disrupted at a later stage of embryonic development. The present experiments demonstrate that the pial basal lamina has an important function during brain morphogenesis in restricting axons to the brain, providing an anchoring of the neuroepithelial cells to the pial surface, and allowing the formation of a defined cytoarchitecture of the brain.

Hair cell development in vivo and in vitro: analysis by using a monoclonal antibody specific to hair cells in the chick inner ear.

The purpose of this study was to establish a hair cell-specific marker and a convenient explant culture system for developing chick otocysts to facilitate in vivo and in vitro studies focusing on hair cell genesis in the inner ear. To achieve this, a hair cell-specific monoclonal antibody, 2A7, was generated by immunizing chick inner ear tissues to a mouse. Through the use of immunofluorescence and immunoelectron microscopy, it was shown that 2A7 immunoreactivity (2A7-IR) was primarily restricted to the apical region of inner ear hair cells, including stereocilia, kinocilia, apical membrane amongst the extending cilia, and superficial layer of the cuticular plate. Although the 2A7 antibody immunolabeled basically all of the hair cells in the posthatch chick inner ear, two different patterns of 2A7-IR were observed; hair cells located in the striolar region of the utricular macula, which consist of two distinct cell types identifiable on the basis of the type of nerve ending, Type I and II hair cells, showed labeling restricted to the basal end of the hair bundles. On the other hand, hair cells in the extrastriolar region, which are exclusively of Type II, showed labeling extending over virtually the entire length of the bundles. These findings raised the possibility that chick vestibular Type II hair cells, characterized by their bouton-type afferent nerve endings, can be divided into two subpopulations. Analysis of developing inner ear by using the 2A7 antibody revealed that this antibody also recognizes newly differentiated immature hair cells. Thus, the 2A7 antibody is able to recognize both immature and mature hair cells in vivo. The developmental potential of embryonic otocysts in vitro was then assessed by using explant cultures as a model. In this study, conventional otocyst explant cultures were modified by placing the tissues on floating polycarbonate filters on culture media, thereby allowing the easy manipulation of explants. In these cultures, 2A7-positive hair cells were differentiated from dividing precursor cells in vitro on the same schedule as in vivo. Furthermore, it was found that hair cells with both types of 2A7-IR were generated in culture as in vivo, indicating that a maturational process of hair cells also occurred. All these results as presented here suggest that the 2A7 monoclonal antibody as a hair cell-specific marker together with the culture system could be a potential tool in analysis of mechanisms underlying hair cell development.

Expression of basal lamina protein mRNAs in the early embryonic chick eye.

Laminin, collagen IV, collagen XVIII, agrin, and nidogen are major protein constituents of the chick retinal basal lamina. To determine their sites of synthesis during de novo basal lamina assembly in vivo, we localized their mRNA expression in the eye during maximum expansion of the retina between embryonic day (E) 2.5 and E6. Our in situ hybridization studies showed that the expression pattern of every basal lamina protein mRNA in the developing eye is unique. Collagen IV and perlecan originate predominantly from the lens epithelium, whereas collagen XVIII, nidogen, and the laminin gamma 1 and beta1 chains are synthesized mainly by the ciliary body. Agrin, collagen XVIII, collagen IV, and laminin gamma 1 also originate from cells of the optic disc. The only basal lamina protein that is synthesized by the neural retina throughout development is agrin with ganglion cells as its main source. Some of the mRNAs have short, transient expressions in the retina, most notably that of collagen IV and laminin gamma 1, both of which appear in the ventral retina between E4 and E5. That most retinal basal lamina proteins originate from extraretinal tissues infers that the basal lamina proteins have to be shed from the lens, optic disc, and ciliary body into the vitreous body. The assembly of the retinal basal lamina then occurs by the binding of these proteins by cellular receptor proteins on the vitreal endfeet of the retinal neuroepithelial cells.

Expression of presynaptic proteins is closely correlated with the chronotopic pattern of axons in the retinotectal system of the chick.

Newly synthesized presynaptic integral membrane proteins in neurons are transported in precursor vesicles from the site of protein biosynthesis in the cell body by fast axonal flow to the presynaptic terminal. We followed the path that presynaptic proteins travel on the way to their central targets of the highly ordered primary visual pathway of the chick and analyzed the developmental changes in the expression of synaptic vesicle protein 2 (SV2), synaptotagmin, and syntaxin. Immunofluorescences revealed that: (1) the onset of protein expression in the retinal ganglion cells occurs in a central to peripheral developmental pattern from embryonic day 4 (E4) onward; (2) the proteins were found first in the inner and later in the outer plexiform layer of the retina; and (3) they were redistributed from the photoreceptor inner segments and cell bodies to the terminals in the outer plexiform layer. From E4 onward, immunopositive axons for SV2, synaptotagmin, and syntaxin were found in the optic nerve, disappearing after E9 for SV2 and synaptotagmin. The optic tract was stained for SV2 and synaptotagmin between E7 and E12, for syntaxin until the posthatching period. Finally, immunoreactivities for the investigated proteins were present at the surface of the tectum from E8 onward, when first retinal axons arrived there. The present study revealed that SV2 and synaptotagmin, but not syntaxin, are, expressed in a transient wave that follows the advancement of optic axons and the proteins towards the optic tectum.

Neurites and growth cones in the chick embryo. Enhanced tissue preservation and visualization of HRP-labeled subpopulations in serial 25-microns plastic sections cut on a rotary microtome.

Study of axonal guidance in developing vertebrates has been hindered by an inability to readily visualize individual growth cones, determine the neuronal population from which they originate, trace their trajectories, and discern their interactions with their embryonic environment. We report a method that combines plastic embedding and serial sectioning with horseradish peroxidase labeling of subpopulations of neurons in the chick embryo. This method labels individual neurites from the soma to the tip of the growth cones, allowing their trajectory to be inferred and their identity to be determined by the position of the somata. As sections are up to 25 micron thick, entire growth cones can often be visualized without laborious reconstruction. Tissue preservation is much better than with similar material embedded in paraffin. Sections are cut relatively quickly using a steel knife on a standard rotary microtome and are suitable for subsequent electron microscopy.

Animal model: dysmorphogenesis and death in a chicken embryo model.

The chicken embryo is a useful animal model for investigating problems in developmental biology and teratology. Here we report data that further define the causes of 2 different patterns of malformation (one associated with amnion abnormalities, the other with isolated neural tube defects) and death induced by making a window in the shell and subshell membranes during the first day of incubation. The interpretation of these data suggests to us the following hypotheses. An early amnion deficit spectrum or syndrome (EADS) in chicken embryos is caused by a brief (less than 10 sec) perturbation that occurs during the windowing procedure. This perturbation results in an acute increase in mechanical tension to the developing embryo and support structures, dehydration localized to the area of the blastoderm, and/or increased friction between the blastoderm and overlying vitelline and shell membranes. Isolated neural tube defects (NTDs) are caused by a longer perturbation (greater than 3 hr) consisting of increased mechanical stress across the blastoderm. The mechanical stress is associated with the introduction of a new air space over the animal pole of the yolk during windowing. The new air space causes the shape of the yolk to change (ie, to be deformed), resulting in an increase in mechanical tension across the vitelline membrane and blastoderm. NTDs involving the head are associated with significant early embryonic mortality, whereas those involving the trunk are not. Death may also be caused by cardiovascular anomalies observed in EADS. It is concluded that disturbances in morphogenesis and death in this model are, therefore, the result of extrinsic forces (eg, mechanical stress, localized dehydration, or friction) acting on different tissue types at various critical times in development. Intensity and duration of these forces on the developing blastoderm are important variables.