High-resolution Light Microscopy Research Articles

Uncovering the Infection Strategy of Phyllachora maydis During Maize Colonization: A Comprehensive Analysis.

Tar spot, a disease caused by the ascomycete fungal pathogen Phyllachora maydis, is considered one of the most significant yield-limiting diseases of maize (Zea mays) within the United States. P. maydis may also be found in association with other fungi, forming a disease complex that is thought to result in the characteristic fisheye lesions. Understanding how P. maydis colonizes maize leaf cells is essential for developing effective disease control strategies. Here, we used histological approaches to elucidate how P. maydis infects and multiplies within susceptible maize leaves. We collected tar spot-infected maize leaf samples from four different fields in northern Indiana at three different time points during the growing season. Samples were chemically fixed and paraffin-embedded for high-resolution light and scanning electron microscopy. We observed a consistent pattern of disease progression in independent leaf samples collected across different geographical regions. Each stroma contained a central pycnidium that produced asexual spores. Perithecia with sexual spores developed in the stomatal chambers adjacent to the pycnidium, and a cap of spores formed over the stroma. P. maydis reproductive structures formed around but not within the vasculature. We observed P. maydis associated with two additional fungi, one of which is likely a member of the Paraphaeosphaeria genus; the other is an unknown fungi. Our data provide fundamental insights into how this pathogen colonizes and spreads within maize leaves. This knowledge can inform new approaches to managing tar spot, which could help mitigate the significant economic losses caused by this disease.

Exploring an innovative decellularization protocol for porcine nerve grafts: a translational approach to peripheral nerve repair.

Peripheral nerves are frequently affected by lesions caused by traumatic or iatrogenic damages, resulting in loss of motor and sensory function, crucial in orthopedic outcomes and with a significant impact on patients' quality of life. Many strategies have been proposed over years to repair nerve injuries with substance loss, to achieve musculoskeletal reinnervation and functional recovery. Allograft have been tested as an alternative to the gold standard, the autograft technique, but nerves from donors frequently cause immunogenic response. For this reason, several studies are focusing to find the best way to decellularize nerves preserving either the extracellular matrix, either the basal lamina, as the key elements used by Schwann cells and axons during the regenerative process. This study focuses on a novel decellularization protocol for porcine nerves, aimed at reducing immunogenicity while preserving essential elements like the extracellular matrix and basal lamina, vital for nerve regeneration. To investigate the efficacy of the decellularization protocol to remove immunogenic cellular components of the nerve tissue and to preserve the basal lamina and extracellular matrix, morphological analysis was performed through Masson's Trichrome staining, immunofluorescence, high resolution light microscopy and transmission electron microscopy. Decellularized porcine nerve graft were then employed in vivo to repair a rat median nerve lesion. Morphological analysis was also used to study the ability of the porcine decellularized graft to support the nerve regeneration. The decellularization method was effective in preparing porcine superficial peroneal nerves for grafting as evidenced by the removal of immunogenic components and preservation of the ECM. Morphological analysis demonstrated that four weeks after injury, regenerating fibers colonized the graft suggesting a promising use to repair severe nerve lesions. The idea of using a porcine nerve graft arises from a translational perspective. This approach offers a promising direction in the orthopedic field for nerve repair, especially in severe cases where conventional methods are limited.

Macroscopic and microscopic characterization of term placentas from nutritionally restricted goats

The aim of this study was to describe the morphological, macroscopic, and microscopic characteristics in term placentas of goats, nutritionally restricted in the prepubertal period and during the second and third of pregnancy gestation. Samples were collected from the placentas of goats separated into 3 treatments: control (C) n= 9; prepubertal feed restriction (PPFR) n = 5; pregnancy feed restriction (PFR) n=5. The placental cellular structure was studied using Masson's trichrome staining. The ultrastructural analysis was performed by high-resolution light microscopy and transmission electron microscopy. The present study revealed the effect of prepubertal and gestational feed restriction on macroscopic and microscopic characteristics in goat placentas with decreases in cotyledonary weight, height, and size in placentas from nutritionally restricted goats in both groups, PPFR and PFR. The smaller size and the change in the shape of the cotyledons could suggest compensatory mechanisms for prepubertal and gestational feed restriction to ensure placental and kid development.

Dynamic Molecular Markers of Otosclerosis in the Human Cochlea.

To investigate the role and distribution of various molecular markers using immunohistochemistry and immunofluorescence to further elucidate and understand the pathogenesis of otosclerosis. Archival celloidin formalin-fixed 20-micron thick histologic sections from 7 patients diagnosed with otosclerosis were studied and compared to controls. Sections in the mid-modiolar region were immunoreacted with rabbit polyclonal antibodies against nidogen-1, β2-laminin, collagen-IX, BSP, and monoclonal antibodies against TGF β-1 and ubiquitin. Digital images were acquired using a high-resolution light and laser confocal microscope. Nidogen-1, BSP, and collagen-IX were expressed in the otospongiotic regions, and to lesser extent, in the otosclerotic regions, the latter previously believed to be inactive. β2-laminin and ubiquitin were uniformly expressed in both otospongiotic and otosclerotic regions. There was a basal level of expression of all of these markers in the normal hearing and sensorineural hearing loss specimens utilized as control. TGF β -1, however, though present in the otosclerosis bones, was absent in the normal hearing and sensorineural hearing loss controls. Our results propose that the activity and function of TGF-1 may play a key role in the development and pathogenesis of otosclerosis. Further studies utilizing a higher number of temporal bone specimens will be helpful for future analysis and to help decipher its role as a potential target in therapeutic interventions.

The histomorphological and stereological assessment of rat dorsal root ganglion tissues after various types of sciatic nerve injury.

Peripheral nerve injuries lead to significant changes in the dorsal root ganglia, where the cell bodies of the damaged axons are located. The sensory neurons and the surrounding satellite cells rearrange the composition of the intracellular organelles to enhance their plasticity for adaptation to changing conditions and response to injury. Meanwhile, satellite cells acquire phagocytic properties and work with macrophages to eliminate degenerated neurons. These structural and functional changes are not identical in all injury types. Understanding the cellular response, which varies according to the type of injury involved, is essential in determining the optimal method of treatment. In this research, we investigated the numerical and morphological changes in primary sensory neurons and satellite cells in the dorsal root ganglion 30days following chronic compression, crush, and transection injuries using stereology, high-resolution light microscopy, immunohistochemistry, and behavioral analysis techniques. Electron microscopic methods were employed to evaluate fine structural alterations in cells. Stereological evaluations revealed no statistically significant difference in terms of mean sensory neuron numbers (p > 0.05), although a significant decrease was observed in sensory neuron volumes in the transection and crush injury groups (p < 0.05). Active caspase-3 immunopositivity increased in the injury groups compared to the sham group (p < 0.05). While crush injury led to desensitization, chronic compression injury caused thermal hyperalgesia. Macrophage infiltrations were observed in all injury types. Electron microscopic results revealed that the chromatolysis response was triggered in the sensory neuron bodies from the transection injury group. An increase in organelle density was observed in the perikaryon of sensory neurons after crush-type injury. This indicates the presence of a more active regeneration process in crush-type injury than in other types. The effect of chronic compression injury is more devastating than that of crush-type injury, and the edema caused by compression significantly inhibits the regeneration process.

Integration of the pollen database PONET into PalDat with new features for light microscopy

Free access to scientific data, knowledge and technology transfer has become more and more important in science, at universities, governmental institutes, and in our society in general. In an interdisciplinary project initialised by palynologists at the University of Vienna and the Austrian Agency for Health and Food Safety Ltd (AGES), the world’s most comprehensive database PalDat has been expanded by more than 2000 datasets from the PONET database, comprising high resolution light microscopy (LM) micrographs of hydrated pollen. The main aim of this fusion of knowledge was to share digital data in a secure way and to ensure users of various disciplines free access to reliable pollen data. The project was finally completed in 2021. In the course of the expansion, additional data entry fields regarding LM description have been added, and new features, such as a search function for orders and families, have been implemented in the new version 3.4 of PalDat. A global online submission and publication tool for palynological data to PalDat, including review and editorial process, assures easy knowledge transfer and facilitates a continually increasing number of datasets and taxa contributed from scientists all over the world. The free accessible online pollen database PalDat is maintained by members of the Division of Structural and Functional Botany (University of Vienna).

Evaluation of the efficacy of micro-Magnetic Resonance Imaging compared with light microscopy to investigate the anatomy of modern and ancient waterlogged wood

Light microscopy is the conventional method used to investigate wood anatomy, identify the wood taxon, and assess its conservation state. It generally requires the mechanical cut of thin sections from a sample to obtain informative images. When dealing with wooden artworks or ancient remains (e.g., archaeological waterlogged wood), it is important to avoid sample destruction. In this work the efficacy of micro-magnetic resonance imaging (μ-MRI) to investigate the anatomy of waterlogged wood is assessed in comparison with light microscopy. Images along the three anatomical directions (transverse, tangential and radial) of six modern wood species and one archaeological specimen of waterlogged wood (from the Neolithic site “La Marmotta”) were obtained both by μ-MRI and light microscopy. μ-MRI images were acquired virtually selecting 2D slices along the three wood anatomical directions. A 3D reconstruction was derived from 2D μ-MRI images. Conventional light microscopy histology was obtained by manually cutting thin sections. To the best of our knowledge, this is the first study in which high-resolution MR images and light microscopy images of the three anatomical directions of seven wood species are compared. The non-destructive μ-MRI approach allows to investigate the 2D and 3D topological organization of the whole waterlogged wood sample up to a resolution of 8 μm. Although the optical microscope attains higher image resolutions and remains superior in the observation of wood diagnostic characters, multi-parametric μ-MRI provides physiological investigation complementary to light microscopy, giving information concerning both a single section and the whole volume of the sample. The presented study may represent a starting point for further improvements of μ-MRI techniques applied to the non-destructive investigation of waterlogged wood samples, especially those of interest for cultural heritage.

Identification of the heterotrophic nanoflagellate Bilabrum latius in the southern Adriatic (Mediterranean Sea)

Heterotrophic flagellates (HF) represent an important protist group in marine ecosystem functioning. Characterised by high taxonomic diversity, identification and classification of HF is often difficult using classical methods of light microscopy (LM). Complementing LM with molecular methods, such as barcoding, enables reliable taxonomic identification of even small size nanoflagellates that share similar or unnoticeable morphological features. The order Bicosoecida is a group of heterotrophic nanoflagellates that are important part of protist plankton and benthic communities of the world oceans. Recently, on the basis of high-resolution light microscopy and barcoding, a new bicosoecid genus, Bilabrum, was described with B. latius sp. as a type species. Our study reports on identification of B. latius from co-culture with the diatom species Chaetoceros affinis isolated from fresh plankton samples collected in the southern Adriatic. This detection of the Adriatic B.latius represents first record of this species outside itś up to now known and described habitat (deep-sea sediment of the South - East Atlantic Ocean) and in diatom co-culture.

In Depth Topological Analysis of Arabidopsis Mid-SUN Proteins and Their Interaction with the Membrane-Bound Transcription Factor MaMYB.

Mid-SUN proteins are a neglected family of conserved type III membrane proteins of ancient origin with representatives in plants, animals, and fungi. Previous higher plant studies have associated them with functions at the nuclear envelope and the endoplasmic reticulum (ER). In this study, high-resolution confocal light microscopy is used to explore the localisation of SUN3 and SUN4 in the perinuclear region, to explore topology, and to study the role of mid-SUNs on endoplasmic reticulum morphology. The role of SUN3 in the ER is reinforced by the identification of a protein interaction between SUN3 and the ER membrane-bound transcription factor maMYB. The results highlight the importance of mid-SUNs as functional components of the ER and outer nuclear membrane.

Vascular characterization and morphogenesis in porcine placenta at day 40 of gestation.

In porcine placenta, abnormal development of the placental vasculature leads to placental insufficiency. The aim of this study was to determine the mRNA expression of angiogenic growth factors and to determine the vascular characteristics in placenta at day 40 of pig gestation. Samples were collected from maternal-chorioallantoic interface (n = 21) for the measurement of mRNA expression of VEGFA, ANGPT1, ANGPT2, FGF2 and its receptors KDR, TEK, FGFR1IIIc, FGFR2IIIb respectively, and for immunohistochemistry analysis of CD31 and VEGFA. Immunohistochemical analysis of CD31 and VEGFA, morphometric measurement of blood vessels, high resolution light microscopy and transmission electron microscopy were performed. Capillary area density, number of blood vessels and capillary area were significantly higher on the maternal side than on the fetal side (P<0.05). The ultrastructural finding of blood vessels demonstrates close contact with the trophoblastic epithelium. The relative mRNA expression of VEGFA and its receptor KDR were higher compared with the other angiogenic genes. In conclusion, a high mRNA expression of VEGFA and its receptor KDR added to the immunohistochemical results suggest a potential role of these genes in this pathway associated with an increase in the density of the capillary area on the maternal side and a reduction in the hemotrophic diffusion distance at the interface for nutrient exchange.

Dynamic self-organization of the human genome during the cell cycle.

The hierarchical organization of the chromatin fiber, the functional form of DNA in cells, has been extensively studied from a single nucleosome to the genome-wide scale. Chromosome conformation capture techniques revealed a detailed static picture of the genome's 3D folded state inside the cell nucleus, revealing presence of loops, topologically associating domains, A/B compartments and chromosome territories [1]. In addition, chromatin organization was found to resemble a fractal globule inside the nucleus [2]. However, chromatin organization is not static but dynamically changes throughout the lifetime of a cell [3]—how it changes during the cell cycle remains an open question. In this work, we study the 3D chromatin organization inside the human cell nucleus and the changes of this organization during the cell cycle. By combining small angle X-ray scattering and high-resolution light microscopy, we investigate chromatin organization across a wide range of length scales during the cell cycle. In addition, we employed targeted biochemical perturbations to identify active cellular processes contributing to chromatin organization at these distinct length scales. Taken together, our data reveal that the 3D chromatin organization changes during the cell cycle in a length scale dependent manner [4]. This research was supported by the National Institutes of Health Grant R01-GM145924 and by the National Science Foundation Grants CAREER PHY-155488, CMMI-1762506 and PHY-2210541.

CNSC-30. IN VIVO DYNAMICS OF ASTROCYTE-GLIOBLASTOMA TUMOR NETWORKS

Abstract As one of the most aggressive incurable primary brain tumors, glioblastomas show a high invasivity and therapeutic resistance caused by their cellular and molecular heterogeneity. In previous studies, we showed that glioma cells interconnect using membrane protrusions called tumor microtubes to form a therapy-resistant malignant network. Here, we extend the concept of tumor networks to heterogeneous connectivity with the glial microenvironment. Using high-resolution light microscopy, ultrastructural tissue imaging, patch-clamp electrophysiology, intravital structural and functional microscopy, we characterize tumor-astrocyte connectivity. First, we used high-resolution light microscopy with immunohistochemistry and ultrastructural imaging to discover and characterize gap junctional connections between tumor cells and astrocytes. Next, we probed functional, heterotypic connections using dye filling with patch-clamp electrophysiology. Employing calcium imaging, we could demonstrate bidirectional communication patterns between tumor cells and astrocytes. We further characterized the stability of these astrocyte-glioma networks by using longitudinal imaging of single cells over several weeks in patient-derived xenograft models. For this, we used sulforhodamine 101 (SR101) as a marker for astrocyte-glioma connectivity to simultaneously visualize tumor cells and their glial microenvironment. The percentage of SR101 uptake in glioma cells increases over time, showing an increasing integration of tumor cells into the tumor-and astrocyte network during tumor evolution. Tumor cells, which are unconnected to other tumor cells or astrocytes, are the main drivers of invasion while a subgroup of glioma cells with stable astrocyte connectivity stay in place over several weeks. Lastly, we showed how these functional, heterotypic connections contribute to therapeutic resistance in the context of radiotherapy. In conclusion, we investigated multicellular networks between glioblastoma cells and astrocytes, their plasticity and role for therapeutic resistance.

Thyreophagus calusorum sp. n. (Acari, Acaridae), a new parthenogenetic species from the USA, with a checklist of Thyreophagus species of the world

The genus Thyreophagus is distributed worldwide, containing species occurring in stored food, house dust, bark, subcortical habitats, scale insect associations, and nests of wasps and bees. As species of this genus prefer concealed habitats, the actual species diversity is currently underappreciated. In addition, some described taxa are poorly known and their species boundaries are not entirely clear. Only a few species are known from both adult and deutonymphal stages. Using high-resolution light microscope and scanning electron microscopy, we describe a new partenogenetic species, Thyreophagus calusorum sp. n. from Florida (USA) based on adult stage (female) and heteromorphic deutonymph. This species was reared in the lab and its morphological variation was also studied. We did not find any substantial variation in the shape of the female spermatheca, but in the heteromorphic deutonymph, we did find extensive variation in the shape of tibial setae hT I (a taxonomically important character). Furthermore, we summarized taxonomic data and compiled an annotated checklist of species of Thyreophagus, with 35 species-level taxa. We clarified the following nomenclatorial questions: type species designation in Monieziella Berlese, 1897 was done by A. Berlese in G. Leonardi 1900 (Tyroglyphus entomophagus Laboulbène) not by A. Jacot (1936) as it was though previously; Monieziella mali Berlese, 1897 is an unjustified emendation and junior objective synonym of the name Acarus malus Shimer, 1868, currently Hemisacoptes malus (Shimer, 1868). Three subspecies of Thyreophagus were elevated to the species status due to their distinct morphology: Thyreophagus italicus Vacante, 1989, stat. nov. (=Thyreophagus entomophagus italicus Vacante, 1989); Thyreophagus mauritianus (Fain, 1982), stat. nov. (=Michaelopus gallegoi mauritianus Fain, 1982), and Thyreophagus ponticus Kadzhaya, 1973, stat. nov. (=Thyreophagus entomophagus ponticus Kadzhaya, 1973).

Widespread retina and optic nerve neuroinflammation in enucleated eyes from glaucoma patients

Neuroinflammation is recognized as a key component of neurodegenerative disease. In glaucoma, a common neurodegenerative disease and the leading cause of irreversible blindness, the evidence for neuroinflammation in patients is lacking. Animal models have demonstrated significant pro-inflammatory activation of resident glia in the retina, as well as influx of blood-derived monocytes and pro-inflammatory factors. Confirmation of this in human donor tissue has been challenging due to a lack of well-preserved and well-characterized post-mortem tissue. To address this we utilize archived, wax embedded eyes fixed immediately following enucleation from living glaucoma patients. We compared glaucoma to control eyes (enucleated for uveal melanoma where the tumor did not impact the central retina or optic nerve). We performed immunolabelling for neurodegenerative and glial markers (CD45, CD163, IBA1, GFAP, Vimentin) which were quantified by high-resolution light microscopy and image analysis in FIJI. Glaucoma eyes demonstrated significant neural loss consistent with advanced neurodegeneration. IBA1 and GFAP were significantly increased in the retina and optic nerve head of the glaucomatous eyes indicating that significant neuroinflammation had occurred which support findings in animal models. Inflammation is a treatable symptom of many diseases and as such, identification of earlier inflammatory processes in glaucoma could be important for potential future treatment options.

Anomalous morphologies in Ixodes scapularis feeding on human hosts

Cases of anomalous morphologies in the blacklegged tick, Ixodes scapularis, have been reported in both field-collected and human-biting specimen in the Northeastern and Midwestern United States, complicating the identification of this medically important tick species. We herein describe four cases of morphological anomalies in I. scapularis females exhibiting nanism and abnormally small genital apertures. We also report a female I. scapularis displaying slight asymmetry in the lower abdomen oriented toward the right side and an abnormal anal groove completely enclosing the anus. The identity of each specimen was confirmed using taxonomic keys, high resolution light and scanning electron microscopy imaging, and DNA sequencing of the 18S rRNA gene. All specimens described in this study were found parasitizing human hosts and were submitted to the Connecticut Agricultural Experiment Station-Tick Testing Laboratory in 2021 for species identification and pathogen screening. Here, we also discuss recent reports of teratological abnormalities in I. scapularis as well as likely causes for such deformities and potential implications.

Advances in studying whole mouse brain vasculature using high-resolution 3D light microscopy imaging.

.SignificanceThe cerebrovasculature has become increasingly recognized as a major player in overall brain health and many brain disorders. Although there have been several landmark studies to understand details of these crucially important structures in an anatomically defined area, brain-wide examination of the whole cerebrovasculature, including microvessels, has been challenging. However, emerging techniques, including tissue processing and three-dimensional (3D) microscopy imaging, enable neuroscientists to examine the total vasculature in the entire mouse brain.AimHere, we aim to highlight advances in these high-resolution 3D mapping methods including block-face imaging and light sheet fluorescent microscopy.ApproachWe summarize latest mapping tools to understand detailed anatomical arrangement of the cerebrovascular network and the organizing principles of the neurovascular unit (NVU) as a whole. ResultsWe discuss biological insights gained from studies using these imaging methods and how these tools can be used to advance our understanding of the cerebrovascular network and related cell types in the entire brain.ConclusionsThis review article will help to understand recent advance in high-resolution NVU mapping in mice and provide perspective on future studies.

Increasing the Image Sharpness of Light Microscope Images Using Deep Learning

Abstract Image-based qualitative and quantitative structural analyses using high-resolution light microscopy are integral parts of the materialographic work on materials and components. Vibrations or defocusing often result in blurred image areas, especially in large-scale micrographs and at high magnifications. As the robustness of the image-processing analysis methods is highly dependent on the image grade, the image quality directly affects the quantitative structural analysis. We present a deep learning model which, when using appropriate training data, is capable of increasing the image sharpness of light microscope images. We show that a sharpness correction for blurred images can successfully be performed using deep learning, taking the examples of steels with a bainitic microstructure, non-metallic inclusions in the context of steel purity degree analyses, aluminumsilicon cast alloys, sintered magnets, and lithium-ion batteries. We furthermore examine whether geometric accuracy is ensured in the artificially resharpened images.

Intestinal goblet cells sample and deliver lumenal antigens by regulated endocytic uptake and transcytosis.

Intestinal goblet cells maintain the protective epithelial barrier through mucus secretion and yet sample lumenal substances for immune processing through formation of goblet cell associated antigen passages (GAPs). The cellular biology of GAPs and how these divergent processes are balanced and regulated by goblet cells remains unknown. Using high-resolution light and electron microscopy, we found that in mice, GAPs were formed by an acetylcholine (ACh)-dependent endocytic event remarkable for delivery of fluid-phase cargo retrograde into the trans-golgi network and across the cell by transcytosis - in addition to the expected transport of fluid-phase cargo by endosomes to multi-vesicular bodies and lysosomes. While ACh also induced goblet cells to secrete mucins, ACh-induced GAP formation and mucin secretion were functionally independent and mediated by different receptors and signaling pathways, enabling goblet cells to differentially regulate these processes to accommodate the dynamically changing demands of the mucosal environment for barrier maintenance and sampling of lumenal substances.

Cover Feature: The Innate Immune Glycoprotein Lactoferrin Represses the Helicobacter pylori cag Type IV Secretion System (ChemBioChem 18/2021)

High-resolution light microscopy of Helicobacter pylori-infected gastric tissue (400× magnification): The picture shows a composite fluorescent image of gastric tissue stained with eosin (pseudo-colored blue) producing lactoferrin (pseudocolored overlay cyan), with the structure of lactoferrin in an inset (circular panel). H. pylori is a Gram-negative bacterial pathogen that infects the stomach and causes ulcers and gastric cancer. The host innate immune system produces the glycoprotein lactoferrin as part of the antimicrobial response to bacterial infection, and this molecule can repress the oncogenic activity of the H. pylori cag T4SS in an iron-dependent manner. More information can be found in the Full Paper by S. D. Townsend, J. A. Gaddy et al.

Epiblast and trophoblast morphogenesis in the pre-gastrulation blastocyst of the pig. A light- and electron-microscopical study.

The epiblast of the amniote embryo is of paramount importance during early development as it gives rise to all tissues of the embryo proper. In mammals, it emerges through segregation of the hypoblast from the inner cell mass and subsequently undergoes transformation into an epithelial sheet to create the embryonic disc. In rodents and man, the epiblast cell layer is covered by the polar trophoblast which forms the placenta. In mammalian model organisms (rabbit, pig, several non-human primates), however, the placenta is formed by mural trophoblast whereas the polar trophoblast disintegrates prior to gastrulation and thus exposes the epiblast to the microenvironment of the uterine cavity. Both, polar trophoblast disintegration and epiblast epithelialization, thus pose special cell-biological requirements but these are still rather ill-understood when compared to those of gastrulation morphogenesis. This study therefore applied high-resolution light and transmission electron microscopy and three-dimensional (3D) reconstruction to 8- to 10-days-old pig embryos and defines the following steps of epiblast transformation: (1) rosette formation in the center of the ball-shaped epiblast, (2) extracellular cavity formation in the rosette center, (3) epiblast segregation into two subpopulations - addressed here as dorsal and ventral epiblast - separated by a "pro-amniotic" cavity. Ventral epiblast cells form between them a special type of desmosomes with a characteristic dense felt of microfilaments and are destined to generate the definitive epiblast. The dorsal epiblast remains a mass of non-polarized cells and closely associates with the disintegrating polar trophoblast, which shows morphological features of both apoptosis and autophagocytosis. Morphogenesis of the definitive epiblast in the pig may thus exclude a large portion of bona fide epiblast cells from contributing to the embryo proper and establishes contact de novo with the mural trophoblast at the junction between the two newly defined epiblast cell populations.