Transient expression of phosphatidylserine at cell-cell contact areas is required for myotube formation.

Cell surface exposure of phosphatidylserine (PS) is shown to be part of normal physiology of skeletal muscle development and to mediate myotube formation. A transient exposure of PS was observed on mouse embryonic myotubes at E13, at a stage of development when primary myotubes are formed. The study of this process in cell cultures of differentiating C2C12 and H9C2 myoblasts also reveals a transient expression of PS at the cell surface. This exposure of PS locates mainly at cell-cell contact areas and takes place at a stage when the structural organization of the sarcomeric protein titin is initiated, prior to actual fusion of individual myoblast into multinucleated myotubes. Myotube formation in vitro can be inhibited by the PS binding protein annexin V, in contrast to its mutant M1234, which lacks the ability to bind to PS. Although apoptotic myoblasts also expose PS, differentiating muscle cells show neither loss of mitochondrial membrane potential nor detectable levels of active caspase-3 protein. Moreover, myotube formation and exposure of PS cannot be blocked by the caspase inhibitor zVAD(OMe)-fmk. Our findings indicate that different mechanisms regulate PS exposure during apoptosis and muscle cell differentiation, and that surface exposed PS plays a crucial role in the process of myotube formation.

Detection of apoptosis in ovarian cells in vitro and in vivo using the annexin v-affinity assay.

The ability of a cell to undergo apoptosis is crucial during development, tissue homeostasis, and in the pathogenesis and treatment of disease (1). To study apoptosis, it is important to be able to detect apoptotic cells reliably. Here we describe a method to detect apoptosis in vitro and in vivo on basis of the changes in phospholipid distribution in the plasma membrane that occur during this process. In healthy cells, phosphatidylserine (PS) is maintained predominantly in the inner plasma membrane surface by an aminophospholipid translocase (2). However, early during apoptosis, PS is translocated from the inner to the outer membrane surface and serves as a trigger for adjacent phagocytes to remove the dying cell (3-5). Exposure of PS can be detected in vitro and in vivo with fluorochrome- or biotin-labeled annexin V, a protein that binds to negatively charged phospholipids in the presence of calcium ions (6,7). In cells that are cultured in suspension, detection of apoptosis on the basis of PS exposure is relatively easy (8). However, sample handling of adherent cell lines, such as the ovarian cell line PA-1, might interfere with reliable detection of PS exposure. Therefore, we developed a method to detect PS exposure in adherent cell lines by labeling cells in a monolayer with annexin V and harvesting the cells afterwards by mechanical scraping (9) (Figs. 1 and 2). Fixation of annexin V-labeled cells also allows the study of the relationship between PS exposure and expression of intracellular antigens (10). We also present a method to detect apoptosis in vivo during follicular maturation in the mouse Fig. 3). This method is based on in vivo studies of viable mouse embryos, which indicate that PS exposure is a pancellular phenomenon of apoptosis during mammalian development (11,12). Fig. 1. Confocal scanning laser microscopy analysis of PA-1 ovary teratocarcinoma cells. Apoptosis was induced by treating the cells for 6 h with 50 µM roscovitine, a cyclin-dependent kinase inhibitor. Cells were labeled with annexin V-Oregon green to detect PS exposure, harvested by mechanical scraping, and labeled with propidium iodide (PI) to detect plasma membrane integrity. A and B show a linear projection of a stack of confocal images of early apoptotic cells after labeling with annexin V. At this stage, the plasma membrane integrity is preserved and, therefore, PI cannot enter the cell. C shows a secondary necrotic PA-1 cell, with clear annexin V staining at the plasma membrane (C1) and with PI staining of the condensed and fragmented chromatin (C2). Fig. 2. Dotplot of bivariate PI/annexin V flow cytometric analysis of adherent ovary cell line PA-1. Plasma membrane integrity is shown on the X-axis and annexin V immunofluorescence is shown on the Y-axis. Cells were treated with 50 µM roscovitine to induce apoptosis. 6 h after roscovitine treatment, cells were labeled with annexin V-Oregon green, harvested by scraping, and labeled with PI. Four populations of cells can be identified: region R1: vital cells (annexin V negative/PI negative), region R2: apoptotic cells (annexin V positive/PI negative), region R3: dead cells (annexin V positive/ PI positive); and region R4: damaged cells (annexin V negative/PI positive). For technical details, see ref. 9. Fig. 3. Micrographs of paraffin sections through mice ovaries that were perfused with biotinylated annexin V (A-F) or HEPES-buffer only (G and H). In A, annexin V labeled early apoptotic cells (arrowhead) and late apoptotic-pyknotic (arrow) granulosa cells are shown. During follicle maturation, initially apoptosis is absent (B). At later phases, annexin V labeled apoptotic granulosa cells (C, arrow) were observed in the primary (C) and secondary (D) follicles. Unstained pyknotic cells were also observed (C, arrowhead), presumably these cells were already located in the phagosomes before perfusion with annexin V. Also in the Graafian follicle, apoptotic cells were present in large numbers (E). F shows an enlargement of the boxed area in E. Labeled apoptotic and postapoptotic necrotic cells that have been shed into the antrum are clearly visible (asterisk), as well as unlabeled late postapoptotic necrotic cells. Labeling of ingested (arrowhead) and noningested (arrow) apoptotic cells was absent in ovaries of specimen that were perfused with HEPES-buffer only (G: overview, H: detail of boxed area in G). Scale bars equal 10 µm (A), 25 µm (C, F, and H), 50 µm (B and D) and 100 µm (E and G).

Phagocytosis of dying chondrocytes by osteoclasts in the mouse growth plate as demonstrated by annexin-V labelling.

Endochondral ossification in the epiphyseal growth plate of long bones is associated with programmed cell death (PCD) of a major portion of the chondrocytes. Here we tested the hypothesis that at the ossification front of the epiphyseal growth plate osteoclasts preferentially phagocytose chondrocytes that are undergoing PCD. We injected biotin-labelled annexin-V (anx-V-biotin, an early marker of PCD) intravenously in young adult mice. After 30 min of labelling, long bones were recovered and the tissue distribution examined of anx-V-biotin-labelled cells in the growth plate using ABC-peroxidase histochemistry. Positive staining for anx-V-biotin was detected in hypertrophic chondrocytes still present in closed lacunae at some distance from the ossification front. At the ossification front, chondrocyte lacunae were opened and close contacts were seen between tartrate-resistant acid phosphatase-positive osteoclasts and hypertrophic cartilage cells. Osteoclasts were significantly more frequently in contact with anx-V-biotin-labelled chondrocytes than with unlabelled chondrocytes. Osteoclasts also contained labelled and unlabelled phagocytic fragments within their cytoplasm. We conclude that in the growth plate osteoclasts preferentially phagocytose hypertrophic chondrocytes that are dying, suggesting these dying cells may signal osteoclasts for their removal.

In situ detection of apoptosis in dental and periodontal tissues of the adult mouse using annexin-V-biotin.

An early event in apoptosis is exposure of phosphatidylserine, an aminophospholipid normally present in the inner leaflet of the plasma membranes, at the outer leaflet of the plasma membrane facing the extracellular space. Annexin V (Anx-V) is a 35-kDa protein with high affinity for phosphatidylserine, which can be applied to detect apoptosis. We injected biotin-labelled Anx-V intravenously in adult mice and examined the tissue distribution of Anx-V-labelled cells in dental and periodontal tissues using ABC-peroxidase histochemistry. In the continuously erupting incisors, strong and frequent immunostaining was observed in transitional stage and late maturation stage ameloblasts with less frequent staining in preameloblasts. Frequency of staining in odontoblasts and pulp cells was low but increased slightly at older stages of dentinogenesis. Labelling was also seen in phagocytic or phagocytic-like cells in the enamel organ and pulp. A positive staining was furthermore found in fibroblasts of the periodontal ligament in continuously erupting incisors and in fully erupted molar teeth. Staining intensity and the number of positive cells were enhanced by antigen retrieval using high-pressure cooking. We conclude that Anx-V-biotin labels dental cells in early stages of cell death and indirectly cells that have ingested labelled apoptotic cells during the course of the experiment. The data confirm that during amelogenesis most cell death occurs in transitional stage and late maturation stage ameloblasts. Thus, labelling with Anx-V is a useful marker for studying cell death and the dynamics of clearance of apoptotic cells during tooth development.

Spatiotemporal distribution of dying neurons during early mouse development.

Apoptosis is a critical cellular event during several stages of neuronal development. Recently, we have shown that biotinylated annexin V detects apoptosis in vivo in various cell lineages of a wide range of species by binding to phosphatidylserines that are exposed at the outer leaflet of the plasma membrane. In the present study, we tested the specificity by which annexin V binds apoptotic neurons, and subsequently investigated developmental cell death in the central and peripheral nervous system of early mouse embryos at both the cellular and histological level, and compared the phagocytic clearance of apoptotic neurons with that of apoptotic mesodermal cells. Our data indicate: (i) that biotinylated annexin V can be used as a sensitive marker that detects apoptotic neurons, including their extensions at an early stage during development; (ii) that apoptosis plays an important part during early morphogenesis of the central nervous system, and during early quantitative matching of brain-derived neurotrophic factor and neurotrophic factor 3 responsive postmitotic large clear neurons in the peripheral ganglia with their projection areas; and (iii) that apoptotic neurons are removed by a process that differs from classical phagocytosis of non-neuronal tissues.

mRNA expression patterns of the IGF system during mouse limb bud development, determined by whole mount in situ hybridization.

During limb development the primary limb bud requires various signals to differentiate. Insulin-like growth factor (IGF)-I and IGF-II serve as ubiquitous cellular growth promoters and are modulated by their binding proteins (IGFBPs), which inhibit or augment IGF bioavailability. This is the first study to give a complete overview of the mRNA expression patterns of Igf-1, Igf-2, type 1 Igf receptor (Igf1r) and six Igf binding proteins (IGFBP-1-6) in embryonic mouse limbs, at various stages of development, by whole mount in situ hybridization (ISH). Our results show that all the members of the Igf system, except Igfbp-1 and -6, have specific spatio-temporal mRNA expression patterns. IGFBP-2 and -5 are found in the apical ectodermal ridge (AER), and IGF-I and IGFBP-4 in the region of the zone of polarizing activity (ZPA). IGF-II and IGF1R are found in regions of pre-cartilage formation. At 13.5 days post coitus (dpc) the IGF system colocalizes with apoptosis areas; IGFBP-2, -4 and -5 are found in the interdigital zone, while IGFBP-3 and IGF-I border this region. Furthermore, IGFBP-3, -4 and -5 are found in the phalangeal joint areas, at an early stage of joint formation. This supports the hypothesis that the IGF system may be involved in chondrogenic differentiation of mesenchyme and the regulation of apoptosis in the developing limb.

Cell surface exposure of phosphatidylserine during apoptosis is phylogenetically conserved.

Exposure of the aminophospholipid phosphatidylserine at the outer leaflet of the plasma membrane by apoptotic cells can trigger phagocytic removal of these dying cells. This functionality of phosphatidylserine exposure in the process of phagocytosis is indicated by in vitro studies of mammalian and insect phagocytes. We have studied the in vivo distribution of cell-surface exposed phosphatidylserine by injecting biotinylated Annexin V, a Ca2+ -dependent phosphatidyl-serine binding protein, into viable mouse and chick embryos and Drosophila pupae. The apparent binding of Annexin V to cells with a morphology which is characteristic of apoptosis and which was present in regions of developmental cell death indicates that phosphatidylserine exposure by apoptotic cells is a phylogenetically conserved mechanism.

Phosphatidylserine plasma membrane asymmetry in vivo: a pancellular phenomenon which alters during apoptosis.

The distribution of phospholipids across the two leaflets of the plasma membrane is important for many cellular processes including phagocytosis and hemostasis. In the present study we investigated the in vivo plasma membrane distribution of the aminophospholipid phosphatidylserine in mouse embryos with a novel technique employing Annexin V, a Ca2+ dependent phosphatidylserine binding protein, conjugated to fluorescein isothiocyanate and biotin. Annexin V directly applied to cryostat sections labeled the plasma membrane of all cells at the interface. In contrast, Annexin V injected intracardially into viable mouse embryos labeled almost exclusively apoptotic cells. These apoptotic cells were visible in all tissues and derived from all germ layers. Our experiments demonstrate that phosphatidylserine is asymmetrically distributed between the two leaflets of the plasma membrane in virtually all cell types in vivo and that this asymmetry is lost early during apoptosis.

In situ detection of apoptosis during embryogenesis with annexin V: from whole mount to ultrastructure.

Apoptosis is of paramount importance during embryonic development. This insight stems from early studies which correlated cell death to normal developmental processes and now has been confirmed by linking aberrant cell death patterns to aberrant development. Linking apoptosis to the phenotype of a developing organism requires spatial information on the localization of the dying cells, making in situ detection essential This prerequisite limits the tools available for such studies (1) to vital dyes, which can be detected at the whole mount level only; (2) to detection based upon apoptotic morphology by routine light microscopy and electron microscopy; and (3) to staining for apoptosis associated DNA fragmentation via, e.g., the TUNEL procedure, which marks cells in a relative late phase of apoptosis. New apoptosis markers need to be specific and should preferably detect cells early during this process. In the present study we show that the recently discovered in vitro marker of apoptosis, Annexin V meets these requirements for in vivo detection. Through intracardiac injections of biotin labeled Annexin V, a Ca2+ dependent phosphatidylserine binding protein, we were able to visualize apoptotic cells derived from each germ layer in the developing mouse embryo from the whole mount level up to the ultrastructural level. Double-labeling on paraffin sections for both this method and TUNEL revealed that cells become Annexin V-biotin labeled early during the process of apoptosis.

Origin of subepicardial cells in rat embryos.

BACKGROUND: The role of the villi and vesicles of the epicardium primordium in the formation of the epicardium has been extensively studied over the last decades. With regard to the cellular contents of the villi and vesicles of the epicardium primordium, in quail the presence of mesenchymal cells in the villi recently has been described. In the present study, we have determined whether the villi and vesicles of the epicardium primordium in rat embryos contain mesenchymal cells that originate from the transverse septum and if so, whether these cells will become part of the subepicardium. METHODS: Mesenchymal cells in the transverse septum of rat embryos were labelled by a method consisting of in vitro whole embryo culture and labelling of the ectoderm and its daughter cells, using wheat germ agglutinin-gold (WGA-Au) as a marker. RESULTS: In concordance with our observations in the standard noncultured rat embryos, labelled cells were present in the transverse septum, extending from the umbilical ring, i.e., the transition of amniotic epithelium to ectoderm, up to the villi, in the villi and vesicles, and subepicardially. CONCLUSIONS: These observations suggest that the epicardium primordium contains mesenchymal cells derived from the transverse septum. These cells reach the subepicardium, using the villi and vesicles of the epicardium primordium as their vehicle.

Changes in inferior vena cava blood flow velocity and diameter during breathing movements in the human fetus.

Breathing movements in the human fetus cause distinct changes in Doppler flow velocity measurements at arterial, venous and cardiac levels. In adults, breathing movements result in a momentary inspiratory collapse of the inferior vena cava vessel wall. The study objective was to quantify the inferior vena cava flow velocity modulation during fetal breathing movements and to evaluate possible inferior vena cava vessel diameter changes in normal third-trimester pregnancies. We studied 57 women after oral administration of dextrose (50 g). In 40 fetuses (n = 19, 27-32 weeks and n = 21, 36-39 weeks), fetal inferior vena cava waveforms were obtained during apnea and fetal breathing activity. In 30 fetuses (27-39 weeks) inferior vena cava vessel diameter changes were studied using the M-mode during apnea and breathing movements. Peak and time-averaged velocities of inferior vena cava flow velocity waveforms showed a gestational age-independent increase of 60-160% during breathing activity. A temporary inferior vena cava vessel wall collapse (range, 50-83%) was recorded, which was significantly different from vessel diameter changes during apnea (range, 11-19%). The marked increase of inferior vena cava flow velocities is due to a raised thoraco-abdominal pressure gradient, which may cause a reduction in vessel size and additional volume flow into the right atrium. The significance of the caval index for recognition of elevated right atrial pressure in abnormal human fetal development needs further investigation.