-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Cell Science 11/2001; 114(Pt 20):3631-42. · 6.11 Impact Factor
-
Journal of Craniofacial Surgery 08/2001; 12(4):399-400. · 0.82 Impact Factor
-
Mammalian Genome 02/2001; 12(1):77-9. · 2.89 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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).
Methods in molecular medicine 01/2001; 39:669-77.
-
[show abstract]
[hide abstract]
ABSTRACT: A histological study was performed on serially sectioned human and mouse embryos to study the influences of programmed cell death (PCD) during morphogenesis for clarifying the existing controversies on the morphology and basic processes involved in the embryonic development of the male anterior urethra. The following new insights into the development of the anterior urethra could be established. The formation of the urethra starts with the early adhesion of the arms of the genital tubercle. In this way an epithelial plate is formed, located in the ventral midline, that is in continuity with the cloacal membrane. Male sex differentiation takes place following rupture of this cloacal membrane through programmed cell death. Fusion of the urogenital swellings with primary luminization gives rise to the penile urethra, whereas the glandular part of the urethra is formed through secondary luminization of the epithelial cord that is formed during fusion of the arms of the genital tubercle, i.e., the glans. In both fusion processes, apoptosis plays a key role. The consequence of fusion of the urogenital swellings is that their mesodermal cores unite on the ventral aspect of the penile urethra, where they differentiate into the integumental structures. The prepuce starts to develop as a fold of ectoderm with a mesodermal core after complete fusion of the entire urethra. Finally, the scrotum was found to develop through merging of the labioscrotal swellings and not by fusion.
Teratology 04/2000; 61(3):172-83.
-
[show abstract]
[hide abstract]
ABSTRACT: Six hundred and ninety-four patients with 993 anomalies of the upper limbs were classified according to the classification of Swanson et al. (1983). The data from these patients were compared with previous studies, and similar discrepancies were found. One explanation for these discrepancies is a lack of uniformity in the classification of Swanson et al., which may be caused by out-dated knowledge of the pathogenesis of congenital limb anomalies. Therefore, it seems necessary to describe the anomalies instead of the diagnoses. A descriptive method is being validated in our outpatient department that records all anomalies of the upper limb.
The Journal of Hand Surgery British & European Volume 03/2000; 25(1):3-7. · 0.04 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A high percentage of craniofacial malformations is ascribed to abnormalities in cell populations derived from the neural crest
and ganglionic placodes. Both cell populations originate from the ectoderm of the head-neck area by means of the mechanism
of epithelio-mesenchymal transformation (EMT). Neural crest cells are multipotent and form the majority of the mesenchymal
compartment of the head-neck area. EMT of the neural crest will stop shortly after closure of the neural tube. The current
opinion about ganglionic placodal cells is that they are derived from the surface ectodermal placodes after closure of the
cranial neural tube, and transform into neurons of the sensory ganglia only. However, additional EMT sites of the cranial
neural and surface ectoderm have also been found to produce multipotent cells. Because of the fact that most craniofacial
malformations develop after closure of the cranial neural tube, in this study we focused on the EMT of the surface ectoderm
of the head-neck area during the stages of outgrowth of facial swellings and branchial arches. The surface ectoderm was labeled
by injecting various tracker dyes into the amniotic cavity of chick embryos after closure of the anterior neuropore, to exclude
labeling of the neural ectoderm and the neural crest. After various incubation periods, ranging from 0–24 h, labeled cells
were found in the mesenchymal compartment scattered over the embryonic face and neck, originating from non-placodal as well
as placodal surface ectoderm. Thus, besides the neural ectoderm and neural crest, it could be shown that the entire surface
ectoderm of the face and neck contributes cells to the underlying mesenchymal tissue, and that this phenomenon occurs in a
higher frequency at sites of outgrowing swellings. As a consequence, the pathogenesis of congenital craniofacial malformations
should be reconsidered.
European Journal of Plastic Surgery 01/2000; 23(4):217-223.
-
[show abstract]
[hide abstract]
ABSTRACT: Unilateral complete cleft lip patients treated with or without a primary nasal correction at the time of cleft lip repair were compared to evaluate the relevance of early surgical correction of the nose by using two assessments: nasal symmetry and morbidity.
The no nasal correction group (NNC, n = 19) was operated by surgeon A using the Millard technique. The primary nasal correction group (PNC, n = 9) was operated by surgeon B combining the modified Millard technique with a columellar lift and alar mobilization. Symmetry was assessed on two sets of standardized photographs at 9 years of age using a computer-assisted analysis. Both cleft groups were compared with normal controls (NC, n = 20). The computer method included area and angular measurements. Morbidity was assessed by the number of procedures on the vermilion, the lip, and/ or nose for revisional surgery up to the age of 9 (NNC, n = 26; PNC, n = 12).
No significant differences in symmetry were found between the NNC and PNC groups regarding the area and angular measurements. With regard to the area measurements, both cleft groups produced a significant asymmetry when compared to the NC group. Concerning the angular measurements, however, the NNC group differed significantly from the NC group, whereas such a difference could not be noted between the PNC group and NC group. With respect to morbidity, no revisional procedures were performed in the PNC group. The number of revisional procedures in the NNC group was 16 in 10 patients.
Results are presented that favor, up to the age of 9 years, a primary nasal correction at the time of cleft lip repair.
The Cleft Palate-Craniofacial Journal 08/1999; 36(4):361-6. · 0.82 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We analysed mRNA and protein localization of the IGF system components in regions with apoptosis during mouse development between 9.5 and 13.5 days post coitum. A spatio-temporal relationship between these expression patterns and the onset of apoptosis in specific areas was sought. The IGFBP mRNA and protein expression patterns were tissue-specific. In most tissues, mRNA expression patterns colocalized with protein localization. Discrepancies between mRNA and protein detection were found in, for example, lens, neural layer of the retina, whiskers and somites. Localization of the IGFs, the type I IGF receptor and IGFBP-2 correlated well with cell death regions. When these genes were expressed no apoptosis occurred and vice versa. Correlation of IGFBP-3, -4 and -5 with apoptosis regions was noticed only at 13.5 days post coitum. In eye muscles, whiskers and somites, the expression of IGF system components preceded the occurrence of apoptotic cells. When IGF-I expression ceased, apoptosis occurred in these areas. In conclusion, our results suggest that IGF-I, the type I IGF receptor and IGFBP-2 inhibit apoptosis. In contrast, IGFBP-3, -4 and -5 may stimulate apoptosis by trapping the IGFs. Tissue-specific modulation of IGF protective action against apoptosis by the different IGFBPs during mouse embryonal development may contribute to organ specific morphology.
Growth Hormone & IGF Research 07/1999; 9(3):195-204. · 2.16 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
European Journal of Neuroscience 03/1999; 11(2):712-24. · 3.63 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Four pregnancies, in two women aged 39 and two women aged 34 years respectively, were complicated by foetal parvovirus B19 infection. First-trimester intrauterine death resulting from multiple congenital anomalies was diagnosed in one patient with proven foetal parvovirus B19 infection. In three patients foetal hydrops was found in the second trimester with variable clinical course. In one of them, foetal hydrops resulted in second-trimester foetal death; in another, foetal hydrops resolved following intrauterine blood transfusion and in a third foetal hydrops resolved spontaneously. Foetal parvovirus B19 infection was diagnosed by polymerase chain reaction (PCR) using foetal cells obtained by amnioscentesis. It is concluded that maternal parvovirus B19 infection is mostly asymptomatic. However, the clinical impact of maternal infection on the foetus is diverse, i.e. infection may result in foetal death or--transient--foetal morbidity, in particular foetal anaemia. In mothers with proven foetal parvovirus B19 infection close monitoring of the foetus by ultrasound is warranted. Occasionally intrauterine transfusion is required. From the literature to date, the estimated incidence of maternal parvovirus B19 infection in pregnancy is 3-7%. The vertical transmission rate approximates 30%. When pregnancy is complicated by foetal hydrops foetal parvovirus B19 infection should be kept in mind.
Nederlands tijdschrift voor geneeskunde 02/1999; 143(1):3-7.
-
[show abstract]
[hide abstract]
ABSTRACT: In the literature, some controversy still exists about the normal and abnormal development of the human anorectum. Therefore, a three-dimensional and histological study was performed on human embryos. In early anorectal development (< or = 49 days postfertilization), the cloaca plays a crucial role, separated from the amniotic cavity by its cloacal membrane. In the cloaca, the yolk sac/primitive hindgut and allantois/primitive urogenital sinus enter. During the embryonic caudal folding process, incorporation of these structures occurs, including their surrounding extraembryonic mesoderm, which fuses to form the urorectal septum. Consequently, this septum does not grow in the direction of the cloacal membrane, and fusion of these structures is likewise never observed. The cloaca remains as such until the cloacal membrane ruptures by apoptotic cell death. The dorsal part of the cloaca then becomes part of the amniotic cavity, and is by no means involved in the development of the anorectum. The tip of the urorectal septum will become the perineal area. Soon after rupture of the cloacal membrane, during late anorectal development (> or = 49 days postfertilization), a secondary occlusion of the anorectal canal occurs, first due to adhesion, followed by formation of an epithelial "plug" at the level of the anal orifice. Recanalization, by apoptotic cell death, of this secondary occluded anal orifice occurs later during development. Based on these embryological observations, congenital anorectal malformations with an abnormal communication to the exterior are best explained as early embryonic defects. The abnormal communications, usually called fistulae, should be regarded as ectopic anal orifices. Anorectal malformations with the anus in normal position are best explained as late embryonic defects.
Teratology 03/1998; 57(2):70-8.
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
APOPTOSIS 02/1998; 3(1):9-16. · 4.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Cytometry 01/1998; 29(4):313-20.
-
[show abstract]
[hide abstract]
ABSTRACT: A prospective clinical study of 30 patients with frontoethmoidal encephaloceles was performed in order to find support for a proposed theory concerning its pathogenesis, based on a previously performed embryological study and relevant findings in the literature. According to this proposed theory the pathogenesis of frontoethmoidal encephaloceles is primarily based on a disturbance in separation of neural and surface ectoderm at the site of final closure of the rostral neuropore during the final phase of neurulation in the 4th week of gestation. An insufficient occurrence of apoptosis might cause this disturbance in separation. The nonseparation of neural and surface ectoderm will result secondarily in a midline mesodermal defect. This mesodermal defect is reflected in the median skull defect at the site of the foramen caecum. The outgrowth of the nasal septum with the concomitant forward displacement of epidermis (surface ectoderm) and attached brain tissue (neural ectoderm) may act as herniating force. The patient study consisted of a clinical investigation, radiological investigations (X skull and CT scans), and surgical treatment in order to obtain specimens which were examined histologically. Clinical findings supportive of the proposed hypothesis are (1) the consistency in the location of the internal skull defect, (2) the close relationship between epidermal structures and glial tissue in 15 out of 29 specimens, and (3) the presence of a normally developed nose in combination with interorbital hypertelorism in all patients. A discussion of these findings is presented with special reference to the embryological aspects.
Pediatric Neurosurgery 12/1997; 27(5):246-56. · 0.70 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Regulation of programmed cell death (apoptosis) is crucial for normal development and growth, both prenatally and postnatally. If its role during normal embryogenesis of a given structure is established, a number of related congenital disorders can be explained by a (local) deregulation of apoptosis. In this study, apoptotic cell death patterns during normal development of the murine coronal suture were investigated. Detection of apoptotic cells was undertaken by labeling with Annexin V. Results showed apoptosis occurring at the same time and place as suture initiation. Apoptotic cells are located along the entire established part of the suture and its developing part. Because apoptosis is shown to be highly associated with sutural genesis, the theory of craniosynostosis being the equivalent of deregulation at this locus seems in line with these findings.
Journal of Craniofacial Surgery 12/1997; 8(6):441-5. · 0.82 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Cell Death and Differentiation 06/1997; 4(4):311-6. · 8.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We describe a case of full monosomy 21 which was prenatally diagnosed in chorionic villi by fluorescent in situ hybridization (FISH). Because of intrauterine fetal death, a curettage was performed and cytogenetic analysis of skin fibroblasts confirmed the presence of monosomy 21 in fetal cells. DNA investigations showed a paternal origin of the single chromosome 21. Inspection and autopsy of the fetus revealed several congenital malformations. Some of them have been reported in earlier studies of monosomy 21; others concern new observations. Regarding the eye, the following abnormalities were microscopically observed: absence of the anterior and posterior eye chambers, aniridy, a hypoplastic ciliary body, Peter's anomaly, and a double retina with secondary dysplasia. In addition, malformations of the extremities were seen: partial, proximal syndactyly of digits 3 and 4 of the right hand; pes varus position of the right foot; and transverse reduction defect at the tarsals of the left foot. To our knowledge, this is the first case in which full monosomy 21 has been proven.
Prenatal Diagnosis 04/1997; 17(3):271-5. · 2.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A fetus with transverse limb reduction defects and jejunal atresia after exposure to chorionic villus sampling (CVS) at 9 weeks of amenorrhoea is described. A theory involving disruption of end-arteries due to the CVS procedure is suggested.
Prenatal Diagnosis 02/1997; 17(1):71-6. · 2.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: There is an increasing number of reports relating chorionic villus sampling (CVS) to transverse limb reduction defects or the oromandibular limb hypogenesis complex. In addition, a correlation has been established between the severity of the defect and the gestational age when CVS is performed. Several hypotheses have been proposed for the increased incidence of congenital malformations after CVS including vascular disruption. Recently, it has been suggested that maternoembryonic transfusion can occur after CVS and that this can lead to a local antibody-mediated reaction, followed by local pathogenetic cell degeneration, i.e., apoptotic cell death, due to vascular disruption. This increased apoptotic cell death will ultimately result in congenital malformations. This paper describes an experimental model that can explain the pathogenesis of congenital malformations after CVS. The model designed uses a whole rat embryo culture technique and intracardiac injection of antisera, mimicking transplacental transfusion after CVS. Injection of antibodies directed against blood group antigens is capable of inducing increased apoptotic cell death. Immunological staining gives evidence of involvement of antibody-mediated reactions in the occurrence of apoptotic cell death. The dorsal aortae in 10-day-old rat embryos of 10-somite stages of development consist of a continuous endothelial cell layer. The effect of intracardiac injection of antisera on the dorsal aortae is only transient. Smaller vessels such as the pharyngeal arch arteries or arteries of the limbs still have fenestrated endothelium and are therefore more vulnerable to the pathogenetic effect of the reaction after transplacental transfusion causing vascular disruption. Development of the vascular pattern and differentiation of the vascular wall reduce the risk of severe malformations later on in pregnancy, although the risk of malformations remains throughout pregnancy. Thus, intracardiac injection of antisera simulating maternoembryonic transfusion such as after CVS can lead to an antibody-mediated reaction with vascular disruption early in pregnancy inducing apoptotic cell death. Increased cell death may ultimately result in congenital malformations, such as transverse limb defects or the oromandibular limb hypogenesis complex.
Prenatal Diagnosis 02/1997; 17(1):59-69. · 2.11 Impact Factor
