Apoptosis in human embryo development: 2. Cerebellum

We analysed the spatial and temporal distribution of apoptosis in human cerebellum development, during embryonic and fetal periods. Cerebella excised from two human embryos (8 weeks old) and eight fetuses (12-22 weeks old), were paraffin embedded and serially sectioned. Apoptotic cells were identified by propidium iodide staining, and TUNEL. In addition, immunohistochemistry for suicide receptor Fas(APO-1/CD95) was performed. We determined the distribution and percentage of apoptotic cells as well as Fas(APO-1/CD95)-positive cells in different regions and stages of development. Apoptotic cells were seen in both proliferative zones and postmitotic regions along the migratory pathways as well as in the developing cerebellar cortex in all examined stages. The Fas(APO-1/CD95) immunoreactivity was present in all examined stages in a small population of apoptotic cells: either neuroblasts or differentiated cells in postmitotic zones. These findings suggest that apoptosis drives the selection of the cells which are committed to differentiate during the early stages of cerebellar development. The differences between apoptotic cells distribution and Fas receptor expression suggest that cell selection is driven by different apoptotic pathways.     

Role of apoptosis and mitosis during human eye development

The spatial and temporal distribution as well as ultrastructural and biochemical characteristics of apoptotic and mitotic cells during human eye development were investigated in 14 human conceptuses of 4-9 postovulatory weeks, using electron and light microscopy. In the 5th developmental week, apoptotic and mitotic cells were found in the neuroepithelium of the optic cup and stalk, being the most numerous at the borderline between the two layers of the optic cup, and at the place of transition of the optic cup into stalk. They were also found at the region of detachment of the lens pit from the surface ectoderm. In the later developmental stages (the 6th-the 9th week), apoptotic and mitotic cells were observed in the neural retina and the anterior lens epithelium. Throughout all stages examined, mitotic cells were found exclusively adjacent to the lumen either of the intraretinal space or the optic stalk ventricle, or were restricted to the superficial epithelial layer of the lens primordium. Unlike mitotic cells, apoptotic cells occurred throughout the whole width both of the neuroepithelium and the surface epithelium. Ultrastructurally, apoptotic cells were characterised by round- or crescent-shaped condensations of chromatin near the nuclear membrane, while in the more advanced stages of apoptosis by apoptotic bodies. The distribution of caspase-3-positive cells coincided with the location of apoptotic cells described by morphological techniques indicating that the caspase-3-dependent apoptotic pathway operates during the all stages of human eye development. The location of cells positive for anti-apoptotic bcl-2 protein was in accordance with the regions of eye with high mitotic activity, confirming the role of bcl-2 in protecting cells from apoptosis. In the earliest stage of eye development, apoptosis and mitosis might be associated with the sculpturing of the walls of optic cup and stalk, while high mitotic activity along the intraretinal space and optic stalk ventricle indicates its role in the gradual luminal closure. These processes also participate in the detachment of the lens pit epithelium from the surface ectoderm as well as in further closure of the lens vesicle. Later on, both processes seem to be involved in the neural retina differentiation, lens morphogenesis and secondary lens fibre differentiation.     

Protein absorption by tubular mesonephric and metanephric structures in the human embryo

Summary The presence of different serum proteins in the cells of the proximal tubule of both meso- and metanephric nephrons in human embryos (7th–12th week of intrauterine life) was investigated using immunohistochemistry. Endogenous lysozyme, alpha-1-antitrpysin and ferritin were detected in mesonephric proximal tubules and, starting from the 8th week, also in metanephric proximal tubules. Our observations provide information concerning the appearance and distribution of tubular protein reabsorption during the early stages of development.     

Proliferation and apoptosis in the epithelium of the developing human cornea and conjunctiva.

To determine the distribution of proliferating and apoptotic cells in the human cornea during prenatal and early postnatal development, we examined sections of the bulbar conjunctiva, the limbus as well as the central and peripheral cornea between 11 weeks of gestation and 6 months after birth. The objective was to localize dividing cells by proliferating cell nuclear antigen-like immunoreactivity (PCNA-LI) and apoptotic cells by terminal transferase-mediated nick-end labeling (TUNEL). Before the 17th gestational week, PCNA-LI was absent in all 4 regions examined, indicating negligible cell proliferation during early development. After 20 weeks, strong PCNA-labeling was observed in all regions examined suggestive of high proliferative activity not only in the limbus and the bulbar conjunctiva, but also in the central and peripheral cornea. This rise in proliferative activity was followed by a steady decline: after 28 weeks, anti-PCNA staining gradually disappeared in the central and peripheral cornea, so that, at 6 months after birth, it was confined to the limbus and the bulbar conjunctiva, resembling the picture described for the adult cornea. TUNEL-positive cells were virtually absent in all 4 regions examined before the 38th gestational week. Apoptotic cells only started to appear at 38 weeks; at this stage, they were confined to the bulbar conjunctival epithelium. At 6 months after birth, TUNEL-positive cells were observed in the bulbar conjunctival epithelium and the entire cornea; the limbus, however remained devoid of apoptotic cells throughout the entire prenatal and early postnatal period. The present study for the first time localizes proliferating and apoptotic cells in the epithelium of the developing human cornea. Three stages of development can be distinguished: Minimal proliferation (until 17th week), vigorous proliferation over the entire cornea including the limbus and the bulbar conjunctiva (until 28th week) and gradual decrease in proliferative activity (after 28th week) accompanied by the appearance of apoptotic cells.     

Monoclonal Antibody to Single-Stranded DNA Is a Specific and Sensitive Cellular Marker of Apoptosis

The most widely used histochemical marker of apoptosis (in situend labeling, TUNEL) detects both apoptotic and necrotic cells and evaluates only late stages of apoptosis. Hence, a specific and sensitive cellular marker of apoptosis is needed to determine the role of apoptotic death in biology and pathology. The present study describes a novel immunohistochemical procedure for the staining of apoptotic cells using a monoclonal antibody (MAb) to single-stranded DNA. This MAb stained all cells with the morphology typical of apoptosis in etoposide-treated HL-60, MOLT-4, and R9 cell cultures, in which apoptosis was accompanied by high, moderate, and low levels of internucleosomal DNA fragmentation, respectively. TUNEL stained all apoptotic cells in HL-60 cultures, nearly 60% of apoptotic cells in MOLT-4 cultures, and only 14% of apoptotic cells in R9 cultures. Apoptotic R9 cells, which progressed into secondary necrosis, retained MAb staining and became TUNEL-positive. Necrotic cells in MOLT-4 cultures treated with sodium azide were stained by TUNEL, but were negative for MAb staining. All floating cells at a late stage of apoptosis in MDA-MB-468 cultures treated with cisplatin were stained by both MAb and TUNEL. However, among adherent cells in the early stages of apoptosis, MAb stained nearly 20 times more cells than TUNEL. In histological sections of human tumor xenografts, MAb detected clusters of apoptotic cells in viable tumor tissue, but did not stain cells in areas of central ischemic necrosis. In contrast, TUNEL stained nuclei in necrotic areas. Thus, MAb to single-stranded DNA is a specific and sensitive cellular marker of apoptosis, which differentiates between apoptosis and necrosis and detects cells in the early stages of apoptosis.     

Apoptosis in human embryo development: 1. Cerebral cortex

We investigated the apoptosis at the beginning of human cerebral cortex development, in the 6th week of embryogenesis, Carnegie stages 16 and 17. Attention was focused on the dorsal wall of the telencephalon to the ventricular zone of proliferation and to the postmitotic zone with beginning of neuronal migration. We identified apoptotic cells in tissue sections by propidium iodide staining, TUNEL and immunohistochemistry for Fas(APO-1/CD95). We determined the distribution and the percentage (reported to the propidium iodide stained nuclei) of apoptotic TUNEL-positive and Fas(APO-1/CD95)-positive cells. TUNEL-positive apoptotic cells in the proliferative zone were 20% in stage 16 and 60% in stage 17. TUNEL-positive apoptotic cells in the postmitotic zone were 8% in stage 16 and 30% in stage 17. CD95-positive apoptotic cells in the proliferative zone were 5% in stage 16 and 2% in stage 17. There were no CD95-positive cells in the postmitotic zone. We evidentiated the presence of the suicide receptor Fas(APO-1/CD95) only on a small population of apoptotic neuroblasts in the proliferative zone. The differences between apoptotic distribution and receptors in early corticogenesis suggest that different apoptotic pathways drive the selection of neuronal populations.     

Disparity of apoptotic response in human breast cancer cells lines MCF-7 and MDA-MB-231 after infection with recombinant adenovirus encoding the VP2 gene of infectious bursal disease virus

Recombinant adenovirus encoding the VP2 gene of infectious bursal disease virus (ADV-VP2) has shown potent anti-tumour effects due to its capability of apoptotic induction in cancer cells. In the present study, human breast cancer cells MCF-7 and MDA-MB-231 were infected with ADV-VP2. The expression of VP2 protein was registered 4 hours post-infection, particularly in MCF-7 cells. Multiple time-point DNA ladder assay demonstrated that ADV-VP2 infected MDA-MB-231 and MCF-7 cells endured apoptosis as early as 8- and 12- hours post-infection, respectively. Apoptosis induction in both MDA-MB-231 and MCF-7 cells, albeit different start points, lasted until 36-hours post-infection. The induction of apoptosis by ADV-VP2 was further shown by the TUNEL assay, with dark brown discoloration of apoptotic cells. The present study also explored the different stages of apoptosis by Annexin V/PI double staining flow cytometry quantification. Treated MCF-7 and MDA-MB-231 cells, respectively detected 25.58 ± 9.02% and 14.51 ± 3.12% of early apoptotic cells, 6.09 ± 4.06% and 77.12 ± 5.09% of late apoptotic cells. Results revealed that there were significant differences in the number of cells of both types which underwent early and late apoptosis. Significant differences were also observed among viable and apoptotic cells which have been post treated with ADV-VP2. The apoptotic effects of ADV-VP2 on human breast cancer cell lines were consistently demonstrated by three apoptosis detection methods. Therefore, a cancer vaccine basing on gene therapy could be developed in the near future using the present construct.     

Physiological and induced apoptosis in sea urchin larvae undergoing metamorphosis

Paracentrotus lividus embryos at the early pluteus stage undergo spontaneous apoptosis. Using a TUNEL (TdT-mediated dUTP Nick-End Labelling) assay on whole mount embryos, we showed that there was a different distribution of the apoptotic cells in different optical sections. Not more than 20% of cells in plutei were spontaneously apoptotic, as confirmed by the counts of dissociated ectoderm and intestine cells. Observation of larva stages closer to metamorphosis confirmed that apoptosis is a physiological event for the development of the adult. In particular, larvae at different developmental stages showed apoptotic cells in the oral and aboral arms, intestine, ciliary band and both apical and oral ganglia. Moreover, we found that the number of apoptotic cells decreased in later larva stages, possibly because in the organism approaching metamorphosis, a smaller number of cells needs to be eliminated. Furthermore, combined phorbol ester (TPA) and heat shock treatment enhanced apoptosis by increasing the number of cells involved in the phenomenon.     

Overexpression of S-adenosylmethionine decarboxylase.（SAMDC） in Xeno-pus embryos activates maternal program of apoptosm as a “fail-safe”mechanism of early embryogenesis

In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or 2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due toactivation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animalside blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of theapoptotic reaction. In the injection at 4-and 8-cell stages, a considerable number of embryos developed intotadpoles and in the injection at 16-and 32-cell stages, all the embryos became tadpoles, although tadpolesobtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cellsof the blastomere injected with SAMDC mRNA at 8-to 32-cell stages are confined within the blastocoel atthe early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continued development of the injected embryos. These results indicate that cells overexpressed with SAMDC undergo apoptotic cell death consistently at the early gastrula stage, irrespective of the timing of the mRNA injection.We assume that apoptosis is executed in Xenopus early gastrulae as a “fall-safe“ mechanism to eliminate physiologically-severely damaged cells to save the rest of the embryo.     

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xeno-pus embryos activates maternal program of apoptosis as a "fail-safe" mechanism of early embryogenesis

In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due toactivation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animal side blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of theapoptotic reaction. In the injection at 4- and 8-cell stages, a considerable number of embryos developed intotadpoles and in the injection at 16- and 32-cell stages, all the embryos became tadpoles, although tadpolesobtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cellsof the blastomere injected with SAMDC mRNA at 8- to 32-cell stages are confined within the blastocoel atthe early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continueddevelopment of the injected embryos. These results indicate that cells overexpress     

Hepatic clearance of apoptotic lymphocytes: simply removal of waste cells?

The in situ liver recognition of apoptotic lymphocytes was studied by using different sources of lymphocytes (i.e. human, rat and mouse) and animal models (i.e. rat and mouse). Lymphocytes were induced to apoptosis using 10(-2)M cycloheximide for up to 24 hours; three types of apoptosing lymphocytes, corresponding to different stages in the apoptotic process, were described: type 1 or early apoptosis, type 2 or mature apoptosis and type 3 or late/necrotic apoptosis. When livers were in situ injected with apoptotic lymphocytes enriched for type 1 (early), 2 (mature) or 3 (late/necrotic) apoptosis, they recognized and internalized apoptosing cells, with an efficiency directly dependent on the stage of the apoptotic process. The highest recognition rate, which was, in all cases, mediated by galactose- and mannose-specific receptors, was obtained with homologous apoptotic cells (i.e. rat lymphocytes and rat liver). Moreover, the drastically reduced efficiency of recognition of human or mouse apoptotic lymphocytes when injected into rat liver, suggested the involvement also of species-specific antigens.     

Simultaneous determination of apoptosis and surface antigen expression in tumor adherent cells

Apoptosis is a physiological, gene-directed form of cell death aimed at controlling cell proliferation in several biological conditions. It plays a crucial role in modulating tissue growth during embryonic development, cell turnover in adult life, and it seems to be the most frequent mechanism of tumor cell deletion by chemotherapy. Flow cytometry is a widely-used technique for checking apoptosis, permitting a multiparametric analysis. It is possible to follow the alterations occurring in the nucleus, mitochondria and plasmatic membrane during the different apoptotic stages using probes such as LDS-751, JC-1 or Annexin V. The potential of these probes to identify the early or late stages of apoptosis has been widely investigated in cells growing in suspension. In order to assess apoptosis in adherent cells, we tested a combination of fluorescein diacetate (FDA), a substrate for non specific esterase whose activity decreases during the early phase of apoptosis, and trypan blue in MCF-7 human breast cancer cells. Apoptotic cells showed a decrease in the green fluorescence emitted by fluorescein, the product of FDA hydrolysis, whereas necrotic cells emitted a red fluorescence due to the trypan blue staining. FDA-trypan blue double-staining was used to investigate the different kinetics of apoptosis induced by taxol, camptothecin and UV-B irradiation in MCF-7 cells. This method is rapid and simple, and can be used for monitoring the process of apoptosis from early stages in adherent cells, for the physical separation of apoptotic and live cells, and for immunophenotyping, including Fas expression.     

Qualitative and quantitative parameters of the growth of the skeleton of the extremities of embryos and early fetuses in man

One hundred and ten human embryos 6-9 weeks of the intrauterine development have been investigated. Sequence of anatomical differentiation of the skeleton in the composition of the upper and lower extremities has been stated. Quantitative changes of the parameters of each anlage at successive stages of the development are analyzed. Equations of regression are obtained, owing to them it is possible to determine age of human embryos and early fetuses in dependence on size of separate fragments of the extremities.     

Suppression of MIP-2 or IL-8 production by annexins A1 and A4 during coculturing of macrophages with late apoptotic human peripheral blood neutrophils

Annexin A1 (ANXA1) is a well-known anti-inflammatory protein that is expressed on the surface of apoptotic cells. Annexin A4 (ANXA4) is also recruited from the nucleus to the cytoplasm in apoptotic cells, although it is not known whether or not ANXA4 is expressed on the surface of apoptotic cells. In this study, we obtained rabbit anti-human ANXA1 and ANXA4 antibodies, and then examined whether or not ANXA1 and ANXA4 are expressed on the surface of early and late human apoptotic cells. ANXA1 and, to a lesser extent, ANXA4 were detected on late but not early apoptotic HeLa cells, whereas ANXA1 and a small amount of ANXA4 were detected on both early and late apoptotic human neutrophils. We then examined the effects of the anti-human ANXA1 and ANXA4 antibodies on the mouse or human macrophage response to human apoptotic cells. Upon coculturing of mouse or human macrophages with late apoptotic human neutrophils, anti-human ANXA1 antibodies and, to a lesser extent, anti-human ANXA4 antibodies increased MIP-2 or IL-8 production significantly, suggesting that ANXA1 and ANXA4 suppress MIP-2 or IL-8 production by macrophages in response to late apoptotic human neutrophils.     

Suppression of MIP-2 or IL-8 production by annexins A1 and A4 during coculturing of macrophages with late apoptotic human peripheral blood neutrophils

Annexin A1 (ANXA1) is a well-known anti-inflammatory protein that is expressed on the surface of apoptotic cells. Annexin A4 (ANXA4) is also recruited from the nucleus to the cytoplasm in apoptotic cells, although it is not known whether or not ANXA4 is expressed on the surface of apoptotic cells. In this study, we obtained rabbit anti-human ANXA1 and ANXA4 antibodies, and then examined whether or not ANXA1 and ANXA4 are expressed on the surface of early and late human apoptotic cells. ANXA1 and, to a lesser extent, ANXA4 were detected on late but not early apoptotic HeLa cells, whereas ANXA1 and a small amount of ANXA4 were detected on both early and late apoptotic human neutrophils. We then examined the effects of the anti-human ANXA1 and ANXA4 antibodies on the mouse or human macrophage response to human apoptotic cells. Upon coculturing of mouse or human macrophages with late apoptotic human neutrophils, anti-human ANXA1 antibodies and, to a lesser extent, anti-human ANXA4 antibodies increased MIP-2 or IL-8 production significantly, suggesting that ANXA1 and ANXA4 suppress MIP-2 or IL-8 production by macrophages in response to late apoptotic human neutrophils.     

Temporospatial patterns of apoptosis in chick embryos during the morphogenetic period of development

We examined the temporospatial pattern of naturally occurring apoptosis in chick embryos to five days of incubation (H.H. stages 1-25; Hamburger and Hamilton, 1951) using TUNEL labeling. The initial TUNEL-positive structure was the embryonic shield at stage 1. Apoptotic cells became ubiquitously present within embryos by stage 3, which is early in gastrulation. Until stage 6, TUNEL-positive cells were restricted to the headfold region. In embryos of stages 7-8, most cell death was localized at the most anterior neural plate. TUNEL-positive neural plate, notochord and somites appeared at stage 9. Otic and optic regions became TUNEL-positive at stage 11. The aggregation of cells from which the tail bud arises contains apoptotic cells from stage 11 onwards. At stage 16, scattered TUNEL-positive cells appeared in the branchial arches. Three streams of apoptotic neural crest cells in the cranial region became most clearly visible at stage 18. The secondary neural tube from which caudal structures develop contains apoptotic cells at stage 14. Apoptotic cells are present in the branchial arches and lateral body wall for extended periods, stages 16-25 and 25 respectively. At stages 24-25, intense positive regions of cell death were confined to the caudal regions of the arches, to limb and tail buds and to the lateral body wall, the latter in relation to body wall closure. The new findings in this study are discussed along with past studies to provide the temporospatial pattern of cell death during early chick development.     

Chronological appearance of spontaneous and induced apoptosis during preimplantation development of rabbit and mouse embryos

This study was undertaken to obtain specific information on the characteristics of spontaneous and induced apoptosis during preimplantation development of rabbit in vivo and in vitro developed embryos and mouse in vitro embryos. After reaching appropriate developmental stages, embryos were transferred into culture media with or without apoptotic inductor (actinomycin D 500 ng/mL) and cultured for 10 h. The identification of apoptotic cells was based on morphological assessment of nuclei and on detection of specific DNA degradation, phosphatidylserine redistribution and active caspase-3 under fluorescence microscope. Our experiments proved that apoptosis is a frequent physiological event occurring during normal preimplantation development. A high number of untreated rabbit and mouse blastocysts contained at least one apoptotic cell. Rabbit embryos showed a lower incidence of spontaneous apoptosis. Treated blastocysts of both species responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and significant increase in the incidence of apoptotic cell death. The occurrence of spontaneous apoptosis during earlier preimplantation development was sporadic and its presence was observed only at stages following embryonic genome activation (at 4-cell stage and later in mouse, at 16-cell and morula stage in rabbit). The susceptibility of embryos at early stages to the apoptotic inductor was much lower. The presence of actinomycin D did not increase the incidence of apoptotic embryos or apoptotic cells. Nevertheless, it slowed down embryo growth and triggered earlier appearance of some apoptotic features (at the 6-cell stage in rabbit). The results show that the occurrence of both spontaneous and induced apoptosis in preimplantation embryos is stage- and species-specific.     

The occurrence of c-myc, p53 and Bcl-2 family proteins in the early phase of development of duodenal epithelium

In last few years, numerous groups of proteins participating in the regulation of cell proliferation, differentiation and death during ontogenesis have been described. In this study we compared the occurrence of Bcl-2, p53 and myc protein families with the level of proliferative activity and apoptosis during development of duodenal epithelium. Paraffin embedded tissues of eight human embryos and foetuses aged from the 6th-18th week of IUD were used. For the detection of apoptotic cells the TUNEL method was performed, the proliferative marker PCNA and all the proteins studied were detected by means of indirect three-step immunohistochemical method. In the 6th and 8th week of intrauterine development we observed isolated TUNEL positive epithelial cells only and this was accompanied by the disperse presence of PCNA as well as by all the studied proteins: Bcl-2, Bax, Bcl-XL, c-myc, N-myc, p53, p63 and p73. In the early foetal period of duodenal development we registered changes in PCNA and TUNEL positivity in accordance with the constitution of the stem cell pool on base of villi, where more numerous Bcl-2 positive cells were also found. The separation of primitive crypts and villi was not accompanied by any differences in distribution of Bax, Bcl-XL, c-myc, N-myc, p63 and p73 proteins between those compartments: all the studied proteins showed dispersed character. P53 rapidly decreased in this period. In the 18th week of intrauterine development the balance between proliferation in crypts and apoptosis of villi epithelium was well established and no p53 positive cells were found. In the presence of Bcl-2, Bax, Bcl-XL, p63 and p73 we did not find any dramatic changes. The myc proteins were restricted within the epithelium of the Lieberkuhn crypts only.     

Difference in the way of macrophage recognition of target cells depending on their apoptotic states.

Dying cells are selectively eliminated from the organism by phagocytosis. Previous studies suggested the existence of some other phagocytosis marker(s) that function together with phosphatidylserine, the best-characterized phagocytosis marker. We obtained here a monoclonal antibody named PH2 that inhibited macrophage phagocytosis of late apoptotic or necrotic cells, but not of early apoptotic cells. On the other hand, phagocytosis of cells at any time during the process of apoptosis was inhibitable by phosphatidylserine-containing liposomes. Inhibition occurred even when target cells were preincubated with PH2 and separated from unbound antibodies. Moreover, PH2 bound to apoptotic cells at late stages more efficiently than to those at early stages, and it did not bind to normal cells unless their plasma membrane was permeabilized. These results suggest that the putative PH2 antigen is a novel phagocytosis marker that translocates to the cell surface at late stages of apoptosis, resulting in maximal recognition and engulfment by macrophages.