RESUMO
Transgenic mice which expressed SV40 large T-antigen under the control of the MSV enhancer and the SV40 promoter were generated. In animals containing an intact MSV enhancer, total lens cataracts and neuroectodermal brain tumors, originating in the pineal organ were observed. In contrast, 5' deletion of the MSV enhancer to a residual 53 bp resulted in a different spectrum of pathologies. Whilst lens cataracts still occurred, no brain tumors could be detected. Instead, fibrosarcomas and adenocarcinomas of the kidneys were induced. In addition, tumors of the endocrine pancreas were observed with both transgene constructs. We conclude that the MSV enhancer element is sufficient to direct the expression of the viral reporter gene to the lens and the pineal organ in transgenic mice. Deletion of the MSV enhancer correlates with the loss of DNA elements responsible for the pineal cell specific expression of SV40 large T-antigen.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Elementos Facilitadores Genéticos , Anormalidades do Olho/genética , Vírus do Sarcoma Murino de Moloney/genética , Neoplasias Experimentais/genética , Vírus do Sarcoma Murino/genética , Vírus 40 dos Símios/genética , Animais , Antígenos Transformantes de Poliomavirus/análise , Neoplasias Encefálicas/etiologia , Deleção Cromossômica , Olho/patologia , Feminino , Genótipo , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , Glândula Pineal/imunologia , Glândula Pineal/patologiaRESUMO
The development of necroses and the course of the mitotic index in the embryonic spinal cord of 10-day-old mouse embryos (NMRI) were investigated between 1.5 h and 4 h after either an intraperitoneal application of 500 mg/kg hydroxyurea (HU) or 1 mg/kg colchicine or a simultaneous application of both substances. One h and 30 min after colchicine application, the mitotic index of the neuroepithelial cells had decreased (5.22%; control 7.7%). Subsequently, the mitotic index increased, reaching a maximum of 10.35% 2 h and 30 min after colchicine application. Between 1 h and 30 min and 4 h after HU application, the mitotic index decreased from 4.6% to 0.61% (control 7.7%). First necroses of neuroepithelial cells were observed 1 h and 50 min after HU application. 4 h after HU treatment, 34.38% of the neuroepithelial cells were necrotic. After simultaneous application of HU and colchicine, the decrease of the mitotic index was similar to the decrease after HU application alone. No necroses were observed in the embryonic spinal cord during a 4 h period after simultaneous application of HU and colchicine. After colchicine, as well as after simultaneous HU and colchicine application, the shape of the nuclei of the neuroepithelial cells changed from oval to round and the chromatin was condensed. Mechanisms for the inhibition of the cytotoxic but not the cytokinetic effects of HU by colchicine are discussed.
Assuntos
Colchicina/farmacologia , Hidroxiureia/farmacologia , Medula Espinal/embriologia , Animais , Núcleo Celular/ultraestrutura , Células Epiteliais , Camundongos , Índice Mitótico/efeitos dos fármacos , Necrose , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacosRESUMO
Transgenic mice expressing the large T-antigen of the simian virus 40 (SV 40) under the control of 1) the enhancer of Moloney murine sarcoma virus (MSV) and 2) the SV 40 promoter develop undifferentiated neuroectodermal tumors located in the midline of the dorsal brain surface, abnormalities in lens fiber differentiation and retinal dysplasia. In this study the brain neoplasms of six adult animals and the brain of one 11-day old mouse were examined by conventional histology and immunocytochemical demonstration of S-antigen, rod-opsin, neuron-specific enolase, neurofilaments (160 and 200 kDa) and glial fibrillary acidic protein. According to histologic criteria the neoplasms were characterized as "primitive" neuroectodermal tumors composed mainly of small cells with scanty and ill-defined cytoplasm. Neoplastic cells displaying immunoreactive S-antigen were found in five brain tumors; three of these tumors also contained a limited number of rod-opsin immunoreactive neoplastic cells. Some tumor cells had neurite-like processes containing immunoreactive neurofilament (200 kDa). No pathologic lesions were found in the brain of the 11-day old animal. Tumors in transgenic mice may resemble pineal cell tumors and a special subtype of medulloblastoma in man. These neoplasms contain S-antigen immunoreactive and also rod-opsin immunoreactive tumors cells in certain cases. The findings suggest that transgenic mice expressing the large T-antigen of SV 40 may become a valuable animal model for analysing the origin, histogenesis and development of primitive neuroectodermal tumors with photoreceptor-like features (pineal cell tumors and certain medulloblastomas).
Assuntos
Antígenos/imunologia , Neoplasias Encefálicas/imunologia , Proteínas do Olho/imunologia , Camundongos Transgênicos/imunologia , Células Fotorreceptoras/imunologia , Animais , Arrestina , Autoantígenos/imunologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Humanos , Imuno-Histoquímica , Masculino , Meduloblastoma/imunologia , Camundongos , Glândula Pineal , Opsinas de BastonetesRESUMO
In vitro laminins stimulate numerous biological effects, such as cell migration, proliferation, attachment and differentiation. In vitro laminins influence immunocompetent cells and in vivo possibly play an important role in graft rejection. To establish how laminins could be involved in the regulation of acute rejection of small bowel allografts (with and without immunosuppression), we investigated laminin distribution in rat small bowel allografts four days after transplantation, i.e., before the onset of histological signs of rejection, using antibodies against alpha1, beta1, gamma1 chain of laminin-1. In immunosuppressed allografts, the ultrastructure of the enterocytic basement membrane appeared normal, but no laminin staining was seen in this membrane, although basement membranes of intramural blood vessels and muscle cells were normally stained. In non-operated immunosuppressed rats, laminin staining was clearly reduced in the enterocytic basement membrane, demonstrating that cyclosporin A is able to affect this membrane. Since only rats in which laminin is altered survive, this laminin alteration in the enterocytic basement membrane presumably plays an important role in overcoming the acute rejection.
Assuntos
Intestino Delgado/transplante , Laminina/análise , Animais , Membrana Basal/química , Membrana Basal/ultraestrutura , Ciclosporina/farmacologia , Terapia de Imunossupressão , Imunossupressores/farmacologia , Intestino Delgado/química , Intestino Delgado/ultraestrutura , Microscopia Eletrônica , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Transplante Homólogo , Transplante IsogênicoRESUMO
In biological tissues, specific carbohydrate moieties of the oligosaccharide chains of glycoproteins can be localized by lectin binding. Such carbohydrate moieties are among the factors that mediate cell-cell or cell-matrix interactions during pre- and postimplantation embryonic development. Binding sites for the lectins RCA I, WGA, and LTA were localized in preimplantation mouse embryos at the ultrastructural level with the help of postembedding lectin gold cytochemistry. WGA and RCA I binding sites, but no LTA binding sites, were present in the zona pellucida. WGA and RCA I binding sites were found at cell surfaces of morulae and in cells of the inner cell mass of blastocysts, suggesting that N-acetylglucosamine-, terminal beta-galactosyl-, and N-acetylgalactosamine-rich glycoproteins might be involved in cell-cell and cell-matrix interactions. WGA binding sites were found predominantly in electron lucid vesicles of the blastomeres, whereas RCA I was detected in electron dense vesicles of the compacted morula and later in the polar trophoblast cells. This allows early identification of blastomere cells that later differentiate into the polar trophoblast.
Assuntos
Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Lectinas/metabolismo , Lectinas de Plantas , Aglutininas do Germe de Trigo/metabolismo , Animais , Sítios de Ligação , Blastocisto/metabolismo , Blastocisto/ultraestrutura , Embrião de Mamíferos/ultraestrutura , Feminino , Camundongos , Mórula/metabolismo , Mórula/ultraestrutura , Gravidez , Zona Pelúcida/metabolismo , Zona Pelúcida/ultraestruturaRESUMO
Nidogen-1, a key component of basement membranes, is considered to function as a link between laminin and collagen Type IV networks and is expressed by mesenchymal cells during embryonic and fetal development. It is not clear which cells produce nidogen-1 in early developmental stages when no mesenchyme is present. We therefore localized nidogen-1 and its corresponding mRNA at the light and electron microscopic level in Day 7 mouse embryos during the onset of mesoderm formation by in situ hybridization, light microscopic immunostaining, and immunogold histochemistry. Nidogen-1 mRNA was found not only in the cells of the ectoderm-derived mesoderm but also in the cytoplasm of the endoderm and ectoderm, indicating that all three germ layers express it. Nidogen-1 was localized only in fully developed basement membranes of the ectoderm and was not seen in the developing endodermal basement membrane or in membranes disrupted during mesoderm formation. In contrast, laminin-1 and collagen Type IV were present in all basement membrane types at this developmental stage. The results indicate that, in the early embryo, nidogen-1 may be expressed by epithelial and mesenchymal cells, that both cell types contribute to embryonic basement membrane formation, and that nidogen-1 might serve to stabilize basement membranes in vivo. (J Histochem Cytochem 48:229-237, 2000)
Assuntos
Embrião de Mamíferos/metabolismo , Glicoproteínas de Membrana/metabolismo , Mesoderma/metabolismo , Animais , Membrana Basal/metabolismo , Colágeno/biossíntese , Colágeno/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Laminina/biossíntese , Laminina/metabolismo , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos , Microscopia Eletrônica , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
To localize the different domains of the laminin-1 molecule in tissues and gain insight into their in vivo relevance, we raised rat anti-mouse monoclonal antibodies (MAbs) against the entire molecule. Then we tested eight of the 20 clones producing anti-laminin-1 MAbs to specify their reactivity towards the alpha 1-, beta 1-, and gamma 1-chains and the elastase-cleaved fragments of the laminin-1 molecule. We found three MAbs with high titers in ELISA that showed good reactivity in embedded tissue. One of these reacted specifically against the E1 fragment, one against the E8 fragment, and one MAb detected the alpha 1-chain of laminin-1 but not the beta 1- or gamma 1-chain. All three MAbs are useful for light immunohistochemical investigations on cryosections and on paraffin-embedded material, and for ultrastructural localization of laminin-1 in LR Gold-embedded mouse tissue. Antibody staining of the E1 and E8 domains of laminin-1 revealed distinct localization of the molecule in the proximal tubule basement membranes of mouse kidney. The short arms (E1) of the laminin-1 molecule are predominantly located in the lamina lucida and the long arms (E8) are oriented towards the lamina fibroreticularis. Therefore, both MAbs are useful for studies of the orientation of the laminin-1 molecule in basement membranes. The distal tubule basement membranes did not show any distinct pattern of laminin-1 distribution. In general, the distal tubules showed the strongest reactions over the entire width of the basement membrane for all three MAbs. In contrast, the proximal tubule basement membranes showed somewhat weaker reactivity but a distinct pattern of laminin-1 distribution, with the E1 fragments oriented towards the adjacent epithelial cell surface.
Assuntos
Anticorpos Monoclonais , Membrana Basal/química , Rim/química , Laminina/análise , Fragmentos de Peptídeos/análise , Animais , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Imuno-Histoquímica , Camundongos , Microscopia EletrônicaRESUMO
Examples of a combined approach using in vivo as well as in vitro methods for the assessment of prenatal toxicity are presented. The topics discussed include the analysis of the possible embryotoxic potential of valproic acid (VPA), female sex hormones, bis(tri-n-butyltin) oxide (TBTO), and acyclovir and the problem of supplementing in vitro systems with drug-metabolizing activity.
Assuntos
Anormalidades Induzidas por Medicamentos/patologia , Aciclovir/toxicidade , Embrião de Mamíferos/efeitos dos fármacos , Estrogênios/toxicidade , Progesterona/toxicidade , Teratogênicos , Compostos de Trialquitina/toxicidade , Ácido Valproico/toxicidade , Animais , Desinfetantes/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos/métodos , RatosRESUMO
Normal liver sinusoids are not lined by a basement membrane (BM). In contrast, in the course of development of liver cirrhosis, a structured BM is formed de novo in the space of Disse. This BM contributes to the inhibition of the metabolic function of the liver but the pathogenic background of the formation of this perisinusoidal BM is still unclear. Integrins of the beta1-class are generally essential for BM stability and some of them (such as alpha2beta1, alpha3beta1 and alpha6beta1) appear de novo in the perisinusoidal space of the cirrhotic liver. Their cellular distribution in capillarized sinusoids as well as the correlation between their cellular distribution and the formation of the microvascular BM in the cirrhotic liver has not been shown at the ultrastructural level. In the present work we aimed to clarify this issue. We focused on integrins alpha3beta1 and alpha6beta1 and localised them ultrastructurally in human cirrhotic liver microvessels using postembedding immunogold which allows the ultrastructural localization of antigens with high resolution in the tissue. The newly formed basement membrane of capillarized sinusoids was visualized by means of fixation with addition of tannic acid, which enables the visualization of structures of the extracellular matrix with the highest resolution. Also, we carried out laminin detection using postembedding immunogold. Our results show that both alpha3beta1 and alpha6beta1 are expressed on the surface of both hepatocytes and endothelial cells, i.e. on both sides of the newly formed basement membrane. This latter shows zones of higher density both in close proximity to the endothelial and to the hepatocytic surfaces which resemble laminae densae. We propose that hepatocytes and endothelial cells may, therefore, by expressing such integrins, contribute to the formation of this pathological BM in the microvessels of the human cirrhotic liver. On stellate cells, which are major producers of BM components, both integrins alpha3beta1 and alpha6beta1 were also localized.
Assuntos
Integrinas/metabolismo , Circulação Hepática , Cirrose Hepática/metabolismo , Subunidades Proteicas/metabolismo , Membrana Basal/patologia , Membrana Basal/ultraestrutura , Endotélio Vascular/química , Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Hepatócitos/química , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Humanos , Imuno-Histoquímica , Integrina alfa3 , Integrina alfa6 , Integrinas/imunologia , Integrinas/ultraestrutura , Cirrose Hepática/patologia , Microscopia Imunoeletrônica , Subunidades Proteicas/genéticaRESUMO
By means of light and electron microscopy we have studied the effect of interferon beta-1a (IFNbeta-1a) in the optic tecta of 20-day-old chick embryos under normal conditions and after exposure to lipopolysaccharide (LPS) which mimics the blood-brain barrier (BBB) disruption in meningoencephalitis. Optic tecta were examined for: (i) ultrastructure by means of transmission electron microscopy; (ii) the immunohistochemical localization of HT7 antigen, a specific marker of differentiation of the brain microvessels; (iii) the brain microvessel permeability, by means of horseradish peroxidase (HRP) tracer; (iv) the expression of microvessel glycoconjugates, by means of lectin histochemistry, using Ricinus communis agglutinin-I (RCA-I), specific for beta-D-galactosyl moieties and Wheat Germ agglutinin (WGA) specific for sialyl and N-acetylglucosaminyl moieties. A morphometric evaluation of brain microvessel permeability and of glycoconjugate expression was also performed. In control- and in IFNbeta-1a-treated embryos, HRP was confined to the vessel lumina which were sealed by the interendothelial tight junctions. RCA-I binding sites were recognizable both in the basal membranes and in the tight junctions, while WGA sites were present on the luminal side of the endothelial cells. HRP was blocked in the vessels lumina by the interendothelial tight junctions. After LPS treatment, HRP showed an extravascular localization and the labeling of microvessels by anti-HT7 antibodies disappeared. RCA-I binding was only found ultrastructurally and appeared as irregularly clustered gold particles, in the cleft of damaged tight junctions, but were no longer detectable in the endothelial basement membranes. After pretreatment of LPS-treated embryos with IFNbeta-1a, the vessel permeability to HRP strongly decreased and the vessels showed the normal pattern of HT7 protein and of the RCA-I binding sites. These results indicate that the changes induced by LPS in the endothelial cells are prevented by IFNbeta-1a.
Assuntos
Antígenos CD , Antígenos de Neoplasias , Antígenos de Superfície , Antineoplásicos/farmacologia , Proteínas Aviárias , Proteínas Sanguíneas , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/embriologia , Circulação Cerebrovascular/efeitos dos fármacos , Interferon beta/farmacologia , Animais , Basigina , Biomarcadores , Encéfalo/irrigação sanguínea , Capilares/efeitos dos fármacos , Capilares/metabolismo , Capilares/ultraestrutura , Embrião de Galinha , Endotélio Vascular/química , Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Peroxidase do Rábano Silvestre/farmacocinética , Interferon beta-1a , Lipopolissacarídeos , Glicoproteínas de Membrana/análise , Microcirculação/efeitos dos fármacos , Microscopia EletrônicaRESUMO
Tight junction biogenesis during blood-brain barrier development (BBB) in mesencephalon microvessels of mouse embryos of day 9, foetuses of day 15 and 19 and new-born (2-day-old) mice was examined by light and electron microscopy, using monoclonal antibodies recognizing the tight junction peripheral membrane protein ZO-1. A faint spot-like staining began to be recognizable under the light microscope in day 15 vessels in which the endothelial cells showed isolated fusion points between the external plasma membrane leaflets under the electron microscope. A stronger labelling was present in microvessels of day 19 foetuses and new-born animals when the endothelial tight junction appeared completely differentiated. In the immunogold study, gold particles were seen scattered throughout the cytoplasm of endothelial cells of day 15 foetuses. In day 19 foetuses and in the new-born mice, gold particles were located only at the cytoplasmic surfaces of the tight junctions. The results indicate that the ZO-1 protein is a specific molecular marker in the developing brain endothelial tight junctions and that its expression takes place parallel to BBB morphofunctional maturation.
Assuntos
Envelhecimento/fisiologia , Barreira Hematoencefálica/fisiologia , Capilares/metabolismo , Desenvolvimento Embrionário e Fetal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Mesencéfalo/irrigação sanguínea , Fosfoproteínas/genética , Animais , Animais Recém-Nascidos , Capilares/embriologia , Capilares/crescimento & desenvolvimento , Capilares/ultraestrutura , Feto , Idade Gestacional , Mesencéfalo/embriologia , Mesencéfalo/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Proteína da Zônula de Oclusão-1RESUMO
Vital staining of neuroblastoma cells with acridine orange produces a bright intracellular red-orange fluorescence most probably due to the occurrence of RNA. The distribution of this fluorescence depends on the state of morphological differentiation. The fluorescence is predominantly found in the perikaryon, the growth cones, and the endings of the processes of differentiated cells. This is of special interest in respect to the biochemistry of differentiation and the function of nerve cells. Comparative autoradiographical studies with 3H-uridine demonstrate that the newly synthesised RNA is transported into the endings of the cell processes.
Assuntos
Acridinas , Neuroblastoma/metabolismo , RNA Neoplásico/metabolismo , Autorradiografia , Diferenciação Celular , Células Clonais , Técnicas In Vitro , Microscopia de Fluorescência , Coloração e Rotulagem , Trítio , Uridina/metabolismoRESUMO
Forelimb buds of mouse embryos (day 11 + 3 h of development) were cultured for 6 days. During this period, the epidermis developed from a two-layered stage (basal and periderm cell layer) to a multilayered squamous epithelium wih stratum granulosum and stratum corneum. To investigate the glycoprotein synthesis during epidermogenesis in vitro, the limb buds were labeled for 30 min or 4 h with 3H-fucose at successive stages of development. Until the development of the stratum intermedium, no different labeling of the individual layers of the epidermis and the periderm was observed. Shortly before the beginning of the stratum granulosum, a strong labeling of the outer region of the intermediate layer, where the shape of the cells had changed from cuboid to flat, was visible. When the stratum granulosum developed, even in this layer and in the upper stratum spinosum, strong glycoprotein synthesis was observed, indicated by strong labeling with 3H-fucose. In the developing stratum corneum, no labeling of this layer was detectable 30 min or 4 h after 3H-fucose application. Labeling of peridermal cells was weak in the early stages of development and ceased in the period of stratum granulosum formation, indicating a stop of glycoprotein synthesis in this embryonal cell layer before its disappearance.
Assuntos
Epiderme/fisiologia , Glicoproteínas/biossíntese , Pele/embriologia , Animais , Autorradiografia , Células Epidérmicas , Epiderme/metabolismo , Técnicas In Vitro , CamundongosRESUMO
The localization of the basement membrane components type IV collagen and laminin was investigated in seven-day-old mouse embryos (NMRI) fixed with formaldehyde, using an immunoperoxidase technique. Posttreatment of the embryos with TBS (trishydroxymethylaminomethane buffered saline) buffer was prerequisite for restoration of the antigenicity after fixation. The localization of the peroxidase (PO) positive reaction after treatment with anti-type IV collagen and anti-laminin antibodies in the embryos has been compared with results obtained after fixating embryos with the addition of tannic acid. Tannic acid stained the basement membrane of the ectodermal cell layer, in particular the lamina densa. After immunostaining for type IV collagen and laminin, a strong PO-positive reaction in the lamina densa of the ectodermal basement membrane was observed. A basement membrane of the endodermal cell layer had not yet been formed at this developmental stage. In this region, which is where a basement membrane was to develop in later stages, a tannic acid positive material consisting of granules with a diameter of about 25 nm was found near the surface of the endoderm. Moreover, PO-positive patches were seen in this part of the embryo after staining for laminin as well as after staining for type IV collagen. These PO-positive patches were mainly localized in areas where mesodermal cells lay adjacent to the surface of the endodermal cell layer. No positive staining for type IV collagen and laminin was found in the cytoplasm of either ectodermal or endodermal cells.
Assuntos
Colágeno/análise , Embrião de Mamíferos/ultraestrutura , Laminina/análise , Animais , Membrana Basal/análise , Feminino , Taninos Hidrolisáveis , Camundongos , Microscopia Eletrônica , GravidezRESUMO
Paraffin sections from vertebral columns of ten human embryos and fetuses ranging from stage 16 to the 12th week were stained with the FITC-coupled lectins PNA, RCA I, Con A and WGA in order to investigate changes in carbohydrate-binding sites during vertebral development. PNA revealed a specific binding site in the vertebral body blastema in the precartilaginous stage of development. Beginning with the 25-mm CRL embryo, PNA-binding sites occurred in the developing fibrous annulus and the inner zone of the intervertebral discs. The first binding sites for RCA I were seen in the extracellular matrix of vertebral bodies during the cartilaginous stage of vertebral development. During early ossification of the vertebrae, staining for RCA I-binding sites in the cytoplasm of the chondrocytes and the area around the future cartilaginous end-plates was observed. Con A bound to the chondrocyte cytoplasm, and also very strongly to notochordal cells in all developmental stages examined. WGA-binding sites appeared simultaneously with cartilage formation. Connective tissue components, e.g. ligaments, were diffusely stained by WGA. Also this lectin showed an affinity for vertebral body chondrocytes. We discuss the biochemical aspects of these lectin-binding sites, and their possible roles in the differentiation process of the human vertebral column. The results of this first lectin histochemical study on human vertebral development are compared with related results in other species.
Assuntos
Embrião de Mamíferos/metabolismo , Lectinas/metabolismo , Coluna Vertebral/metabolismo , Sítios de Ligação , Concanavalina A/metabolismo , Feto/metabolismo , Imunofluorescência , Idade Gestacional , Histocitoquímica , Humanos , Aglutinina de Amendoim , Ricina/metabolismo , Coluna Vertebral/embriologia , Aglutininas do Germe de Trigo/metabolismoRESUMO
The role of extracellular matrix molecules in ontogenetic differentiation processes is a matter of increasing interest. In cartilage development, type II collagen is suspected of promoting chondrogenic differentiation, since its expression has been demonstrated in a range of precartilaginous tissues of vertebrate species. Up to now, no studies supplying a coherent description of type II collagen expression in the skeletogenesis of human embryos including early embryonic stages have been published. In this work, we examine the temporal and spatial distribution of type II collagen mRNA during vertebral column development in human embryos from 4 to 12 weeks of gestation using non-radioactive in situ hybridization. The onset of gene expression was demonstrable in the 5th week in precartilaginous mesenchymal cells and in notochordal cells. Additionally, we found a weaker hybridization signal in the mesenchymal precursors of the intervertebral discs. In the anlagen of the axial skeleton, type II collagen expression decreased during osteogenic reconstruction in the 11th/12th week, whereas expression continued in the notochordal remnants of the future nuclei pulposi. The results suggest a relatively late occurrence of type II collagen in human vertebral development compared with other vertebrate species. The distribution of gene expression is concordant with a possible role of this molecule in promoting differentiation of mesenchymal cells into chondrocytes. The mechanism of this influence remains to be established.
Assuntos
Colágeno/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , RNA Mensageiro/biossíntese , Coluna Vertebral/embriologia , Cartilagem/embriologia , Colágeno/genética , Matriz Extracelular , Feminino , Humanos , Hibridização In Situ , Modelos Biológicos , Notocorda/anatomia & histologia , Notocorda/embriologia , Gravidez , Coluna Vertebral/anatomia & histologiaRESUMO
Although the composition of Reichert's membrane, a thick multilayered basement membrane between the parietal endoderm cells and the trophoblast cells of rodents, has often been investigated, the site of its production remains a subject of controversial discussion. In particular, the role of the trophoblast cells is unclear. In the present work we examined the initial development of Reichert's membrane in the early mouse embryo, using glutaraldehyde fixation with tannic acid. In the early blastocyst the occurrence of a tannic-acid-positive layer located at the inner surface of the mural trophoblast indicated the onset of basement membrane formation by the trophoblast cells. In the peri-implantation phase, this basement membrane extended into lateral areas of the inner cell mass separating the newly differentiated ectoderm and endoderm cells from each other. In these lateral regions, where the recently formed primitive endoderm cells had been attached to the monolayered basement membrane of the mural trophoblast cells, followed by an apposition of basement membrane material, probably synthesized by primitive endoderm cells, along this primary membrane.
Assuntos
Membrana Basal/embriologia , Endoderma , Trofoblastos , Animais , Blastocisto , Camundongos , Microscopia EletrônicaRESUMO
Mouse embryos of the blastocyst stage (day 0 of culture) were cultured in human cord serum (HCS) for 8 days. Until day 7 in vitro, the embryos had developed to the 4-5 somite stage. Apart from the occurrence of some cell necroses in the embryonic ectoderm and vacuoles in the yolk sac entoderm and to a lesser degree even in the embryonic entoderm, the in vitro development of the embryos was indistinguishable from the in vivo development. However, between days 7 and 8 in vitro the embryonic development was heavily disturbed. The most pronounced disturbances were visible in the central nervous system (CNS) of the embryos. A great number of the neuroepithelial cells of the entire CNS were necrotic. Even mesodermal structures, mainly the axial structures in the posterior part of the embryos, were affected, leading to disorganization of somites and notochord in this region. Since in the presence of rat serum embryos develop normally between days 7 and 8 in vitro (Hsu 1981; Chen and Hsu 1982), it was stated that HCS lacks an embryonic growth and differentiation factor (EGDF-4) present in rat serum which regulates the development of the CNS and the organization of the axial structures between days 7 and 8 in vitro, i.e., during the 4-5 somite stage and the time of the turning of the embryos (8-12 somite stage).
Assuntos
Técnicas de Cultura , Embrião de Mamíferos , Animais , Sistema Nervoso Central/patologia , Sangue Fetal , Crescimento , Camundongos , Necrose , Fatores de TempoRESUMO
The paraxial mesenchyme in seven human embryos aged between Carnegie stages 12 and 17 was studied by lectin histochemistry with the lectins AIA, Con A, GSA II, LFA, LTA, PNA, RCA I, SBA, SNA, WGA. The paraxial mesenchyme was found to be segmented into sclerotomes by intersegmental vessels and from late stage 12 by intrasclerotomal clefts dividing each sclerotome into a cranial and caudal half. The lectins Con A, GSA II, LFA, LTA, SBA and SNA did not react at all in the paraxial mesenchyme. Staining for AIA, PNA, RCA I and WGA was found in the developing sclerotomes. However, no differences in the staining pattern between the two sclerotomal halves could be seen. It was striking that in contrast to the chick embryo no differences in binding for PNA between the cranial and caudal sclerotomal parts was observed. These findings reveal that PNA-binding sites do not play the same functional role in segmented axonal outgrowth and neural crest immigration into cranial sclerotomal halves in the human embryo, as found in chick embryonic development. Beginning with the stage 16-embryo, the already condensed caudal sclerotomal halves express Con A-, RCA- and PNA-binding sites. The staining for PNA in particular marked the differentiation of chondrogenous structures developing in this half. From the late stage 12 or stage 13, the walls of intersegmental and other vessels showed binding sites for AIA, PNA, RCA I, SNA and WGA.
Assuntos
Desenvolvimento Embrionário e Fetal , Lectinas/metabolismo , Mesoderma/metabolismo , HumanosRESUMO
Oncoproteins and tumour-suppressor proteins are thought to possess an antagonistic function in the regulation of growth and differentiation processes during embryonic and fetal development. In contrast, in the adult, tumour growth is associated with the overexpression of oncoproteins or the malfunction of tumour-suppressor proteins. We examined the occurrence of the tumour proteins c-erb-B2 and c-fos and the tumour-suppressor protein p53 in 17 human embryos and fetuses with the help of immunohistochemistry. C-erb-B2 was detected mainly in embryonic tissue that are not known for c-erb-B2-overexpression in tumours in the adult. In contrast, c-fos was almost always located in fetal tissues corresponding to its location in adult tumours. Staining for p53 was found in a wide variety of embryonic and fetal tissues. C-erb-B2 and p53 were localized in the same tissue structures of the developing skin, heart and muscle. In other tissues, e.g. muscle and bone, c-fos was found together with p53, suggesting an antagonistic action of these proliferative and antiproliferative factors. Furthermore, c-erb-B2. c-fos and p53 appear to be important for growth and differentiation processes in human development as the occurrence of these proteins was not only restricted to specific tissues but also to specific stages of development of these tissues.