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Preliminary investigation on the submicroscopical structure of blood capillaries of the pineal body in the embryonic and adult domestic fowl.

The morphological changes of the blood network capillaries in the pineal gland of the domestic fowl during embryonic development and in the first post-hatching year have been analyzed. Our preliminary results indicate that the first intrinsic vessels, showing an immature aspect, appear among the epithelial vesicles on 7th-8th incubation day (i.d.), when the pinealoblasts are actively proliferating; then, they drastically increase in number (10th i.d.), but they begin to undergo significant maturative changes only on the 19th i.d., in parallel with the unfolding of the pinealocytes differentiation. Finally they reach the definitive set-up within the first post-hatching year, while the pineal is acquiring its full morphological and functional maturity.

Aging↗

Structure of the murine homeobox gene cdx-2. Expression in embryonic and adult intestinal epithelium.

To gain insight into the mechanisms which govern cellular identity in the intestinal epithelium we have begun a detailed study of the murine cdx-2 homeobox gene. We isolated and sequenced both cDNA and genomic clones in order to define the open reading frame and mature transcript. A detailed analysis of cdx-2 transcript levels late in embryogenesis showed that they increased 6-7-fold at a time when the gut undergoes a major developmental transition. In the adult, cdx-2 was expressed in colon in a region-specific manner with transcripts some 5-fold more abundant in the cecum as compared with the rectum. In situ hybridization and immunohistochemical experiments showed cdx-2 transcripts and protein were present in all epithelial cells in the proximal colon irrespective of their degree of differentiation. In distal colon, however, transcripts were most abundant in undifferentiated cells at the bottom of crypts, whereas the highest protein levels were present in mature cells in the upper half of crypts. We conclude that cdx-2 is expressed specifically in gut epithelium where it is not restricted to a particular cell lineage. Rather, the rostrocaudal expression gradient suggests that it may play a role in specifying positional identity.

Amino Acid Sequence↗

Localization of H blood group antigen in ectoblastic derivatives of murine teratocarcinoma.

Ectoblastic derivatives (ectodermal and neuroectodermal components) constitute more than 90% of all structures in the murine teratocarcinoma derived from the PCC4-aza-1 line. This tumor was labeled immunocytochemically with fluoresceinated antibodies to A, B and H blood groups. A and B antigens were always noted to be absent from all structures of the three germ layers. With anti-H, however, embryonic and fetal endodermal components, e.g. alimentary or respiratory duct-like structures, gave positive staining. Mesodermal components, i.e. bone and cartilage structures, fibroblasts and myocytes of mature or embryonal type, were negative. Immature and mature ectodermal components, viz. epidermoid cysts or islets, were always positive. Neuroectodermal components, neuroblastic cysts and differentiated neuronal, glial and ependymal elements were always negative. Ectoblastic and/or neuroectoblastic individual cells or cell clusters, observed in the vicinity of positive differentiating ectodermal and neuroectodermal structures, were positive. Similar cells or clusters were negative when located close to negative neuroectodermal components. The undifferentiated, embryonal carcinoma cells and structures were always negative. These observations are compared to the staining patterns of H antigen in murine embryos and adult mice and in human teratocarcinomas. It is suggested that poorly differentiated, morphologically similar ectoblastic and/or neuroectoblastic structures are positive, if they concern ectodermal components, and negative, if they belong to neuroectodermal components, with the exception of some primary sensory cells that are positive. These last cells are also positive in normal fetal and adult tissues.

ABO Blood-Group System↗

DNA oxidation as a potential molecular mechanism mediating drug-induced birth defects: phenytoin and structurally related teratogens initiate the formation of 8-hydroxy-2'-deoxyguanosine in vitro and in vivo in murine maternal hepatic and embryonic tissues.

A considerable number of teratogens, including the anticonvulsant drug phenytoin and structurally related drugs and environmental chemicals, may be bioactivated by peroxidases, such as prostaglandin H synthase (PHS) and lipoxygenases (LPOs), to a reactive free radical intermediate that initiates birth defects. However, the molecular targets of the reactive free radical intermediates mediating chemical teratogenesis, and hence the fundamental determinants of susceptibility, are poorly understood. In these studies, a teratogenic dose of phenytoin (65 mg/kg), when injected into pregnant CD-1 mice during organogenesis on gestational day 12, initiated the oxidation of DNA in maternal hepatic and embryonic nuclei, forming 8-hydroxy-2'-deoxyguanosine. Significant maternal and embryonic DNA oxidation occurred at 6 and 3 h, respectively, suggesting relative embryonic deficiencies in free radical-related cytoprotective enzymes, although the rates appeared similar. Maximal DNA oxidation in both maternal and embryonic tissues occurred at 6 h, presumably reflecting the balance of DNA oxidation and repair, the latter of which appeared similar in both tissues. Inhibition of phenytoin-initiated embryonic DNA oxidation by the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (41.5 mg/kg), and by acetylsalicylic acid (10 mg/kg), an inhibitor of the cyclooxygenase component of PHS, was consistent with the previously reported reduction by these inhibitors of phenytoin-initiated murine birth defects. In vitro studies using a horseradish peroxidase (0.5 mg/ml)-H2O2 (5.45 micrograms/ml) bioactivating system for drug-initiated oxidation of 2'-deoxyguanosine (3.74 mM), indicated that the potency of xenobiotic-initiated formation of 8-hydroxy-2'-deoxyguanosine for the structurally related drugs and metabolites phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin, trimethadione, dimethadione, l-mephenytoin, l-nirvanol, d-nirvanol (80 microM each), or thalidomide (64 microM), reflected their murine teratogenic potency. Given the relatively low activities of cytochromes P450, compared to PHS and LPOs, in human and rodent embryonic tissues, these data support the potential teratological importance of peroxidase-catalysed bioactivation of xenobiotics with structural similarities to phenytoin. These studies provide the first evidence that peroxidase-catalysed embryonic DNA oxidation may constitute a critical molecular mechanism mediating the teratogenicity of phenytoin and related drugs and environmental chemicals, and suggest the potential teratological importance of additional embryonic processes, such as DNA repair and tumor suppressor genes, as determinants of susceptibility.

8-Hydroxy-2'-Deoxyguanosine↗

[Morphohistochemical development of mesenchyme in human fetal labial glands].

The structural and histochemical patterns of the salivary mesenchyma were analysed in relation to the epithelium of the labial glands during the embryonic development to correlate the structural and histochemical characteristics in both tissues during the histogenesis. Samples of human fetal lips were analysed using H/E, PAS, Cason, Alcian blue, Toluidine blue and Methenamine/silver. The process of glandular histogenesis begun between 8 to 10 weeks. The mesenchyma surrounding the glandular buds had PAS positive granulations which were also alcianophilic, metachromatic and periodatoreactive. Periodatoreactive collagenous fibrillae, reticular cells and nervous fibers of considerable diameter were observed. Basement membranes were PAS positive, alcianophilic and discontinuous. At 12 weeks the mesenchyma differentiated as loose connective tissue which produced a delicate periglandular capsule with fibroblast and collagenous fibrillae. From 20 to 24 weeks the acini were structurally and histochemically differentiated as serous, mucous and mixed. It was postulated that the periglandular mesenchyma would play and important role in the morphogenetic process in relation to the histochemical identification of molecules which have a specific biological functions in the epithelium-mesenchyma interactions during organogenesis.

Histocytochemistry↗

Structural analysis and subunit interaction of insulin receptor from membranes of cultured embryonic chick heart cells.

In previous studies of cultured embryonic chick heart, insulin hyperpolarized cells and slowed their beat rate through occupancy of a high affinity receptor. In the present studies, we chemically characterize the native structure and subunit interactions of this insulin receptor. A stokes radius of 87 A and an apparent molecular mass of 350,000 daltons were found for membrane proteins specifically cross-linked to [125I] insulin by disuccinimidyl suberate. A primary subunit of 125,000 daltons in dithiothreitol or 115,000 daltons in its absence (alpha-subunit) was heavily cross-linked. A smaller subunit had a size of 90,000 daltons (beta-subunit). This beta-subunit was not readily labeled by [125I]insulin cross-linking, but insulin enhanced 32P incorporation from [gamma-32P]ATP, enabling its visualization. Subunit interaction could be studied, because alkaline conditions produced dissociation of the native 350,000-dalton receptor. This spontaneous dissociation was not a result of proteolysis and was prevented by acid conditions, oxidants, or N-ethylmaleimide. Using alkaline conditions followed by chemical reduction in two-way gels, we directly visualized the native complex of 350,000 daltons dissociating into combinations of subunits of apparent sizes of 290,000 (alpha 2 beta), 220,000 (alpha alpha), and 195,000 (alpha beta) daltons. Dithiothreitol produced combinations of subunits, which differed from alkaline dissociation. In alkaline conditions, the 290,000 (alpha 2 beta) and 220,000 (alpha alpha) dalton combinations predominated, whereas dithiothreitol produced 190,000 (alpha beta)-dalton proteins, which suggested that alpha-S-S-alpha disulfide bonds existed and were susceptible to chemical reductants, while alpha-S-S-beta disulfide bonds were more sensitive to alkaline lysis. We conclude from these observations that the native insulin receptor of embryonic chick heart cell exists on the sarcolemmal membrane as a relatively homogeneous tetramer of nonhomologous subunits in an alpha 2 beta 2 configuration. The alpha-subunits are the primary sites for insulin binding, and a beta-subunit is autophosphorylated. alpha-S-S-alpha and alpha-S-S-beta bonding exist, and these disulfide bonds have different sensitivities to chemical reducing agents and alkaline lysis.

Adenosine Triphosphate↗

Gene therapy of murine teratocarcinoma: separate functions for insulin-like growth factors I and II in immunogenicity and differentiation.

Teratocarcinoma is a germ-line carcinoma giving rise to an embryoid tumor with structures derived from the three embryonic layers: mesoderm, endoderm, and ectoderm. Teratocarcinoma is widely used as an in vitro model system to study regulation of cell determination and differentiation during mammalian embryogenesis. Murine embryonic carcinoma (EC) PCC3 cells express insulin-like growth factor I(IGF-I) and its receptor, while all derivative tumor structures express IGF-I and IGF-II and their receptors. Therefore the system lends itself to dissect the role of these two growth factors during EC differentiation. With an episomal antisense strategy, we define a role for IGF-I in tumorigenicity and evasion of immune surveillance. Antisense IGF-I EC transfectants are shown to elicit a curative anti-tumor immune response with tumor regression at distal sites. In contrast, IGF-II is shown to drive determination and differentiation in EC cells. Since IGF-I and IGF-II bind to type I receptor and antisense sequence used for IGF-II cannot form duplex with endogenous IGF-I transcripts, it follows that this receptor is not involved in determination and differentiation.

Animals↗

Protein-protein interactions and structural entities within the sea urchin extraembryonic matrix, the hyaline layer.

We have investigated the effects of a variety of experimental conditions on the structural integrity of the sea urchin extraembryonic matrix, the hyaline layer. Removal of Ca2+ resulted in the quantitative release of hyalin from isolated layers. Protein gel blot analyses indicated that, in the absence of Ca2+, hyalin was also quantitatively released from the layers surrounding 1-h-old embryos. However, no other polypeptides of the hyaline layer were released in significant amounts. The layers remaining after removal of hyalin were refractory to digestion with proteinase K and dissociated only in the presence of chaotropes. These results provide insights into the structural organization within the hyaline layer as well as the role of the embryonic cell surface in maintaining the structural integrity of this extraembryonic matrix.

Animals↗

Cardiomyopathy in infancy: observations in an epidemiologic study.

Cardiomyopathy (Cm), a rare form of cardiac disease in infancy, is receiving increasing attention stimulated by the availability of endocardial biopsy and new forms of therapy. Population-based information on frequency of occurrence, types, and maternal and infant characteristics of this diverse group of heart muscle disorders has been obtained in the course of an etiologic study on cardiovascular disease in infancy. The Baltimore-Washington Infant Study (BWIS) enrolled 2659 infants with heart disease and 2801 control infants between January 1, 1981 and March 31, 1987, a 6-year prevalence of 4.46/1000 live births. Fifty-six infants had cardiomyopathy, in the absence of a structural defect (prevalence 1/10,000). The cases were classified clinicopathologically as follows: dilated Cm (n = 17), hypertrophic Cm (n = 26), tumor (n = 5), endocardial fibroelastosis (n = 5), glycogen storage (n = 1), mucocutaneous lymph node syndrome (n = 1), and infarction (n = 1). Eleven syndromic associations and six metabolic disturbances indicate genetic risk factors. Some of the same syndromes occurred in other infants who had structural cardiac abnormalities. This overlap suggests that embryonic myocardial disease might sometimes be responsible for altered cardiac structures, possibly secondary to hemodynamic changes. Familial myocardial disease occurred in two infants with hypertrophic Cm. The Cm group did not differ by race and sex from controls, but the mothers were of lower educational and occupational status with less private care and with later registration for pregnancy care. The descriptive epidemiology of this population-based case group provides evidence of greater etiologic heterogeneity than has been shown in clinical reports.

Cardiomyopathies↗

In vitro secretion of gastrin, insulin, and glucagon in tissue cultures of pancreas from a child with neonatal intractable hypoglycemia.

Small pieces of pancreatic tissue were obtained at surgery from a subtotal pancreatectomy performed in a 45-day-old child suffering from intractable neonatal hyperinsulinic hypoglycemia. Histological examination was performed using aldehyde fuchsin, Grimelius', and Hellerström-Hellman's stainings, and immunoperoxidase labeling of insulin and gastrin. The pancreatic tissue before explantation showed numerous and sometimes hyperplastic islets, together with isolated insulin-, glucagon-, and gastrin-containing cells scattered among the exocrine tissue, in aspects similar to "B cell nesidioblastosis." These features could be interpreted as an acinoinsular transformation and/or an embryonic malformation. Extralobular endocrine islet formation by budding from ductular structures was evoked, suggesting the persistency of embryonic properties. The pieces were cultivated on rooster plasma coagulum covered with culture medium. In vitro, endocrine cells survived for 43 days, with outgrowth from the explant and with retention of their secretory abilities. After each medium renewal, radioimmunoassays were performed on the culture medium; they showed that insulin and glucagon secretions decreased with time. On the contrary, secretion of immunoreactive gastrin progressively increased, and kept up to 43 days, with subcultures. Some explants developed in a peculiar way, outgrowing as epithelial layers rich in gastrin-secreting cells as indicated by radioimmunoassays performed after they were reexplanted.

Cell Movement↗

The dynamics of structural modifications of mitochondria at the early stages of sea urchin embryonic development.

The organization of the chondriome and the ultrastructure of mitochondria have been studied in eggs and embryos of the sea urchin Paracentrotus lividus. The egg chondriome is characterized by an arrangement in well-delimited clusters. Analysis of mitochondrial clusters on electron micrographs of ultrathin serial sections shows two kinds of mitochondria of different shapes, the rod-shaped and the spherical. The egg mitochondria have a dense matrix and a well-ordered arrangement of cristae which, in rod-shaped variety, are perpendicular to the major axis. Cell division is accompanied by significant changes in intracellular distribution of mitochondria and in their structure. At the stage of 2-4 blastomeres, the clusters break up and numerous mitochondrial rods show signs of fragmentation; most of the observable mitochondria are of spherical shape. At the same time, the matrix becomes less dense, and the orderly arrangement of the cristae disappears. From the blastula to the gastrula stage, the observed modifications are reversed: the number of spherical-shaped mitochondria decreases, while that of the rod-shaped increases; the diameter of the latter is almost equal to the initial diameter of the spherical forms, the matrix becomes dense again and the cristae resume their orderly arrangement.

Animals↗

Genetic control of epidermis differentiation in Drosophila.

In arthropods, the animal body is isolated from the external environment by a protective exoskeleton called the cuticle. The cuticle of young larvae has certainly been the most scrutinized structure in Drosophila and genetic studies of the pattern of cuticular extensions has provided the main source of our comprehension of the control of embryonic development. However, the complex structure of the cuticle remains poorly understood and analysis of the underlying epidermis has started only recently. Here I review different aspects of epidermis differentiation with the aim of presenting an integrated view of the organisation of the Drosophila integument. Although profound differences in epidermis organisation are observed across species, accumulated results suggest that epidermis formation and differentiation might share an unsuspected number of homologies between Drosophila and vertebrates.

Animals↗

Embryonic gene expression in nonoverloaded ventricles of hereditary hypertrophic cardiomyopathic hamsters.

Current information regarding the molecular and biochemical mechanisms of myocardial hypertrophy, as obtained from isolated cardiomyocytes and/or healthy animals with aortic banding, does not permit dissection of the hierarchical relationship among different steps and triggers of the pathogenic process in vivo. The aim of the present study was to depict the temporal relationship among myocardial structural and functional characteristics, the embryonic gene program, and transforming growth factor (TGF) beta 1 expression in euthyroid hereditary hypertrophic cardiomyopathic hamsters (CMPH). This investigation was performed using Western and Northern blot and in situ hybridization techniques. The results show that in CMPH, the severity of the hemodynamic overload is not related to any modification in structural myocardial characteristics (cardiac mass, cardiomyocyte dimensions, total RNA, and protein content), whereas an early activation of the embryonic gene program occurs in not yet overloaded 90-day-old CMPH (left ventricular end diastolic pressure < 15 mm Hg). In these animals, a 30% to 90% decrease in the alpha myosin heavy chain (alpha MHC) relative content was found in ventricles, whereas beta MHC increased 5-fold. In addition, the alpha skeletal actin expression was enhanced 2-fold versus age-matched controls. No modifications were observed in myosin function evaluated by in vitro motility assay, whereas the administration of L-thyroxine (100 micrograms/kg intraperitoneally daily) to CMPH was able to reinduce the ventricular expression of the alpha MHC isoform (5-fold increase). Conversely, no changes were found in alpha cardiac actin and myosin light chain 2 (MLC2) expression. A close temporal relationship occurred in CMPH ventricles between the re-expression of the embryonic gene program and a 3-fold enhancement of the expression of TGF beta 1. These results indicate that the CMPH provides a useful model for investigating the expression of embryonic genes in hypertrophic ventricles in the absence of mechanical and hormonal stimuli, and that TGF beta 1 is involved in regulating in vivo the "embryonic step" of myocardial hypertrophy. Furthermore, the study offers new insights into the pathophysiologic mechanisms leading to heart failure.

Actins↗

Structural characteristics of heparin-line domain required for interaction of midkine with embryonic neurons.

Midkine is a heparin binding growth/differentiation factor with neurite promoting activity. We examined inhibitory activities of various heparin derivatives toward interaction of midkine with neurons and elucidated the structural requirements of the heparin-like domain necessary for the interaction. All of the three sulfate groups in the heparin disaccharide unit (6-O-sulfate, 2-O-sulfate and N-sulfate) were necessary for full inhibitory activity. Among these, the N-sulfate group was critically important. The minimum size with inhibitory activity was approximately 7,000 Da. Thus, the highly sulfated region in cell surface heparan sulfate proteoglycan is required for neurons to interact with midkine.

Animals↗

[Morphological study of the basement membrane of the developing lung in rats].

In order to know the significance of the basement membrane in the developing lung, an immunohistochemical study and an electron microscopical study were conducted. Wistar-Imamichi rats, which were 15, 18 and 20 day-old embryos and 5 day-old newborns, were used. The ABC method (labeled antibody method) and immunofluorescence method (FITC-labeled antibody method) were introduced, using antisera against laminin, type IV collagen and fibronectin which are the major components of the basement membrane. The basement membrane of 15 day-old embryos (pseudo-glandular period) showed very wide fine structures: the lamina lucida and lamina densa could not be defined. The width of the basement membrane became narrow in 18 and 20 day-old embryonic lungs, and the fine structural appearance of lamina lucida and lamina densa became visible after birth. There was no difference between laminin and type IV collagen immunostainability. The width of the type IV collagen and laminin immunoreactions gradually became narrow with the fine structural maturation of the basement membrane. Fibronectin immunostaining with the FITC-labeled method presented a diffuse nodular pattern, while that with the peroxidase labeled antibody method displayed a notched linear pattern. In 15 day-old embryos, fibronectin was mainly localized in the basement membrane zone of pseudo-glandular tubules and in the mesenchymal tissue. This immunoreaction in the mesenchymal tissue gradually disappeared.

Animals↗

Mutations affecting skeletal muscle myofibril structure in the zebrafish.

We describe embryonic lethal mutations in the zebrafish, Brachydanio rerio, which affect organization of skeletal muscle myofibrils. The mutations, fub-1(b45) and fub-1(b126), were independently isolated from progeny of gamma-irradiated females. Each segregates as a single recessive gene: b45 is located about 23 map units from its centromere. The b126 mutation has a similar but slightly larger apparent gene-centromere distance and a less severe phenotype. The two mutations fail to complement, suggesting that they are allelic. Homozygous b45 mutant embryos are paralyzed, and their axial skeletal muscle cells are unstriated, containing severely disorganized myofibrillar components. Gel-electrophoretic comparisons of b45 mutant and wild-type muscle proteins failed to reveal absent or altered major myofibrillar proteins. Embryos genetically mosaic for b45 were also phenotypically mosaic, suggesting that the defect is cell-autonomous. We suggest that these mutations identify a gene required for proper organization of skeletal muscle myofibrils, and that the more severe mutation may represent a null allele.

Animals↗