Developmentally regulated changes in the glycoproteins of the equine embryonic capsule

The embryonic capsule, which covers the equine blastocyst after it loses its zona pellucida, is composed of mucin-like glycoproteins. In the present study, we investigated both macroscopic and molecular changes in the capsule during development. The weight of the capsule increased from day 11-12 of pregnancy and reached a maximum at about day 18, coinciding with the time during which the conceptus migrates extensively throughout the uterus. The sialic acid content of the capsule declined markedly from about day 16, the time of conceptus 'fixation' in the uterus, which suggests a unique developmentally regulated mechanism for the control of embryo mobility. These results lead us to propose that the capsule may have an anti-adhesion function in the developing conceptus, and that this effect could be regulated by the sugar side chains of the capsular glycoproteins. The glycosylation characteristics of the blastocyst coverings also underwent changes at about day 9 of pregnancy, which may be related to loss of the zona pellucida. An anti-capsule monoclonal antibody was raised and shown to recognize a tissue-specific antigen present only on the capsule and trophoblast. This antigen was present on the trophoblastic cells soon after the blastocyst is formed, reached a maximum concentration at about day 18, and was absent after day 22, coinciding with the disappearance of the capsule. Immunohistochemical studies indicate that the mucin-like capsular glycoproteins are secreted, at least in major part, by the trophoblast.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmentally regulated spontaneous activity in the embryonic chick retina

Even before birth and the onset of sensory experience, neural activity plays an important role in shaping the vertebrate nervous system. In the embryonic chick visual system, activity in the retina before vision has been implicated in the refinement of retinotopic maps, the elimination of transient projections, and the survival of a full complement of neurons. In this study, we report the detection of a physiological substrate for these phenomena: waves of spontaneous activity in the ganglion cell layer of the embryonic chick retina. The activity is robust and highly patterned, taking the form of large amplitude, rhythmic, and wide-ranging waves of excitation that propagate across the retina. Activity waves are most prominent and organized between embryonic days 13-18, coinciding with the developmental period during which retinal axons refine their connections in their targets. The spatial and temporal features of the patterns observed are consistent with the role of activity patterns in shaping eye-specific projections and retinotopic maps but inconsistent with the hypothesis that they specify lamina-specific projections in the tectum. Antagonists of glutamatergic and glycinergic transmission and of gap junctional communication suppress spontaneous activity, whereas antagonists to GABAergic transmission potentiate it. Based on these results, we propose that spontaneous activity in the ganglion cells is regulated by chemical inputs from both bipolar and amacrine cells and by gap junctional coupling involving ganglion cells.     

Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos

Embryonic lethality of thrombomodulin-deficient mice has indicated an essential role for this regulator of blood coagulation in murine development. Here, the embryonic expression pattern of thrombomodulin was defined by surveying beta-galactosidase activity in a mouse strain in which the reporter gene was placed under the regulatory control of the endogenous thrombomodulin promoter via homologous recombination in embryonic stem cells. The murine trophoblast was identified as a previously unrecognized anatomical site where TM expression is conserved between humans and mice and may exert a critical function during postimplantation development. Targeted reporter gene expression in mesodermal precursors of the endothelial cell lineage defined thrombomodulin as an early marker of vascular differentiation. Analysis of the thrombomodulin promoter in differentiating ES cells and in transgenic mice provided evidence for a disparate and cell type-specific gene regulatory control mechanism in the parietal yolk sac. The thrombomodulin promoter as defined in this study will allow the targeting of gene expression to the parietal yolk sac of transgenic mice and the initiation of investigations into the role of parietal endoderm in placental function.     

Autoantigen Ku in the brain. Developmentally regulated expression and subcellular localization

A double-stranded DNA end-binding factor with high levels of expression in brain and testis of adult mice was identified as the Ku protein, earlier described as an autoantigen in connective tissue diseases and found to be essential for recombination of the immunoglobulin genes and DNA repair. High Ku levels were found in the cerebellum and pituitary gland, lower levels in the hippocampus, hypothalamus and white matter structures. Ku levels were much higher in embryonic rat brain than in the adult brain, suggesting a role of the Ku protein in brain development. In embryonic rat brain, Ku was associated with cell nuclei, but was predominantly located in the cytosol in the adult rat cerebellum and hippocampus. The abundant expression of Ku in the brain suggests the involvement of Ku autoantibodies in the pathogenesis of neuropsychiatric complications in connective tissue diseases.     

Developmentally regulated and spatially restricted antigens of radial glial cells

Radial glial cells, present in many parts of the embryonic vertebrate central nervous system (CNS), have been implicated in the guidance of neuroblasts from the ventricular zone to their laminar destinations. Moreover, radial glial cells may be progenitors of some CNS neurons and glia. To gain new insight into the structure and development of these cells, we have generated and characterized a panel of monoclonal antibodies that recognize radial glial cells of the chick optic tectum. Mice were immunized with homogenates of embryonic day (E) 10 tectum, and antibodies were analyzed by immunofluorescence and immunoblotting. We describe here three pairs of antibodies. 1) H5 and a previously generated antibody, R5 (Dr?ger et al., J. Neurosci. 4:2025, 1984), stain the whole extent of the radial glial cell from E7 to E20. In cultures prepared from E10 tecta, both stain a filamentous meshwork in glial cells but not in neurons. On immunoblots, both recognize a protein of approximately 52 kD that is closely related (or identical) to vimentin. 2) H28 and H29 stain radial glia between E7 and E14, but not later. Moreover, H28 and H29 staining is markedly more intense in the ventricular and intermediate zones than in the laminae of the tectal plate. Both of these antibodies recognize an intracellular epitope in cultured glial cells and a protein of approximately 35 kD on immunoblots. 3) H2 and H27 recognize antigens concentrated in the most superficial processes and endfeet of radial glia in late (E16-E20) embryos. They stain distinct structures in cultured glia, suggesting that they recognize distinct antigens. H27 recognizes a protein of approximately 29 kD on immunoblots. Thus antibodies H5 and R5 are good markers of radial glial cells at all stages, whereas the others define antigens that are developmentally regulated and localized to discrete domains. Together, these antibodies can be used to study temporal and spatial specializations of radial glia.     

Developmentally regulated expression of interleukin-1 beta by peri-implantation conceptuses in swine

Interleukin-1 beta (IL-1 beta) is one of the critical inflammatory cytokines involved in many physiological processes. This study investigated the temporal expression of IL-1 beta in pig conceptuses. A human IL-1 beta cDNA probe and a anti-human IL-1 beta antibody were used to examine IL-1 beta gene and protein expression in pig conceptuses on days 11, 12, 13, 14, 15, 20, 30, 45, 60, 75, 90, 105 and 112 of pregnancy using Northern, Slot and Western blot analyses. A human cell line (A375.S2) was used to determine IL-1 activity in pig conceptuses. High levels of IL-1 beta mRNA were detected in days 11, 12 and 13 conceptuses. IL-1 beta protein was also detected in conceptuses on days 11, 12, 13, and 14, but not in conceptuses recovered after day 15 of pregnancy. IL-1 beta biological activity was demonstrated in days 11 and 12 conceptus homogenates, but not in homogenates of days 112 allantochorion. Low levels of IL-1 beta mRNA were detected by Northern blot analysis in Day 15 conceptuses, endometrium and myometrium only when poly(A+) RNA was used. The production of IL-1 beta by peri-implantation pig conceptuses was temporally associated with maternal recognition of pregnancy. The results suggest that conceptus IL-1 beta may be important for conceptus development and establishment of pregnancy.     

Developmentally regulated expression of persyn, a member of the synuclein family, in skin

Synucleins constitute a group of unique, evolutionarily conserved proteins that are expressed predominantly in neurons of the central and peripheral nervous system. Although the normal cellular functions of synucleins are not clear, these proteins have been implicated in various neurodegenerative conditions in humans. We found that persyn, a recently characterized member of the synuclein family, is expressed not only in the nervous system but also in the stratum granulosum of the epidermis of neonatal and adult mice. This finding together with our recent observations that persyn influences neurofilament network integrity in sensory neurons raises the possibility that persyn in skin could be involved in modulation of the keratin network.     

Developmentally regulated mRNA splicing of clathrin assembly protein 3 (AP-3)

Clathrin assembly protein 3 (AP-3) is a neuron-specific component of clathrin coated vesicles. Because it promotes the assembly of uniform clathrin cages, AP-3 may play a regulatory role in synaptic vesicle recycling. Previously, using the monoclonal antibody MabR-18, we demonstrated that AP-3 expression starts from the embryonic stage and is maintained at high levels from the early postnatal stages through adult. In order to study the expression of AP-3 during early postnatal development at the mRNA level, RT-PCR analysis was performed. We divided the coding region of AP-3 into 10 regions and designed primers to amplify each region. As a result, developmentally regulated splicing sites were found in two regions. In one region, a PCR product with a 108-bp deletion was detected from postnatal day 10 (P10). In the other region, a product with a 15-bp deletion was increased compensating for the decrease of the undeleted product. The expression of isoforms changed mainly from around P7 to P10, whereas the level of AP-3 protein remained relatively constant throughout postnatal development. These results suggest that the expression of AP-3 isoforms with mRNA splicing is developmentally regulated in the brain and may be involved in the maturation of synaptic vesicle recycling.     

Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.     

Electroencephalographic changes in rats received antigens of different parasites

The prevalence of epilepsy in developing countries is much higher than in developed ones. Some of the highest prevalence rates in developing countries have been reported from tropical Africa where parasitic infections are endemic. This work was carried out to assess the effect of different parasitic antigens on the activity of cerebral cortex in rats. Nine crude antigens were used: Adult Fasciola, adult S. mansoni, hydatid cyst, T. spiralis, E. histolytica, Acanthamoeba spp. G. lamblia, Cryptosporidium spp. and crude antigen of T. gondii. All the parasitic antigens induced electroencephalographic changes compared with baseline tracings.