Developmentally regulated expression of cell surface carbohydrates during mouse embryogenesis

Cell surface carbohydrates undergo marked alterations during mouse embryogenesis. In preimplantation embryos, many carbohydrate markers show stage-specific expression in diverse ways. In early postimplantation embryos, certain carbohydrate markers are localized in defined regions in the embryo. Important carriers of stage-specific carbohydrates are the lactoseries structure (Gal beta 1----4GlcNAc) and the globoseries structure (Gal alpha 1----4Gal). Notably, the glycoprotein-bound large carbohydrate of poly-N-acetyllactosamine-type ([Gal beta 1----4GlcNAc beta 1----3]n) carries a number of markers preferentially expressed in early embryonic cells. These markers are of practical value in analyzing embryogenesis and cell differentiation. For example, in order to monitor in vitro differentiation of multipotential embryonal carcinoma cells, stage-specific embryonic antigen-1 (SSEA-1) and the Lotus agglutinin receptor have been used as markers of the undifferentiated cells, and the Dolichos agglutinin receptor has been used as a marker of extraembryonic endoderm cells. Developmental control of cell surface carbohydrates is attained by controlled expression of activities of key glycosyltransferases; for example, the activity of N-acetylglucosaminide alpha 1----3 fucosyltransferase is lost during in vitro differentiation of embryonal carcinoma cells to parietal endoderm cells, in parallel to the disappearance of SSEA-1. Accumulating evidence suggests that poly-N-acetyllactosamine-type glycans that are abundant in early embryonic cells are involved in cell surface recognition of these cells.     

Developmentally regulated expression of the P2X3 receptor in the mouse cochlea

ATP-gated non-selective cation channels assembled from P2X₃ receptor subunits contribute to transduction and neurotransmitter signaling in peripheral sensory systems and also feature prominently in the development of the central nervous system. In this study, P2X₃ receptor expression was characterized in the mouse cochlea from embryonic day 18 (E18) using confocal immunofluorescence. From E18 to P6, spiral ganglion neuron cell bodies and peripheral neurites projecting to the inner and outer hair cells were labeled. The inner spiral plexus associated with the inner hair cell synapses had a stronger fluorescence signal than outer spiral bundle fibers which provide the afferent innervation to the outer hair cells. Labeling in the cell bodies and peripheral neurites diminished around P6, and was no longer detected after the onset of hearing (P11, P17, adult). In opposition to the axiom that P2X₃ expression is neuron-specific, inner and outer sensory hair cells were labeled in the base and mid turn region at E18, but at P3 only the outer hair cells in the most apical region of the cochlea continued to express the protein. These data suggest a role for P2X₃ receptor-mediated purinergic signaling in cochlear synaptic reorganization, and establishment of neurotransmission, which occurs just prior to the onset of hearing function.     

Molecular cloning,nucleotide sequence and expression of a cDNA encoding an intracellular protein tyrosine phosphatase,PTPase-2, from mouse testis and T-cells

The PTP-2 cDNA encoding an intracellular protein tyrosine phosphatase (PTPase-2) was isolated and sequenced from mouse testis and T-cell cDNA libraries. This PTP-2 cDNA was found to be homologous to human PTP-TC and rat PTP-S, and contained 1,551 nucleotides, including 1,146 nucleotides encoding 382 amino acids as well as 5 (61 nucleotides) and 3 (344 nucleotides) non-coding regions. Northern blot analysis indicated that PTP-2 mRNA of 1.9 Kb was most abundant in testis and kidney, although it was also present in spleen, muscle, liver, heart and brain.Abbreviations PTPase Protein Tyrosine Phosphatase (EC3.1.3.48) - PTKase Protein Tyrosine Kinase (EC2.7.1.112)     

Developmentally regulated expression of a cell surface class I nuclease in Leishmania mexicana

Leishmania mexicana, like other trypanosomatid parasites, is a purine auxotroph and must obtain these essential nutrients from its sandfly and mammalian hosts. A single copy gene encoding its unique externally oriented, surface membrane, purine salvage enzyme 3'-nucleotidase/nuclease, was isolated. Structural features of the deduced protein included: an endoplasmic reticulum-directed signal peptide, several conserved class I catalytic and metal co-factor (Zn(2+)) binding domains, transmembrane anchor sequence and a C-terminal cytoplasmic tail. 3'-Nucleotidase/nuclease gene (mRNA) and protein (enzyme activity) expression were examined in three different L. mexicana developmental forms: procyclic promastigotes, metacyclic promastigotes and amastigotes. Results of both approaches demonstrated that the 3'-nucleotidase/nuclease was a stage-specific enzyme, being expressed by promastigote forms (stages restricted to the insect vector), but not by amastigotes (which produce disease in mammalian hosts). Starvation of these parasites for purines resulted in the significant up-regulation of both 3'-nucleotidase/nuclease mRNA and enzyme activity in promastigotes, but not in amastigotes. These results underscore the critical role that the 3'-nucleotidase/nuclease must play in purine salvage during the rapid multiplicative expansion of the parasite population within its insect vector. To our knowledge, the L. mexicana 3'-nucleotidase/nuclease is the first example of a nutrient-induced and developmentally regulated enzyme in any parasitic protozoan.     

Ciliogenesis and centriole formation in the mouse embryonic nervous system. An ultrastructural analysis

Serial ultrathin sections were used to study the formation of the primary cilium and the centriolar apparatus, basal body, and centriole in the neuroepithelial primordial cell of the embryonic nervous system in the mouse. At the end of mitosis, the centrioles seem to migrate toward the ventricular process of the neuroepithelial cell, near the ventricular surface. One of these centrioles, the nearest to the ventricular surface, begins to mature to form a basal body, since its tip is capped by a vesicle probably originating in the cytoplasm. This vesicle fuses with the plasmalemma and the cilium growth by the centrifugal extension of the 9 sets of microtubule doublets. These 9 sets invade the thick base of the cilium which is initially capped by a ball-shaped tip with the appearance of a mushroom cilium. The secondary extension of 7, then 5, and finally 2 sets of microtubule doublets contribute to form the tip of the mature cilium, which is associated with a mature centriolar apparatus formed by a basal body and a centriole. Centriologenesis occurs before mitosis and is concomitant with the progressive resorption of the cilium. The daughter centriole, or procentriole, begins to take form near the tips of fibrils that extend perpendicularly and at a short distance from the wall of the parent centriole. Osmiophilic material accumulates around these fibrils, and gives rise to the microtubules of the mature daughter centriole. These centrioles formed by a centriolar process are further engaged in mitosis, after the total resorption of the cilium. This pattern of development suggests that in the primordial cells of the embryonic nervous system, centriologenesis and ciliogenesis are 2 independent phenomena.     

Comparison of the nervous system of the rhabdocoel Mesostoma ehrenbergii with that of the polyclad Notoplana acticola

The neuromuscular organization of the free-living marine polyclad Notoplana acticola (Boone) can be compared with that of the fresh water rhabdocoel Mesostoma ehrenbergii (Focke). These examples act as outgroups for each other, belonging to different clades of the Turbellaria and similarities between the two can be taken as synapomorphies to indicate features that might be primitive and general features of flatworm nervous systems. The fluorescent dye Lucifer Yellow was used to fill cells in the brains of the two species and the neuronal anatomies were photographed or drawn. Many different cell types have been found in both species. The predominant cell types in Mesostoma were heteropolar bipolar and heteropolar multipolar cells with isopolar bipolar and isopolar multipolar cells in much lower numbers; only a few unipolar cells were found. In Notoplana, many of the cells penetrated were also heteropolar bi- or multipolar cells. There were also neurons and architectural features that were morphologically very similar to each other, in the two animals, such as the BRA cells and commissural tracts. These shared features suggest that many of the peculiarities of polyclad nervous systems may be general flatworm features.     

Differential expression of the Tmem132 family genes in the developing mouse nervous system

The family of novel transmembrane proteins (TMEM) 132 have been associated with multiple neurological disorders and cancers in humans, but have hardly been studied in vivo. Here we report the expression patterns of the five Tmem132 genes (a, b, c, d and e) in developing mouse nervous system with RNA in situ hybridization in wholemount embryos and tissue sections. Our results reveal differential and partially overlapping expression of multiple Tmem132 family members in both the central and peripheral nervous system, suggesting potential partial redundancy among them.     

Molecular cloning of the cDNA coding for regucalcin and its mRNA expression in mouse liver: The expression is stimulated by calcium administration

The molecular cloning of the cDNA coding for a Ca2+-binding proteinregucalcin and its mRNA expression in mouse liver were investigated. ThecDNA clone encoding a regucalcin was isolated from a mouse liver cDNAlibrary and sequenced. Analysis of the sequence of the cloned cDNA showedthat the cDNA encoded the complete amino acid sequence of the mouseregucalcin molecule; the cDNA had an open reading frame of 897 bp. Mouseregucalcin was composed of 299 amino acid residues, and its molecular weightwas estimated to be 33,406 Da. The amino acid sequence of mouse regucalcinhad 94% homology, as compared with that of rat regucalcin. Northernblot analysis with the mouse liver cDNA probe revealed that mouse regucalcinmRNA was mainly present in the liver but only slightly in the kidney with asize of 1.8 kb. Hepatic regucalcin mRNA level of male mouse was higher thanthat of female mouse. A single intraperitoneal administration of calciumchloride (5, 15, and 30 mg Ca2+/100 g body weight) to mice induced aremarkable increase in regucalcin mRNA in the liver; the increase inregucalcin mRNA levels at 30 min after calcium administration wasdose-dependent. The present results demonstrate that regucalcin mRNA in miceis uniquely expressed in the liver, and that its expression is stimulated bycalcium administration.     

Niemann-Pick C1 expression is not regulated by the amount of cholesterol flowing through cells in the mouse

The Niemann-Pick C1 (NPC1) protein functions to regulate the transport of cholesterol from late endosomes/lysosomes to other cellular compartments after lipoprotein uptake through the coated-pit pathway. The present study examines the relative expression of NPC1 mRNA and NPC1 protein in different tissues of the mouse in relation to the uptake of total cholesterol carried in chylomicron remnants (CMr-TC), low density lipoproteins (LDL-TC), cholesteryl ester carried in high density lipoproteins (HDL-CE), and cholesterol synthesis. Results from this study demonstrate that the highest relative expression of NPC1 is in the liver, which is also the tissue with the highest uptake of CMr-TC, LDL-TC, HDL-CE, and cholesterol synthesis. However, there was no similar relation in the remaining tissues. To examine the relative expression of NPC1 in relation to the amount of cholesterol that flowed through the coated-pit pathway, mice were fed a diet supplemented with increasing amounts of cholesterol or cholestyramine. The results from this study demonstrated that there was no relation between the relative expression of NPC1 and the amount of cholesterol that flowed through the coated-pit pathway. We conclude that the relative expression of NPC1 is not regulated by the flow of cholesterol through cells in the mouse and is therefore constitutive.     

GPI‐anchored human placental alkaline phosphatase has a nonpolarized distribution on the cell surface of mouse cerebellar granule neurons in vitro

The glycosyl phosphatidylinositol (GPI) lipid anchor, which directs GPI‐anchored proteins to the apical cell surface in certain polarized epithelial cell types, has been proposed to act as an axonal protein targeting signal in neurons. However, as several GPI‐anchored proteins have been found on both the axonal and somatodendritic cell‐surface domains of a variety of neuronal cell types, the role of the GPI anchor in protein localization to the axon remains unclear. To begin to address the role of the GPI anchor in neuronal protein localization, we used a replication‐incompetent retroviral vector to express a model GPI‐anchored protein, human placental alkaline phosphatase (hPLAP), in early postnatal mouse cerebellar granule neurons developing in vitro. Purified granule neurons were cultured in large mitotically active cellular reaggregates to allow retroviral infection of undifferentiated, proliferating granule neuron precursors. To more easily visualize hPLAP localization during the sequence of differentiation of single postmitotic granule neurons, reaggregates were dissociated following infection, plated as high‐density monolayers, and maintained for 1–9 days under serum‐free culture conditions. As we previously demonstrated for uninfected granule neurons developing in monolayer culture, hPLAP‐expressing granule neurons likewise developed in vitro through a series of discrete temporal stages highly similar to those observed in situ. hPLAP‐expressing granule neurons first extended either a single neurite or two axonal processes, and subsequently attained a mature, well‐polarized morphology consisting of multiple short dendrites and one or two axons that extended up to 3 mm across the culture substratum. hPLAP was expressed uniformly on the entire cell surface at each stage of granule neuron differentiation. Thus, it appears that the GPI anchor is not sufficient to confer axonal localization to an exogenous GPI‐anchored protein expressed in a well‐polarized primary neuronal cell type in vitro; other signals, such asthose present in the extracellular domain of these proteins, may be necessary for the polarized targeting or retention of axon‐specific GPI‐anchored proteins. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 119–141, 1999     

Developmentally regulated 75-kilodalton protein expressed in LLC-PK1 cultures is a component of the renal Na+/glucose cotransport system

Na+/D-glucose symport is a secondary active glucose transport mechanism expressed only in kidney proximal tubule and in small intestine. A monoclonal antibody that recognized the Na+/glucose symporter of pig renal brush border membranes also recognized a 75-kD protein in apical membranes isolated from highly differentiated LLC-PK1 cultures, an epithelial cell line of pig renal proximal tubule origin. The 75-kD antigen was enriched from solubilized LLC-PK1 apical membranes by means of high-pressure liquid chromatography. The symporter antigen became apparent on the apical membrane surface after the development of a confluent monolayer in correlation with the expression of transport activity. Long-term treatment of cultures with the differentiation inducer hexamethylene bisacetamide was accompanied by a dramatically increased expression of the symporter antigen as detected quantitatively by Western blot analysis and qualitatively by immunofluorescence staining. The number of symporter-positive cells was dramatically increased after inducer treatment as predicted for differentiation-regulated expression. These results identify a 75-kD protein as a component of a developmentally regulated renal Na+/glucose symporter expressed in cell culture.     

Cell cycle dependent expression of a glucose regulated cell surface glycoprotein

Summary A cell surface associated “glucose regulated protein” has been described on nontransformed human fibroblasts. To examine the distribution of that protein on human fibroblasts specific antisera were used. The antisera was used in conjunction with indirect immunofluorescence and revealed that the glucose regulated protein was present as fibers on spread cells. Further, the antisera was used in complement mediated cytotoxicity assays to examine cells during specific stages of the mitotic cell cycle. Fibroblasts were synchronized by serum starvation, hydroxyurea inhibition, or colcemid inhibition followed by mitotic selection. The results demonstrated that the glucose regulated protein was maximally displayed during the G₁ phase of the cell cycle and minimally displayed during the S and M phases. Research was supported by contract AG00697 from the National Institutes of Health.     

Developmentally regulated expression of a sunflower 11S seed protein gene in transgenic tobacco

Summary The trifunctional TRP1 gene from Neurospora crassa (N-TRP1) was subcloned into the yeast-Escherichia coli shuttle vector YEp13 and expressed in Saccharomyces cerevisiae. The three activities of the N-TRP1 gene product were detected in yeast mutants that lacked either N-(5-phosphoribosyl) anthranilate (PRA) isomerase or both the glutamine amidotransferase function of anthranilate synthase and indole-3-glycerol phosphate (InGP) synthase. The protein was detected on immunoblots only as the full length 83 kda product indicating that the trifunctional gene product was expressed in yeast primarily in a fully active, undegraded form. By placing the subcloned N-TRP1 gene under the control of the inducible PHO5 promoter from yeast, the expression of all three activities was increased to more than ten fold that of wild-type yeast and the overproduced protein could be visualized by SDS-polyacrylamide gel electrophoresis of crude extract and Coomassie Blue staining. Using the expression system described the effect of selective deletion of regions of the coding sequence of the N-TRP1 gene on expression of the three activities was tested. Expression of either the F- or C-domains, catalyzing respectively the PRA isomerase or InGP synthase activities, did not depend on the presence of the other domain in the active polypeptide. Furthermore, normal dimer formation occurred with a protein active for InGP synthase in a deletion derivative lacking most of the PRA isomerase domain, ruling out the hypothesis that interaction between the active site regions for PRA isomerase and InGP synthase accounted for dimer formation in the trifunctional product.Abbreviations PRA N-(5'-phosphoribosyl)anthranilate - InGP indole-3-glycerol phosphate - SDS sodium dodecyl sulfate     

Baculovirus expression of mouse lactoferrin receptor and tissue distribution in the mouse

Lactoferrin (Lf) has been shown to have a role in the immune system and in early development of the mouse embryo. A specific receptor for Lf has been suggested to mediate the functions of Lf. We have recently identified a Lf receptor (LfR) in human fetal small intestine. We therefore hypothesized that the mouse homologue of this protein functions as a LfR. We expressed mouse Lf (MLf) and the mouse homologue (MLfR) in a baculovirus-insect cell system. The recombinant MLfR (rMLfR) was purified by immobilized recombinant MLf (rMLf) affinity chromatography, demonstrating an interaction between rMLf and the rMLfR. RT-PCR revealed that MLfR was expressed in various tissues and during embryonic development. Immunohistochemical analysis revealed that the MLfR was localized in various tissues including small intestinal epithelium, stomach, kidney, ovary, and various regions of brain. In summary, the MLfR functions as a receptor for MLf, is expressed and localized in various tissues, and may be involved in the indispensable function of MLf during early embryonic development.     

Control of the neuronal cell attachment by functionality manipulation of diazo-naphthoquinone/novolak photoresist surface

The surface of the photosensitive Diazo-Naphto-quinone/novolak film was chemically manipulated through UV exposure and subsequent thermal processes to obtain different surface functionalities (DNQ, carboxylic, imidazole, indene, silylated and charged groups) and hydrophobicities. The neuronal cell attachment is sensitive to chemical functionalization, with favourable influence from charged, imidazole and carboxylic groups, while the hydrophobic/hydrophilic balance of the photoresist surface plays at best a secondary role. The microlithographic techniques assessed (standard positive tone, negative and positive tone image reversal, and surface imaging based on silylation) can be used to gain insight into the cell attachment mechanisms. The positive tone DNQ/novolak/imidazole system was found to be a suitable candidate for cell patterning.