Distinct patterns of expression of the β-1,4-galactosyltransferases during testicular development in the mouse

Glycosylation is one of the most important post-translational modifications and it is clear that the single step of -1,4-galactosylation is performed by a family of -1,4-galactosyltransferases (4-GalTs) and that each member of this family may play a distinct role in different tissues and cells. In this study, we characterized the gene expression of six 4-GalTs in mouse testis and analyzed the changes of galactosylation of testis glycoproteins during postnatal development. Northern blot analysis revealed that 4-GalT-I and 4-GalT-IV were expressed mainly in newborn mouse testis and that the expression of 4-GalT-II increased markedly and persisted at the highest levels in adult mouse testis. The expression of 4-GalT-III and 4-GalT-V, however, remained relatively at low levels during mouse testicular development. In contrast, the expression of 4-GalT-VI was undetectable in mouse testis. The gene expression of 4-GalT-II in mouse testis was further analyzed by in situ hybridization due to its unique expression pattern. Strong hybridization signals were detected in the seminiferous tubules and the expression varied among the different stages of spermatogenic differentiation. The distinct gene expression patterns of 4-GalTs in mouse testis could affect the differential galactosylation of testis glycoproteins, as revealed by lectin histochemistry analysis.     

Heterogeneity and differential expression of the γ-aminobutyric acidA (GABAA)/benzodiazepine receptor in the avian brain during development

Summary 1. The changes in the GABA_A/benzodiazepine receptor in chicken brain during development has been studied by using³H-flunitrazepam as the probe for the benzodiazepine modulator site and the antibodies recognizing the receptor protein. In the telencephalon and optic tectum, the proteins of 48, 50, and 51 kD were markedly labeled by³H-flunitrazepam from embryonic day 18 to postnatal days, as revealed by photoaffinity labeling and SDS-PAGE of the brain membranes; the 51-kD protein appeared to be the predominant one in labeling intensity except at embryonic day 18 and postnatal days 14 and 28, whereas the 47- and 50-kD proteins were dominant in the cerebellum. However, the 47- and 48-kD proteins were faintly seen after postnatal day 28 in the three regions examined.2. Immunoblotting using a monoclonal antibody against the 50- and 51-kD proteins showed that the straining pattern in the developing telecephalon or optic tectum was similar to the 50 kD/51 kD pattern obtained from fluorography. The antibody also stained the 50- and 51-kD proteins in the cerebellum despite the fact that the 51-kD protein was barely seen in the fluorogram. Moreover, the 50-kD protein was recognized by an antiserum raised against a partial sequence of the 1 subunit of the receptor expressed in bacteria. The staining levels for the 50-kd protein by the antiserum on immunoblots of the brain regions were low in embryonic animals but higher during postnatal stages, consistent with that seen in fluorograms.3. Receptor binding autoradiography using³H-flunitrazepam exhibited that varying degrees of labeling intensity occurred among various brain areas at different ages. High densities of binding were present in the olfactory bulb, paleostriatum, optic tectum, and midbrain. These results support the diversity of the GABA_A/benzodiazepine receptor in the vertebrate CNS.     

Changes in expression of two endogenous β-galactoside-binding isolectins in the dermis of chick embryonic skin during development in ovo and in vitro

In order to elucidate the roles of metal-independent animal lectins, we systematically investigated changes in expression of 2 kinds of -galactoside-binding isolectins (MW 14 and 16 kDa) in the dermis of chick embryonic tarsometatarsal skin during the course of development. These lectins were immunohistochemically located at different stages of development both in ovo and in vitro by light and electron microscopy. Light-microscopic observation showed that while positive staining for the 14-kDa lectin was weak at days 8 and 10 it became intense after day 13. In contrast, staining for the 16-kDa lectin was intense at days 8, 10, and 13, but it became weak after day 17 when keratinization of the epidermis was completed. Immuno-electron-microscopic observation revealed that both the 14 and 16-kDa lectins were located on the basement membrane, in the extracellular matrix, and in both the cytoplasm and the nucleus of dermal fibroblasts. Distribution of the 2 isolectins was also examined in cultured skin explants in vitro. The results were almost the same as those obtained in ovo when the skin explant was keratinized in the presence of hydrocortisone. However, in the skin explant where keratinization was prevented and mucous metaplasia was induced by the addition of vitamin A, the distribution of the 14-kDa lectin in the epidermis was significantly affected. These results indicate that (1) the expression of the 2 isolectins is differently regulated in both the dermis and epidermis, (2) the 16-kDa lectin is involved in the early stage of the formation of the dermis and the basement membrane and is replaced by the 14-kDa lectin as keratinization of the epidermis occurs, and (3) the expression of the 2 isolectins in the dermis is not significantly affected by the induction of mucous metaplasia, in contrast to their drastic changes in the epidermis.     

The expression of β-glucuronidase during preimplantation development of mouse embryos

β-Glucuronidase activity was measured in mouse embryos during the preimplantation period of development by using a microfluorometric assay. A 100-fold increase in activity was observed between 57 (8-cell stage) and 84 hr (morulae) of development. Activity changes between 30 and 60 hr were also significant. Genetic variants of β-glucuronidase occur between the strains of mice $\text{C57BL}\text{6J}$ and $\text{C3H}\text{HeJ}$ which differ in levels of activity and heat denaturation kinetics. Activity changes and heat denaturation kinetics of β-glucuronidase in $\text{C57BL}\text{6}\text{,}\text{C3H}\text{HeJ}$ and F₁ hybrid embryos were compared, and it was demonstrated that paternal genes were expressed during the 100-fold increase in activity and that embryonic genes may be functioning between 30 and 60 hr of development.     

Subcellular localization of β-catenin and cadherin expression in the cap-stage enamel organ of the mouse molar

We analyzed the subcellular distribution of -catenin in the cap-stage enamel organ and compared it with the expression of E- and P-cadherin by using confocal laser microscopy. The amounts of the molecules in the cytoplasm and the nucleus showed regional variations in the enamel organ, whereas cell surface-associated -catenin was ubiquitous. In both the enamel knot and the inner dental epithelium, -catenin was detected in the cytoplasm and in the nucleus. However, the amount of nuclear -catenin was apparently higher in the enamel knot than in the inner dental epithelium. P-cadherin also gave a stronger signal in the enamel knot than in other parts of the enamel organ. In the stellate reticulum, where E-cadherin was preferentially expressed, as well as in the cervical loop and outer dental epithelium, -catenin was localized in the cytoplasm but not in the nucleus. The nuclear localization of -catenin in the enamel knot suggests a specific activation of the canonical Wnt signaling pathway. A coincident upregulation of P-cadherin was observed in this area. Altogether, these observations suggest the possibility of a linkage between cell adhesion and Wnt signaling in the enamel knot.     

Developmental changes in functional expression and β-adrenergic regula-tion of If in the heart of mouse embryo

The hyperpolarization-activated current (If) plays an important role in determining the spontaneous rate of cardiac pacemaker cells. The automatic rhythmicity also exists in working cells of embryonic heart,therefore we studied developmental changes in functional expression and β-adrenergic regulation of If in embryonic mouse heart. The expression of If is high in early developmental stage (EDS) (10.5 d after coitus) ventricular myocytes, low in intermediate developmental stage (IDS) (13.5 d) atrial or ventricular myocytes and even lower in late developmental stage (LDS) (16.5 d) atrial or ventricular myocytes, indicating that these cells of the EDS embryonic heart have some properties of pacemaker cells, β-adrenergic agonist isoproterenol (ISO) stimulates If in LDS but not in EDS cardiomyocytes, indicating that the β-adrenergic regulation of If is not mature in EDS embryonic heart. But forskolin (a direct activator of adenylate cyclase) and 8-Br-cAMP (a membrane-permeable analogue of cAMP) increase the amplitude of If in EDS cells,indicating that adenylate cyclase and cAMP function fairly well at early stage of development. Furthermore,the results demonstrate that If is modulated by phosphorylation via cAMP dependent PKA both in EDS and LDS cells.     

Developmental changes in functional expression and β-adrenergic regulation of If in the heart of mouse embryo

The hyperpolarization-activated current (If) plays an important role in determining the spontaneousrate of cardiac pacemaker cells. The automatic rhythmicity also exists in working cells of embryonic heart,therefore we studied developmental changes in functional expression and β-adrenergic regulation of If inembryonic mouse heart. The expression of If is high in early developmental stage (EDS) (10.5 d after coitus)ventricular myocytes, low in intermediate developmental stage (IDS) (13.5 d) atrial or ventricular myocytesand even lower in late developmental stage (LDS) (16.5 d) atrial or ventricular myocytes, indicating thatthese cells of the EDS embryonic heart have some properties of pacemaker cells. β-adrenergic agonistisoproterenol (ISO) stimulates If in LDS but not in EDS cardiomyocytes, indicating that theβ-adrenergicregulation of If is not mature in EDS embryonic heart. But forskolin (a direct activator of adenylate cyclase)and 8-Br-cAMP (a membrane-permeable analogue of cAMP) increase the amplitude of If in EDS cells,indicating that adenylate cyclase and cAMP function fairly well at early stage of development. Furthermore,the results demonstrate that If is modulated by phosphorylation via cAMP dependent PKA both in EDSand LDS cells.     

Regional expression and subcellular localization of the voltage-gated calcium channel β subunits in the developing mouse brain

J. Neurochem. (2012) 122, 1095-1107. ABSTRACT: Ca(2+) channel β subunits determine the maturation, biophysical properties and cell surface expression of high voltage-activated channels. Thus, we have analysed the expression, regional distribution and subcellular localization of the Ca(v) β subunit family in mice from birth to adulthood. In the hippocampus and cerebellum, Ca(v) β(1) , Ca(v) β(3) and Ca(v) β(4) protein levels increased with age, although there were marked region- and developmental stage-specific differences in their expression. Ca(v) β(1) was predominantly expressed in the strata oriens and radiatum of the hippocampus, and only weakly in the cerebellum. The Ca(v) β(3) subunit was mainly expressed in the strata radiatum and lucidum of the hippocampus and in the molecular layer of the cerebellum. During development, Ca(v) β(3) protein expression in the cerebellum peaked at postnatal days (P) 15 and 21, and had diminished drastically by P60, and in the hippocampus increased with age throughout all subfields. Ca(v) β(4) protein was detected throughout the cerebellum, particularly in the molecular layer, and in contrast to the other subunits, Ca(v) β(4) was mainly detected in the molecular layer and the hilus of the hippocampus. At the subcellular level, Ca(v) β(1) and Ca(v) β(3) were predominantly located post-synaptically in hippocampal pyramidal cells and cerebellar Purkinje cells. Ca(v) β(4) subunits were detected in the pre-synaptic and post-synaptic compartments of both regions, albeit more strongly at post-synaptic sites. These results shed new light on the developmental regulation and subcellular localization of Ca(v) β subunits, and their possible role in pre- and post-synaptic transmission.     

Distinct subcellular localization of β-tubulin epitopes in the adult mouse brain

 A panel of monoclonal antibodies specific of α-tubulin (TU-01, TU-09) and β-tubulin (TU-06, TU-13) subunits was used to study the location of N-terminal structural domains of tubulin in adult mouse brain. The specificity of antibodies was confirmed b immunoblotting experiments. Immunohistochemical staining of vibratome sections from cerebral cortex, cerebellum, hippocampus, and corpus callosum showed that antibodies TU-01, TU-09, and TU13 reacted with neuronal and glial cells and their processes, whereas the TU-06 antibody stained only the perikarya. Dendrites and axons were either unstained or their staining was very weak. As the TU-06 epitope is located on the N-terminal structural domain of β-tubulin, the observed staining pattern cannot be interpreted as evidence of a distinct subcellular localization of β-tubulin isotypes or known post-translational modifications. The limited distribution of the epitope could, rather, reflect differences between the conformations of tubulin molecules in microtubules of somata and neurites or, alternatively, a specific masking of the corresponding region on the N-terminal domain of β-tubulin by interacting protein(s) in dendrites and axons. Accepted: 11 November 1996     

Disruption of β-arrestins blocks glucocorticoid receptor and severely retards lung and liver development in mice

In this study, the role of β-arrestin 1 and β-arrestin 2 in fetal lung and liver development was examined using Arrb1(-/-)Arrb2(-/-) mouse embryos. β-Arrestin 1/2 dual-null mice died shortly after birth and morphological examination revealed an obvious pulmonary hypoplasia and severe hepatic impairment. Western blot analysis demonstrated that GR protein levels in Arrb1(-/-)Arrb2(-/-) lung and liver tissues were significantly decreased compared to wild type embryos. Expression of GR proteins was confirmed in the nuclei of type II pneumocytes of 18.5 day embryos (E18.5) by immunofluorescence. The production of hepatic glucose and mRNA level of gluconeogenic enzymes were dramatically reduced in E18.5 Arrb1(-/-)Arrb2(-/-) liver. These results suggest that GR is an important downstream effector of the β-arrestin signaling pathway involved in regulation of lung and liver development. However, no obvious changes in GR expression following in vitro modulation of β-arrestin 1/2 indicated the existence of an indirect regulatory relationship between GR and the β-arrestin signaling pathway.     

Identification of residues involved in homodimer formation located within a β-strand region of the N-terminus of a Yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) are members of a superfamily of cell surface signaling proteins that play critical roles in many physiological functions; malfunction of these proteins is associated with multiple diseases. Understanding the structure–function relationships of these proteins is important, therefore, for GPCR-based drug discovery. The yeast Saccharomyces cerevisiae tridecapeptide pheromone α-factor receptor Ste2p has been studied as a model to explore the structure–function relationships of this important class of cell surface receptors. Although transmembrane domains of GPCRs have been examined extensively, the extracellular N-terminus and loop regions have received less attention. We have used substituted cysteine accessibility method to probe the solvent accessibility of single cysteine residues engineered to replace residues Gly20 through Gly33 of the N-terminus of Ste2p. Unexpectedly, our analyses revealed that the residues Ser22, Ile24, Tyr26, and Ser28 in the N-terminus were solvent inaccessible, whereas all other residues of the targeted region were solvent accessible. The periodicity of accessibility from residues Ser22–Ser28 is indicative of an underlying structure consistent with a β-strand that was predicted computationally in this region. Moreover, a number of these Cys-substituted Ste2p receptors (G20C, S22C, I24C, Y26C, S28C and Y30C) were found to form increased dimers compared to the Cys-less Ste2p. Based on these data, we propose that part of the N-terminus of Ste2p is structured and that this structure forms a dimer interface for Ste2p molecules. Dimerization mediated by the N-terminus was affected by ligand binding, indicating an unanticipated conformational change in the N-terminus upon receptor activation.     

Identification of residues involved in homodimer formation located within a β-strand region of the N-terminus of a Yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) are members of a superfamily of cell surface signaling proteins that play critical roles in many physiological functions; malfunction of these proteins is associated with multiple diseases. Understanding the structure-function relationships of these proteins is important, therefore, for GPCR-based drug discovery. The yeast Saccharomyces cerevisiae tridecapeptide pheromone α-factor receptor Ste2p has been studied as a model to explore the structure-function relationships of this important class of cell surface receptors. Although transmembrane domains of GPCRs have been examined extensively, the extracellular N-terminus and loop regions have received less attention. We have used substituted cysteine accessibility method to probe the solvent accessibility of single cysteine residues engineered to replace residues Gly20 through Gly33 of the N-terminus of Ste2p. Unexpectedly, our analyses revealed that the residues Ser22, Ile24, Tyr26, and Ser28 in the N-terminus were solvent inaccessible, whereas all other residues of the targeted region were solvent accessible. The periodicity of accessibility from residues Ser22-Ser28 is indicative of an underlying structure consistent with a β-strand that was predicted computationally in this region. Moreover, a number of these Cys-substituted Ste2p receptors (G20C, S22C, I24C, Y26C, S28C and Y30C) were found to form increased dimers compared to the Cys-less Ste2p. Based on these data, we propose that part of the N-terminus of Ste2p is structured and that this structure forms a dimer interface for Ste2p molecules. Dimerization mediated by the N-terminus was affected by ligand binding, indicating an unanticipated conformational change in the N-terminus upon receptor activation.     

The Higher Structure of Chromatin in the LCR of the β-Globin Locus Changes during Development

The β-globin locus control region (LCR) is able to enhance the expression of all globin genes throughout the course of development. However, the chromatin structure of the LCR at the different developmental stages is not well defined. We report DNase I and micrococcal nuclease hypersensitivity, chromatin immunoprecipitation analyses for histones H2A, H2B, H3, and H4, and 3C (chromatin conformation capture) assays of the normal and mutant β-globin loci, which demonstrate that nucleosomes at the DNase I hypersensitive sites of the LCR could be either depleted or retained depending on the stages of development. Furthermore, MNase sensitivity and 3C assays suggest that the LCR chromatin is more open in embryonic erythroblasts than in definitive erythroblasts at the primary- and secondary-structure levels; however, the LCR chromatin is packaged more tightly in embryonic erythroblasts than in definitive erythroblasts at the tertiary chromatin level. Our study provides the first evidence that the occupancy of nucleosomes at a DNase I hypersensitive site is a developmental stage-related event and that embryonic and adult cells possess distinct chromatin structures of the LCR.