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PubMed1994

Modulation of cell surface-associated mannoprotein antigen expression in experimental candidal vaginitis.

The monoclonal antibody (MAb) AF1 recognizes an oligosaccharide epitope present on highly immunogenic and immunomodulatory mannoproteins (MP) of Candida albicans. The expression of this epitope (AF1-MP) during experimental candidal vaginitis was studied in two strains of C. albicans (3153 and CA-2) which were equally vaginopathic but differed in the mode of hypha formation in the vagina. In both strains, immunofluorescence of vaginal samples, taken 1 h after challenge, revealed an intense, MAb AF1-specific labelling of the yeast cells. This labelling was very scarce in fungal cells taken at 24 h and on subsequent days during the development of filamentous forms. Electron-microscopic gold immunolabelling observations showed that molecules carrying AF1-MP spanned the entire cell wall in the initial yeast cells but were absent on the cell surface and in the outermost, capsular layer of the cell wall of the germ tubes and filamentous forms. In both strains, at any time and for any form of intravaginal growth, AF1-MP was clearly expressed in the cytoplasm and cytoplasmic vesicles, and was fully incorporated into the inner layers of the cell wall. As seen by immunofluorescence, the vaginal fluid from C. albicans-infected rats did not hinder the expression of AF1-MP on the yeast cells surface in vitro. In electron-microscopic gold immunolabelling, a hypha-specific MAb (3D9) labelled the surface of the hyphal but not of the yeast cells of C. albicans harvested from rat vagina. Overall, these data strongly suggest that cell surface expression of MP antigen is modulated during intravaginal growth and morphogenesis of C. albicans.

Animals↗
PubMed1989

Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice.

A construct containing approximately 2500 base pairs (bp) of 5' upstream and approximately 700 bp of 3' downstream sequence was used to drive the expression of an intronless human K14 gene in vitro and in vivo. To track the expression of the gene, a small sequence encoding the antigenic portion of neuropeptide substance P was inserted in frame 5' to the TGA translation stop codon of the gene. Surprisingly, this gene was expressed promiscuously in a wide variety of cultured cells transiently transfected with the construct. In contrast, when introduced into the germ line of transgenic mice, the construct was expressed in a fashion analogous to the endogenous K14 gene--namely, in the basal layer of stratified squamous epithelia. Our results suggest that some regulatory mechanism is overridden as a consequence of transient transfection but that sequences that can control proper K14 expression are present in the construct. The appropriate tissue-specific and differentiation-specific expression of K14.P in transgenic mice is an important first step in characterizing a promoter that could be employed to drive the foreign expression of drug-related genes in the epidermis of skin grafts.

Animals↗
PubMed1995

Molecular cloning of tyrosine kinases in the early Xenopus embryo: identification of Eck-related genes expressed in cranial neural crest cells of the second (hyoid) arch.

Growth factors and their receptors play an important role in controlling cellular proliferation, migration, and differentiation during vertebrate embryogenesis. We have used the reverse transcription-polymerase chain reaction to survey the repertoire of receptor tyrosine kinases (TK) expressed during early embryogenesis of Xenopus laevis. Twelve distinct Xenopus TK cDNA classes were identified among a total of 352 cDNAs screened. A single TK cDNA class has been described previously and encodes the fibroblast growth factor receptor FGFR-A1. The remaining 11 TK cDNA classes appear to encode novel genes of the FGFR, platelet-derived growth factor receptor (PDGFR), Eph, Csk, Tyk2, and Klg subfamilies. By RNase protection assays, Xenopus TK mRNAs are rare transcripts (< 10(7) mRNA molecules/embryo), and are usually found to be expressed also maternally in the embryo. Most Xenopus TK genes examined by whole-mount in situ hybridization were expressed widely in tissues derived from multiple germ layers. Two Eck-related genes, however, were found to be restricted in their expression to neural crest of the second (hyoid) arch. Our findings are consistent with the proposed function of TKs in the regulation of specification and differentiation of embryonic tissues.

Amino Acid Sequence↗
PubMed1997

Transcription factors and induction in Xenopus laevis embryos.

Studies with amphibian embryos have contributed major insights into the molecular basis of induction processes and the formation of germ layers during vertebrate embryogenesis. Primary signals that have been identified as growth factors or growth factor-related ligands act as inducing factors on their target cells and, by a change of the genetic program, evoke a specification of the cellular differentiation pathways. While at present the signal transduction mechanisms leading from the ligands via cognate receptors to the nuclei are still poorly understood, there is growing information on transcription factors which are activated upon induction. They govern the expression of other regulatory molecules and co-ordinate the expression of cell type-specific structural genes. Meanwhile, it is generally accepted that development and cellular differentiation in all multicellular organisms depends upon a cascade of evolutionarily conserved transcription factors. Striking structural similarities within their DNA-binding domains allow many of these factors to be subdivided into different transcription factor families. Most of the basic knowledge on these factors emerged from the pioneering work done with Drosophila embryos which was greatly facilitated by the availability of numerous mutants. Despite the fact that Drosophila development until the blastoderm stage proceeds in a multinuclear syncytium and thus is significantly different from that in vertebrate organisms, the primary structures of many embryonic transcription factors have been conserved in higher organisms. This especially holds true for the various DNA binding motifs and it facilitated the isolation and characterization of vertebrate homologues to factors previously identified in lower organisms.

Animals↗
PubMed1998

Testicular expression of the mouse histone H1.1 gene.

The replication-dependent H1 histone subtype H1.1 is the predominant H1 histone subtype in germ cell chromatin during early spermatogenesis. H1.1 gene transcription was assigned by in situ hybridization with H1.1-specific RNA probes to the basal layer of germ cells within the seminiferous tubule, but not to the somatic cells of the testis. In situ hybridization and immunohistochemistry with testis from 5-, 9-, and 20-day-old mice showed that H1.1 mRNA and protein was already present in spermatogonia of the prepuberal testis. In testis from 20-day-old and adult mice, the H1.1 mRNA remained confined to the most peripheral layer of germ cells. In contrast to the H1.1 mRNA, the H1.1 protein persisted in the germ cell chromatin with decreasing concentration throughout meiosis and in postmeiotic cells. These data demonstrate that in situ hybridization with H1.1 mRNA-specific probes can serve to identify germ cells with ongoing DNA replication. Comparison of results obtained from in situ hybridization and immunocytochemistry indicates that the period of H1.1 gene transcription is restricted to the proliferative phase in which cells undergo successive divisions, whereas the H1.1 protein is a component of the proliferative, meiotic, and spermiogenic phases.

Animals↗
PubMed1999

Pleiotropic features of syndromic craniosynostoses correlate with differential expression of fibroblast growth factor receptors 1 and 2 during human craniofacial development.

Mutations in FGFR1, -2, and -3 are linked to five human craniosynostosis syndromes. In addition to premature fusion of cranial sutures, nonskeletal manifestations in skin, and teeth together with CNS abnormalities, reflect widespread effects of these mutations. To understand this pleiotropy, we have assessed craniofacial FGFR1 and -2 expression in the human embryo from 6 wk postfertilization. We found that both genes are expressed in sheets of condensed mesenchyme before overt chondrogenic differentiation and that distinct patterns of expression are established by 8 wk. Thus, FGFR2(BEK) is expressed evenly throughout developing cartilage and bone, whereas FGFR1 transcripts predominate in perichondria and periostea. Complementary patterns of FGFR1 and FGFR2(BEK and KGFR) expression are also observed in the enamel epithelium and papilla mesenchyme of the tooth germ, at a stage when morphogenetic tissue interactions ensue. Both genes are expressed in the cortical layer of the brain, but expression levels vary significantly within the choroid plexus and wall of the fourth ventricle. Similarly, tissue-specific differences in receptor expression are found in both the skin and salivary glands. These expression data are consistent with the pleiotropic manifestations of syndromic craniosynostoses and provide the basis for a new paradigm to explain the associated CNS problems.

Case-Control Studies↗
PubMed2005

The intron 5/6 promoter region of the ship1 gene regulates expression in stem/progenitor cells of the mouse embryo.

The s-SHIP protein is a shorter isoform of the longer SHIP1 protein and lacks the N-terminal SH2 domain region contained in SHIP1. s-SHIP is expressed in ES cells and in enriched bone marrow stem cells, and may be controlled by a promoter within intron 5 of the ship1 gene. We therefore examined the potential specificity of promoter activity in ES cells of an intron 5/intron 6 ship1 genomic segment and its tissue specificity within transgenic mice expressing GFP from this promoter region. The results indicate that s-SHIP promoter activity is specific for ES cells in vitro and for known and presumptive stem/progenitor cells throughout embryo development of the transgenic mice. Specific GFP expression was observed in the blastocyst, primordial germ cells, thymus, arterioles, osteoblasts, and skin epidermis. The epidermis/epithelium is the progenitor for hair follicles, mammary tissue, and prostate. Interestingly, each of these latter tissues acquired a few GFP-positive cells in the course of their development from the epithelial layers, and these cells express marker proteins for stem/progenitor cells. These results identify potential stem cell populations, mark these cells for analyses in normal and cancer development, and implicate s-SHIP as an important protein in stem/progenitor cell function.

Animals↗
PubMed2006

Derivation and characterization of four new human embryonic stem cell lines: the Danish experience.

In September 2003, legislation approved in Denmark legalized work on surplus human embryos from IVF for clinical purposes to establish human embryonic stem (ES) cell cultures. The aim of this study was to establish such stem cell lines. Fresh surplus embryos were donated after informed consent from the donors. Embryos were cultured into blastocysts and using the immunosurgery procedure, inner cell masses were isolated and cultured on irradiated human foreskin fibroblasts in KnockOut D-MEM supplemented with KnockOut Serum Replacement, bFGF, and LIF. Within a period of 12 months, 198 embryos were donated. Four isolated inner cell masses developed into putative ES cell lines, CLS1, CLS2, CLS3, CLS4, which have now been continuously cultured for eight months, corresponding to 30 passages. These cells expressed markers for undifferentiated human ES cells: stage-specific embryonic antigen-4, tumour-related antigen (TRA)-1-60, TRA-1-81, OCT4, NANOG, SOX2, and FGF4. The cells expressed high levels of telomerase activity, had a normal karyotype, and have been successfully cryopreserved and thawed. Finally, the cells displayed the potential to differentiate in vitro into cell types originating from all three germ layers. It is thought that the cell lines described in this study are the first human ES cells established in Denmark.

Antigens, Surface↗
PubMed1988

An in vitro model of gonad differentiation in the chick embryo. Roller cultures in gas permeable biofoil bags.

Embryonic gonads of 6 1/2 to 12 days old chick embryos were enzymatically dissociated. The cell suspensions were cultured in small gas permeable bags of foil (Biofolie Heraeus) in a roller culture apparatus. The cells formed multiple small aggregates, in which sex specific differences developed within two days. In cell suspensions of embryonic testes smooth spheric aggregates formed with well delineated testicular cords in the center and a tunica albuginea-like mesenchymal layer at the outside. Most of the male germ cells were incorporated in the central cords. A number of germ cells were barred from entering the cords by the tunica albuginea-like mesenchymal layer and populated the outer surface of the aggregates. The aggregates of left ovary were irregular in shape and characterized by clusters of germ cells residing in an outer cortical zone. The aggregates of the right ovary, which regresses in vivo, showed poor growth and did not differentiate, thus, indicating that the suppression of right ovary was not removed in culture. In the roller cultures of dissociated embryonic gonads male and female morphogenesis was mimicked in a reproducible manner, so that the system can be used for further experimental studies of gonadal development.

Animals↗
PubMed1999

High frequency of tissue-specific mosaicism in Turner syndrome patients.

Interphase fluorescent studies of X chromosome aneuploidy in cultured and uncultured blood lymphocytes and oral mucosa epithelial cells using X centromere-specific DNA probe in addition to standard karyotype analysis were performed in 50 females with a clinical suspicion of Turner syndrome. All the patients were previously screened for the presence of 'hidden' Y chromosome mosaicism, using the primers DYZ3 and DYZ. The use of fluorescence in situ hybridization (FISH) analysis of interphase nuclei of tissues from different germ layers (lymphocytes from mesoderm and buccal epithelial cells from ectoderm) improves the accuracy of detection of low-level mosaicism. FISH studies on interphase nuclei revealed that 29% of patients with a pure form of monosomy X detected by metaphase analysis are, in fact, mosaics. The level of cells with the normal chromosomal constitution in lymphocytes of these cases as a rule was low, ranging from 3 to 18%, with an average of 7%. Two false-positive cases and one false-negative case of X monosomy mosaicism determined by standard cytogenetic approach were detected using FISH analysis. The majority of patients (92%) with mosaic form of Turner syndrome have considerable tissue-specific differences in levels of X aneuploidy. Our data indicate that in cases when mosaic aneuploidy with low-level frequency is questionable (approximately 10% and lower), the results of standard metaphase analysis should be supplemented with additional FISH studies of interphase nuclei. Tissue-specific differences in contents of different cell lines in the same patients point to the necessity of studying more than one tissue from each patient.

Adolescent↗
PubMed1998

Opposite phenotypes of hypomorphic and Y766 phosphorylation site mutations reveal a function for Fgfr1 in anteroposterior patterning of mouse embryos.

Intercellular communication is needed for both the generation of the mesodermal germ layer and its division into distinct subpopulations. To dissect the functions of fibroblast growth factor receptor-1 (FGFR1) during mouse gastrulation as well as to gain insights into its possible roles during later embryonic development, we have introduced specific mutations into the Fgfr1 locus by gene targeting. Our results show functional dominance of one of the receptor isoforms and suggest a function for the autophosphorylation of site Y766 in the negative regulation of FGFR1 activity. Y766F and hypomorphic mutations in Fgfr1 generate opposite phenotypes in terms of homeotic vertebral transformations, suggesting a role for FGFR1 in patterning the embryonic anteriorposterior axis by way of regulation of Hox gene activity.

Alleles↗
PubMed1987

Expression of Xenopus N-CAM RNA in ectoderm is an early response to neural induction.

We have isolated Xenopus laevis N-CAM cDNA clones and used these to study the expression of N-CAM RNA during neural induction. The results show that the first marked increase in N-CAM RNA levels occurs during gastrulation when mesoderm comes in contact with ectoderm and induces neural development. In situ hybridization results show that the early expression of N-CAM RNA is localized to the neural plate and its later expression is confined to the neural tube. Induction experiments with explanted germ layers show that N-CAM RNA is not expressed in ectoderm unless there is contact with inducing tissue. Together these results suggest an approach to studying how ectoderm is committed to form neural rather than epidermal tissue. Specifically, the data suggest that neural commitment is marked and perhaps mediated by the transcriptional activation of genes, like N-CAM, in the neural ectoderm.

Animals↗
PubMed1980

[Staining of cysts of Entamoeba invadens, Entamoeba histolytica and Entamoeba coli with wheat germ agglutinin labelled with colloidal gold].

Cysts of Entamoeba invadens obtained under axenic conditions were stained with wheat germ agglutinin labelled with colloidal gold. Microscopic observation of encysting cultures revealed that upon staining, cysts acquired a red coloration, while trophozoites remained unstained. E. histolytica and E. coli cysts obtained from asymptomatic patients were also stained red by this technique. Electronmicroscopic examination of stained cells showed a layer of colloidal gold granules attached to the surface of cysts, while trophozoites remained free of gold granules. The results of the staining procedure developed, agreed with the known facts that wheat germ agglutinin is a lectin that binds specifically to chitin and its oligomers, and that chitin is present in the cyst wall of E. invadens. The results suggested that chitin is also present in the wall of cysts of E. histolytica and E. coli.

Entamoeba↗
PubMed2000

Conservation of the WD-repeat, microtubule-binding protein, EMAP, in sea urchins, humans, and the nematode C. elegans.

The echinoderm microtubule-associated protein (EMAP) is the most abundant microtubule-binding protein in the first cleavage mitotic apparatus in sea urchin embryos. The first goal of this study was to determine whether there is sufficient EMAP in the egg and embryo to modify microtubule dynamics during the early cleavages divisions and whether EMAP functions at a specific time or place in the embryo. To accomplish this goal, we examined the relative abundance, tissue distribution, and temporal pattern of EMAP expression during embryonic development. The second goal of this study was to identify important functional domains within the EMAP coding sequence. A conserved sequence might reveal a potential microtubule-binding domain. We cloned, sequenced and compared overlapping EMAP cDNAs from two different sea urchin species that diverged approximately 80 million years ago, and compared these with cDNA sequences from a vertebrate and nematode species. From quantitative immunoblots, we determined the EMAP concentration in eggs to be 4 microM. The steady-state levels of EMAP mRNA and protein accumulated during development, and all three germ layers expressed EMAP. During the early stages of development, EMAP and tubulin were both abundant in the ectoderm, mesoderm and endoderm. However, during late gastrulation and the formation of the early pluteus larvae, EMAP was enriched in the mesoderm, while tubulin staining was most abundant in the archenteron. These results indicate that EMAP may have tissue-specific functions in the late stage embryo. To identify conserved functional domains, we compared the predicted amino acid sequence encoded by Strongylocentrotus purpuratus and Lytechinus variegatus EMAP cDNAs, and determined that these two sea urchin EMAPs were 95% conserved and shared an identical domain organization. A parsimonious analysis of these sea urchin protein sequences, as well as human and C. elegans EMAP sequences was used to construct a gene tree. Together these results suggest that EMAP is an important microtubule protein required at all developmental stages of sea urchins, and whose cellular function may be conserved amongst metazoans.

Amino Acid Sequence↗
PubMed1997

Simultaneous anterograde labeling of axonal layers from lateral superior olive and dorsal cochlear nucleus in the inferior colliculus of cat.

The laminar organization of the central nucleus of inferior colliculus includes layers of axons that may be important in shaping the responses of neurons. Depending on their source, some layered axons are afferents that are superimposed and terminate on the same postsynaptic neurons, while other layered afferents, such as those from the ipsilateral and contralateral lateral superior olive, terminate side-by-side. The specific pattern of convergence may dictate which populations of axons are presynaptic to layered disc-shaped neurons in the central nucleus. We compared the distribution of afferent axons from the dorsal cochlear nucleus and the lateral superior olive to the contralateral inferior colliculus in the cat. Injection sites in cochlear nucleus and superior olive were physiologically characterized by extracellular recordings of single and multiple units in response to monaural and binaural acoustic stimulation. Two separate injections were made in each case, and both injection sites contained units with overlapping best frequencies. Biotinylated dextran, fluorescent dextran, 3H-leucine, and wheat germ agglutinin conjugated to horseradish peroxidase were used as anterograde tracers. The present results show that layered axons from the dorsal cochlear nucleus and lateral superior olive are superimposed in part of the contralateral central nucleus. Both projections were arranged in rostro-caudally oriented axonal layers that converged in the ventral part of the central nucleus. However, in the dorsal part of the central nucleus, the same layer of axons from the dorsal cochlear nucleus did not terminate with afferents from the lateral superior olive. Within the overlapping layers in the ventral central nucleus, the overlap of axons from the dorsal cochlear nucleus and the lateral superior olive was uniform except for small patches that were usually smaller than the dendritic fields of disc-shaped neurons. These data suggest that the layers may create specific functional zones in the central nucleus of the inferior colliculus. One zone may contain neurons with binaural responses that combine the properties of the inputs from the contralateral lateral superior olive and the dorsal cochlear nucleus. A second zone may contain inputs from the cochlear nucleus but lack those of the lateral superior olive.

Afferent Pathways↗
PubMed2005

Mouse ES cell lines show a variable degree of chondrogenic differentiation in vitro.

Pluripotent mouse embryonic stem (ES) cells differentiate in vitro spontaneously into cell types of all three primary germ layers when cultivated as cell aggregates, so-called 'embryoid bodies'. Many reports have shown that this system recapitulates cellular developmental processes and gene expression patterns of early embryogenesis. During ES cell differentiation, efficient and directed differentiation into a specific cell type is influenced by many parameters, for example, the batch of the serum used or the application of growth factors and signalling molecules. Because all ES cell lines are considered to be pluripotent, one should not expect remarkable differences regarding their spontaneous differentiation efficiencies. However, here we show that different ES cell lines exhibit a variable degree of spontaneous chondrogenic differentiation indicating that lines with a specific differentiation capacity could be selected. This is an important aspect if ES cells are applied for tissue regeneration.

Alcian Blue↗
PubMed1997

Mechanism of infertility in male guinea pigs immunized with sperm PH-20.

PH-20, a testis-specific protein first expressed in haploid germ cells, is present on the posterior head plasma membrane and inner acrosomal membrane of mature guinea pig sperm. PH-20 is bifunctional, having a hyaluronidase activity that allows sperm to penetrate the cumulus layer and a separate activity required for binding of acrosome-reacted sperm to the zona pellucida. The immunization of male guinea pigs with PH-20 reproducibly results in infertility with a duration of 6-12 mo or longer. In this study, we analyzed the immunopathology in the reproductive tract of PH-20-immunized males to probe the mechanism(s) responsible for the induced infertility and found two separate effects. Remarkably, in almost all infertile, PH-20-immunized males, the caudae epididymides were empty (contained no sperm) or contained only abnormal sperm. The complete loss of normal sperm in the epididymis apparently results in infertility. A second effect was the induction of experimental autoimmune orchitis (EAO), representing the first report of EAO induced by a purified testis/sperm molecule of known functions. PH-20-induced EAO differed from EAO induced by crude testis antigens in two respects: 1) an absence of epididymitis with abscess and granuloma and 2) the presence of antibody on germ cells within seminiferous tubules and inside the cauda epididymidis. The former suggests that crude testis antigens other than PH-20 are responsible for epididymitis, and the latter suggests a possible role of antibody in EAO pathogenesis and infertility induction. Return to fertility, after 6-12 mo, was accompanied by regression of EAO and reappearance of spermatozoa in the caudae epididymides.

Animals↗
PubMed2002

Thalamic relay nuclei of the basal ganglia form both reciprocal and nonreciprocal cortical connections, linking multiple frontal cortical areas.

Thalamic relay nuclei transmit basal ganglia output to the frontal cortex, forming the last link in corticobasal ganglia circuitry. The thalamus regulates cortical activity through differential laminar connections, providing not only feedback, but also initiating "feedforward" loops, via nonreciprocal projections, that influence higher cortical areas. This study examines the organization of thalamic connections with cortex from basal ganglia relay nuclei, including ventral anterior (VA), ventral lateral (VL), and mediodorsal (MD) nuclei, in the Macaque monkey. Anterograde and bidirectional tracer injections ([3H]-amino acids, dextran conjugates of Fluorescein, Lucifer Yellow or FluoroRuby, or wheat germ agglutinin) into discrete VA/VL, MD, and frontal cortical sites demonstrate specific thalamocortical connections. VL projections target caudal motor areas (primary, supplementary, and caudal premotor areas), whereas VA projections target more rostral premotor areas (including cingulate and presupplementary motor areas) and MD projects to dorsolateral and orbital prefrontal cortices. Thalamocortical projections innervate cortical layers I and III, and to a lesser extent, layer V. In motor areas layer I projections are more extensive than those to layer III (and V). The complex laminar organization of projections from specific thalamic sites suggests differential regulation of cortical function. Injections of bidirectional tracers into thalamic and frontal cortical sites also show that in comparison to thalamocortical projections, corticothalamic projections to VA-VL and MD are more widespread. These findings demonstrate both reciprocal and nonreciprocal components to the thalamo-cortico-thalamic relay. Together, these experiments indicate a dual role for VA-VL and MD nuclei: (1) to relay basal ganglia output within specific cortical circuits and (2) to mediate information flow between cortical circuits.

Amino Acids↗