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PubMed1992

Multiple gap junction genes are utilized during rat skin and hair development.

The expression of four different gap junction gene products (alpha 1, beta 1, beta 2, and beta 3) has been analysed during rat skin development and the hair growth cycle. Both alpha 1 (Cx43) and beta 2 (Cx26) connexins were coexpressed in the undifferentiated epidermis. A specific, developmentally regulated elimination of beta 2 expression was observed in the periderm at E16. Coinciding with the differentiation of the epidermis, differential expression of alpha 1 and beta 2 connexins was observed in the newly formed epidermal layers. alpha 1 connexin was expressed in the basal and spinous layers, while beta 2 was confined to the differentiated spinous and granular layers. Large gap junctions were present in the basal layer, while small gap junctions, associated with many desmosomes, were typical for the differentiated layers. Although the distribution pattern for alpha 1 and beta 2 expression remained the same in the neonatal and postnatal epidermis, the RNA and protein levels decreased markedly following birth. Hair follicle development was marked by expression of alpha 1 connexin in hair germs at E16. Following beta 2 detection at E20, the expression increased for both alpha 1 and beta 2 in developing follicles. A cell-type-specific expression was detected in the outer root sheath, in the matrix, in the matrix-derived cells (inner root sheath, cortex and medulla) and in the dermal papilla. In addition, alpha 1 was specifically expressed in the arrector pili muscle, while sebocytes expressed both alpha 1 and beta 3 (Cx31) connexin. beta 1 connexin (Cx32) was not detected at any stage analysed. The results indicate that multiple gap junction genes contribute to epidermal and follicular morphogenesis. Moreover, based on the utilization of gap junctions in all living cells of the surface epidermis, it appears that the epidermis may behave as a large communication compartment that may be coupled functionally to epidermal appendages (hair follicles and sebaceous glands) via gap junctional pathways.

Animals↗
PubMed2004

Macrophages in the small intestinal muscularis externa of embryos, newborn and adult germ-free mice.

Previously, we demonstrated the presence of a constant and regularly distributed macrophage population of ramified cells in the intestinal muscle layers of smaller rodents. The function of these resident macrophages under normal conditions remains unknown. Histochemistry, immunohistochemistry and electron microscopy were applied to the muscularis externa of 15- and 17-day-old embryos, 2-day-old mice, adult germ-free and conventional mice. Since lipopolysaccharides (LPS) activates macrophages and inflammation affects gut motility, LPS-treated mice were also included in the study. Two macrophage antibodies, F4/80 and 2F8 were used to demonstrate the presence of macrophages in the muscle layers. The localization was confirmed by electron microscopy. In contrast to conventional adult mice, the muscle layers in embryos, newborn and germ-free adult mice were devoid of class II MHC antigen reactive cells. The acid phosphatase reaction and antibodies directed towards a lysosomal protein (Lamp-2) were used in order to verify other activation markers. None of these showed specific staining of the muscularis macrophages. Only LPS-treated adult mice showed iNOS-positive cells in whole mounts. We conclude that the characteristic organization and distribution of muscularis macrophages in adult mice are also present in embryos, newborn and germ-free mice and thus develop independently of foreign antigens. Further, these macrophages are truly resident and appear to have differential responses to exogene stimuli.

Animals↗
PubMed1986

Gastric inhibitory polypeptide receptor in hamster pancreatic beta cells. Direct cross-linking, solubilization and characterization as a glycoprotein.

125I-labelled gastric inhibitory polypeptide (125I-GIP) is directly cross-linked to its specific receptor in hamster pancreatic beta cell membranes by using an ultraviolet irradiation procedure. This approach results in the identification of a GIP-protein complex of apparent Mr 64,000. The labelling of this protein species is specific since it is inhibited when incubating the membranes with increasing doses of native GIP (0.1 nM-1 microM) together with 125I-GIP, half-maximal inhibition being elicited by 5 nM peptide. Reduction of the GIP-protein complex by 100 mM dithiothreitol induces a decrease of the electrophoretic mobility of the complex. Alternatively pretreatment of membranes with dithiothreitol (up to 1 M) does not prevent the binding of 125I-GIP to its receptor. When prelabelled membranes are extracted by 0.5% Triton X-100 (v/v) and the extract is layered on a Sephadex G-50 column, a high peak of radioactivity is eluted with the void volume of the column. Treatment of this peak by 10 min ultraviolet irradiation followed by SDS-PAGE leads to identification of a major band of Mr 64,000. When the peak is further layered on Sephacryl S-200 it yields a single peak of radioactivity corresponding to a protein species with a Stokes radius of 3.2 nm and an apparent Mr of 65,000. The solubilized GIP-receptor complex is specifically adsorbed by Sepharose coupled to wheat germ agglutinin and concanavalin A and eluted from these lectins by their respective sugars. In conclusion the GIP receptor in pancreatic beta cells is a protein monomer of apparent Mr 59 000; its structure is maintained by intrachain disulfide bridges, these bonds being, however, not involved in the interaction of GIP with its receptor; the GIP receptor is a glycoprotein containing N-acetylglucosamine, mannose and probably sialic acid in its carbohydrate moiety.

Adsorption↗
PubMed1990

Lectin-binding sites in the retina of the pig, baboon and cat.

There are notable interspecific differences in the lectin binding sites in the retina of mammals. In the porcine retina a distinct labeling of all the neurons of the stratum ganglionare is obtained by use of succinylated wheat-germ-agglutinin (WGA-S). In the baboon the stratum ganglionare does not label, though the entire inner plexiform layer can be demonstrated with WGA-S. In the cat WGA-S binds exclusively to the outer segments of the rods and cones. These specific differences will be compared with findings in the human retina [10] and discussed in connection with biochemical results obtained during our studies of the porcine retina.

Animals↗
PubMed1993

[Embryology of vessels].

Endothelial emergence is the best known aspect of vessel formation during embryogenesis. It has been analyzed in an avian model of chimeras at the time of organogenesis or morphogenesis. These chimeras involve two species, chick and quail, whose cells may be distinguished on the basis of distinct nuclear heterochromatin patterns or through the use of antibodies that are species and lineage specific. QH1, a monoclonal antibody obtained in our group, whose affinity is restricted to the quail hemangioblastic lineage (endothelial and hemopoietic cells), has been a sensitive probe to study the origin of these cells in various chimeric patterns. By transplanting organ rudiments or primordial germ layers, we have shown that endothelium emergence in organ rudiments occurs through two different mechanisms, vasculogenesis or in situ differentiation, and angiogenesis or colonization by extrinsic precursors. Vasculogenesis occurs in the mesoderm of internal organ rudiments while angiogenesis occurs in external rudiments. The conclusion is that associated endoderm exerts a positive influence on the emergence of endothelial progenitors from mesodermal precursors.

Animals↗
PubMed1980

Partial purification and characterization of oestrogen receptors in subfractions of hepatocyte plasma membranes.

To assess the subcellular distribution of oestrogen-binding components in their native state, plasma membrane and other cell fractions were prepared from hepatocytes in the absence of [(3)H]oestradiol-17beta. Cells from livers of ovariectomized rats were disrupted, with submaximal homogenization in buffered isotonic sucrose with CaCl(2) and proteinase inhibitor, and fractionated by using isotonic media. Fractions were characterized by determinations of enzyme activities, biochemical constituents and ligand binding. Specific binding of 2nm-[(3)H]oestradiol-17beta to intact cells and their fractions was detemined after equilibration for 1.5h at 4 degrees C. More than 92% of the radioactivity from representative preparations was verified as authentic oestradiol by thin-layer chromatography. Activities of plasma-membrane marker enzymes as well as binding sites for oestrogen and for wheat germ agglutinin were present principally in particulate fractions, rather than in 105000g-supernatant fractions. However, by using alternative homogenization procedures (i.e. hypotonic media), known to fragment and strip structural components, oestradiol-binding sites and activities of plasma-membrane marker enzymes were distributed predominantly into cytosol. By using the more conservative procedures, plasma membranes of low (rho=1.13-1.16) and high (rho=1.16-1.18) density were purified from crude nuclear fractions. A second low-density subfraction of plasma membrane was prepared from microsome-rich fractions. Activities of plasma-membrane marker enzymes were enriched to about 28 and four times that of the homogenate in plasma membranes of low and high density respectively. Binding sites for wheat germ agglutinin and oestradiol were concentrated in low-density plasma membranes to 46-63 times that of the homogenate. Specific binding of oestrogen in low-density plasma membranes purified from crude nuclei was saturable, with an apparent association constant of 3.5nm. At saturation, such oestradiol receptors corresponded to 526fmol/mg of membrane protein. A Hill plot showed a moderate degree of positive co-operativity in the interaction of hormone with plasma membranes. Specific binding of [(3)H]oestradiol-17beta was reduced by a 200-fold molar excess of unlabelled oestradiol-17beta, oestriol or diethylstilbestrol, but not by oestradiol-17alpha, cortisol, testosterone or progesterone. Binding was also blocked by prior exposure of membranes to trypsin or to 60 degrees C, but remained essentially undiminished by extraction of membranes with either hypotonic or high-salt buffers. Extraction with 0.1% (v/v) Triton X-100 partially solubilized the oestrogen-binding component(s) of plasma membranes. Particle-free extracts were resolved on 5-20% (w/v) sucrose density gradients with either 0.01m- or 0.4m-KCl, and the fractions were analysed by adsorption to hydroxyapatite. In low-salt gradients macromolecule-bound oestrogen sedimented at predominantly 7.4S and binding was 1560 times that of the homogenate. Under high-salt conditions oestradiol-binding activity occurred at both 3.6S and 4.9S.

Animals↗
PubMed2003

No tail co-operates with non-canonical Wnt signaling to regulate posterior body morphogenesis in zebrafish.

The vertebrate posterior body is formed by a combination of the gastrulation movements that shape the head and anterior trunk and posterior specific cell behaviors. Here, we investigated whether genes that regulate cell movements during gastrulation [no tail (ntl)/brachyury, knypek (kny) and pipetail (ppt)/wnt5] interact to regulate posterior body morphogenesis. Both kny;ntl and ppt;ntl double mutant embryos exhibit synergistic trunk and tail shortening by early segmentation. Gene expression analysis in the compound mutants indicates that anteroposterior germ-layer patterning is largely normal and that the tail elongation defects are not due to failure to specify or maintain posterior tissues. Moreover, ntl interacts with ppt and kny to synergistically regulate the posterior expression of the gene encoding bone morphogenetic protein 4 (bmp4) but not of other known T-box genes, fibroblast growth factor genes or caudal genes. Examination of mitotic and apoptotic cells indicates that impaired tail elongation is not simply due to decreased cell proliferation or increased cell death. Cell tracing in ppt;ntl and kny;ntl mutants demonstrates that the ventral derived posterior tailbud progenitors move into the tailbud. However, gastrulation-like convergence and extension movements and cell movements within the posterior tailbud are impaired. Furthermore, subduction movements of cells into the mesendoderm are reduced in kny;ntl and ppt;ntl mutants. We propose that Ntl and the non-canonical Wnt pathway components Ppt and Kny function in parallel, partially redundant pathways to regulate posterior body development. Our work initiates the genetic dissection of posterior body morphogenesis and links genes to specific tail-forming movements. Moreover, we provide genetic evidence for the notion that tail development entails a continuation of mechanisms regulating gastrulation together with mechanisms unique to the posterior body.

Animals↗
PubMed1998

Binding specificities of lectins to immobilized glycoproteins and oligosaccharides differ from those of immobilized lectins to oligosaccharides.

The carbohydrate-binding specificities of lectins in solution to glycoproteins and neoglycolipids immobilized on a solid phase were analyzed in order to establish a simple, rapid method for structural analysis of the carbohydrate moieties of small amounts of individual glycoproteins blotted on membrane. Eight glycoproteins containing typical O-linked tetrasaccharides or a series of typical N-linked oligosaccharides of the high-man-nose type, hybrid type, and complex type and 6 neoglycoproteins containing mono- or di-saccharides were dot blotted on membranes and the membranes were then reacted with 8 kinds of horseradish peroxidase-conjugated lectins before and after heat treatment. Neoglycolipids containing the glycoprotein-derived oligosaccharides immobilized on a thin layer chromatography plate were also reacted with lectins. The heat treatment of the membrane increased lectin reactivity toward the glycoproteins. The carbohydrate-binding behavior of lectins, Phaseolus vulgaris erythroagglutinin, wheat germ agglutinin, and concanavalin A in solution toward glycoproteins and neoglycolipids immobilized on a solid phase differed from that of immobilized lectins toward oligosaccharides in solution. This difference should be noted in lectin detection of specific carbohydrates of individual glycoproteins on membrane.

Glycoproteins↗
PubMed1998

Chromatin structure and functional analysis of the mouse HNF3alpha gene.

The transcription factor HNF3alpha is a member of the winged-helix family of regulatory proteins. It is expressed in the definitive endoderm, notochord, and neural tube in embryos, but in the adult is expressed primarily in endoderm-derived tissues such as liver, lung, and pancreas. We present here the cloning of the mouse HNF3alpha gene and a characterization of its chromatin structure and regulatory sequences. The HNF3alpha gene is encoded by two exons and its transcription initiates at multiple start sites at a TATA-less promoter that is highly conserved between mouse and rat. We found different patterns of DNaseI hypersensitive sites in HNF3alpha gene chromatin in different adult tissues in which HNF3alpha is expressed, suggesting distinct regulatory mechanisms occurring within different tissue derivatives of the endoderm germ layer. Cell transfection data indicate that sequences spanning certain upstream hypersensitive sites can enhance transcription from the HNF3alpha promoter, but only when stably integrated into chromatin and not when transiently transfected. The results suggest a complex regulatory interplay between distinct genetic regulatory sequences that function specifically in chromatin.

Animals↗
PubMed1993

Implications of dynamic patterns of Delta and Notch expression for cellular interactions during Drosophila development.

Delta and Notch function are required for cell fate specification in numerous tissues during embryonic and postembryonic Drosophila development. Delta is expressed by all members of interacting cell populations within which fates are being specified and is subsequently down-regulated as cells stably adopt particular fates. Multiphasic expression in the derivatives of many germ layers implies successive requirements for Delta function in a number of tissues. At the cellular level, Delta and Notch expression are generally coincident within developing tissues. At the subcellular level, Delta and Notch are localized in apparent endocytic vesicles during down-regulation from the surfaces of interacting cells, implying an interaction consistent with their proposed roles as signal and receptor in cellular interactions during development.

Animals↗
PubMed1976

In vitro development of secondary blastodiscs from dispersed blastoderm cells of Gallus domesticus.

A study of the in vitro growth of embryonic structures from dispersed blastoderm cells is reported. The specific type of blastoderm cells with the capability of growing on the coverslip developed into the discoidal embryonic structures resembling those of avian species. The glass surface was apparently an initiator of differentiation into at least three types of cells specifically distributed in the blastodisc. Groups of structures formed were evaluated at 6, 12, 24 and 36 hours to study the developmental pattern in vitro and to estimate the number of cells per structure. Microscopic examination of the area pellucida revealed that all three basic germ layers were established after 24 hours of incubation in vitro. The 36 hour stage was represented by bulky growth of mesodermal-like cells and changes in hypoblast layer where some of the cells degenerated and some were transformed to mesenchymal spindle-like cells.

Animals↗
PubMed1998

Expression of the small heat shock protein HSP 27 in developing human skin.

The 27 kDa heat shock protein (HSP 27) is expressed in keratinocytes of the upper epidermal layers, and recent evidence suggests that this protein is involved in the regulation of epidermal differentiation. The expression of HSP 27 was investigated in developing human skin by immunohistochemistry utilizing a specific monoclonal antibody. We used formalin-fixed, paraffin-embedded tissue of abdominal skin obtained from 34 human fetuses ranging between 13 and 30 weeks estimated gestational age (EGA). We found that HSP 27 is not expressed in keratinocytes until week 14 EGA. At this stage staining is observed in the periderm and the upper intermediate cells but not in hair germs. During further development, HSP 27 expression correlates with increasing epidermal differentiation, i.e. shedding of the periderm and beginning of keratinization. HSP 27 expression is confined to the upper cell layers and sparse basal cells. In hair follicles, HSP 27 can be detected in the innermost cell layer of the outer root sheath and in keratinocytes of the bulge identical to what is observed in adult skin. The hair papilla, matrix cells and sebaceous glands are negative for HSP 27 and remain so during further development. In eccrine sweat glands of the 24th week EGA, HSP 27 is confined to the superficial cell layer of the sweat ducts. In the present report we demonstrate differentiation-related expression of HSP 27 in developing human skin. Further in vitro studies will address the molecular function of HSP 27 in epidermal differentiation and development.

Eccrine Glands↗
PubMed2006

Adult pancreatic stem/progenitor cells spontaneously differentiate in vitro into multiple cell lineages and form teratoma-like structures.

Cells isolated from pancreas have a remarkable potential for self-renewal and multilineage differentiation. We here present a comprehensive characterisation of stem/progenitor cells derived from exocrine parts of the adult rat pancreas. Using purified cells from either single colonies or even single-cell clones, we specifically demonstrate: (i) the cells contain the typical stem/progenitor cell markers alkaline phophatase, SSEA-1, Oct-4, CD9, Nestin, Pax6, CD44, a-Fetoprotein and Brachyury, demonstrated by immunocytochemistry and RT-PCR; (ii) the cells have the potential to differentiate into lineages of all three germ layers in vitro; (iii) a clonal analysis revealed that even cell lines derived from a single cell have stem/progenitor cell properties such as self-renewal and spontaneous differentiation into various cell lineages; (iv) the cells have the propensity to form three-dimensional, teratoma-like structures in vitro, which contain cells of different lineages; and (v) external stimuli can activate the generation of certain cell types. For instance, cells treated with retinoic acid show an increased expression of alpha-smooth muscle actin. These results suggest that exocrine glands, such as pancreas may be a potential source of adult stem/progenitor cells, suitable for cell therapy of degenerative diseases.

Actins↗
PubMed2004

Non-canonical Wnt signals are modulated by the Kaiso transcriptional repressor and p120-catenin.

Gastrulation movements are critical for establishing the three principal germ layers and the basic architecture of vertebrate embryos. Although the individual molecules and pathways involved are not clearly understood, non-canonical Wnt signals are known to participate in developmental processes, including planar cell polarity and directed cell rearrangements. Here we demonstrate that the dual-specificity transcriptional repressor Kaiso, first identified in association with p120-catenin, is required for Xenopus gastrulation movements. In addition, depletion of xKaiso results in increased expression of the non-canonical xWnt11, which contributes to the xKaiso knockdown phenotype as it is significantly rescued by dominant-negative Wnt11. We further demonstrate that xWnt11 is a direct gene target of xKaiso and that p120-catenin association relieves xKaiso repression in vivo. Our results indicate that p120-catenin and Kaiso are essential components of a new developmental gene regulatory pathway that controls vertebrate morphogenesis.

Animals↗
PubMed2002

Differentiation of pluripotent embryonic stem cells into cardiomyocytes.

Embryonic stem (ES) cells have been established as permanent lines of undifferentiated pluripotent cells from early mouse embryos. ES cells provide a unique system for the genetic manipulation and the creation of knockout strains of mice through gene targeting. By cultivation in vitro as 3D aggregates called embryoid bodies, ES cells can differentiate into derivatives of all 3 primary germ layers, including cardiomyocytes. Protocols for the in vitro differentiation of ES cells into cardiomyocytes representing all specialized cell types of the heart, such as atrial-like, ventricular-like, sinus nodal-like, and Purkinje-like cells, have been established. During differentiation, cardiac-specific genes as well as proteins, receptors, and ion channels are expressed in a developmental continuum, which closely recapitulates the developmental pattern of early cardiogenesis. Exploitation of ES cell-derived cardiomyocytes has facilitated the analysis of early cardiac development and has permitted in vitro "gain-of-function" or "loss-of-function" genetic studies. Recently, human ES cell lines have been established that can be used to investigate cardiac development and the function of human heart cells and to determine the basic strategies of regenerative cell therapy. This review summarizes the current state of ES cell-derived cardiogenesis and provides an overview of how genomic strategies coupled with this in vitro differentiation system can be applied to cardiac research.

Animals↗
PubMed2005

Microarray analysis of LIF/Stat3 transcriptional targets in embryonic stem cells.

Mouse embryonic stem (ES) cells can be propagated in vitro while retaining their properties of pluripotency and self-renewal under the continuous presence of leukemia inhibitor factor (LIF). An essential role has been attributed to subsequent activation of the Stat3 transcription factor in mediating LIF self-renewal response. To date, however, downstream target genes of Stat3 in ES cells are still unknown. To isolate these genes, we performed a microarray-based kinetic comparison of LIF-stimulated (undifferentiated) ES cells versus ES cells induced to differentiate by shutting down Stat3 activity through either LIF deprivation or, more specifically, expression of a Stat3 dominant-negative mutant. In each case, we chose the earliest time at which ES cells lose their self-renewal properties, as illustrated by a decrease in the number of embryoid bodies and blast cell colony formation as well as germ layer marker expression. Comparison of the two independent approaches revealed similarly regulated genes that are likely to be involved in the Stat3 effects on ES cell self-renewal. For instance, upregulation of growth factors such as the transforming growth factor-beta relative Lefty1 or transcriptional regulators such as Id1 and Id2 and down-regulation of the groucho-like protein Aes1 (grg5) were found. Promoter analysis of the aes1 gene revealed three functional Stat3 consensus sites, as shown by luciferase assays. Furthermore, chromatin immunoprecipitation experiment demonstrated that Stat3 is recruited to the promoter of aes1 in ES cells. These data demonstrated that the aes1 gene is a direct transcriptional target of Stat3 in ES cells.

Animals↗
PubMed2000

An analysis of the teratagenic effects that could possibly be due to alcohol consumption by pregnant mothers.

It can be concluded that alcohol is definitely harmful to the developing fetus. The effect can manifest in various ways, the most extreme of which is a condition called Fetal Alcohol Syndrome (FAS). The diagnosis of maternal alcoholism leading onto cases of FAS is difficult due to absence of accurate diagnostic tests. The diagnosis of FAS in a child is easier by a proper examination. There is no specific treatment of FAS in a child. The only management is by institution of corrective and rehabilitative measures. The exact mechanism of the teratogenic action of alcohol is not known. It is probably due to the harmful effect of alcohol on the epiblast layer of the bilaminar germ disc. In the absence of adequate knowledge regarding FAS, not much can be done to remedy the deleterious effects of alcohol. Hence, a word of advice to all pregnant women is to avoid drinking during pregnancy.

Alcohol Drinking↗
PubMed2005

Plant lectins as carriers for oral drugs: is wheat germ agglutinin a suitable candidate?

Wheat germ agglutinin (WGA) is a plant protein that binds specifically to sugars expressed also by gastrointestinal epithelial cells. WGA is currently investigated as an anti-tumor drug and as a carrier for oral drugs. Information on whether it can cross the gastrointestinal epithelium and on its possible effects on the integrity of the epithelial layer is however scanty or lacking, and herein we address these issues. Differentiated Caco2 cells have been used as a model of polarized intestinal epithelium. WGA concentration at both the apical and the basolateral side of the epithelium has been quantified using a sensitive ELISA assay (sensitivity threshold 0.84 nM). Trans epithelial electrical resistance (TEER) has been measured to evaluate the integrity of the epithelium upon treatments with WGA. (3)H-Mannitol (182.2 Da) and FITC-dextran (3000 Da) have been used to measure the permeability of the epithelium. Cell viability has been measured by the MTT, by 7-AAD uptake, and Annexin-V binding assays. Up to a concentration of 5.6 microM, approximately 0.1% of intact WGA molecules only could cross the epithelial layer. WGA perturbed the integrity of the epithelium and increased the permeability of the tissue in a dose- and time-dependent manner. WGA did not induce cell death but increased the permeability of individual cells to 7-AAD which is normally not uptaken by viable cells. These data allowed us to define a toxicity threshold for WGA on epithelial cells. WGA suitability as a carrier for oral drugs can therefore be evaluated on a rational basis.

Caco-2 Cells↗