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PubMed1983

Localization of axonally transported 125I-wheat germ agglutinin beneath the plasma membrane of chick retinal ganglion cells.

The distribution of 125I-wheat germ agglutinin (WGA) transported by axons of chick retinal ganglion cells to layer d of the optic tectum was studied by electron microscopic autoradiography. We found that 52% of the radioactivity was located in axons and axon terminals in the contralateral optic tectum 22 h after intravitreal injection of affinity-purified 125I-WGA. Axons comprised 43% of the volume of layer d. Dendrites, glial cells, and neuron cell bodies contained 20%, 17%, and 3% of the label, whereas these structures comprised 24%, 21%, and 2% of the tissue volume, respectively. We also measured the distances between the autoradiographic silver grains and the plasma membranes of these profiles, and compared observed distributions of grains to theoretical distributions computed for band-shaped sources at various distances from the plasma membranes. This analysis revealed that the radioactive source within axons was distributed in a band of cytoplasm extending in from the plasma membrane a distance of 63 nm. Because WGA is known to bind to specific membrane glycoconjugates, we infer that at least some glycoconjugates may be concentrated within an annular region of cytoplasm just beneath the axonal plasma membrane after axoplasmic transport from the neuron cell body.

Animals↗

PubMed2004

Afferent and efferent connections of the nucleus geniculatus lateralis ventralis demonstrated by WGA-HRP in the chick.

Fibre connections of the chick nucleus geniculatus lateralis ventralis (GLv) were investigated using the axonal tracing method with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). After an injection of WGA-HRP into the GLv, many labelled neurons were observed in layer i of the stratum griseum et fibrosum superficiale (SGFS) in the ipsilateral tectum opticum (TO) and in the nucleus lentiformis mesencephali (LM). In the TO-GLv projection, cells of origin were located in the deeper part of layer i of the TO and were topographically distributed along the direction from the rostrodorsal part to the caudoventral part of the TO relating to a rostrocaudal axis of the GLv. In the LM-GLv connection, the dorsal and ventral parts of the LM connected reciprocally with the rostral and caudal halves of the GLv, respectively. In contrast, in the GLv efferent connection, labelled axon terminals spread widely in the ipsilateral area pretectalis without any clear topographical arrangement.

Afferent Pathways↗

PubMed1979

Microbial interference and colonization of the murine gastrointestinal tract by Listeria monocytogenes.

Two strains of Listeria monocytogenes, one that formed smooth colonies on agar surfaces and a varient of it that formed rough colonies, colonized the gastrointestinal tracts of germfree mice. Within 24 h after mice were inoculated orally with about 100 bacteria, the population levels per gram (wet weight) of tissue of both strains were 10(5) to 10(7) in the stomach and ileum and 10(8) to 10(9) in the cecum and colon, respectively. As detected in Gram-stained histological sections, in such gnotobiotes, the bacteria colonized the lumen in all areas of the tract and much of the mucus layer on the epithelial surface in the proximal colon. The strain that formed smooth colonies did not colonize the tracts of specific-pathogen-free mice, but did colonize, to the same levels as in germfree mice, the stomachs and bowels of ex-germfree mice previously associated with two members of the indigenous flora (Bacteroides and Clostridium). In the latter animals, however, the listeria did not form layers on the colonic epithelium as efficiently as they did in monoassociated gnotobiotes.

Animals↗

PubMed1980

The vascular pattern in the papillary region of rat incisor and molar tooth enamel organ.

Scanning electron microscopy of the enamel organ of rat incisor and molar teeth in the maturation stage of amelogenesis revealed two vascularization patterns of the papillary layer. In one pattern, the anastomosing capillaries formed loops of varying sizes around spherical or somehwat oblong papillae. In the second pattern, the capillaries were parallel to each other embedded in furrows between long ridges of papillary cells. It is postulated that each of these two patterns may be associated with a specific stage in the process of enamel maturation.

Animals↗

PubMed1992

Intraepithelial lymphocytes and Langerhans cells in the oral mucosa--dynamic aspects.

In the oral epithelium a constant population of non-epithelial immunocompetent cells--lymphocytes and Langerhans cells exists. 3025 such cells of the murine and 542 of the normal human oral mucosa were documented photographically at the ultrastructural level. Location within the epithelium, morphological signs of locomotion, membrane configuration and labeling of the intercellular substance by ruthenium red were registered. About 40 per cent of the lymphocytes and 70 per cent of the Langerhans cells show signs of locomotion, predominantly in a lateral and basal direction. Occasional cells crossing the basal lamina are seen. Close apposition, or rarely a denticulated surface and lysis of desmosomes were present, both probably indications of cellular interactions. Ruthenium red labeling of the glycocalix is continuous forming a homogeneous layer between epithelial and non-epithelial cells and between intraepithelial cells, suggesting an even distribution of antigens and the lack of preformed spaces for intraepithelial cells. Murine and human intraepithelial cells exhibited no major differences.

Animals↗

PubMed1999

Gastrulation in zebrafish: what mutants teach us.

A major approach to the study of development is to compare the phenotypes of normal and mutant individuals for a given genetic locus. Understanding the development of a complex metazoan therefore requires examination of many mutants. Relatively few organisms are being studied this way, and zebrafish is currently the best example of a vertebrate for which large-scale mutagenesis screens have successfully been carried out. The number of genes mutated in zebrafish that have been cloned expands rapidly, bringing new insights into a number of developmental pathways operating in vertebrates. Here, we discuss work on zebrafish mutants affecting gastrulation and patterning of the early embryo. Gastrulation is orchestrated by the dorsal organizer, which forms in a region where maternally derived beta-catenin signaling is active. Mutation in the zygotic homeobox gene bozozok disrupts the organizer genetic program and leads to severe axial deficiencies, indicating that this gene is a functional target of beta-catenin signaling. Once established, the organizer releases inhibitors of ventralizing signals, such as BMPs, and promotes dorsoanterior fates within all germ layers. In zebrafish, several mutations affecting dorsal-ventral (D/V) patterning inactivate genes functioning in the BMP pathway, stressing the central role of this pathway in the gastrula embryo. Cells derived from the organizer differentiate into several axial structures, such as notochord and prechordal mesoderm, which are thought to induce various fates in adjacent tissues, such as the floor plate, after the completion of gastrulation. Studies with mutants in nodal-related genes, in one-eyed pinhead, which is required for nodal signaling, and in the Notch pathway reveal that midline cell fate specification is, in fact, initiated during gastrulation. Furthermore, the organizer coordinates morphogenetic movements, and zebrafish mutants in T-box mesoderm-specific genes help clarify the mechanism of convergence movements required for the formation of axial and paraxial mesoderm.

Animals↗

PubMed2006

Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs.

During embryogenesis, multipotent progenitors within the single-layered surface epithelium differentiate to form the epidermis and its appendages. Here, we show that microRNAs (miRNAs) have an essential role in orchestrating these events. We cloned more than 100 miRNAs from skin and show that epidermis and hair follicles differentially express discrete miRNA families. To explore the functional significance of this finding, we conditionally targeted Dicer1 gene ablation in embryonic skin progenitors. Within the first week after loss of miRNA expression, cell fate specification and differentiation were not markedly impaired, and in the interfollicular epidermis, apoptosis was not markedly increased. Notably, however, developing hair germs evaginate rather than invaginate, thereby perturbing the epidermal organization. Here we characterize miRNAs in skin, the existence of which was hitherto unappreciated, and demonstrate their differential expression and importance in the morphogenesis of epithelial tissues within this vital organ.

Animals↗

PubMed1997

Differentiation of embryonic stem cells into adipocytes in vitro.

Embryonic stem cells, derived from the inner cell mass of murine blastocysts, can be maintained in a totipotent state in vitro. In appropriate conditions embryonic stem cells have been shown to differentiate in vitro into various derivatives of all three primary germ layers. We describe in this paper conditions to induce differentiation of embryonic stem cells reliably and at high efficiency into adipocytes. A prerequisite is to treat early developing embryonic stem cell-derived embryoid bodies with retinoic acid for a precise period of time. Retinoic acid could not be substituted by adipogenic hormones nor by potent activators of peroxisome proliferator-activated receptors. Treatment with retinoic acid resulted in the subsequent appearance of large clusters of mature adipocytes in embryoid body outgrowths. Lipogenic and lipolytic activities as well as high level expression of adipocyte specific genes could be detected in these cultures. Analysis of expression of potential adipogenic genes, such as peroxisome proliferator-activated receptors gamma and delta and CCAAT/enhancer binding protein beta, during differentiation of retinoic acid-treated embryoid bodies has been performed. The temporal pattern of expression of genes encoding these nuclear factors resembled that found during mouse embryogenesis. The differentiation of embryonic stem cells into adipocytes will provide an invaluable model for the characterisation of the role of genes expressed during the adipocyte development programme and for the identification of new adipogenic regulatory genes.

Adipocytes↗

PubMed2003

Independent projection streams from macaque striate cortex to the second visual area and middle temporal area.

The interareal wiring of the neocortex is usually depicted as a network of single point-to-point connections, often side-stepping the possibility that some neurons may project to multiple cortical areas. The prevalence of such neurons is unknown; if they are abundant, cortical circuits are more likely to be connectionally diffuse. We used a dual-tracer approach to determine whether single neurons in the macaque primary visual cortex (V1) project to two extrastriate areas, the second visual area (V2) and the middle temporal area (MT). We found two large intermingled groups of single-labeled neurons in layer 4B of V1 projecting independently to either V2 or MT. A third, sparser group of double-labeled neurons projected to both areas; we termed these manifold neurons. We also found that MT-projecting cells were distributed indiscriminately with respect to cytochrome oxidase compartment in layer 4B, revealing a subpopulation that provides a potential source of patch input from V1 to MT. The results demonstrate that primary sensory cortices can use multiple projection strategies to distribute signals to higher areas, and suggest that feedforward projections may route signals with more specificity than feedback pathways.

Animals↗

PubMed2003

Nonhuman primate parthenogenetic stem cells.

Parthenogenesis is the biological phenomenon by which embryonic development is initiated without male contribution. Whereas parthenogenesis is a common mode of reproduction in lower organisms, the mammalian parthenote fails to produce a successful pregnancy. We herein describe in vitro parthenogenetic development of monkey (Macaca fascicularis) eggs to the blastocyst stage, and their use to create a pluripotent line of stem cells. These monkey stem cells (Cyno-1 cells) are positive for telomerase activity and are immunoreactive for alkaline phosphatase, octamer-binding transcription factor 4 (Oct-4), stage-specific embryonic antigen 4 (SSEA-4), tumor rejection antigen 1-60 (TRA 1-60), and tumor rejection antigen 1-81 (TRA 1-81) (traditional markers of human embryonic stem cells). They have a normal chromosome karyotype (40 + 2) and can be maintained in vitro in an undifferentiated state for extended periods of time. Cyno-1 cells can be differentiated in vitro into dopaminergic and serotonergic neurons, contractile cardiomyocyte-like cells, smooth muscle, ciliated epithelia, and adipocytes. When Cyno-1 cells were injected into severe combined immunodeficient mice, teratomas with derivatives from all three embryonic germ layers were obtained. When grown on fibronectin/laminin-coated plates and in neural progenitor medium, Cyno-1 cells assume a neural precursor phenotype (immunoreactive for nestin). However, these cells remain proliferative and express no functional ion channels. When transferred to differentiation conditions, the nestin-positive precursors assume neuronal and epithelial morphologies. Over time, these cells acquire electrophysiological characteristics of functional neurons (appearance of tetrodotoxin-sensitive, voltage-dependent sodium channels). These results suggest that stem cells derived from the parthenogenetically activated nonhuman primate egg provide a potential source for autologous cell therapy in the female and bypass the need for creating a competent embryo.

Animals↗

PubMed1989

Expression of cell adhesion molecule E-cadherin in Xenopus embryos begins at gastrulation and predominates in the ectoderm.

The expression of the Ca2+-dependent epithelial cell adhesion molecule E-cadherin (also known as uvomorulin and L-CAM) in the early stages of embryonic development of Xenopus laevis was examined. E-Cadherin was identified in the Xenopus A6 epithelial cell line by antibody cross-reactivity and several biochemical characteristics. Four independent mAbs were generated against purified Xenopus E-cadherin. All four mAbs recognized the same polypeptides in A6 cells, adult epithelial tissues, and embryos. These mAbs inhibited the formation of cell contacts between A6 cells and stained the basolateral plasma membranes of A6 cells, hepatocytes, and alveolar epithelial cells. The time of E-cadherin expression in early Xenopus embryos was determined by immunoblotting. Unlike its expression in early mouse embryos, E-cadherin was not present in the eggs or early blastula of Xenopus laevis. These findings indicate that a different Ca2+-dependent cell adhesion molecule, perhaps another member of the cadherin gene family, is responsible for the Ca2+-dependent adhesion between cleavage stage Xenopus blastomeres. Detectable accumulation of E-cadherin started just before gastrulation at stage 9 1/2 and increased rapidly up to the end of gastrulation at stage 15. In stage 15 embryos, specific immunofluorescence staining of E-cadherin was discernible only in ectoderm, but not in mesoderm and endoderm. The ectoderm at this stage consists of two cell layers. The outer cell layer of ectoderm was stained intensely, and staining was localized to the basolateral plasma membrane of these cells. Lower levels of staining were observed in the inner cell layer of ectoderm. The coincidence of E-cadherin expression with the process of gastrulation and its restriction to the ectoderm indicate that it may play a role in the morphogenetic movements of gastrulation and resulting segregation of embryonic germ layers.

Age Factors↗

PubMed1994

Androgen receptor distribution in rat testis: new implications for androgen regulation of spermatogenesis.

The distribution of the androgen receptor (AR) in the adult rat testis was determined by biotin-streptavidin immunoperoxidase, employing tissue embedded in polyester wax which preserves antigenicity without compromising tissue preservation. The antibody probe used, which has been characterized previously, was an affinity purified, rabbit polyclonal antibody raised to the amino terminus peptide of the rat AR. Within the interstitial compartment, AR immunostaining was detected in some Leydig cells and all smooth muscle cells forming the walls of blood vessels, but endothelial cells of blood vessels were negative. Furthermore, in those Leydig cells that were clearly identified as exhibiting AR immunostaining, the intensity of the reaction varied. In the seminiferous tubules AR immunostaining was observed in all peritubular myoid cell nuclei, but not in the distal layer of lymphatic endothelial cells. In Sertoli cells, nuclear AR immunostaining was stage specific. Moderate AR immunostaining first became evident at late stage IV or early stage V of the cycle, reached a robust peak at stages VII-VIII, and then disappeared completely. Specific AR immunostaining was also discerned in the nuclei of stage XI elongated spermatids, the spermatids in which nuclear elongation is apparent but chromatin condensation has not yet begun. Next, with onset of chromatin condensation, nuclear AR immunostaining in elongated spermatids was not discerned concomitant with its detection in the cytoplasm of the germ cells. These results are interpreted in the following manner: 1) The presence of AR in Leydig cells is consistent with the hypothesis that androgens modify Leydig cell activity in an autocrine fashion. Further, that not all Leydig cells exhibited AR immunostaining at steady state suggests a differential, functional activity of these cells within the population. 2) The intense AR immunostaining of smooth muscle cells present in the interstitium indicates that these cells are targets for androgens. 3) AR immunoreactivity in both Sertoli and peritubular myoid cells suggests their involvement in the androgenic control of spermatogenesis. The stage specific AR immunoreactivity in Sertoli cells, however, may be more indicative of a specific androgen response during these stages, whereas peritubular cells may participate in the tonal maintenance of spermatogenesis. 4) The specific presence of AR in step 11 elongated spermatids may suggest that androgens can act directly on germ cells to regulate spermatogenesis.

Androgens↗

PubMed1998

Amelogenin protein in tooth germs of the snake Elaphe quadrivirgata, immunohistochemistry, cloning and cDNA sequence.

In the snake, Elaphe quadrivirgata, the occurrence of amelogenin was immunohistochemically demonstrated in the enamel of developing tooth germs. Teeth of the snake are covered with a thin true enamel layer about 1-2 microns in thickness. Light and electron microscopic immunohistochemistry indicated an intense amelogenin immunoreactivity occurring in the enamel layer during the secretory stage of tooth development. Cloning and cDNA sequence of snake amelogenin was performed by RT-PCR. The amino acid sequence of the snake amelogenin cDNA--in its portion corresponding to the area from exon 5 to exon 7 of human X189 amelogenin gene--showed 45% homology with humans. Regions of both the N-terminus and C-terminus were well conserved. Furthermore, the positions of prolin in the amino acid alignment of the snake amelogenin corresponded well with those of human amelogenin. It is suggested that prolin is an essential constituent of amelogenin and therefore its positions in the molecule have been conserved after the evolutionary divergence of reptiles and mammals. This study using reptiles is the first detection of specific amelogenin immunoreactivity by high resolutional immunoelectron microscopy and the first cloning of amelogenin cDNA in a non-mammalian animal.

Amelogenin↗

PubMed1986

The projection of frontal cortical oculomotor areas to the superior colliculus in the domestic cat.

The projection of frontal cortical oculomotor areas to separate laminae of the superior colliculus was examined in adult domestic cats. Following the placement of WGA-HRP/HRP injections within the superficial, intermediate, and deep layers of the superior colliculus, several observations may be made regarding the frontal corticotectal projection in the cat. Large injections including all of the collicular layers result in the retrograde labeling of neurons in frontal cortical areas that have been suggested previously to be analogous to the frontal eye fields in monkeys. These areas are a portion of the ventral bank and lip of the cruciate sulcus, including a small portion of the medial wall of the hemisphere, and the medial and lateral banks and fundus of the presylvian sulcus. The same pattern of retrograde labeling in frontal cortical areas is seen when the injection sites are restricted to either the intermediate or deep collicular layers. That is, different frontal cortical areas of the cat project separately but not differentially upon different collicular laminae. The greatest numbers of labeled neurons within frontal cortical oculomotor areas are observed following injections placed laterally within the caudal portion of the superior colliculus, regardless of whether the injection sites are located within the intermediate or deep collicular layers. Injections restricted to the superficial gray layer do not result in retrograde labeling in any of the frontal cortical oculomotor areas of the cat. These results are discussed in terms of the patterns of visuomotor integration specific to the cat, as well as in relation to previous evidence showing similarities between frontal cortical areas in cats and monkeys.

Animals↗

PubMed1997

Blockade of afferent impulse activity disrupts on/off sublamination in the ferret lateral geniculate nucleus.

The ferret retinogeniculate projection is organized into on/off sublaminae within each eye-specific layer. The role of afferent activity in the formation of these sublaminae was examined. We report that when the activity of retinal ganglion cells is blocked with tetrodotoxin during postnatal weeks three and four, on/off sublamination is disrupted, supporting a role for afferent activity in the formation of this pattern.

Afferent Pathways↗

PubMed2001

Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells.

The resetting of a somatic epigenotype to a totipotential state has been demonstrated by successful animal cloning, via transplantation of somatic nuclei into enucleated oocytes. We have established an experimental system, which reproduces the nuclear reprogramming of somatic cells in vitro by fusing adult thymocytes with embryonic stem (ES) cells. Analysis of the lymphoid-cell-specific V-(D)-J DNA rearrangement of the T cell receptor and immunoglobin genes shows that the ES cells have hybridized with differentiated cells. In these ES cell hybrids, the inactivated X chromosome derived from a female thymocyte adopts some characteristics of an active X chromosome, including early replication timing and unstable Xist transcription. We also found that an Oct4-GFP transgene, which is normally repressed in thymocytes, is reactivated 48 hr after cell fusion. The pluripotency of the ES-thymocyte hybrid cells is shown in vivo, since they contribute to all three primary germ layers of chimeric embryos. The somatic DNA methylation pattern of the imprinted H19 and Igf2r genes is maintained in these hybrids, unlike hybrids between ES and EG (embryonic germ) cells in which the differential methylation is erased. Thus, ES cells have the capacity to reset certain aspects of the epigenotype of somatic cells to those of ES cells.

Animals↗

PubMed1986

Wheat germ agglutinin binding sites in the adult mouse cerebellum: light and electron microscopic studies.

The binding properties of derivatized wheat germ agglutinin (WGA) have been examined in fixed tissue sections from the adult mouse cerebellum and also in axonal tracing paradigms following cerebellar injections. The aim of these studies is to begin to distinguish the roles different binding sites may play in generating diverse biological activities which lead to neuronal uptake and axonal transport of lectins or glycoconjugates. Vibratome sections from aldehyde-fixed cerebellum were incubated in N-[acetyl-3H] WGA or WGA conjugated to horseradish peroxidase (WGA-HRP). Sections from this in vitro binding paradigm and those from cerebellar pressure injection cases using those tracers (in vivo binding paradigm) were processed for light microscopic autoradiography, histochemistry, and electron microscopy. Blocking experiments were also performed with various sugar haptens to confirm the binding specificity of these lectin preparations. Light microscopy of lectin binding patterns within the cerebellar cortex has revealed that both derivatized WGA preparations bind most intensely to the molecular layer. Within the deep cerebellar nuclei, binding is unique and produces a punctate delineation of cell bodies and dendrites. Electron microscopy revealed that these binding sites are associated with glial processes which abut the plasma membrane of deep nuclei cells. Cerebellar WGA-HRP injection sites contain labeled profiles involved in uptake and axonal transport of the labeled lectin (e.g., multivesicular and dense bodies) in addition to label associated with synapses, glia, undetermined components of the extracellular space, and neuronal plasma membranes. These sites are therefore presumed to possess a high affinity or capacity for binding derivatized WGA. Binding studies performed here thus reveal, for the first time, the existence of discrete glial sites that display an extraordinary attraction for lectins such as WGA. The roles such glial glycoconjugates play in diverse biological activities including neuronal uptake and transport of macromolecules need further study.

Animals↗

PubMed1985

Laminar organization of geniculocortical projections in Galago senegalensis and Aotus trivirgatus.

The projections of the lateral geniculate nucleus to striate cortex were traced by anterograde and retrograde transport of WGA-HRP in two primates, Galago and Aotus. The goal was to determine the laminar organization of the terminals of individual layers of the lateral geniculate nucleus. The results show that in both species the magnocellular layers project to cortical layer IV alpha, the parvicellular layers project to IV beta, and the intercalated geniculate layers (which term includes layers 4 and 5 in Galago) project to layers III and I. The distribution of terminals in layer III is periodic, which is to say, there are regularly spaced regions of terminals separated by regions devoid of terminals. When the two species are compared to Saimiri, it is clear that the basic organization of the three pathways relaying in the lateral geniculate nucleus is common to all three primates. At the same time, there are some significant species differences. The total pattern of projections in Galago is compressed in comparison to that in the monkey, and this compression seems to be related to the poorer differentiation of cortical layers in the prosimian. For example, layer IIIC is distinct only in the two monkeys and at the same time it is spared from geniculate terminations. Both Galago and Aotus differ from Saimiri in that they lack a projection from the parvicellular geniculate layers to the layer IIIB. These species comparisons are relevant to the questions of the functional significance of the three pathways and the evolution of the primate striate cortex.

Animals↗