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PubMed1985

Immunohistochemical studies with a monoclonal antibody on the distribution of phosphophoryn in predentin and dentin.

A monoclonal antibody was raised against phosphophoryn, a unique noncollagenous phosphoprotein in dentin. Mouse myeloma NS-I cells were fused with spleen cells obtained from BALB/c mice immunized with phosphophoryn from fetal calf tooth germs. Mice inoculated with the hybridoma produced ascites fluid containing the antibody and this reacted only with a band of phosphophoryn transblotted from polyacrylamide gel. Immunohistochemical studies with the antibody showed that phosphophoryn was present in odontoblasts, odontoblastic processes and dentin, but not in the matrix of predentin, and that the phosphophoryn content of the dentin layer was high at and around the predentin-dentin junction and gradually decreased toward the enamel layer. The area corresponding to mantle dentin was not stained with the antibody.

Animals↗
PubMed2006

Epigenetic conversion of human adult bone mesodermal stromal cells into neuroectodermal cell types for replacement therapy of neurodegenerative disorders.

Tissue-specific stem cells, such as bone marrow-derived mesodermal stromal cells (MSCs), are thought to be lineage restricted and, therefore, could only be differentiated into cell types of the tissue of origin. Several recent studies, however, suggest that these types of stem cells might be able to break barriers of germ layer commitment and differentiate in vitro and/or in vivo into cells of different tissues, such as neuroectodermal cell types. Recently, protocols for high-yield generation of undifferentiated neural stem cell (NSC)-like cells from MSCs of primate and human origin were reported. Undifferentiated NSCs are commonly used and are more suitable for neurotransplantation compared with fully differentiated neural cells, as differentiated neural cells are well known to poorly survive detachment and subsequent transplantation procedures. These human MSC-derived NSC-like cells (MSC-NSCs) grow in neurosphere-like structures and express high levels of early neuroectodermal markers, but lose characteristics of MSCs. In the presence of selected growth factors, human MSC-NSCs can be differentiated into the three main neural phenotypes: astroglia, oligodendroglia and neurons. Compared with direct differentiation of human MSCs into mature neural cells, the conversion step seems to be essential to generate mature functional neuroectodermal cells. This review describes the techniques for the conversion of human MSCs into NSCs and summarises the data on epigenetic conversion of human MSCs into immature neuroectodermal cells. These cells provide a powerful tool for investigating the molecular mechanisms of neural differentiation, and might serve as an autologous cell source to treat acute and chronic neurodegenerative diseases.

Adult↗
PubMed1991

The response of female urogenital tract epithelia to mesenchymal inductors is restricted by the germ layer origin of the epithelium: prostatic inductions.

The epithelium of the mammalian vagina arises from two distinct germ layers, endoderm from the urogenital sinus and mesoderm from the Müllerian ducts. While neonatal vaginal epithelium can be induced to form prostate which is normally an endodermal derivative, it has not been determined whether this ability to form prostate is shared by both mesoderm- and endoderm-derived vaginal epithelia. To test the competence of vaginal epithelia we have isolated sinus-derived and Müllerian-derived vaginal epithelia from newborn mice, combined them with rat urogenital sinus mesenchyme, and grown the tissue recombinants for 4 weeks in male athymic nude mice. Endoderm-derived sinus vaginal epithelium was induced to form prostatic tissue which expressed prostate-specific secretory proteins in 21 of 23 tissue recombinants. Müllerian-derived vaginal epithelium formed small ducts and cysts lined by a simple epithelium. These latter tissue recombinants lacked any evidence of prostatic secretory proteins. Similarly, endoderm-derived urethral epithelium was induced to form prostate (17 of 17 cases), while mesoderm-derived uterine epithelium was not (0 of 13 cases). Therefore, the ability to form prostatic epithelium was limited to endodermal derivatives of the urogenital tract.

Androgen-Binding Protein↗
PubMed2002

Production of germline chimeras by transfer of chicken gonadal primordial germ cells maintained in vitro for an extended period.

We previously reported that germline chimeras could be produced by transfer of chicken gonadal primordial germ cells (gPGCs) cultured for a short term (5 days). This study was subsequently undertaken to examine whether gPGCs maintained in vitro for an extended period could retain their specific characteristics to induce germline transmission. Chicken (White Leghorn, WL) gPGCs were retrieved from embryos at stage 28 (5.5 days of incubation) and continuously cultured for 2 months in modified Dulbecco's minimal essential medium without subpassage and changing of the feeder cell layer. After the identification of gPGC characteristics using Periodic acid-Shiff's (PAS) reaction and anti stage-specific embryonic antigen-1 (SSEA-1) antibody staining at the end of the culture, cultured gPGCs were injected into the dorsal aorta of Korean Ogol Chicken (KOC) recipient embryos at stage 17 (2.5 days of incubation). Nineteen chickens (13 males and 6 females) were hatched, grown to sexual maturity, and subsequently subjected to testcross analysis employing artificial insemination with adult KOC. Of these, four (three males and one female) hatched chickens with white coat color. The percentage of germline chimerism was 21% (4/19). The results of this study demonstrated that gPGCs could maintain their specific characteristics for up to 2 months in vitro, resulting in the birth of germline chimeras following transfer to recipient embryos.

Animals↗
PubMed2000

Cells remain competent to respond to mesoderm-inducing signals present during gastrulation in Xenopus laevis.

During gastrulation, the vertebrate embryo is patterned and shaped by complex signaling pathways and morphogenetic movements. One of the first regions defined during gastrulation is the prospective notochord, which exhibits specific cell behaviors that drive the extension of the embryonic axis. To examine the signals involved in notochord formation in Xenopus laevis and the competence of cells to respond to these signals, we performed cell transplantation experiments during gastrulation. Labeled cells from the prospective notochord, somitic mesoderm, ventrolateral mesoderm, neural ectoderm, and epidermis, between stages 9 (pregastrulation) and 12 (late gastrulation), were grafted into the prospective notochord region of the early gastrula. We show that cells from each region are competent to respond to notochord-inducing signals and differentiate into notochordal tissue. Cells from the prospective neural ectoderm are the most responsive to notochord-inducing signals, whereas cells from the ventrolateral and epidermal regions are the least responsive. We show that at the end of gastrulation, while transplanted cells lose their competence to form notochord, they remain competent to form somites. These results demonstrate that at the end of gastrulation cell fates are not restricted within germ layers. To determine whether notochord-inducing signals are present throughout gastrulation, grafts were made into progressively older host embryos. We found that regardless of the age of the host, grafted cells from each region give rise to notochordal tissue. This indicates that notochord-inducing signals are present throughout gastrulation and that these signals overlap with somite-inducing signals at the end of gastrulation. We conclude that it is the change of competence that restricts cells to specific tissues rather than the regulation of the inducing signals.

Animals↗
PubMed1983

[Ultrastructure of intercellular bridges of male germ cells].

The morphology of intercellular bridges between spermatogonia, spermatocytes and spermatids has been examined by electron microscopy on different species and on man. Bridges between spermatocytes and spermatids exhibit species specificity with regard to size, structure and density of the electron dense bridge substance. In each species, intercellular bridges differentiate from the basal compartment to the adluminal compartment. This is seen in structural changes within a layer of dense substance in the subplasmalammal layer. Common to all species are structures such as membranous systems, microtubules or midbodies, associated with the bridges. There is evidence that intercellular bridges have organelle character.

Animals↗
PubMed1985

An anterograde HRP-WGA study of aberrant corticorubral projections following neonatal lesions of the rat sensorimotor cortex.

Anterograde transport of horseradish peroxidase - wheat germ agglutinin (HRP-WGA) was used to examine the effect of unilateral neonatal ablation of the sensorimotor cortex on the remaining corticofugal projections to the midbrain in the rat. In unlesioned animals, the sensorimotor cortical efferents to the midbrain were entirely ipsilateral, terminal labeling being evident in the red nucleus, the midbrain reticular formation, the periaqueductal gray, the intermediate gray layer of the superior colliculus, the nucleus parafascicularis prerubralis and the perilemniscal area. Corticorubral fibers were seen to reach the midbrain through the thalamus or the cerebral peduncle. In the red nucleus, terminal labeling was essentially restricted to the parvocellular region. In neonatally lesioned adults, aberrant corticofugal fibers crossed the midline to terminate in the contralateral red nucleus, the midbrain reticular formation, the periaqueductal gray, the nucleus parafascicularis prerubralis and the intermediate gray layer of the superior colliculus. The aberrant projections maintained the topographic specificity of the normal ipsilateral projections. This was most evident in the corticorubral projection, where the aberrant contralateral fibers terminated in the parvocellular area of the red nucleus.

Aging↗
PubMed1999

Vertebrate endoderm development.

Endoderm, one of the three principal germ layers, contributes to all organs of the alimentary tract. For simplicity, this review divides formation of endodermal organs into four fundamental steps: (a) formation of endoderm during gastrulation, (b) morphogenesis of a gut tube from a sheet of cells, (c) budding of organ domains from the tube, and (d) differentiation of organ-specific cell types within the growing buds. We discuss possible mechanisms that regulate how undifferentiated endoderm becomes specified into a myriad of cell types that populate the respiratory and gastrointestinal tracts.

Animals↗
PubMed2005

Prefrontal cortex in the rat: projections to subcortical autonomic, motor, and limbic centers.

This paper describes the quantitative areal and laminar distribution of identified neuron populations projecting from areas of prefrontal cortex (PFC) to subcortical autonomic, motor, and limbic sites in the rat. Injections of the retrograde pathway tracer wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) were made into dorsal/ventral striatum (DS/VS), basolateral amygdala (BLA), mediodorsal thalamus (MD), lateral hypothalamus (LH), mediolateral septum, dorsolateral periaqueductal gray, dorsal raphe, ventral tegmental area, parabrachial nucleus, nucleus tractus solitarius, rostral/caudal ventrolateral medulla, or thoracic spinal cord (SC). High-resolution flat-map density distributions of retrogradely labelled neurons indicated that specific PFC regions were differentially involved in the projections studied, with medial (m)PFC divided into dorsal and ventral sectors. The percentages that WGA-HRP retrogradely labelled neurons composed of the projection neurons in individual layers of infralimbic (IL; area 25) prelimbic (PL; area 32), and dorsal anterior cingulate (ACd; area 24b) cortices were calculated. Among layer 5 pyramidal cells, approximately 27.4% in IL/PL/ACd cortices projected to LH, 22.9% in IL/ventral PL to VS, 18.3% in ACd/dorsal PL to DS, and 8.1% in areas IL/PL to BLA; and 37% of layer 6 pyramidal cells in IL/PL/ACd projected to MD. Data for other projection pathways are given. Multiple dual retrograde fluorescent tracing studies indicated that moderate populations (<9%) of layer 5 mPFC neurons projected to LH/VS, LH/SC, or VS/BLA. The data provide new quantitative information concerning the density and distribution of neurons involved in identified projection pathways from defined areas of the rat PFC to specific subcortical targets involved in dynamic goal-directed behavior.

Animals↗
PubMed2004

Ectoplasmic specialization, a testis-specific cell-cell actin-based adherens junction type: is this a potential target for male contraceptive development?

The seminiferous tubule of the mammalian testis is largely composed of Sertoli and germ cells, which coordinate with Leydig cells in the interstitium and perform two major physiological functions, namely spermatogenesis and steroidogenesis respectively. Each tubule is morphologically divided into (i) the seminiferous epithelium composing Sertoli and germ cells, and (ii) the basement membrane (a modified form of extracellular matrix); underneath this lies the collagen fibril network, the myoid cell layer, and the lymphatic vessel, which collectively constitute the tunica propia. In the seminiferous epithelium, of rodent testes each type A1 spermatogonium (diploid, 2n) differentiates into 256 elongated spermatids (haploid, 1n) during spermatogenesis. Additionally, developing germ cells must migrate progressively from the basal to the luminal edge of the adluminal compartment so that fully developed spermatids can be released into the lumen at spermiation. Without this timely event of cell movement, spermatogenesis cannot reach completion and infertility will result. Yet developing round elongating/elongated spermatids must remain attached to the epithelium via a specialized Sertoli-germ cell actin-based adherens junction (AJ) type known as ectoplasmic specialization (ES), which is crucial not only for cell attachment but also for spermatid movement and orientation in the epithelium. However, the biochemical composition and molecular architecture of the protein complexes that constitute the ES have only recently been studied. Furthermore, the signalling pathways that regulate ES dynamics are virtually unknown. This review highlights recent advances in these two areas of research. It is expected that, if adequately expanded, these studies should yield new insights into the development of novel contraceptives targeted to perturb ES function in the testis. The potential to specifically target the ES may also mean that contraceptive action could be achieved without perturbing the hypothalamic-pituitary-testicular axis.

Actins↗
PubMed1995

Local projections of GABAergic neurons in the dentate gyrus and CA1 region in the rat hippocampal formation.

Using retrograde transport of wheat germ agglutinin conjugated colloidal gold (WGA-gold) combined with immunoreactivity for glutamate decarboxylase (GAD), a specific synthesizing enzyme for gamma-aminobutyric acid (GABA), local projections of GABAergic neurons in the dentate gyrus and CA1 were examined. In the hilus of the dentate gyrus, it was found that GABAergic neurons in the granule cell layer projected to the ipsilateral upper leaf of the molecular layer, with a mediolateral extension of more than 1.2 mm and a rostrocaudal extension of over 0.8 mm. Non-GABAergic neurons in nearly the entire hilar area were found to project to the ipsilateral upper leaf of the molecular layer. In the dorsal CA1 region, GABAergic neurons in the stratum pyramidale and radiatum converged onto the ipsilateral stratum pyramidal/oriens, with a mediolateral extension of over 1 mm and a rostrocaudal extension of over 0.7 mm. These results provide direct evidence that in both the dentate gyrus and CA1, GABAergic interneurons from a fairly large field converge onto a very small target area. This suggests that the output signals from GABAergic neurons in the dentate gyrus and CA1, and non-GABAergic neurons in the dentate gyrus, may propagate beyond the anatomical limits contained in conventional slice preparations of the hippocampal formation.

Animals↗
PubMed1989

Intron and upstream sequences regulate expression of the Drosophila beta 3-tubulin gene in the visceral and somatic musculature, respectively.

The morphogenetic programs involved in the differentiation of internal organs, such as the muscle system, during Drosophila embryogenesis have remained largely obscure. beta 3-tubulin has proved to be a good marker for mesoderm development as this tubulin isotype is detectable soon after mesoderm formation and during the process of mesoderm differentiation. The beta 3-tubulin gene is expressed in the somatic and pharyngeal musculature, the dorsal vessel, and the visceral musculature. To learn more about the programs underlying mesodermal differentiation, we have started to dissect the regulatory elements of the beta 3-tubulin gene by means of P-element-mediated transformation experiments. We show that expression of the beta 3-tubulin gene in the somatic muscles, the pharyngeal muscles, and the dorsal vessel is mediated by far upstream sequences. We also demonstrate that the first intron of the beta 3-tubulin gene bears a tissue-specific enhancer element that is required for expression in the visceral muscles and that also functions efficiently when cloned downstream of an indicator gene. The separability of elements driving beta 3-tubulin expression in the somatic and visceral mesoderm facilitates the investigation of the different programs involved in regulating the early differentiation of this germ layer.

Animals↗
PubMed1997

A specific cell surface antigen of Streptococcus gordonii is associated with bacterial hemagglutination and adhesion to alpha2-3-linked sialic acid-containing receptors.

A Ca2+-independent lectin activity for alpha2-3-linked sialic acid-containing receptors is associated with Streptococcus gordonii DL1 (Challis) but not with a spontaneous mutant, strain D102, that specifically lacks hemagglutinating activity. Comparison of crossed-immunoelectrophoresis patterns of parent and mutant sonicated cell extracts identified a unique antigen (Hs antigen) in the parent cell extract that was purified by DEAE Sephacel column chromatography and by a wheat germ agglutinin (WGA) lectin affinity column. The purified antigen formed a single arc in crossed immunoelectrophoresis with anti-DL1 serum and migrated as a diffuse band above the 200-kDa marker in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoelectron microscopy with specific anti-Hs antibody revealed labeling of structures in the fibrillar layer of strain DL1 and no labeling of fibrillar structures on strain D102. Rabbit anti-DL1 serum and anti-Hs Fab inhibited the hemagglutinating activity of strain DL1, and the inhibition was specifically neutralized by purified Hs antigen. Anti-Hs Fab did not inhibit the hemagglutinating activities of several heterologous S. gordonii strains; however, these bacteria were agglutinated by anti-Hs immunoglobulin G and also by WGA. In contrast, two S. gordonii strains that lacked hemagglutinating activity did not react with anti-Hs antibody or with WGA. These findings associate the sialic acid-binding lectin activity of S. gordonii DL1 with a specific fibrillar antigen, which is composed of protein and WGA reactive carbohydrate, and indicate that cross-reactive antigens occur on other strains of this species that possess hemagglutinating activity.

Animals↗
PubMed1993

Characterization of the unusually rapid cell cycles during rat gastrulation.

The onset of gastrulation in rodents is associated with the start of differentiation within the embryo proper and a dramatic increase in the rate of growth and proliferation. We have determined the duration of the cell cycle for mesodermal and ectodermal cells of rat embryos during gastrulation (days 8.5 to 9.5 of gestation) using a stathmokinetic analysis. These embryonic cells are the most rapidly dividing mammalian cells yet described. Most cells of the ectoderm and mesoderm had a cell cycle time of 7 to 7.5 hours, but the cells of the primitive streak divided every 3 to 3.5 hours. Total cell cycle time was reduced by shortening S and G2, as well as G1, in contrast to cells later in development, when cell cycle duration is modulated largely by varying the length of G1. In the ectoderm and mesoderm, G1 was 1.5 to 2 hours, S was 3.5 to 4 hours, and G2 was 30 to 40 minutes. G1, S and G2 were shortened even further in the cells of the primitive streak: G1 was less than 30 minutes, S was 2 to 2.75 hours, and G2 was less than 20 minutes. Thus, progress of cells through all phases of the cell cycle is extensively modified during rodent embryogenesis. Specifically, the increased growth rate during gastrulation is associated with radical changes in cell cycle structure and duration. Further, the commitment of cells to become mesoderm and endoderm by entering the primitive streak is associated with expression of a very short cell cycle during transit of the primitive streak, such that developmental decisions determining germ layer fate are reflected in differences in cell cycle regulation.

Animals↗
PubMed2006

Concise review: embryonic stem cells: a new tool to study osteoblast and osteoclast differentiation.

Bone remodeling involves synthesis of organic matrix by osteoblasts and bone resorption by osteoclasts. A tight collaboration between these two cell types is essential to maintain a physiological bone homeostasis. Thus, osteoblasts control bone-resorbing activities and are also involved in osteoclast differentiation. Any disturbance between these effectors leads to the development of skeletal abnormalities and/or bone diseases. In this context, the determination of key genes involved in bone cell differentiation is a new challenge to treat any skeletal disorders. Different models are used to study the differentiation process of these cells, but all of them use pre-engaged progenitor cells, allowing us to study only the latest stages of the differentiation. Embryonic stem (ES) cells come from the inner mass of the blastocyst prior its implantation to the uterine wall. Because of their capacity to differentiate into all germ layers, and so into all tissues of the body, ES cells represent the best model by which to study earliest stages of bone cell differentiation. Osteoblasts are generated by two methods, one including the generation of embryoid body, the other not. Mineralizing cells are obtained after 2 weeks of culture and express all the specific osteoblastic markers (alkaline phosphatase, type I collagen, osteocalcin, and others). Osteoclasts are generated from a single-cell suspension of ES cells seeded on a feeder monolayer, and bone-resorbing cells expressing osteoclastic markers such as tartrate-resistant alkaline phosphatase or receptor activator of nuclear factor kappaB are obtained within 11 days. The aim of this review is to present recent discoveries and advances in the differentiation of both osteoblasts and osteoclasts from ES cells.

Bone Diseases↗
PubMed2003

Development of innervation in primary incisors in the foetal period.

Sections from the frontal part of the mandible of 43 human foetuses from 9 to 39 weeks of prenatal age, which contained two, three and sometimes four lower incisors were immunohistochemically examined using protein gene product and neuron specific enolase (NSE) antibodies in order to establish the time of appearance of nerve fibres in the developing tooth germ and to define their topography. Nerve fibres were first detected in the dental follicle in the 11th week of intrauterine life. Their presence in the dental papilla was confirmed in the 18th week when the first layers of dentine and enamel were deposited. In the 24th week of intrauterine life, the nerve fibres first reached the subodontoblastic region. In the subsequent weeks, an increase in the number of nerve fibres accompanying blood vessels in the central portion of the dental papilla resulted in the formation of neuro-vascular bundles. Moreover, the progressive deposition of enamel and dentine was accompanied by branching of papillary nerves, which thereby formed a fan-pattern. In the foetal period, no evidence was found for the formation of a subodontoblastic plexus. However, we did observe single nerve fibres in close proximity to the odontoblast layer at the end of intrauterine life. Nerve fibres were not detected in either predentine or dentine throughout foetal life.

Dental Papilla↗
PubMed2006

Insulin-producing cells.

Embryonic stem (ES) cells offer great potential for cell replacement and tissue engineering therapies because of their almost unlimited proliferation capacity and the potential to differentiate into cellular derivatives of all three primary germ layers. This chapter describes a strategy for the in vitro differentiation of mouse ES cells into insulin-producing cells. The three-step protocol does not select for nestin-expressing cells as performed in previous differentiation systems. It includes (1) the spontaneous differentiation of ES cells via embryoid bodies and (2) the formation of progenitor cells of all three primary germ layers (multilineage progenitors) followed by (3) directed differentiation into the pancreatic lineage. The application of growth and extracellular matrix factors, including laminin, nicotinamide, and insulin, leads to the development of committed pancreatic progenitors, which subsequently differentiate into islet-like clusters that release insulin in response to glucose. During differentiation, transcript levels of pancreas-specific transcription factors (i.e., Pdx1, Pax4) and of genes specific for early and mature beta cells, including insulin, islet amyloid pancreatic peptide, somatostatin, and glucagon, are upregulated. C-peptide/insulin-positive islet-like clusters are formed, which release insulin in response to high glucose concentrations at terminal stages. The differentiated cells reveal functional properties with respect to voltage-activated Na+ and ATP-modulated K+ channels and normalize blood glucose levels in streptozotocin-treated diabetic mice. In conclusion, we demonstrate the efficient differentiation of murine ES cells into insulin-producing cells, which may help in the future to establish ES cell-based therapies in diabetes mellitus.

Animals↗
PubMed1989

Expression of the cell-cell adhesion glycoprotein cell-CAM 120/80 in normal human tissues and tumors.

Polyclonal and monoclonal antibodies raised to the 80 kd glycoprotein component of the cell to cell adhesion molecule cell-CAM 120/80 were used to map its distribution immunohistochemically in normal human tissues and in benign and malignant tumors. Cell-CAM 120/80 was found in all normal epithelial tissues, but was not expressed on neural, lymphoid, smooth, striated and cardiac muscle, connective tissue, or the germ cells in either sex. The expression of this adhesion molecule was polarized in ductal and glandular epithelia and evenly circumferential in squamous and transitional epithelia. Some organs, such as the kidney, liver and endocrine glands, showed unique organ to tissue specific patterns. Maturation-dependent loss of cell-CAM 120/80 was noticed in superficial layers of squamous epithelium and the placenta. Benign epithelial tumors expressed cell-CAM 120/80 in a manner comparable with their tissue of origin. Malignant tumors expressed cell-CAM 120/80 either in a manner similar to the tissue of their origin or assumed a less polarized phenotype. Overall, the immunoreactivity in many malignant tumors appeared weaker and the polarization was less pronounced. Thus, cell-CAM 120/80 is a universal marker of human epithelial cells, but its mode of expression differs in various anatomic sites, and may be influenced by maturation or malignant transformation of cells.

Adenocarcinoma↗