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PubMed1980

Quantitative analysis of developing epiglottal taste buds in sheep.

Epiglottal taste buds of the sheep increase in number during development, and continue to increase until the epiglottis has reached its adult size. However, since the increase in taste bud numbers is paralleled by increase in the surface area of the epiglottis, the density of taste buds decreases progressively in the fetus and newborn. After birth the density remains relatively constant. From examination of the morphological stages of epiglottal taste bud development, we conclude that taste buds are originally produced by de novo formation, but that further increase in numbers is effected by division of existing, developing taste buds. Because of the large number of taste buds, and the maintenance of their density on the epiglottis, it is concluded that they have an important functional role in upper airway protective reflexes.

Aging↗

PubMed1996

Unilateral innervation of guinea pig vallate taste buds as determined by glossopharyngeal neurectomy and HRP neural tracing.

The innervation pattern by primary afferent nerve fibres and the neurotrophic effect on taste cells were investigated in the guinea pig vallate taste bud by means of glossopharyngeal neurectomy and horseradish peroxidase (HRP) or wheat germ agglutinin-horseradish peroxidase (WGA-HRP) tracing. In the glossopharyngeal neurectomy study, taste buds in the vallate papillae of adult guinea pigs were denervated by unilateral resection of the right glossopharyngeal nerve. Denervated animals were killed on days 1, 3 and 5 and weeks 1-9, 12 and 24 postneurectomy. The results showed that, on the denervated side, the taste buds decreased significantly in number during the 1st 2 wk, and disappeared completely by wk 3; no mature taste buds were present even 24 wk after neurectomy. This suggests that the vallate taste buds disappear in the absence of the glossopharyngeal nerve. In the neural tracing study, HRP or WGA-HRP was injected into the proximal end of the right glossopharyngeal nerve, near the jugular foramen. After a survival time of 24 h, the vallate papillae were sectioned and examined by light and electron microscopy. Light microscopy revealed that the HRP or WGA-HRP-labelled fibres innervated the vallate taste buds of the injected side. Most of the taste cells in the buds were labelled with HRP or WGA-HRP reaction products from the basal to the apical region. At the ultrastructural level, the reaction products were confined to the cytoplasm of the labelled cells, which were identified as type I, II and III cells, but not basal cells. Labelled intragemmal nerve profiles were seen among the taste cells. No synapse formation was seen with nerve profiles abutting on type I and II cells, whereas on certain type III cells, typical synapses were formed. We conclude that both the right and left vallate papilla in the guinea pig are unilaterally innervated by the glossopharyngeal nerve without cross-innervation.

Animals↗

PubMed1998

Uptake and release of neurotransmitter candidates, [3H]serotonin, [3H]glutamate, and [3H]gamma-aminobutyric acid, in taste buds of the mudpuppy, Necturus maculosus.

Neurotransmitters in vertebrate taste buds have not yet been identified with confidence. Serotonin, glutamate, and gamma-aminobutyric acid (GABA) have been postulated, but the evidence is incomplete. We undertook an autoradiographic study of [3H]serotonin, [3H]glutamate, and [3H]GABA uptake in lingual epithelium from the amphibian, Necturus maculosus, to determine whether taste bud cells would accumulate and release these substances. Lingual epithelium containing taste buds was incubated in low concentrations (0.4-6 microM) of these tritiated transmitter candidates and the tissue was processed for light microscopic autoradiography. Merkel-like basal taste cells accumulated [3H]serotonin. When the tissue was treated with 40 mM K+ after incubating the tissue in [3H]serotonin, cells released the radiolabelled transmitter. Furthermore, depolarization (KCl)-induced release of [3H]serotonin was Ca-dependent: if Ca2+ was reduced to 0.4 mM and 20 mM Mg2+ added to the high K+ bathing solution, Merkel-like basal cells did not release [3H]serotonin. In contrast, [3H]glutamate was taken up by several cell types, including non-sensory epithelial cells, Schwann cells, and some taste bud cells. [3H]glutamate was not released by depolarizing the tissue with 40 mM K+. [3H]GABA uptake was also widespread, but did not occur in taste bud cells. [3H]GABA accumulated in non-sensory epithelial cells and Schwann cells. These data support the hypothesis that serotonin is a neurotransmitter or neuromodulator released by Merkel-like basal cells in Necturus taste buds. The data do not support (nor rule out) a neurotransmitter role for glutamate or GABA in taste buds.

Animals↗

PubMed1997

Shh expression in developing and regenerating limb buds of Xenopus laevis.

The zone of polarizing activity (ZPA) is a specialized region involved in the antero-posterior (A-P) axis formation in chick and mouse limb buds. The existence of ZPA in the posterior margin is suggested in Xenopus hindlimb buds because 180 degree rotation of the distal limb tip induces the supernumerary limb. In this study, we investigated the expression of Sonic hedgehog (shh), a molecular marker for ZPA, in Xenopus developing limb buds and regenerating blastemas by whole-mount in situ hybridization. Although shh was expressed in the posterior margin of the limb bud like in chicks, its expression domain did not correspond to the ZPA map of Xenopus hindlimb buds. shh expression was distant from the ZPA at stage 53 in particular. To clarify the difference between the shh expression domain and ZPA, we examined shh expression in 180 degree rotated limb buds. As a result, ectopic shh expression was newly induced in the proximal region to its original expression domain. These results suggest that ZPA is accompanied by shh expression as in chick limb buds. Furthermore we examined shh expression in regenerating blastemas. shh was reexpressed in the posterior margin of the blastema. This result supports the possibility that ZPA also exists in the regenerating blastema.

Animals↗

PubMed2006

Macrophages promote collagen fibrillogenesis around terminal end buds of the developing mammary gland.

Development of the ductal network in the mammary gland is dependent in part on the presence of macrophages. Here we utilize multi-photon microscopy and second harmonic generation to describe terminal end bud 3-dimensional structure and the organization of the surrounding collagen matrix. We have applied this approach to analyze the effect of macrophage deficiency on terminal end bud structure and collagen organization, using mice homozygous for a null mutation in the colony stimulating factor-1 gene (Csf1op/Csf1op). Primary terminal end buds have an oblong shape, with long collagen I fibers close to the neck of the terminal end bud and radiating upwards in the direction of growth. Around the terminal end buds, the amount of total collagen I detected by antibody staining was not affected by macrophage deficiency. However the amount of collagen I organized into long fibers, detected by second harmonic generation signal, was reduced in Csf1op/Csf1op mice. Macrophage deficiency also caused terminal end buds to be rounder and shorter. These studies reveal a role for macrophages in collagen fibrillogenesis and in organization of the structure of terminal end buds.

Animals↗

PubMed1984

On the possible origin of extra-epithelial enterochromaffin cells by budding from the crypts of Lieberkuhn.

This study was undertaken to test Pierre Masson's still unconfirmed theory that extra-epithelial enterochromaffin cells in the gut arise in adult life by budding from the crypts of Lieberkuhn under conditions of low grade inflammation. Appendices (900) were reviewed and 19 were selected for serial section study because in random sections they showed lateral fusion of the crypts, one of the key features described by Masson. Ten specimens without crypt fusion served as controls. Sixteen of the 19 study specimens and one of the control specimens showed budding, averaging one bud in every 88 sections. Most buds were in direct contact with Schwann cells in the adjacent lamina propria and 45 per cent of them contained enterochromaffin cells. There was also histologic evidence linking buds and lateral crypt fusion to low grade inflammation. Masson's ideas are, therefore, confirmed insofar as the existence of buds and their relationship to enterochromaffin cells, Schwann cells, and inflammation is concerned. The actual separation of buds from the crypts to form extra-epithelial enterochromaffin cells has yet to be proved.

Adolescent↗

PubMed1993

Induction of polarizing activity by retinoic acid occurs independently of duplicate formation in developing chick limb buds.

Retinoic acid (RA) is known to mimic the action of the zone of polarizing activity (ZPA) in inducing the formation of anteroposterior duplicates in the chick limb bud. Although RA had been thought to be a morphogen produced by the ZPA, recently we (Noji et al., 1991) and Wanek et al. (1991) independently concluded that RA induces polarizing activity in cells at the anterior margin of the chick limb bud. In this study, we examined the distribution of RA-induced polarizing activity in the limb bud. At first, RA-containing beads were implanted into various regions of stages 20-23 limb buds. After 24 hr, we grafted tissue fragments from sites adjacent to the RA beads into the anterior margin of host limb buds. High polarizing activity was induced in anterior-distal and apical mesoderm regions immediately under the apical ectodermal ridge (AER) when beads presoaked in 1 mg/ml RA were grafted to anterior sites at stages 19-20. However, in mesoderms distant from the AER, even if adjacent to the implanted RA bead, only low activity was induced. Although implantation of beads soaked in a high concentration (10 mg/ml) of RA did not result in duplications but rather in truncations of the limbs, the apical mesodermal cells in these limbs were converted to ZPA cells, indicating that induction of polarizing activity by RA can occur independently of duplicate formation. The ability to respond to RA treatment was rapidly lost in the cells along the anterior margin of stage 22 limb buds. When the anterior AER was removed, the induction of polarizing activity in the anterior margin of limb buds was markedly reduced. Thus, the AER seems to be necessary for the RA-directed induction of polarizing activity.

Animals↗

PubMed1993

MRC-5, human embryonic lung fibroblasts, induce the duplication of the developing chick limb bud.

The anteroposterior (A-P) axis pattern of the chick limb is likely to be controlled by a small region of mesenchyme cells at the posterior margin of the limb bud (ZPA, zone of polarizing activity). In this study, we found that MRC-5 fibroblast cells had the capacity for duplicated-pattern formation of the chick limb along the A-P axis when grafted to the anterior region of the limb bud. MRC-5 cells were effective only during pre-limb bud stages, and the leg bud was more responsive than the wing bud. Grafted cells remained at the base of the limb bud when limb development proceeded. These results suggest that the products of MRC-5 cells are involved in three possible processes of duplicated-pattern formation: induction of the polarizing activity; maintenance of this activity, which is present weakly at pre-limb bud stages; and determination of A-P axis as the ZPA factor(s). By allowing the use of a small number of cells of embryonic tissues with polarizing activity, the analysis of duplicate formation with the MRC-5 cell line provides a powerful tool for elucidating the molecular nature of the ZPA.

3T3 Cells↗

PubMed1994

Expression of the connexin43 gap junctional protein in tissues at the tip of the chick limb bud is related to the epithelial-mesenchymal interactions that mediate morphogenesis.

The pattern of connexin43 expression in developing chick limb buds was examined using a site-specific polyclonal antibody and confocal microscopy. Connexin43 is expressed at stages of limb development when epithelial-mesenchymal interactions are occurring that mediate morphogenesis. Extensive labeling was observed in the apical ectodermal ridge and labeling was also found in underlying mesenchyme cells at the tip of the bud. In mouse limb buds, the same gap junction protein is expressed only in the apical ridge. Manipulations of developing chick wing buds show that mesenchymal expression of connexin43 appears to be controlled by the apical ectodermal ridge. When the apical ridge is surgically removed and limb truncations result, mesenchymal labeling is markedly reduced and conversely the grafting of an additional ridge induces connexin43 expression between underlying mesenchymal cells which do not normally show expression at this stage of development. In addition, a treatment with retinoic acid that flattens the apical ridge and inhibits bud outgrowth reduces expression in both mesenchymal and epithelial tissues. The abolition of connexin43 expression in mesenchymal and epithelial domains when bud outgrowth is halted suggests that synthesis of this gap junction protein is related to the epithelial-mesenchymal interactions that mediate morphogenesis of the bud.

Animals↗

PubMed1984

Neurotrophic and neuronotrophic effects in the regenerating newt limb bud after electrical stimulation of brachiospinal nerves.

An earlier work demonstrated that electrical stimulation of newt brachiospinal nerves produces a 20% increase in protein synthesis in the regenerating limb bud at 6 h post-stimulation. The present study shows that if stimulation of nerve cell bodies is prevented by placing procaine between the cell bodies and the stimulating electrode, there is no increase in limb bud protein synthesis compared to the non-stimulated, contralateral control limb bud. Similarly, if colchicine is applied to the brachiospinal nerves at the site of and prior to stimulation, there is no increase in limb bud protein synthesis after stimulation. Colchicine applied to brachiospinal nerves in the absence of stimulation results in a reduction of limb bud protein synthesis that is of the same magnitude as the increase seen with stimulation. The results suggest that the neurotrophic increase in limb bud protein synthesis after stimulation is under the control of the cell body and that this control is mediated by changes in fast axonal transport. A neuronotrophic increase in axonal density in the stimulated side limb bud is seen at the same time as the increase in protein synthesis after stimulation.

Animals↗

PubMed1984

Genetic analysis of developmental mechanisms in hydra. IX. Effect of food on development of a slow-budding strain (L4).

A mutant hydra strain L4 produces buds at a much lower rate than the standard wild-type strain (105) when fed with brine shrimp nauplii (T. Sugiyama and T. Fujisawa, 1979b, Dev. Growth Differ. 21, 361-375). It was found that addition of a small amount of tubifex worm tissue to the normal brine shrimp diet significantly improved the budding rate of L4 but not of 105. Detailed examination and comparison of food effect on various developmental processes in L4 and 105 have provided the following observations. (1) L4 development is strongly affected by food and has significantly lower rates than 105 in all the developmental parameters examined which involve the bud initiation process. In contrast, such effects and differences are not observed in parameters not involving bud initiation. These observations suggest that L4 has a defect(s) in its bud initiating mechanisms and that the expression of this defect is somehow strongly affected by food. (2) Epithelial cells proliferate in L4 nearly as rapidly as in 105 and without food effect. Epithelial cells produced by cell division are mostly utilized to form buds in mature 105 polyps. These cells, however, are not fully utilized for this purpose in L4 polyps which bud very slowly. Instead, they appear to be somehow lost from tissue in these animals.

Animals↗

PubMed1985

Changes in the pericellular matrix during differentiation of limb bud mesoderm.

Mesodermal cells in the developing chick embryo limb bud appear morphologically homogeneous until stage 21. At stage 22 the prechondrogenic and premyogenic areas begin to condense, culminating in the appearance of cartilage and muscle by stage 25-26. We have examined changes in the hyaluronate-dependent pericellular matrices elaborated by mesodermal cells of the limb bud from different developmental stages and the corresponding changes in production of cell surface-associated and secreted glycosaminoglycans. When placed in culture, most early mesodermal cells (stage 17 lateral plate and stage 19 limb bud) exhibited pericellular coats as visualized by the exclusion of particles. These coats were removed by treatment of the cultures with Streptomyces hyaluronidase. Cells from stage 20-21 limb buds (precondensation) had smaller coats, whereas cells derived from stage 22, 24, and 26 limb buds (condensed chondrogenic and myogenic regions) lacked coats. However, coats were reformed during subsequent cytodifferentiation of chondrocytes; chondrocytes from stage 28 and 30 limb buds, and more mature chondrocytes from stage 38 tibiae, had pericellular coats. Thus, cytodifferentiation of cartilage is accompanied by extensive intercellular matrix accumulation in vivo and reacquisition of pericellular coats in vitro. Although their structure was still dependent on hyaluronate, chondrocyte coats were associated with increased proteoglycan content compared to the coats of early mesodermal cells. The amount of incorporation of [3H]acetate into cell surface hyaluronate remained relatively constant from stages 17 to 38, whereas in the medium compartment, incorporation into hyaluronate was more than 4-fold greater by stage 17 and 19 mesodermal cells than by cells from stages between 20 and 38. However, there was a progressive increase in incorporation into cell surface and medium chondroitin sulfate throughout these developmental stages. Thus, at the time of cellular condensation in the limb bud in vivo, we have observed a reduction in size of hyaluronate-dependent pericellular coats and a dramatic change in the relative proportion of hyaluronate and chondroitin sulfate produced by the mesodermal cells in vitro.

Acetates↗

PubMed1988

Effective stage in the cell cycle for control of the budding direction of cdc mutants of Saccharomyces cerevisiae using electric stimulus.

Cell division cycle (cdc) mutants of Saccharomyces cerevisiae were used to determine the most effective stage for the directional control of cell budding using an electric stimulus. The selected mutants were cdc 35 and cdc 28, which could be reversibly arrested before spindle pole body satellite formation (SPBSF) and spindle pole body duplication (SPBD), respectively. The budding direction (theta) was defined so that the direction parallel to that of the electric field was 0 degree. Considering the symmetry of the experimental conditions, the range of theta was defined as 0-90 degrees. The electric stimulus applied in the present study was alternating pulses (pulse height, +/- 15 V; pulse width at half pulse height, 5 microseconds; frequency; 10 kHz). The peak height of the cross membrane potential was estimated as 472 mV, which was sufficient to induce considerable strain in the cell membrane. In the case of cdc 35, the 95% confidence interval (95% CI) of the budding direction was 7-25 degrees when subjected to electric stimulus, while the 95% CI of the budding direction without electric stimulus was 35-57 degrees. In the case of cdc 28, 95% CI values of the budding direction with and without electric stimulus were 1229 degrees and 23-56 degrees, respectively. These results demonstrate that the stage after SPBD is effective for the directional control of yeast cell budding using an electric stimulus. Simultaneously, an electric stimulus reduced the cell budding time of both the cdc mutants used. Therefore, the electric stimulus was also effective in promoting cell cycle progression under the present conditions.

Cell Cycle↗

PubMedDate not supplied

In vitro culture of preimplantation mouse embryos and day 12 limb-buds: effects of serum and albumin.

The effects of three different protein sources at different concentrations on the growth and development of preimplantation mouse embryos and day 12 mouse limb-buds in culture were studied. Mouse embryos and forelimb-buds were cultured with a range of concentrations (5.5 to 42%) of either donor bovine serum (DBS) or fetal bovine serum (FBS), or (0.2 to 0.8%) bovine serum albumin (BSA). After 48 h in culture, the rate of embryo development was significantly higher in 5.5% DBS than in all other groups (P < 0.05). The embryo hatching rate was higher in 21% FBS, 42% FBS, and all DBS groups than in serum-free medium, and all BSA groups (P < 0.05). Morphologic analysis of cultured limb-buds at 72 h revealed that total, paw, and cartilage area were greater (P < 0.05) in the serum-free medium than in all other groups. Shape factor analysis suggested that 5.5% DBS was most beneficial to mouse limb-bud development. No differences were seen in DNA or protein content of limb-buds among groups. Results suggest that mouse forelimb-buds can be successfully cultured in serum-free medium and that high concentrations of FBS and DBS may be detrimental for preimplantation embryo and/or limb-bud growth and development.

Animals↗

PubMed2003

Direct observations of the redox states of frozen cherry buds by a unique in vivo ESR.

Low temperatures cause cellular damage in flower buds of the sweet cherry (Prunus avium L. cv. Satohnishiki). In this study, the redox states within the cherry buds suffering freezing damage were non-destructively observed by a unique in vivo electron spin resonance (ESR) technique with a spin probe such as carbamoyl-PROXYL. The ESR signals of carbamoyl-PROXYL-treated bud were continuously recorded under freezing and thawing condition, which was decreased to approximately -4 degrees C and maintained for 1.5h, and then returned to room temperature. Most of the buds began to freeze at -2.5 to -3.9 degrees C. The peak areas of the ESR signals significantly increased during the period of temperature rise. These results show that the reduced carbamoyl-PROXYL within the frozen bud was re-oxidized and became ESR-detectable while the bud was thawing. Our in vivo ESR technique has confirmed the oxidative transition of the redox states within the buds during thawing.

Electron Spin Resonance Spectroscopy↗

PubMed2006

Budding invasive margin and prognosis in colorectal cancer--no direct association with beta-catenin expression.

Cancer cell budding at the invasive margin has been associated with poor prognosis in rectal cancer. beta-Catenin is an adhesion protein involved in the nuclear Wnt/beta-catenin pathway, and mesenchymal transition of colorectal cancer cells. Hence, we investigated the relationship between cancer cell budding at the invasive margin, beta-catenin expression, and 5-year-survival in colorectal cancer. Four hundred and sixty six colorectal cancer specimens were analysed for budding margin, and 108 specimens from the same set for beta-catenin by immunohistochemistry. A budding margin was present in 24.0% of the cases and predicted a poor 5-year-survival (15.4%, P < 0.00001). Nuclear beta-catenin expression increased from the central area towards the invasive margin (P < 0.001), but did not predict budding. Budding margin is an independent factor associated with poor prognosis in colorectal cancer, and could be utilised in diagnostic pathology. Nuclear beta-catenin was often found at the invasive margin, but is unlikely to be the sole cause of budding.

Colorectal Neoplasms↗

PubMed2005

Cdc42-dependent localization of polarisome component Spa2 to the incipient bud site is independent of the GDP/GTP exchange factor Cdc24.

Cdc42, a member of the Rho subfamily of small GTPases, is highly conserved in both sequence and function across eukaryotic species. In budding yeast, Cdc42 triggers polarized growth necessary for bud emergence via rearrangement of the actin cytoskeleton. It has been shown that the role of Cdc42 in bud emergence requires both Cdc28-Cln (G1) kinase and the passage through START. In this report, we show that Cdc42 also serves an essential function in the establishment of bud site prior to START by catalyzing the translocation of bud-site components such as Spa2 to the cell cortex. Our analysis of various conditional alleles of CDC42 suggests that these two functions (bud site establishment and bud emergence) are genetically separable. Surprisingly, the role of Cdc42 in the cortical localization of Spa2 appears to be independent of its well known GTP/GDP exchange factor Cdc24. We also provide evidence that this role of Cdc42 requires the function of the COPI coatomer complex.

Alleles↗

PubMed2006

A luciferase-based budding assay for Ebola virus.

The VP40 matrix protein of Ebola virus (EBOV) is capable of budding from mammalian cells as a virus-like particle (VLP) and is the major protein involved in virus egress. A functional budding assay has been developed based upon this characteristic of VP40 to assess the contributions of VP40 sequences as well as host proteins to the budding process. This well-defined assay has been modified for potential use in a high-throughput format in which the detection and quantification of firefly luciferase protein in VLPs represents a direct measure of VP40 budding efficiency. Luciferase was found to be incorporated into budding VP40 VLPs. Furthermore, co-expression of EBOV glycoprotein (GP) enhances release of VLPs containing VP40 and luciferase. In contrast, when luciferase is co-expressed with a budding deficient mutant of VP40, luciferase levels in the VLP fraction decrease significantly. This assay represents a promising high-throughput approach to identify inhibitors of EBOV budding.

Cell Line↗