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PubMed1985

Loss of supernumerary axons during neuronal morphogenesis.

The morphogenesis of individual neurons was investigated in the cardiac ganglion of the frog. Intracellular injection of horseradish peroxidase shows that mature neurons lack dendrites and have a single axon. Early in development, more than half of the neurons are multipolar and have as many as four processes emanating from their cell body. The most likely mechanism for the developmental transformation of larval neurons is that the supernumerary processes are pruned from the cell body. Supernumerary processes in larval neurons have features characteristic of axons. The processes of larval neurons can be highly branched and extend throughout the target with distinctive varicosities along the length of each process. Electron microscopy shows that all processes of individually injected cells contain clusters of vesicles apposed to active zones. Thus, the larval cardiac ganglion neuron is capable of extending more than one axon from its cell body.

Animals↗
PubMed1983

A re-examination of the osteocytic network of interdental bone.

A new method for removal of bone minerals and matrix was used in this study to expose osteocytes and osteocytic processes for examination by techniques of scanning electron microscopy. Osteocytes demonstrated more numerous Type II junctions with osteocytic processes than had previously been recognized. The network of osteocytic processes was extensive and exhibited several structural modifications for more efficient transport: rings and branching. Adjacent osteocytes were connected by osteocytic processes suggesting a functional syncytium. The study suggests that the network of osteocytes and osteocytic processes is more extensive and complex than previously recognized.

Animals↗
PubMed1998

Dendritic mast cells in the human nasal mucosa.

Human mast cells can be divided into two subtypes: MCTC cells, which contain tryptase and chymase, and MCT cells, which contain tryptase only. Herein we have used a combination of histamine, tryptase and chymase immunohistochemistry as a novel approach to the study of mast cells. Using this technique, we have discovered a new type of MCTC mast cell in biopsies of the nasal mucosa from healthy subjects and allergic patients. These mast cells have histamine-positive, dendrite-like cellular processes. Some cells have only one slender process, whereas other cells have several long processes extending from different parts of the cell body. Some of the cellular processes divide into two or three terminal branches, and histamine is sometimes found in small swellings along the course of the processes. Our findings contribute new aspects to the concept of mast cell heterogeneity. Thus, human mast cells may vary not only with respect to mediator content, but also with respect to gross morphologic features such as the presence of dendrite-like cellular processes. The recognition of this extreme heterogeneity may be an important step toward a better understanding of mast cell biology.

Adult↗
PubMed1997

EGF receptor ligands are a large fraction of in vitro branching morphogens secreted by embryonic kidney.

Much attention has recently focused upon hepatocyte growth factor (HGF) as a potential regulator of epithelial branching morphogenesis. However, since neither the HGF nor c-met "knockout" mice show abnormal kidney branching morphogenesis, we sought to analyze the relative importance of HGF in in vitro branching morphogenesis compared with other factors secreted by the embryonic kidney. Exploiting an assay that employs kidney epithelial cells (murine inner medullary collecting duct, mIMCD3) seeded in collagen cocultured with the embryonic kidney, we found that a tyrosine kinase inhibitor that is highly specific for the epidermal growth factor (EGF) receptor (EGFR), tyrphostin AG1478, inhibited mIMCD3 cell process formation (an early step in branching tubulogenesis) by 40%, whereas high concentrations of neutralizing anti-HGF antibodies had a lesser effect (20% inhibition), suggesting that EGFR ligands account for a larger fraction of branching morphogens secreted by the embryonic kidney than HGF. In addition, when an embryonic epithelial cell line derived from c-met (-/-) mice was cocultured with the embryonic kidney, these c-met (-/-) cells underwent process formation. EGFR ligands but not HGF were able to induce branching tubulogenesis in these cells. All EGFR ligands tested, including EGF, transforming growth factor-alpha, heparin-binding EGF, betacellulin, and amphiregulin, induced mIMCD3 cell tubulogenesis. EGFR ligands caused upregulation of urokinase, urokinase receptor, and matrix metalloprotease-1, and tubulogenesis could be inhibited by the metalloprotease inhibitor 1,10-phenanthroline. Our results support the notion that multiple parallel and potentially redundant growth factor-dependent pathways regulate branching tubulogenesis.

Animals↗
PubMed1992

Effects of the steroid hormone, 20-hydroxyecdysone, on the growth of neurites by identified insect motoneurons in vitro.

During metamorphosis in the hawkmoth, Manduca sexta, identified larval leg motoneurons survive the degeneration of their larval targets to innervate new muscles of the adult legs. The dendrites and axon terminals of these motoneurons regress at the end of the larval stage and then regrow during adult development. Previous studies have implicated the insect steroid, 20-hydroxyecdysone (20-HE), in similar examples of dendritic reorganization during metamorphosis. The present studies were undertaken to test whether 20-HE acts directly on the leg motoneurons to regulate dendritic growth. Larval leg motoneurons were labeled with a fluorescent dye to permit their identification in culture following the dissociation of thoracic ganglia at later stages of development. Leg motoneurons isolated from early pupal stage animals (just before the normal onset of dendritic regrowth) survived in vitro and grew processes regardless of whether 20-HE was added to the culture medium. The extent of process outgrowth, however, as measured by the total length of all processes and the number of branches, was significantly greater for motoneurons maintained in the presence of 20-HE. The enhancement could be blocked by the addition of a juvenile hormone analog. By contrast, larval leg motoneurons that were isolated just before the normal period of dendritic regression did not show enhanced growth of neurites in the presence of 20-HE. The results suggest that 20-HE acts directly on the leg motoneurons to regulate the growth of processes during metamorphosis.

Animals↗
PubMed1984

[Spatial organization and interrelation of structural elements of the 1st layer of the neocortex].

Investigating topography and structure of elements in the layer I of the fields 4, 3, 1 of the human neocortex (4 cases) and M1, Ep zones in the cat neocortex (5 cases) by means of modified methods of Golgi, Peters and Kluver-Barerr, it became possible to work out an original classification of astrocytes and to reveal the neurono-glio-vascular complexes consisting of the neuronal base and two glial-vascular links. Three main varieties of protoplasmic astrocytes are determined: short-rayed with a long dendrite-like process, short-rayed cap-like and long-rayed with a double bush of branchings. In the latter forms axon-like processes are revealed, some of them make complex basket-like branchings and each of them surrounds a group of neuronal bodies, predominantly the pyramidal ones in the layer III. Distribution of the marginal glia and the three mentioned varieties of astrocytes is subjected to a single plan. The raws of gliocytes along the horizontal and vertical lines are connected with each other and with the neuronal elements. Peculiar receptive apparatuses performing interrelation are ball-like formations revealed on the apical dendrites of the pyramidal neurons. The information that gets into them is processed automatically. Problems concerning the importance of the ball-like formations in integration of the layer I is discussed, and the role of the glial cells with axon-like processes in the active transport of various substances and in regulating metabolism of the pyramidal cells of the layer III is also dealt with.

Animals↗
PubMed1991

Sensory neurons of the rat sciatic nerve.

Experiments have been undertaken in this laboratory over recent years to accurately determine the numbers and sizes of somatic neurons which contribute to the normal sciatic nerve, at mid-thigh levels, of the adult, albino rat. This article is concerned with the dorsal root ganglion (DRG) neuron population of the sciatic nerve whose cell bodies were identified through retrograde labeling of cut branches of the sciatic with horseradish peroxidase (HRP) and/or its wheat germ conjugate (WGA-HRP). It is essential to understand the neuronal composition of the normal rat sciatic nerve if the consequences of aging, nerve injury, and surgical repair to improve functional regeneration are to be properly evaluated. Neuron counts were determined from camera-lucida paper drawings of all labeled profiles in DRGs L3-L6 at 100 x magnification. The profiles, obtained by labeling individual branches of the sciatic nerve (sural, lateral sural, tibial, peroneal, medial, and lateral gastrocnemius/soleus nerves) were traced from 40-microns-thick, serial, frozen sections. The sizes of the perikarya, areas and diameters, were determined by tracing the perimeters of the drawn profiles on a digitizing tablet. The tablet's output was inputted directly into a specially designed computer spreadsheet which contained a mathematical table for correcting the split-cell error inherent to the sectioning process. Afferents from any given branch of the sciatic normally occupied two to three adjacent ganglia. Sciatic DRG neurons were normally located in lumbar ganglia L3-L6. Nearly 98-99% of all sciatic DRG perikarya resided in the L4 and L5 DRGs. The L6 DRG, traditionally regarded as an important contributor to the rat sciatic, contained merely 0.4% of its afferent neurons while the L3 ganglion, frequently overlooked as a contributor, contained 1.2% of the mid-thigh sciatic afferents. The mean size of rat DRG neurons was about 29 microns (550-600 microns2). The corrected counts revealed that the normal sciatic nerve (at mid-thigh levels), in rats between 2 and 12 months of age, contained a mean, total DRG neuron population of about 10,500 neurons. This is probably an underestimate by 3-5% of the true number due to occasional unreliable labeling of some of the small DRG neurons. It is estimated that the normal, mean number of sciatic DRG neurons of young to middle-aged rats lies somewhere between 10,500 and 11,000 +/- 2000. The data suggest that nearly 20% of all DRG neurons in the sciatic nerve supply muscle afferents. The vast majority of the remaining neurons are involved with innervation of the skin.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Evidence for the rare decay B-->K*l+l- and measurement of the B-->Kl+l- branching fraction.

We present evidence for the flavor-changing neutral current decay B-->K*l+l- and a measurement of the branching fraction for the related process B-->K l+l-, where l+l- is either an epsilon+epsilon- or a mu+mu- pair. These decays are highly suppressed in the standard model, and they are sensitive to contributions from new particles in the intermediate state. The data sample comprises 123 x 10(6) Upsilon(4S)-->B(-)B decays collected with the BABAR detector at the SLAC PEP-II epsilon+epsilon- storage ring. Averaging over K(*) isospin and lepton flavor, we obtain the branching fractions B(B-->Kl+l-)=(0.65(+0.14)(-0.13)+/-0.04)x10(-6) and B(B-->K*l+l-)=(0.88(+0.33)(-0.29)+/-0.10)x10(-6), where the uncertainties are statistical and systematic, respectively. The significance of the B-->Kl+l- signal is over 8sigma, while for B-->K*l+l- it is 3.3sigma.

Journal Article↗
PubMed1985

Isolated follicular dendritic cells: cytochemical antigen localization, Nomarski, SEM, and TEM morphology.

The objectives of the present study were to determine the cytological features of isolated follicular dendritic cells (FDC), which distinguish them from other leukocytes or dendritic cell types. Consequently, we have developed methods for the fixation, peroxidase cytochemistry, and visualization of FDC, which are applicable to cytological evaluations by Nomarski optics, scanning, and transmission electron microscopy. A functionally supported identification of FDC in vitro was made possible by utilizing, in conjunction with the dendritic morphology, the cytochemically identifiable antigen, horseradish peroxidase (HRP), and the known capacity of FDC to sequester immune complexes (i.e. HRP-anti-HRP) on their plasma membranes. The observations showed that FDC constitute a relatively pleomorphic, nonphagocytic group, distinct from other dendritic type cells such as lymphoid dendritic cells, Langerhans cells, and interdigitating cells (LDC, LC, and IDC), as well as typical leukocytes. Morphologically distinct FDC were identified as cells either with filiform dendrites or with "beaded" dendrites. FDC possessed a single or sometimes a double, lymphocyte-size cell body, which contained an irregular, lobated nucleus, Golgi apparatus, numerous small vesicles, and some mitochondria. Mitochondria were not abundant in the dendritic processes. Filiform dendrites tended to branch and anastomose near the cell body and form a radiating "sunburst"-like pattern. On the average, dendrites measured 15-20 microns in length and 0.1-0.3 micron in diameter. Occasional dendrites were extremely elongated, reached several hundred microns in length, and terminated in an enlargement measuring nearly a micron in diameter. Other filiform dendrites usually had a club-shaped terminal enlargement. The microspheres of "beaded" dendrites ranged between 0.3 and 0.6 micron in diameter. The dendritic processes were also shown to have a highly ordered pattern of immune complex attachment on their surface, suggestive of a periodic arrangement of receptor sites.

Animals↗
PubMed1998

Epimorphin functions as a key morphoregulator for mammary epithelial cells.

Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.

Animals↗
PubMed2002

Digital blood flow analysis from microscopic images of mesenteric microvessel with multiple branching.

Blood flow through frog mesenteric microvessel with multiple branching, at Reynolds number 0.022, is analyzed. After pre-processing the images the velocity and erythrocyte distribution profiles by image velocimetry and axial tomography are obtained, respectively. The vascular parameters and shape descriptors are obtained by image processing techniques. The axial velocity shows the variation around branching areas associated with an increase in radial velocity, which is up to 10% of axial velocity. From the erythrocyte distribution profiles at various locations in the microvessel the tomograms are reconstructed. Based on these the contours of equal concentration are reconstructed. From these contours the point-to-point variation in cell concentration throughout the microvessel is observed and regions where the cellular concentration varies from 3 to 5%, at zero axial velocity, are identified. The change in erythrocyte shape, determined by the shape descriptors, while passing through the capillary is significant. This passage of cells, depending on their orientation, also displaces the capillary walls by 1.5 to 2.5 microm.

Animals↗
PubMedDate not supplied

Glucocorticoids, TGF-beta, and embryonic mouse salivary gland morphogenesis.

Branching morphogenesis is a fundamental embryologic process in many developing organs: salivary gland, lung, pancreas, mammary gland, and kidney. Glucocorticoids (CORT) are known to regulate morphogenesis in several branching organs (e.g., lung); we hypothesize that CORT is also important to embryonic mouse salivary gland morphogenesis. We studied the CORT-glucocorticoid receptor (GR) signal transduction pathway during embryonic mouse submandibular gland development. Western analysis demonstrates that the 96-kDa GR is present in E14 to E18 submandibular glands. The embryonic GR is functional, as defined by its ability to bind a DNA CORT response element, for all gestational ages evaluated (E14 to E18); increasing GR levels are observed with progressive development. The level of endogenous corticosterone in embryonic submandibular glands was also determined. Using radioimmunoassays corticosterone is first detected on day 15 of gestation; there is a progressive increase in steroid levels from day 15 to 18 of gestation. In addition, we investigated the effect of exogenous CORT on submandibular gland morphogenesis in vivo and in vitro. In vivo experiments were performed using maternal injection of CORT or sham injection on day 12 of gestation with sacrifice 72 hr post-injection (E15). CORT treatment significantly increases embryonic submandibular gland growth in vivo. To further analyze the effect of exogenous CORT on embryonic submandibular gland morphogenesis, E13 mouse submandibular glands were cultured under serum-free, chemically-defined conditions. A significant (P < 0.05) enhancement of mean branching ratios (72 hr/0 hr) is detected in CORT-supplemented explants (10(-5) M to 10(-8) M) compared to control explants; doses more dilute than 10(-8) M CORT do not significantly (P > 0.10) increase the induction of branching. In addition, CORT administration to embryonic mice in utero enhances the expression of acinar-cell-specific mucin protein. These data indicate that the CORT-GR signal transduction pathway plays an important role in salivary gland morphogenesis. Northern analysis of TGF-beta 1, TGF-beta 2, and TGF-beta 3 transcript levels in vivo confirms the presence of TGF-beta 1 and TGF-beta 2 and identifies TGF-beta 3 in embryonic submandibular glands. To begin to identify a molecular mechanism of CORT-GR mediated submandibular gland morphodifferentiation, we studied the effect of CORT on growth factor gene expression, specifically TGF-beta. Northern analysis suggests that the CORT-GR signal transduction pathway modulates the rate of morphogenesis by regulating TGF-beta 2 and TGF-beta 3 mRNA expression.

Analysis of Variance↗
PubMed2003

Chorioallantoic morphogenesis and formation of the placental villous tree.

The placenta is a highly specialized organ whose primary function is to promote the exchange of nutrients and oxygen between maternal and fetal blood, essential for survival and growth of the baby. The surface area for nutrient transport is a highly convoluted villous structure that forms by branching morphogenesis. In mice, this process begins after embryonic day 8.5, following attachment of allantoic mesoderm to the chorion, and continues through the end of gestation. Gene targeting studies in mice have identified a large number of genes that are essential for chorioallantoic development to give rise to the layer of the placenta called the labyrinth. Collectively, these studies reveal that a number of signaling pathways regulate four distinct phases of labyrinth development: chorioallantoic attachment (involving VCAM1 and its receptor alpha4 integrin, Bmp5/7, and Wnt7b, as well as the cochaperone Mrj), initiation of branching (involving the Gcm1 transcription factor to select sites of branch initiation), extension of villous branching (involving FGF, EGF, and HGF/Met signaling, through the Grb2/Sos1/Mek1/p38alpha MAPK pathway), followed by vascularization of the villous tree. The restricted expression and/or action of the signaling components indicate that a series of intercellular interactions regulate chorioallantoic development.

Allantois↗
PubMed1975

Extrinsic nerve supply of the naked rhinarium in the tree shrew.

The extrinsic nerve supply of the median strip of skin in the naked rhinarium of the tree shrews (Tupaia glis) was studied by means of nerve section. Experiments were designed, where the external nasal nerves, infraorbital nerves or a combination of both were sectioned. The cholinesterase and silver impregnation techniques were employed to demonstrate intraepithelial nerves, mammalian end-organs, and the dermal nerve plexus before and after denervation. These experiments have given results contrary to what was expected, as the skin in this region, which develops from the frontonasal process, was found to be supplied by the infraorbital nerves (branches of the maxillary division of the fifth cranial nerve, which is the nerve of the maxillary process) and not the external nasal nerves (branches of the ophthalmic division of the fifth cranial nerve, the nerve of the frontonasal process).

Animals↗
PubMed2005

Automated nomenclature of bronchial branches extracted from CT images and its application to biopsy path planning in virtual bronchoscopy.

We propose a novel anatomical labeling algorithm for bronchial branches extracted from CT images. This method utilizes multiple branching models for anatomical labeling. In the actual labeling process, the method selects the best candidate models at each branching point. Also a special labeling procedure is proposed for the right upper lobe. As an application of the automated nomenclature of bronchial branches, we utilized anatomical labeling results for assisting biopsy planning. When a user inputs a target point around suspicious regions on the display of a virtual bronchoscopy (VB) system, the path to the desired position is displayed as a sequence of anatomical names of branches. We applied the proposed method to 25 cases of CT images. The labeling accuracy was about 90%. Also the paths to desired positions were generated by using anatomical names in VB.

Algorithms↗
PubMed1984

A Golgi-type study of the hypothalamus of the lizard, Calotes versicolor.

The hypothalamus of the garden lizard, Calotes versicolor, was investigated with the use of the rapid Golgi and Golgi-Cox techniques. Individual neurons belonging to different nuclear groups were identified and studied with respect to their position, orientation, nature, pattern of arborization and connections. Magnocellular neurons in the preoptic region (rapid Golgi method) are large, lobular, bipolar or multipolar elements characterized by smooth contours of their somata, and processes of differing caliber and pattern of branching. Parvocellular neurons were found to belong to the primitive and generalized isodendritic type displaying pyriform, fusiform or spindle-shaped somata. Each soma gives rise to two to four straight, sometimes curved processes, which show varying patterns of arborization. The branches extend toward the 'integrative area', comprised of medial and lateral neuropil, or among major hypothalamic tracts; they are suggested to establish extrahypothalamic contacts or to extend to adjoining nuclear zones where they may establish specific intrahypothalamic connections. CSF-contacting processes from some magnocellular and parvocellular neurons were encountered. In addition to drawing correlations between information obtained from Golgi- and Nissl preparations, an attempt has been made to understand the precise organization of neuronal elements and to study the scope and pattern of arborization in the hypothalamus of Calotes versicolor.

Animals↗
PubMed2002

Intermediary glucan structures formed during starch granule biosynthesis are enriched in short side chains, a dynamic pulse labeling approach.

The formation of intermediary glucans, mature starch, and phytoglycogen was studied using leaves of Arabidopsis thaliana wild type and dbe mutant, which lacks plastidic isoamylase (Zeeman, S. C., Umemoto, T., Lue, W. L., Au-Yeung, P., Martin, C., Smith, A. M., and Chen, J. (1998) Plant Cell 10, 1699-1711). A new approach to the study of starch biosynthesis was developed based on "very short pulse" labeling of leaf starch through photosynthetic fixation of (14)CO(2). This allowed selective analysis of the structure of starch formed within a 30-s period. This time frame is shorter than the period required for the formation of a single crystalline amylopectin lamella and consequently permits a direct analysis of intermediary structures during granule formation. Analysis of chain length distribution showed that the most recently formed outer layer of the granules has a structure different from the mature starch. The outer layer is enriched in short chains that are 6-11 glucose residues long. Side chains with 6 glucose residues are the shortest abundant chains formed, and they are formed exclusively by transfer from donor chains of 12 glucose residues or longer. The labeling pattern shows that chain transfer resulting in branching is a rapid and efficient process, and the preferential labeling of shorter chains in the intermediary granule bound glucan is suggested to be a direct consequence of efficient branching. Although similar, the short chain intermediary structure is not identical to phytoglycogen, which is an even more highly branched molecule with very few longer chains (more than 40 glucose residues). Pulse and chase labeling profiles for the dbe mutant showed that the final structure is more highly branched than the intermediary structures, which implies that branching of phytoglycogen occurs over a longer time period than branching of starch.

Arabidopsis↗
PubMed1980

Structure of developing frog neuromuscular junctions.

Developing neuromuscular junctions in the cutaneous pectoris muscle from tadpoles and and postmetamorphic frogs were studied in the light microscope. Presynaptic nerve terminals and postsynaptic acetylcholinesterase (AChE) activity simultaneously demonstrated at these developing junctions using the NBT-AChE method. The earliest nerve contacts studied were small enlargements at the ends of unmyelinated axons. As development progressed, single nerve contacts, often with growth cones, grew in length, generally parallel to the long axis of the myotube. Further endplate maturation was characterized by individual terminal processes developing secondary and tertiary branches, ultimately leading to highly complex terminal arborizations. The initial synaptic contact at developing neuromuscular junctions was made by a single axon, but with further development these same synaptic sites became multiply innervated. The occurrence of multiple innervation was a transient phenomena; the multiple synaptic inputs were eliminated during further maturation. The time course of synapse elimination was protracted, with some multiple innervation even persisting in relatively large adult frogs. The first nerve contacts were generally devoid of observable endplate AChE activity. Early appearance of AChE activity was sometimes graded and in some cases portions of the nerve terminal processes were associated with AChE while other regions of the same terminal arborization were not.

Acetylcholinesterase↗