SOME OBSERVATIONS ON THE FINE STRUCTURE OF THE LATERAL LINE ORGAN OF THE JAPANESE SEA EEL LYNCOZYMBA NYSTROMI

The fine structure of the lateral line organ of the Japanese sea eel Lyncozymba nystromi has been studied with the electron microscope. The sensory epithelium of the lateral line organ consists of a cluster of two major types of cells, the sensory hair cells and the supporting cells. The sensory cell is a slender element with a flat upper surface provided with sensory hairs, Two different types of synapses are distinguished on the basal surface of the receptor cell. The first type is an ending without vesicles and the second type is an ending with many vesicles. These are presumed to correspond to the afferent and the efferent innervations of the lateral line organ. The fine structure of the supporting cells and the morphological relationship between the supporting cells and the receptor cells were observed. The possible functions of the supporting cells are as follows: (a) mechanical and metabolic support for the receptor cell; (b) isolation of the individual receptor cell; (c) mucous secretion and probably cupula formation; (d) glial function for the intraepithelial nerve fibers. Both myelinated and unmyelinated fibers were found in the lateral line nerve. The mode of penetration of these fibers into the epithelium was observed.     

STRUCTURE OF THE MACULA UTRICULI WITH SPECIAL REFERENCE TO DIRECTIONAL INTERPLAY OF SENSORY RESPONSES AS REVEALED BY MORPHOLOGICAL POLARIZATION

The anatomy of the labyrinth and the structure of the macula utriculi of the teleost fish (burbot) Lota vulgaris was studied by dissection, phase contrast, and electron microscopy. The innervating nerve fibers end at the bottom of the sensory cells where two types of nerve endings are found, granulated and non-granulated. The ultrastructure and organization of the sensory hair bundles are described, and the finding that the receptor cells are morphologically polarized by the presence of an asymmetrically located kinocilium in the sensory hair bundle is discussed in terms of directional sensitivity. The pattern of orientation of the hair cells in the macula utriculi was determined, revealing a complicated morphological polarization of the sensory epithelium. The findings suggest that the interplay of sensory responses is intimately related to the directional sensitivity of the receptor cells as revealed by their morphological polarization. The problem of efferent innervation is discussed, and it is concluded that the positional information signaled by the nerve fibers innervating the vestibular organs comprises an intricate pattern of interacting afferent and efferent impulses     

THE ULTRASTRUCTURE OF THE KINOCILIUM OF THE SENSORY CELLS IN THE INNER EAR AND LATERAL LINE ORGANS

The bundle of sensory hairs protruding from the top of each receptor cell in the vestibular and lateral line organs in the teleost fish (burbot) Lota vulgaris is composed of a number of stereocilia and one kinocilium located in the periphery of the bundle. The ultrastructure of the kinocilium and its basal body is described. It is found that the kinocilium is morphologically polarized by the asymmetric arrangement of its component fibers and of the basal body by the presence of a basal foot. Peripheral fibers 5 and 6 of the kinocilium and the basal foot of the basal body are oriented away from the stereocilia; that is, in a direction coinciding with the direction of excitatory stimulation. The findings are discussed in terms of directional sensitivity.     

Effect of cell polarization on hepatitis C virus entry

The primary reservoir for hepatitis C virus (HCV) replication in vivo is believed to be hepatocytes within the liver. Three host cell molecules have been reported to be important entry factors for receptors for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI), and the tight-junction (TJ) protein claudin 1 (CLDN1). The recent discovery of a TJ protein as a critical coreceptor highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. The colorectal adenocarcinoma Caco-2 cell line forms polarized monolayers containing functional TJs and was found to express the CD81, SR-BI, and CLDN1 proteins. Viral receptor expression levels increased upon polarization, and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins ZO-1 and occludin 1 were unchanged upon polarization. HCV infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry was neutralized by anti-E2 monoclonal antibodies, demonstrating glycoprotein-dependent entry. HCV pseudoparticle infection and recombinant HCV E1E2 glycoprotein interaction with polarized Caco-2 cells occurred predominantly at the apical surface. Disruption of TJs significantly increased HCV entry. These data support a model where TJs provide a physical barrier for viral access to receptors expressed on lateral and basolateral cellular domains.     

SYNAPTIC STRUCTURES IN THE LATERAL LINE CANAL ORGAN OF THE TELEOST FISH LOTA VULGARIS

This paper is a preliminary report on some of the results of electron microscopic studies on the lateral line canal organ of the teleost fish Lota vulgaris. It deals with the ultrastructure of the synaptic area on the hair cells of the sensory epithelium and describes the nerve endings as well as a complicated system of foldings of the hair cell plasma membranes enclosing portions of the hair cell cytoplasm in the synaptic area. These findings are discussed in the light of present knowledge of the ultrastructure of other receptoneuronal synapses.     

Hepatoma polarization limits CD81 and hepatitis C virus dynamics

Many viruses target the polarized epithelial apex during host invasion. In contrast, hepatitis C virus (HCV) engages receptors at the basal surface of hepatocytes in the polarized liver parenchyma. Hepatocyte polarization limits HCV entry by undefined mechanism(s). Given the recent reports highlighting a role for receptor mobility in pathogen entry, we studied the effect(s) of hepatocyte polarization on viral receptor and HCV pseudoparticle (HCVpp) dynamics using real‐time fluorescence recovery after photobleaching and single particle tracking. Hepatoma polarization reduced CD81 and HCVpp dynamics at the basal membrane. Since cell polarization is accompanied by changes in the actin cytoskeleton and CD81 links to actin via its C‐terminus, we studied the dynamics of a mutant CD81 lacking a C‐terminal tail (CD81_ΔC) and its effect(s) on HCVpp mobility and infection. CD81_ΔC showed an increased frequency of confined trajectories and a reduction of Brownian diffusing molecules compared to wild‐type protein in non‐polarized cells. However, these changes were notobserved in polarized cells. HCVpp showed a significant reduction in Brownian diffusion and infection of CD81_ΔC expressing non‐polarized cells. In summary, these data highlight the dynamic nature of CD81 and demonstrate a role for CD81 lateral diffusion to regulate HCV infection in a polarization‐dependent manner.     

Brain-derived neurotrophic factor (BDNF) promotes molecular polarization and differentiation of immature neuroblastoma cells into definitive neurons

Throughout development, neuronal progenitors undergo complex transformation into polarized nerve cells, warranting the directional flow of information in the neural grid. The majority of neuronal polarization studies have been carried out on rodent-derived precursor cells, programmed to develop into neurons. Unlike rodent neuronal cells, SH-SY5Y cells derived from human bone marrow present a sub-clone of neuroblastoma line, with their transformation into neuron-like cells showing a range of highly instructive neurobiological characteristics. We applied two-step retinoic acid (RA) and brain-derived neurotrophic factor (BDNF) protocol to monitor the conversion of undifferentiated SH-SY5Y into neuron-like cells with distinctly polarized axon-dendritic morphology and formation of bona fide synaptic connections. We show that BDNF is a key driver and regulator of the expression of axonal marker tau and dendritic microtubule-associated protein-2 (MAP2), with their sorting to distinct cellular compartments. Using selective kinase inhibitors downregulating BDNF-TrkB signaling, we demonstrate that constitutive activation of TrkB receptor is essential for the maintenance of established polarization morphology. Importantly, the proximity ligation assay applied in our preparation demonstrates that differentiating neuron-like cells develop elaborate synaptic connections enriched with hallmark pre- and postsynaptic proteins. Described herein findings highlight several fundamental processes related to neuronal polarization and synaptogenesis in human-derived cells, which are of major relevance to neurobiology and translational neuroscience.     

FINE STRUCTURE OF THE PINEAL ORGANS OF THE ADULT FROG, RANA PIPIENS

Frontal organs and epiphyses of the pineal system from the adult frog, Rana pipiens, were fixed in s-collidine-buffered osmium tetroxide, embedded in Epon 812, and examined by electron microscopy. Epiphyseal material was also fixed in a variety of ways and subjected to a series of cytochemical tests for light microscopy. An ultrastructure resembling that of lateral eye retina is confirmed in this species. Photoreceptor cells of the epiphysis and frontal organ display many cytological features similar to those of retinal rods and cones in the arrangement of their outer and inner segments and synaptic components. However, in these pineal organs the outer segments are disoriented relative to each other and may display a disarranged internal organization unlike normal retinal photoreceptors. Furthermore, other pineal outer segments often appear degenerate. Since immature stages in the development of new outer segments also appear to be present, adult pineal photoreceptors are probably engaged in a constant renewal of outer segment membranes. The evidence further suggests that macrophages are involved in phagocytosis of degenerated outer segments. Postulated photoreceptor activities and the possibility of secondary pineal functions, such as secretion, are discussed in view of current morphological and cytochemical findings.     

THE PHOTOTROPIC EFFECT OF POLARIZED LIGHT

For the growing cell of Phycomyces, a difference in the phototropic effect of light is described depending on its plane of polarization with reference to the axis of the cell. The difference which is found is primarily due to differences in the reflection losses at the cell surface. The magnitude of the effect approximates that deduced from the theory of phototropism suggested for this system. No specific effect of plane polarized light on the growth processes of the cell need be postulated.     

A NOTE ON THE RELATIVE PHOTOSENSORY EFFECT OF POLARIZED LIGHT

Experiments were made to compare the stimulating effectiveness of vertically and horizontally polarized lights and non-polarized lights of equal intensity upon phototropic movements of the beetle Tetraopes tetraopthalmus; and to compare the effectiveness of two light beams polarized at right angles to one another upon phototropic orientation of the land isopod Cylisticus convexus. Tetraopes is positively, and Cylisticus, negatively phototropic. Tests were also made of the intensities of horizontally and of vertically polarized light required to inhibit stereotropism in larvæ of Tenebrio. Under the conditions of the tests, no certain qualitative effect connected with polarization could be detected.     

THE ULTRASTRUCTURE OF THE SENSORY HAIRS AND ASSOCIATED ORGANELLES OF THE COCHLEAR INNER HAIR CELL, WITH REFERENCE TO DIRECTIONAL SENSITIVITY

From the apical end of the inner hair cell of the organ of Corti in the guinea pig cochlea protrude four to five rows of stereocilia shaped in a pattern not unlike the wings of a bird. In the area devoid of cuticular substance facing toward the tunnel of Corti lies a consistently present centriole. The ultrastructure of this centriole is similar to that of the basal body of the kinocilium located in the periphery of the sensory hair bundles in the vestibular and lateral line organ sensory cells and to that of the centrioles of other cells. The physiological implications of the anatomical orientation of this centriole are discussed in terms of directional sensitivity.     

Organization of the olfactory system of the Indian major carp <Emphasis Type="Italic">Labeo rohita</Emphasis> (Ham.): A scanning and transmission electron microscopy study

Catla catla, Labeo rohita, and Cirrhinus mrigala represent important alimentary fish in India. Their reproduction/breeding depends on seasons. Fish perceive external factors-stimuli and chemical signals through the olfactory system that plays the key role in central regulation of reproduction. However, no electron microscopy data are available on organization of olfactory components of these fish. We studied organization of the olfactory organ in male L. rohita using scanning (SEM) and transmission electron microscopy (TEM). This organ consists of olfactory epithelium, a short nerve, and olfactory bulb. The olfactory organ is ovoid in shape and consists of about 47–52 lamellae in adults and about 14–20 lamellae in fingerlings. These lamellae originate from the midline raphe. By SEM, microvillar sensory and ciliated non-sensory cells were observed in the lamellae. TEM revealed microvillar receptor cell with rough endoplasmic reticulum and Golgi apparatus towards apical end. Basal cells were present at the base of receptor cell, supporting cells were located adjacent to the olfactory receptor neurons, while epithelial cells—in the nonsensory part of olfactory epithelium. Mast, blastema, and macrophage cells were also found at the basement membrane. This work is the first publication on ultrastructural organization of the olfactory system of the Indian major carp, which provides information about morphological and ultrastructural organization of the olfactory system and opens new avenues for further investigation of chemical neuroanatomy, sensory signal processing, and neural regulation of reproduction in the Indian major carp.     

CD73 Activity is Dispensable for the Polarization of M2 Macrophages

The ectoenzyme CD73 catalyzes the hydrolysis of AMP, and is one of the most important producers of extracellular adenosine. On regulatory T cells, CD73 is necessary for immunosuppressive functions, and on Th17 cells CD73-generated adenosine exerts anti-inflammatory effects. However, the expression and function of CD73 in pro-inflammatory M1 and in immunosuppressive M2 macrophages is largely unknown. Here we show that CD73 expression and enzyme activity were induced in in vitro polarized pro-inflammatory human M(LPS+TNF) monocytes/macrophages, while CD73 was absent from immunosuppressive M(IL-4+M-CSF)-polarized macrophages. Inhibition of CD73 activity with the inhibitor AMPCP did not affect the polarization of human monocytes. In mice, CD73 was present on resident peritoneal macrophages. In striking contrast, elicited peritoneal macrophages remained CD73 negative regardless of their polarization towards either a pro-inflammatory M(LPS) or anti-inflammatory M(IL-4c) direction. Finally, the ability of peritoneal macrophages to polarize to pro- and anti-inflammatory cells was perfectly normal in CD73-deficient mice in vivo. These data indicate that, in contrast to other major leukocyte subpopulations, CD73 activity on macrophages does not play a major role in their polarization and that in mice host CD73 on any cell type is not required in vivo for peritoneal macrophage polarization towards either a pro- or an anti-inflammatory direction.     

BDNF-TrkB Axis Regulates Migration of the Lateral Line Primordium and Modulates the Maintenance of Mechanoreceptor Progenitors

BDNF and its specialized receptor TrkB are expressed in the developing lateral line system of zebrafish, but their role in this organ is unknown. To tackle this problem in vivo, we used transgenic animals expressing fluorescent markers in different cell types of the lateral line and combined a BDNF gain-of-function approach by BDNF mRNA overexpression and by soaking embryos in a solution of BDNF, with a loss-of-function approach by injecting the antisence ntrk2b-morpholino and treating embryos with the specific Trk inhibitor K252a. Subsequent analysis demonstrated that the BDNF-TrkB axis regulates migration of the lateral line primordium. In particular, BDNF-TrkB influences the expression level of components of chemokine signaling including Cxcr4b, and the generation of progenitors of mechanoreceptors, at the level of expression of Atoh1a-Atp2b1a.     

西伯利亚鲟侧线神经丘发育过程的观察

研究对西伯利亚鲟(Acipenser baerii)的胚后幼鱼进行石蜡切片HE染色,同时利用荧光染料DiA4-(4-Diethylaminostyryl)-1-methylpyridinium iodide对侧线管中侧线神经丘毛细胞特异性标记的特点,示踪了西伯利亚鲟胚后仔鱼各个时期侧线神经丘分化发育的过程。结果显示,西伯利亚鲟侧线管内侧线神经丘毛细胞如纤毛状,呈竖立紧密排列。出膜3d仔鱼眼眶后神经基板发育分化活动剧烈,出膜10d的仔鱼眼眶后方的神经基板分化出眼眶上下侧线神经丘的两个分支,同时眼眶后神经基板进一步向后分化发育在眼眶后部形成躯干侧线神经丘,但整个侧线神经丘还未完全发育完成,待出膜15d时,眼眶上下和躯干侧线神经丘已基本发育完全,出膜22d的仔鱼侧线神经丘发育基本完成。研究为今后深入研究西伯利亚鲟侧线发育过程中的神经分化发育、细胞迁移奠定了初步形态学基础。       

BREFELDIN A DISTORTS DISCOID AND POLARIZED MORPHOLOGICAL ORGANIZATIONS OF EPITHELIAL CELLS IN CULTURE

Brefeldin A (BFA), an agent disorganizing organelle traffic, was used to find out the role of this traffic in the maintenance of shape and cytoskeletal organization of cultured epithelial MDCK cells. Cell shape was assessed using dispersion and elongation indices (Dunn and Brown, 1986,J Cell Sci83: 313–340). BFA reversibly transformed discoid MDCK cells with circular actin pattern into moderately polarized cells with straight actin bundles. In other experiments the same MDCK cells were first transformed into highly polarized fibroblast-like cells by incubation with ‘scatter factor’ (HGF/SF) and then treated with BFA. In these conditions BFA significantly decreased the degree of polarization. It is suggested that both highly polarized and discoid morphological organizations are controlled by organelle traffic.     

Compartmentalized Notch signaling sustains epithelial mirror symmetry

Bilateral symmetric tissues must interpret axial references to maintain their global architecture during growth or repair. The regeneration of hair cells in the zebrafish lateral line, for example, forms a vertical midline that bisects the neuromast epithelium into perfect mirror-symmetric plane-polarized halves. Each half contains hair cells of identical planar orientation but opposite to that of the confronting half. The establishment of bilateral symmetry in this organ is poorly understood. Here, we show that hair-cell regeneration is strongly directional along an axis perpendicular to that of epithelial planar polarity. We demonstrate compartmentalized Notch signaling in neuromasts, and show that directional regeneration depends on the development of hair-cell progenitors in polar compartments that have low Notch activity. High-resolution live cell tracking reveals a novel process of planar cell inversions whereby sibling hair cells invert positions immediately after progenitor cytokinesis, demonstrating that oriented progenitor divisions are dispensable for bilateral symmetry. Notwithstanding the invariably directional regeneration, the planar polarization of the epithelium eventually propagates symmetrically because mature hair cells move away from the midline towards the periphery of the neuromast. We conclude that a strongly anisotropic regeneration process that relies on the dynamic stabilization of progenitor identity in permissive polar compartments sustains bilateral symmetry in the lateral line.     

DNA ORGANIZATION IN THE MATURE SPERM OF SEVERAL ORTHOPTERA BY THE METHOD OF POLARIZED FLUORESCENCE MICROSCOPY

Mature sperm of Acheta domesticus, Acheta assimilis, Nemobius fasciatus, Nemobius confusus, Melanoplus femur-rubrum, Romalea microptera, Scudderia curvicauda, and Ceuthophilus nigricans were examined for DNA configuration by means of polarized fluorescence microscopy. In all cases, the results suggest that the DNA lies in unsupercoiled array, predominantly parallel to the elongate sperm head axes. Detailed calculations of the factors influencing the degree of polarization of the fluorescent emission from supercoiled DNA are contained in an appendix.     

RAPID DEGENERATION OF AMPULLARY ELECTRORECEPTOR ORGANS AFTER DENERVATION

Electroreceptors (ampullary organs) of the transparent catfish (Kryptopterus bicirrhus) lie in the epidermis, and contain spherical receptor cells that receive purely afferent innervation from the lateral line nerve. Section of this nerve causes rapid degenerative changes to occur in the receptors. Fine structural alterations occur in the receptor cell synapses and nerve fiber 6–12 h postoperatively. Disruption of the receptor cells begins by 18 h and most are lost by 48 h. By 72 h supporting cells and secretory cells also show marked degeneration, and by 96 h they may be totally lost. The rapid degeneration of the electroreceptor organs of Kryptopterus should make them a useful preparation for analysis of neurotrophic functions.     

Isolation and characterization of a novel plasma membrane protein, osteoblast induction factor (obif), associated with osteoblast differentiation

Background

The exocrine pancreas is composed of a branched network of ducts connected to acini. They are lined by a monolayered epithelium that derives from the endoderm and is surrounded by mesoderm-derived mesenchyme. The morphogenic mechanisms by which the ductal network is established as well as the signaling pathways involved in this process are poorly understood.

Results

By morphological analyzis of wild-type and mutant mouse embryos and using cultured embryonic explants we investigated how epithelial morphogenesis takes place and is regulated by chemokine signaling. Pancreas ontogenesis displayed a sequence of two opposite epithelial transitions. During the first transition, the monolayered and polarized endodermal cells give rise to tissue buds composed of a mass of non polarized epithelial cells. During the second transition the buds reorganize into branched and polarized epithelial monolayers that further differentiate into tubulo-acinar glands. We found that the second epithelial transition is controlled by the chemokine Stromal cell-Derived Factor (SDF)-1. The latter is expressed by the mesenchyme, whereas its receptor CXCR4 is expressed by the epithelium. Reorganization of cultured pancreatic buds into monolayered epithelia was blocked in the presence of AMD3100, a SDF-1 antagonist. Analyzis of sdf1 and cxcr4 knockout embryos at the stage of the second epithelial transition revealed transient defective morphogenesis of the ventral and dorsal pancreas. Reorganization of a globular mass of epithelial cells in polarized monolayers is also observed during submandibular glands development. We found that SDF-1 and CXCR4 are expressed in this organ and that AMD3100 treatment of submandibular gland explants blocks its branching morphogenesis.

Conclusion

In conclusion, our data show that the primitive pancreatic ductal network, which is lined by a monolayered and polarized epithelium, forms by remodeling of a globular mass of non polarized epithelial cells. Our data also suggest that SDF-1 controls the branching morphogenesis of several exocrine tissues.