Neurturin-GFRalpha2 signaling controls liver bud migration along the ductus venosus in the chick embryo

During chick liver development, the liver bud arises from the foregut, invaginates into the septum transversum, and elongates along and envelops the ductus venosus. However, the mechanism of liver bud migration is only poorly understood. Here, we demonstrate that a GDNF family ligand involved in neuronal outgrowth and migration, neurturin (NRTN), and its receptor, GFRalpha2, are essential for liver bud migration. In the chick embryo, we found that GFRalpha2 was expressed in the liver bud and that NRTN was expressed in the endothelial cells of the ductus venosus. Inhibition of GFRalpha2 signaling suppressed liver bud elongation along the ductus venous without affecting cell proliferation and apoptosis. Moreover, ectopic expression of NRTN perturbed the directional migration along the ductus venosus, leading to splitting or ectopic branching of the liver. We showed that liver buds selectively migrated toward an NRTN-soaked bead in vitro. These data represent a new model for liver bud migration: NRTN secreted from endothelial cells functions as a chemoattractant to direct the migration of the GFRalpha2-expressing liver bud in early liver development.     

大鼠输精管平滑肌细胞的体外培养及形态学特征(英文）

建立大鼠输精管平滑肌细胞的培养方法。取大鼠输精管,剥离外膜和内膜,用组织块法进行体外培养。用抗α-SMA(anti α-smooth muscle actin)免疫组化染色的方法鉴定培养的细胞。结果显示,在倒置显微镜下观察细胞形态多样,表现为长梭形或星形,细胞伸出突起互相接触,彼此融合,部分区域细胞多层重叠,部分区域细胞单层高低起伏,呈"峰-谷"状生长。免疫组化染色鉴定呈阳性反应,用该方法所分离、培养的输精管平滑肌细胞纯度达99%以上。应用组织块法培养大鼠输精管平滑肌细胞,操作简单,结果稳定。     

Immunohistochemistry of the cytoskeleton in the excurrent ducts of the testis in birds of the Galloanserae monophyly

The presence, location and degree of immunoexpression of various microfilament (MF) and intermediate filament (IF) systems (actin, cytokeratins, desmin, vimentin) were studied in the excurrent ducts of the testis in sexually mature and active galliform (Japanese quail, domestic fowl, turkey) and anseriform (duck) birds. These proteins were variably expressed between the epithelia and periductal tissue (periductal smooth muscle cell layer and interductal connective tissue) types and between species. Variable heterogeneous co-expression of filament systems was also found in the various duct epithelia and periductal tissue types: co-expression of filament systems was the rule rather than the exception. In the duck, neither vimentin nor cytokeratin was present in any of the tissues, whereas actin and desmin (absent in the rete testis) were co-expressed in the efferent ducts and epididymal duct unit (comprising the ductus conjugens, ductus epididymidis and ductus deferens). Actin, desmin and vimentin were generally co-expressed in the rete testis, efferent ducts and epididymal duct unit of the quail, domestic fowl and turkey, with vimentin being more strongly immunoreactive than actin and desmin in the epididymal duct unit, but more weakly immunoexpressed in the efferent ducts. Cytokeratin was present and co-expressed with actin, desmin and vimentin in the rete testis, efferent ducts and epididymal duct unit of the domestic fowl and turkey, but not in the quail and duck. The periductal smooth muscle cell layer and interductal tissue co-expressed actin, desmin and vimentin variably in all birds. Luminal spermatozoa of both the turkey and duck were immunonegative for all protein systems, whereas those of the quail and domestic fowl co-expressed actin, desmin and vimentin moderately or strongly. The tissues of the reproductive tract of male birds thus contain cytoskeletal protein systems that are variably but mostly co-expressed and whose contractile ability appears necessary and sufficient for transportation through the various excurrent ducts of the voluminous testicular fluid and its high sperm content, characteristic features of male avian reproduction.     

Elektronenmikroskopische Untersuchungen an Zellen des Ductus thoracicus der Ratte nach Osmium-Zink-Jodid (OZI) Imprägnation

Zusammenfassung Die Lymphozyten des Ductus thoracicus von unbehandelten weißen Ratten und von Ratten nach Sensibilisierung mit Meerrettichperoxydase wurden nach einer zweistündigen Vorfixierung in 2,5% Glutaraldehyd in dem Osmium-Zink-Jodid-Gemisch nach Maillet (1959) inkubiert und elektronemikroskopisch untersucht. Es zeigte sich, daß bei den unbehandelten Tieren nur wenige Lymphozyten keine Reaktion im Kernspalt aufwiesen — ER und Golgi waren nicht ausgebildet —, während die Mehrzahl der Zellen eine starke OZI-Imprägnation des Kernspalts, des ER, des Golgifeldes und der Mitochondrienmatrix zeigte. Die Membransysteme und Mitochondrien der Plasmazellen, die in der Ductuslymphe nach Sensibilisierung auftraten, waren ebenfalls stark OZI-positiv. Auf Grund dieser Befunde halten wires für sehr wahrscheinlich, daß das OZI-Gemisch mit bestimmten Proteinen reagiert, auch mit Antikörpern. Bei einer Erhöhung des pH-Werte auf 6,5 findet in den genannten Systemen keine Reaktion mehr statt.

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Electron microscopic studies of ductus thoracicus cells of the rat after osmium-zinc-jodide (OZI) impregnation

Summary Ductus thoracicus lymphocytes from untreated white rats and from rats after sensibilisation with horseradish peroxydase were incubated in Maillet's (1959) osmiumzinc-jodide mixture after prefixation in 2.5% glutaraldehyd, and examined electron microscopically. It is demonstrated that among the untreated lymphocytes only few cells show no reaction product in the perinuclear space—ER and Golgi A. are not to be observed—while the majority exhibits strong reaction in the nuclear envelope, the ER, the Golgi field and the matrix of the mitochondria. All plasma cells, present in the thoracic duct lymph after sensibilisation with horseradish peroxidase show also a strong OZI-reaction in their cisternal elements and in the mitochondria. According to these findings there is strong evidence that the OZI-solution reacts with certain proteins also with antibodies. Setting the pH of the OZI-solution to 6.5 no more reaction is found in the formentioned systems.

     

Developmental changes in the effects of prostaglandin E<Subscript>2</Subscript> in the chicken ductus arteriosus

Prostaglandin E₂ (PGE₂) is the major vasodilator prostanoid of the mammalian ductus arteriosus (DA). In the present study we analyzed the response of isolated DA rings from 15-, 19- and 21-day-old chicken embryos to PGE₂ and other vascular smooth muscle relaxing agents acting through the cyclic AMP signaling pathway. PGE₂ exhibited a relaxant response in the 15-day DA, but not in the 19- and 21-day DA. Moreover, high concentrations of PGE₂ (≥3 μM in 15-day and ≥1 μM in 19-day and 21-day DA) induced contraction of the chicken DA. The presence of the TP receptor antagonist SQ29,548, unmasked a relaxant effect of PGE₂ in the 19- and 21-day DA and increased the relaxation induced by PGE₂ in the 15-day DA. The presence of the EP receptor antagonist AH6809 abolished PGE₂-mediated relaxation. The relaxant responses induced by PGE₂ and the β-adrenoceptor agonist isoproterenol, but not those elicited by the adenylate cyclase activator forskolin or the phosphodiesterase 3 inhibitor milrinone, decreased with maturation. High oxygen concentrations (95%) decreased the relaxation to PGE₂. The relaxing potency and efficacy of isoproterenol and milrinone were higher in the pulmonary than in the aortic side of the DA, whereas no regional differences were found in the response to PGE₂. We conclude that, in contrast to the mammalian situation, PGE₂ is a weak relaxant agent of the chicken DA and, with advancing incubation, it even stimulates TP vasoconstrictive receptors.     

Morphological analysis of the hagfish heart. II. The venous pole and the pericardium

The morphological characteristics of the venous pole and pericardium of the heart were examined in three hagfish species, Myxine glutinosa, Eptatretus stoutii, and Eptatretus cirrhatus. In these species, the atrioventricular (AV) canal is long, funnel‐shaped and contains small amounts of myocardium. The AV valve is formed by two pocket‐like leaflets that lack a papillary system. The atrial wall is formed by interconnected muscle trabeculae and a well‐defined collagenous system. The sinus venosus (SV) shows a collagenous wall and is connected to the left side of the atrium. An abrupt collagen‐muscle boundary marks the SV‐atrium transition. It is hypothesized that the SV is not homologous to that of other vertebrates which could have important implications for understanding heart evolution. In M. glutinosa and E. stoutii, the pericardium is a closed bag that hangs from the tissues dorsal to the heart and encloses both the heart and the ventral aorta. In contrast, the pericardium is continuous with the loose periaortic tissue in E. cirrhatus. In all three species, the pericardium ends at the level of the SV excluding most of the atrium from the pericardial cavity. In M. glutinosa and E. stoutii, connective bridges extend between the base of the aorta and the ventricular wall. In E. cirrhatus, the connections between the periaortic tissue and the ventricle may carry blood vessels that reach the ventricular base. A further difference specific to E. cirrhatus is that the adipose tissue associated with the pericardium contains thyroid follicles. J. Morphol. 277:853–865, 2016. © 2016 Wiley Periodicals, Inc.     

A centrosomal inclusion (striped nebulous body) in pinealocytes of the golden hamster

Summary The postnatal maturation of regions of the epididymis and intragonadal segment of the deferens duct was studied in the rat by light-and transmission electron microscopy. Maturation of the genital duct starts in the distal cauda epididymidis and ductus deferens after one week of life, and one week later, in the more cranial segments of the epididymis. Epithelial principal cells and peritubular contractile cells are structurally mature 35 days after birth. The synchronous changes of these cells indicate that the same factors control their postnatal maturation. The epithelial principal cells obtain an endocytotic apparatus and long stereocilia, whereas peritubular cells acquire contractile features. These changes are associated with a progressive increase in the immunoreaction for smooth muscle actin in both cell types. Smooth muscle myosin is detected in the apical region of the epithelial cells and the peritubular cell cytoplasm by day one of postnatal development. The differentiation of contractile cells in the wall is accompanied by progressive organization of the pericellular matrix into a continuous basement membrane. Although fibronectin is visible at birth, it is gradually removed from the tubule wall.     

The basement membranes of cryofixed or aldehyde-fixed,freeze-substituted tissues are composed of a lamina densa and do not contain a lamina lucida

When tissues are processed for electron microscopy by conventional methods, such as glutaraldehyde fixation followed by rapid dehydration in acetone, basement membranes show two main layers: the electron-lucent lamina lucida (or rara) and the electrondense lamina densa. In an attempt to determine whether this subdivision is real or artefactual, two approaches have been used. Firstly, rat and mouse seminiferous tubules, mouse epididymis and associated tissues, and anterior parts of mouse eyes were subjected to cryofixation by instant freezing followed by freeze substitution in a-80° C solution of osmium tetroxide in dry acetone, which was gradually warmed to room temperature over a 3-day period. The results indicate that, in areas devoid of ice crystals, basement membranes consist of a lamina densa in direct contact with the plasmalemma of the associated cells without an intervening lamina lucida. Secondly, a series of tissues from mice perfused with 3% glutaraldehyde were cryoprotected in 30% glycerol, frozen in Freon 22 and subjected to a 3-day freeze substitution in osmium tetroxide-acetone as above. Under these conditions, no lamina lucida accompanies the lamina densa in the basement membranes of the majority of tissues, including kidney, thyroid gland, smooth and skeletal muscle, ciliary body, seminiferous tubules, epididymis and capillary endothelium. Thus, even though these tissues have been fixed in glutaraldehyde, no lamina lucida appears when they are slowly dehydrated by freeze substitution. It is concluded that the occurrence of this lamina in conventionally processed tissues is not due to fixation but to the rapid dehydration. However, in this series of experiments, the basement membranes of trachea and plantar epidermis include a lamina lucida along their entire length, while those of esophagus and vas deferens may or may not include a lamina lucida. To find out if the lamina lucida appearing under these conditions is a real structure or an artefact, the trachea and epidermis were fixed in paraformaldehyde and slowly dehydrated by freeze substitution. Under these conditions, no lamina lucida was found. Since this result is the same as observed in other tissues by the previous approaches, it is proposed that the lamina lucida is an artefact in these as in the other investigated basement membranes. Thus, basement membranes are simply composed of a lamina densa that closely follows the plasmalemma of the associated cells. At high magnification, the lamina densa consists of a tridimensional network of cords, while the plasmalemma is covered by a glycocalyx; close contact is observed between cords and glycocalyx and is interpreted by assuming that the laminin present in the cords binds to laminin receptors in the glycocalyx.     

Tachykinin-like immunoreactivity in the sinus venosus of the dogfish (Scyliorhinus canicula)

The sinus venosus of the elasmobranch heart is characterized by the presence of large bundles of unmyelinated nerve fibres that bulge into the cardiac lumen, below the endocardium. In the dogfish (Scyliorhinus canicula), these fibres contain numerous dense-core membrane-bounded granules of about 200 nm in diameter. Most intramural ganglion cells of the sinus venosus also show densely packed granules similar to those found in the subendocardial fibres. We have observed strong substance-P-like immunoreactivity in the large fibre bundles and in the perikarya of the ganglion cells. Preabsorption of the antisera with fragment 7–11 of substance P has shown that the antisera recognize the tachykinin canonic sequence. Our findings suggest that an undetermined tachykinin is secreted in the elasmobranch heart, and that it is probably released into the blood stream in the context of a little-known neuroendocrine system.     

The response of the lamb ductus arteriosus to endothelin: developmental changes and influence of light

Endothelin-1 (ET-1) is a putative messenger of oxygen in the ductus arteriosus. Since the ability of the vessel to contract to oxygen increases with gestation, we wished to ascertain whether ET-1 action is also developmentally regulated. A corollary objective was to assess whether any gestational variation in the ET-1 contraction is due to a change in the ET(A)-mediated action or to a shift in the balance between opposing, contractile (ET(A) - mediated) and relaxant (ET(B)-mediated), actions. Experiments were performed with isolated ductal strips from preterm (0.7 gestation) and near-term fetal lambs. ET-1 contracted the ductus dose-dependently (10(-10)-10(-7) M) at both ages; however, the peak contraction was about double in magnitude at term. Regardless of age, ET-1 contraction was greater with preparations kept in the dark compared to those exposed to light. This effect of light was not seen after removing the endothelium or when treating the intact tissue with the ET(B) antagonist BQ788 (1 microM). In the dark, however, BQ788 did not modify significantly the ET-1 response at either age. We conclude that ET-1 becomes a stronger ductus constrictor with fetal age, conceivably by acting on ET(A) receptors. Hence, the concept of ET-1 mediating the oxygen contraction is further validated. Peculiarly, the ET-1 contraction is curtailed by light through a hitherto undefined ET(B) receptor-linked process.