Cell type complexity in the basal metazoan Hydra is maintained by both stem cell based mechanisms and transdifferentiation

Understanding the mechanisms controlling the stability of the differentiated cell state is a fundamental problem in biology. To characterize the critical regulatory events that control stem cell behavior and cell plasticity in vivo in an organism at the base of animal evolution, we have generated transgenic Hydra lines [Wittlieb, J., Khalturin, K., Lohmann, J., Anton-Erxleben, F., Bosch, T.C.G., 2006. Transgenic Hydra allow in vivo tracking of individual stem cells during morphogenesis. Proc. Natl. Acad. Sci. U. S. A. 103, 6208-6211] which express eGFP in one of the differentiated cell types. Here we present a novel line which expresses eGFP specifically in zymogen gland cells. These cells are derivatives of the interstitial stem cell lineage and have previously been found to express two Dickkopf related genes [Augustin, R., Franke, A., Khalturin, K., Kiko, R., Siebert, S. Hemmrich, G., Bosch, T.C.G., 2006. Dickkopf related genes are components of the positional value gradient in Hydra. Dev. Biol. 296 (1), 62-70]. In the present study we have generated transgenic Hydra in which eGFP expression is under control of the promoter of one of them, HyDkk1/2/4 C. Transgenic Hydra recapitulate faithfully the previously described graded activation of HyDkk1/2/4 C expression along the body column, indicating that the promoter contains all elements essential for spatial and temporal control mechanisms. By in vivo monitoring of eGFP+ gland cells, we provide direct evidence for continuous transdifferentiation of zymogen cells into granular mucous cells in the head region. We also show that in this tissue a subpopulation of mucous gland cells directly derives from interstitial stem cells. These findings indicate that both stem cell-based mechanisms and transdifferentiation are involved in normal development and maintenance of cell type complexity in Hydra. The results demonstrate a remarkable plasticity in the differentiation capacity of cells in an organism which diverged before the origin of bilaterian animals.     

Rho GTPase controls invagination and cohesive migration of the Drosophila salivary gland through Crumbs and Rho-kinase

Coordinated cell movements shape simple epithelia into functional tissues and organs during embryogenesis. Regulators and effectors of the small GTPase Rho have been shown to be essential for epithelial morphogenesis in cell culture; however, the mechanism by which Rho GTPase and its downstream effectors control coordinated movement of epithelia in a developing tissue or organ is largely unknown. Here, we show that Rho1 GTPase activity is required for the invagination of Drosophila embryonic salivary gland epithelia and for directed migration of the internalized gland. We demonstrate that the absence of zygotic function of Rho1 results in the selective loss of the apical proteins, Crumbs (Crb), Drosophila atypical PKC and Stardust during gland invagination and that this is partially due to reduced crb RNA levels and apical localization. In parallel to regulation of crb RNA and protein, Rho1 activity also signals through Rho-kinase (Rok) to induce apical constriction and cell shape change during invagination. After invagination, Rho-Rok signaling is required again for the coordinated contraction and dorsal migration of the proximal half of the gland. We also show that Rho1 activity is required for proper development of the circular visceral mesoderm upon which the gland migrates. Our genetic and live-imaging analyses provide novel evidence that the proximal gland cells play an essential and active role in salivary gland migration that propels the entire gland to turn and migrate posteriorly.     

乳腺特异性表达172~（HIS）突变型p53转基因鼠的建立

ｐ５３基因突变几乎存在于所有人类肿瘤中,尤其与严重危害妇女健康的恶性肿瘤──乳腺癌关系密切。研究表明：野生型ｐ５３基因是抑癌基因,而突变型ｐ５３基因则是癌基因──呈显性负调控突变型。本研究首次构建了这种类型的１７２ＨＩＳ突变型ｐ５３基因的转基因鼠,用以研究ｐ５３基因突变与乳腺癌发生的关系。用重组ＰＣＲ法把小鼠ｐ５３基因的第１７２个密码子ＣＧＣ突变成ＣＡＣ（由编码精氨酸突变为编码组氨酸）,得到１７２ＨＩＳ突变型ｐ５３基固。将其插入到大鼠乳清酸性蛋白（ＷＡＰ）启动子与ＳＶ４０ｐｏｌｙ（Ａ）信号序列之间。获得置于载体ｐＢＬ１１９（Ｆｉｇ．１）上的表达结构ＷＡＰ－１７２ＨＩＳｍｔｐ５３－ＳＶ４０。经过ＢｓｓＨＩＩ酶切,纯化该表达结构部分。通过显微注射,将其分别导入ＦＶＢ和Ｃ５７ＢＬ品系小鼠受精卵中,获得３２只Ｆ０代鼠。经ＰＣＲ鉴定,其中９只为转基因阳性鼠（Ｆｉｇ．２）。对其进行的Ｓｏｕｔｈｅｒｎ分析（Ｆｉｇ．３）证实了ＰＣＲ的鉴定,并得出了每只鼠中整合的转基因拷贝数（Ｔａｂｌｅ１）。当这９只鼠哺乳第二天时,取乳腺制备ＲＮＡ,进行Ｎｏｒｔｈｅｒｎ杂交分析,其中５只的乳腺中有转基因表达,其中３只呈较高水平表达（Ｆｉｇ．     

Tracheal submucosal gland serous cells stimulated in vitro with adrenergic and cholinergic agonists A morphometric study

Summary A morphometric analysis was made of alterations in serous cell structure induced by adrenergic and cholinergic agonists. Ferret tracheal rings were exposed for 30 min in vitro to one of the following agonists: phenylephrine, terbutaline, or methacholine (all at 10^–5 M). Controls were incubated similarly in medium containing no drugs or medium containing both the agonist and an excess of the appropriate antagonist (phentolamine, propranolol or atropine, all at 10^–4 M).Electron microscopic observation and stereological analysis of the incubated samples revealed that the volume density of serous cell granules in controls (0.30 ± 0.02, mean ± SE, n = 4) was significantly reduced by phenylephrine (0.19 ± 0.03, n = 4) and methacholine (0.17 ± 0.01, n = 4), but not by terbutaline (0.27 ± 0.04, n = 4). The presence of antagonists in the medium prevented the observed changes (phenylephrine/phentolamine: 0.29 ± 0.03, n = 3 and methacholine/atropine: 0.33 ± 0.06, n = 3). In addition, the volume density of intracellular vacuoles in controls (0.02 ± 0.005, n = 4) was increased in response to methacholine stimulation (0.12 + 0.05, n = 4), but not in response to the other agonists. This effect was blocked by atropine (0.01 ± 0.00, n = 3).We conclude that serous-cell granules are discharged by both alpha-adrenergic and cholinergic, but not beta-adrenergic stimulation. In addition, cholinergic stimulation evokes the formation of intracellular vacuoles, a possible indication of active ion and water transport.     

Autophagie in den Drüsenzellen des Magens bei jungen Mäusen

Zusammenfassung Autophagische Prozesse in verschiedenen Drüsenzellen des Magens neugeborener bis 8 Tage alter Mäuse wurden elektronenmikroskopisch untersucht. Es zeigte sich dabei, daß die intrazellulären Abbauprozesse in jeder Zellart typisch verlaufen. In den mukoiden Nebenzellen findet man große autophagische Vakuolen mit zahlreichen Mitochondrien, in denen beträchtliche Cytoplasmaregionen sequestriert sein können. Stärker abgebaute Mitochondrien liegen oft im Drüsenlumen oder in den interzellulären Räumen. Anscheinend wird der Inhalt autophagischer Vakuolen in sie ausgeschieden. Alle Zellen mit Autophagosomen enthalten gleichzeitig viele und formenreiche Lysosomen. Einige abgenutzte Zellen des mukoiden Oberflächenepithels scheinen durch Heterophagie abgebaut zu werden. In kleinen Autophagosomen dieser Zellen findet man nur einzelne Organellen. In den Belegzellen ist die Autophagie viel seltener. Typisch für die Belegzellen sind stark osmiophile multivesikuläre Körperchen, die sehr wahrscheinlich Lysosomen verkörpern. In den Hauptzellen, die erst nach der Geburt ausdifferenziert werden, trifft man nur ausnahmsweise sequestrierte Zellpartien.Die Ergebnisse werden unter Berücksichtigung von Beobachtungen anderer Autoren, die die intrazellulären Abbauprozesse analysierten, und eigener früher erzielter Resultate diskutiert.

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Autophagy in gastric gland cells of young mice

Summary Autophagy in different gastric gland cells of newborn till 8 days old mice were studied electronmicroscopically. Intracellular digestion goes on in a way typical for each cell type. In the mucous neck cells, big autophagic vacuoles occur in which a large part of cytoplasm with numerous mitochondria is separated. The damaged mitochondria lie very often in the gland lumen or in the intercellular space. It seems, that the contents of autophagic vacuoles can be extruded in these places. All cells with autophagic vacuoles contain also numerous lysosomes of different shape. Some involuted mucous cells of the surface epithelium seem to be digested in a heterophagic process. Autophagy does not appear often in parietal gastric cells. The autophagosomes of these cells are small, and they contain only some organelles. Typical for these elements are osmiophilic multivesicular bodies, probably lysosomes. Chief cells, that differentiate only after birth, have autophagic vacuoles, but seldom. The results are discussed in comparison with results of other authors that worked on the problems of intracellular digestion.

Der Jugoslawischen Forschungsgemeinschaft Savezni Fond (Belgrad) und Sklad Borisa Kidria (Ljubljana) danken wir für finanzielle Unterstützung, Frau B. Zorman und Herrn F. Kovai für technische Hilfe.     

The relationship between the morphology of cell organelles and kinetics of the secretory process in male sex accessory glands of mice

Summary Two male sex accessory glands of the mouse, seminal vesicle and coagulating gland, were compared with the aim of relating differences in the morphology of organelles to the kinetics of the secretory process. The epithelial cells of the two glands were assessed by morphometric analysis, cytochemical staining, and electron-microscopic autoradiography after administration of a labeled amino acid. The rough endoplasmic reticulum of the seminal vesicle comprised narrow parallel cisternae, while that of the coagulating gland was greatly distended and occupied a much larger percentage of the cytoplasmic volume. Radioactively labeled products were secreted much more rapidly in the seminal vesicle than in the coagulating gland. The primary point of difference in kinetics of intracellular transport between the two glands was in exit of material from the rough endoplasmic reticulum. The more rapid drainage of the rough endoplasmic reticulum may be related to its relatively greater membrane surface density and lesser internal volume. In contrast, similarities in size and cytochemical staining in the Golgi apparatus of the two glands were accompanied by similar kinetics of intracellular transport of secretory protein through this organelle.     

Organogenesis of the exocrine gland

Morphogenesis of exocrine glands is a complex stepwise process of epithelial ingrowth, ductal elongation, ductal branching, and alveolar or acinar differentiation. Emerging from an increasing number of mouse gene knockout, dominant-negative, and antisense models is the identification of a remarkable collection of cell adhesion molecules, growth factors, and their receptors whose time-dependent contributions to glandular organogenesis are essential. Many have cryptically overlapping and interdependent but noncompensatory roles. Discoidin domain receptor 1 tyrosine kinase (DDR1) and the ErbB1 receptor of amphiregulin are, for example, required for ductal branching and elongation. Each is in turn dependent on the Wnt family of morphogenic factors for autophosphorylation or transactivation, respectively. Here we review the current cast of exocrine glandular morphogens, as a foundation for a global or systems biology appreciation of the interweaving signaling pathways that underlie mammalian glandular morphogenesis.     

Fine structure of the pseudobranch of the flounder Paralichthys lethostigma

Summary The free or non-glandular pseudobranch of the flounder Paralichthys lethostigma was studied with the electron microscope. Cells typical of glandular type pseudobranchs are found. This indicates that, at least in the flounder, the free pseudobranch should be called glandular. In addition, the chloride-type cells, commonly found in the gill, buccal epithelium, and surface epithelium of other fish, have been found in the pseudobranch, where they have not been described previously. The fine structure of both the chloride-type and the pseudobranch-type cell has been characterized and contrasted. We have not been able to confirm previous reports that the specific cells in both pseudobranch and gill are identical in the flounder.Supported by grant-in-aid from the USPHS (General Medical Sciences Institute, GM-06836) and the National Science Foundation (GB-676).