Rhou maintains the epithelial architecture and facilitates differentiation of the foregut endoderm

Rhou encodes a Cdc42-related atypical Rho GTPase that influences actin organization in cultured cells. In mouse embryos at early-somite to early-organogenesis stages, Rhou is expressed in the columnar endoderm epithelium lining the lateral and ventral wall of the anterior intestinal portal. During foregut development, Rhou is downregulated in regions where the epithelium acquires a multilayered morphology heralding the budding of organ primordia. In embryos generated from Rhou knockdown embryonic stem (ES) cells, the embryonic foregut displays an abnormally flattened shape. The epithelial architecture of the endoderm is disrupted, the cells are depleted of microvilli and the phalloidin-stained F-actin content of their sub-apical cortical domain is reduced. Rhou-deficient cells in ES cell-derived embryos and embryoid bodies are less efficient in endoderm differentiation. Impaired endoderm differentiation of Rhou-deficient ES cells is accompanied by reduced expression of c-Jun/AP-1 target genes, consistent with a role for Rhou in regulating JNK activity. Downregulation of Rhou in individual endoderm cells results in a reduced ability of these cells to occupy the apical territory of the epithelium. Our findings highlight epithelial morphogenesis as a required intermediate step in the differentiation of endoderm progenitors. In vivo, Rhou activity maintains the epithelial architecture of the endoderm progenitors, and its downregulation accompanies the transition of the columnar epithelium in the embryonic foregut to a multilayered cell sheet during organ formation.     

Differential distributions of CSE1L/CAS and E-cadherin in the polarized and non-polarized epithelial glands of neoplastic colorectal epithelium

Colorectal glands are important functional organs in colorectal tissue and are also the origin of colorectal carcinomas. Epithelial cell polarization of colorectal glands is related to structural integrity and physiological functions of colorectal glands as well as colorectal carcinoma formation. The cellular apoptosis susceptibility (CSE1L/CAS) protein has been shown to induce polarity formation of human colorectal cells in cell culture. E-cadherin expression in epithelial cells is crucial for the establishment and maintenance of epithelial cell polarity. In this study we examined the distributions of CSE1L and E-cadherin in the epithelial glands of normal and neoplastic colorectal epithelium and correlated these to polarity formation in the colorectal glands. Our results showed that CSE1L was differentially stained in the epithelial glands of neoplastic colorectal epithelium, and the staining was related to gland epithelial cell polarization and E-cadherin distribution. CSE1L was associated E-cadherin in GST pull-down experiments and immunoprecipitation assays. Basolateral staining of CSE1L and E-cadherin were seen in the polarized glands of normal and neoplastic colorectal epithelium. Absence of basolateral CSE1L staining in neoplastic epithelium glands was associated with loss of gland epithelial cell polarity, and this was parallel with E-cadherin staining. The non-polarized areas in epithelium glands showed a patchy staining for CSE1L and E-cadherin. These results indicate that examination of CSE1L and E-cadherin distribution in colorectal epithelium glands may be valuable for evaluating the malignance of colorectal disease.     

Evaluation of the glyoxal-bis-(2-hydroxyanil)-method for staining of calcium in model gelatin films and pancreatic islets.

The nature of tissue calcium, detectable with glyoxal-bis-(2-hydroxyanil), (GBHA), was investigated using gelatin films as model. The results indicate that in the films the procedure detects only the calcium fraction which was ionized in the original gelatin solution. The GBHA staining intensity (absorbance) appeared to be linear with the amount of ionized calcium in the range from 0 to 2 micrograms/cm2. The method allows detection of amounts of ionized calcium as low as 0.15 micrograms/cm2 or 0.0015 pg/mu2. For the measurement of calcium in pancreatic tissue of fed rats, the tissue was subjected to freeze-substitution at -80 degree C in acetone containing 1% oxalic acid. Adjacent sections were stained with either GBHA or aldehyde-fuchsin (AF). Exocrine tissue hardly stained with GBHA whereas islet tissue stained intensely. For GBHA as well as for AF a variation in staining intensity (visual evaluation) between islets was observed. Islet GBHA- and AF-staining intensities did not correlate. The AF-staining intensity but not the GBHA-staining intensity decreased with increasing islet diameter. Also in pancreatic islet tissue the GBHA method appears to be very sensitive and reproducible and small differences in islet GBHA-staining intensity can be detected. The results indicate that between islets differences in ionized calcium content exist. These differences do not correlate with the degree of B-cell granulation.     

Cytologic study of the tissue repair cells of the uterine cervix. With special reference to their origin.

In order to characterize tissue repair cells (TR) of the uterine cervix and clarify their origin, exfoliated cells obtained after laser conization for early cervical lesions were examined. One specimen was first examined by Papanicolaou staining and then examined for epithelial membrane antigen (EMA) and vimentin by immunocytochemical staining, using a restaining method. The other specimen was observed by phosphotungstic acid-hematoxylin (PTAH) staining. Morphologic findings on TR were investigated together with the histologic findings on the wound healing process. TR were classified morphologically into three groups: stromal (STR), epithelial (ETR) and of unknown origin (UTR). Validity of this classification was confirmed by the findings of immunocytochemical staining with EMA and vimentin. These cells appeared one to eight weeks after laser conization. TR with relatively large nuclei, or atypical TR (ATR), appeared when each type of TR was most plentiful, at two to five weeks. Regarding the origins of each TR, cytologic and histologic findings could be considered to offer evidence that ETR originated with hyperplasia of immature cells of the squamous epithelium or reserve cells below the columnar epithelium. The presence of myofibrils in cytoplasm, demonstrated by PTAH staining for STR and some UTR, strongly suggested the possibility that these cells were myofibroblasts in granulation tissue.     

Collagenase-3 expression in periapical lesions: an immunohistochemical study

Collagenase-3 (matrix metalloproteinase-13) is a metalloproteinase (MMP) that is associated with bone lesions and exhibits variable expression patterns in odontogenic cysts; it may play a role in regulating focal proliferation and maturation of jaw cyst epithelium. We studied the localization, staining intensity and distribution of collagenase-3 in 13 periapical granulomas with epithelium, 16 periapical granulomas without epithelium and 10 radicular cysts using archived formalin fixed, paraffin embedded tissues. A monoclonal antibody against human collagenase-3 was used to evaluate its expression. Immunohistochemical staining intensities of collagenase-3 in all periapical lesions were (?), 4 (10%); (+), 1 (3%); (++), 22 (56%) and (+++), 12 (31%); differences were not statistically significant. Immunohistochemical distribution of collagenase-3 in epithelial cells was (?), 17 (44%); (+), 17 (44%); (++), 5 (13%); in fibroblasts it was (?), 8 (20%); (+), 23 (59%); (++), 8 (21%); in plasma cells it was (?), 7 (18%); (+), 22 (56%); (++), 10 (26%); in macrophages it was (?), 7 (18%); (+), and 15 (38%); and (++), 17 (44%). Statistically significant differences were found in epithelial cells (p = 0.00) and fibroblasts (p = 0.02), whereas differences were not statistically significant for plasma cells and macrophages. Collagenase-3 may play a role in the conversion of a periapical granuloma with epithelium to radicular cyst. MMP's influence not only epithelial rest cell migration, but also invasion of various stromal cells into granulomatous tissue.     

Genetic Analysis of the Early Natural History of Epithelial Ovarian Carcinoma

Background

The high mortality rate associated with epithelial ovarian carcinoma (EOC) reflects diagnosis commonly at an advanced stage, but improved early detection is hindered by uncertainty as to the histologic origin and early natural history of this malignancy.

Methodology/Principal Findings

Here we report combined molecular genetic and morphologic analyses of normal human ovarian tissues and early stage cancers, from both BRCA mutation carriers and the general population, indicating that EOCs frequently arise from dysplastic precursor lesions within epithelial inclusion cysts. In pathologically normal ovaries, molecular evidence of oncogenic stress was observed specifically within epithelial inclusion cysts. To further explore potential very early events in ovarian tumorigenesis, ovarian tissues from women not known to be at high risk for ovarian cancer were subjected to laser catapult microdissection and gene expression profiling. These studies revealed a quasi-neoplastic expression signature in benign ovarian cystic inclusion epithelium compared to surface epithelium, specifically with respect to genes affecting signal transduction, cell cycle control, and mitotic spindle formation. Consistent with this gene expression profile, a significantly higher cell proliferation index (increased cell proliferation and decreased apoptosis) was observed in histopathologically normal ovarian cystic compared to surface epithelium. Furthermore, aneuploidy was frequently identified in normal ovarian cystic epithelium but not in surface epithelium.

Conclusions/Significance

Together, these data indicate that EOC frequently arises in ovarian cystic inclusions, is preceded by an identifiable dysplastic precursor lesion, and that increased cell proliferation, decreased apoptosis, and aneuploidy are likely to represent very early aberrations in ovarian tumorigenesis.     

Gross and histological characterization of endometrial tissues from SLA miniature pigs with cystic endometrial hyperplasia

Cystic endometrial hyperplasia (CEH) is a uterine disorder characterized by the formation of large numbers of cysts in the endometrium. The purpose of this study was to examine and characterize cell types in the endometrium associated with the cysts and uterine glands. No apparent histological differences between CEH-involved and normal uterine columnar epithelium were found. Endometrial glands in CEH-involved and normal uteri were lined with simple or ciliated columnar epithelial cells and surrounded by lamellar connective tissue. The cyst epithelium appeared to be stretched obliquely and compressed so that both the cells and nuclei were horizontally oriented relative to the cyst lumen and were surrounded by lamellar connective tissue. Electron microgaphs revealed an abnormally high number of mitochondria in the cystic cells as compared to normal glandular cells. In conclusion, CEH is characterized by the formation of cysts which develop from the uterine glandular tissue. Epithelial cells lining the glands appeared to be distorted, possibly in response to internal pressure from increased volume due to high metabolic activity, and/or no uterine luminal opening.     

Cell Proliferation in Epithelial and Lympho-hematopoietic Tissues of Cyclostomes

The cyclostomes, hagfishes and lamprey, represent modern agnathans,the most primitive vertebrates. They are therefore of specialinterest from the phylogenetic view point with regard to proliferativeactivities of epithelial and of lympho-hematopoietic tissues.The animals, kept in aquaria at 15 C, were given 1.0 µCiof ³H-thymidine per gram of body weight intramuscularly, killed2 hr later, different organs prepared for autoradiograms usingthe liquid emulsion technique, and the labeling indices determined.In peripheral blood, cell proliferation occurred mainly in thehemocytoblast group of cells in both species. Both lympho-hematopoieticcells and epithelial cells proliferated in the lamprey, althoughat a relatively low rate, perhaps attributable to senescence.In the hagfish, blood-forming and epithelial cells were rapidlyproliferating, with the dramatic exception of intestinal epithelium,where the proliferative activity was extremely low. This Findingmay well explain the documented high resistance of hagfishesto irradiation and alkylating agents, in contrast to the lamprey,which is approximately as sensitive to these agents as mostadvanced vertebrates.     

Morphology of the jaw apparatus in 8 species of Patellogastropoda (Mollusca, Gastropoda) with special reference to Testudinalia tesulata (Lottiidae)

The fine structure of the jaw apparatus was studied by scanning electron microscopy in eight species of Patellogastropoda. The jaw apparatus is an unpaired two-layered dorsolateral structure with anterior and posterior wings attached to the odontophore by muscles. The jaw of Testudinalia tesulata (O.F. Müller, 1776) is a derivative of the cuticle typical for the foregut. The tissue forming the jaw is a specialized foregut epithelium (gnathoepithelium), consisting of a special type of cells called gnathoblasts. The jaw grows in areas of the epithelium characterized by high concentration of electron-dense vesicles, ER and long microvilli that penetrate deep into the jaw plate. This indicates that the gnathoblasts take an active part in jaw growth. In most cases, these areas of the gnathoepithelium are highly folded. The main differences between the species studied are form and thickness of the frontal edge of the jaw. These differences do not correlate with the systematic position of the species studied but likely depend more on the feeding mode. The transmission electron microscopy studies yielded new morphological criteria for comparison between various gastropod species and other members of Trochozoa, in particular, Annelida. The jaws of Annelida are cuticular structures formed on the surface of specialized epithelial cells, often also called gnathoblasts. The jaw of Patellogastropoda can be attributed to the first type of annelid jaw formation characterized by an epithelium with long microvilli and continuous growth.     

Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells

Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas.     

Effects of some pesticides on the vital organs of juvenile rainbow trout (Oncorhynchus mykiss)

Gill, trunk kidney, spleen, and liver of rainbow trout (Oncorhynchus mykiss) were examined after exposure to different sublethal concentrations of carbosulfan (25, 50 and 200 μg L⁻¹), propineb (3, 6 and 24 mg L⁻¹), and benomyl (2, 5 and 20 mg L⁻¹) for 14 days. Lesions were observed in gill, trunk kidney, spleen, and liver of rainbow trout exposed to either concentration of pesticides. The most important lesions were determined in the highest concentrations of pesticides. Lamellar fusion, lamellar hyperplasia, epithelial lifting, vacuolization of epithelial tissue, epithelial necrosis, hypertrophy and sloughing of epithelium were observed on fish exposed to carbosulfan, propineb and benomyl. Fish had cell necrosis, degeneration and oedemas in liver, trunk kidney and spleen. None of these lesions were seen in control fish.     

Normal and abnormal epithelial differentiation in the female reproductive tract

In mammals, the female reproductive tract (FRT) develops from a pair of paramesonephric or Müllerian ducts (MDs), which arise from coelomic epithelial cells of mesodermal origin. During development, the MDs undergo a dynamic morphogenetic transformation from simple tubes consisting of homogeneous epithelium and surrounding mesenchyme into several distinct organs namely the oviduct, uterus, cervix and vagina. Following the formation of anatomically distinctive organs, the uniform MD epithelium (MDE) differentiates into diverse epithelial cell types with unique morphology and functions in each organ. Classic tissue recombination studies, in which the epithelium and mesenchyme isolated from the newborn mouse FRT were recombined, have established that the organ specific epithelial cell fate of MDE is dictated by the underlying mesenchyme. The tissue recombination studies have also demonstrated that there is a narrow developmental window for the epithelial cell fate determination in MD-derived organs. Accordingly, the developmental plasticity of epithelial cells is mostly lost in mature FRT. If the signaling that controls epithelial differentiation is disrupted at the critical developmental stage, the cell fate of MD-derived epithelial tissues will be permanently altered and can result in epithelial lesions in adult life. A disruption of signaling that maintains epithelial cell fate can also cause epithelial lesions in the FRT. In this review, the pathogenesis of cervical/vaginal adenoses and uterine squamous metaplasia is discussed as examples of such incidences.     

Toll-Like Receptor 4 Expression in the Epithelium of Inflammatory Periapical Lesions.: An immunohistochemical study

Toll-like receptors (TLR) are essential for the innate immune response against invading pathogens and have been described in immunocompetent cells of areas affected by periapical disease. Besides initiating the inflammatory response, they also directly regulate epithelial cell proliferation and survival in a variety of settings. This study evaluates the in situ expression of TLR4 in periapical granulomas (PG) and radicular cysts, focusing on the epithelial compartment.Twenty-one periapical cysts (PC) and 10 PG were analyzed; 7 dentigerous non-inflamed follicular cyst (DC) served as control. TLR4 expression was assessed by immunohistochemistry. TLR4 immunoreaction products were detected in the epithelium of all specimens, with a higher percentage of immunostained cells in PG. Although TLR4 overexpression was detected in both PG and PC, there were differences that seemed to be related to the nature of the lesion, since in PG all epithelial cells of strands, islands and trabeculae were strongly immunoreactive for TLR4, whereas in PC only some areas of the basal and suprabasal epithelial layers were immunostained. This staining pattern is consistent with the action of TLR4: in PG it could promote formation of epithelial cell rests of Malassez and in epithelial strands and islands the enhancement of cell survival, proliferation and migration, whereas in PC TLR4 could protect the lining epithelium from extensive apoptosis. These findings go some way towards answering the intriguing question of why many epithelial strands or islands in PG and the lining epithelium of apical cysts regress after non-surgical endodontic therapy, and suggest that TLR4 plays a key role in the pathobiology of the inflammatory process related to periapical disease.Key words: TLR4, periapical inflammatory granulomas, radicular cysts     

Lectin-binding patterns in epithelial lining of jaw cysts

Lectin-binding patterns were examined in epithelial walls of 65 jaw cysts (30 post-operative maxillary cysts: POMCs, 20 radicular and 15 follicular cysts), and characteristic lectin staining for each kind of jaw cysts is presented. Between squamous and columnar epithelia, the staining intensity of WGA, Con A and UEA-I was not different, but SBA bound more remarkably to squamous than to columnar epithelia. In both epithelia the outer layers did react more strongly with the lectins examined. Concerning odontogenic cysts, the lectin-binding affinities of outer and intermediate layer cells were nearly the same in both follicular and radicular cysts. Basal cells of radicular cyst walls were however, more markedly positive for lectin binding than of follicular cysts. Furthermore, basal cells of keratinized (RKSE 60 keratin-positive) epithelium were inferior to those of non-keratinized linings in the bindings. Lectin-binding patterns of metaplastic squamose epithelia of POMCs which were positive for RGE53-keratin (principally columnar epithelium-specific keratin) were similar to originally squamous linings of odontogenic cysts. Columnar linings of unusual radicular cysts were positively stained with SBA. By these results, lectin-binding sugar residues of the epithelium seem to be related to the epithelial morphology.     

Myoid fibrils in epithelial cells: studies of intestine, biliary and pancreatic pathways, trachea, bronchi, and testis.

Cytoplasmic filaments have been studied extensively by electron microscopy, but the histochemical nature of such fibrils in non-keratinizing epithelia has not been systematically investigated. During studies of early arterial lesions we observed structures with the staining properties of myosins in epithelial cells of various organs. The configurational staining, polarization and fluorescence microscopic properties of these myoid structures were compared with those of myofibrils in smooth muscle and classical myoepithelial cells. The following structures showed the characteristics of myofibrils: the terminal web in columnar epithelial cells of intestine, trachea, bronchi, bile ducts, pancreatic ducts and ductus epididymidis, the pericanalicular layer of bile and pancreatic canaliculi, fibers in the caudal tube of spermatids and the flagella of spermatozoa. Cilia, e.g. of respiratory epithelium, tonofibrils in squamous epithelium and nerve axons did not react. These studies indicate significant histochemical differences between cytoplasmic filaments. Different types of intracellular fibrils can be found in the same cell, e.g. in respiratory epithelium.     

Immunocytochemical localization of insulin and somatostatin in the endocrine pancreas of the sea lamprey Petromyzon marinus L., at various stages of its life cycle

Summary Light-microscopic immunocytochemistry and routine staining techniques were used to localize insulin and somatostatin-immunoreactive cells within the endocrine pancreatic tissue of the lamprey, Petromyzon marinus, during various stages of the life cycle. The endocrine pancreas of larvae consists solely of follicles of insulin-immunoreactive cells surrounding the junction of oesophagus, intestine and bile duct. Somatostatin-immunoreactive cells are restricted to the intestinal epithelium. In both parasitic and upstream-migrating adults the endocrine pancreas consists of cranial and caudal portions, both containing separate populations of insulin and somatostatin-immunoreactive cells.Supported by NSERC of Canada grant no. A5945 to JHY     

Langerhans cells in odontogenic tumours and cysts as detected by S-100 protein immunohistochemistry

Immunohistochemical demonstration of S-100 protein in Langerhans cells (LCs) was made in odontogenic epithelial tumours (71 cases), radicular cysts (40 cases), follicular cysts (28 cases), odontogenic keratocysts (11 cases), primordial cysts (7 cases) and fissual cysts (6 cases). With the use of polyclonal antiserum against S-100 protein, positive LCs, dendrical or irregular in shape were found in tumour or cystic epithelia, and sometimes in stromal connective tissue. Incidence of positive S-100 staining LCs was 11 cases out of 61 ameloblastomas, 22 cases out of 40 radicular cysts, 3 cases of 28 follicular cysts, and other lesions in both odontogenic tumours and cystic diseases lacked LCs. The cases with S-100 protein positive LCs were usually accompanied with a high degree of inflammatory infiltration in their lesions; on the contrary, the negative cases also generally lacked inflammatory responses.     

Dynamic analysis of filopodial interactions during the zippering phase of Drosophila dorsal closure

Dorsal closure is a paradigm epithelial fusion episode that occurs late in Drosophila embryogenesis and leads to sealing of a midline hole by bonding of two opposing epithelial sheets. The leading edge epithelial cells express filopodia and fusion is dependent on interdigitation of these filopodia to prime formation of adhesions. Since the opposing epithelia are molecularly patterned there must exist some mechanism for accurately aligning the two sheets across this fusion seam. To address this, we generated a fly in which RFP-Moesin and GFP-Moesin are expressed in mutually exclusive stripes within each segment using the engrailed and patched promoters. We observe mutually exclusive interactions between the filopodia of engrailed and patched cells. Interactions between filopodia from matching cells leads to formation of tethers between them, and these tethers can pull misaligned epithelial sheets into alignment. Filopodial matching also occurs during repair of laser wounds in the ventral epithelium, and so this behaviour is not restricted to leading edge cells during dorsal closure. Finally, we characterise the behaviour of a patched-expressing cell that we observe within the engrailed region of segments A1-A5, and provide evidence that this cell contributes to cell matching.