Expression and function of myc during asexual reproduction of the budding ascidian Polyandrocarpa misakiensis

The budding ascidian Polyandrocarpa misakiensis proliferates asexually by budding. The atrial epithelium is a multipotent but differentiated tissue, which transdifferentiates into various tissues and organs after the bud separates from the parental body. We isolated cDNA clones homologous to the myc proto-oncogene from P. misakiensis. The cDNA, named Pm-myc, encoded a polypeptide of 639 amino acid residues, containing Myc-specific functional motifs, Myc box I and Myc box II, and the basic helix-loop-helix domain. Expression of Pm-myc was observed in the atrial epithelium in the organ-forming region of the developing bud, where the epithelial cells dedifferentiate and re-enter the cell cycle. The expression was also observed in fibroblast-like cells, which are known to participate in the organogenesis together with the epithelial cells. Unexpectedly, the atrial epithelium expressed Pm-myc more than one day before the dedifferentiation. The organogenesis was disturbed by Pm-myc-specific double-stranded RNA. In situ hybridization revealed that Pm-myc-positive fibroblast-like cells disappeared around the organ primordium of the dsRNA-treated bud. The results suggest that the mesenchymal-epithelial transition of fibroblast-like cells is important for the organogenesis in this budding ascidian species.     

Development of irradiated tunicate buds: Is cell division cycle required for morphallaxis?

In the tunicate, Polyandrocarpa misakiensis , transdifferentiation occurs in the multipotent atrial epithelium during morphallactic bud development. Irradiation (10–80 Gy) or aphidicolin (10 μg/mL) blocked this process severely, although the atrial epithelium could form organ placodes. The placodes consisted of cuboidal cells with a high nucleus : cytoplasm ratio and were lacking the alkaline phosphatase antigen from the cell surface, suggesting that the atrial epithelium might undergo dedifferentiation without initiating cell cycling. Irradiated buds could resume organogenesis in temporal accordance with the restoration of mitotic activity. Bud pieces irradiated at 40 Gy were juxtaposed with unirradiated counterparts. In the operated buds, irradiated, non-dividing cells participated in organogenesis at the site of juxtaposition in cooperation with the unirradiated, dividing cells. These results have shown that in P. misakiensis the cell division cycle, probably DNA replication, is indispensable for transdifferentiation of the atrial epithelium, although every cell in the organ rudiment need not enter cell cycling. We suggest that homoiogenetic induction occurs between dividing cells and non-dividing cells.     

Identification of a novel protein (G210) specific to the Golgi apparatus

A monoclonal antibody, 3C9, has enabled the detection of a novel Golgi-specific protein in bovine tissues. Immunohistochemical studies at the light microscopic level have detected the 3C9 antigen only in certain cells: exocrine pancreas, gut epithelium, and thymus epithelium. Examination of gut and pancreas by immunoelectron microscopy showed a localization exclusive to the Golgi apparatus. The relative molecular weight of the antigen detected by immunoblotting is 210,000 daltons. The antigen is not extracted from microsomal membranes of bovine gut epithelium by sodium carbonate solutions. Furthermore, the 3C9 antigen enters into the detergent phase when Triton X-114 partitioning methods are used. These data strongly suggest that this novel antigen is an intrinsic membrane protein, resident in the Golgi apparatus of certain cells. Moreover, they enhance the hypothesis that the distribution of enzymes and polypeptides in the Golgi apparatus is cell specific.     

Mucus-associated antigen in epithelial cells of the chicken digestive tract: Developmental change in expression and implications for morphogenesis-function relationships

The embryonic chicken digestive tract consists of endodermal epithelium and mesenchyme derived from splanchnic mesoderm. Interactions between these two tissues are important for the establishment of regionality and the subsequent differentiation of digestive organs. In the present study we obtained a monoclonal antibody that reacted with mucus-associated antigen and named it the MA antibody. From 6 days of incubation, this antibody reacted with the esophageal, proventricular and gizzard epithelia. In the proventriculus, the MA antigen was expressed in luminal epithelial cells, while pepsinogen-producing gland cells became MA antigen-negative. The intestinal goblet cells, which secrete mucus, became positive to the antibody from day 13 of incubation. When the esophageal, proventricular or gizzard epithelium of a 6 day embryo was associated and cultivated with the proventricular mesenchyme, the luminal epithelial cells remained reactive to the MA antibody while gland cells were negative or only weakly positive. If the small-intestinal epithelium was cultivated with the proventricular or gizzard mesenchyme, the antigen was detected on the apical surface of the epithelium, suggesting that the expression of the MA antigen was induced by mesenchymal influences in the small-intestinal epithelium. These results suggest that spatio-temporally regulated expression of the MA antigen is controlled by the epithelial-mesenchymal interactions.     

Detection and localization of antibody ingested with a mosquito bloodmeal

Mouse immunoglobulins were found to persist in Aedes aegypti (L.) mosquito bloodmeals for 2-3 days after ingestion. Immunoenzyme labelling revealed mouse antibody specifically bound to the mosquito midgut epithelium after ingestion; immunogold labelling of thin sections revealed mouse antibody within the cytoplasm of the microvilli on the midgut epithelium. Ingested mouse antibody was not conclusively demonstrated bound to tissues outside the gut, though antibody was detected in mosquito haemolymph using a sensitive ELISA test. Possible mechanisms by which antibody may cross mosquito gut barriers and actions that in vivo antibody:antigen reactions may have on these bloodfed insects are discussed.     

Cellular distribution of smooth muscle actins during mammalian embryogenesis: expression of the alpha-vascular but not the gamma- enteric isoform in differentiating striated myocytes

The cellular distribution of the alpha-vascular and gamma-enteric smooth muscle actin isoforms was analyzed in rat embryos from gestational day (gd) 8 through the first neonatal week by in situ antigen localization using isoactin specific monoclonal antibodies. The alpha-vascular actin isoform was first detected on gd 10 in discrete cells lining the embryonic vasculature. By gd 14, this isoform was also present in the inner layers of mesenchymal cells condensing around the developing airways and gut. The gamma-enteric actin, however, was not detected until gd 15 when cells surrounding the developing aorta, airways, and gut labeled with the gamma-enteric-specific probe. There was continued expression of these two actin isoforms in regions of developing smooth muscle through the remainder of gestation and first neonatal week at which time their distribution coincided with that found in the adult. In addition to developing smooth muscle, the alpha- vascular actin isoform was expressed in differentiating striated muscle cells. On gd 10, there was intense labeling with the alpha-vascular specific probe in developing myocardiocytes and, within 24 h, in somitic myotomal cells. Although significant levels of this smooth muscle actin were present in striated myocytes through gd 17, by the end of the first postnatal week, alpha-vascular actin was no longer detectable in either cardiac or skeletal muscle. Thus, the normal developmental sequence of striated muscle cells includes the transient expression of the alpha-vascular smooth muscle actin isoform. In contrast, the gamma-enteric smooth muscle actin was not detected at any time in embryonic striated muscle. The differential timing of appearance and distribution of these two smooth muscle isoforms indicates that their expression is independently regulated during development.     

Vertebrate phylogeny of antigen D10: identification of a conserved foregut cell lineage

The monoclonal antibody 5HL-5D11-D10 to antigen D10 identifies a cell lineage that is restricted to certain tissues of the human foregut. We investigated the tissue distribution of antigen D10 in mammals, birds, reptiles, amphibians and fish by immunohistochemical staining. Tissue from human and each of ten other mammalian species showed staining of gastric mucous neck cells and glands of the cardia and antrum, Brunner's glands, peribiliary glands and periductal glands of the pancreas. Six of the mammalian species also expressed antigen D10 in mucosa of the larger bronchi, and five expressed it to varying degree in small bowel distal to the duodenum and in colon (three of these five species). Antigen was not detected in any of the three species of bird studied. Both reptiles and amphibians showed strong staining for antigen D10 in the gastric mucous neck cells and pyloric glands, and in a subpopulation of secretory cells in the oesophagus, with the amphibian also expressing antigen in some epithelial cells of the mouth and lung. Although absent from two species of bony fish, antigen D10 was expressed by small groups of epithelial cells of the intestine of a shark, and generally by the epithelial and connective tissue cells of the gut and gills, and hepatocytes of one species of ray. The presence of antigen D10 in different tissues and species was confirmed by both an indirect ELISA and immunoblot analysis of tissue extracts. Our observations suggest that the D10 epitope characterises a subpopulation of mucus-secreting cells, predominantly of the foregut and associated organs, which has been conserved throughout terrestrial vertebrate evolution.     

Reactivity of a Monoclonal Antibody Raised against Human Leukemic Cells to Embryonic and Adult Tissues of the Mouse and Teratocarcinomas

C-9-1, a monoclonal IgM antibody raised against human null cell acute lymphocytic leukemia cells reacted with restricted regions of embryonic and adult tissues of the mouse. The antigen positive sites in the embryos included embryonic ectoderm, visceral endoderm, trophoblastic cells invading the maternal decidua of 5∼7-day embryos, primordial germ cells of 10∼12-day embryos, epithelium of nasal chamber, the bronchus, Mullerian duct, epididymis and bladder of 12∼17-day embryos. In the adult mice, C-9-1 antigen was detected in renal tubules, a part of stomach, bladder, endometrium and epididymal sperm. Embryonal carcinoma cells, but not endodermal cells of teratocarcinoma expressed the antigen. Thus, C-9-1 antigen showed distribution similar to SSEA-1. However, C-9-1 antigen was not detected in preimplantation embryos, nor in oviduct, both of which are positive for SSEA-1.     

EMA,an epithelial membrane-associated antigen during early development and morphogenesis ofXenopus laevis

Summary We raised monoclonal antibodies against a membrane fraction ofXenopus neurulae in order to detect tissue-specific cell-surface markers. Here we describe a monoclonal antibody that recognizes an epithelial membrane-associated antigen (EMA) in immunohistological stainings. The tissue-specific and membrane-associated antigen detected in immunohistological stainings could serve as useful marker in epithelium differentiation and membrane organization of the early embryo. In tadpoles and adults EMA was found in specific epithelial tissues derived from different germ layers such as kidney, skin, gut, pancreas, epiphysis and choroid plexus. In the cleaving embryo this antibody stained newly formed membranes between blastomeres from the two-cell stage onwards. Cytoplasmic staining in large oocytes and early embryos was also observed. The possibility that the cytoplasmic signal represents a maternal store of membrane material is discussed.     

A determinant of Mr 34,000 expressed by hamster epididymal epithelium binds specifically to spermatozoa in co-culture

A murine monoclonal antibody raised against hamster cauda epididymal spermatozoa was shown to recognize an Mr 34,000 component of epididymal epithelium. Antigen was localized by immunocytochemistry on the surface and in the apical cytoplasm of principal cells in the proximal corpus epididymidis but not in the caput or initial segment regions. Spermatozoa from the corpus epididymidis expressed antigen on their post-acrosomal plasma membrane and annulus. Epididymal principal cells from the proximal corpus region when cultured in vitro bound antibody on their apical surface for at least 5 days. Spermatozoa from the caput epididymidis co-cultured with epithelium expressed antigen after incubation for 8 and 24 h. These results suggest that a surface change to epididymal spermatozoa during maturation in vivo may also be elicited during in-vitro culture.     

A novel antigen is common to the dome epithelium of gut- and bronchus-associated lymphoid tissues

Summary The dome epithelium (DE) covering bronchus- and gut-associated lymphoid tissues (BALT and GALT) is composed of columnar cells, groups of lymphocytes, M cells, and pre-M cells. Although the cell biology and immunologic processes of this tissue are likely important in the afferent arm of secretory immune responses, virtually nothing is known about biochemical constituents of the DE. Therefore, a monoclonal antibody, 30E5, was used to study the distribution of a novel antigen, common to dome epithelia of GALT and BALT. 30E5 was secreted by a hybridoma, prepared by fusing murine splenocytes, immunized against dome epithelial cells, with P3×68/Ag8 myeloma cells. Reactivity of antigens was defined by indirect immunocytochemistry on sections of rabbit tissues or with dissociated epithelial cells. In situ, 30E5-reactive antigen circumscribed each group of dome epithelial lymphocytes, most or all of which were T cells, in rabbit appendix, sacculus rotundus, cecal patch, Peyer's patch, and BALT. In the DE this antigen was associated with the apical surface and the supranuclear or perinuclear regions of epithelial cells, but it was not associated with epithelial cells of villi, epithelium, or with individual lymphocytes. In peripheral lymph nodes, spleen, and in domes and follicles of GALT or BALT, 30E5-reactive antigen was visualized in linear wisps, primarily in regions populated by thymocytes. In other adult tissues, 30E5-reactive antigen was associated with involuntary muscle, myoepithelial cells of lactating mammary gland and with what appeared to be neural dendrites; but it was not found in epithelia other than DE. In neonatal rabbit appendix, this antigen first appeared in the upper dome epithelium two days after birth, a period coinciding with T cell infiltration and M cell maturation. The histologic distribution of 30E5-reactive antigen suggested that it might be a contractile filament, a receptor, or a differentiation antigen. Since 30E5 was associated with DE of both GALT and BALT, results support the concept of a molecule common to all mucosa-associated lymphoid tissues.In conducting the research described in this report, the investigators adhered to standards set forth in the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23) as promulgated by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, USALimited quantities of ascites containing monoclonal antibody 30E5 will be distributed to interested investigators until such time as the hybridoma is available from American Type Culture CollectionAbbreviations ABC avidin-biotin-horseradish peroxidase complex - BALT bronchus-associated lymphoid tissues - DMEM Dulbecco's modified Eagle medium - GALT gut-associated lymphoid tissues - DE dome epithelium - DEL dome epithelial lymphocytes - MAb monoclonal antibody - MALT mucosal-associated lymphoid tissues The views of the authors expressed here do not purport to reflect the position of the Department of the Army or the Department of Defense Send offprint requests to: Department of Experimental Pathology, Division of Pathology, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100, USA     

Cell surface proteoglycan expression correlates with epithelial-mesenchymal interaction during tooth morphogenesis

Tooth morphogenesis and differentiation of the dental cells are guided by interactions between epithelial and mesenchymal tissues. Because the extracellular matrix is involved in these interactions, the expression of matrix receptors located at the cell surface may change during this developmental sequence. We have examined the distribution of an epithelial cell surface proteoglycan antigen, known to behave as a receptor for interstitial matrix, during tooth morphogenesis. Intense staining was seen around the cells of the embryonic oral epithelium as well as the dental epithelium at the early bud stage. With development, expression was greatly reduced in the enamel organ. Differentiation of these cells into ameloblasts was associated with the loss of expression, while the epithelial cells remaining in the stratum intermedium and stellate reticulum regained intense staining. The PG antigen was weakly expressed in the loose neural crest-derived jaw mesenchyme but it became strongly reactive in the condensed dental papilla mesenchyme when extensive morphogenetic movements took place. With development, the PG antigen disappeared from the advanced dental papilla mesenchyme but persisted in the dental sac mesenchyme, which gives rise to periodontal tissues. The PG antigen was not expressed by odontoblasts. Hence, the expression of the PG antigen changes during the epithelial-mesenchymal interactions of tooth development and is lost during terminal cell differentiation. The expression follows morphogenetic rather than histologic boundaries. The acquisition and loss of expression in epithelial and mesenchymal tissues during tooth development suggest that this proteoglycan has specific functions in the epithelial-mesenchymal interactions that guide morphogenesis.     

Novel heterophile chicken antigen: immunohistochemical localization using antisera to Mycobacterium smegmatis and possible association with lymphocyte maturation

Summary A novel heterophile antigen shared byMycobacterium smegmatis and chicken tissues was demonstrated by the indirect immunoperoxidase method using antisera raised in rabbits immunized with a complete Freund's adjuvant containing killedMycobacterium smegmatis as an immunostimulating component. This antigen was strongly expressed in medullary lymphocytes of the thymus and bursa of Fabricius, but was undetectable in lymphoid cells of the cortical regions of these organs. Only a few lymphocytes stained positively for the antigen in T- and B-cell areas of the spleen. These data suggest that the heterophile antigen is associated with the intrathymic and intrabursal maturation of chicken lymphocytes. The antigen was also detected in some nonlymphoid cells. It was not found in sheep erythrocytes, human and rat tissues or in killed bacillus Culmette—Guerin.     

Growth of intestinal epithelium in organ culture is dependent on EGF signalling

Differentiation of endoderm into intestinal epithelium is initiated at E13.5 of mouse development when there are significant changes in morphology resulting in the conversion of undifferentiated stratified epithelium into a mature epithelial monolayer. Here we demonstrate that monolayer formation is associated with the selective apoptosis of superficial cells lining the lumen while cell proliferation is progressively restricted to cells adjacent to the basement membrane. We describe an innovative embryonic gut culture system that maintains the three-dimensional architecture of gut and in which these processes are recapitulated in vitro. Explants taken from specific regions of the gut and placed into organ culture develop and express molecular markers (Cdx1, Cdx2 and A33 antigen) in the same spatial and temporal pattern observed in vivo indicating that regional specification is maintained. Inhibition of the epidermal growth factor receptor (EGFR) tyrosine kinase using the specific inhibitor AG1478 significantly reduced the proliferation and survival of cells within the epithelial cell layer of cultured gut explants. This demonstrates an essential role for the EGF signalling pathway during the early stages of intestinal development.     

Cellular prion protein is expressed in a subset of neuroendocrine cells of the rat gastrointestinal tract.

Prion diseases are believed to develop from the conformational change of normal cellular prion protein (PrPc) to a pathogenic isoform (PrPsc). PrPc is present in both the central nervous system and many peripheral tissues, although protein concentration is significantly lower in non-neuronal tissues. PrPc expression is essential for internalization and replication of the infectious agent. Several works have pointed to the gastrointestinal (GI) tract as the principal site of entry of PrPsc, but how passage through the GI mucosa occurs is not yet known. Here we studied PrPc expression using Western blot, RT-PCR, and immunohistochemistry in rat GI tract. PrPc mRNA and protein were detected in corpus, antrum, duodenum, and colon. Immunoreactivity was found in scattered cells of the GI epithelium. With double immunofluorescence, these cells have been identified as neuroendocrine cells. PrPc immunostaining was found in subsets of histamine, somatostatin (Som), ghrelin, gastrin (G), and serotonin (5HT) cells in stomach. In small and large bowel, PrPc cells co-localized with subpopulations of 5HT-, Som-, G-, and peptide YY-immunolabeled cells. Our results provide evidence for a possible and important role of endocrine cells in the internalization of PrPsc from gut lumen.     

A novel C-type lectin regulating cell growth, cell adhesion and cell differentiation of the multipotent epithelium in budding tunicates

We have isolated two Ca(2+)-dependent, galactose-binding polypeptides from the budding tunicate, Polyandrocarpa misakiensis. Based on their partial amino acid sequences, full-length cDNAs were cloned. One of them was identical with a tunicate C-type lectin (TC14-2) reported previously. The other was a novel C-type lectin, referred to as TC14-3. In living animals, they appeared to be coupled. This complex of lectins, when applied in vitro to tunicate multipotent cells of epithelial origin, blocked cell proliferation and induced cell aggregation. The aggregates expressed a homolog of the integrin alpha-chain and other differentiation markers specific for epithelial cells. Recombinant TC14-3 could reproduce all the activities of native lectins by itself, which was accelerated by recombinant TC14-2. The inhibitory activity of TC14-3 on cell growth was completely abolished by the addition of 50 microM D-galactose. Anti-TC14-3 monoclonal antibody showed that the antigen was expressed constitutively by the multipotent epithelial and mesenchymal cells. These results provide evidence that in P. misakiensis a C-type lectin plays a novel, cytostatic role in regulating cell growth, cell adhesion and cell differentiation during asexual reproduction.     

Water-soluble luminal contents of the gut of the earthworm Lumbricus terrestris L. and their physiological significance.

In the post-gizzard gut of the earthworm Lumbricus terrestris, distinguishing the functions of the luminal epithelium from those of the chloragogenous tissue has been hindered by the close apposition of these two tissues. Moreover, both tissues may have different functions from the anterior to the posterior of the animal. We analyzed the gut luminal contents of L. terrestris so as to gain a better understanding of the function of the luminal epithelium. The intestine was divided into four regions from anterior to posterior, and the water-soluble portion of the luminal contents of these four regions was analyzed for protease and amylase activity, calcium and ammonium ions, and protein. The same four regions of the gut wall were analyzed for glutamate dehydrogenase (GDH) and serine dehydratase (SDH) to determine their location with reference to the site of ammonia production. We observed high levels of proteases, amylase, protein and calcium ions in the gut luminal contents of the first two regions, and a significant decline of all four variables in region III. Conversely, ammonia was low in the gut contents of regions I and II but rose sharply in region III, which was also the region to which the tissue enzymes GDH and SDH were localized. The ammonia content of earthworm casts was observed to be much higher than that of the surrounding soil. These data are presented as partial evidence for the proposal that the excretory ammonia produced by feeding earthworms is a product of the luminal epithelium of region III of the gut. It is also proposed that ammonia and calcium may function as ion-exchangers in the absorptive function of the earthworm gut.     

Characterization of matriptase expression in normal human tissues.

Matriptase is a type II transmembrane serine protease that has been implicated in the progression of epithelium-derived tumors. The role of this protease in the biology of normal epithelial cells remains to be elucidated. Matriptase mRNA has been detected by Northern analysis in tissues rich in epithelial cells, and the protein is expressed in vivo in normal and cancerous breast, ovarian, and colon tissues. However, a systematic analysis of the distribution of matriptase protein and mRNA in normal human tissues rich in epithelium has not been reported. In this study we characterized the expression of the protease in a wide variety of normal human tissues using a tissue microarray and whole tissue specimens. Significant immunoreactivity and mRNA expression were detected in the epithelial components of most epithelium-containing tissues. Matriptase expression was found in all types of epithelium, including columnar, pseudostratified columnar, cuboidal, and squamous. Distinct spatial distributions of reactivity were observed in the microanatomy of certain tissues, however. This suggests that although matriptase is broadly expressed among many types of epithelial cells, its activity within a tissue may be regulated in part at the protein and mRNA levels during the differentiation of selected epithelia.