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Differential expression of cytokeratin during rat gonadal morphogenesis
Biol. Cell (1991), 73 CYTOKERATIN PATTERNS OF HUMAN GINGIVAL EPITHELIUM RECONSTITUTED IN VITRO USING TWO CELL CULTURE PROCEDURES
Fr(~d6dque GOSSELINand Marie-Madeleine PORTIER Laboratoire de Biochimie Cellulaire du Colldge de France. 11, place Marcelin Berthelot. 75231 Paris Cedex 05 France
Since cytokeratins are considered to be accurate tools (I) at the molecular level to investigate epithelial differentiation, they can be used in the evaluation of the precise differentiation stage of human gingival epithelium reconstituted in vitro following two different gingival keratinocyte culture procedures in order to select the most suitable experimental model system. Human trypsin-dissociated gingival keratinocytes were seeded either on a feeder layer of irradiated mouse 3T3 fibroblasts or on a connective tissue equivalent 0attice) made up of fibroblasts included in a collagen gel (2). The cytokeratins were extracted and analysed by two-dimensional gel electmphoresis (NEPHGE)and identified on western blots using a broad range of very specific antibodies to cytokeratins. Both methods showed on I~istologicalsections that cultured gingival keratinocytes formed a multilayered non-keratinizing epithelium but the cytokeratin patterns are different. The gingival epithelium-like structure reconstituted on 3T3 feeder layer expressed some cytokerafins characteristic of the in situ gingival epithelium (K 5, 6, 14, 16, 17) and some which do not exist in the normal tissue (K 7, 18, 19, traces of 13 and 15) and are specific for embryonic, simple turnout epithelia. The gingival epithelium reconstituted on connective tissue equivalent expressed all the cytokeratins present in the normal tissue (K 5, 6, 14, 16, 17) except those specific for terminal differentiation (K 1, 2 and 10/11). In conclusion, the growth of gingival keratinocytes on connective tissue equivalents allows them to reproduce physiological stages of gingival differentiation, (I) KOPANR. and FUCHSE.. OenesDev.. 3. !-I5 (1989) (2) GOSSELINF. and PORTIERM.-M.,CJ~.Acad.$ci2nris, 309 i!1,323-329(1989) PARIETAL ENDODERM CELLS OF ELONGATING OVINE BLASTOCYSTS ARE PLURINUCLEATE. OBSERVATIONS ON THE CYTOSKELETON. Jacques E. FLECHON (1), Bernadette FLECHON (1), Jedl DEGROLARD (1), Solange DELASALLE (1), Michel GUILLOMOT (2). (1) Laboratoire de Biologie Cellulaire et MolcSculaire(2) Laboratoire d'Endocrinolosiede l'Embryon,INRA, 78352 Jouy.en.Josas,Cedex,France. The pdmitive endoderm is the first cell layer differenciated from the inner cell mass in the early blastocyst. We have analysed these cells by scanning and transmission electron microscopy andby immunofluorescence. The endoderm cells have the character of epithelial cells : they contain intermediate filaments made of cytokeratins 8 and 18. The cells are polarized, carrying microvilli on their face oriented toward the blastocoele. During the elongation of the blastocyst, the parietal endoderm (that is all the endoderm surrounding the blastocoele except under the embryonic disc) follows the growth of the trophectoderm, Whereas the trophectoderm cells multiply by mitosis (many metaphases and mid bodies), the endodenn cells become fusiform and contain several nuclei, These syncitial elements elongate parallel to the length of the blastocyst and frequently contain pairs of nuclei regularly distributed. It is supposed that the pludnuclear cells arise from nuclear divisions without cytokinesis. Each nucleus is still surrounded by microtubules and by circular and radiating bundles of cytokeratin filaments. These.filam..en_tsare bound to the lateral plasma membranes by desmesomes characterized by monoclonal antibodies oesmopiagin I + II. Tile staining of F-Actin with rhodamine-phalloidin underlines the long stretches of parallel plasma membranes of the syncitiai elements connected by gap junctions stained by anti connexin 43. These results suggest that the strategy of the elongation in the ovine blastocyst as far as endoderm is concerned, is different from that in porcine blastocyst where we have observed previously that the parietal ondoderm cells are mononucleated and have characters of mobile cells. However the ovine endoderm also produces cytoplasmic fibronectin (recognized with a speci,c monoclonal antibody) which is secreted in the extracellular matrix (ECM) between endoderm and Irophectoderm; as in the pig blastocyst, the trophectoderm cells are bound to the ECM by receptors of fibronectin, detected with monoclonal antibodies to integdns (b-subunit). So the tactics of trcphectoderm is well conserved in the two species. The endoderm of the ovine blastocyst appears to be an interesting model for the study of the nucleocytoplasmic relationships in pludnucleated cells, Differential
expression
of c y t o k e r a t [ n
during
rat
gonadal
morphogenesis.
Valdrle Fridmacher, Odette Locquet and Solange Magre. Laboratoirede Physiolo$iedu D~eloppement, Coll~.$ede France, 11, place Marcelin Bertheiot,75005 PARIS. Cytokeratin (CK) intermediate filaments are heteropolymers of type I (acidic) and type II (basic) CK proteins which are expressed in a variety of epithelia in tissue specific patterns. In fetal and prepubertal testis, expression of CK8 (type II CK) and CK18 (type I CK) was shown in 5ertoli cells (1, 2). In adult testis, no cytokeratins were detected (1, 2, 3). In the present work, using immunofluorescence and immunoblotting technics with specific monoclonal antibodies, we show that, in the undifferentiated gonad, CK 19 (type I CK) (clone LP2K) is expressed in addition to CK8 (clone LE41), whereas no significant immunoreactivity to CK 18 (clones LE 65 and RGE 53) is detected. As testicular morphogenesis takes place, CK 18 becomes evident in 5ertoli cells while CK 19 decreases and is no longer observed at the end of gestation. In fetal and neonatal ovaries studied up to two weeks after birth, CK 18 has never been detected; CK 8 and CK 19 are present in somatic cells of the ovigerous cords and in primordial but not in growing follicles. This study shows that, during the morphogenesis of the testis, a CK 18KX 19 shift in expression occurs which has not been observed in the differentiating ovary. The understanding of mechanisms responsible for this differential CK expression should provide additional insights into the sexual differentiation of the gonads as well as the processes involved in epithelial differentiation. (1) Paranko et ai., 1986, Dev. Biol. 117 : 35-44; (2) 5tosiek et ai., 1990, Differentiation 43 : 66-70; (3) Franke et ai., 1979, Eur. ]. Cell i3ioi.19-- 269-275. 1Oa
Fr(~d6dque GOSSELINand Marie-Madeleine PORTIER Laboratoire de Biochimie Cellulaire du Colldge de France. 11, place Marcelin Berthelot. 75231 Paris Cedex 05 France
Since cytokeratins are considered to be accurate tools (I) at the molecular level to investigate epithelial differentiation, they can be used in the evaluation of the precise differentiation stage of human gingival epithelium reconstituted in vitro following two different gingival keratinocyte culture procedures in order to select the most suitable experimental model system. Human trypsin-dissociated gingival keratinocytes were seeded either on a feeder layer of irradiated mouse 3T3 fibroblasts or on a connective tissue equivalent 0attice) made up of fibroblasts included in a collagen gel (2). The cytokeratins were extracted and analysed by two-dimensional gel electmphoresis (NEPHGE)and identified on western blots using a broad range of very specific antibodies to cytokeratins. Both methods showed on I~istologicalsections that cultured gingival keratinocytes formed a multilayered non-keratinizing epithelium but the cytokeratin patterns are different. The gingival epithelium-like structure reconstituted on 3T3 feeder layer expressed some cytokerafins characteristic of the in situ gingival epithelium (K 5, 6, 14, 16, 17) and some which do not exist in the normal tissue (K 7, 18, 19, traces of 13 and 15) and are specific for embryonic, simple turnout epithelia. The gingival epithelium reconstituted on connective tissue equivalent expressed all the cytokeratins present in the normal tissue (K 5, 6, 14, 16, 17) except those specific for terminal differentiation (K 1, 2 and 10/11). In conclusion, the growth of gingival keratinocytes on connective tissue equivalents allows them to reproduce physiological stages of gingival differentiation, (I) KOPANR. and FUCHSE.. OenesDev.. 3. !-I5 (1989) (2) GOSSELINF. and PORTIERM.-M.,CJ~.Acad.$ci2nris, 309 i!1,323-329(1989) PARIETAL ENDODERM CELLS OF ELONGATING OVINE BLASTOCYSTS ARE PLURINUCLEATE. OBSERVATIONS ON THE CYTOSKELETON. Jacques E. FLECHON (1), Bernadette FLECHON (1), Jedl DEGROLARD (1), Solange DELASALLE (1), Michel GUILLOMOT (2). (1) Laboratoire de Biologie Cellulaire et MolcSculaire(2) Laboratoire d'Endocrinolosiede l'Embryon,INRA, 78352 Jouy.en.Josas,Cedex,France. The pdmitive endoderm is the first cell layer differenciated from the inner cell mass in the early blastocyst. We have analysed these cells by scanning and transmission electron microscopy andby immunofluorescence. The endoderm cells have the character of epithelial cells : they contain intermediate filaments made of cytokeratins 8 and 18. The cells are polarized, carrying microvilli on their face oriented toward the blastocoele. During the elongation of the blastocyst, the parietal endoderm (that is all the endoderm surrounding the blastocoele except under the embryonic disc) follows the growth of the trophectoderm, Whereas the trophectoderm cells multiply by mitosis (many metaphases and mid bodies), the endodenn cells become fusiform and contain several nuclei, These syncitial elements elongate parallel to the length of the blastocyst and frequently contain pairs of nuclei regularly distributed. It is supposed that the pludnuclear cells arise from nuclear divisions without cytokinesis. Each nucleus is still surrounded by microtubules and by circular and radiating bundles of cytokeratin filaments. These.filam..en_tsare bound to the lateral plasma membranes by desmesomes characterized by monoclonal antibodies oesmopiagin I + II. Tile staining of F-Actin with rhodamine-phalloidin underlines the long stretches of parallel plasma membranes of the syncitiai elements connected by gap junctions stained by anti connexin 43. These results suggest that the strategy of the elongation in the ovine blastocyst as far as endoderm is concerned, is different from that in porcine blastocyst where we have observed previously that the parietal ondoderm cells are mononucleated and have characters of mobile cells. However the ovine endoderm also produces cytoplasmic fibronectin (recognized with a speci,c monoclonal antibody) which is secreted in the extracellular matrix (ECM) between endoderm and Irophectoderm; as in the pig blastocyst, the trophectoderm cells are bound to the ECM by receptors of fibronectin, detected with monoclonal antibodies to integdns (b-subunit). So the tactics of trcphectoderm is well conserved in the two species. The endoderm of the ovine blastocyst appears to be an interesting model for the study of the nucleocytoplasmic relationships in pludnucleated cells, Differential
expression
of c y t o k e r a t [ n
during
rat
gonadal
morphogenesis.
Valdrle Fridmacher, Odette Locquet and Solange Magre. Laboratoirede Physiolo$iedu D~eloppement, Coll~.$ede France, 11, place Marcelin Bertheiot,75005 PARIS. Cytokeratin (CK) intermediate filaments are heteropolymers of type I (acidic) and type II (basic) CK proteins which are expressed in a variety of epithelia in tissue specific patterns. In fetal and prepubertal testis, expression of CK8 (type II CK) and CK18 (type I CK) was shown in 5ertoli cells (1, 2). In adult testis, no cytokeratins were detected (1, 2, 3). In the present work, using immunofluorescence and immunoblotting technics with specific monoclonal antibodies, we show that, in the undifferentiated gonad, CK 19 (type I CK) (clone LP2K) is expressed in addition to CK8 (clone LE41), whereas no significant immunoreactivity to CK 18 (clones LE 65 and RGE 53) is detected. As testicular morphogenesis takes place, CK 18 becomes evident in 5ertoli cells while CK 19 decreases and is no longer observed at the end of gestation. In fetal and neonatal ovaries studied up to two weeks after birth, CK 18 has never been detected; CK 8 and CK 19 are present in somatic cells of the ovigerous cords and in primordial but not in growing follicles. This study shows that, during the morphogenesis of the testis, a CK 18KX 19 shift in expression occurs which has not been observed in the differentiating ovary. The understanding of mechanisms responsible for this differential CK expression should provide additional insights into the sexual differentiation of the gonads as well as the processes involved in epithelial differentiation. (1) Paranko et ai., 1986, Dev. Biol. 117 : 35-44; (2) 5tosiek et ai., 1990, Differentiation 43 : 66-70; (3) Franke et ai., 1979, Eur. ]. Cell i3ioi.19-- 269-275. 1Oa