Browsing by Subject "Morphogenesis"

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    Alterations induced by E-cadherin and ß-catenin antibodies during the development of Buto arenarum (Anura-Bufonidae)
    (F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2001) Izaguirre, M. F.; Adur, J. F.; Peralta Soler, A.; Casco, V. H.
    E(e pi theli al)-ca dh erin is a member of a ca lcium-dependent famil y of cell surface glycoproteins in vo lved in ce ll- ce ll adh esion and mo rph oge nesis. Catenins are a large family of proteins that connect the cad herins to the cytoskeleton. They are import ant for cadherin fUll ction and for transducing signals involved in specification of ce ll fate during embryogenesis. The best charac teri zed ca tenin s in clude U - , /3- , Yo, and p1 20- ca tenin. Usin g spec ifi c antibodi es, we studi ed th e ex pression and distribution of E-cadherin, and u- and /3 - catenin in developmental stages of Bufo arellarum toad. The three proteins were fo und co-locali zed in stages 19 to 41 of development. Surprisingly, E-cadherin was the only of these three proteins fo und earlier than stage 19. To test whe th e r E-ca dh e rin a nd /3- ca te nin have a functional role in B/./fo arenarlIIn embryogenesis, stage 17 whole embryos were incubated with anti-E-cadherin and /3-catenin antibodies. Both anti-E-cadherin and anti- /3- ca tenin antibodi es indu ced seve re morph ological alterations. However, while alterations produced by the anti-/3 -catenin antibody, showed some vari ability from the most severe (neural tube and notochord duplication) to a simple delay in deve lopment, the alterations with anti-E-cadherin were homogeneous. These observa tions suggest a critica l role for E-cadherin and/3-catenin in the ea rl y embryo ni c development of the Bufo arenarum toad. Our resul ts are consistent with the development al role of these proteins in other species. One of the most surprising findin gs was th e blockage with the anti-Bcat enin antibodi es on late r embr yo stages, and we hypothesize that the partial axes duplica tion could be medi ated by the notochord induction.
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    Morphogenesis of normal human salivary gland cells in vitro
    (Murcia : F. Hernández, 1994) Azuma, M.; Sato, M.
    Primary cultured human salivary gland cells were transfected with ori-defective mutant DNA of SV40. Following 2-3 weeks of transfection, slowly expanding colonies consisting of small compact cells emerged, while mock-transfected cells did not grow any more and eventually entered crisis, followed by cell death. Using limited dilution technique, we isolated 4 cell clones with distinct morphology from a single colony. Morphological observation of cells cultured on plastic dishes precisely revealed the characteristics of the constituent cells of salivary gland; i.e., three cell clones showing cuboidal- (NS-SV-DC), spindle- (NS-SV-MC), and flattened morphology (NS-SV-SC) were similar to duct-, myoepithelial-, and squamous phenotype, respectively. A remaining cell clone, polygonal in shape and with numerous secretory granules (NS-SV-AC), resembled an acinar cell. Characterization of cell clones by ultrastructural exarnination and search for specific antigens showed the similarity of NS-SV-DC, NS-SV-MC, NS-SV-AC, and NS-SV-SC to duct, myoepithelial, acinar, and squamous cells, respectively. Anchorage-independent growth in soft agar and tumorigenicity in nude mice were not recognized in al1 cell clones. These results demonstrate that establishment of cell clones with duct-, myoepithelial-, acinar-, or squamous phenotype was accomplished in the in vitro system, and that based on the evaluation of colony-forming ability in soft agar and tumorigenicity in nude mice these cell clones can be considered to be non-tumorigenic. Using the above in vitro system, we examined the effect of a reconstituted basement membrane extract, Matrigel, on the morphogenesis of cultured normal human salivary gland cells. When NS-SV-DC or NS-SV-MC were seeded on Matrigel in serum-free culture conditions, they formed round or zona1 clusters on day 1; failing however, to develop into a salivary gland morphogenesis. Semithin sections of cell clones cultured on Matrigel exhibited multicellular aggregates on day 1, while on days 2 and 3 these cells lost both cell-cell and cell-Matrigel interactions and eventually entered crisis. In an attempt to understand the mechanism involved in this phenomenon, we have investigated proteolytic enzymes and their inhibitors secreted by cell clones. Although cell clones produced almost identical levels of gelatinases, they released increased amounts of plasminogen activators (PAs) as compared with a neoplastic human salivary gland cell line (HSG), which had already been demonstrated to differentiate into acinar cells when cultured on Matrigel. Obvious difference of expression leve1 of tissue inhibitor of metalloproteinases-1 (TIMP- 1) was not observed in these cells. However, secretion of PA inhibitors was elevated in NS-SV-MC when compared to NS-SV-DC. Neutralization of excess PAs by exogenously-added serine protease inhibitors corrected the aberrant morphogenesis of NS-SV-DC, but not that of NS-SV-MC, and allowed NS-SV-DC to form glandular-like structures. Thus, these results may suggest that a tightly regulated proteolytic activity is, in part, essential for in vitro morphogenesis of normal salivary gland duct cells.