Cell Tissue Res (1990) 259:455461 Cell and Tmsue Research © Springer-Verlag 1990 Morphological changes in the zonula adhaerens during embryonic development of chick retinal pigment epithelial cells Martin Sandig and Vitauts I. Kalnins Department of Anatomy, University of Toronto, Toronto, Ontario, Canada Summary. Retinal pigment epithelial cells from chicks at various stages of development were examined by transmis- sion electron microscopy to determine how the adult form of the zonula adhaerens, composed of subunits termed zon- ula adhaerens complexes, is acquired. During early stages of development, between embryonic day 4 and embryonic day 7, the intermembrane discs of zonula adhaerens com- plexes appear to be formed from material already present between the junctional membranes of the zonulae adhaer- entes. In contrast, the cytoplasmic plaque material of the zonulae adhaerentes is difficult to detect before hatching; it is seen as a dense band along the junctional membranes at hatching and as individual subunits in register with the intermembrane discs in adult retinal pigment epithelial cells. After embryonic day 16, when the zonulae adhaerentes in- crease dramatically in size, single zonula adhaerens com- plexes are also present basal to the zonulae adhaerentes along the lateral cell membrane. This suggests that, during later stages of development, the junctions grow in size and/ or turn over by the addition of pre-assembled zonula ad- haerens complexes. Key words: Adherens-type junction - Development - Mi- crofilaments - Retinal pigment epithelium - Chick A characteristic feature of epithelial cells is an apical junc- tional complex commonly composed of a continuous tight junction, a continuous ZA and desmosomes (Farquhar and Palade 1963; Staehelin 1974). Whereas tight junctions prin- cipally form a seal between different environments (Steven- son et al. 1988), desmosomes and ZAs mediate strong adhe- sion between the adjacent epithelial cells (Cowin et al. 1985). The RPE is a single layer of cuboidal cells situated between the neural retina and the choroid (Nguyen-Legros 1978; Zinn and Marmor 1979). In chickens its junctional complexes lack desmosomes (Docherty et al. 1984; Owaribe et al. 1988), but include a tight junction and a large ZA associated with prominent CMBs (Hudspeth and Yee 1973; Crawford 1979; Kuwabara 1979; Owaribe and Masuda Send offprint requests to: Martin Sandig, Department of Anatomy, University of Toronto, Medical Sciences Building, Toronto, On- tario, Canada, M5S 1A8 Abbreviations: CMB Circumferential microfilament bundle; ZA Zonula adhaerens; ZAC Zonula adhaerens complex;RPE Retinal pigment epithelium 1982; Philp and Nachmias 1985; Turksen and Kalnins 1987; Sandig and Kalnins 1988). Recent examination by electron microscopy of the orga- nization of the ZA-CMB-complex in RPE cells in adult chickens revealed that the ZA in these cells is composed of structural subunits, termed ZACs (Sandig and Kalnins 1988). Each ZAC is about 40 nm in diameter, and is com- posed of an electron-dense extracellular intermembrane disc situated in the space between the two junctional mem- branes, and of two electron-dense cytoplasmic plaques, one in each of the neighbouring cells. Throughout most of the junctional region the ZACs are densely packed in a hexago- nal array, although in some areas of the junction, especially near the edges, single ZACs can also be seen. The intermem- brane discs of ZACs probably represent the extracellular domains of integral membrane glycoproteins whereas the cytoplasmic plaques appear to contain the proteins vinculin and alpha-actinin (Opas and Kalnins 1985; Geiger et al. 1985) which are involved in binding the microfilaments of the associated CMB to the plasma membrane. Relatively little is known about the development of ad- herens-type junctions. In the RPE of the chick the ZAs increase about sixfold in size during development. We have therefore studied the organization and growth of the ZA junctions in the RPE of the chick during this period. Materials and methods Fertilized eggs of white leghorn chickens were obtained from Glen Fenelon Farms (Toronto, Ontario) and incu- bated in a humidified atmosphere at 37.8 ° C until the em- bryos reached the desired ages. Adult white leghorn chick- ens were obtained from a local slaughter house. The eyes of chicks at embryonic day 4 (E4), E7, E8, E12, E14, El6, El8, and from newly-hatched and adult chickens were excised from the animals after sacrifice by decapitation, placed into fixative, and dissected into smaller pieces, except for eyes of E4 embryos which were fixed intact. The tissue was fixed for 1 h at room temperature in 2.5% glutaraldehyde in 0.1 M phosphate buffer, with or without 4% tannic acid and 0.05 mg/ml saponin (Mau- pin and Pollard 1983). After postfixation for 0.5 h in 0.5% osmic acid in 0.1 M phosphate buffer, the specimens were dehydrated in ethanol and embedded in a mixture of Aral- dite and Epon. Ultrathin sections were collected on copper grids, stained with uranyl acetate and lead citrate and