On the role of metal cations in cellular adhesion: Cation specificity

Reprinted from THE JOURNAL or EXPERIMENTAL Zoomcv Vol. 167, No. 3, March 1968 © The Wistar Institute 1968 On the Role of Metal Cations in Cellular Adhesion: Cation Speciﬁcity PETER B. ARMSTRONG AND DANIEL P. JONES Department of Zoology, University of California, Davis Campus, Davis, California ABSTRACT Of the four common alkaline earth cations, only Ca++ is able to protect Rana pipiens embryos from dissociation of the epidermis into isolated cells by EDTA. Presumably this is because only Ca++ is able to satisfy the divalent cation require- ment for cellular adhesion in this tissue. Although early stage embryos are completely dissociable by EDTA, the axial musculature, brain and spinal cord become resistant to dissociation by stage 17. Tetraphenyl boron is also effective in disaggregating am- phibian embryo tissues. An important attribute of most cell types of multicellular organisms is their mutual adhesiveness. This permits the formation of tissues from collections of individual cells. Cell surface events are presumed to be the factors most likely involved in adhe- sion mechanisms since adhesions are estab- lished and maintained between cell sur- faces. Almost all of the various hypotheses which have been concerned with mecha- nisms of adhesion have dealt principally with cell surface phenomena. One phenomenon common to adhesion of almost every cell type which has been studied is a requirement for divalent alka- line earth cations. Removal of divalent cations promotes disaggregation of tissues into isolated cells. The cells of tissues dis- persed in this manner can often be caused to reaggregate into tissue masses but only if divalent cations are added back to the medium (for references see Armstrong, ’66). The data presented in this paper deal with the speciﬁcity of cation involvement. MATERIALS AND METHODS The eggs of Hyla regilla were collected in the Vaca Hills west of Davis. They were stored at 4°C until needed and then al- lowed to develop at 16°C in pond water. The eggs of Rana pipiens and Xenopus laevis were obtained by hormonally in- duced ovulation (Wright and Flathers, &#39;61). The jelly layers of prehatched em- bryos of the ﬁrst two species were removed with watchmakers forceps, and those of Xenopus were removed by incubation with J. Exr. ZooL., 167: 275-282. cysteine (Gusseck and Hendrick, ’67). The hyaline membrane could then be removed with forceps. Disaggregation was carried out at room temperature in the wells of three—depression spot plates (Pyrex cat. no. 7740) placed in 100 mm Petri dishes to retard evaporation. All solutions were made up in doubly distilled water, the second dis- tillation being from a glass still. Stages 19—20 (newly hatched embryos — Shum- way, ’40) Rana embryos were used most extensively, but most of the results ob- tained with Rana were conﬁrmed with Xenopus embryos (Nieuwkoop and Faber Stages 8-30, in New, ’66). Hyla embryos were used for a few experiments. Calcium concentrations were determined using a Beckman atomic absorption spectrometer. RESULTS The metal cation chelating agent diso- dium ethylene diamine tetraacetate (Naz- EDTA) was very effective in disaggregat- ing several of the tissues of amphibian embryos into single cells. In later stage embryos (for example, stage 19 Rana pipiens embryos), EDTA disaggregated epidermis and the underlying mesenchyme and yolky endoderm but did not affect brain, spinal cord or somite tissues (ﬁg. 1). In the concentrations employed (see table 1), the ﬁrst signs of disaggregation were evident within a few minutes as one or a few foci of loosened and disag- gregating epidermal cells. Disaggregation continued by the enlargement of these foci until the entire ectoderm was dissoci- 275