Multiple interactions in juxtaposed monolayers of amphibian neuronal, epidermal, and mesodermal limb blastema cells

Abstract

We have utilized a novel cell culture system to probe nerve-epidermal-blastema cell interactions, in which the cellular participants are plated as compartmentalized juxtaposed monolayers. A mesoderm monolayer is attached to a culture plate insert and placed into a tissue culture well, the bottom of which is seeded with a second cell type(s); the two monolayers are separated by a distance of 1 mm. An examination of the multiple interactions occurring during blastemal growth can thus be accomplished by deletion of one or more of the cellular participants and employment of a replacement strategy utilizing substances with putative growth-promoting activity. [3H]Thymidine incorporation into DNA of urodele amphibian blastemal mesoderm cells was evaluated in response to the influence of juxtaposed brain or epidermal cell monolayers or both cultured in paired combinations. Brain cells were resolved by differential adhesive selectively into enriched neuronal and glial subpopulations. In the presence of enriched neuronal cells, radiolabel incorporation into blastemal mesoderm DNA was significantly greater than that achieved by coculturing with glial or unresolved brain cell populations. The effect of epidermal cells was not overtly expressed in the absence of neuronal cells. The effect of growth factors [fibroblast growth factor (FGF), epidermal growth factor (EGF), nerve growth factor (NGF)] on radiolabel incorporation into blastemal mesoderm DNA was also assessed in both the presence and absence of neuronal cells. Both EGF and FGF enhanced radiolabel incorporation into DNA when blastemal mesoderm was cultured in the absence of nerve, but the effect was significantly less pronounced than that achieved by neuronal cells alone. This system has provided us with an additional means of characterizing the dialogue between epidermis, nerve, and mesodermal blastema cells, and determining the identity of growth signals.