238 The Nature of Biological Diversity 



epithelial one continues the ilichotomous branching characteristic of 

 normal morphogenesis. When, however, the mesenchymal component 

 is cultured over a 20-day period, its capacity to support the dichoto- 

 nious branching of freshly isolated epithelium gradually declines. 

 Accompanying the decline there is a change in the outgrowth pattern 

 of the mesenchyme, from sheet-like behavior with cohesive boundary 

 to increasingly dispersive behavior with individually migrating fibro- 

 blast-like cells at the boundary (Fig. 19). Here a behavioral change, 

 which may well conform with Moscona's observation of decline in 

 aggregability and production of extracellular material, is accompanied 

 by altered morphogenetic effect on the associated dependent epi- 

 thelium. 



Note that in Moscona's experiments aggregative and differentiative 

 behavior are simultaneously altered during the period in monolayer 

 culture. This is, of course, in conformity with many observations that 

 culturing, under conditions which promote cell dispersal, tends to be 

 antidifferentiative ( Grobstein, 1959). A well-documented example of 

 this has just been reported by Holtzer et al. ( 1960 ) . They examined 

 chondrocytes freed from already formed cartilage. Over a period of 

 10 days of culture in a disposed state, these cells lost all detectable 

 cartilage properties, as judged by their ability to incorporate S 35 or to 

 form recognizable matrix when returned to conditions under which 

 freshly dissociated cells undergo chondrogenesis. Although changes in 

 aggregative behavior were not stressed in the study, it is interesting 

 that freshly liberated chondrocytes on a clot spontaneously aggregate 

 within 24 hours and show little spreading tendency. After several 

 passages in culture, while the differentiative properties were declining, 

 the cells failed to aggregate spontaneously, forming a thin, rapidly 

 spreading sheet. 



There is, therefore, good reason to suspect a relationship between 

 the mucoid, microenvironmental material and differentiation. Results 

 reported by Wilde (1960) may offer the most direct demonstration 

 to date that such a relationship actually exists. Working with 1 to 15 

 cells from early amphibian embryos in restricted, microdrop nutrient 

 environments, he found that under particular conditions none of the 

 cells differentiated. When, however, the microdrops containing similar 

 cells also included "the opalescent, slightly viscous material which 

 poured off the cells in the disaggregating fluid," differentiation of the 

 cells was normal and similar to that seen in organotypic cultures of 

 the source tissue. Wilde reports further data, involving combination 

 of cells with extracellular material from heterotypic sources, suggest- 

 ing that the material may not only be necessary for differentiation but 



