Microenvironmental Influences in Cytodifferentiation 237 



These results fortify the notion, earlier advanced hy Baitsell (1925 I , 

 Weiss (1933), Huzella (1941 I. and others, that microenvironmental 

 materials produced hy cells have important developmental signifi- 

 cance. Additional support is afforded by data from the second major 

 line of investigation to which I referred earlier — the reaggregation 

 of dissociated cells. This phenomenon was studied first, among ani- 

 mals, in sponges and other relatively uncomplicated invertebrates. 

 More recently it has been under investigation in slime molds, in 

 amphibia, and even in higher vertebrates. Observations on reaggregat- 

 ing mouse and chick cells in the last several years, reported hy Mos- 

 cona ( 1960 ) , are particularly relevant and striking. Moscona finds 

 that at an early stage in the reaggregation of freshly dispersed chick 

 embryonic cells a transparent mucoid material, "evidently an exudate 

 of cellular origin," makes its appearance. The reaggregating cells ap- 

 pear to move within fine strands of this material, which collects on 

 introduced cotton fibers and on the glass floor of the vessel and ap- 

 pears to be the "immediate substratum or microenvironment" for 

 migration and aggregation. Staining reactions and susceptibility to 

 enzymatic digestion suggest that the material probably is a muco- 

 protein. 



Freshly disaggregated cells produce considerable quantities of 

 mucoid; it can be accumulated in strands or ropes by gently swirling 

 reaggregating cells toward a central vortex. Cells which are grown as 

 monolayer cultures for a period of time undergo change in the qual- 

 ity, and reduction in the quantity, of the mucoid material they 

 produce. Concomitantly, aggregative behavior and cell coherence 

 gradually decline. Thus, dissociated mouse embryonic skin cells, after 

 more than 10 days in monolayer culture, form only small clusters, 

 even when they are swirled into proximity or centrifugally packed to 

 favor aggregation. Such clusters show very limited histogenesis and 

 differentiation. The aggregation and histogenesis are little affected 

 when the cells are intermixed with freshly dissociated chick or mouse 

 precartilage cells (heterotypic), but are markedly improved on inter- 

 mixing with freshly dissociated chick skin cells ( homotypic ) . Under 

 the latter conditions the mouse cells are found grouped together and 

 participating in the formation of follicle-like structures. 



The decline in aggregability and histogenesis with culturing de- 

 scribed by Moscona recalls and illuminates an observation made some 

 years ago on embryonic mouse salivary rudiments in vitro. The sali- 

 vary rudiment is a two-component structure like the metanephros and, 

 similarly, the components can be separated by trypsin treatment. 

 When the two components are recombined immediately, the inner 



