Fig. 1. (A,B) Dependence of the outer zone depth on temperature in 2-D cultures. a, outer zone; b, inner zone. The dark outermost edge is a meniscus. Freshly starved NC-4 cells at (A) 30°C, 10 min after preparation, and (B) 4.5°C, 15 min after preparation. (C) Freshly starved NC-4 and Ax-2 cells were two-dimensionally cultured in an isothermal vessel. Each plot represents a 1-2 h time average of the outer zone depth L_out. Plots are fitted by L_outTexp(E/2RT), where T is absolute temperature (K) and R is the gas constant, giving activation energy of E=65 kJ mol^-1, E=69 kJ mol^-1 for Ax-2 and NC-4 cells, respectively. Plots at 40°C have been omitted for the curve fitting. (D—G) Temperature dependence on cell differentiation. Earlymound stage cells were subjected to 2-D culture. Cells at the outermost region of the outer zone show D19 expression at both 22°C, t=4 h (D) and 10°C, t=4 h (E). The ecmB expression observed near the border at 22°C (t=2 h) (F) is suppressed at 10°C (t=3 h) (G). Scale bars, 200 µm (A,B); 100 µm (C,D).