Microenvironmental Influences in Cytodifferentiation 233 



were placed in trypsin, the spot gradually disappeared over a 10- 

 minute interval (Fig. 15 ) . Curiously, the spinal cord could he removed 

 from the filter relatively easily hefore fixation, and such filters follow- 

 ing fixation showed no sign of a spot. In general, the behavior sug- 

 gested deposition in the filter of a mucoid slime. Neither unspotted 

 filters, however, nor spotted filters following careful washing to re- 

 move fixative had any inductive effect on metanephrogenic mesen- 

 chyme subsequently added. 



The impression that protein is a constituent of the interspace 

 material is strengthened by recent autoradiographic studies in which 

 dorsal spinal cord was labeled by exposure to tritiated amino acids, 

 before being put into culture on the membrane in the presence or 

 absence of metanephrogenic mesenchyme. After varying periods of 

 incubation, the cultures were fixed in alcohol-formalin, sectioned, 

 and processed for autoradiography. The presence of silver grains in 

 the overlying radiation-sensitive film marks the location of the tritium 

 and, because tritium emission has low energy and a short emission 

 path, the resolution of the method is high. Dorsal spinal cord becomes 

 very heavily labeled during incubation for 2 hours in the tritiated 

 amino acid solution. This labeling, presumably primarily of proteins 

 in the tissue, is indicated by marked blackening of the film immedi- 

 ately over the cord ( Fig. 16 ) . More interesting, however, is the fact that 

 in cultures incubated for 24 hours there is evident radioactivity in 

 the filter interspace beyond the tissue, as well as in the metanephro- 

 genic mesenchyme on the opposite side. Grain counts over the filter 

 gave values 7 to 16 times background count, and these indications of 

 radioactivity were limited to the general region previously identified 

 as the "spot." It thus appears that administration of labeled protein 

 precursors to the spinal cord deposits label in the filter in a form 

 fixable by alcohol-formalin. It remains to be demonstrated that the 

 tritium-labeled material and the trypsin-sensitive material are one 

 and the same, but it will be more surprising if they are not than if 

 they are. 



Using the grain-counting procedure, it is possible to get an estimate 

 of the amount of labeled material with increasing distance from the 

 spinal cord. For this purpose labeled cord was incubated on a thicker 

 filter, 125 to 150 microns, to provide a distance greater than that 

 which inductive activity will cross. After incubation for varying 

 periods such cultures were autoradiographed, and counts were made 

 on successive 13-micron ranks across the thickness of the filter. Omit- 

 ting details, it is clear that radioactivity falls off sharply in the first 

 60 microns and approaches background in the vicinity of 100 microns 



