and they reveal the microscopic appearance of 

 RS cells at various stages in their development 

 in a synchronized culture. During ontogeny, a 

 point is reached beyond which the cell becomes 

 irreversibly committed to RS formation. This 

 is the morphogenetic point of no return. It is 

 figuratively represented by the point of 

 dichotomy of the two arrows, and it amounts to 

 43% of the RS generation time - chronologically, 

 36 hours under the conditions we use for growth. 

 Before this point is reached, the cell's morpho- 

 logical potential displays an inherent plasticity; 

 if the bicarbonate is removed from its environ- 

 ment, it reverses "direction" and embarks upon 

 the alternate morphogenetic pathway. In other 

 words, functionally it turns into an OC cell. 

 However, beyond this point of no return, removal 

 of bicarbonate does not cause morphogenetic 

 reversal; the cell continues on its way toward 

 the RS type whether or not the bicarbonate is 

 still present. 



This feature further emphasizes the fact 

 that a synchronized culture of Blastocladiella 

 emersonii represents an easily exploitable sys- 

 tem for experimental studies of morphogenesis. 

 Indeed, it is for this reason that I and my associ- 

 ates, past and present, have been trying to track 

 the various events - intracellular and extracel- 

 lular - which are associated with the genesis of 



a. 

 o 



20-- 



30-- 



Fig. 7. 



Uptake of h'^COJ by an RS cell as it approaches and 

 passes the morphological point of no return. 



a resistant sporangium as it approaches, passes 

 through and then departs again from its point of 

 no return. Many of these events have been fol- 

 lowed on a per-cell basis, and a superficial 

 digest of the results is seen in Fig. 9. You will 

 note that many things begin to increase at an 

 exponential rate - albeit not all of them at 

 identical rates - after spore germination, and 

 cease to do so at the point of no return. Others, 

 glucose uptake for example, begin much later 

 but still end at this same point of no return. 

 However, other features, such as weight, lipid, 

 total nitrogen, chitin, polysaccharide, RNA, 

 melanin, etc., continue to increase to different 

 stages in ontogeny beyond the point of no return. 

 Then again, there are still other events which 

 commence only at or beyond the point of no 

 return. Clearly, then, it was of interest to find 

 out which, if any, of the qualities associated with 

 an RS cell before its point of no return would 

 change to a new state more characteristic of an 

 OC cell if morphological reversal were induced. 

 We have done tests of this sort, and I would 

 like to show you the results that were obtained 

 from one such experiment that Dr. Lovett and I 

 did some years ago. Figure 10 shows what hap- 

 pens to isocitric dehydrogenase and ketoglutaric 

 dehydrogenase during development along the RS 

 path up to a stage well beyond the point of no 

 return. For convenience in making comparisons, 

 the total units per cell at the spore stage were 

 set at one for both enzymes, and all other 

 values were then related to this and plotted 

 accordingly. When bicarbonate was removed 

 from RS cells a few hours before the point 

 of no return, thus inducing morphological re- 

 versal, the total units/cell of isocitric dehydro- 

 genase dropped sharply (whereas without re- 

 versal, it continued to rise) and the total units/ 

 cell of ketoglutaric dehydrogenase rose sharply 

 (whereas without reversal, it did not do so). 

 These results are represented by the dotted 

 lines in Fig. 10. When this same kind of experi- 

 ment is done with cells which have gone beyond 

 the point of no return - and which, therefore, 

 have lost the capacity for morphological re- 

 versal - the total units/cell of these two en- 

 zymes is not influenced by removal of bicarbon- 

 ate. In summary, before the point of no return, 

 morphological plasticity is associated with a 

 corresponding plasticity of two key enzyme sys- 

 tems thought to be directly involved in RS 

 formation; after the point of no return, this 

 plasticity is lost. Analyses of this sort have 

 thus provided additional direct evidence for 

 the biochemical nature of the bicarbonate trigger 



154 



