roughly twice the number of cells as did diploid 

 embryos at a comparable developmental stage. 

 It has been shown elsewhere that triploid cells 

 are 3/2 as large (10) and contain 3/2 as much 

 DNA as diploid cells. This implies that triploid 

 and diploid embryos of the same size contain the 

 same amount of DNA, since triploid embryos 

 contain two-thirds as many cells as diploid 

 embryos and each triploid cell has 3/2 as much 

 DNA as a diploid cell. 



Indirect evidence suggests that the amount 

 of DNA necessary to reach any developmental 

 stage is a function of the volume of the egg 

 from which the embryo originated. Frog embryos 

 originating from small eggs, consisted of a 

 reduced number of normal sized cells as com- 

 pared to control embryos originating from 

 normal sized eggs (12). The smaller embryos 

 contained fewer cells and, therefore, probably 

 less DNA than did the larger embryos at the 

 same developmental stage. A reduction in the 

 amount of cytoplasm per embryo thus produced 

 a proportional decrease in the DNA content per 

 embryo as compared to normal sized controls. 

 These considerations suggest that during de- 

 velopment the extent of DNA synthesis is 

 regulated by the amount of cytoplasm present 

 in the embryo and that this regulation is re- 

 flected by the similar DNA/ cytoplasm ratios of 

 haploid, diploid and triploid embryos. 



Since haploid and diploid embryos of the 

 same developmental stage contain about the same 

 amount of DNA, it is possible that the stage of 

 an embryo is dependent on the DNA content of 

 that embryo. That is, a certain quantity of DNA 

 (relative to the amount of cytoplasm present) 

 must be present in an embryo before the 

 embryo can attain a specific developmental 

 stage. 



We have seen that the DNA content seems 

 to be controlled by the amount of cytoplasm 

 present in the egg and that R-RNA synthesis 

 is apparently stage dependent. With these ob- 

 servations in mind a hypothesis concerning the 

 gross regulation of R-RNA synthesis during 

 development follows. Specifically I would sug- 

 gest that during development the extent of DNA 

 synthesis is controlled by the amount of cyto- 

 plasm present in the embryo and that an inter- 

 action between the DNA and the cytoplasm 

 somehow regulates the synthesis of R-RNA. 

 It is now possible to design experiments to 

 directly test this hypothesis. 



POLLARD: Have you tried any microinjec- 

 tions? You could just mash up an ordinary 

 embryo, one that won't arrest in two or three 

 days, separate out the enzyme part and inject 



it into the mutant. This is based on the possi- 

 bility that a "transcriptase" for making ribos- 

 omal RNA is missing. 



KOHNE: Usually when you inject anything 

 into these embryos, they arrest all by them- 

 selves. It's very difficult to put anything into an 

 egg because you get chromosomal abnormalities. 



POLLARD: If these are already arrested, 

 you've got nothing to lose. 



KOHNE: There is something that more or 

 less approximates what you're asking. I haven't 

 the vaguest idea what it means but Briggs at 

 Indiana has an axolotol mutant that he calls 

 the "00" or something similar. This mutant 

 even looks different during early development, 

 but it will develop into a gastrula and then 

 become arrested. However, if you take normal 

 egg cytoplasm and inject it into this mutant, it 

 develops beautifully. 



POLLARD: Maybe that "loosens up" the 

 transcription. 



GROSS: With this technique you get a gas- 

 trula arrest and a failure of the ribosomal 

 RNA synthesis to turn on? However, you're not 

 suggesting that it's the failure of ribosomal 

 RNA synthesis to turn on that is responsible 

 for the gastrula arrest, are you? 



KOHNE: No, I think the evidence from the 

 anucleolate mutant says that ribosomal RNA 

 is not needed yet, at least until stage 21. 



PAPACONSTANTINOU: Are you familiar 

 with the experiments that Stanley Cohen did a 

 few years ago in regard to this arrest? He 

 looked at the respiratory cycle intermediates 

 and found an accumulation of malonic acid in 

 these embryos. I don't know if anybody has 

 repeated them, but I know they are in the 

 literature. You may have a lesion in the res- 

 piratory function and, if this is the case, you 

 may be able to repeat this with your controls 

 by adding malonate. 



KOHNE : There' s one other comment on this 

 that I'd like to make with respect to hybrids 

 of the Rana catesbeiana sperm x Rana pipiens 

 egg cross, which have a haploid set of Rana 

 chromosomes but arrest at gastrula. Haploids 

 which have the Rana pipiens chromosomes de- 

 velop almost normally until the swimming tad- 

 poles. Thus, the catesbeiana chromosomes are 

 doing something that is poisoning the system. 



PAPACONSTANTINOU: Does it always 

 have to be the catesbeiana rasile and the pipiens 

 female? Can it be the other way around? 



KOHNE: Yes, but they arrest, too. There 

 are a lot of hybrid embryos it would be inter- 

 esting to work with but the problem is getting 

 the material. Rana sylxxitica is one where you 



42 



