Thomas J. King and Robert Briggs

Lankenau Hospital Research Institute and Institute for Cancer Research, Philadelphia, Pennsylvania

Nuclei of late gastrula endoderm, transplanted to enucleated eggs (Rana pipiens) promote the following general types of development: 1) arrest at gastrula stages, 2) normal gastrulation followed by deficient development at later stages, especially in ectodermal derivatives, 3) normal development throughout.

In order to determine if the nuclear changes responsible for the deficient development are stable, serial transfers of endoderm nuclei were carried out. Indiviual nuclei were transplanted to enucleated eggs, which cleaved and produced blastulae. In a given test, one of these blastulae was sacrificed to provide nuclei for transfer to a new group of eggs. Such a group represents a clone, all members of which are nucleated by descendents of one original endoderm nucleus. One member of such a clone may be sacrificed at the blastula stage to provide nuclei for transfer to a new group of enucleated eggs, giving in effect a second blastula generation of the clone. The same process may be repeated to provide several generations.

Analysis of the development of 18 clones revealed the following: Whereas test eggs containing different endoderm nuclei developed in the different ways mentioned above, eggs within one clone developed more uniformly. In some clones all embryos were arrested at the gastrula stage, in others they displayed a fairly uniform set of deficiencies in post-gastrula development, and in a few clones almost all embryos developed normally throughout. Furthermore, within any given clone the development in the second and later generations were generally of the same type as that observed in the first generation. In a few clones the deficiencies became more severe in the later generations, but no case of reversal to a more normal type of development was noted.

Chromosome studies on donor embryos in the clonal experiments showed that chromosome number generally remained unchanged at the diploid value (26). In three clones, all consisting of embryos arresting at early gastrula stage, a few small ring chromosomes were present. Otherwise, no chromosome changes were detected even though the clones exhibited quite different types of development.

These experiments show that the descendants of individual endoderm nuclei have a fairly uniform expression with respect to differentiation, which does not reverse to a more normal expression in the course of the serial transfers. In other words, compared with nuclei of undifferentiated cells, the endoderm nuclei show stabilized changes in capacity to promote differentiation. How these changes arise, whether they are specific, and which of the nuclear or peri-nuclear structures are involved, are problems remaining to be worked out.

1. Meisner LF, and Frenster JH, "In Vivo Evolution within Radiation-Induced Clones of Human Lymphocytes", J. Cell Biol. vol. 39, no. 2, part 2, p. 155 a (1968).

2. Frenster JH, and Herstein PR, "Gene De-Repression", New Eng. J. Med. vol. 288, pp. 1224-1229 (June 7, 1973).

3. Frenster JH, "Activation of DNA Transcription within Repressed Chromatin", 14th John Innes Symposium, Sept. 5-8, 2001.