The other major objection to a genetically based embryology remained: How could nuclear genes direct development when they were the same in every cell type? The existence of this genomic equivalence was not so much proved as assumed (because every cell is the mitotic descendant of the fertilized egg), so one of the first problems of developmental genetics was to determine whether every cell of an organism indeed had the same set of genes, or genome, as every other cell.
The ultimate test of whether the nucleus of a differentiated cell has undergone any irreversible functional restriction is to have that nucleus generate every other type of differentiated cell in the body. If each cell’s nucleus is identical to the zygote nucleus, then each cell’s nucleus should also be totipotent (capable of directing the entire development of the organism) when transplanted into an activated enucleated egg. As early as 1895, the embryologist Yves Delage predicted that “If, without deterioration, the egg nucleus could be replaced by the nucleus of an ordinary embryonic cell, we should probably see this egg developing without changes” (Delage 1895, p. 738). Before such an experiment could be done, however, three techniques for transplanting nuclei into eggs had to be perfected: (1) a method for enucleating host eggs without destroying them; (2) a method for isolating intact donor nuclei; and (3) a method for transferring such nuclei into the host egg without damaging either the nucleus or the oocyte.
All three techniques were developed in the 1950s by Robert Briggs and Thomas King (see Mc Kinnell 1978; Di Berardino and Mc Kinnell 2004). First, they combined the enucleation of the host egg with its activation. When an oocyte (a developing egg cell) from the leopard frog Rana pipiens is pricked with a clean glass needle, the egg undergoes all the cytological and biochemical changes associated with fertilization. The internal cytoplasmic rearrangements of fertilization occur, and the completion of meiosis takes place near the animal pole of the cell. The meiotic spindle can easily be located as it pushes away the pigment granules at the animal pole, and puncturing the oocyte at this site causes the spindle and its chromosomes to flow outside the egg (Figure 1). The host egg is now considered both activated (the fertilization reactions necessary to initiate development have been completed) and enucleated.
The transfer of a nucleus into the egg is accomplished by disrupting a donor cell and transferring the released nucleus into the oocyte through a micropipette. Some cytoplasm accompanies the nucleus to its new home, but the ratio of donor to recipient cytoplasm is only 1:105, and the donor cytoplasm does not seem to affect the outcome of the experiments. In 1952, Briggs and King, using these techniques, demonstrated that blastula cell nuclei could direct the development of complete tadpoles when transferred into the cytoplasm of an activated enucleated frog egg. This procedure is called somatic nuclear transfer, or more commonly, cloning.
What happens when nuclei from more advanced developmental stages are transferred into activated enucleated oocytes? King and Briggs (1956) found that whereas most blastula nuclei could produce entire tadpoles, there was a dramatic decrease in the ability of nuclei from later stages to direct development to the tadpole stage (Figure 2). When nuclei from the somatic cells of tailbud-stage tadpoles were used as donors, normal development did not occur. However, nuclei from the germ cells of tailbud-stage tadpoles (which could give rise to a complete organism after fertilization) were capable of directing normal development in 40 percent of the blastulae that developed (Smith 1956). Thus, most somatic cells appeared to lose their ability to direct development as they became determined and differentiated.
Is it possible that some differentiated cell nuclei differ from others in their ability to direct development? John Gurdon and his colleagues, using slightly different methods of nuclear transplantation on the frog Xenopus, obtained results suggesting that the nuclei of some differentiated cells can remain totipotent. Gurdon, too, found a progressive loss of potency with increasing developmental age, although Xenopus cells retained their potencies for a longer period than did the cells of Rana (Figure 3).
To clone amphibians from the nuclei of cells known to be differentiated, Gurdon and his colleagues cultured epithelial cells from adult frog foot webbing. These cells were shown to be differentiated: each of them contained a specific keratin, the characteristic protein of adult skin cells. When nuclei from these cells were transferred into activated enucleated Xenopus oocytes, none of the first-generation transfers progressed further than the formation of the neural tube shortly after gastrulation. By serial transplantation (i.e., taking nuclei from the cloned blastulas), however, numerous tadpoles were generated (Gurdon et al. 1975). Although the tadpoles all died prior to feeding, they showed that a single differentiated cell nucleus still retained incredible potencies. A nucleus of a skin cell could produce all the cells of the young tadpole.