15 July 2013
ByAppeared in BioNews 713
It is not given to many to overturn a central dogma of scientific faith. But last year, John Gurdon was awarded the Nobel Prize for Physiology or Medicine for doing it.
Fifty-four years earlier, in the Zoology Department of Oxford University, he had, with Michael Fischberg and Tom Elsdale, published in Nature, 'Sexually Mature Individuals of Xenopus laevis from the Transplantation of Single Somatic Nuclei'.
Running to less than two columns (about the length of this piece), the ramifications of what it described have been legion.
Well-entrenched in the 1940s and 1950s, the dogma was this: that once a cell is 'fully differentiated' its functional identity is fixed and irreversible. It is fair to say that, as dogmas go, it was a good one. Were it not true then the cells of a particular tissue, the brain say, might take it into their heads (as it were) to become other sorts of cell - bone, maybe. The Oxford research showed that on the contrary, a differentiated cell could not only give rise to another different, differentiated, cell type but to any and all the cell types in a sexually mature adult animal, genetically identical to the one from which the mature cell had been taken. The motivation for the award of the Nobel Prize, rather laconically, put it like this, '…for the discovery that mature cells can be reprogrammed to become pluripotent'.
A 'slip' cutting from a plant can grow into a genetically identical plant. Greek for slip (or twig) is 'klōn', similar to the modern term 'clone'. 'Clone' itself was first used in the early 20th century to describe plants that had reproduced 'vegetatively'. In 1963, JBS Haldane used the word with reference to animals. In the 1960s pictures of clones (that is, 'families' of genetically identical individuals) of Xenopus laevis, the species of frog that John Gurdon used, assumed iconic status and could be seen almost everywhere. A word of warning though: cloning was not the aim of the game. But clones were the necessary (and compelling) evidence that the game had been won, at least in that round.
Newly 'discovered' scientific territory consists not only of what is now known that was not known before, but also the way in which it was discovered. Here, the way was conceptually simple. Inject into the egg a nucleus taken from a mature differentiated cell ─ and wait to see what the egg did with it. Would the cell divide and embark on a normal developmental trajectory? The cell did (or at least some cells did); and, moreover, followed the trajectory all the way to a healthy, sexually mature, fully developed frog.
Although the Nobel citation referred to a mature cell being reprogrammed, it might equally well have said 'de-programmed'. Stability is what is needed in a fully differentiated somatic cell. It is 'designed' (programmed if you like) to stay put and do the particular job it has been given; and definitely not start embarking on new and unsuitable careers. In a striking contrast an egg cell is designed to initiate development. Its first job on encountering the package of tightly packed DNA that constitutes the head of a sperm is to destabilise, unpack and de-programme it. The success of the Gurdon experiment depended absolutely on the egg cytoplasm doing to a somatic cell nucleus (something that had never happened naturally, remember) what evolution had fitted it to do to the head of a sperm. After all, a sperm is an extremely specialised cell, arguably even more specialised than, say, a nerve cell. The interaction between egg and sperm might have been very specific.
Evidence may be convincing, but that does not necessarily guarantee its reliability. For a start it could not be absolutely certain that the transferred-in nucleus was 'fully differentiated'. Indeed did the term have a defined, demonstrable, universally applicable meaning? Science waited until 1975 for what it regarded as final proof. An antibody-producing lymphocyte assumed paradigm status for a fully differentiated cell in Gurdon-type experiments.
And there again could it be beyond any shadow of doubt that the clone had not developed from the undifferentiated egg 'nucleus'? Yes. But if and only if there was a means of discriminating between cells that had developed from egg (if any) and somatic nucleus (if any). Enter the 'nucleolus'; the cell's 'ribosome factory'. It is the most apparent sub-structure in the nucleus and easily seen under a light microscope. The 'wild type' nucleus has two. The Oxford embryology lab, courtesy of Michael Fischberg, had a colony of mutant Xenopus with only one. By taking the donor nucleus from mutant strains and the recipient egg from wild type ones, it was straightforward to identify the source of the developing embryos in their early stages.
Modern science likes its explanations to be mechanistic. Today, thanks to molecular biology, we know a lot about the machinery the egg uses to unpack the transplanted nucleus, including the three-dimensional structures of many of its individual proteins. John Gurdon shared the Nobel Prize with Shinya Yamanaka, who in the 1990s, unpacked a differentiated nucleus not in vivo using egg cytoplasm but in vitro with a cocktail of (some of) those proteins.
What was reported in Nature in 1958 was neither the start nor finish of a research enterprise. Indeed, whether the words have much meaning in the evolutionary process we call science is doubtful. It owed much both conceptually and methodologically to the experiments of Robert Briggs and Thomas King in Philadelphia and reported six years earlier. Their experiments, though, had given the opposite result to Gurdon's, concluding that nuclei do not retain their full potentiality for development. In research terms they had come to a dead end. Science awards Nobel Prizes to those who have created the greatest opportunities for other scientists to do interesting research. Enough said.