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SummarySir John Gurdon and Professor Shinya Yamanaka were therecipients of the 2012 Nobel Prize for Physiology or Medicine.This Spotlight article is a commentary on the early nucleartransplant work in Xenopus, which was very important for theNobel award in 2012, and the influence of this work on thereprogramming field.

BackgroundAs a brand new graduate student starting in October 1956, mysupervisor Michail Fischberg, a lecturer in the department ofZoology at Oxford, suggested that I should try to make somatic cellnuclear transplantation work in the South African frog Xenopuslaevis. There were good reasons for wanting to do this (see below).The very important question to be addressed at that time waswhether all cell types in the body have the same set of genes. Thisquestion had been asked by embryologists since 1886 (Rauber,1886), and Spemann (Spemann, 1938) had demonstrated by an eggligation experiment that the nuclei of an eight-cell frog embryo aredevelopmentally totipotent. It was clear that a definitive experimentrequired the replacement of a zygote nucleus by a somatic cellnucleus, asking whether the somatic nucleus could functionallyreplace the zygote nucleus by eliciting normal development of theenucleated recipient egg (Fig. 1)? Briggs and King (Briggs andKing, 1952) had already succeeded in transplanting a blastula cellnucleus into an enucleated egg and obtaining normal tadpoles in thefrog Rana pipiens. However, Briggs and King (Briggs and King,1957) had also found that the nucleus of an endoderm cell from aneurula embryo could no longer support normal development(Fig. 2). They drew the reasonable conclusion that, as developmentproceeds from a blastula to a neurula stage (about 24 hours), somegenes needed for normal development had either been lost orirreversibly repressed.

To this extent, the aim of my proposed PhD work had alreadybeen done and the answer to the primary question already obtained.Why then did it make sense for me to try to repeat this work on arelated species? There appeared to be two possible outcomes. Onewas that I might obtain a different result from Briggs and King andso the primary question would be re-opened and subject to fruitfulinvestigation. The other was that I might obtain the same result asBriggs and King and this would then open the important question ofwhat the mechanism could be by which a somatic cell nucleusalready committed to a specific (in this case endoderm) fate isresistant to reprogramming by exposure to egg cytoplasm.

Preliminary investigation showed that there would be substantialtechnical difficulties in achieving somatic cell nuclear transfer withXenopus, in the way that Briggs and King succeeded for Rana

pipiens. The first of these was that, unlike Rana, the Xenopus eggis covered with a dense and extremely elastic jelly, which iscompletely impenetrable by even the finest of micropipettes (Fig. 3).The second was that this jelly covering made it very hard, even ifpossible, to remove metaphase egg chromosomes by causingextrusion of them with a needle, the method used in Rana pipiens.There were, on the other hand, very good reasons to wish to makethis technique succeed in Xenopus. How Xenopus laevis, a native ofSouth Africa, came into use for developmental biology has anamusing and serendipitous history (Gurdon and Hopwood, 2000).First, Xenopus can respond to the injection of commerciallyavailable mammalian hormones (follicle stimulating hormone andluteinising hormone) by laying eggs the next day and this procedureworked throughout the year. In contrast, frogs of the European andNorth American Rana species will lay eggs only in the spring ofeach year unless they are injected with frog pituitary gland extract,and this requires about five pituitary glands from killed frogs toobtain one egg ovulation. In the past, European embryologists hadthe use of living frog eggs for only a month or two in the year andhad to do other things, such as histology etc., for the rest of the year.Xenopus, in principle, permitted experiments on living embryos tobe done throughout the year. Second, Xenopus is an aquatic frogand therefore easy to maintain in the laboratory in water tanks ratherthan having to clean a terrarium as was necessary with Rana.Furthermore, Xenopus species can be reared from the fertilised eggto sexual maturity in less than one year (compared with three to fouryears for Rana), thereby making it realistic to propagate geneticmutants and make use of them. A further advantage of Xenopuslaevis is that this species lives in highly infected cattle sewage pondsand has built up an extraordinary resistance to infection anddiseases. Michail Fischberg therefore concluded that it was sensibleto have me try, at least for a while, to achieve successful somatic cellnuclear transfer in Xenopus.

The aim of this article is to recount the early history of nucleartransfer in Xenopus as a result of which the recent Nobel award wasmade (Jaenisch, 2012). Further work in Xenopus that has led onfrom this up to the present time is covered only briefly, and has beenreviewed elsewhere (Jullien et al., 2011; Pasque et al., 2011b).

The technique of nuclear transfer in XenopusThere is no doubt that I was blessed with a considerable amount ofluck. But the phrase that ‘luck favours the prepared mind’ may wellhave been true. My supervisor had just acquired a microscopeequipped with ultraviolet light illumination. There was reason tobelieve that ultraviolet light would destroy DNA in the eggchromosomes, which, very fortunately, are located right on thesurface of the animal pole of amphibian eggs. Aiming the ultravioletlight source onto the animal pole of unfertilised eggs was successfulin destroying the egg chromosomes, as shown by fertilising suchirradiated eggs with sperm and obtaining haploid embryos. If theegg chromosomes had not been located on the surface of a largeamphibian egg, ultraviolet light (having very low penetration)would not have reached them (Gurdon, 1960a). Perhaps even morefortunate was our finding that the particular ultraviolet lamp just

1Wellcome Trust/Cancer Research UK Gurdon Institute, The Henry WellcomeBuilding of Cancer and Developmental Biology, University of Cambridge, TennisCourt Road, Cambridge CB2 1QN, UK. 2Department of Zoology, University ofCambridge, Cambridge, UK.

*Author for correspondence (j.gurdon@gurdon.cam.ac.uk)

©The Nobel Foundation

SPOTLIGHT

Development 140, 2449-2456 (2013) doi:10.1242/dev.097170© 2013. Published by The Company of Biologists Ltd

The egg and the nucleus: a battle for supremacyJ. B. Gurdon1,2,*

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bought for microscopy progressively denatured (dissolved) theelastic jelly around the egg. After ultraviolet light exposure,unfertilised eggs became easily penetrable by a micropipette, andthis happened in a dose-dependent way, making it possible toenucleate the egg, leaving just enough jelly to help seal thepenetration wound made by a micropipette. It was not known, atthat time, that this egg jelly could be removed by an alkalinesolution of cysteine hydrochloride, but good luck, or mysupervisor’s wisdom, or both, did not stop at this point. Crucial tothe validity of these early experiments was proof that the eggchromosomes had in fact been destroyed and did not contribute tothe development of the nuclear transplant embryos. Another PhDstudent of my supervisor, namely Sheila Smith, was studying themorphology of haploid development in Xenopus. She was advisedto use a single nucleolus per nucleus as a measure of haploidy. Sheencountered an inexplicable result, namely that embryos carryingonly one nucleolus per nucleus developed entirely normally,whereas haploids (which have only one nucleolus per nucleus orchromosome set) always die before feeding. Most supervisorswould have told the student to repeat the experiment the next week,starting with completely different material, to see if the result wasreproducible. Michail Fischberg (Fig. 4), however, had the wisdomor intuition to tell the student to find out which frog had been usedto give the eggs that yielded normal one-nucleolated embryos.Amazingly, the student’s result was reproducible. Michail Fischbergconcluded that there must have been a mutation in one chromosomeset so that it was unable to make a nucleolus (Elsdale et al., 1960).Later work showed that this strain of Xenopus had indeed lost all the

ribosomal genes located in one nucleolus organiser and thereforethat heterozygotes for this deletion would never carry more thanone nucleolus per diploid chromosome set (Brown and Gurdon,1964). This mutation gave us an extraordinarily valuable nuclearmarker for nuclear transfer experiments (Fig. 5). Some years later,an albino strain of Xenopus laevis provided a more visually strikingmarker (Fig. 6).

Using the benefits of ultraviolet radiation, combined with agenetic marker, it was possible, rather rapidly, to show that somaticcell nuclear transfer in Xenopus worked well. Within one year ofstarting work, I had found that the nucleus of an endoderm cell froman advanced tadpole was able to yield some normal development upto the nuclear transplant tadpole stage. This was not in agreementwith the results of Briggs and King (Fig. 2).

Normal development from the nuclei ofdifferentiated intestinal epithelium cellsWithin another year, now 1958, I found that it was technicallypossible to transplant single nuclei from the intestinal epithelium offeeding tadpoles. I found it best to distort the donor cells as little aspossible (Gurdon, 1960b), so that at least some of them had thenucleus in a ruptured cell wall, even though other such donor nucleimay have been transplanted in whole non-permeabilised cells,which would not be able to respond to the egg cytoplasm or beginto cleave. It seemed important not to expose the nucleus of aruptured cell to the simple saline medium used for nuclear transfer.It was later found that treating small donor cells with StreptolysinO was a great deal easier than cell rupture in a narrow pipette (Chanand Gurdon, 1996).

The success of these intestinal epithelium nuclear transfersdiffered from one experiment to another. However, some femalessupplying recipient eggs gave significantly better developmentthan others. Egg quality was therefore a factor. Nevertheless, theseresults showed that starting with nuclei from differentiatedintestinal epithelium cells, with a striated border, some of the

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Somatictissue

Somaticcells

Somaticnucleusin egg

Adult nucleartransplant frog

Egg enucleated

by UV irradiation

Fig. 1. Design of a somatic cell nuclear transfer experiment usingunfertilised eggs as first designed by Briggs and King (Briggs andKing, 1952) for Rana pipiens and as used subsequently in Xenopus. InRana, enucleation is by hand with a needle, and in Xenopus by ultravioletlight irradiation (Gurdon, 1960a).

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Developmental stage from which nuclei were taken

Xenopus normal feeding tadpoles

Rana swimming tadpoles

Fig. 2. Survival of nuclear transplant embryos in Rana pipiens andXenopus laevis. Even advanced donor cells from the endoderm ofXenopus tadpoles have nuclei that can sometimes yield normalindividuals after nuclear transfer [data taken from Briggs and King (Briggsand King, 1957) for Rana and from Gurdon (Gurdon, 1962) for Xenopus].

A B

C

BBBB

D

B

Fig. 3. The Xenopus egg is surrounded by a dense elastic jelly so thatit is not possible to penetrate into the egg cytoplasm with amicropipette, unless the jelly is removed or denatured by ultravioletlight. (A) Side view. (B) ‘Animal’ pole; the white area in the middle of theblack area is where the egg chromosomes are located. (C,D) If the egg isnot de-jellied, a micropipette depresses the jelly coat (C), eventuallydragging the pipette, still surrounded by jelly, through the egg withoutentering the cytoplasm (D).

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nuclear transplant embryos developed entirely normally to thefeeding tadpole stage and were progressing towardsmetamorphosis. Furthermore, these tadpoles carried only onenucleolus per nucleus with a diploid set of chromosomes. Thisshowed that the transplanted nucleus that gave normaldevelopment did indeed derive from an intestinal epithelium cell.Although the percentage of intestinal epithelium cell nucleartransfers that yielded entirely normal feeding tadpoles was low(1.5%) (Gurdon, 1962) many such individuals were obtained andthey all carried the nuclear marker.

My supervisor and his assistant looked after my nucleartransplant tadpoles, which had by now metamorphosed into youngfrogs, during my absence for postdoctoral work in another field. Onmy return, these intestinal nuclear transplant tadpoles had becomeadult male and female frogs, and their fertility and ability to generatenormal embryos was tested. This yielded, in 1966, our paper entitled“Fertile” intestine nuclei (Gurdon and Uehlinger, 1966). Thistherefore gave the opposite conclusion to that of the Briggs andKing work with Rana pipiens. Of course, there was criticism that agraduate student, working almost alone, had not been able to repeatthe results of well-established and highly respected workers Briggsand King. The use of the one-nucleolated genetic marker was crucialin persuading scientists that this Xenopus work was valid. In thecourse of time, it became accepted in scientific circles that cells canundergo complete differentiation, to the point of making intestinalepithelium cells of a feeding tadpole, without any loss or stableinactivation of genes needed for entirely unrelated cell lineages andindeed for every cell type.

After these early experiments, the main conclusion that, duringthe course of cell differentiation, the genome is conserved andrepressed quiescent genes can be reactivated was confirmed. Withvarious colleagues, and especially R. A. Laskey, we were able toobtain normal tadpoles from adult foot web skin and from a rangeof adult organs, such as heart, lung, etc., from cells grown out inculture from these tissues (Laskey and Gurdon, 1970). Although wewere able to obtain normal sexually mature male and female adultanimals from the intestinal epithelium cells of a feeding tadpole andwe were able to obtain feeding tadpoles from the nuclei of adultcells, we never obtained a sexually mature adult animal startingfrom the nucleus of another adult cell. We think that the intensely

rapid cell division and DNA replication enforced on an amphibiantransplanted nucleus by an activated egg has a high probability ofintroducing replication defects, as is seen in Rana pipiens (DiBerardino and King, 1967), thereby greatly reducing the chance ofobtaining entirely normal development from the nucleus of an adultcell.

Epigenetic memoryIn addition to the rapid DNA replication and cell division enforcedon a transplanted somatic nucleus, there are other ways in whichwe may account for the progressively decreasing success rate ofnuclear transfers from differentiating and differentiated cells. Oneof these is that there may be a memory of a pattern of geneexpression characteristic of the differentiated state. One obviouspossibility is that there could be a resistance to the reactivation ofthose genes that are needed for early development but which havebecome quiescent or repressed during cell differentiation. Thispossibility is discussed below under the heading of ‘Resistance toreprogramming by oocytes’.

Another interesting possibility is that there could be a memory ofan active gene state. For example, those genes that are stronglyexpressed in specialised cells might fail to be switched off afternuclear transfer and might then interfere with new directions oflineage selection by nuclear transplant embryos. Methods that werenot available when amphibian nuclear transfers were first carriedout have now made it possible to test this idea. Nuclei weretransplanted from muscle or other lineage-specific progenitor cellsof the resulting nuclear transplant embryos. Although many of theresulting nuclear transplant embryos develop abnormally, it waspossible to collect enough material from partially cleaved embryosto carry out gene-specific transcript assays. The surprising resultwas obtained that a considerable memory of an active gene statepersisted through many cell cycles of inactive transcriptioncharacteristic of early amphibian embryos. We found that theneurectoderm and endoderm lineages of nuclear transplant embryosderived from muscle progenitor nuclei often continued to expressmuscle genes to an excessive extent (Ng and Gurdon, 2005). Thememory was imperfect, in that about half of the nuclear transplantembryos from muscle progenitor cells showed an excessive,

1-nucleolated strain 2-nucleolated strain

Fig. 4. Michail Fischberg, 1918-1998. Michail Fischberg was born in StPetersburg, educated in Switzerland and completed his PhD under E.Hadorn. A distinguished line of mentorship traces back from Hadorn toBaltzer to Boveri. Michail Fischberg was my graduate supervisor in Oxford,England, from where he moved to Geneva.

Fig. 5. A nucleolar genetic marker for Xenopus laevis. Heterozygotesof the one-nucleolated strain have only one nucleolus per diploidnucleus (left), compared with the wild-type 2-nucleolated form for whichmost nuclei have two nucleoli (right; examples indicated by arrows)(Elsdale et al., 1960). The one-nucleolated strain has a deletion ofribosomal genes on one chromosome (Brown and Gurdon, 1964). D

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sometimes very large, overexpression of muscle genes ininappropriate tissues whereas the other half did not (Fig. 7). Genescharacteristically expressed in a certain lineage became repressedfor the early cleavage divisions of a nuclear transplant embryo butthen became re-expressed throughout the embryo after the stage oftranscriptional activation at the late blastula stage. This result wasalso found in other, non-muscle lineages (Ng and Gurdon, 2005). Itwas also found that this ‘memory’ of an active gene state wasassociated with histone H3.3, an abundant protein in eggs and earlyembryos. The explanation offered for this phenomenon was that thevery high level of histone H3.3 normally present in oocytes andeggs (Cho and Wolffe, 1994) enhances transcription of any genethat is in an active state at the time of nuclear transplantation (Ngand Gurdon, 2008). Histone H3.3 is known to be associated withactive transcription. Memory of an active gene state wassubsequently described in iPSC experiments (Polo et al., 2010). Theobservation that about 50% of nuclear transplant embryos show thismemory that is not seen in the other 50% exemplifies the concept(see later) that there is a conflict between components of an egg thatare designed to change gene expression to that characteristic of an

egg and embryo and the resistance of the nucleus of determined orspecialised cells to resist any change thereby stabilising the pathwayof differentiation on which an embryonic cell has set out.

Nuclear transfer in mammalsFor these early results in Xenopus to be reproduced in mammals tooknearly 40 years (Campbell et al., 1996; Wilmut et al., 1997) in sheep.A very important feature of these first successful mammalian nucleartransfers in sheep was the use of unfertilised eggs, as was actuallyused in amphibia. Earlier work with mice (McGrath and Solter, 1984)used fertilised eggs. Although fertilised eggs can be used (Egli et al.,2007), synchronisation between nucleus and egg is harder to achievethan with the use of unfertilised eggs. A very elegant and importantexperiment that confirmed the general principle that celldifferentiation proceeds with the retention of a complete set of geneswas carried out using nuclei with a rearranged genome from maturemouse B or T donor cells (Hochedlinger and Jaenisch, 2002). In thecourse of time, somatic cell nuclear transfer to eggs has beensuccessful in the eggs of mice and other mammals (Wakayama et al.,1998). In each species there seem to be some technical requirementsthat have to be identified and overcome. In mammals, the early celldivisions after fertilisation are extremely slow compared withamphibians (20 hours from fertilisation to two-cell stage in themouse). It is therefore unlikely that the chromosome damage seen inamphibian work (above) takes place in mammals. Nevertheless, thedecreasing success rate of nuclear transplant embryo development isabout the same in mice and frogs (Fig. 8). There must be other reasonsfor this resistance to reprogramming by eggs.

This brief history of successful somatic cell nucleartransplantation does not do justice to the many importantcontributions made after the early Xenopus work. For a review ofthe early Xenopus work see Gurdon (Gurdon, 1986). Subsequentreviews that also cover the early work have been published byMcKinnell (McKinnell, 1978), Di Berardino and Hoffner (DiBerardino and Hoffner, 1983) and Gurdon (Gurdon, 2006).

Mechanisms of nuclear reprogramming by eggsThe second question raised when embarking on a PhD thesis onnuclear transfer in Xenopus concerned mechanisms ofreprogramming. There are two parts to this question. First, how doesan egg reverse the differentiation state of a somatic nucleus to enableit to behave like a zygote nucleus when it leads to entirely normaldevelopment? The second question asks in what way do the nucleiof somatic cells become progressively resistant to thereprogramming conditions of an egg?

To approach these questions it was clearly necessary to focus onthe transcription of individual kinds of genes. At that time, in the1960s, genes had not been cloned and it was only possible to workwith genes that were present in multiple copies per genome, such as28S, 18S and 5S ribosomal genes. Working with ribosomal genes,transfer RNA genes, and the gross class of genes whose basecomposition resembled the average of the genome, it was shownthat transplanted somatic nuclei in blastula and gastrula stages ofnuclear transplant embryos had reverted to an embryonic pattern oftranscription (Woodland and Gurdon, 1969). However, this did notlead to understanding the mechanism by which this rejuvenationtook place. It took a few decades before single genes expressed inearly Xenopus development had been cloned and the necessaryprobes and procedures developed by which the expression of thesegenes could be monitored in nuclear transplant embryos.

At this time it seemed useful to investigate the mechanisms thatguide early embryo development when nuclear transplant embryos,

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Fig. 6. A clone of albino male frogs obtained by transplanting nucleifrom cells of an albino embryo to enucleated eggs of the wild-typefemale shown. The albino frogs are genetically identical and will acceptskin grafts from each other.

Donor tadpole

Muscle cell

Nuclear Blastula Endoderm

Neurectoderm

No transcription

Highexpressionof muscle

genes

56%

52%

Transcription

transfer

Fig. 7. Epigenetic memory in nuclear transplant embryos. Nucleartransplant embryos derived from muscle nuclei were grown to theblastula stage, and then depleted of the mesoderm region (musclelineage). The remaining regions (neurectoderm for nerve/skin cells andendoderm for intestine lineages) express the muscle gene marker MyoDto an excessive extent in about half of all such embryos (Ng and Gurdon,2008). D

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especially from advanced donor stages, often develop veryabnormally. I wondered whether the nuclear transfer techniquescould be used to introduce purified macromolecules into an egg,and hence into embryonic cells. Thanks to my scientific friendshipwith Jean Brachet of Belgium, a major contributor to the field ofdevelopmental biology (Brachet, 1957), I was able to acquire asmall sample of purified globin mRNA from the laboratory of DrChantrenne. He was one of the first to purify any animal mRNA.Even a few micrograms of this was more precious than gold dust,and anything that came in contact with it had to be chromic acidcleaned for fear of RNase. A highlight of my career at this time wasthe discovery that purified mRNA could be extremely efficientlytranslated into protein when injected directly into an egg orembryonic cell (Gurdon et al., 1971). Interestingly, this finding wascompletely unexpected because of the very high ribonucleaseactivity present in eggs. For this reason, a grant application to permitsuch an experiment would not have succeeded. I was fortunate tohave enough other funding to do this work without specific grantsupport. Amazingly, it was possible to inject rabbit globin mRNAinto the fertilised Xenopus egg, grow that egg to a tadpole stage anddemonstrate that tissues such as muscle were still translating highlevels of globin, wholly inappropriate to that cell type, without anyinterference in normal development (Woodland et al., 1974). Theinjection of mRNA, and other macromolecules, into an egg has nowbecome a very widely used procedure in developmental biology. Iam still amazed at how well this works. We can now understandthat the injection of an egg with a micropipette is sufficientlyharmless that ribonuclease is not released from egg cytoplasm. Itmay be that the penetration of an egg with a micropipette is no moreharmful than the penetration of an egg by sperm after fertilisation.These early experiments led to the widespread use of mRNAinjection for over- and under-expression experiments indevelopmental biology.

A key mechanism in early development is the concentration-dependent response of cells to signalling molecules, an area knownas morphogen gradient interpretation. Even twofold differences inligand concentration are enough to make competent embryonic cellschoose which cell type lineage to follow (Dyson and Gurdon, 1998).We now know that small quantitative deficiencies in signalling canadversely affect nuclear transplant embryo development, as shownin cross-species nuclear transplantation (Narbonne et al., 2011).Another particularly interesting aspect of concentration-dependentsignalling is illustrated by the community effect (Gurdon, 1988).Resulting from single-cell transplantations, the concept developedby which a group of similar cells can contribute a high enough

concentration of signal molecule to exceed a threshold neverproduced by a single cell. This community effect seems tocontribute to the normal development of multicellular tissues indevelopment. It later turned out that the principle behind thecommunity effect had already been proposed, as ‘quorum sensing’,to be involved in bacteria-dependent light emission in predatory fishand in other examples (Lamb, 2012).

To progress with the analysis of reprogramming by eggcytoplasm, an obviously desirable route would be to achievesuccessful reprogramming of somatic nuclei by extracts of eggs;this could lead to the identification of such components byfractionation and selective depletion. Somatic nuclei transplantedto eggs are almost immediately induced to commence DNAreplication (Graham et al., 1966). Extracts of eggs are remarkablysuccessful in inducing DNA replication in isolated nuclei (Laskeyet al., 1989; Méchali, 2010), but such extracts are notoriouslydifficult to make in such a way that transcription proceedsmeaningfully. This difficulty in making functional extracts of cellsis in marked contrast to the long-lasting success of injecting mRNA,genes, etc., into living cells. The injection of components into eggsand early embryo cells can be regarded as ‘living biochemical testtubes’ (Gurdon, 1974).

The analysis of nuclear reprogramming by oocytesIt was evident from the earliest amphibian nuclear transferexperiments that the replication of DNA and chromosomes insomatic nuclei transplanted to eggs is very often defective (DiBerardino and King, 1967). Once penetrated and activated by spermor an injection pipette, amphibian eggs immediately enter a phaseof some ten or more rapid cell division cycles. It is very difficultfor a somatic cell nucleus, which might normally divide once everytwo days, to switch immediately to a division cycle of 30 minutes.As a result, the DNA of transplanted nuclei or their daughters isoften torn apart at cell division when incompletely replicated. Thisleads to major chromosome loss and other defects, especially whenthe nucleus of a slow-dividing somatic cell is transplanted. It wasclear, at this point, that we had to find a way of analysing thereprogramming of somatic cell nuclei without the disadvantage anddamaging effect of enforced rapid DNA replication and celldivision. This led to the introduction of amphibian oocytes insomatic cell nuclear transfer experiments.

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Fig. 8. Survival of nuclear transplant embryos in Xenopus andmouse. Xenopus data taken from Gurdon (Gurdon, 1962) and mouse datafrom Wakayama et al. (Wakayama et al., 1998).

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Fig. 9. The Xenopus oocyte grows in the ovary from a germ cell overmany months while it is in first meiotic prophase. When fully grown itcan respond to hormones, such as progesterone, to complete firstmeiosis and arrest in second meiotic metaphase. Once fertilised, itprogresses to the blastula stage in 8 hours and somatic lineages alreadystart to appear. D

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The amphibian oocyte is the growth phase of an early germ cellto a full-sized egg progenitor with lampbrush chromosomes over aperiod of several months (Callan, 1982). These cells are in theprophase of first meiosis (Fig. 9). These full-sized egg progenitorsare normally induced by hormone levels to complete first meiosisand progress to the metaphase of second meiosis, after which theycan respond to fertilisation. While still in my PhD graduate work, Ideveloped the use of Xenopus oocytes to analyse the origin of theDNA replication-inducing capacity of eggs. Even sperm nuclei canbe converted to lampbrush chromosomes after injection to oocytes(Gall and Murphy, 1998). It became clear that somatic nuclei, oreven pure DNA, would be efficiently and correctly transcribed wheninjected into the germinal vesicle (=nucleus) of an oocyte (Mertzand Gurdon, 1977; Brown and Gurdon, 1977). It is important toappreciate that the germinal vesicle of an amphibian oocyte containsan enormous reserve of developmentally essential components,which are distributed to the egg cytoplasm during completion ofmeiosis (Fig. 10). These reserves are necessary for normalembryonic development. Fortunately for developmental biologists,these components, specified by the intensely active lampbrushchromosomes, are accumulated in the specialised germinal vesiclewhere they represent a high concentration of components that laterenter the egg cytoplasm. Since DNA replication and cell divisiondo not take place in these growing oocytes, the oocyte germinalvesicle provides an accessible accumulation of transcriptionallyactive components.

Somatic nuclei, chromatin or DNA, of amphibian or mammalianorigin, can, with practice, be injected into the invisible germinalvesicle of an intact oocyte (Halley-Stott et al., 2010). Xenopusoocytes show selective transcription of somatic nuclei fromunrelated species (De Robertis and Gurdon, 1977). Transcription ofinjected nuclei or genes takes place at a high rate, with as much asseveral hundred re-initiations of transcription on a gene per day(Fig. 11). Two hundred to 300 somatic nuclei can be injected intoone oocyte’s germinal vesicle, so that one injected oocyte providesthe same amount of nuclear material as 250 eggs injected with asingle nucleus (Fig. 12). This makes it realistic to carry out onoocytes those molecular techniques that normally require largeamounts of material. When injecting purified DNA, this becomeschromatinised (Wyllie et al., 1977). Oocytes continue to transcribeinjected nuclei or chromatin for several days. It is possible tomanually isolate the germinal vesicle from an oocyte, containinginjected somatic nuclei, and to carry out antibody binding, FRAPassays, etc., on individual transplanted nuclei. After injection intothe germinal vesicle, somatic nuclei undergo a massive chromatin

decondensation as does sperm in an egg. After transfer to oocytes,some genes are transcribed extensively, and continue to accumulatelarge numbers of transcripts. These activated genes include someof those that are active in embryos, including the well-knownpluripotency genes, such as Oct4, Sox2, Nanog, etc. Thesecharacteristics make the injected first meiotic prophase oocyte ofXenopus very suitable for analysing both the activation of genesduring reprogramming as well as the basis of resistance by thenuclei of differentiated somatic cells (Halley-Stott et al., 2010).

Transcriptional activationSeveral necessary early steps have now been identified. The first ofthese is the movement of a special linker histone, known as B4 inamphibia or H1foo in mammals, into transplanted nuclei. Thishistone protein is very abundant in the germinal vesicle ofamphibian oocytes and a large amount of it is incorporated into thechromatin of injected nuclei within 2-3 hours at 17°C. This step isnecessary for subsequent transcriptional activation, as shown by theuse of antibodies and overexpressed dominant-negative forms ofthis histone that inhibit subsequent pluripotency gene activation(Jullien et al., 2010). When B4 histone invades transplanted nuclei,these nuclei lose the somatic form of linker histone. Thissubstitution of linker histone in chromatin is likely to be animportant part of the striking decondensation of chromosomes thattakes place soon after nuclear injection. This early event is thoughtto give access of other oocyte components, including transcriptionfactors, to injected chromatin. B4 histone is abundant in oocytes butis not present in normal development after the blastula stage (Smithet al., 1988). The next important events include the movement ofanother oocyte-specific histone, namely histone H3.3, into injected

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Oocyte Egg Embryo First

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Maturation

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Fig. 10. A Xenopus oocyte has a huge (420 μm diameter) germinalvesicle, which includes its tetraploid chromosome set. Aftercompletion of meiosis, the germinal vesicle contents are distributed tothe egg and subsequently to the embryo.

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Fig. 11. Somatic nuclei injected into the germinal vesicle of anoocyte transcribe genes that are quiescent in the donor cells but arerapidly transcriptionally activated. The most specialised donor nuclei(mouse thymus) showed temporal resistance to transcriptional activation.

A

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Fig. 12. Multiple somatic nuclei can be injected into the germinal vesicle of an oocyte. (A) Somatic nuclei immediately afterinjection into the germinal vesicle (outlined in white). (B) Decondensednuclei two days after transplantation to a germinal vesicle. D

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nuclei. Histone H3.3 is present in somatic cells but at a much higherconcentration in oocytes, and is generally associated with activetranscription. We have noted above that histone H3.3 may becausally associated with epigenetic memory in somatic nucleitransplanted to second metaphase eggs. If histone H3.3 has a generalrole of enhancing transcription, this would help to account both forepigenetic memory in nuclear transplant embryos as well as for theincreasing level of transcription seen in somatic nuclei transplantedto oocytes (Gurdon, 1986). A later event is the polymerisation ofnuclear actin in oocytes and in nuclei transplanted to their germinalvesicles (Miyamoto et al., 2011). This seems to enhance the level oftranscription of transplanted nuclei during the first two days. Thissequence of events leads to a high level of transcriptionalreprogramming, and takes place at a remarkably fast rate. Withintwo days, most of the somatic nuclei transplanted to oocytes havestrongly activated transcription of the pluripotency gene Sox2; thishappens at 17°C, the metabolic equivalent of 12 hours at 37°C.

Although the transcription of some genes, after nuclear transfer tooocytes, is enormously increased from a somatic level, up to 100times for Sox2, the oocyte germinal vesicle does not cause a globaltranscriptional enhancement of all genes. RNA seq analysis showsthat most genes in a mouse somatic cell are not changed intranscription, some remaining at a high level and others remaining ata repressed level. A minority of genes that were active in somatic cellsbecome repressed after transfer to oocytes and a smaller fractionundergoes a great enhancement of transcription. Thus, thereprogramming of somatic nuclei by the oocyte germinal vesicle ishighly selective (J. Jullien, unpublished). Those genes that aretranscriptionally activated include ones that are strongly expressedand important in early mammalian development, including Sox2,Oct4 and Nanog. The germinal vesicle of an oocyte seems to beendowed with components that induce intense transcriptional activity,as seen in lampbrush chromosomes, for all genes that are accessible(Jullien et al., 2011). However, some genes in somatic nuclei do notrespond to the transcription-inducing conditions of the egg.

Resistance to reprogramming by oocytesTo me, resistance to transcriptional activation is now the mostinteresting aspect of nuclear reprogramming. This increasingresistance associated with development seems to reflect theremarkable stability of cell differentiation. Hardly ever does a cell ofone specialised type switch to another cell type, or produce daughtercells that do so. Resistance to reprogramming is also evident in cellfusion experiments (Blau et al., 1983), and even more so in iPSCwork (Yamanaka, 2012). The transplantation of mammalian somaticnuclei containing a repressed X chromosome has identified one kindof molecule responsible. Mouse embryo fibroblasts containing aninactive X chromosome are highly resistant to the transcription ofthese genes after nuclear transfer to oocytes. However, the nuclei ofmouse embryo blastocyst cells that also contain an inactive Xchromosome are strongly reactivated transcriptionally by oocytes.This difference between blastocyst and adult nuclei has turned out tobe attributable to the chromosomal component macroH2A whoseremoval or inactivation in adult Mef nuclei results in transcription ofpluripotency genes (Pasque et al., 2011a). At present, we envisagethe female mammalian X inactivation process as a set of steps thatprogressively stabilise the inactive state. Thus, as development andcell differentiation proceed, successive levels of inactivation,involving histone modifications such as H3K27Me2/3 andmacroH2A absorption into chromatin, and finally methylation ofDNA, cause a gene to become stably repressed and highly resistantto reprogramming (Pasque et al., 2011b).

To analyse other ways in which genes become resistant toreprogramming, two experimental routes are likely to prove useful.One is to progressively remove components of isolated nuclei, andthen test their transcription in injected oocytes until resistance islost (R. P. Halley-Stott, unpublished). This procedure is provingsuccessful in depleting nuclei of all RNA, including non-codingRNA. Increasing concentrations of NaCl with Triton canprogressively deplete isolated nuclei of chromosomal proteins. Ifresistance can be restored by adding back defined fractions ofreleased proteins, this could lead to the identification of thosechromosomal proteins that confer resistance on individual genes.Another potentially valuable experimental approach is tooverexpress, by mRNA injection to oocytes, those enzymes that addor remove modifications to histones. It should then be possible torelate a particular histone or other chromosomal modification to theresistance of a gene to reprogramming by oocytes. By thesemethods, there is a prospect of understanding, in reasonable detail,the mechanisms of nuclear reprogramming and resistance in nucleitransplanted to amphibian oocytes.

Overview and prospectsThe process of nuclear reprogramming by eggs and oocytes can beseen as a conflict between the cytoplasm of an egg, whosecomponents are designed to promote rapid DNA replication andthen transcription, and the components of differentiated cell nuclei,whose function is to maintain a stable state. The cytoplasm of anegg is specially designed to activate the highly condensed andspecialised nucleus of sperm, with 100% efficiency. Notsurprisingly, the same components are effective at activating thenucleus of a somatic cell. The difference is that a somatic cellnucleus has become, during the process of cell differentiation,highly resistant to activation by egg cytoplasm in a way that isdifferent from sperm nuclei. These nuclei of differentiated cells areprovided with molecules that stabilise their differentiated state andresist reversal or rejuvenation. If differentiated cell nuclei could betoo easily switched to an embryonic state, this could permit thereversal of differentiation and lead to cancer.

The experimental work described here has centred on the use ofamphibian eggs and oocytes because of the abundance of materialand ready availability offered by them, an advantage that was veryclear to developmental biologists up to the 1950s. The generalprinciples that have emerged from work on amphibia seem also toapply to mammals and other vertebrate species. A full understandingof nuclear reprogramming by amphibian eggs and oocytes may wellfacilitate nuclear reprogramming in mammals, including humans,and hence contribute to the eventual therapeutic application of cellreplacement.

AcknowledgementsI thank my PhD supervisor Dr Michail Fischberg and members of Dr Fischberg’sgroup in the 1960s, and I thank the Nobel Foundation for their recognition ofthis work.

FundingThis work was supported by grants from the Wellcome Trust, the MedicalResearch Council and Cancer Research UK.

A video of the lecture presented in Stockholm in December 2012 is availableonline at http://www.nobelprize.org/nobel_prizes/medicine/laureates/2012/gurdon-lecture.html

ReferencesBlau, H. M., Chiu, C. P. and Webster, C. (1983). Cytoplasmic activation of

human nuclear genes in stable heterocaryons. Cell 32, 1171-1180.Brachet, J. (1957) Biochemical Cytology. New York, NY: Academic Press. D

EVELO

PMENT

2456

Briggs, R. and King, T. J. (1952). Transplantation of living nuclei from blastulacells into enucleated frogs’ eggs. Proc. Natl. Acad. Sci. USA 38, 455-463.

Briggs, R. and King, T. J. (1957). Changes in the nuclei of differentiatingendoderm cells as revealed by nuclear transplantation. J. Morphol. 100, 269-312.

Brown, D. D. and Gurdon, J. B. (1964). Absence of ribosomal-RNA synthesis inthe anucleolate mutant of Xenopus laevis. Proc. Natl. Acad. Sci. USA 51, 139-146.

Brown, D. D. and Gurdon, J. B. (1977). High-fidelity transcription of 5S DNAinjected into Xenopus oocytes. Proc. Natl. Acad. Sci. USA 74, 2064-2068.

Callan, H. G. (1982). The Croonian Lecture, 1981. Lampbrush chromosomes.Proc. R. Soc. Lond. B Biol. Sci. 214, 417-448.

Campbell, K. H., McWhir, J., Ritchie, W. A. and Wilmut, I. (1996). Sheep clonedby nuclear transfer from a cultured cell line. Nature 380, 64-66.

Chan, A. P. and Gurdon, J. B. (1996). Nuclear transplantation from stablytransfected cultured cells of Xenopus. Int. J. Dev. Biol. 40, 441-451.

Cho, H. and Wolffe, A. P. (1994). Xenopus laevis B4, an intron-containingoocyte-specific linker histone-encoding gene. Gene 143, 233-238.

De Robertis, E. M. and Gurdon, J. B. (1977). Gene activation in somatic nucleiafter injection into amphibian oocytes. Proc. Natl. Acad. Sci. USA 74, 2470-2474.

Di Berardino, M. A. and King, T. J. (1967). Development and cellulardifferentiation of neural nuclear-transplants of known karyotype. Dev. Biol. 15,102-128.

DiBerardino, M. A. and Hoffner, N. J. (1983). Gene reactivation in erythrocytes:nuclear transplantation in oocytes and eggs of Rana. Science 219, 862-864.

Dyson, S. and Gurdon, J. B. (1998). The interpretation of position in amorphogen gradient as revealed by occupancy of activin receptors. Cell 93,557-568.

Egli, D., Rosains, J., Birkhoff, G. and Eggan, K. (2007). Developmentalreprogramming after chromosome transfer into mitotic mouse zygotes.Nature 447, 679-685.

Elsdale, T. R., Gurdon, J. B. and Fischberg, M. (1960). A description of thetechnique for nuclear transplantation in Xenopus laevis. J. Embryol. Exp.Morphol. 8, 437-444.

Gall, J. G. and Murphy, C. (1998). Assembly of lampbrush chromosomes fromsperm chromatin. Mol. Biol. Cell 9, 733-747.

Graham, C. F., Arms, K. and Gurdon, J. B. (1966). The induction of DNAsynthesis by frog egg cytoplasm. Dev. Biol. 14, 349-381.

Gurdon, J. B. (1960a). The effects of ultraviolet irradiation on uncleaved eggs ofXenopus laevis. Q. J. Microsc. Sci. 101, 299-312.

Gurdon, J. B. (1960b). Factors responsible for the abnormal development ofembryos obtained by nuclear transplantation in Xenopus laevis. J. Embryol.Exp. Morphol. 8, 327-340.

Gurdon, J. B. (1962). The developmental capacity of nuclei taken from intestinalepithelium cells of feeding tadpoles. J. Embryol. Exp. Morphol. 10, 622-640.

Gurdon, J. B. (1974). Molecular biology in a living cell. Nature 248, 772-776.Gurdon, J. B. (1986). Nuclear transplantation in eggs and oocytes. J. Cell Sci. 4

Suppl., 287-318.Gurdon, J. B. (1988). A community effect in animal development. Nature 336,

772-774.Gurdon, J. B. (2006). From nuclear transfer to nuclear reprogramming: the

reversal of cell differentiation. Annu. Rev. Cell Dev. Biol. 22, 1-22.Gurdon, J. B. and Hopwood, N. (2000). The introduction of Xenopus laevis into

developmental biology: of empire, pregnancy testing and ribosomal genes.Int. J. Dev. Biol. 44, 43-50.

Gurdon, J. B. and Uehlinger, V. (1966). “Fertile” intestine nuclei. Nature 210,1240-1241.

Gurdon, J. B., Lane, C. D., Woodland, H. R. and Marbaix, G. (1971). Use of frogeggs and oocytes for the study of messenger RNA and its translation in livingcells. Nature 233, 177-182.

Halley-Stott, R. P., Pasque, V., Astrand, C., Miyamoto, K., Simeoni, I., Jullien,J. and Gurdon, J. B. (2010). Mammalian nuclear transplantation to GerminalVesicle stage Xenopus oocytes – a method for quantitative transcriptionalreprogramming. Methods 51, 56-65.

Hochedlinger, K. and Jaenisch, R. (2002). Monoclonal mice generated bynuclear transfer from mature B and T donor cells. Nature 415, 1035-1038.

Jaenisch, R. (2012). Nuclear cloning and direct reprogramming: the long andthe short path to Stockholm. Cell Stem Cell 11, 744-747.

Jullien, J., Astrand, C., Halley-Stott, R. P., Garrett, N. and Gurdon, J. B. (2010).Characterization of somatic cell nuclear reprogramming by oocytes in which alinker histone is required for pluripotency gene reactivation. Proc. Natl. Acad.Sci. USA 107, 5483-5488.

Jullien, J., Pasque, V., Halley-Stott, R. P., Miyamoto, K. and Gurdon, J. B.(2011). Mechanisms of nuclear reprogramming by eggs and oocytes: adeterministic process? Nat. Rev. Mol. Cell Biol. 12, 453-459.

Lamb, R. F. (2012). Amino acid sensing mechanisms: an Achilles heel in cancer?FEBS J. 279, 2624-2631.

Laskey, R. A. and Gurdon, J. B. (1970). Genetic content of adult somatic cellstested by nuclear transplantation from cultured cells. Nature 228, 1332-1334.

Laskey, R. A., Fairman, M. P. and Blow, J. J. (1989). S phase of the cell cycle.Science 246, 609-614.

McGrath, J. and Solter, D. (1984). Inability of mouse blastomere nucleitransferred to enucleated zygotes to support development in vitro. Science226, 1317-1319.

McKinnell, R. G. (1978) Cloning: Nuclear Transplantation In Amphibia.Minneapolis, MN: University of Minnesota Press.

Méchali, M. (2010). Eukaryotic DNA replication origins: many choices forappropriate answers. Nat. Rev. Mol. Cell Biol. 11, 728-738.

Mertz, J. E. and Gurdon, J. B. (1977). Purified DNAs are transcribed aftermicroinjection into Xenopus oocytes. Proc. Natl. Acad. Sci. USA 74, 1502-1506.

Miyamoto, K., Pasque, V., Jullien, J. and Gurdon, J. B. (2011). Nuclear actinpolymerization is required for transcriptional reprogramming of Oct4 byoocytes. Genes Dev. 25, 946-958.

Narbonne, P., Simpson, D. E. and Gurdon, J. B. (2011). Deficient inductionresponse in a Xenopus nucleocytoplasmic hybrid. PLoS Biol. 9, e1001197.

Ng, R. K. and Gurdon, J. B. (2005). Epigenetic memory of active genetranscription is inherited through somatic cell nuclear transfer. Proc. Natl. Acad.Sci. USA 102, 1957-1962.

Ng, R. K. and Gurdon, J. B. (2008). Epigenetic memory of an active gene statedepends on histone H3.3 incorporation into chromatin in the absence oftranscription. Nat. Cell Biol. 10, 102-109.

Pasque, V., Gillich, A., Garrett, N. and Gurdon, J. B. (2011a). Histone variantmacroH2A confers resistance to nuclear reprogramming. EMBO J. 30, 2373-2387.

Pasque, V., Jullien, J., Miyamoto, K., Halley-Stott, R. P. and Gurdon, J. B.(2011b). Epigenetic factors influencing resistance to nuclear reprogramming.Trends Genet. 27, 516-525.

Polo, J. M., Liu, S., Figueroa, M. E., Kulalert, W., Eminli, S., Tan, K. Y.,Apostolou, E., Stadtfeld, M., Li, Y., Shioda, T. et al. (2010). Cell type of origininfluences the molecular and functional properties of mouse inducedpluripotent stem cells. Nat. Biotechnol. 28, 848-855.

Rauber, A. (1886). Personaltheil und germinaltheil des individuum. Zool. Anz. 9,166-171.

Smith, R. C., Dworkin-Rastl, E. and Dworkin, M. B. (1988). Expression of ahistone H1-like protein is restricted to early Xenopus development. Genes Dev.2, 1284-1295.

Spemann, H. (1938) Embryonic Development And Induction. New Haven, CT: YaleUniversity Press.

Wakayama, T., Perry, A. C., Zuccotti, M., Johnson, K. R. and Yanagimachi, R.(1998). Full-term development of mice from enucleated oocytes injected withcumulus cell nuclei. Nature 394, 369-374.

Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J. and Campbell, K. H. S.(1997). Viable offspring derived from fetal and adult mammalian cells. Nature385, 810-813.

Woodland, H. R. and Gurdon, J. B. (1969). RNA synthesis in an amphibiannuclear-transplant hybrid. Dev. Biol. 20, 89-104.

Woodland, H. R., Gurdon, J. B. and Lingrel, J. B. (1974). The translation ofmammalian globin mRNA injected into fertilized eggs of Xenopus laevis. II.The distribution of globin synthesis in different tissues. Dev. Biol. 39, 134-140.

Wyllie, A. H., Gurdon, J. B. and Price, J. (1977). Nuclear localisation of anoocyte component required for the stability of injected DNA. Nature 268, 150-152.

Yamanaka, S. (2012). Induced pluripotent stem cells: past, present, and future.Cell Stem Cell 10, 678-684.

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PMENT