Nuclear transplantation involves the replacement of the nucleus of one cell with that of another. The original purpose of this technique was to test whether the nucleus of a somatic cell can replace the zygote nucleus of a fertilized egg, and so determine whether cell differentiation and morphogenesis are accompanied by stable changes in the genome. The need for such a test was evident since Weismann’s (1892) germ-plasm hypothesis in which it was supposed that genetic determinants are segregated into different cells as development proceeds. The technique of nuclear transfer was first achieved in single-celled organisms, such as Acetabularia (Hammerling, 1934) ?n?Amoeba (Comandon & De Fonbrune, 1939). The delayed-nucleation experiment of Spemann (1938) showed that nuclei are genetically equivalent up to at least the eight-cell stage in amphibia, but was not able to test the reasonable possibility that nuclei might change at later stages when cells begin to differentiate.

The first successful nuclear transfer experiment in animals was achieved in 1952 by Briggs & King who obtained normal feeding stage larvae from transplanted blastula nuclei of Rana pipiens. This was soon followed by the discovery that nuclei transplanted from the mesoderm or endoderm of R. pipiens late gastrulae can no longer support normal development (King & Briggs, 1955). This result and much subsequent nuclear transfer work with R. pipiens and the axolotl (e.g. see Briggs & King, 1957, 1960; Briggs, Signoret & Humphrey, 1964), led to the suggestion that as cells differentiate their nuclei undergo restrictions in developmental capacity reflecting some kind of nuclear differentiation. The next significant step came from the serial nuclear transplantation experiments of King & Briggs (1956), which showed that the developmental restrictions of individual Rana gastrula nuclei differ from each other and are stable over many nuclear divisions. The nature of these developmental restrictions was greatly clarified by detailed chromosome analyses carried out over several years. It became evident that nuclear-transplant embryos that develop abnormally most often have microscopically visible chromosome abnormalities, which are not present in the donor nuclei (Briggs, King & Di Berardino, 1960; Briggs et al. 1964; Di Berardino & King, 1965), and that these chromosome defects are a sufficient explanation for the developmental abnormalities observed. It was clear that these developmental and chromosomal abnormalities are not a direct reflection of the properties of somatic nuclei but rather a consequence of the inability of those nuclei to accelerate their replication cycle to coincide with the exceptionally rapid pace imposed by eggs.

Well before this level of understanding had been reached, the first nuclear transfer results were obtained with Xenopus, this species being easier to rear to sexual maturity than Rana. Two significant results came from these early experiments (Fischberg, Gurdon & Elsdale, 1958; Gurdon, Elsdale & Fischberg, 1958). First, nuclear transplant embryos were reared to sexually mature individuals, demonstrating that the transplanted nuclei were totipotent, rather than pluripotent as shown by the production of larvae. Second, sexually mature frogs were obtained from transplanted nuclei of endoderm cells of tail-bud larvae, a very different result from that obtained in Rana. Nuclear transfers from the endoderm of Xenopus were pursued, and culminated in the finding that fertile adult nuclear-transplant frogs could be obtained from nuclei from the intestine of feeding larvae (Gurdon, 1962c; Gurdon & Uehlinger, 1966). Although the proportion of intestinal nuclei shown by nuclear transfer to be pluripotent or totipotent was small, these results generated the interpretation (see Gurdon, 1962c) that cell differentiation does not require or depend upon any irreversible change in nuclear genetic material, a view contrasting with that generated at that time by work on R. pipiens. This interpretation was reinforced several years later by transplanting nuclei from the terminally differentiated, keratinized skin cells of adult foot web (Gurdon, Laskey & Reeves, 1975); these yielded feeding-stage tadpoles (but not adults) and therefore contained pluripotential nuclei.

The purpose of this article is to review the conclusions that can be drawn from nuclear transfer experiments, to examine their validity, and to consider the prospects for further work in this field.

NUCLEAR TRANSFER PROCEDURE

The procedure for transplanting nuclei in amphibia, as first used successfully by Briggs & King (1952), is as follows. Donor cells are dissociated, and a single cell sucked into a micropipette of such a size that the cell is ruptured but its nucleus is still surrounded by cytoplasm. The whole broken cell is injected into an unfertilized egg that has been enucleated, and if necessary activated. The egg should then develop by division of the transplanted nucleus, which has therefore replaced the nuclear material normally present in the zygote.

The technique involves three main steps: the preparation of donor cells, the preparation of recipient eggs, and the transplantation procedure itself (Fig. 1). Eggs of amphibia, unlike those of mammals, do not need to be fertilized before nuclear transfer, and only the egg’s chromosomes, which are present near the animal pole on the second meiotic metaphase spindle, have to be removed or destroyed. This is done either manually with a needle (Rana) or by ultraviolet irradiation (Xenopus). In the case of Anura (Rana, Xenopus), unfertilized eggs do not need activation beyond what is already achieved by penetration with an injection pipette. In Urodeles (Ambystoma, Pleurodeles), the additional activation stimulus required is provided, usually before enucleation, by an electric shock.

Fig. 1.

Nuclear transfer procedure lorXenopus, illustrated with transfers from adult foot skin cells. Nuclear transfers from specialized cells often yield a substantial number of partially cleaved blastulae, which can be used, as shown here, as a source of nuclei for serial transfers. The nuclear transfer procedure for other amphibian species differs from that shown mainly by the method of enucleation (UV, ultraviolet irradiation, in this figure) and in some cases by the inclusion of an egg activation step.

Fig. 1.

Nuclear transfer procedure lorXenopus, illustrated with transfers from adult foot skin cells. Nuclear transfers from specialized cells often yield a substantial number of partially cleaved blastulae, which can be used, as shown here, as a source of nuclei for serial transfers. The nuclear transfer procedure for other amphibian species differs from that shown mainly by the method of enucleation (UV, ultraviolet irradiation, in this figure) and in some cases by the inclusion of an egg activation step.

To prepare nuclei for injection, donor cells are dissociated in a calcium-free medium supplemented with EDTA, or trypsin for more differentiated tissues, and maintained in a medium containing serum albumin, spermine, or other components found to improve results. The nuclear transfer step requires a micropipette made with great precision. It must be small enough to rupture a cell sucked into it; it must also be large enough and must have a sufficiently smooth contour on its opening not to disperse the cytoplasm, which protects the nucleus from the adverse effects of all media so far used. It must also have a point sharp enough not to damage the recipient egg. Such a pipette, whose tip resembles that of a hypodermic needle, is made with a microforge. Since the tip of the pipette cannot be seen once it is in an egg, an air bubble is used to monitor ejection of the nucleus.

This procedure requires considerable practice for the best results. Detailed requirements differ for each species used, and these are described in the following papers: Rana:King (1966); Xenopus:Gurdon (1977); Ambystoma: Signoret, Briggs & Humphrey (1962); Pleurodeles: Signoret & Picheral (1962).

NORMAL DEVELOPMENT OF NUCLEAR-TRANSPLANT EMBRYOS

The most conclusive outcome of a nuclear-transplant experiment is the development of a normal adult frog that is fertile, and which carries a genetic marker proving its derivation from the transplanted nucleus and not from the recipient egg pronucleus. Fertile adults, carrying a genetic marker, have been most readily obtained in Xenopus, and are listed in Table 1, to exemplify the cell types from which they have been obtained. It can be seen that nuclei of endoderm and of intestine at the stage when its cells are first differentiated (feeding stage) have yielded fertile adult frogs. The same is true of other Xenopus cell types, including hatched tadpole epidermis and presumptive neural-fold brain cells (Table 1). In R. pipiens adults have been obtained from transplanted blastula nuclei (Table 1). All of these cases provide direct evidence for the totipotency of nuclei during cell differentiation, as discussed in detail at the end of this section. The success of nuclear transplantation with the nuclei of embryonic cells has made it possible to produce clones of genetically identical animals from the nuclei of one embryo (Gurdon, 1962J; McKinnell, 1962; Gallien & Aimar, 1966). Such multiple identical twins are all the same sex, they accept each other’s skin grafts, and differ only in the detailed speckling of their skin and in growth rate if crowded. No-one has yet obtained a normal adult nuclear-transplant frog from the transplanted cell nucleus of another adult frog. A very small number of nuclear transfer larvae from adult skin started feeding, the longest survivor dying just after metamorphosis (Gurdon, 1974, page 23).

Table 1.

Adult nuclear-transplant frogs and their cell type of origin

Adult nuclear-transplant frogs and their cell type of origin
Adult nuclear-transplant frogs and their cell type of origin

Many nuclear-transplant embryos that fail to become fertile adults nevertheless develop far enough to give valuable information about the developmental capacity of transplanted nuclei. One such level of development is a heart-beat larva, in which all major cell types, including muscle, nerve, blood, lens, etc., are fully functional and are furthermore organized into morphogenetically normal tissues and organs (Fig. 2B,C). The only obvious defect in such larvae is that they fail to feed or grow, eventually becoming oedematous and dying. Transplanted nuclei that promote development this far are usually described as pluripotential. An earlier stage at which it is also useful to assess nuclear-transplant embryo development is the muscular response stage (Fig. 2A). In Xenopus, this is reached 2 days after fertilization or nuclear transfer, and reflects the formation of sufficiently normal muscle and nerve for lateral body movements to take place spontaneously and after stimulation.

Fig. 2.

Examples of muscular response and heart-beat nuclear transplant larvae. A. 2-Day abnormal larva, with well-organized functional myotomes, and capable of swimming movements. B. 4-Day larva, not completely normal as seen by small eyes and heart oedema; it has a beating heart, with circulating blood cells, and swims actively, c. A histological section through an eye of a heart-beat stage larva, derived in this case from the transplanted nucleus of an adult epidermal skin cell. All figures are of A. laevis. c is from Gurdon et al. (1975).

Fig. 2.

Examples of muscular response and heart-beat nuclear transplant larvae. A. 2-Day abnormal larva, with well-organized functional myotomes, and capable of swimming movements. B. 4-Day larva, not completely normal as seen by small eyes and heart oedema; it has a beating heart, with circulating blood cells, and swims actively, c. A histological section through an eye of a heart-beat stage larva, derived in this case from the transplanted nucleus of an adult epidermal skin cell. All figures are of A. laevis. c is from Gurdon et al. (1975).

Macroscopic and histological examination shows the presence of differentiated muscle cells organized into characteristic blocks of myotomes. As seen in Table 2, heart-beat stage larvae have been obtained with transplanted nuclei taken from all adult cell types that have been well tested in Xenopus and from many terminally specialized cell types including skin, muscle and melanophore. With A. pipiens, such larvae have also been obtained from transplanted nuclei of adult erythrocytes (Table 2). The proportion of nuclear transplants reaching the heart-beat larval stage is small, though their significance is clear when the donor cell population has been well characterized. A substantially higher proportion of embryos derived from the same donor nuclei reach the muscular response stage (Table 2). Even though many of these embryos become abnormal before reaching the heart-beat stage, the formation of functional and histologically normal muscle cells from the nuclei of such cells as adult skin and erythrocytes demonstrates a dramatic change in gene expression.

Table 2.

Nuclear transfers from differentiated and adult cells

Nuclear transfers from differentiated and adult cells
Nuclear transfers from differentiated and adult cells

Before drawing definitive conclusions from these experiments, certain possible reservations about their interpretation and validity need discussion.

Egg enucleation and genetic markers of donor nuclei

It is essential to be sure that there has been no contribution of the recipient egg chromosomes to nuclear-transplant embryo development. The most direct and convincing evidence comes from the use of genetic markers. Xenopus, the \-nu nucleolar mutation (Elsdale, Gurdon & Fischberg, 1960) can be seen from the gastrula stage onwards, and the ?p albino mutant (Hoperskaya, 1975) in a stage 40 swimming tadpole and thereafter. In Rana, where genetic markers visible at early stages of development are not yet available, evidence for non-participation of the egg nucleus has depended on finding the egg nucleus in sectioned egg jellies containing an exovate (Di Berardino & Hoffner, 1983). In the best cases, this has been coupled with the use of triploid donor nuclei (McKinnell, Deggins & Labat, 1969; Di Berardino, Mizell, Hoffner & Friesendorf, 1983). In all nuclear-transplant experiments, it is normal practice to show that fertilized eggs subjected to enucleation treatment yield haploids, i.e. that the egg nucleus has been eliminated. However, in the absence of other evidence this is not completely convincing, especially if conclusions are drawn from a very small proportion of total nuclear transplants; cases of this kind (Muggleton-Harris & Pezzella, 1972) are not included in Table 1.

The most widely used genetic marker in nuclear-transplant experiments is the \-nu mutant of Xenopus. How reliable is this? Extensive tests were reported by Gurdon et al. (1975) to show that a 1-?w diploid chromosome set does not arise by the combined effects of ultraviolet irradiation of the egg pronucleus and a doubling of its chromosome set. In recent work, the albino mutation was used in conjunction with the \-nu marker (Gurdon, Brennan, Fairman & Mohun, 1984). 2-nu nuclei are sometimes seen in \-nu embryos, but when investigated these have turned out to be tetrapioid cells (Gurdon, 1959) and therefore to be valid derivatives of the transplanted nucleus, which must have undergone a doubling in some somatic cells.

The differentiated state of donor cells

The proportion of nuclear-transplant embryos that develop normally or nearly so is small, and it is essential to be sure that these are not derived from a minority of non-specialized cells that differ from the well-characterized majority. Ideally, the specialized characteristics of individual donor cells can be seen under a microscope, and only such cells taken for nuclear transfer, as has been done for melanocytes (Kobel, Brun & Fischberg, 1973), erythrocytes (Di Berardino & Hoffner, 1983), and spindle-shaped muscle cells (Gurdon et al. 1984). When donor cells cannot be individually recognized, it is necessary to have a fairly homogeneous donor cell population, and to determine what proportion of these cells are not of the differentiated kind. For adult skin cells and lymphocytes, this has been done with antibodies and a simple statistical assessment used to prove that the embryos reaching a certain stage of development could not have come from the small minority of donor cells that did not react with antibody (Gurdon et al. 1975; Wabl, Brun & Du Pasquier, 1975). Under one or other of these conditions just discussed, there is no doubt about the differentiated state of the donor cells from which tadpoles were obtained.

Is new gene activity required for nuclear-transplant embryo development?

Amphibian eggs contain a large store of maternal mRNA (Rosbash & Ford, 1974), and we must consider the possibility that development as far as the muscular response stage, and perhaps even to the heart-beat larval stage, could be achieved by the use of maternal components and might not depend on gene activity by transplanted nuclei. All information at present available strongly indicates the specific requirement of nuclear gene activity if development is to proceed beyond the late blastula stage. Thus nuclear-transplant hybrids between species die at this stage (see below), and the earliest cell types to differentiate, such as muscle, make their proteins from newly transcribed nuclear genes (Mohun et al. 1984). Therefore, it seems clear that all stages of development beyond a late blastula are a meaningful test of the genetic activity of transplanted nuclei.

Cancer nuclei

A final comment on the transplantation of cancer cell nuclei is justified. All experiments have been carried out using the Lucké adenocarcinoma, a herpes virus induced and transplantable epithelial cancer of R. pipiens kidney. Since the first experiments of King & McKinnell (1960), nuclear transfer results have steadily improved; King & Di Berardino (1965) and McKinnell et al. (1969) have all obtained muscular response, and in a few cases heart-beat, larvae from adenocarcinoma nuclear transfers. In the last of these papers, triploid donor nuclei were used to provide direct evidence of successful recipient egg enucleation. No tumours were observed in the nuclear-transplant embryos, though it would have been surprising if they had appeared so early in development. A crucial question of interpretation in these experiments is whether the larvae were derived from the transfer of nuclei from true cancer cells or from non-cancerous cells present in the tumour. McKinnell et al. (1976) argue convincingly that the donors of nuclear-transplant tadpoles were epithelial and not stromal or blood cells. Di Berardino et al. (1983) selected only epithelial cells of Lucké tumours for nuclear transfer, and obtained larvae that died somewhere between the muscular response and feeding stage; but even in this case it is hard to eliminate entirely the possibility that non-cancerous epithelial cell nuclei gave the best development. In conclusion, it seems very likely, though not conclusively proved, that nuclei from at least one kind of cancer cell can promote nuclear-transplant embryo development to about the same extent as the nuclei of normal adult cells.

Conclusion

In summary, the following conclusions can be drawn from the extent to which transplanted nuclei promote normal development. Totipotency, or the ability to promote the formation of fertile adult frogs, has been demonstrated for the nuclei of many kinds of embryonic cells and for one source of differentiated cells of early larvae. Pluripotency, as judged by the development of heart-beat larvae has been documented for the nuclei of all cell types thoroughly tested, including terminally specialized adult cells, though only in a small proportion of total nuclear transfers. A higher proportion of nuclei from specialized cells promote development to the muscular response than to the heart-beat stage. A substantial change in nuclear gene activity is demonstrated by the development of muscular response stage (motile) embryos. All these conclusions rest on experiments in which the differentiated state of donor cells has been established, and in which recipient egg enucleation is documented, usually with a genetic marker.

Most nuclear-transplant embryos develop abnormally, not only in post-blastula stages that depend on nuclear gene expression, but also during early cleavage before gene transcription has started. We now ask to what extent we can account for these abnormalities and whether they give useful information about the nuclei of differentiating cells.

Nuclear-transplant abnormalities occur most frequently, and are most severe, when nuclei are taken from the more differentiated and least actively dividing cells. This result was first described in R. pipiens (King & Briggs, 1955; Briggs & King, 1957), and has been subsequently confirmed in all other species tested (e.g. see Gurdon, 1960, ior Xenopus \Briggs et al. 1964, for axolotl; Picheral, 1962; Aimar, 1972, for Pleurodeles). The effect is most pronounced in R. pipiens and least in Xenopus (Fig. 3), though later experiments of Hennen (1970) withwz?? endoderm nuclei, using improved procedures, gave results equivalent to those typical of Xenopus. Nuclear transfers from germ cells, by definition totipotent, develop more normally than those from endoderm nuclei (8 % larvae versus 0 %) though less normally than those from blastula nuclei (55 % larvae); these results of Smith (1965) with R. pipiens argue that the reduction in nuclear transfer success is more likely to be due to a lower donor cell division rate than to loss of nuclear totipotency.

Fig. 3.

Nuclear transplant embryo development in relation to increasing donor stage (endoderm nuclei). (&2022;—&2022;) X. laevis (Gurdon, 1960); (▫—▫) R. pipiens (Briggs & King, 1957); (▪) R. pipiens (Hennen, 1970); (▴— ▴) Pleurodeles (Aimar, 1972).

Fig. 3.

Nuclear transplant embryo development in relation to increasing donor stage (endoderm nuclei). (&2022;—&2022;) X. laevis (Gurdon, 1960); (▫—▫) R. pipiens (Briggs & King, 1957); (▪) R. pipiens (Hennen, 1970); (▴— ▴) Pleurodeles (Aimar, 1972).

Nuclear-transplant abnormalities are propagatable and stable. It was first found by King & Briggs in 1956 and confirmed in all subsequent work that serial nuclear-transfer clones tend to be morphologically homogeneous within each clone, but substantially different between clones (Fig. 4). This implies that there are stable differences between the nuclei that populate a first-transfer embryo; since all of these nuclei were derived from a single originally transplanted nucleus, the stable differences evidently arose after the first transfer, but were little if at all enhanced during serial transfer. The developmental defects are alleviated neither by parabiosis (fusion) with normal embryos (Briggs et al. 1960), nor by allowing a transplanted nucleus to fuse with the haploid nucleus of a non-enucleated egg thereby creating triploid embryos (Subtelny, 1965); thus the deficiencies of nuclei in abnormal embryos are ‘rescued’ neither by circulating components of embryos nor by a normal set of chromosomes.

Fig. 4.

Variation between serial nuclear transfer clones derived from nuclei of a first transfer embryo. Endoderm nuclei were transplanted from a muscular response, stage 25, embryo. When the resulting embryos had become late blastulae, three were used to provide nuclei for serial transfers. All other first-transfer embryos, and all serial transfer embryos, were allowed to develop to the feeding larva stage if they were able. Most of the normal larvae shown would have become frogs if maintained. Two of the serial transfer clones developed very uniformly within each clone but very differently between clones. (From Gurdon, 1960.)

Fig. 4.

Variation between serial nuclear transfer clones derived from nuclei of a first transfer embryo. Endoderm nuclei were transplanted from a muscular response, stage 25, embryo. When the resulting embryos had become late blastulae, three were used to provide nuclei for serial transfers. All other first-transfer embryos, and all serial transfer embryos, were allowed to develop to the feeding larva stage if they were able. Most of the normal larvae shown would have become frogs if maintained. Two of the serial transfer clones developed very uniformly within each clone but very differently between clones. (From Gurdon, 1960.)

Chromosome abnormalities

The basis of these stable nuclear changes is now known to lie in chromosomal aberrations that arise after the first transfer of somatic nuclei. Though early results described chromosome defects in only the most abnormal embryos (King & Briggs, 1956), subsequent more detailed studies have shown that all embryos dying before the feeding stage have visibly abnormal chromosomes, that the most severe developmental abnormalities have the most severe chromosomal defects, and hence that chromosome abnormalities are a sufficient explanation for the developmental abnormalities (Di Berardino & King, 1965; Briggs et al. 1964; Di Berardino & Hoffner, 1970; Aimar, 1972). This at once provides an explanation for the irreversibility of nuclear-transplant embryo defects observed in serial nuclear transfer experiments.

A particularly interesting feature of these chromosomal defects is that they occur not only at the first mitosis after nuclear transfer (Briggs et al. 1960; Gurdon, 1962c), but continue to take place during many mitoses thereafter (Briggs et al. 1964). For example, fragments of chromosomes without centromeres (which means that they must have arisen in the last few divisions) are seen in late blastulae, at least 12 divisions after the original nuclear transfer (Fig. 5). It seems that major chromosome defects occur at the first post-transfer mitosis, a situation readily understandable in view of the gross disparity in division rate between donor cells and recipient egg. What is unexpected and unexplained is the continued shedding of chromosome fragments long after the transplanted nucleus has been forced to adopt the cell division cycle of an egg.

Fig. 5.

Abnormal chromosomes in nuclear transplant embryos. Each figure shows an abnormal chromosome set in nuclear-transplant embryos of R. pipiens. The stained squash preparations were made from blastulae, about 10 divisions after nuclear transfer. If chromosome abnormalities were to arise only at the first division after nuclear transfer, the ring chromosome and acentromeric fragments (A) would have been lost by the 10th division. Chromosomes that do not separate properly at division (B) are usually lost before the next mitosis. (From Briggs et al. (1964).)

Fig. 5.

Abnormal chromosomes in nuclear transplant embryos. Each figure shows an abnormal chromosome set in nuclear-transplant embryos of R. pipiens. The stained squash preparations were made from blastulae, about 10 divisions after nuclear transfer. If chromosome abnormalities were to arise only at the first division after nuclear transfer, the ring chromosome and acentromeric fragments (A) would have been lost by the 10th division. Chromosomes that do not separate properly at division (B) are usually lost before the next mitosis. (From Briggs et al. (1964).)

The key question that now arises is whether the chromosomal and developmental abnormalities of nuclear-transplant embryos bear any relationship to the tissue origin of the transplanted nuclei. Embryos prepared from transplanted endoderm nuclei first show defects in their ectoderm and mesoderm (Briggs & King, 1957); this is probably due to the later differentiation of endoderm compared to other germ layers and the same defects are also observed in embryos derived from ectodermal and mesodermal nuclei. Di Berardino & King (1967) made a detailed study of over 1200 nuclear transfers from neural nuclei; these yielded only four embryos with normal chromosome sets, and three of these had deficiencies in their mesoderm and endoderm; but even these had histologically and morphogenetically recognizable mesoderm and endoderm tissues such as muscle, notochord and gut. In all other work involving several species, no relationship has been observed between the nature of the developmental abnormalities and the tissue of origin of the transplanted nuclei (e.g. see Gurdon, 1962c; and Simnett, 1964, tor Xenopus;Aimar, 1972, for Pleurodeles). It is important to recognize this point. Readers should not be confused by the description of developmental abnormalities as being “consistent with the donor tissue of origin”. This situation is expected for endoderm nuclei (see above) but is not significant unless nuclei of different origins can be shown to give different kinds of abnormality.

Conclusion and prospects

The simplest interpretation of abnormal nuclear-transplant embryo development, at the present time, is the following. All abnormal development is attributable to chromosome aberrations that occur soon after the transfer of a somatic cell nucleus to an egg, and to a lesser extent in subsequent cell divisions. The more differentiated, and the less active in cell division, are donor cells, the more abnormal are the chromosomal, and hence developmental, defects. There is no evidence that these defects are related to the cell type from which nuclei are taken. As Briggs et al. (1964) pointed out, the chromosomal changes that follow nuclear transfer, like those that can occur in cell culture, need to be understood or controlled, before full advantage can be taken of this experimental system.

Future work on the developmental capacity of transplanted nuclei is likely to concentrate on attempts to obtain adult animals from the nuclei of adult cells, and to increase the proportion of transplanted nuclei from differentiated cells that yield larvae. A way has to be found of derepressing the chromatin of somatic nuclei so that replication can be completed within the first cell cycle of the egg. The simplest concept is that, as development proceeds, non-specific repressors such as histones become tightly associated with all chromosomal DNA that has not already been bound by gene-specific transcription factors (Brown, 1984). Two experimental approaches have given some encouragement. One is to add spermine to the nuclear transfer medium; this has dramatically improved nuclear transfer results with R. pipiens endoderm nuclei (Hennen, 1970). The other is to transplant nuclei to R. pipiens meiotic oocytes, thereby giving them some 24 h of exposure to cytoplasm before they are required to replicate (Di Berardino & Hoffner, 1983). Spermine has a more beneficial effect in A??a than m Xenopus, whose eggs appear to have stronger natural derepressing properties, and so to yield more normal nuclear transfer results. Since spermine has already been used in all recent Xenopus work, some additional procedures are needed. One that does not make much difference is to transplant nuclei synchronized at different stages of the cell cycle (McAvoy, Dixon & Marshall, 1975; Ellinger, 1978; von Beroldingen, 1981). If a means can be found of substantially improving nuclear transfer results, this would be important in its own right, and might also suggest the nature of the developmental mechanisms that restrict replication and, perhaps, transcription of somatic nuclei.

Enucleated eggs of one species, when fertilized with sperm of another, develop as androgenetic haploids in which all of the genetic material is of one (paternal) species, and all of the cytoplasm is of the other (maternal) species. Nucleocytoplasmic combinations of this kind are useful for showing the earliest time of nuclear gene expression as well as the longest lasting cytoplasmic effects, and hence the relative contribution of nucleus and cytoplasm to normal development. They also show how far cytoplasmic signals that regulate nuclear activity are species specific. Fertilization cannot be achieved between distantly related species, and nuclear transplantation, which has no such limitations, has opened up a more extensive analysis of nucleocytoplasmic interactions.

Nuclear transfers between members of the same species (subspecies, strain or other variant) develop entirely normally, becoming fertile adults. In contrast, the nucleus of one species with the cytoplasm of another is nearly always lethal; the more distantly related the species, the earlier does development arrest (Fig. 6). The general rule is that nuclear transfers between any amphibian species (even between Urodeles and Anura) will form a regularly cleaved late blastula. However, more distant combinations, such as an insect or mammalian nucleus in frog egg cytoplasm, do not permit more than a few replications, development being arrested as irregular blastulae (Brun, 1973).

Fig. 6.

Developmental arrest of hybrid nuclear-transplant embryos. The greater the taxonomic difference between nucleus and cytoplasm, the earlier in development does arrest take place, and the more severe are chromosome abnormalities. Brackets signify an enucleated and unfertilized egg.

Fig. 6.

Developmental arrest of hybrid nuclear-transplant embryos. The greater the taxonomic difference between nucleus and cytoplasm, the earlier in development does arrest take place, and the more severe are chromosome abnormalities. Brackets signify an enucleated and unfertilized egg.

For assessing the contribution of nucleus and cytoplasm to early development, the most informative nuclear transfers are those between closely related forms, in which distinguishing characteristics are known, and in which development is normal enough for these to show. Gene expression in nuclear-transplant hybrids has been reviewed by Gallien (1979), who cites previous work. The main conclusions are these. The only cytoplasmic or maternally determined characteristics of nuclear-transplant embryos are those directly attributable to components of the egg. These include pigmentation derived from the unfertilized egg, and the size of the prefeeding embryo, itself determined by the size of the egg; both properties can still be seen several days after fertilization. However, there is no persistent effect of egg cytoplasm in viable intraspecific combinations; even when a nucleus of one subspecies was serially transplanted three times (a total of 45 replications) into eggs of another subspecies, and the progeny of the resulting nuclear-transplant frogs examined, no cytoplasmic effect was seen (Gurdon, 1961). In nearly all respects, the characteristics of embryos are determined entirely by the expression of nuclear genes. Furthermore, the activation of genes takes place at the same stage in development as it does in normally fertilized eggs of the nuclear species (e.g. lactate dehydrogenase isozymes in Pleurodeles, Gallien, Aimar & Guillet, 1973). The earliest nuclear gene expression in a nuclear-transplant hybrid has been described for certain proteins that were seen at the late blastula and early gastrula stages in a Pleurodeles/Ambystoma combination (Aimar, Desvaux & Chalumeau-le-Foulgoc, 1976). The observation that nuclear-transplant hybrids between distantly related species are arrested at the late blastula stage (Fig. 6) reinforces the conclusion that new gene activity not only takes place but is already necessary at this very early stage.

The results of nuclear transfers between species that are not closely related are hard to interpret on account of the chromosomal abnormalities that commonly arise. Soon after the technique of nuclear transplantation was established, some surprising results were described by Moore (1958, 1960). An enucleated egg of Rana sylvatica was fertilized with R. pipiens sperm. When nuclei from the resulting blastula were back-transferred to enucleated eggs of their own R. pipiens type, all of the resulting embryos arrested by the early gastrula stage. This result was interpreted, at the time, as incorporation of genetic material during replication in foreign cytoplasm (Moore, 1962). However, Hennen (1963) repeated the Rana experiments, and found that back-transfers were arrested at all developmental stages from a late blastula to abnormal larvae, and that the developmental abnormalities of the back-transfer embryos were correlated with chromosomal abnormalities. Evidently, nuclear replication in the cytoplasm of a foreign species (but not subspecies) causes chromosome, and hence developmental, abnormalities. Why this should be so is obscure, though it is known that abnormal chromosomes can be easily induced by injecting adult liver nuclear proteins into normal fertilized eggs of R. pipiens (Markert & Ursprung, 1963; Ursprung & Markert, 1963).

Nuclear transfers between closely related species may escape chromosomal damage and are potentially informative. For example, Hennen (1965, 1972, 1974) found that nuclei of Rana palustris in cytoplasm of R. pipiens develop as abnormal neurulae, and yet back-transfers of these nuclei to their own R. palustris cytoplasm develop normally. The developmental arrest of the palustris-pipiens combination is not therefore due to chromosome defects; it could result from either the failure of the cytoplasm of one species to activate genes of the other or from an incompatibility of gene products of one species with the cytoplasm of the other. Either situation would be of value if it turns out that some genes or gene products function correctly and others not at all, since this could provide a means of investigating the activation or activity of individual genes operative in early development. It has indeed been found that some genes are more inhibited than others in a lethal nucleocytoplasmic combination (Woodland & Gurdon, 1969). This approach could be seen as a partial substitute for mutants that are hard to collect in amphibia; its main benefit will be felt when the activation of many different genes can be accurately probed as is now beginning to be done (Sargent & Dawid, 1983; Mohun et al. 1984).

Somatic cell nuclei can be injected into oocytes (the growing egg cells present in an amphibian ovary), or into oocytes undergoing meiotic division, i.e. those that have been released from the ovary through a hormone effect and are passing along the oviduct. Nuclei injected into these kinds of oocytes soon conform in morphology to that of the host cell nuclei or chromosomes (Gurdon, 1968), as they also do when injected into eggs. Whether deposited in the cytoplasm of an oocyte or in its nucleus (germinal vesicle), somatic nuclei gradually enlarge; they usually develop prominent nucleoli if of amphibian, but not of mammalian, origin (Fig. 7A). In contrast, somatic nuclei injected into oocytes in meiotic division are soon resolved into condensed chromosomes on multiple spindles (Fig. 7?). In contrast again, the same kind of nuclei injected into eggs undergo a massive and rapid swelling, as do sperm nuclei, but nucleoli do not appear until later (Fig. 7c).

Fig. 7.

Changes in adult brain nuclei injected into oocytes or eggs. About 300 adult Xenopus brain nuclei were injected into each oocyte or egg, which was incubated for the time stated. The same kind of injected nuclei undergo substantial changes in morphology and activity to conform to that characteristic of the host cell to whose cytoplasm they are exposed. (From Graham et al. (1966), Gurdon (1968), and Gurdon (1977).)

Fig. 7.

Changes in adult brain nuclei injected into oocytes or eggs. About 300 adult Xenopus brain nuclei were injected into each oocyte or egg, which was incubated for the time stated. The same kind of injected nuclei undergo substantial changes in morphology and activity to conform to that characteristic of the host cell to whose cytoplasm they are exposed. (From Graham et al. (1966), Gurdon (1968), and Gurdon (1977).)

The most important consequence and advantage of injecting somatic nuclei into oocytes is that they do not divide, nor do their genes increase in number. A single nucleus transplanted into an egg will have increased to 104 nuclei in 7 h, whereas 500 somatic nuclei injected into an oocyte will still be 500 nuclei after several days. Another major difference between eggs and oocytes is that an egg, once activated by injection, will always proceed through embryonic development as far as it can. In contrast, an ovarian oocyte shows no visible response to numerous injected nuclei, and can be maintained in culture, in a metabolically active but unchanged state, for as long as 3 weeks. The main value of injecting somatic nuclei into oocytes is to study changes in gene expression unaccompanied by cell division or DNA replication (see next section).

The fact that transplanted nuclei of terminally differentiated cells can lead to the formation of feeding-stage tadpoles demonstrates the important point that genes rendered inactive in the course of cell differentiation can be re-expressed after nuclear transfer. This is not an obvious result, since cell differentiation is remarkably stable. There is no known way of converting a keratinized skin cell into a blood cell, or muscle cell into a lens cell. Yet the transplanted nuclei of skin and muscle cells generate cells of totally different kinds, including blood, lens, etc. The nature of these induced changes is of considerable interest, since they may throw light on the mechanisms regulating genes in specialized cells.

Nuclear transfers to eggs

How soon after transplantation do changes in nuclear activity take place? The first change is DNA synthesis; this is initiated in nuclei from all cell types including adult erythrocytes within lh of injection into eggs (Graham, Arms & Gurdon, 1966; Leonard, Hoffner & Di Berardino, 1982). This is not a traumatic response to nuclear isolation or injection since the ability to induce DNA synthesis is absent from oocytes and is acquired during the maturation process (Gurdon, 1967a).

Changes in transcription are also evident very soon after nuclear transfer. It has been known since the work of Bachvarova & Davidson (1966) that no nuclear RNA synthesis is detectable in Xenopus embryos for the first few hours after fertilization. It starts suddenly at about the 12th division as the cell cycle lengthens at the so-called mid-blastula transition (Newport & Kirschner, 1982). When a neurula endoderm nucleus, very active in RNA synthesis, is transplanted to an egg, no [3H]uridine incorporation into RNA is detected by autoradiography 1 h later (Gurdon, 19676; Gurdon & Woodland, 1969), though the same procedure shows massive incorporation of [3H]thymidine into DNA over the same period. Thus a major switch from transcription to replication takes place almost immediately after nuclear transfer.

When transcription recommences at the mid-blastula transition, do transplanted nuclei adopt an embryonic pattern of RNA synthesis or do they resume the transcriptional activity of their original tissue? As far as current analysis permits, it is clear that the former is the case. Using sucrose gradients and column chromatography, it was clear that transplanted nuclei follow the same sequence of nucleic acid activation events as normal embryos reared from fertilized eggs; these include a synthesis of high molecular weight (non-ribosomal, but at that time undefined) RNA in mid-blastulae (stage 8), followed by 4 S (believed to be transfer) RNA synthesis in late blastulae, followed in turn by 18 S and 28 S RNA synthesis during gastrula and neurula stages (Gurdon & Brown, 1965; Gurdon & Woodland, 1969). Current methods of analysis are more sensitive, and synthesis of these classes of RNA can now be detected at earlier stages of development than by older methods (review by Woodland & Old, 1984). Nevertheless, the rates of synthesis of these classes of RNA relative to each other and to DNA clearly differ in blastulae and early gastrulae compared to later stages; in all experiments nuclear transplant embryos change their pattern of synthesis to coincide exactly with that of fertilized controls at the same stage.

More recently it has been possible to confirm these results with more precision and in respect of single kinds of regulated genes. Oocyte-type 5 S genes are not transcribed in somatic cells except very briefly at the late blastula stage (Wakefield & Gurdon, 1983; Wormington & Brown, 1983). Embryos prepared from transplanted nuclei of neurulae, in which 5 Sooc genes are inactive, transcribe these genes briefly as they pass through the late blastula stage (Wakefield & Gurdon, 1983). Of genes that show cell-type specific expression, one of the earliest to be expressed inXenopus, and one for which a cloned probe is available, is a muscle-specific actin gene first expressed at the late gastrula stage and only in prospective myotome cells (Mohun et al. 1984). Nuclear-transplant embryos prepared from muscle cell nuclei show inactivation of muscle actin gene transcription as soon as measurements can be made (mid-blastula), and reactivation of it at the normal stage (late gastrula) (Gurdon et al. 1984) (Fig. 8). In conclusion, it is evident that nuclei transplanted to eggs undergo rapid changes in the expression of all genes whose transcription has been measured.

Fig. 8.

Changes in gene transcription induced by nuclear transfer to eggs. Nuclei from differentiated muscle cells of a Xenopus muscular response embryo were transplanted to enucleated eggs. When these had reached the mid-blastula (stage 8), late blastula (stage 9), mid-gastrula (stage 12), or muscular response (stage 26) stages, representative samples (nt) and controls (c) were frozen and RNA extracted. Part of each embryo was analysed by S1 nuclease protection for muscle actin gene transcripts (250, 130, 120); another part of each embryo was analysed independently with a probe for 5 S gene transcripts (120), as a measure of the amount of maternal 5 S RNA and hence of total material in each sample. (From Gurdon et al. (1984).)

Fig. 8.

Changes in gene transcription induced by nuclear transfer to eggs. Nuclei from differentiated muscle cells of a Xenopus muscular response embryo were transplanted to enucleated eggs. When these had reached the mid-blastula (stage 8), late blastula (stage 9), mid-gastrula (stage 12), or muscular response (stage 26) stages, representative samples (nt) and controls (c) were frozen and RNA extracted. Part of each embryo was analysed by S1 nuclease protection for muscle actin gene transcripts (250, 130, 120); another part of each embryo was analysed independently with a probe for 5 S gene transcripts (120), as a measure of the amount of maternal 5 S RNA and hence of total material in each sample. (From Gurdon et al. (1984).)

Nuclear transfers to oocytes

We can ask whether the reprogramming of gene expression seen in somatic nuclei transplanted to eggs depends upon DNA replication; perhaps genes in the originally transferred nucleus remain repressed, and only new genes formed by DNA synthesis are expressed. This can be tested by transplanting multiple nuclei into oocytes in which no nuclear division takes place. As mentioned above, somatic nuclei injected into oocytes enlarge and are very active in RNA synthesis, as seen autoradio-graphically, for up to a few weeks. Changes in gene expression induced by oocytes were first seen with HeLa nuclei; of 25 proteins synthesized by HeLa cells but not by Xenopus oocytes, 22 were extinguished and three were expressed in oocytes containing HeLa nuclei (Gurdon, De Robertis & Partington, 1976?; De Robertis, Partington, Longthorne & Gurdon, 1977). Direct evidence that an oocyte causes injected nuclei to switch to an oocyte type of gene expression came from experiments in which Xenopus kidney cell nuclei were injected into oocytes of another amphibian, Pleu?odeles. This was done because several oocyte-specific proteins of Xenopus differ, by two-dimensional gel analysis, from the oocyte specific proteins of Pleu?odeles. It was found that kidney nuclei in oocytes ceased synthesizing kidney -specific proteins, but started to synthesize oocyte-specific proteins (De Robertis & Gurdon, 1977; see Fig. 9). These changes take place in nuclei deposited in the cytoplasm of an oocyte, and relatively slowly (3&2013;5 days) compared to events in eggs and embryos. A similar type of experiment has been carried out with oocyte-type 5 S genes (Korn & Gurdon, 1981) and with another class of repetitive genes called OAX (Wakefield, Ackerman & Gurdon, 1983). In each case, these genes are inactive in somatic cells, but are extensively transcribed when somatic nuclei containing these genes are injected into oocytes. Using lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH) isozyme assays, Etkin (1976) found that liver nuclei injected into oocytes adopted an oocyte pattern of expression.

Fig. 9.

Changes in gene expression induced by injecting nuclei into oocytes. The nuclei of cultured Xenopus kidney cells were injected into oocytes of Pleu?odeles, and the labelled proteins of the injected oocytes analysed by two-dimensional gel electrophoresis. The diagrams of the two-dimensional gel results show only the most abundant two or three newly synthesized proteins. (Based on De Robertis & Gurdon (1977).)

Fig. 9.

Changes in gene expression induced by injecting nuclei into oocytes. The nuclei of cultured Xenopus kidney cells were injected into oocytes of Pleu?odeles, and the labelled proteins of the injected oocytes analysed by two-dimensional gel electrophoresis. The diagrams of the two-dimensional gel results show only the most abundant two or three newly synthesized proteins. (Based on De Robertis & Gurdon (1977).)

To date all results mentioned emphasize the rapid effect of egg or oocyte cytoplasm on nuclear gene activity. There are, however, two apparent exceptions. 5 Sooc genes are activated by the oocytes of most females, as mentioned above, but not by oocytes of certain ‘non-activating’ females (Korn & Gurdon, 1981). This seems to be due to individual variation in the amount of activating substance in oocytes (Korn, personal communication). The injection of 500 nuclei, with 40 000 5 Sooc genes each, seems to exceed the activating capacity of some oocytes, since each of these genes is individually repressed in somatic cells (Gurdon, Dingwall, Laskey & Korn, 1982), though all oocytes have sufficient activating substance to de-repress their own endogenous 5 S genes during oogenesis. The activation of 5 S genes by oocytes is not therefore a real exception to the general rule.

The other apparent exception to the rule of cytoplasmic dominance comes from experiments on the maternal effect o mutant of the axolotl, in which fertilized eggs laid by a homozygous mutant (o/o female) fail to activate RNA synthesis as usual at the mid-blastula stage and die as early gastrulae (Briggs, 1972). Brothers (1976) found that enucleated mutant eggs (of a o/o female) are arrested in development if injected with a mid-blastula (pre-activation) nucleus, but develop normally with a post-activation (late blastula) nucleus. Once a nucleus has undergone activation, this state is inherited in o/o eggs, as deduced from the following experiment (Brothers, 1976). A post-activation +/+ nucleus was transplanted to o/o eggs, which were grown to a pre-activation stage, when serial nuclear transfers were made to other o/o eggs. These embryos were able to develop normally, indicating that the activated state of the original +/+ nucleus can be propagated through several o/o embryos in which activation does not take place. Unfortunately, confirmation of these remarkable results seems to be precluded by the loss of the o mutant stock (Malacinski, personal communication). If the interpretation of these results is correct, this would be a unique situation in which transplanted nuclei are not rapidly reprogrammed by egg cytoplasm.

In conclusion, gene expression by transplanted nuclei appears to be fully under the control of the surrounding cytoplasm, an influence exerted within a few hours or less in eggs. The huge volume excess of cytoplasm over nucleus in eggs may account for the rapidity and strength of cytoplasmic control, which is also evident, though less clearly, in hybrid somatic cells. The cytoplasmic effect seems not to require DNA synthesis or cell division, as is also the case with hybrid somatic cells (Blau, Chiu & Webster, 1983).

The clear objective of this type of work is to understand at the molecular level how components of egg and oocyte cytoplasm regulate nuclear or gene activity. It seems essential to simplify the assay for cytoplasmic effects, ideally to the point of analysing the transcription of a single gene or type of gene. However, even the most sensitive assays cannot detect less than 105 molecules of a gene transcript, and it is therefore highly desirable to be able to work with cloned genes. While it is undesirable to inject more than a few hundred nuclei, it is easy to inject 108 copies of a cloned gene into an egg or oocyte, and extensive work of this kind has been done over the last 10 years (review by Gurdon & Melton, 1981). If injected genes come under correct regulation, it is a matter of technology to define ‘"s-acting’ sequences required for their regulation, and so work towards identifying cytoplasmic components that interact with these sequences. If genes are not correctly transcribed after injection, this opens up the possibility of rescue or complementation analysis, by co-injecting extracts of cells in which the genes work correctly (Weisbrod, Wickens, Whytock & Gurdon, 1982; Korn, Gurdon & Price, 1982; Galli, Hofstetter, Stunnenberg & Birnstiel, 1983). Oocytes are particularly favourable cells for this type of analysis, since fairly large amounts of crude cell extract can be injected into the cytoplasm, from where appropriate molecules migrate into the nucleus (references just cited above). Eventually it will be necessary to develop cell-free systems in which genes or nuclei respond meaningfully enough to cytoplasmic preparations to permit these to be fractionated and to identify active components.

The magnitude and rapidity of changes in gene activity undergone by transplanted nuclei encourage special interest in the molecular mechanisms involved. Nothing detailed can be said in this respect, except that some events that follow nuclear transfer suggest processes likely to be involved. One event is a dramatic swelling that occurs within an hour of nuclear transfer to eggs, and within a few days of transfer to oocytes. The enlargement can reach 50-fold in eggs and in oocytes (Subtelny & Bradt, 1963; Graham etal. 1966), as seen in Fig. 7, and the use of nuclei with prelabelled DNA shows that the chromatin of the injected nuclei is dispersed throughout the enlarged nuclei (Gurdon, 1976). In this respect nuclei transplanted to eggs simulate the natural behaviour of a sperm nucleus after fertilization.

The other apparently significant event associated with nuclear transfer is an exchange of proteins between nucleus and cytoplasm. By prelabelling the proteins of donor nuclei or recipient cytoplasm, a loss of acidic proteins is observed in nuclei injected into eggs (Gurdon, 1970; Gurdon & Woodland, 1969; Leonard et al. 1982) or into oocytes (Gurdon, Partington & De Robertis, 1976b), and both acidic and basic proteins migrate from the cytoplasm into injected nuclei.

The enlargement and chromatin dispersion seen in somatic nuclei injected into oocytes might be regarded as a non-specific effect of manipulation and could involve a gross derangement of chromatin structure. Some of these possibilities have been tested by injecting nuclei containing labelled core histones, as well as by reisolating injected nuclei and looking for changes in structure (Weisbrod et al. 1982). Such tests have shown that the reprogramming of somatic nuclei in oocytes is not accompanied by a general displacement of core histones or of the non-histone protein group known as HMG proteins. Furthermore chromatin assembled in vitro with an incorrect 145 base-pair nucleosome spacing is not corrected after injection to the normal 200 base-pair spacing (which is in fact formed by injecting purified DNA). These results argue that reprogramming in oocytes does not involve a general destruction or gross rearrangement of chromatin structure, but rather a more specific and perhaps physiological change. As more is learnt about the structure of chromatin and as methods are developed for correctly assembling chromatin in vitro with known labelled components, experiments along the lines of those just described above should provide a fruitful route towards an analysis of molecular changes associated with nuclear transfer.

The swelling and chromatin dispersion described above take place in eggs and in oocytes whether DNA or RNA synthesis is induced. Presumably, oocyte and egg cytoplasm contains chromatin decondensing factors that remove Hl histone and other components, so that genes are responsive to specific cytoplasmic signals. Some of the cytoplasmic proteins that enter transplanted nuclei may help to direct subsequent nuclear activity. It is interesting that similar events to those just described also take place in hybrid cells when changes in DNA synthesis, RNA synthesis, or gene expression are induced (Ringertz & Savage, 1976).

Nuclei have been successfully transplanted in many species, including Amoeba, ciliates, Acetabularia, Neurospora (for references, see Gurdon, 1964), and perhaps in fish and ascidians (Tung et al. 1973, 1977). In some of these cases, nuclear markers have not been used, and in none have nuclei been taken from differentiated cells. These results do not therefore extend conclusions reached with amphibia. More extensive experiments have been carried out with insects and mammals.

Two nuclear transfer procedures have been used in insects. In one, multiple donor nuclei and associated cytoplasm are sucked out of an embryo and injected into another genetically different (host) embryo at a syncytial (pre-cellular) stage. Parts of the resulting mosaic embryos are then transferred to an adult abdomen for growth, and subsequently to a larval abdomen, for metamorphosis into adult structures. In all such work (e.g. see Zalokar, 1973; Okada, Kleinman & Schneiderman, 1974; Santamaria, 1975) it is found that nuclei from preblastoderm and even early blastoderm stages can participate in all kinds of adult differentiation, including the formation of gametes, irrespective of their normal developmental fate. The transplanted nuclei seem to conform to the type of differentiation expected of the host region in which they are deposited, and are therefore influenced by host cytoplasmic factors as in amphibia. The only work to give a different result is that of Kauffman (1980), who found that groups of 10&2013;20 nuclei, and associated cytoplasm, give their own type of (anterior) differentiation when transferred to other (posterior) regions of a host. A possible explanation for this discordant result is that .nuclei were transferred to much later (late pre-blastoderm) hosts, and may have been cellularized with their own cytoplasm soon after transfer, thus escaping the cytoplasmic control factors of the host. None of these experiments provides a rigorous test of nuclear potentiality for development, since host cells are always present in an embryo, and could compensate for deficiencies in the transplanted nuclei.

The other design of nuclear transfer experiment in insects is close to that used for amphibia, and involves the injection of one or a few nuclei into an unfertilized egg. Though not enucleated, it is assumed that the egg pronucleus does not fuse with the injected nuclei. In work of this kind (Geyer-Duszynska, 1967; Illmensee, 1972, 1973, 1976; Schubiger & Schneiderman, 1971) larvae have been obtained from five different regions of an early gastrula, no differences in survival or types of defects being observed between nuclei from the various regions. These experiments, assuming no egg-nucleus participation, clearly establish pluripotentiality of early gastrula nuclei. Totipotency has not been demonstrated since none of the nuclear-transplant larvae became adults.

Success with nuclear transplantation in mammals was first described by Illmensee & Hoppe (1981), and Hoppe & Illmensee (1982), who reported that an inner cell mass nucleus can support early development of a one-cell mouse egg, subsequently enucleated. Recently, McGrath & Solter (19846) have carried out a detailed investigation of nuclear transfers in mice, and found that transplanted nuclei from one-cell and two-cell stages can support blastocyst formation by enucleated eggs, but nuclei from four-cell and later stages cannot. They suggest that the results of Illmensee & Hoppe may depend on persistent and functional fragments of the host egg pronuclei. Since it is known that each of the first four cells of a mouse egg can form an embryo (Kelly, 1977), nuclei must be totipotent at this stage and yet unable to cooperate harmoniously with the cytoplasm of a one-cell embryo. The special difficulty that seems to afflict nuclear transplantation in mammals may well be connected with the surprising requirement for both maternaLand paternal genomes for normal mouse development, and with the very early transcriptional activity of the genome (see McGrath & Solter, 1984?). These special properties apply to mouse and probably other mammalian development, but not to the great majority of eggs of other species.

It will be interesting to see whether future nuclear transfer experiments with invertebrate and other species give results similar to those with amphibia, and whether they are affected by chromosomal damage. If results resemble those in mice, where even an eight-cell nucleus cannot replace the zygote nucleus, this could open a way of identifying gene products required for early development. Some mouse genes start to be actively transcribed at the two-cell stage; the injection of two-cell RNA, together with an eight-cell nucleus into a one-cell egg might be informative.

The author is grateful to S. Brennan, S. Cascio, R. A. Laskey, T. J. Mohun and J. O. Thomas for comments on the manuscript.

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