Use this URL to cite or link to this record in EThOS:
Title: Studies on nucleocytoplasmic relationships during differentiation in vertebrates
Author: Gurdon, J. B.
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1961
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
1. In 1952 Briggs and King devised a method of transplanting a living embryonic nucleus into an enucleated egg; the development of the resulting embryo provides information about the developmental potentialities of the transplanted nucleus. These authors used Rana pipiens for their experiments. The present thesis describes how this techique has been modified for use with Xenopus laevis which is in some respects more suitable than Rana pipiens for the kind of problems which can be investigated by this technique. The description and analysis of the technique is summarised in section 2 below. Section 3 describes the results of transplanting nuclei from differentiating endoderm cells; it also summarises the development of of embryos, as well as the adult characters and reproductive capacity of frogs, derived from these nuclei. Section 4 concerns an incomplete series of experiments in which nuclei are transferred into eggs of different species and subspecies of frogs. 2. Nuclear transplantation in Xenopus laevis is carried out by a similar method to that of Briggs and King for Rana pipiens. When embryonic cells have been disaggregated chemically, each is sucked into a pipette just narrow enough to break the cell wall and is then injected into an enucleated egg. The main differences in the technique for the two species are: (i) a nuclear marker is used which provides proof that the nuclei of the resulting transplant-embryos and frogs are derived from the injected nucleus and not the egg nucleus. (ii) the recipient eggs are injected while not immersed in a fluid medium; this greatly facilitates the operation, and is harmless to the eggs which are not exposed to the air long enough for their surface to become dry. (iii) the unfertilised recipient eggs are "enucleated" by a short exposure of their animal hemisphere to ultraviolet irradiation. There are many reasons for believing that this irradiation treatment has no effect on the egg cytoplasm. It has a most useful result of weakening the vitelline membrane sufficiently to enable the egg to be penetrated by a micro-pipette. Lastly it inactivates the egg nucleus which does not then participate in the subsequent development of the egg. The fate of the egg nucleus has been followed. It sinks towards the centre of the egg and comes to lie adjacent to the developing nucleus; it does not fuse with it but eventually disappears as a pyenetic clump in the centre of the spindle formed for the first mitotic division of the developing nucleus. There is no reason to believe that the degenerating egg nucleus has any effect on the subsequent development of the egg. When eggs are u.v. irradiated soon after fertilisation, they develop as androgenetic haploids, none of which survive beyond typically microcephalic and oedematous tadpoles. Each part of the technique has been analysed in order to find out whether it can account for any developmental abnormalities of trans- plant-embryos. This was done by taking each step in the technique in turn, varying the way it is carried out, and then looking for any correlation with developmental abnormalities of transplant-embryos. Only one part of the technical treatment of donor nuclei and recipient eggs was related to abnormal transplant-embryo development. A direct correlation was found to exist between the degree of donor cell distortion to the pipette and the proportion of total transplantations which became regular late blastulae. As the degree of cell distortion cannot be kept entirely constant, a variation of up to 20 in the proportion of late blastulae formed may be due to unavoidable inconsistency in the technique. This effect can be excluded from the results by drawing conclusions from the further development of blastula transplant-embryos, rather than from the development of all transplantations. Eggs laid by laboratory frogs vary greatly in their ability to withstand the treatment to which they are subjected during transplantation; thus poor egg quality is indirectly responsible for abnormal transplant-embryo development. Egg quality can only be recognised by the frequency of abnormalities among the resulting embryos. As the quality of all eggs laid during one ovulation of a frog is about the same, control transplantations using undifferentiated donor nuclei show whether egg quality was good in each series of experiments. The clearest results can therefore be obtained by considering only those "selected" experiments in which egg quality was subsequently found to have been good. This analysis of the technique has shown that, if attention is restricted to selected experiments, then the way in which blastula transplant-embryos develop gives a good indication of the developmental potentialities of the transplanted nuclei. 3. A survey has been made of the developmental potentialities of nuclei from differentiating endoderm or vegetal cells; donor nuclei were taken from this region of a series of embryos ranging from late blastulae to swimming tadpoles. All endoderm nuclei appear to remain totipotent until the early neurula stage and there are still a few nuclei in the mid-gut region of swimming tadpoles which have given rise to normal tadpoles and frogs. However endoderm nuclei tend to become changed genetically as the tissue from which they are taken becomes differentiated. This is shown by a progressive increase in the frequency and severity of developmental abnormalities derived from transplanted nuclei, the older the stage from which they are taken. The capacity of endoderm nuclei for normal cleavage (i.e. the proportion of total transplantations which become regular late blastulae} first shows a decline in muscular response embryos, and decreases progressively from this stage onwards. On the other hand blastula transplant-embryos develop increasingly abnormally from the early neuruala stage onwards. There is no clear indication that the kind of abnomalities from which those transplant-embryos suffer are specific for the endodermal origin of their nuclei. Serial transplantation experiments have shown that nuclear differentiation is heritable, and at least to a large extent, irreversible. These results are compared with those of Briggs and King who transplanted endoderm nuclei in Rana pipiens. There is a considerable difference In the rate and time of onset of endodermal nuclear differentiation in the two species. This difference shows that nuclear differentiation is not directly related to the morpholo- gical differentiation of endoderm tissue which is similar in the two species. It is suggested that nuclear differentiation is related to the fully differentiated state of individual cells. Normal transplant-embryos derived from endoderm nuclei have been reared into adult frogs; the study of these frogs and their reproductive capacity gives further information about the genetic qualities of the nuclei from which they are derived. Most of these frogs are normal and give rise to normal offspring, but a few are abnormal in some way, and in these cases it is found that all frogs derived from the nuclei of one embryo suffer from the same type of abnormality. There is an indication that endoderm nuclei from advanced embryos are deficient for genes concerned in oogenesis, so that some of the resulting frogs are either sterile or sex-reversed into males. All transplant-frogs derived from the single or serial trans- plAantation of undifferentiated (e.g. blastula) nuclei should be genetically identical. A series of 19 such frogs have been reared to sexual maturitv; they differ to some extent in size (which is not only controlled by the genetic quality of their nuclei), but in other respects appear to be identical.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available