The Science Of The Lambs
The Science Of The Lambs
by The advent of mammalian cloning extends key observations made over thirty
years ago by John Gurdon using the frog Xenopus laevis. (2) Gurdon used an
earlier strategy for nuclear transplantation developed by Robert Briggs and
Thomas King (3) to unequivocally demonstrate that nuclei from tadpole
intestinal epithelial cells could direct the development of fertile adult
frogs. Over the subsequent decade, these experiments were repeated in ever
increasing detail by a dedicated group of investigators until the pluripotency
of adult cell nuclei was definitively established. (4, 5) Most importantly, a
firm scientific foundation was laid for the future work on mammalian cloning.
Nevertheless, in spite of much effort, no single adult nucleus has ever given
rise to an adult frog by nuclear transplantation in amphibia. The use of an
adult cell derived from the mammary epithelium of a sheep by Wilmut and
colleagues as a donor in the nuclear transplantation experiment that gave rise
Early embryogenesis requires the totipotent egg nucleus to cleave during cell
division and progressively acquire all of the separate identities that exist
in the tissues of an organism. This involves the precisely staged association
of transcription factors and specialized chromosomal proteins with the
regulatory elements of genes. As development proceeds, an increasing number
of cells exists in the embryo, and the regulatory nucleoprotein complexes that
establish lineages or identity become more elaborate and resistant to physical
and biochemical perturbation. This functional specialization of chromatin and
chromosomes also becomes more difficult to reverse when an embryonic cell
nucleus is transplanted into an enucleated egg. The more differentiated
the cell from which a donor nucleus is taken, the more unlikely it is that
correct development will proceed. However, persistence leads to dividends
since in amphibia, nuclei taken from adult keratinocytes or reticulocytes can
be shown in a few but notable instances to support the development of all the
cell types found in a tadpole. (4, 5) Therefore all of the regulatory
nucleoprotein complexes that control the specific patterns of gene activity in
these differentiated cells can be disassembled.
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(Issue 4; posted March 21, 1997; archived April 4, 1997)
An industrial-strength scientific effort is devoted to understanding the
molecular events that make individuals and their cellular components different
from each other. The mapping of individual genes, their mutations, and
activity states underpins the great advances of molecular medicine. Individual
cells differ dependent on the constellation of active and inactive genes that
each express, which is in turn determined by their developmental history. It
is remarkable that under certain circumstances, this developmental history is
reversible. The clearest and most compelling demonstration of this
reversibility is the recent cloning of the lamb Dolly by Ian Wilmut and
colleagues. (1)
to Dolly now indicates that adult nuclei can also become totipotent. What
does acquisition of totipotency imply for the molecular mechanisms that
establish cell fate?
The first experiments that examined specific gene activity within transplanted
somatic nuclei revealed that nucleoli disappeared and that previously active
ribosomal RNA genes were inactivated within the egg. (6) Nucleoli are an
example of the compartmentalization of a particular biosynthetic event, the
synthesis of rRNA, to a specific chromosomal structure. The
inhibition of ribosomal RNA transcription in eggs clearly demonstrated the
capacity of egg cytoplasm to influence nuclear function. As development of
the embryo containing the transplanted nucleus proceeds, the ribosomal genes
were reactivated and nucleoli reappeared. This influence of cytoplasm on
nuclear function could be better understood once it was shown that a
considerable movement of proteins from the egg cytoplasm to the somatic
nucleus occurred following transplantation. (7, 8) This movement was
concomitant with nuclear swelling and with a significant reduction in the
amount of transcriptionally inactive heterochromatin within the nucleus.
Subsequent work examined the association of specific components of the transcriptional machinery with individual genes. Both the regulatory nucleoprotein complexes that activated transcription and those that repressed transcription were found to be unstable in an egg environment. (9, 10) This was in marked contrast to their stability in the nuclei of differentiated cells. (11) Molecular chaperones that are stored in the egg have been shown to have a causal role in directing the loss of chromosomal proteins specific to somatic nuclei and thus the remodeling of somatic nuclei following exposure to egg cytoplasm. (12) However, this only part of the process. The somatic nucleus progressively acquires the proteins and modifications normally found with the embryonic chromosome. Many of these specialized modifications are also characteristic of the transformed cell nuclei found in many tumors.
The orchestrated exchange of somatic nuclear proteins for egg cytoplasmic components takes time, and it is the failure to effect the restructuring of chromatin, chromosomes, and nuclei before cell division that most probably leads to chromosomal damage and the developmental abnormalities apparent in many nuclear transplant embryos. In this regard the mammalian cloning experiments provide additional insight. (1) These investigators made use of adult somatic cells that they synchronized in G0 - a quiescent state within the cell cycle. This state of quiescence is normally achieved by starving cells for serum, causing cells in G1 to leave the cell cycle. This exit can be reversed by adding back serum in culture, or evidently by transplanting a G0 cell nucleus into the egg. This might facilitate the remodeling of chromatin by attuning the nuclear and cytoplasmic cell cycles just before entry into S phase. DNA replication itself will further facilitate the disruption of regulatory nucleoprotein complexes. (13) Therefore using the strategy of Wilmut and colleagues, (1) an embryonic chromosomal structure might be established before cell division occurs, thereby preventing chromosomal damage. If this hypothesis is true, then simple manipulations to somatic cell nuclei that would facilitate nuclear remodeling, such as preincubation with the molecular chaperone nucleoplasmin, would greatly facilitate the efficiency of animal cloning.
The original interpretation of nuclear transplantation experiments in amphibia
suggested that the genetic material was not irreversibly altered as
development proceeds. This concept had a major impact on developmental
science at the time. However, we now know that this is not necessarily true,
since cell type specific rearrangement of immunoglobulin genes and generalized
loss of telomeric sequences occurs, dependent on differentiation and aging.
Moreover, specific patterns of cytosine methylation and demethylation
correlate with gene activity and repression in particular cells. The mammary
epithelial cell nucleus used by Wilmut did not encounter VDJ recombination to
define the antibody repertoire; however, loss of telomeric sequences will
presumably have occurred. Hence the aging of mammalian clones may differ from
those animals derived from the fusion of gametes. As for DNA methylation, it
must either be reversible or be unimportant for the establishment of
differential states of gene activity. No doubt future studies will explore
these possibilities.
A final technical point is that much of early embryonic development is driven
through the activity of proteins and messenger RNAs stored in the egg. Masked
maternal mRNA is translationally silent until fertilization (or nuclear
transplantation). Recruitment to the translational machinery then initiates
the determinative events that restrict the fates of embryonic cells. The same
molecular chaperones that facilitate the remodeling of somatic nuclei
following transplantation also facilitate the unmasking of maternal mRNA. (14)
Importantly the maternal determinants will differ from egg to egg. Thus a
true clone from a nuclear transplant embryo is not achievable in the sense
that monozygotic identical twins are clonal.
Recognition of the success of mammalian cloning will surely have a major
impact on many aspects of basic developmental biology. The experiments are
simple and powerful. They answer a crucial biological question with respect
to development by clearly demonstrating the reversibility of determinative
mechanisms. Differential gene activation is what drives development, not the
irreversible alteration of the genetic material itself. The dramatic
affirmation of this conclusion should help restore the balance of science
towards understanding what represents the developmental ground state of
totipotency and how specific patterns of gene activity can be lost.
Understanding these processes in molecular terms will undoubtedly have
general relevance for human disease.
A.P. Wolffe is Chief of the Laboratory of Molecular Embryology and of the Section on Molecular Biology at the National Institute of Child Health and Human Development, NIH, Bethesda, Maryland.