326
Xenopus
f eld. In 1893, Weismann put forth the germ-plasm hypothesis, in which he suggested that as development proceeds,
genetic determinants are segregated into cells based on
their type. It was quite intuitively supposed that genes that
are not required for the intended fate of the cell are either
permanently deactivated or, more likely, disposed of. An
experiment in which the totipotent nucleus of the zygote is
replaced by a differentiated, somatic nucleus was considered a defnitive approach to solving this question, because
proper development of the transplanted embryo would confrm that the genetic material remains intact and that differentiation does not irreversibly affect the plasticity of the
nucleus.
23.2.1. EARLY SCNT EXPERIMENTS ESTABLISH
THE REPROGRAMMING FIELD
Between 1952 and 1957, Briggs and King revisited this
crucial biological question and performed nuclear transfer
(NT) experiments using the frog Rana pipiens as a model
system. They introduced a blastula nucleus into an enucleated egg and obtained normal, living tadpoles (Briggs and
King, 1952). This demonstrated that, at least until the blastula stage, the derived nuclei were able to support normal
development and could give rise to complete organisms.
Following this experiment, Briggs and King attempted to
introduce nuclei of further differentiated endoderm cells
into enucleated Rana pipiens eggs. The embryos resulting
from this NT experiment did not survive, due to which they
concluded that the ability of a nucleus to support normal
development either decreases with, or is permanently discontinued at some point during their own differentiation.
However, the curiosity of many scientists was not satisf ed
by these results, and one group in particular decided to
tackle this problem once more.
John Gurdon began his Ph.D. in 1956 in the lab of Michail
Fischberg with the same question in mind: Do all cell types
have the same set of genes? For this, it was crucial to pick
up where Briggs and King left off—with a repetition of the
SCNT experiment. Gurdon performed a series of NTs using
donor nuclei isolated from successive developmental stages of
Xenopus laevis. The donor nuclei were derived from the endoderm, taken either from the vegetal cell mass at blastula or
gastrula stages or the foor of gut-lumen in hatched tadpoles.
He showed that, contrary to the earlier results obtained by
Briggs and King, donor nuclei from more advanced stages of
development can also support normal growth via NT (Gurdon,
1962). In fact, the organisms resulting from similar experiments most strikingly developed into sexually mature adults
that could produce normally developing embryos themselves
(Gurdon and Uehlinger, 1966).
The discrepancy between these fndings and those of the
widely respected and recognized scientists Briggs and King
led to a certain amount of incredulity among the scientif c
population. However, there were certain noteworthy differences between the two studies. First, choosing the optimal
model organism is extremely important. The use of Rana
pipiens brings with it a number of technical disadvantages,
one of which is its seasonal reproductive cycle. Xenopus
laevis can be induced to lay eggs at any point of the year
using mammalian hormones such as gonadotropic hormone.
Furthermore, Xenopus has high disease resistance, and the
growth of Xenopus laevis to sexual maturity occurs frequently
in laboratory conditions and within one year. Second, a key
diffculty of NT experiments at the time was the inability to
safely penetrate the protective jelly surrounding eggs, which
was necessary for enucleation. Gurdon used UV irradiation
to destroy the chromosomes inside the egg, also making the
egg jelly more penetrable in the process for microinjection
during NT. Third, experimental outcomes were corrected
for variations caused by technical damage to the oocyte during the NT procedure and for varying egg quality. This left
innate properties of the donor nuclei as the only signif cant
variable that might hinder effciency of reprogramming in
NT experiments (Gurdon, 1960). The fourth reason, which
ultimately ensured the irrefutability of the study, was the use
of marked donor nuclei. Nuclei obtained from a single-nucleolus strain of Xenopus were used as donors for NT, and the
resulting successively transferred embryos were easily distinguishable from wild-type 2-nucleolated strains (Elsdale et
al., 1960). This marker was crucial to validate Gurdon’s NT
experiments against all doubt.
The most puzzling outcome of these experiments was
that the eff ciency of survival and appropriate development
of the NT embryo decreased with an increase in the differentiation state of their respective donor nuclei. For example,
the effciency of NT from a donor of an early developmental stage, that is, the blastula stage, was rather high (60%
of cleaving NT-embryos), whereas the effciency of NT
from a donor of highly differentiated state was extremely
low (~1–2% of cleaving NT-embryos) (Gurdon, 1962). This
observation pointed to an existing resistance of cells to
reprogramming that becomes more prominent with differentiation. This can now be considered one of the earliest
experimental indicators of epigenetics—that a separate regulatory mechanism might exist on top of DNA sequence to
specifcally supervise the expression of the genome based
on its cell type. The implications of this fnding for the
mechanism’s underlying cell-fate stability and reprogramming resistance will be discussed in the following sections.
Regardless, his work answered a long-standing open question in developmental biology—it showed that genes unnecessary for the intended cell type are not lost or irreversibly
inactivated through the course of cellular differentiation.
In fact, differentiated nuclei retain the complete genome
throughout development, including the genes important for
forming functional germ cells, as evident from the fertile
frogs resulting from NT (Gurdon and Uehlinger, 1966).
Importantly, this new possibility of reprogramming the
nucleus of a cell to totipotency or even a different cell fate
has major implications for the feld of regenerative medicine. It suggests that, in theory, any cell of our bodies can
be changed to any other type to replace damaged or irreversibly lost cells.
Xenopus
f eld. In 1893, Weismann put forth the germ-plasm hypothesis, in which he suggested that as development proceeds,
genetic determinants are segregated into cells based on
their type. It was quite intuitively supposed that genes that
are not required for the intended fate of the cell are either
permanently deactivated or, more likely, disposed of. An
experiment in which the totipotent nucleus of the zygote is
replaced by a differentiated, somatic nucleus was considered a defnitive approach to solving this question, because
proper development of the transplanted embryo would confrm that the genetic material remains intact and that differentiation does not irreversibly affect the plasticity of the
nucleus.
23.2.1. EARLY SCNT EXPERIMENTS ESTABLISH
THE REPROGRAMMING FIELD
Between 1952 and 1957, Briggs and King revisited this
crucial biological question and performed nuclear transfer
(NT) experiments using the frog Rana pipiens as a model
system. They introduced a blastula nucleus into an enucleated egg and obtained normal, living tadpoles (Briggs and
King, 1952). This demonstrated that, at least until the blastula stage, the derived nuclei were able to support normal
development and could give rise to complete organisms.
Following this experiment, Briggs and King attempted to
introduce nuclei of further differentiated endoderm cells
into enucleated Rana pipiens eggs. The embryos resulting
from this NT experiment did not survive, due to which they
concluded that the ability of a nucleus to support normal
development either decreases with, or is permanently discontinued at some point during their own differentiation.
However, the curiosity of many scientists was not satisf ed
by these results, and one group in particular decided to
tackle this problem once more.
John Gurdon began his Ph.D. in 1956 in the lab of Michail
Fischberg with the same question in mind: Do all cell types
have the same set of genes? For this, it was crucial to pick
up where Briggs and King left off—with a repetition of the
SCNT experiment. Gurdon performed a series of NTs using
donor nuclei isolated from successive developmental stages of
Xenopus laevis. The donor nuclei were derived from the endoderm, taken either from the vegetal cell mass at blastula or
gastrula stages or the foor of gut-lumen in hatched tadpoles.
He showed that, contrary to the earlier results obtained by
Briggs and King, donor nuclei from more advanced stages of
development can also support normal growth via NT (Gurdon,
1962). In fact, the organisms resulting from similar experiments most strikingly developed into sexually mature adults
that could produce normally developing embryos themselves
(Gurdon and Uehlinger, 1966).
The discrepancy between these fndings and those of the
widely respected and recognized scientists Briggs and King
led to a certain amount of incredulity among the scientif c
population. However, there were certain noteworthy differences between the two studies. First, choosing the optimal
model organism is extremely important. The use of Rana
pipiens brings with it a number of technical disadvantages,
one of which is its seasonal reproductive cycle. Xenopus
laevis can be induced to lay eggs at any point of the year
using mammalian hormones such as gonadotropic hormone.
Furthermore, Xenopus has high disease resistance, and the
growth of Xenopus laevis to sexual maturity occurs frequently
in laboratory conditions and within one year. Second, a key
diffculty of NT experiments at the time was the inability to
safely penetrate the protective jelly surrounding eggs, which
was necessary for enucleation. Gurdon used UV irradiation
to destroy the chromosomes inside the egg, also making the
egg jelly more penetrable in the process for microinjection
during NT. Third, experimental outcomes were corrected
for variations caused by technical damage to the oocyte during the NT procedure and for varying egg quality. This left
innate properties of the donor nuclei as the only signif cant
variable that might hinder effciency of reprogramming in
NT experiments (Gurdon, 1960). The fourth reason, which
ultimately ensured the irrefutability of the study, was the use
of marked donor nuclei. Nuclei obtained from a single-nucleolus strain of Xenopus were used as donors for NT, and the
resulting successively transferred embryos were easily distinguishable from wild-type 2-nucleolated strains (Elsdale et
al., 1960). This marker was crucial to validate Gurdon’s NT
experiments against all doubt.
The most puzzling outcome of these experiments was
that the eff ciency of survival and appropriate development
of the NT embryo decreased with an increase in the differentiation state of their respective donor nuclei. For example,
the effciency of NT from a donor of an early developmental stage, that is, the blastula stage, was rather high (60%
of cleaving NT-embryos), whereas the effciency of NT
from a donor of highly differentiated state was extremely
low (~1–2% of cleaving NT-embryos) (Gurdon, 1962). This
observation pointed to an existing resistance of cells to
reprogramming that becomes more prominent with differentiation. This can now be considered one of the earliest
experimental indicators of epigenetics—that a separate regulatory mechanism might exist on top of DNA sequence to
specifcally supervise the expression of the genome based
on its cell type. The implications of this fnding for the
mechanism’s underlying cell-fate stability and reprogramming resistance will be discussed in the following sections.
Regardless, his work answered a long-standing open question in developmental biology—it showed that genes unnecessary for the intended cell type are not lost or irreversibly
inactivated through the course of cellular differentiation.
In fact, differentiated nuclei retain the complete genome
throughout development, including the genes important for
forming functional germ cells, as evident from the fertile
frogs resulting from NT (Gurdon and Uehlinger, 1966).
Importantly, this new possibility of reprogramming the
nucleus of a cell to totipotency or even a different cell fate
has major implications for the feld of regenerative medicine. It suggests that, in theory, any cell of our bodies can
be changed to any other type to replace damaged or irreversibly lost cells.