Amphibian embryos were used in the very first embryological experiments, when

Wilhelm Roux conducted his "hot needle" experiment in an attempt to prove his
concept of qualitative division. Amphibian embryos remained the embryos of choice
for experimental embryologists for many decades. European embryologists used
predominantly urodele embryos (such as Triturus) and embryos of the frog Rana
temporaria, which is related to the North American species Rana pipiens. Amphibian
embryos are large, can be obtained in large numbers and can be maintained easily and
inexpensively in the laboratory. They are relatively easy to manipulate with
microsurgical instruments, and they heal readily after surgery. In the early days, those
instruments were made by hand. Studies on embryos were complemented by studies
on oocytes, which are readily accessible by simple surgery on females.
One disadvantage of traditional amphibian species is that they are seasonal breeders.
This meant that investigators could not do their experiments throughout the
year. Xenopus laevis, the South African Clawed Frog, is a notable exception. In fact, it
was its ability to spawn when induced with an injection of gonadotropic hormone that
led to its common usage for human pregnancy tests in the 1950s: An injection of
pregnancy urine (which contains chorionic gonadotropin) would induce spawning.
This led investigators to consider its use in experimental embryology. In fact, it was
commonly used for experimental embryology in South Africa before its utility was
recognized elsewhere. B.I. Balinsky was one of the South African embryologists who
usedXenopus.
During the 1950s, Fischberg's laboratory in Geneva used Xenopus for studying
development. His student, John Gurdon, brought attention to Xenopus by
demonstrating that transplantation of tadpole intestinal epithelial nuclei into
enucleated eggs could promote development to the adult, thus extending Briggs and
King's earlier nuclear transplantation research that utilized Rana pipiens. Shortly
thereafter, Don Brown, Igor Dawid and Ron Reeder in the United States began to
use Xenopus for biochemical research. In fact, Brown was the first to isolate a
eukaryotic gene: the Xenopus ribosomal RNA genes from oocytes. Gradually, the ease
of obtaining eggs on a year-round basis, the rapid rate of development and the
availability of mutants such as the anucleolate and albino mutant caused investigators
began to forsake their favorite frogs and newts for the homely Xenopus.

Another impetus for investigators to adopt Xenopus came in 1971, when Gurdon and
his colleagues demonstrated that the Xenopus oocyte will translate messenger RNA
injected into it (Gurdon et al., 1971). This has proven to be a valuable system for the
expression of RNA. Later, when recombinant DNA technology made it possible to
clone individual Xenopus genes, injections of synthetic RNA into zygotes allowed
investigators to overexpress RNA or to express antisense RNA to evaluate the role of
a transcript during development.
Meanwhile, in the Netherlands in the late 1960s and early 1970s, P.D. Nieuwkoop was
investigating inductive interactions during early development of urodeles and,
later, Xenopus. Using his ingenious animal cap assay, he demonstrated that the
mesoderm is induced by cells of the vegetal hemisphere. This assay has proven to be a
valuable weapon in developmental biologists' armory. The animal cap of the blastula
will respond to the appropriate signals to produce a variety of tissues. This assay has
enabled investigators to hone in on the most intractable problem in developmental
biology: embryonic induction. We now know that growth factors in the TGF- and
FGF families provide the signals for embryonic induction.
Clearly, Xenopus has a number of advantages that have enabled investigators to use it
to study many aspects of development. However, one of the disadvantages has been
the lack of a dependable technique for making transgenic embryos. You can clone its
genes, and you can inject RNA into zygotes. However, RNA is relatively short-lived.
Therefore, the study of molecular events after the mid-blastula transition remained
problematic. Attempts to inject genes to be expressed in the embryo were frustrated by
the fact that they do not integrate into the frog chromosomes during cleavage and are
then unequally distributed in embryonic cells and, therefore, are always expressed
mosaically. This remained the case until 1996, when Kroll and Amaya developed a
technique for making stable, non-mosaic transgenic Xenopus embryos. This technique
has the potential to boost the utility of Xenopus tremendously. One big advantage over
transgenesis in mice is that one can make first generation transgenics; you don't need
to wait until the next generation to examine the effects of the exogenous gene on
development.
The transgenesis technique has several steps, and each step is fraught with problems.
However, when it works, it is very powerful. Because exogenous genes are not
incorporated into the zygotic genome, Kroll and Amaya decided to attempt to
introduce them into sperm nuclei. Sperm nuclei are treated with lysolecithin to

demembranate them before incubation with linearized plasmid containing the

exogenous gene to be incorporated into them, along with restriction enzyme to create
nicks in the sperm nuclear DNA. The nicks facilitate incorporation of the plasmid
DNA. The nuclei are then placed in an interphase egg extract, which causes the nuclei
to swell as if they were male pronuclei.
The swollen nuclei are then injected into unfertilized eggs. The suspension of nuclei is
diluted to optimize the probability that a single nucleus is injected. The injection
process mimics sperm entry, activating development. In response, the egg nucleus
completes the second meiotic division and forms a pronucleus, which fuses with the
transplanted nucleus. If a single sperm nucleus is transplanted, a single pair of
centrioles facilitates normal cleavage. However, if multiple nuclei are transplanted,
supernumerary centrioles would be present, resulting in abnormal cleavage. This
would be comparable to the effects of dispermy on sea urchin development
that Boveri observed.
To demonstrate the effectiveness of the transgenesis procedure, Kroll and Amaya
made transgenic embryos from sperm nuclei into which they had introduced
introduced reporter genes. The results are shown in Figures 1 and 2. Figure 1
illustrates the non-mosaic expression of introduced plasmids. In Figure 1A and E, the
expression of green fluorescent protein (GFP) under the control of cytomegalovirus
(CMV) promoter is shown. This contrasts to the mosaic expression of this gene is
plasmic-injected embryos (Fig. 1C). The expression of the chloramphenicol
acetyltransferase (CAT) gene under the control of a neural-specific promoter (Ntubulin) is shown in Figure 1F and H (transgenic) and Figure 1G (plasmid-injected).
The transgenic embryo shows expression in the neural tube, whereas the plasmidinjected embryo shows sparse, mosaic expression.
A muscle-specific actin promoter was also used to drive expression of GFP and CAT.
As shown in Figure 1J-L and N, appropriate expression in somites and cardiac muscle
is observed in transgenics, whereas mosaic expression is seen in plasmid-injected
embryos (Fig. 1M and O).
Confirmation of integration of plasmids into the genome of transplantation-derived
embryos was obtained by probing Southern blots of DNA from one month-old
tadpoles with a GFP probe. Typically, unintegrated plasmids would be lost by this
stage. As shown in Figure 2, GFP sequences were found in transplantation-derived

tadpoles that expressed pCARGFP but were not found in their non-expressing
siblings.

Figure 1. Plasmid expression in transgenic embryos from sperm nuclear transplantations compared to mosaic
expression of plasmids injected into embryos.
A: Trunk of transgenic embryo expressing pCMVnGFP.
B: DAPI staining of the region shown in A. (DAPI stains DNA.)
C. Expression of GFP in trunk of embryo injected with pCMVnGFP.
D. DAPI staining of region shown in C.
E. Transgenic embryo expressing pCMVnGFP.
F. Transgenic embryo expressing the N-tubulin/CAT plasmid.
G. Expression of CAT in embryo injected with the N-tubulin/CAT plasmid.
H. Cross-section of transgenic embryo showing expression of the N-tubulin/CAT plasmid in primary neurons.
I. Bright-field image of transgenic tadpole expressing pCARGFP.
J. Fluorescent image of tadpole shown in I. GFP expression can be seen in somites and heart muscle.
K. Transgenic expression of pCARGFP in somites.
L. Transgenic expression of pCARGFP in heart.
M. Mosaic expression of pCARGFP in plasmid-injected embryo.
N. Transgenic expression of pRLCAR (CAT driven by muscle-specific actin promoter).
O. Mosaic expression of pRLCAR in plasmid-injected embryo.
(Figure from Kroll and Amaya, 1996. Reproduced with permission of The Company of Biologists.)

Confirmation of integration of plasmids into the genome of transplantation-derived

embryos was obtained by probing Southern blots of DNA from one month-old
tadpoles with a GFP probe. Typically, unintegrated plasmids would be lost by this

stage. As shown in Figure 2, GFP sequences were found in transplantation-derived

tadpoles that expressed pCARGFP but were not found in their non-expressing
siblings.

Figure 2. Tadpoles produced by sperm nuclear transplantation contain integrated plasmid. (A) Schematic of
linearized pCARGFP plasmid: cardiac actin promoter (open box), GFP sequences (gray box), SV40

polyadenylation site (solid box) and bacterial sequences (thin line). Below, products expected after pCARGFP
concatemerization in the embryo. N, NotI. (B) Southern blot of genomic DNA from 1-month-old tadpoles
produced using pCARGFP nuclear transplantations (lanes 4-11). Tadpoles expressing GFP non-mosaically (+)
and tadpoles not expressing GFP (-) are designated. pCARGFP was detected in HindIII (H)-digested genomic
DNA using probe sequences designated in A. Lanes 1-3, pCARGFP plasmid was added to genomic DNA from
control tadpoles (not produced using nuclear transplantation) just prior to Hind III digestion. (Figure from
Kroll and Amaya, 1996. Reproduced with permission of The Company of Biologists.)