Dynamic Development

CONTENTS

Main Page Dynamic Development

The Foundations of Developmental Biology

Gametogenesis

From Sperm and Egg to Embryo

Genetic Regulation of Development

Organizing the Multicellular Embryo

Generating Cell Diversity


Dynamic Development at a Glance


Learning Resources

Research Resources

The Developmental Biology Journal Club

Developmental Biology Tutorial

Xenopus as a Model System in Developmental Biology

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 used Xenopus.

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-ß 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 (N-tubulin) is shown in Figure 1F and H (transgenic) and Figure 1G (plasmid-injected). The transgenic embryo shows expression in the neural tube, whereas the plasmid-injected 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).

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.)

We have used the Kroll and Amaya technique to generate transgenic animals in my own laboratory (Fig. 3). Click here for details about research in the Browder laboratory.


Figure 3. Transgenic Xenopus laevis tadpole expressing the Green Fluorescent Protein gene under control of the cytomegalovirus promoter. The tadpole was generated by Jill Johnston in Leon Browder's laboratory using the technique of Kroll and Amaya.

A recent paper from Sylvia Evans' laboratory describes a more direct approach for generation of transgenics (Fu et al., 1998). These two techniques, combined with the accessibility and ease of manipulation of Xenopus embryos, should provide investigators with unprecedented opportunities to examine the molecular basis of early development.


Digging Deeper

The Xenopus Molecular Marker Resource


References

Fu, Y., Wang, Y. and Evans, S.M. 1998. Viral sequences enable efficient and tissue-specific expression of trangenes in Xenopus. Nature Biotechnology 16: 253-257.

Gurdon, J.B., Lane, C.D., Woodland, H.R. and Marbaix, G. 1971. Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells. Nature 233: 177-182.

Kroll, K.L. and Amaya, E. 1996. Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 122: 3173-3183.


Tutorial Exercise

Amphibian Tutorial


Dynamic Development at a Glance
Main Page Dynamic Development

Dynamic Development is a Virtual Embryo learning resource

This material may be reproduced for educational purposes only provided credit is given to the original source.
Leon Browder & Laurie Iten (Ed.) Dynamic Development
Last revised Wednesday, March 4, 1998