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Settling a hotly debated issue in the field of cloning, a team of researchers from the Whitehead Institute and the University of Hawaii has shown that the egg can reset the developmental clock of a female adult cell, first reversing and then faithfully reproducing an early genetic event called X-inactivation. X-inactivation is a process by which one of two X chromosomes in female embryos is randomly silenced during development.
The findings, published in the November 24 issue of Science, provide the first molecular evidence for the egg's ability to reprogram an adult cell back to its embryonic state and show for the first time that the process of X-inactivation in clones occurs in a manner similar to that in normal development.
Since Dolly the sheep was first cloned in 1997, researchers have debated whether she has random X-inactivation as normal females do, or the same X chromosome inactivated in all her cells (the X that was inactive in the mammary cell nucleus from which she had been cloned).
In this study, scientists from Professor of Biology Rudolf Jaenisch's laboratory at Whitehead and Ryuzo Yanagimachi's laboratory at the University of Hawaii used their expertise in cloning mice to answer this question. Their results show that X-inactivation is random in the cells of cloned animals. The egg therefore is able to reprogram the X chromosome of an adult cell to a state that is appropriate for an embryonic one.
"Scientists have suggested that cloning of mammals by nuclear transfer requires reprogramming of the differentiated state of donor cell to an embryonic ground state, but there was no direct molecular evidence for such reprogramming. The process of X-inactivation is an example. It has never been clear whether this process is reversible during cloning," said Professor Jaenisch.
Cloning is a very inefficient process, and it is possible that faulty reprogramming contributes to the low success rate. "It will therefore be crucial to determine whether the expression state of other genes of the adult donor cell can be faithfully reset during the cloning process," he said.
THE WHY OF X-INACTIVATION
X-inactivation is nature's way of making sure that males and females have equal numbers of X-linked genes, explained biology graduate student Kevin Eggan, the first author on the paper. All human embryos inherit 23 pairs of chromosomes: one pair of sex chromosomes and 22 pairs of non-sex, or autosomal, chromosomes. Embryos that inherit an X chromosome from their mother and a Y chromosome from their father develop into males. Embryos that inherit two X chromosomes (one from each parent) develop into females.
At the earliest stages of embryonic development, both X chromosomes are active in female embryos, but just before implantation, one chromosome is chosen for inactivation and silenced. The X-inactivation is random in the embryonic tissue, but in the tissue that ultimately becomes the placenta, the inactivation is thought to happen by a process called imprinting in which the paternal X is marked for inactivation.
All female adult cells therefore have one X chromosome silenced, and the authors wondered whether cloning can reverse it. To do this, they used green fluorescent protein to label a gene on the X chromosome subject to inactivation and followed its course in different lineages of cloned embryos. This allowed them to tell, based on whether the cells were dark or fluorescent green, which X was inactivated and when.
The researchers then created various populations of donor cells with known X-inactivation states (which could be color coded as dark or green donor cells) and used these donor cells to clone new embryos, transferring the nuclei of these cells into enucleated eggs.
When researchers analyzed the results, they found that X-inactivation is random in the embryonic lineage of cloned mice. "This random inactivation indicates that the epigenetic marks that distinguish the active and inactive X in adult cells can be removed and re-established on either X during the cloning process, resulting in random X-inactivation in the cloned animal," Mr. Eggan said. Epigenetic phenomena are heritable traits that aren't necessarily directly encoded by changes in the sequence of DNA.
In contrast, the epigenetic marks on the active and inactive X in the donor cell are not removed in the early placental tissue of cloned mice and predispose the active X of the donor cell to be active and the inactive X to be inactive. However, just as in normal development, this somatically acquired mark is ignored during early stages of development as both X chromosomes are expressed, he said.
"Our results imply that the epigenetic marks acquired during normal random X-inactivation in the embryo are functionally equivalent to the marks acquired during sperm and egg development, as they both can determine which X will be active and which will be inactive in the placenta," Professor Jaenisch said.
"Although the egg reprograms some aspects of X-inactivation, for instance, turning on genes in the silent X chromosome, there clearly are other aspects that the egg ignores, namely the marks that lead to imprinted X-inactivation in the placenta," Mr. Eggan said.
"It is expected that genes on other chromosomes are also reprogrammed to their embryonic state within the egg. If the marks on the genes on the X chromosome and other chromosomes are faulty in the cell cloned, the egg may not have the ability to fix them, leading perhaps to abnormal development and death of embryos," Dr. Yanagimachi said.
Other Whitehead authors of the Science paper were research assistant Konrad Hochedlinger and postdoctoral fellow William Rideout.
A version of this article appeared in MIT Tech Talk on December 6, 2000.