John B. Gurdon

Morphogen gradient interpretation

A morphogen gradient is an important concept in developmental biology, because it describes a mechanism by which the emission of a signal from one part of an embryo can determine the location, differentiation and fate of many surrounding cells. The value of this idea has been clear for over half a century, but only recently have experimental systems and methods of analysis progressed to the point where we begin to understand how a cell can sense and respond to tiny changes in minute concentrations of extracellular signalling factors.

Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis.

Using an expression cloning strategy that relies on a functional assay, we have cloned a novel Xenopus homeobox-containing gene, Siamois. Embryos injected in a ventral-vegetal blastomere with as little as 5 pg of Siamois mRNA develop a complete secondary axis, but the progeny of the injected cells do not participate in the secondary axis formation. In normal development, Siamois mRNA is first detected shortly after the midblastula transition, which is earlier than mRNAs for goosecoid or Xbrachyury, and is present most abundantly in the dorsal endoderm of early gastrulae. The activation of this gene can be obtained cell autonomously in dispersed embryo cells. These results indicate that Siamois may play an important role in the formation of the Nieuwkoop center.

Adult frogs derived from the nuclei of single somatic cells.

Abstract A survey has been made of over 150 adult frogs which have been obtained by the transplantation of nuclei from endoderm cells of Xenopus laevis donors ranging from late blastulae to swimming tadpoles. Many of the transplant-frogs from donors of all ages were entirely normal, and many of the abnormalities observed were not connected with the condition of the transplanted nuclei. Frogs derived from the nuclei of differentiating cells were more often abnormal than those derived from embryonic cells. Out of 27 frogs derived from nuclei of hatched tadpole gut cells, 7 were sterile. The results demonstrate that at least 30% of blastula nuclei and at least 4% of hatched tadpole gut-cell nuclei contain a complete range of the genetic information necessary for the formation and functioning of a normal adult individual.

DNA DEMETHYLATION IS NECESSARY FOR THE EPIGENETIC REPROGRAMMING OF SOMATIC CELL NUCLEI

Nuclear transplantation experiments in amphibia and mammals have shown that oocyte and egg cytoplasm can extensively reprogram somatic cell nuclei with new patterns of gene expression and new pathways of cell differentiation; however, very little is known about the molecular mechanism of nuclear reprogramming. Here we have used nuclear and DNA transfer from mammalian somatic cells to analyse the mechanism of activation of the stem cell marker gene oct4 by Xenopus oocytes. We find that the removal of nuclear protein accelerates the rate of reprogramming, but even more important is the demethylation of somatic cell DNA. DNA demethylation seems to precede gene reprogramming, and is absolutely necessary for oct4 transcription. Reprogramming by oocytes occurs in the absence of DNA replication and RNA/protein synthesis. It is also selective, operating only on the promoter, but not enhancers, of oct4; both a putative Sp1/Sp3 and a GGGAGGG binding site are required for demethylation and transcription. We conclude that the demethylation of promoter DNA may be a necessary step in the epigenetic reprogramming of somatic cell nuclei.

Nuclear Reprogramming in Cells

Nuclear reprogramming describes a switch in gene expression of one kind of cell to that of another unrelated cell type. Early studies in frog cloning provided some of the first experimental evidence for reprogramming. Subsequent procedures included mammalian somatic cell nuclear transfer, cell fusion, induction of pluripotency by ectopic gene expression, and direct reprogramming. Through these methods it becomes possible to derive one kind of specialized cell (such as a brain cell) from another, more accessible, tissue (such as skin) in the same individual. This has potential applications for cell replacement without the immunosuppression treatments that are required when cells are transferred between genetically different individuals. This article provides some background to this field, a discussion of mechanisms and efficiency, and comments on prospects for future nuclear reprogramming research.

Epigenetic memory of an active gene state depends on histone H3.3 incorporation into chromatin in the absence of transcription

The remarkable stability of gene expression in somatic cells is exemplified by the way memory of an active gene state is retained when an endoderm cell nucleus is transplanted to an enucleated egg. Here we analyse the mechanism of a similar example of epigenetic memory. We find that memory can persist through 24 cell divisions in the absence of transcription and applies to the expression of the myogenic gene MyoD in non-muscle cell lineages of nuclear transplant embryos. We show that memory is not explained by the methylation of promoter DNA. However, we demonstrate that epigenetic memory correlates with the association of histone H3.3 with the MyoD promoter in embryos that display memory but not in those where memory has been lost. The association of a mutated histone H3.3 (H3.3 E4, which lacks the methylatable H3.3 lysine 4) with promoter DNA eliminates memory, indicating a requirement of H3.3 K4 for memory. We also show that overexpression of H3.3 can enhance memory in transplanted nuclei. We therefore conclude that the association of histone H3.3 with the MyoD promoter makes a necessary contribution to this example of memory. Hence, we suggest that epigenetic memory helps to stabilize gene expression in normal development; it might also help to account for the inefficient reprogramming in some transplanted nuclei.

The generation of diversity and pattern in animal development

Experimental embryology started in the late 1800s asking such questions as whether one of the first two blastomeres of a 2-cell newt egg can compensate if the other is killed. Landmarks in the early history of developmental biology include cell culture (Harrison, 1910), the discovery of embryonic induction and the organizer (Spemann and Mangold, 1924), and the ability of dissociated cells to reorganize themselves when reaggregated (Townes and Holtfreter, 1955). Intense activity in the 1930s followed the discovery that partially purified tissue extracts could act as inducers by causing ectoderm to change its fate and differentiate as neurogenic tissue (reviewed by Hamburger, 1988 and Saxen, 1989). However, enthusiasm faded when it was realized that the kind of response made by embryonic tissue to such inducers was primarily a property of the cells and not of the inducing substances. Over the last few decades, methods of molecular biology and genetics have been applied to problems of development with increasing success. The most significant recent advances have centered on the identification of single genes of major developmental importance, particularly those whose products activate or repress other genes in sequential steps that lead to cell typeor region-specific gene expression. In addition, there has been agreatly increased appreciation of the widespread importance of intercellular interactions in all kinds of embryos, and this aspect of development is emphasized here. The aim of this article is to provide a brief and therefore necessarily somewhat personal survey of concepts of early development and of the principles so far recognized, as a background for the more detailed reviews that follow. Most of our current understanding of early development comes from work on a small number of species whose characteristics make them especially suitable for analysis by embryological, biochemical, or genetic methods. Although the morphology and timing of development is quite different in these organisms (Figure l), their regulatory genes and fundamental mechanisms of development seem broadly similar (Akam, 1989; McGinnis and Krumlauf, 1992 [this issue of Cell]). The major question addressed in this article is how the fertilized egg-a single cell bearing no resemblance to the organism it eventually forms-can generate the diversity of cell types and complexity of pattern seen only a few days later in an embryo or larva. The first section considers when the main axes of the embryo are laid down. Are they already fixed in the undivided egg, or do they appear as Review

Eomesodermin, a Key Early Gene in Xenopus Mesoderm Differentiation

Eomesodermin (Eomes) is a novel Xenopus T-domain gene. In normal development, it is expressed in mesodermal cells in a ventral-to-dorsal gradient of increasing concentration. It reaches its peak expression 1-2 hr before any other known panmesodermal gene. It is strongly inducible by normal vegetal cells and by mesoderm-inducing factors. Ectopic expression of Eomes in animal caps induces the transcription of nearly all mesodermal genes in a concentration-dependent way. Overexpression of Eomes dorsalizes ventral mesoderm, inducing gsc and changing cell fate to muscle and notochord. Blocking the function of Eomes causes gastrulation arrest and defective mesoderm-dependent gene activation. We propose that Eomes fulfills an essential function in initiating mesoderm differentiation and in determining mesodermal cell fate.

Nuclei of adult mammalian somatic cells are directly reprogrammed to oct-4 stem cell gene expression by amphibian oocytes

Nuclear reprogramming by the transplantation of somatic cell nuclei to eggs (in second meiotic metaphase) is always followed by a phase of chromosome replication and cell division before new gene expression is seen. To help understand the mechanism of nuclear reprogramming, we have asked whether the nuclei of normal, nontransformed, nondividing, and terminally differentiated mammalian cells can be directly reprogrammed, without DNA replication, by Xenopus oocytes. We find that nuclei of adult mouse thymocytes and of adult human blood lymphocytes, injected into Xenopus oocytes, are induced to extinguish a differentiation marker and to strongly express oct-4, the most diagnostic mammalian stem cell/pluripotency marker. In the course of 2 days at 18 degrees C, the mammalian oct-4 transcripts are spliced to mature mRNA. We conclude that normal mammalian nuclei can be directly reprogrammed by the nucleus (germinal vesicle) of amphibian oocytes to express oct-4 at a rate comparable to that of oct-4 in mouse ES cells. To our knowledge, this is the first demonstration of a stem cell marker being induced in a differentiated adult human cell nucleus. This is an early step toward the long-term aim of developing a procedure for reprogramming readily accessible human adult cells for cell replacement therapy.

A homeobox-containing marker of posterior neural differentiation shows the importance of predetermination in neural induction

A homeobox sequence has been used to isolate a new Xenopus cDNA, named XIHbox6. A short probe from this gene serves as an early marker of posterior neural differentiation in the Xenopus nervous system. The gene recognized by this cDNA sequence is first transcribed at the late gastrula stage and solely in the posterior neural cells. The gene is expressed when ectodermal and mesodermal tissues of an early gastrula are placed in contact, but not by either tissue cultured on its own. However, gene expression is most easily inducible in ectoderm from the dorsal region, i.e., in ectoderm normally destined to form neural structures. This establishes the principle, in contrast to previous belief, that the induction of the embryonic nervous system involves a predisposition of the ectoderm and does not depend entirely on an interaction with inducing mesoderm.