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J B Gurdon

Publications and source records attributed to J B Gurdon.

At least 19 recordsLinked to original sources
PubMed2006

Two-dimensional morphogen gradient in Xenopus: boundary formation and real-time transduction response.

Morphogen gradients play an important role in pattern formation in embryo. However, the interpretation of position in a morphogen gradient is not well understood. Because it is hard to analyze morphogen gradients especially in opaque embryos such as those of Xenopus, it is necessary to fix and section the embryo, thereby eliminating the possibility of real-time observation, and making more difficult the interpretation of events that take place in three dimensions. We describe here a two-dimensional preparation of cells from a Xenopus blastula animal cap, in which an activin concentration gradient appears to be formed and interpreted at the same rate and in the same way as in normal embryos. We use two-dimensional preparations of this kind to contribute the following new information about gradient formation and interpretation in embryo. We determine the dynamics of formation of an activin activity gradient in real time. We demonstrate that this gradient is established by diffusion of activin through intercellular space and does not require internalization of receptor or ligand. We also show that the generation of a boundary of gene expression depends on the interpretation, rather than a change of composition, of the concentration gradient.

Animals↗
PubMed2006

From nuclear transfer to nuclear reprogramming: the reversal of cell differentiation.

This is a personal historical account of events leading from the earliest success in vertebrate nuclear transfer to the current hope that nuclear reprogramming may facilitate cell replacement therapy. Early morphological evidence in Amphibia for the toti- or multipotentiality of some nuclei from differentiated cells first established the principle of the conservation of the genome during cell differentiation. Molecular markers show that many somatic cell nuclei are reprogrammed to an embryonic pattern of gene expression soon after nuclear transplantation to eggs. The germinal vesicles of oocytes in first meiotic prophase have a direct reprogramming activity on mammalian as well as amphibian nuclei and offer a route to identify nuclear reprogramming molecules. Amphibian eggs and oocytes have a truly remarkable ability to transcribe genes as DNA or nuclei, to translate mRNA, and to modify or localize proteins injected into them. The development of nuclear transplant embryos depends on the ability of cells to interpret small concentration changes of signal factors in the community effect and in morphogen gradients. Many difficulties in a career can be overcome by analyzing in increasing depth the same fundamentally interesting and important problem.

Animals↗
PubMed2005

Sinistral snails and gentlemen scientists.

A single gene mutation can cause a complete left-to-right and right-to-left inversion of the body axis. This mutation exemplifies a large class of so-called maternal effect genes that regulate key events during early development. This principle was first established nearly one hundred years ago, principally by the work of amateur naturalists.

Animals↗
PubMed2005

Maintenance of epigenetic memory in cloned embryos.

Different cell types have characteristic patterns of gene expression. Once a cell has differentiated, its daughter cells nearly always differentiate in the same way. The maintenance of cell lineage involves either instructions from a cell's surroundings or the inheritance of memory from a parent cell. In normal development, the differentiation state of a cell is remarkably stable and irreversible. However the transplantation of a somatic cell nucleus to an enucleated egg often leads to a complete reprogramming of gene expression. We summarize here the results of some Amphibian nuclear transfer experiments that reveal a memory of gene expression. This and some other experiments exemplify epigenetic memory that persists through many cell divisions. In the case of nuclear transfer experiments, the actively transcribed state of a gene can be propagated through many cell divisions in the absence of the stimulus that first induced the activity of this gene. We discuss the possible basis of these two examples of persistent epigenetic memory, namely changes at DNA methylation, and histone modifications.

Animals↗
PubMed2005

Changing cell fate by nuclear reprogramming.

The differentiated state of somatic cells is remarkably stable, but nuclear transfer can efficiently override the stability of cell differentiation by nuclear reprogramming. Genes that were silent in the differentiated state are activated, whereas genes specific for the differentiated state are switched off during the reprogramming process. The epigenetic changes that occur after nuclear transfer to Xenopus oocytes involve chromatin remodelling and DNA demethylation. In particular, we have reported that reactivation of the mouse stem cell specific gene oct-4 depends on demethylation of CpG:s in the proximal oct-4 promoter. Here we discuss molecular mechanisms of nuclear reprogramming, with special emphasis on DNA demethylation.

Animals↗
PubMed2005

Nuclear reprogramming by xenopus oocytes.

The aim of this contribution is to relate amphibian nuclear transplantation work to prospects for stem cell creation and hence to the long-term aim of cell replacement in humans. The methods used include the transplantation of single somatic cell nuclei to enudeated unfertilized eggs of Xenopus, and also the transfer of multiple somatic cell nuclei to the nucleus (germinal vesicle) of a growing ovarian oocyte. A key difference between these types of recipient cell is that eggs immediately induce DNA replication in transplanted nuclei, whereas an oocyte induces no DNA replication, but directly reprograms an injected nucleus to a new pattern of transcriptional activity. We summarize the extent and success of past and current nuclear reprogramming in experiments with enucleated frog eggs, and also those carried out with growing oocytes. We discuss possible mechanisms of nuclear reprogramming, and the possible contribution of such knowledge for stem cell creation and cell replacement in humans.

Animals↗
PubMed2005

The use of Xenopus oocytes and embryos as a route towards cell replacement.

When nuclei of somatic cells are transplanted to enucleated eggs of Xenopus, a complete reprogramming of nuclear function can take place. To identify mechanisms of nuclear reprogramming, somatic nuclei can be transplanted to growing meiotic oocytes of Xenopus, and stem cell genes activated without DNA replication. The combination of somatic cell nuclear transfer with morphogen signalling and the community effect may lead towards the possibility of cell replacement therapy. When mechanisms of nuclear reprogramming are understood, it may eventually be possible to directly reprogramme human somatic cell nuclei without the use of eggs.

Animals↗
PubMed2005

Reproductive cloning: past, present and future.

This brief outline in reproductive cloning describes the background to these studies and then discusses successive aspects of the subject. These include abnormalities in cloned animals, therapeutic cloning and the ethics of this subject. A reference to further reading is provided.

Animals↗
PubMed2004

Many ways to make a gradient.

A recent publication describes a novel mechanism by which a morphogen gradient might be established. These results concern a gradient of FGF8 expression along the longitudinal axis of the chick embryo with a high level of transcripts at the tail, fading off in an anterior direction. Assaying for intron transcripts, it is shown that fgf8 is transcribed only in the tail cells and that the gradient of fgf8 transcripts is produced by growth and mRNA degradation. This possible mechanism of gradient formation can operate only when growth is involved, as is the case in many examples including the longitudinal axis formation of vertebrates, but is not in some other systems.

Animals↗
PubMed2003

Nuclear reprogramming and stem cell creation.

The transplantation of a somatic cell nucleus to an enucleated egg results in a major reprogramming of gene expression and switch in cell fate. We review the efficiency of nuclear reprogramming by nuclear transfer. The serial transplantation of nuclei from defective first-transfer embryos and the grafting of cells from such embryos to normal host embryos greatly increases the proportion of nuclei that can be seen to have been reprogrammed. We discuss possible reasons for the early failure of most nuclear transfers from differentiated cells and describe the potential value of growing oocytes, rather than unfertilized eggs, as a source of nuclear reprogramming molecules and for the eventual identification of these molecules. Nuclear transfer provides a possible route for the creation of stem cells from adult somatic cells.

Animals↗
PubMed2003

The first half-century of nuclear transplantation.

Fifty years after Briggs and King first succeeded in obtaining normal tadpoles from transplanted embryo nuclei in vertebrates, two general principles have emerged from work in amphibia and mammals. One is the conservation of the genome during cell differentiation. A small percentage of adult or differentiated cells have totipotent nuclei, and a much higher percentage of cells committed to one pathway of cell differentiation have multipotent nuclei. The other is the remarkable reprogramming capacity of cell, and especially egg, cytoplasm. The eventual identification of reprogramming molecules and mechanisms could facilitate a route toward cell replacement therapy in humans.

Amphibians↗
PubMed2002

From intestine to muscle: nuclear reprogramming through defective cloned embryos.

Nuclear transplantation is one of the very few ways by which the genetic content and capacity for nuclear reprogramming can be assessed in individual cells of differentiated somatic tissues. No more than 6% of the cells of differentiated tissues have thus far been shown to have nuclei that can be reprogrammed to elicit the formation of unrelated cell types. In Amphibia, about 25% of such nuclear transfers form morphologically abnormal partial blastulae that die within 24 h. We have investigated the genetic content and capacity for reprogramming of those nuclei that generate partial blastulae, using as donors the intestinal epithelium cells of feeding Xenopus larvae. We have analyzed single nuclear transplant embryos obtained directly from intestinal tissue, thereby avoiding any genetic or epigenetic changes that might accumulate during cell culture. The expression of the intestine-specific gene intestinal fatty acid binding protein is extinguished by at least 10(4) times, within a few hours of nuclear transplantation. At the same time several genes that are normally expressed only in early embryos are very strongly activated in nuclear transplant embryos, but to an unregulated extent. Remarkably, cells from intestine-derived partial blastulae, when grafted to normal host embryos, contribute to several host tissues and participate in the normal 100-fold increase in axial muscle over several months. Thus, cells of defective cloned embryos unable to survive for more than 1 day can be reprogrammed to participate in new directions of differentiation and to maintain indefinite growth, despite the abnormal expression of early genes.

Animals↗
PubMed2002

Regulatory principles of developmental signaling.

Signaling between cells is a widely used mechanism by which cell fate and tissue patterning is determined in development. We review the mechanisms by which signaling between cells is regulated so that a cell receives the right amount of signal, at the right time, to achieve its intended developmental fate and position. In nearly all cases, we find that the supply of signal factor (ligand) is the limiting step in initiating a signaling process. Ligand supply is regulated by the transcription and localization of RNA, the spread of ligand from a source, and by inhibitors that operate at several different levels. We emphasize the different regulatory strategies that operate for threshold as opposed to concentration-dependent (morphogen) signaling. Threshold signaling is extensively regulated by feedback mechanisms. Morphogen signaling is regulated quantitatively by receptor loading and transduction flow.

Animals↗
PubMed2002

Nuclear exclusion of Smad2 is a mechanism leading to loss of competence.

Controlling the duration of a signalling process in development by loss of competence is important because too strong an induction can change cell fate. To understand some of the mechanisms that underlie loss of competence, we have analysed the transduction of transforming growth factor-beta (TGF-beta) signalling during mesoderm formation, which is thought to be induced by TGF-beta-like signalling, in embryos of the frog Xenopus laevis. Here we show that gastrula ectoderm has the ability to express mesodermal marker genes in response to the TGF-beta signalling molecule activin for many hours, but then loses this ability within 1 h for all mesodermal genes tested. This loss of mesodermal competence correlates with the inability of Smad2, the principal intracellular signal transducer of activin, to accumulate in the nucleus. Mutating three phosphorylation sites within Smad2 abrogates the temporal restriction of Smad2 to accumulate in the nucleus. Overexpression of this mutant form of Smad2 can prolong the competence of endogenous mesodermal genes to respond to activin signalling. Thus, restricting the subcellular localization of an intracellular signal transducer constitutes a mechanism that leads to loss of mesodermal competence. This mechanism operates within less than an hour, and is therefore well suited to control an orderly sequence of inductions.

Active Transport, Cell Nucleus↗
PubMed2002

A changing morphogen gradient is interpreted by continuous transduction flow.

In vertebrate development, most signalling factors behave as morphogens, eliciting divergent cell fates according to their concentration. We ask how cells interpret morphogen concentration as it changes during the establishment of a gradient. Using dissociated blastula cells of Xenopus exposed to activin for only 10 minutes, we have followed the phosphorylation of tagged Smad2, the principal activin transducer, from a cytoplasmic pool to the nucleus in real time. We show that a changing concentration of extracellular activin is rapidly and continuously transduced to provide a corresponding nuclear concentration of Smad2, even though gene response may be delayed for several hours. Nuclear Smad2 concentration changes up as the extracellular concentration of activin increases. We conclude that cells interpret a changing extracellular concentration by maintaining a continuous flow of activated transducer from a large cytoplasmic pool to the nucleus where it is degraded. The volume of this flow determines the steady state concentration of Smad2 in the nucleus and this is used by cells to interpret extracellular morphogen concentration.

Activins↗
PubMed2001

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.

Animals↗
PubMed2001

Nonradioactive in situ hybridization to xenopus tissue sections.

We describe a protocol for the localization of specific messenger RNAs in Xenopus laevis embryo tissue sections using a nonradioactive detection method. After fixation, embryos are embedded in paraffin wax, sectioned, mounted on slides, and subjected to a series of prehybridization treatments which improve the accessibility of the probe to the target mRNA and reduce nonspecific binding. These treatments are followed by hybridization in situ with single-stranded antisense RNA probe generated by in vitro transcription and labeled with digoxigenin. The hybridization products are detected with preabsorbed alkaline phosphatase-coupled digoxigenin antibody and subsequently localized using a chromogenic substrate that generates a colored precipitate at the hybridization site. The nonradioactive in situ hybridization method we describe is reproducible and has a detection sensitivity akin to those methods that use antisense RNA probes labeled with radioisotopes; however, it is faster, safer, and easier to perform. Sectioning of prestained whole-mount X. laevis embryos does not always show the complete expression pattern of many genes, particularly those in deep endodermal structures, due to inadequate probe penetration. Therefore thorough analysis of gene expression patterns often requires in situ hybridization on presectioned material whereby probe has equal accessibility to all tissue.

Animals↗