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J B Gurdon

Publications and source records attributed to J B Gurdon.

At least 91 records · Page 5Linked to original sources
PubMed1990

Activation of muscle genes without myogenesis by ectopic expression of MyoD in frog embryo cells.

The sequence-specific DNA-binding protein, MyoD, can activate muscle-specific gene expression in some cells in culture. Xenopus MyoD (XMyoD) transcription is activated as a consequence of mesoderm induction in the early myotomes, from which the axial musculature develops. XMyoD RNA accumulates about two hours before muscle-specific actin transcripts first appear, and so is expressed at the right time and in the right place to play a part in activating muscle-specific gene expression in normal development. To test this idea, we have expressed XMyoD ectopically in early Xenopus embryos. We find that injection of XMyoD RNA can strongly activate muscle genes in embryo cells normally destined to form ectoderm. Nevertheless, these cells fail to differentiate as muscle, suggesting that additional factors are required for complete and stable myogenesis.

Amino Acid Sequence↗
PubMed1990

The induction of anterior and posterior neural genes in Xenopus laevis.

We have investigated the interactions between mesoderm and ectoderm that result in the formation of a regionally differentiated nervous system in Xenopus embryos. We have used genes expressed at different positions along the neural tube as regional markers of neural induction in both whole, and in experimentally manipulated embryos. By comparing transcription from the anterior marker, XIF3, with that from the posterior marker, X1Hbox6, and the general neural marker XIF6, we have shown that the normal induction process requires interactions between ectoderm and mesoderm that persist through gastrulation into the late neurula stages. We have found that competence of the ectoderm to respond to induction is lost at the same early neurula stage for all three marker genes. Using rhodamine dextran-labelled mesoderm, we have established that the duration of contact between ectoderm and mesoderm required for gene activation in conjugates is the same for each of the markers. We have, however, identified regions of the mesoderm that can induce different combinations of neural marker gene expression. The anterior mesoderm induces expression of the anterior marker, XIF3, and the later migrating posterior mesoderm induces the ectoderm overlying it to express the posterior marker X1Hbox6. It has been proposed that neural inducing signals reach the ectoderm by two different routes: from mesoderm lying directly beneath the ectoderm or along the plane of the ectoderm. We have assessed the contribution of each route in respect of our three neural markers and find that a signal passing directly from mesoderm to ectoderm fully accounts for neural gene expression. We were unable to detect an inducing signal that passes along the plane of the ectoderm.

Animals↗
PubMed1989

A Xenopus mRNA related to Drosophila twist is expressed in response to induction in the mesoderm and the neural crest.

We have cloned a Xenopus cDNA related to the twist gene, which is required for mesodermal differentiation in Drosophila. Northern blots of dissected embryos and in situ hybridization show that the corresponding mRNA, called Xtwi, first appears in early gastrulae, and is present only in mesodermal cells. Within the mesoderm, Xtwi is expressed in the notochord and lateral plate, but not in the myotome; therefore there is a complementary pattern of Xtwi and muscle-specific gene expression in the mesoderm. Xtwi expression therefore marks the subdivision of the mesoderm. Xtwi is also activated a few hours later in the early development of the neural crest. This gene is thus expressed in response to two sequential early inductions in frog development.

Amino Acid Sequence↗
PubMed1989

The Florey lecture, 1988. From egg to embryo: the initiation of cell differentiation in Amphibia.

Some of the principles by which different cell types first arise at the beginning of animal development are illustrated by muscle cell formation in Amphibia. If the nucleus of a differentiated muscle cell is transplanted to an enucleated egg, some of the resulting embryos develop into tadpoles with a wide range of normally differentiated cells. These experiments show that genes undergo major changes in activity as a response to components of egg cytoplasm. Two fundamental mechanisms account for the regional activation of genes in early embryos. One involves the effect of localized 'determinants' in egg cytoplasm, and the other concerns cell interactions or embryonic induction. Both these mechanisms seem to be responsible for muscle cell formation in amphibian development. The old problem of embryonic induction has recently become accessible to analysis at the molecular level, especially in the case of the mesoderm or muscle-forming induction. This has been greatly facilitated by using a sensitive and quantitative assay to detect the first transcripts of muscle genes a few hours after the start of induction. The role of early events and of interactions among like cells during response to induction is discussed. In analysing specific gene activation following induction, DNA injection into fertilized eggs has shown that a very small part of the cardiac actin gene promoter is sufficient to enable it to respond to induction. Although the experimental work summarized here has been done on amphibian embryos, which are more suitable than other embryos for embryological manipulation, the conclusions reached are believed to be generally applicable to the development of other organisms.

Amphibians↗
PubMed1989

The CArG promoter sequence is necessary for muscle-specific transcription of the cardiac actin gene in Xenopus embryos.

Promoter sequences required for activation of the Xenopus cardiac actin gene in embryonic muscle were analysed by micro-injecting chimeric actin/beta-globin genes into the two-cell Xenopus embryo. Transcription was monitored during subsequent differentiation of embryonic muscle and non-muscle tissues. The effect of a variety of mutations including internal deletions and linker scan mutations between -64 and -396 within the cardiac actin promoter were tested. This region contains four copies of a conserved motif, the CArG box, common to vertebrate striated muscle acting gene promoters. In the Xenopus cardiac actin gene, the most proximal of these motifs (CArG box 1) located at -80, was essential for muscle-specific transcription. Other CArG motifs could functionally substitute for CArG box 1 when placed in this position. CArG boxes 3 and 4 bound the same activity in a neurula embryo nuclear extract as CArG box 1 and the amount of this binding activity was constant through early development.

Actins↗
PubMed1989

MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos.

We describe the cloning, cDNA sequence and embryonic expression of a Xenopus homologue of MyoD, a mouse gene encoding a DNA-binding protein that can activate muscle gene expression in cultured cells. The predicted Xenopus MyoD protein sequence is remarkably similar to mouse MyoD. Zygotic expression of MyoD begins in early gastrulae, but there is a low level of unlocalized maternal message. Northern blot analysis of dissected embryos and in situ hybridization show that MyoD RNA is restricted to the gastrula mesoderm and to the somites of neurulae and tailbud embryos. The time and place of MyoD expression are consistent with a role for MyoD in the activation of other muscle genes in the somites of the frog embryo. However, MyoD is skeletal muscle-specific and is not expressed even in the early embryonic heart, which co-expresses cardiac and skeletal actin isoforms. Striated muscle genes can therefore be activated in some embryonic tissues in the absence of MyoD. The concentration of MyoD in the somites falls once they have formed, suggesting that MyoD may act there transiently to establish muscle gene expression. MyoD transcription is activated following mesoderm induction, and is the earliest muscle-specific response to mesoderm-inducing factors so far described.

Amino Acid Sequence↗
PubMed1989

Embryonic induction and muscle gene activation.

Embryonic induction, a process in which the differentiation of a cell is determined by its proximity to other kinds of cells, is of major importance in animal development. We review here what is known of the steps by which a muscle-specific actin gene is first activated by embryonic induction in early amphibian embryos.

Actins↗
PubMed1989

Loss of competence in amphibian induction can take place in single nondividing cells.

The ability of ectodermal tissue to be induced to form mesoderm is lost during gastrula stages in Xenopus embryos. We have examined the extent to which this loss of competence depends on intercellular interactions, cell division, or protein synthesis. We find that ectoderm, when separated from a whole embryo as soon as the early blastula stage, and even when dissociated into its component cells, loses its competence at the normal time. When cell division was arrested by culturing isolated cells in solid medium, the time of competence loss was unaffected. To test whether protein synthesis is required for competence loss, ectoderm was treated with cycloheximide during the normal time that competence is lost; in some cases, this treatment had no effect and in others it prolonged competence, but only slightly. We conclude that the loss of mesodermal competence is a highly autonomous process in ectodermal cells, taking place in the absence of cell communication or cell division.

Animals↗
PubMed1989

The localization of an inductive response.

Combinations of tissues from Xenopus blastulae have been used to identify several mechanisms that limit the number of animal cells forming muscle after induction by vegetal cells. The results disagree with a model in which direct physical contact or very close proximity between animal and vegetal cells restricts the number of cells that receive the inductive signal. Rather it seems that a diffusible inducer is released by vegetal cells, and spreads through 4-8 animal cell diameters, equivalent to a distance of 80 microns, from the nearest vegetal cells. Several factors seem to cooperate to prevent the further spread of the mesoderm-forming induction. These include the slow diffusion and/or instability of the inducer, the time of loss of competence of animal cells to respond to induction, and the amount of vegetal tissue that releases inducer for a limited time. The combination of these, and perhaps other, processes seems to ensure that a consistent minority of animal cells are induced to form muscle, thereby leaving other animal cells available to form the nervous system and epidermis.

Animals↗
PubMed1989

XIF3, a Xenopus peripherin gene, requires an inductive signal for enhanced expression in anterior neural tissue.

A full-length cDNA clone for the Xenopus intermediate filament gene XIF3 has been isolated. It is very similar in sequence to the rat intermediate filament cDNA clone 73 that is thought to encode the neuronal intermediate filament protein 'peripherin'. By analysing dissected embryos, we show that XIF3 is expressed predominantly in anterior and dorsal structures and most strongly in the brain of the tailbud (stage 26) embryo. In situ hybridization shows XIF3 transcripts to be localized in neural tissue and especially in regions that most probably correspond to the motor neurones of the neural tube and to some cranial nerve ganglia. New XIF3 transcripts are first found at the start of gastrulation at a low level throughout the ectoderm and are not localized to the presumptive neurectoderm. Expression subsequently increases by about 10-fold in neural tissue, and requires an interaction of the mesoderm with overlying ectoderm. Because new transcripts are found predominantly in neural tissue of the head, this response can be used as a marker of anterior neural induction.

Amino Acid Sequence↗
PubMed1988

The origin of cell-type differences in early embryos.

Differences between cells first arise in embryonic development by two principal mechanisms. One is the asymmetric distribution of cytoplasmic substances at mitosis in eggs and early embryos. The other is cell interaction or embryonic induction. Certain aspects of these major mechanisms are considered, and emphasis is placed on the value of molecular markers. The effects of unequal cell division on the concentration of cytoplasmic determinants are discussed. In embryonic induction, the nature and timing of response is determined more by properties of the responding tissue than by those of the inducing molecules. Possible future directions of work are discussed in relation to experience with amphibian eggs and oocytes.

Animals↗
PubMed1987

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.

Animals↗
PubMed1987

Functional gap junctions are not required for muscle gene activation by induction in Xenopus embryos.

Muscle gene expression is known to be induced in animal pole cells of a Xenopus blastula after 2-3 h of close contact with vegetal pole cells. We tested whether this induction requires functional gap junctions between vegetal and animal portions of an animal-vegetal conjugate. Muscle gene transcription was assayed with a muscle-specific actin gene probe and the presence or absence of communication through gap junctions was determined electrophysiologically. Antibodies to gap junction protein were shown to block gap junction communication for the whole of the induction time, but did not prevent successful induction of muscle gene activation. The outcome was the same whether communication between inducing vegetal cells and responding animal cells was blocked by introducing antibodies into vegetal cells alone or into animal cells alone. We conclude that gap junctions are not required for this example of embryonic induction.

Animals↗
PubMed1987

The initiation of new gene transcription during Xenopus gastrulation requires immediately preceding protein synthesis.

The incubation of Xenopus embryo fragments in cycloheximide at 5 or 10 micrograms ml-1 rapidly inhibits protein synthesis to 10% or less of control levels. In most batches of embryos, treatment with cycloheximide for up to 1 h causes no obvious cellular damage and protein synthesis is fully restored to normal levels 5 h later. Transcript analysis with RNA probes shows that the inhibition of protein synthesis at late blastula or early gastrula stages completely suppresses the normal initiation of actin gene transcription at the mid-late gastrula stage. This applies to muscle-specific actin genes, whose transcription is initiated by induction, as well as to cytoskeletal actin genes not activated by induction. Two-dimensional gel protein analysis shows that cycloheximide irreversibly inhibits only 10% of all genes normally expressed at a postneurula stage and that all of these are genes whose expression is normally initiated during or soon after gastrulation. Cycloheximide treatment causes a limited reduction of DNA synthesis, and no reduction of overall RNA synthesis. We conclude that the initiation of new gene transcription during gastrulation in Xenopus is dependent on the immediately preceding synthesis of certain proteins.

Actins↗
PubMed1986

Upstream sequences required for tissue-specific activation of the cardiac actin gene in Xenopus laevis embryos.

The entire DNA sequence of the Xenopus laevis cardiac actin gene was determined. A recombinant plasmid comprising the cardiac actin gene promoter fused to the bacterial chloramphenicol acetyl transferase (CAT) gene is correctly regulated after introduction into fertilized Xenopus eggs. The fusion gene shows a temporal and tissue-specific pattern of expression in the early embryo which is indistinguishable from that of the endogenous cardiac actin gene. The fusion gene is also activated in cultured embryo fragments that are induced by cell interactions to form embryonic muscle tissue. Tissue-specific expression of the recombinant requires sequences between 217 and 416 nucleotides upstream from the transcription initiation site. In contrast, both the chimaeric gene and the entire cardiac actin gene are expressed at a basal level after microinjection into Xenopus oocytes, requiring only the presence of a TATA box upstream from the cap site.

Actins↗
PubMed1986

Muscle gene activation by induction and the nonrequirement for cell division.

We are investigating the mechanism by which animal cells of an amphibian blastula are induced to differentiate as muscle after contact with blastula vegetal cells. After briefly summarizing previous work on this system, we have asked whether this response of animal cells to vegetal induction requires cell division. Animal and vegetal parts of a blastula were placed in contact with each other, and the resulting conjugates cultured in medium containing a sufficient concentration of colchicine or cytochalasin B to inhibit cell division. Muscle differentiation, as indicated by cardiac actin gene transcription, is induced when cell division is inhibited, though at a substantially reduced rate. However, cytoskeletal actin gene transcription, which does not depend on induction, is also much reduced under the same inhibitory conditions. We conclude that, although the cell division inhibitors seem to reduce all gene transcription, they have no preferential effect on the response to induction, and therefore that this process does not require cytoplasmic or nuclear division.

Actins↗