The generation of diversity and pattern in animal development.
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Biomedical subjects
J B Gurdon
Publications and source records attributed to J B Gurdon.
The DNA-binding protein E12 co-operates with XMyoD in the activation of muscle-specific gene expression in Xenopus embryos.
Two alternatively spliced products of the human E2A gene, E12 and E47, encode helix-loop-helix DNA-binding proteins. Here we describe the isolation of two Xenopus cDNAs; one, XE12, is structurally similar to human E12 and the other contains a sequence similar to E47. Transcripts of both cDNAs are present at all the stages of Xenopus development tested and in all regions of the embryo. The DNA binding properties of in vitro translated XE12 are indistinguishable from those of human E12. We have shown previously that an embryonic muscle DNA-binding activity, EMF1, that binds to a promoter sequence required for the expression of the cardiac actin gene, contains the Xenopus myogenic factor XMyoD. Here we show that it also contains protein that interacts with an anti-E12 antiserum, suggesting that XE12 and XMyoD proteins, or very similar ones, are present in EMF1. We have addressed the functional role of XE12 in muscle gene transcription in Xenopus embryos by injecting in vitro synthesized RNA into the two cell embryo. Overexpression of XE12 and XMyoD augments by greater than 10-fold the ectopic activation of the endogenous cardiac actin gene that can be produced by XMyoD alone. Our DNA binding results strongly suggest that this effect is mediated through a direct interaction of the XE12-XMyoD complex with specific sites in the cardiac actin promoter. We suggest that XE12 is functionally important in muscle gene activation in embryonic development.
Amino Acid Sequence↗
Expression of XMyoD protein in early Xenopus laevis embryos.
A monoclonal antibody specific for Xenopus MyoD (XMyoD) has been characterized and used to describe the pattern of expression of this myogenic factor in early frog development. The antibody recognizes an epitope close to the N terminus of the products of both XMyoD genes, but does not bind XMyf5 or XMRF4, the other two myogenic factors that have been described in Xenopus. It reacts in embryo extracts only with XMyoD, which is extensively phosphorylated in the embryo. The distribution of XMyoD protein, seen in sections and whole-mounts, and by immunoblotting, closely follows that of XMyoD mRNA. XMyoD protein accumulates in nuclei of the future somitic mesoderm from the middle of gastrulation. In neurulae and tailbud embryos it is expressed specifically in the myotomal cells of the somites. XMyoD is in the nucleus of apparently every cell in the myotomes. It accumulates first in the anterior somitic mesoderm, and its concentration then declines in anterior somites from the tailbud stage onwards.
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Muscle gene activation in Xenopus requires intercellular communication during gastrula as well as blastula stages.
In Xenopus an early morphological marker of mesodermal induction is the elongation of the mesoderm at the early gastrula stage (Symes and Smith, 1987). We show here that the elongation of equatorial (marginal) tissue is dependent on protein synthesis in a mid blastula, but has become independent of it by the late blastula stage. In animal caps induced to become mesoderm, the time when protein synthesis is required for subsequent elongation immediately follows the time of induction, and is not related to developmental stage. For elongation, intercellular communication during the blastula stage is of primary importance. Current experiments involving cell transplantation indicate a need for further cell:cell interactions during gastrulation, and therefore after the vegetal-animal induction during blastula stages. These secondary cell interactions are believed to take place among cells that have already received a vegetal induction, and may facilitate some of the later intracellular events known to accompany muscle gene activation.
Animals↗
Mechanisms of gene activation in early vertebrate development.
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Nuclear transplantation in Xenopus.
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Xenopus embryos contain a somite-specific, MyoD-like protein that binds to a promoter site required for muscle actin expression.
We identify the "M region" of the muscle-specific Xenopus cardiac actin gene promoter from -282 to -348 as necessary for the embryonic expression of a cardiac actin-beta-globin reporter gene injected into fertilized eggs. Four DNA-binding activities in embryo extracts, embryonic M-region factors 1-4 (EMF1-4), are described that interact specifically with this region. One of these, EMF1, is detected in extracts from microdissected somites, which differentiate into muscle, but not in extracts from the adjacent neurectoderm, which differentiates into a variety of other cell types. Moreover, EMF1 is detected in embryo animal caps induced to form mesoderm, which includes muscle, and in which the cardiac actin gene is activated, but not in uninduced animal caps. EMF1 is also first detectable when cardiac actin transcripts begin to accumulate; therefore, both its temporal and spatial distributions during Xenopus development are consistent with a role in activating cardiac actin expression. Two lines of evidence suggest that EMF1 contains the myogenic factor Xenopus MyoD (XMyoD): (1) XMyoD synthesized in vitro can bind specifically to the same site as EMF1; and (2) antibodies raised against XMyoD bind to EMF1. DNA-binding studies indicate that EMF1 may be a complex between XMyoD and proteins found in muscle and other tissues. Our results suggest that the myogenic factor XMyoD, as a component of somite EMF1, regulates the activation of the cardiac actin gene in developing embryonic muscle by binding directly to a necessary region of the promoter.
Actins↗
Xenopus Myf-5 marks early muscle cells and can activate muscle genes ectopically in early embryos.
We have cloned a Xenopus cDNA that encodes a homologue of the human myogenic factor, Myf-5. Xenopus Myf-5 (XMyf5) transcripts first accumulate in the prospective somite region of early gastrulae. The pattern of XMyf5 expression is similar to that of the Xenopus MyoD (XMyoD) gene, except that XMyf5 transcripts are largely restricted to posterior somitic mesoderm even before any somites have formed. Transient ectopic expression of XMyf5 activates cardiac actin and XMyoD genes in animal cap cells, but does not cause full myogenesis, even in combination with XMyoD. These results suggest that XMyf5 acts together with XMyoD as one of the set of genes regulating the earliest events of myogenesis, additional factors being required for complete muscle differentiation.
Amino Acid Sequence↗
Gene activation in the amphibian mesoderm.
Cell potency is progressively restricted in amphibian development by a series of cellular interactions called inductions. The mesoderm is believed to develop in response to the earliest known induction, in which vegetal cells of the blastula divert overlying animal hemisphere cells away from epidermal and towards mesodermal fates. We describe two early markers of mesodermal differentiation in Xenopus laevis, both mRNAs that encode DNA-binding proteins of the helix-loop-helix family. One is a frog homologue of MyoD, a gene that in transfection experiments can convert cultured fibroblasts into myoblasts. Xenopus MyoD (XMyoD) is expressed in the early myotomes, from which the axial musculature develops. The accumulation of XMyoD RNA precedes that of transcripts from the cardiac actin gene, until now the earliest known marker of the muscle lineage, this result indicating that XMyoD could play a role in initiating muscle differentiation in normal development. We show by microinjection of synthetic RNA that XMyoD can indeed activate muscle-specific gene expression in animal cap cells, which would normally form only ectoderm. However, the XMyoD-injected animal caps did not produce differentiated muscle, suggesting that additional specific factors are required for full myogenesis. The other mRNA is a relative of the twist gene of Drosophila, which is required for mesodermal differentiation in flies. This gene (Xtwi) is expressed widely in the early frog mesoderm, but not, however, in the myotomes, where XMyoD is expressed. Later, the Xtwi gene is activated, in response to a second induction, in the developing neural crest.
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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↗
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.
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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↗
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↗
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↗
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↗
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↗
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.
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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.
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