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.

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.

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.

Expression of a mRNA related to c-rel and dorsal in early Xenopus laevis embryos.

We describe a Xenopus mRNA, Xrel1, that is related to the avian protooncogene c-rel, the embryonic pattern gene dorsal of Drosophila, and the mammalian transcription factor NK-kappa B/KBF1. The sequence of Xrel1 is homologous to the other rel-related proteins in the large amino-terminal region that defines this class of transcriptional regulators, but the carboxyl-terminal part of the protein is quite different. Xrel1 mRNA is present throughout oogenesis and during early embryogenesis at 4 x 10(5) transcripts per oocyte or embryo. Xrel1 transcripts are present in all of the dissected parts of early embryos that we have examined. They are enriched in the animal hemisphere compared to the vegetal hemisphere of oocytes and blastulae.

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.

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.

Cellular and genetic responses to mesoderm induction in Xenopus.

Mesodermal cell differentiation begins in response to an inductive interaction early in frog development. In parallel with the recent finding that certain peptide growth factors can induce mesoderm, early cellular and genetic responses to the induction have been discovered. I review here recent work on these responses, work that aims to understand how cells respond to inducers to form the complex pattern of the vertebrate mesoderm.

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.

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.

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.