Synergistic induction of mesoderm by FGF and TGF-beta and the identification of an mRNA coding for FGF in the early Xenopus embryo

The primary patterning event in early vertebrate development is the formation of the mesoderm and its subsequent induction of the neural tube. Classic experiments suggest that the vegetal region signals the animal hemisphere to diverge from the pathway of forming ectoderm to form mesoderm such as muscle. Here we show that bovine basic FGF has a limited capacity to induce muscle actin expression in animal hemisphere cells. This level of expression can be raised to levels normally induced in the embryo by another mammalian growth factor, TGF-beta, which by itself will not induce actin expression. We show that the Xenopus embryo contains an mRNA encoding a protein highly homologous to basic FGF. These results together with the identification of a maternal mRNA with strong homology to TGF-beta, suggest that molecules closely related to FGF and TGF-beta are the natural inducers of mesoderm in vertebrate development.     

A community effect in muscle development

BACKGROUND: Most vertebrate tissues arise by embryonic induction, as a result of which new cell layers are formed. These are subsequently subdivided into discrete groups of homogeneous cell populations, each containing different cell-types with specific gene expression. There is preliminary evidence from previous work that the mesoderm-forming induction in amphibian development may be followed by a further interaction among some of the induced mesoderm cells, and that this could be required for muscle gene activation in uniform cell populations. RESULTS: We have established the existence, time and place of this further cell interaction by transplanting muscle progenitor cells from Xenopus mid-gastrulae into ectoderm sandwiches, and then culturing these constructs until the time of muscle gene activation. We find that cells implanted as reaggregates, but not those implanted as single cells, activate early myogenic genes and later muscle-specific genes. More than 100 cells must be near each other for muscle gene activation. These cells can induce non-muscle mesoderm cells to express muscle genes by emitting a signal that differs from the preceding mesoderm induction signal. Muscle gene activation under these conditions does not require gap junction communication. CONCLUSION: Cells within the muscle progenitor region of a Xenopus embryo need to interact with each other in order to activate muscle genes in homogeneous cell groups. This exemplifies the 'community effect', which may be a widespread developmental mechanism used to increase the homogeneity within, and demarkation between, embryonic tissues.     

The role of Xmsx-2 in the anterior-posterior patterning of the mesoderm in Xenopus laevis

Many molecules are involved in defining mesodermal patterning of the Xenopus embryo. In this paper, evidence is provided that a member of the msx family of genes, the Xmsx-2 gene, is involved in anterior-posterior patterning of the mesoderm. A comparison of its sequence to another previously cloned msx-2 Xenopus homolog, Xhox-7.1' [45] showed that they are closely related. The Xmsx-2 gene is first expressed at midgastrulation predominantly in the dorsal part of the embryo. It showed a complex pattern of spatial expression, consistent with a role in patterning of the anterior-posterior axis. This inference is confirmed by gain-of-function experiments in which overexpressed msx-2 mRNA in developing Xenopus embryos resulted in embryos lacking anterior structures. Analysis of markers in mutant embryos showed that genes involved in ventral-posterior patterning such as Xhox-3, Xwnt-8, and Xvent-1 were upregulated, confirming the posteriorized nature of the embryos. We believe that the Xmsx-2 gene is involved in refining the patterning of the anterior-posterior part of the dorsal mesoderm after the initial signals determining the dorsal or ventral nature of the mesoderm have been specified.     

Neural crest induction by paraxial mesoderm in Xenopus embryos requires FGF signals

At the border of the neural plate, the induction of the neural crest can be achieved by interactions with the epidermis, or with the underlying mesoderm. Wnt signals are required for the inducing activity of the epidermis in chick and amphibian embryos. Here, we analyze the molecular mechanisms of neural crest induction by the mesoderm in Xenopus embryos. Using a recombination assay, we show that prospective paraxial mesoderm induces a panel of neural crest markers (Slug, FoxD3, Zic5 and Sox9), whereas the future axial mesoderm only induces a subset of these genes. This induction is blocked by a dominant negative (dn) form of FGFR1. However, neither dnFGFR4a nor inhibition of Wnt signaling prevents neural crest induction in this system. Among the FGFs, FGF8 is strongly expressed by the paraxial mesoderm. FGF8 is sufficient to induce the neural crest markers FoxD3, Sox9 and Zic5 transiently in the animal cap assay. In vivo, FGF8 injections also expand the Slug expression domain. This suggests that FGF8 can initiate neural crest formation and cooperates with other DLMZ-derived factors to maintain and complete neural crest induction. In contrast to Wnts, eFGF or bFGF, FGF8 elicits neural crest induction in the absence of mesoderm induction and without a requirement for BMP antagonists. In vivo, it is difficult to dissociate the roles of FGF and WNT factors in mesoderm induction and neural patterning. We show that, in most cases, effects on neural crest formation were parallel to altered mesoderm or neural development. However, neural and neural crest patterning can be dissociated experimentally using different dominant-negative manipulations: while Nfz8 blocks both posterior neural plate formation and neural crest formation, dnFGFR4a blocks neural patterning without blocking neural crest formation. These results suggest that different signal transduction mechanisms may be used in neural crest induction, and anteroposterior neural patterning.     

VegT, eFGF and Xbra cause overall posteriorization while Xwnt8 causes eye-level restricted posteriorization in synergy with chordin in early Xenopus development

We examined several candidate posterior/mesodermal inducing molecules using permanent blastula-type embryos (PBEs) as an assay system. Candidate molecules were injected individually or in combination with the organizer factor chordin mRNA. Injection of chordin alone resulted in a white hemispherical neural tissue surrounded by a large circular cement gland, together with anterior neural gene expression and thus the development of the anterior-most parts of the embryo, without mesodermal tissues. When VegT, eFGF or Xbra mRNAs were injected into a different blastomere of the chordin -injected PBEs, the embryos elongated and formed eye, muscle and pigment cells, and expressed mesodermal and posterior neural genes. These embryos formed the full spectrum of the anteroposterior embryonic axis. In contrast, injection of CSKA-Xwnt8 DNA into PBEs injected with chordin resulted in eye formation and expression of En2 , a midbrain/hindbrain marker, and Xnot , a notochord marker, but neither elongation, muscle formation nor more posterior gene expression. Injection of chordin and posteriorizing molecules into the same cell did not result in elongation of the embryo. Thus, by using PBEs as the host test system we show that (i) overall anteroposterior neural development, mesoderm (muscle) formation, together with embryo elongation can occur through the synergistic effect(s) of the organizer molecule chordin , and each of the 'overall posteriorizing molecules' eFGF , VegT and Xbra ; (ii) Xwnt8-mediated posteriorization is restricted to the eye level and is independent of mesoderm formation; and (iii) proper anteroposterior patterning requires a separation of the dorsalizing and posteriorizing gene expression domains.     

Post-transcriptional repression of the Drosophila midkine and pleiotrophin homolog miple by HOW is essential for correct mesoderm spreading

The even spreading of mesoderm cells in the Drosophila embryo is essential for its proper patterning by ectodermally derived signals. In how germline clone embryos, defects in mesoderm spreading lead to a partial loss of dorsal mesoderm derivatives. HOW is an RNA-binding protein that is thought to regulate diverse mRNA targets. To identify direct HOW targets, we implemented a series of selection methods on mRNAs whose levels were elevated in how germline clone embryos during the stage of mesoderm spreading. Four mRNAs were found to be specifically elevated in the mesoderm of how germline clone embryos, and to exhibit specific binding to HOW via their 3' UTRs. Importantly, overexpression of three of these genes phenocopied the mesoderm-spreading phenotype of how germline clone embryos. Further analysis showed that overexpressing one of these genes, miple (a Drosophila midkine and pleiotrophin heparin-binding growth factor), in the mesoderm led to abnormal scattered MAPK activation, a phenotype that might explain the abnormal mesoderm spreading. In addition, the number of EVE-positive cells, which are responsive to receptor tyrosine kinase (RTK) signaling, was increased following Miple overexpression in the mesoderm and appeared to be dependent on Heartless function. In summary, our analysis suggests that HOW downregulates the levels of a number of mRNA species in the mesoderm in order to enable proper mesoderm spreading during early embryogenesis.     

Xwnt-8 modifies the character of mesoderm induced by bFGF in isolated Xenopus ectoderm.

In Xenopus, growth factors of the TGF-beta, FGF and Wnt oncogene families have been proposed to play a role in generating embryonic pattern. In this paper we examine potential interactions between the bFGF and Xwnt-8 signaling pathways in the induction and dorsal-ventral patterning of mesoderm. Injection of Xwnt-8 mRNA into 2-cell Xenopus embryos does not induce mesoderm formation in animal cap ectoderm isolated from these embryos at the blastula stage, but alters the response of this tissue to mesoderm induction by bFGF. While animal cap explants isolated from non-injected embryos differentiate to form ventral types of mesoderm and muscle in response to bFGF, explants from Xwnt-8 injected embryos form dorsal mesodermal and neural tissues in response to the same concentration of bFGF, even if the ectoderm is isolated from the prospective ventral sides of embryos or from UV-ventralized animals. Our results support a model whereby dorso-ventral mesodermal patterning can be attained by a single mesoderm inducing agent, possibly bFGF, which is uniformly distributed across the prospective dorsal-ventral axis, and which acts in concert with a dorsally localized signal, possibly a Wnt protein, which either alters the response of ectoderm to induction or modifies the character of mesoderm after its induction.     

Interaction of Wnt and activin in dorsal mesoderm induction in Xenopus.

Both the activin and Wnt families of peptide growth factors are capable of inducing dorsal mesoderm in Xenopus embryos. Presumptive ventral ectoderm cells isolated from embryos injected with Xwnt8 mRNA were cultured in the presence of activin A to study the possible interactions between these two classes of signaling proteins. We find that overexpression of Xwnt8 RNA alters the response of ventral ectoderm to activin such that ventral explants differentiate dorsoanterior structures including notochord and eyes. This response is similar to the response of dorsal ectoderm to activin alone. When embryos are irradiated with uv light to inhibit dorsal axis formation, ectodermal explants differentiate notochord when they are induced by a combination of both signaling factors, but not when cells receive only one inducing signal (activin or Xwnt8). This result is further supported by the observation that goosecoid (gsc) mRNA, an early marker for dorsal mesoderm, is expressed in these explants only when they are injected with Xwnt8 mRNA followed by exposure to activin. Early morphogenetic movements of the induced cells and activation of muscle-specific actin and Brachyury (Xbra) genes also reveal a cooperation of activin A and Xwnt8 in mesoderm induction.     

A mesoderm-inducing factor is produced by Xenopus cell line

Inductive interactions play a major role in the diversification of cell types during vertebrate development. These interactions have been extensively studied in amphibian embryos (usually Xenopus laevis) where the earliest is mesoderm induction, in which an equatorial mesodermal rudiment is induced from the animal hemisphere under the influence of signal from the vegetal hemisphere. The molecular basis of mesoderm induction is unknown, although Tiedemann has isolated a protein form 9- to 13-day chick embryos that has the properties one would expect of a mesoderm-inducing factor. However, the relevance of this molecule to the events of early amphibian development is unclear, and it is a matter of some importance to discover a Xenopus mesoderm-inducing factor. In this paper I show that the Xenopus XTC cell line secretes mesoderm-inducing activity into the culture medium. Isolated animal pole regions cultured in XTC-conditioned medium differentiate into muscle and notochord, while controls form 'atypical epidermis'. Three different cell lines -XL, XL177 and KR- secrete no such activity indicates that the active principle is heat stable, trypsin sensitive, nondialysable, and has an apparent relative molecular mass of about 16,000. Work is in progress to characterize the activity further and to discover whether the mesoderm-inducing factor is also present in normal embryos.