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.     

Hemopoietic differentiation potential of cultured lateral plate mesoderm explanted from Rana pipiens embryos at successive developmental stages

Ventral blood island mesoderm and dorsal lateral plate mesoderm were removed from Rana pipiens embryos at successive developmental stages (stages 13-19; 50-118 h) and cultured as individual explants in serum-free medium. After 5-7 days, the cultures were harvested, and differential counts were made of Wright-Giemsa-stained cells. Ventral blood island explants gave rise to cells of the myeloid lineage, suggesting that ventral blood island mesoderm was committed to hemopoiesis at the time of explant. Although erythrocytes were present in the cultures, granulocytes and monocyte/macrophages predominated. This differentiation profile occurred without the addition of any exogenous humoral factors. Monocyte/macrophages and immature precursor cells exhibited recurring inverse fluctuations with respect to one another. In all cases examined, cultures of dorsal lateral plate mesoderm showed marginal hemopoietic cell differentiation, suggesting a requirement for exogenous humoral factors and/or cell-cell interactions. When viewed in the context of previous studies from our laboratory, these results demonstrate that, in the amphibian embryo, there are two sources of hemopoietic stem cells separated both in space and time.     

Synergistic principles of development: overlapping patterning systems in Xenopus mesoderm induction.

The first inductive event in Xenopus development establishes the mesoderm at the equator of the developing embryo. As part of this process, the dorsal-ventral and anterior-posterior axes of the embryo are initially established. A number of signalling molecules which may play a role in mesodermal induction and patterning have been identified in the last several years, including members of the FGF, TGF-beta and Wnt gene families. A variety of experiments, using either purified factors or injection of RNA encoding these factors, have added to the wealth of classical embryological experimental data collected over the last century. We have synthesized some recent results with the classical data to provide a framework for examining the process of mesoderm induction, and to formulate putative roles for some of the different factors. We incorporate these ideas into a working model of mesoderm induction that provides a basis for future experimental directions. Finally, we suggest that mesoderm induction may not be a discrete set of well separated events, but instead may be a process involving partially overlapping signals that produce the same pattern.     

Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction.

The Brachyury (T) gene is required for mesoderm formation in the mouse. In this paper we describe the cloning and expression of a Xenopus homolog of Brachyury, Xbra. As with Brachyury in the mouse, Xbra is expressed in presumptive mesodermal cells around the blastopore, and then in the notochord. We show that expression of Xbra occurs as a result of mesoderm induction in Xenopus, both in response to the natural signal and in response to the mesoderm-inducing factors activin A and basic FGF. Expression of Xbra in response to these factors is rapid, and will occur in dispersed cells and in the presence of a protein synthesis inhibitor, indicating that this is an "immediate-early" response to mesoderm induction.     

The role of mesodermal signals during liver organogenesis in zebrafish

Three germ cell layers, the ectoderm, mesoderm and endoderm, are established during the gastrulation stage. All cell types in different organs and tissues are derived from these 3 germ cell layers at later stages. For example, skin epithelial cells and neuronal cells are derived from the ectoderm, while endothelial cells and muscle cells from the mesoderm and lung, and intestine epithelial cells from the endoderm. While in a normal situation different germ cells are destined to specific cell fates in differ...     

Scanning electron microscopy of the aggregation of head mesoderm cells from chick embryos

Head mesoderm cells from chick embryos at different stages of development were dissociated and cultured on plastic coverslips. In all cultures several cellular aggregates were described by means of scanning electron microscopy. Isolated cells present filopodia and lamellipodia. However, when mesoderm cells make contact with one another the filopodial and lamellipodial activity in the contact cellular edge disappear. Thus, the cells into cellular clusters do not present projections. The clusters were circular and bidimensional in character. The scanning electron microscopic observations showed that it is the type 1 variant of "contact inhibition of locomotion" which occurs. By means of these mechanisms the bidimensional aggregates are formed and cellular overlapping is not present. Since the behaviour of the mesoderm cells "in vitro" in some way could be comparable to their behaviour "in situ", the results here observed are discussed in relation to the conduct of mesoderm cells "in vivo".     

Myoblast determination in the somatic and visceral mesoderm depends on Notch signalling as well as on milliways(mili(Alk)) as receptor for Jeb signalling

The visceral muscles of the Drosophila midgut consist of syncytia and arise by fusion of founder and fusion-competent myoblasts, as described for the somatic muscles. A single-step fusion results in the formation of binucleate circular midgut muscles, whereas a multiple-step fusion process produces the longitudinal muscles. A prerequisite for muscle fusion is the establishment of myoblast diversity in the mesoderm prior to the fusion process itself. We provide evidence for a role of Notch signalling during establishment of the different cell types in the visceral mesoderm, demonstrating that the basic mechanism underlying the segregation of somatic muscle founder cells is also conserved during visceral founder cell determination. Searching for genes involved in the determination and differentiation of the different visceral cell types, we identified two independent mutations causing loss of visceral midgut muscles. In both of these mutants visceral muscle founder cells are missing and the visceral mesoderm consists of fusion-competent myoblasts only. Thus, no fusion occurs resulting in a complete disruption of visceral myogenesis. Subsequent characterisation of the mutations revealed that they are novel alleles of jelly belly (jeb) and the Drosophila Alk homologue named milliways (mili(Alk)). We show that the process of founder cell determination in the visceral mesoderm depends on Jeb signalling via the Milliways/Alk receptor. Moreover, we demonstrate that in the somatic mesoderm determination of the opposite cell type, the fusion-competent myoblasts, also depends on Jeb and Alk, revealing different roles for Jeb signalling in specifying myoblast diversity. This novel mechanism uncovers a crosstalk between somatic and visceral mesoderm leading not only to the determination of different cell types but also maintains the separation of mesodermal tissues, the somatic and splanchnic mesoderm.     

Patterning of mouse embryonic stem cell-derived pan-mesoderm by Activin A/Nodal and Bmp4 signaling requires Fibroblast Growth Factor activity

Abstract Embryonic stem (ES) cells have the potential to differentiate into all cell types of the adult body, and could allow regeneration of damaged tissues. The challenge is to alter differentiation toward functional cell types or tissues by directing ES cells to a specific fate. Efforts have been made to understand the molecular mechanisms that are required for the formation of the different germ layers and tissues from ES cells, and these mechanisms appear to be very similar in the mouse embryo. Differentiation toward mesoderm and mesoderm derivatives such as cardiac tissue or hemangioblasts has been demonstrated; however, the roles of Activin A/Nodal, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) signaling in the early patterning of ES cell-derived pan-mesoderm and anterior visceral endoderm (aVE) have not been reported yet. We therefore analyzed the roles of Activin A/Nodal, BMP, and FGF signaling in the patterning of ES cell-derived mesoderm as well as specification of the aVE by using a dual ES cell differentiation system combining a loss-of-function with a gain-of-function approach. We found that Activin A or Nodal directed the nascent mesoderm toward axial mesoderm and mesendoderm, while Bmp4 was inducing posterior and extraembryonic mesoderm at the expense of anterior primitive streak cells. FGF signaling appeared to have an important role in mesoderm differentiation by allowing an epithelial-to-mesenchymal transition of the newly formed mesoderm cells that would lead to their further patterning. Moreover, inhibition of FGF signaling resulted in increased expression of axial mesoderm markers. Additionally, we revealed that the formation of aVE cells from ES cells requires FGF-dependent Activin A/Nodal signaling and the attenuation of Bmp4 signaling.     

The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm

Nodal signals, a subclass of the TGFbeta superfamily of secreted factors, induce formation of mesoderm and endoderm in vertebrate embryos. We have examined the possible dorsoventral and animal-vegetal patterning roles for Nodal signals by using mutations in two zebrafish nodal-related genes, squint and cyclops, to manipulate genetically the levels and timing of Nodal activity. squint mutants lack dorsal mesendodermal gene expression at the late blastula stage, and fate mapping and gene expression studies in sqt(-/-); cyc(+/+) and sqt(-/-); cyc(+/-) mutants show that some dorsal marginal cells inappropriately form hindbrain and spinal cord instead of dorsal mesendodermal derivatives. The effects on ventrolateral mesendoderm are less severe, although the endoderm is reduced and muscle precursors are located nearer to the margin than in wild type. Our results support a role for Nodal signals in patterning the mesendoderm along the animal-vegetal axis and indicate that dorsal and ventrolateral mesoderm require different levels of squint and cyclops function. Dorsal marginal cells were not transformed toward more lateral fates in either sqt(-/-); cyc(+/-) or sqt(-/-); cyc(+/+) embryos, arguing against a role for the graded action of Nodal signals in dorsoventral patterning of the mesendoderm. Differential regulation of the cyclops gene in these cells contributes to the different requirements for nodal-related gene function in these cells. Dorsal expression of cyclops requires Nodal-dependent autoregulation, whereas other factors induce cyclops expression in ventrolateral cells. In addition, the differential timing of dorsal mesendoderm induction in squint and cyclops mutants suggests that dorsal marginal cells can respond to Nodal signals at stages ranging from the mid-blastula through the mid-gastrula.     

The FGF8-related signals Pyramus and Thisbe promote pathfinding, substrate adhesion, and survival of migrating longitudinal gut muscle founder cells

Fibroblast growth factors (FGFs) frequently fulfill prominent roles in the regulation of cell migration in various contexts. In Drosophila, the FGF8-like ligands Pyramus (Pyr) and Thisbe (Ths), which signal through their receptor Heartless (Htl), are known to regulate early mesodermal cell migration after gastrulation as well as glial cell migration during eye development. Herein, we show that Pyr and Ths also exert key roles during the long-distance migration of a specific sub-population of mesodermal cells that migrate from the caudal visceral mesoderm within stereotypic bilateral paths along the trunk visceral mesoderm toward the anterior. These cells constitute the founder myoblasts of the longitudinal midgut muscles. In a forward genetic screen for regulators of this morphogenetic process we identified loss of function alleles for pyr. We show that pyr and ths are expressed along the paths of migration in the trunk visceral mesoderm and endoderm and act largely redundantly to help guide the founder myoblasts reliably onto and along their substrate of migration. Ectopically-provided Pyr and Ths signals can efficiently re-rout the migrating cells, both in the presence and absence of endogenous signals. Our data indicate that the guidance functions of these FGFs must act in concert with other important attractive or adhesive activities of the trunk visceral mesoderm. Apart from their guidance functions, the Pyr and Ths signals play an obligatory role for the survival of the migrating cells. Without these signals, essentially all of these cells enter cell death and detach from the migration substrate during early migration. We present experiments that allowed us to dissect the roles of these FGFs as guidance cues versus trophic activities during the migration of the longitudinal visceral muscle founders.     

Inhibitory effect on limb morphogenesis by cells of the polarizing zone coaggregated with pre- or postaxial wing bud mesoderm.

The influence of cells of the polarizing zone mesoderm on the morphogenesis of recombinant chick limbs was studied. The recombinant buds were composed of leg bud ectoderm and different regions of the wing bud mesoderm, which had been dissociated and reaggregated. In any case where the polarizing zone mesoderm was coaggregated with the wing mesoderm the morphogenetic capabilities of the recombinant were reduced. This was the case with postaxial mesoderm, preaxial mesoderm plus polarizing tissue, and postaxial mesoderm from which a piece of the nonpolarizing mesoderm (comparable in size to the polarizing zone) had been removed. All of these gave outgrowths with digits in only a very low percentage of cases. In contrast, those recombinants without polarizing mesoderm developed outgrowths with digits in a high percentage of cases, indicating good morphogenesis. Finally, if the polarizing zone were removed prior to dissociation, the recombinant limb, composed of the total remaining wing bud mesoderm plus leg bud ectoderm, exhibited a higher percentage of complete morphogenesis than if the polarizing zone had been part of the recombinant.It is clear that cells of the polarizing zone, when dissociated, and coaggregated with wing mesoderm, are inhibitory to the morphogenetic performance of that mesoderm in the recombinant limb situation.     

Regionalisation of cell fate and morphogenetic movement of the mesoderm during mouse gastrulation

The developmental fate of cells in the epiblast of early-primitive-streak-stage mouse embryos was assessed by studying the pattern of tissue colonisation displayed by lac Z-expressing cells grafted orthotopically to nontransgenic embryos. Results of these fate-mapping experiments revealed that the lateral and posterior epiblast contain cells that will give rise predominantly to mesodermal derivatives. The various mesodermal populations are distributed in overlapping domains in the lateral and posterior epiblast, with the embryonic mesoderm such as heart, lateral, and paraxial mesoderm occupying a more distal position than the extraembryonic mesoderm. Heterotopic grafting of presumptive mesodermal cells results in the grafted cells adopting the fate appropriate to the new site, reflecting a plasticity of cell fate determination before ingression. The first wave of epiblast cells that ingress through the primitive streak are those giving rise to extraembryonic mesoderm. Cells that will form the mesoderm of the yolk sac and the amnion make up a major part of the mesodermal layer of the midprimitive-streak-stage embryo. Cells that are destined for embryonic mesoderm are still found within the epiblast, but some have been recruited to the distal portion of the mesoderm. By the late-primitive-streak-stage, the mesodermal layer contains only the precursors of embryonic mesoderm. This suggests that there has been a progressive displacement of the midstreak mesoderm to extraembryonic sites, which is reminiscent of that occurring in the overlying endodermal tissue. The regionalisation of cell fate in the late-primitive-streak mesoderm bears the same spatial relationship as their ancestors in the epiblast prior to cell ingression. This implies that both the position of the cells in the proximal-distal axis and their proximity to the primitive streak are major determinants for the patterning of the embryonic mesoderm. © 1995 Wiley-Liss, Inc.