Change of the differentiation pattern of amphibian ectoderm after the increase of the initial cell mass

Summary Early amphibian gastrula ectoderm (Triturus alpestris) has been treated with vegetalizing factor. While normal sandwiches (animal caps of two eggs) differentiated mainly into endoderm derived tissues, giant-sandwiches (a combination of 8 animal caps) formed mesodermal and neural tissues in addition. The results support the interpretation that ectoderm will differentiate into endoderm derived tissues when all or nearly all cells are induced (presumably depending on certain threshold concentrations of the inducer). This is the case in the normal sandwich after treatment with high concentrations of vegetalizing factor for 24 h. However, in a giantsandwich it must be assumed that only the cells in the vicinity of the inducer will be triggered to differentiate into endoderm derived tissues. Mesodermal structures will be formed by secondary interactions between the induced ectoderm (endoderm) and non induced ectodermal cells. The induction of neural structures could be explained as a further interaction between mesodermalized and non induced ectodermal cells. This chain of events is compared with the steps of determination in normogenesis.     

The formation of mesodermal derivatives after induction with vegetalizing factor depends on secondary cell interactions

Early amphibian gastrula ectoderm induced with vegetalizing factor for 6 h using the sandwich-method and cultured for up to 12 days, differentiated into mesodermal and endodermal tissues. Explants which were dissociated into single cells after the induction followed by immediate reaggregation and then cultured for 12 days likewise differentiated into mesodermal and endodermal tissues. However, if after induction and dissociation of the tissue, single cells are cultured for 20 h prior to the reaggregation, the reaggregated cell mass mainly differentiated into endoderm (liver and intestine) and irregularly shaped epidermis (formerly called ‘atypical epidermis’). Blood cells and, in few cases, heart structures were the only mesodermal structures found in this series. The results suggest that an endodermal anlage is induced first. The differentiation of mesodermal derivatives depends on secondary cell interactions between endodermal induced and non-induced ectoderm. For this process permanent cell-to-cell contacts are necessary.     

The inducing capacity of the presumptive endoderm of Xenopus laevis studied by transfilter experiments

Summary The inducing capacity of the vegetal hemisphere of early amphibian blastulae was studied by placing a Nucleopore filter (pore size 0.4 m) between isolated presumptive endoderm and animal (ectodermal) caps. The inducing effect was shown to traverse the Nucleopore membrane. The reacting ectoderm differentiated into mainly ventral mesodermal derivatives. Expiants consisting of five animal caps also formed dorsal mesodermal and neural structures. Those results together with data published elsewhere suggest that, in addition to a vegetalizing factor, different mesodermal factors must be taken into consideration for the induction of either the ventral or the dorsal mesodermal derivatives. The neural structures are thought to be induced by the primarily induced dorsal mesodermal tissue. Electron microscopic (TEM) examination did not reveal any cell processes in the pores of the filter. The results indicate that transmissible factors rather than signals via cytoplasmic contacts or gap junctions are responsible for the mesodermal induction of ectodermal cells. The data support the view that in normogenesis the mesoderm is determined by the transfer of inducing factors from vegetal blastomeres to cells of the marginal zone (presumptive mesodermal cells).     

Endoderm patterning by the notochord: development of the hypochord in Xenopus

The patterning and differentiation of the vertebrate endoderm requires signaling from adjacent tissues. In this report, we demonstrate that signals from the notochord are critical for the development of the hypochord, which is a transient, endodermally derived structure that lies immediately ventral to the notochord in the amphibian and fish embryo. It appears likely that the hypochord is required for the formation of the dorsal aorta in these organisms. We show that removal of the notochord during early neurulation leads to the complete failure of hypochord development and to the elimination of expression of the hypochord marker, VEGF. Removal of the notochord during late neurulation, however, does not interfere with hypochord formation. These results suggest that signals arising in the notochord instruct cells in the underlying endoderm to take on a hypochord fate during early neural stages, and that the hypochord does not depend on further notochord signals for maintenance. In reciprocal experiments, when the endoderm receives excess notochord signaling, a significantly enlarged hypochord develops. Overall, these results demonstrate that, in addition to patterning neural and mesodermal tissues, the notochord plays an important role in patterning of the endoderm.     

The possible role of mesodermal growth factors in the formation of endoderm inXenopus laevis

We have raised a monoclonal antibody, 4G6, against gut manually isolated from stage 42Xenopus laevis embryos. It is specific for endoderm and recognises an epitope that is first expressed at stage 19 and which persists throughout subsequent development. The antibody maintains gut specificity through metamorphosis and into adulthood. The epitope is conserved in the mouse, where it is also found in the gut. Isolated vegetal poles fromXenopus blastula stage embryos express the epitope autonomously after culturing to the appropriate stage. This shows that certain aspects of endoderm differentiation do not require germ layer interactions. Animal cap cells from stage 9 blastulae cultured in the presence of the mesodermal growth factors FGF, XTC-MIF and PIF form both endodermal and mesodermal tissues, assessed by the binding of tissue-specific monoclonal antibodies. Endoderm is typically found in those caps which form intermediate and ventral forms of mesoderm, that is muscle and lateral plate. Correspondence to: E.A. Jones     

Inhibitory and stimulatory influences on mesodermal erythropoiesis in the early chick blastoderm

We use a standing-drop culturing method to investigate the effect on mesodermal erythropoiesis of ectoderm and endoderm from the area opaca vasculosa (AOV) and area pellucida (AP) of stage-4 chick blastoderms. We find that ectoderm from the AOV and ectoderm and endoderm from the AP exert an inhibitory influence on mesodermal erythropoiesis. This inhibitory influence is coupled with the tendency of the explants to spread out and become flattened in culture. In contrast, endoderm from the AOV is found to be stimulatory, in agreement with previous studies. We correlate these in vitro inhibitory and stimulatory influences with the morphogenetic patterns that occur during normal development.     

The role of growth factors in the embryogenesis and differentiation of the eye.

The vertebrate eye is composed of a variety of tissues that, embryonically, have their derivation from surface ectoderm, neural ectoderm, neural crest, and mesodermal mesenchyme. During development, these different types of cells are subjected to complex processes of induction and suppressive interactions that bring about their final differentiation and arrangement in the fully formed eye. With the changing concept of ocular development, we present a new perspective on the control of morphogenesis at the cellular and molecular levels by growth factors that include fibroblast growth factors, epidermal growth factor, nerve growth factor, platelet-derived growth factor, transforming growth factors, mesodermal growth factors, transferrin, tumor necrosis factor, neuronotrophic factors, angiogenic factors, and antiangiogenic factors. Growth factors, especially transforming growth factor-beta, have a crucial role in directing the migration and developmental patterns of the cranial neural-crest cells that contribute extensively to the structures of the eye. Some growth factors also exert an effect on the developing ocular tissues by influencing the synthesis and degradation of the extracellular matrix. The mRNAs for the growth factors that are involved in the earliest aspects of the growth and differentiation of the fertilized egg are supplied from maternal sources until embryonic tissues are able to synthesize them. Subsequently, the developing eye tissues are exposed to both endogenous and exogenous growth factors that are derived from nonocular tissues as well as from embryonic fluids and the systemic circulation. The early interaction between the surface head ectoderm and the underlying chordamesoderm confers a lens-forming bias on the ectoderm; later, the optic vesicle elicits the final phase of determination and enhances differentiation by the lens. After the blood-ocular barrier is established, the internal milieu of the eye is controlled by the interactions among the intraocular tissues; only those growth factors that selectively cross the barrier or that are synthesized by the ocular tissues can influence further development and differentiation of the cells. An understanding of the tissue interactions that are regulated by growth factors could clarify the precise mechanism of normal and abnormal ocular development.     

Primary differentiation and ectoderm-specific gene expression in the animalized sea urchin embryo

Primary differentiation in sea urchin embryos, animalized by zinc, has been gauged by the formation of characteristic endodermal and mesodermal tissue derivatives and by the accumulation of the ectoderm-specific Spec 1 mRNA. Increasing the dosage of zinc diminishes the differentiation of secondary mesenchyme, primary mesenchyme, endoderm, and ectoderm, in decreasing order. Treatment is effective only during the blastula stages, involving successive periods of sensitivity for these tissues. Removal of zinc with chelator results in the resumption of differentiation to increasing degree for this series of tissues. The developmental initiation of Spec 1 gene expression, normally at the earliest blastula stage, can be delayed by zinc for at least 30 hr before being implemented by treatment of the animalized embryos with a chelator. We conclude (1) that those processes in the blastula which are required for differentiation and are suppressed by zinc are distinguishable from the determinative processes, which are not affected by the animalizing agent and occur earlier during midcleavage; (2) that animalization by zinc involves a graded failure of primary tissues to form; and (3) that animalization involves a pause in the schedule of differentiation, which can be reinstated by removal of the animalizing agent, thereby providing a survival value inherent in a flexible schedule of development.     

An FGF signal from endoderm and localized factors in the posterior-vegetal egg cytoplasm pattern the mesodermal tissues in the ascidian embryo

The major mesodermal tissues of ascidian larvae are muscle, notochord and mesenchyme. They are derived from the marginal zone surrounding the endoderm area in the vegetal hemisphere. Muscle fate is specified by localized ooplasmic determinants, whereas specification of notochord and mesenchyme requires inducing signals from endoderm at the 32-cell stage. In the present study, we demonstrated that all endoderm precursors were able to induce formation of notochord and mesenchyme cells in presumptive notochord and mesenchyme blastomeres, respectively, indicating that the type of tissue induced depends on differences in the responsiveness of the signal-receiving blastomeres. Basic fibroblast growth factor (bFGF), but not activin A, induced formation of mesenchyme cells as well as notochord cells. Treatment of mesenchyme-muscle precursors isolated from early 32-cell embryos with bFGF promoted mesenchyme fate and suppressed muscle fate, which is a default fate assigned by the posterior-vegetal cytoplasm (PVC) of the eggs. The sensitivity of the mesenchyme precursors to bFGF reached a maximum at the 32-cell stage, and the time required for effective induction of mesenchyme cells was only 10 minutes, features similar to those of notochord induction. These results support the idea that the distinct tissue types, notochord and mesenchyme, are induced by the same signaling molecule originating from endoderm precursors. We also demonstrated that the PVC causes the difference in the responsiveness of notochord and mesenchyme precursor blastomeres. Removal of the PVC resulted in loss of mesenchyme and in ectopic notochord formation. In contrast, transplantation of the PVC led to ectopic formation of mesenchyme cells and loss of notochord. Thus, in normal development, notochord is induced by an FGF-like signal in the anterior margin of the vegetal hemisphere, where PVC is absent, and mesenchyme is induced by an FGF-like signal in the posterior margin, where PVC is present. The whole picture of mesodermal patterning in ascidian embryos is now known. We also discuss the importance of FGF induced asymmetric divisions, of notochord and mesenchyme precursor blastomeres at the 64-cell stage.     

derrière: a TGF-beta family member required for posterior development in Xenopus

TGF-beta signaling plays a key role in induction of the Xenopus mesoderm and endoderm. Using a yeast-based selection scheme, we isolated derrière, a novel TGF-beta family member that is closely related to Vg1 and that is required for normal mesodermal patterning, particularly in posterior regions of the embryo. Unlike Vg1, derrière is expressed zygotically, with RNA localized to the future endoderm and mesoderm by late blastula, and to the posterior mesoderm by mid-gastrula. The derrière expression pattern appears to be identical to the zygotic expression domain of VegT (Xombi, Brat, Antipodean), and can be activated by VegT as well as fibroblast growth factor (FGF). In turn, derrière activates expression of itself, VegT and eFGF, suggesting that a regulatory loop exists between these genes. derrière is a potent mesoderm and endoderm inducer, acting in a dose-dependent fashion. When misexpressed ventrally, derrière induces a secondary axis lacking a head, an effect that is due to dorsalization of the ventral marginal zone. When misexpressed dorsally, derrière suppresses head formation. derrière can also posteriorize neurectoderm, but appears to do so indirectly. Together, these data suggest that derrière expression is compatible only with posterior fates. In order to assess the in vivo function of derrière, we constructed a dominant interfering Derrière protein (Cm-Derrière), which preferentially blocks Derrière activity relative to that of other TGFbeta family members. Cm-derrière expression in embryos leads to posterior truncation, including defects in blastopore lip formation, gastrulation and neural tube closure. Normal expression of anterior and hindbrain markers is observed; however, paraxial mesodermal gene expression is ablated. This phenotype can be rescued by wild-type derrière and by VegT. Our findings indicate that derrière plays a crucial role in mesodermal patterning and development of posterior regions in Xenopus.