The expression of the imprinted gene Ipl is restricted to extra-embryonic tissues and embryonic lateral mesoderm during early mouse development

Genes with restricted expression within the developing embryo represent valuable tools as they allow distinct tissue types to be distinguished and studied. In order to identify genes that are expressed within a particular germ layer, a differential screen was performed using germ layer-specific cDNA libraries derived from gastrulation stage mouse embryos. The gene expression profiles of the germ layers were compared following the hybridisation of some 20,000 cDNA clones with probes derived from germ layer-specific Ectoderm, Mesoderm and Endoderm libraries. A cDNA clone (50c15) was identified that hybridised with the Mesoderm-derived probe but not Ectoderm or Endoderm. 50c15 derives from Ipl/Tssc3/BWR1C, an imprinted gene which in human maps to chromosome 11p15.5. This region has been associated with Beckwith-Weidemann Syndrome, Wilms' tumour and ovarian, breast and lung cancer. In the gastrulating mouse embryo, wholemount RNA in situ hybridisation revealed that Ipl expression is restricted not only to the mesodermal germ layer, but specifically to lateral mesoderm and the most posterior extent of the primitive streak from which lateral and extra-embryonic mesoderm is derived. Moreover, Ipl is expressed in extra-embryonic tissues prior to gastrulation and afterwards in extra-embryonic mesoderm, ectoderm and endoderm. This expression profile indicates that Ipl is a good molecular marker for embryonic mesoderm and extra-embryonic tissues. In addition heterotopic grafting studies indicate that nascent mesoderm, which expresses Ipl, is restricted in its potential and therefore may be committed to its fate.     

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...     

Spatial patterns of metallothionein mRNA expression in the sea urchin embryo

Metallothioneins (MTs) are small, cysteine-rich proteins that bind heavy metals which induce their synthesis. Tissue fractionation of embryos at pluteus stage previously demonstrated that in the absence of added zinc, basal expression of MT mRNA is confined to ectoderm, whereas induction by zinc results in increased expression in the endoderm + mesoderm tissue fraction. Using in situ hybridization we now show that expression in the pluteus larva is restricted almost exclusively to the single cell type comprising the aboral ectoderm. Induction by Zn results in a marked accumulation of MT mRNA in gut and oral ectoderm to levels at least as high as that in aboral ectoderm. MT mRNA is also expressed in presumptive aboral ectoderm at earlier stages of normal development. In addition it is transiently expressed at variable levels in oral ectoderm and, to a lesser extent, in presumptive gut.     

The expression patterns of c-kit and Sl in chicken embryos suggest unexpected roles for these genes in somite and limb development

We have used whole-mount in situ hybridization to investigate the patterns of c-kit and Sl expression in stage 11-22 chicken embryos. Our analysis shows that c-kit and Sl are expressed quite differently in chicken embryos compared to the reported expression patterns of these genes in embryos of other taxa. Most notably, chicken c-kit is expressed in primordial germ cells as well as in the developing somite, the apical ectodermal ridge, and in the early foregut endoderm. Sl is expressed in the lateral and intermediate mesoderm and in extraembryonic membranes. These data suggest that chicken c-kit and Sl may play novel and unexpected roles in somitogenesis, limb development, and foregut development in avian embryos.     

Changing patterns of cytokeratins and vimentin in the early chick embryo

The distribution of cytokeratins and vimentin intermediate filaments in the first 48 h of chick development has been determined using immunofluorescent labelling. During formation of the germ layers, cytokeratin expression is associated with the appearance of an integral epithelium (ectoderm), whereas vimentin expression is associated with cells that detach and migrate from this epithelium to form endoderm and mesoderm. Subsequently, vimentin persists in the endoderm and mesoderm and the tissues derived therefrom, such as the somites and developing heart, throughout the period of study. The appearance of cytokeratins at later stages of development occurs in some epithelia such as the ectoderm, endoderm, lateral plate and epimyocardium but not others including the neural plate, neural tube and somites. Expression of cytokeratins in endoderm and mesenchymal tissues occurs in tandem with vimentin. In conclusion, vimentin expression is related to its distribution in the epiblast before germ layer formation. Its initial appearance may be related to the motile behaviour of cells about to ingress through the primitive streak. The appearance of cytokeratin filaments, however, does not reflect germ layer derivation but rather the need for an epithelial sheet.     

Phospholipid and nucleic acid gradients in the developing amphibian embryo

Rana pipiens embryos at the end of the blastula stage were dissociated and the cell suspension was separated into presumptive ectoderm, mesoderm, light endoderm, and heavy endoderm cells by a discontinuous density gradient centrifugation technique. The isolated germ layers were analyzed for total lipid, lipid phosphorus, plasmalogen, RNA, and DNA. Per gram dry weight, DNA showed a threefold decrease from ectoderm to heavy endoderm. On the same basis, the RNA content of the mesoderm was 34 per cent higher than that of ectoderm, and 320 and 570 per cent higher than that of light and heavy endoderm, respectively. In addition to the RNA and DNA gradients, there were at least two superimposed lipid gradients: a neutral lipid gradient decreasing from ectoderm to endoderm, and a total phospholipid gradient increasing from ectoderm to endoderm. In contrast to total phospholipid, a specific phospholipid class, ethanolamine plasmalogen, decreased from ectoderm to endoderm. The total lipid content per gram dry weight was the same in all the germ layers. Total phospholipids were analyzed quantitatively by thin layer chromatography. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and inositol phospholipid constituted 34, 13, 12, and 34 per cent, respectively, of the total lipid phosphorus. The phospholipid composition was different in each germ layer. The possible role of specific lipids in embryonic induction and differentiation is discussed.     

The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis

Orthotopic grafts of [3H]thymidine-labelled cells have been used to demonstrate differences in the normal fate of tissue located adjacent to and in different regions of the primitive streak of 8th day mouse embryos developing in vitro. The posterior streak produces predominantly extraembryonic mesoderm, while the middle portion gives rise to lateral mesoderm and the anterior region generates mostly paraxial mesoderm, gut and notochord. Embryonic ectoderm adjacent to the anterior part of the streak contributes mainly to paraxial mesoderm and neurectoderm. This pattern of colonization is similar to the fate map constructed in primitive-streak-stage chick embryos. Similar grafts between early-somite-stage (9th day) embryos have established that the older primitive streak continues to generate embryonic mesoderm and endoderm, but ceases to make a substantial contribution to extraembryonic mesoderm. Orthotopic grafts and specific labelling of ectodermal cells with wheat germ agglutinin conjugated to colloidal gold (WGA-Au) have been used to analyse the recruitment of cells into the paraxial mesoderm of 8th and 9th day embryos. The continuous addition of primitive-streak-derived cells to the paraxial mesoderm is confirmed and the distribution of labelled cells along the craniocaudal sequence of somites is consistent with some cell mixing occurring within the presomitic mesoderm.     

Sonic hedgehog in the pharyngeal endoderm controls arch pattern via regulation of Fgf8 in head ectoderm

Fgf8 signalling is known to play an important role during patterning of the first pharyngeal arch, setting up the oral region of the head and then defining the rostral and proximal domains of the arch. The mechanisms that regulate the restricted expression of Fgf8 in the ectoderm of the developing first arch, however, are not well understood. It has become apparent that pharyngeal endoderm plays an important role in regulating craniofacial morphogenesis. Endoderm ablation in the developing chick embryo results in a loss of Fgf8 expression in presumptive first pharyngeal arch ectoderm. Shh is locally expressed in pharyngeal endoderm, adjacent to the Fgf8-expressing ectoderm, and is thus a candidate signal regulating ectodermal Fgf8 expression. We show that in cultured explants of presumptive first pharyngeal arch, loss of Shh signalling results in loss of Fgf8 expression, both at early stages before formation of the first arch, and during arch formation. Moreover, following removal of the endoderm, Shh protein can replace this tissue and restore Fgf8 expression. Overexpression of Shh in the non-oral ectoderm leads to an expansion of Fgf8, affecting the rostral-caudal axis of the developing first arch, and resulting in the formation of ectopic cartilage. Shh from the pharyngeal endoderm thus regulates Fgf8 in the ectoderm and the role of the endoderm in pharyngeal arch patterning may thus be indirectly mediated by the ectoderm.     

Early endoderm development in vertebrates: lineage differentiation and morphogenetic function

Gastrulation of the vertebrate embryo culminates in the formation of three primary germ layers: ectoderm, mesoderm and endoderm. The endoderm contributes to the lining of the gut and the associated organs. New components of the molecular pathway for endoderm specification have been identified in the zebrafish and Xenopus. In the mouse, the activity of orthologous factors is involved with the allocation and differentiation of the definitive endoderm. Morphogenetic interactions between the endoderm and the other germ layer derivatives are critical for the morphogenesis of head structures and organogenesis of gut derivatives.     

Indian hedgehog signaling in extraembryonic endoderm and ectoderm differentiation in ES embryoid bodies

We previously demonstrated that a member of the Hedgehog gene family, Indian hedgehog (Ihh), is expressed in the visceral endoderm of EC and ES cell embryoid bodies and mouse embryos. Overexpression studies suggested that Ihh was involved in visceral endoderm differentiation. We now provide evidence for a Hh response in the embryoid body core and in the mesothelial layer of the visceral yolk sac. We also demonstrate that treatment of ES embryoid bodies with the Hh antagonists cAMP and forskolin results in downregulation of the Hh response and altered embryoid body differentiation. The outer endoderm layer undergoes a transition to parietal endoderm while formation of an embryonic ectoderm layer surrounding a cavity is inhibited. These treatments also result in a decrease in the expression of markers for the mesoderm derivatives, blood and endothelial cells. We present a model to explain how Ihh and BMP signaling may regulate extraembryonic endoderm and embryonic ectoderm differentiation.     

Polarity in the rabbit embryo

The main aim of the gastrulation process is commonly regarded to be the generation of the definitive germ layers known as mesoderm, endoderm and ectoderm. Here we discuss how the topography of gene expression, cellular migration and proliferative activity in the preliminary germ layers (hypoblast and epiblast) of the rabbit embryo reveal the sequence of events that establishes the three major body axes. We present a testable model in which a combination of cellular movement in the hypoblast with a morphogen gradient created by the (extraembryonic) trophoblast creates morphological polarity in the embryo and, hence, the co-ordinates for germ layer formation.     

Spatially distinct head and heart inducers within the Xenopus organizer region.

BACKGROUND: The mouse anterior visceral endoderm, an extraembryonic tissue, expresses several genes essential for normal development of structures rostral to the anterior limit of the notochord and has been termed the head organizer. This tissue also has heart-inducing activity and expresses mCer1 which, like its Xenopus homolog cerberus, can induce markers of cardiac specification and anterior neural tissue when ectopically expressed. We investigated the relationship between head and heart induction in Xenopus embryos, which lack extraembryonic tissues. RESULTS: We found three regions of gene expression in the Xenopus organizer: deep endoderm, which expressed cerberus; prechordal mesoderm, which showed overlapping but non-identical expression of genes characteristic of the murine head organizer, such as XHex and XANF-1; and leading-edge dorsoanterior endoderm, which expressed both cerberus and a subset of the genes expressed by the prechordal mesoderm. Microsurgical ablation of the cerberus-expressing endoderm decreased the incidence of heart, but not head, formation. Removal of prechordal mesoderm, in contrast, caused deficits of anterior head structures. Finally, although misexpression of cerberus induced ectopic heads, it was unable to induce genes thought to participate in head induction. CONCLUSIONS: In Xenopus, the cerberus-expressing endoderm is required for heart, but not head, inducing activity. Therefore, this tissue is not the topological equivalent of the murine anterior visceral endoderm. We propose that, in Xenopus, cerberus is redundant to other bone morphogenetic protein (BMP) and Wnt antagonists located in prechordal mesoderm for head induction, but may be necessary for heart induction.