Fibroblast growth factor during mesoderm induction in the early chick embryo

A chick genomic clone that reveals a high degree of homology to the mammalian and Xenopus bFGF gene has been isolated. The pattern of expression of bFGF has been examined during early chick embryogenesis. RNA blot analysis revealed that chick bFGF is already transcribed at pregastrula stages. Immunolabeling analysis indicated that bFGF protein is present at these early developmental stages and is distributed evenly in the epiblast, hypoblast and marginal zone of the chick blastula. Substances that can inhibit FGF action were applied to early chick blastoderms grown in vitro under defined culture conditions (DCM). Both heparin and suramin were capable of blocking the formation of mesodermal structures in a dose-dependent manner. Our results indicate that FGF-like substances may need to be present for axial structures to develop although they may be acting earlier during the induction of non-axial mesoderm.     

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.     

Cell coherence during production of the presomitic mesoderm and somitogenesis in the mouse embryo

In this study, we investigated (in the early mouse embryo) the clonal properties of precursor cells which contribute to the segmented myotome, a structure derived from the somites. We used the laacZ method of single cell-labelling to visualise clones born before segmentation and bilateralisation. We found that clones which contribute to several segments both unilateral and bilateral were regionalised along the mediolateral axis and that their mediolateral position was maintained in successive adjacent segments. Furthermore, clones contributed to all segments, from their most anterior to their most posterior borders. Therefore, it appears that mediolateral regionalisation of myotomal precursor cells is a property established before bilateralisation of the presomitic mesoderm and that coherent clonal growth accompanies cell dispersion along both the mediolateral and anteroposterior axes. These findings in the mouse correlate well with what is known in the chick, suggesting conservation of the mode of production and distribution of the cells of the presomitic mesoderm. However, in addition, we also found that the mediolateral contribution of a clone is already determined in the pool of self-renewing cells that produces the myotomal precursor cells and thus that this pool is itself regionalised. Finally, we found that bilateral clones exhibit symmetry in right and left sides in the embryo at all levels of the mediolateral axis of the myotome. All these properties indicate synchrony and symmetry of formation of the presomitic mesoderm on both sides of the embryo leading to formation of a static embryonic structure with few cell movements. We suggest that sequential production of groups of cells with an identical clonal origin for both sides of the embryo from a single pool of self-renewing cells, coupled with acquisition of static cell behaviour, could play a role in colinearity of expression of Hox genes and in the segmentation system of higher vertebrates.     

Cephalic neurulation and optic vesicle formation in the early mouse embryo.

The overall pattern of cephalic neurulation and the concomitant early development of the optic vesicles in mouse embryos were examined by scanning electron microscopy. Paraffin-sectioned specimens were also examined. The overall pattern of closure of the cephalic neural folds accords well with earlier observations of this process. The earliest indication of optic placode formation was seen in histological sections of embryos at the 4-somite stage, while optic pit formation was first observed at the 5- to 6-somite stage. The upper halves of the optic vesicles were formed in 10- to 15-somite embryos by the fusion of the neural folds at the junction between the mesencephalon and prosencephalon, while closure of the lower halves was associated with the closure of the rostral neuropore, and was usually completed by about the 20-somite stage. By the 25- to 30-somite stage, a rapid increase in the volume of the forebrain was observed, so that the optic vesicles were displaced laterally. An overall increase in the volume of the optic vesicles and decrease in the diameter of the optic stalks were also observed at this time. This account of cephalic neurulation and optic organogenesis provides useful baseline data relevant to the study of the normal early development of the mouse. A comparison is made between similar events in the rat, the hamster, and the human embryo.     

Morphology of the mouse embryo,from the time of implantation to mesoderm formation

Summary The preparation technique of electron microscopy was adapted to light microscopy, in attempts to obtain well preserved implantation sites. The most appropriate technique comprised perfusion fixation in glutaraldehyde, post-fixation in osmium acid, Epon-embedding, ultramicrotomy, and staining with toluidine blue.The morphology of the early mouse embryo from the time of nidation to mesoderm formation is described: the formation of Reichert's membrane occurs already at 6 1/2 days, by which time free trophoblast cells are to be found in the uterine cavity.     

Expression of lbx1 involved in the hypaxial musculature formation of the mouse embryo

Somites are the source of hypaxial musculature including skeletal muscles of the limb, tongue, and trunk. To get insight into the function of mouse Lbx1 homeobox gene in early somitic mesoderm differentiation, in situ hybridization analyses were performed. At the 4-6 somite stage (8 dpc), Lbx1 was first expressed in the lateral portion of the epithelial somite and dermomyotomal epithelium. This was in contrast to the expression of myf-5 in the medial region of the somite. The lateral expression of Lbx1 in somitic mesoderm then occurred regionally along the anterior-posterior body axis. Later, at 10 dpc (stage 1 of limb bud development), Lbx1-positive migrating cells originated in the lateral dermomyotomal lips at occipital, forelimb, and hindlimb levels. They also expressed Pax-3 and c-met, known as markers of the migrating limb muscle precursor cells. In stage 4 hindlimb bud (11.5 dpc), the dorsal and ventral muscle precursor populations expressed Lbx1. In stage 8 forelimb buds (12.5 dpc), Lbx1 expression was reduced in the proximal muscle masses, where the high expression of myogenin accompanying muscle differentiation was detected. These results suggest that mouse Lbx1 might be involved in the commitment or determination of a muscle cell subpopulation during hypaxial musculature development. J. Exp. Zool. 286:270-279, 2000.     

The allocation of cells in the presomitic mesoderm during somite segmentation in the mouse embryo

Orthotopic grafts of wheat germ agglutinin-colloidal gold conjugate (WGA-gold) labelled cells were used to demonstrate differences in the segmental fate of cells in the presomitic mesoderm of the early-somite-stage mouse embryos developing in vitro. Labelled cells in the anterior region of the presomitic mesoderm colonized the first three somites formed after grafting, while those grafted to the middle region of this tissue were found mostly in the 4th-7th newly formed somites. Labelled cells grafted to the posterior region were incorporated into somites whose somitomeres were not yet present in the presomitic mesoderm at the time of grafting. There was therefore an apparent posterior displacement of the grafted cells in the presomitic mesoderm. Colonization of somites by WGA-gold labelled cells was usually limited to two to three consecutive somites in the chimaera. The distribution of cells derived from a single graft to two somites was most likely due to the segregation of the labelled population when cells were allocated to adjacent meristic units during somite formation. Further spreading of the labelled cells to several somites in some cases was probably the result of a more extensive mixing of mesodermal cells among the somitomeres prior to somite segmentation.     

Combinatorial activities of Smad2 and Smad3 regulate mesoderm formation and patterning in the mouse embryo

TGFbeta/activin/Nodal receptors activate both Smad2 and Smad3 intracellular effector proteins. The functional activities of these closely related molecules have been extensively studied in cell lines. We show both are expressed in the early mouse embryo from the blastocyst stage onwards and mediate Foxh1-dependent activation of the Nodal autoregulatory enhancer in vitro. Genetic manipulation of their expression ratios reveals that Smad3 contributes essential signals at early post-implantation stages. Thus, loss of Smad3 in the context of one wild-type copy of Smad2 results in impaired production of anterior axial mesendoderm, while selective removal of both Smad2 and Smad3 from the epiblast additionally disrupts specification of axial and paraxial mesodermal derivatives. Finally, we demonstrate that Smad2;Smad3 double homozygous mutants entirely lack mesoderm and fail to gastrulate. Collectively, these results demonstrate that dose-dependent Smad2 and Smad3 signals cooperatively mediate cell fate decisions in the early mouse embryo.     

Single cell analysis of mesoderm formation in the Xenopus embryo

We have examined the developmental specification of individual cells in the Xenopus blastula using a new in vitro culture system. Regional differences are apparent at the mid-blastula stage when animal hemisphere cells form only ectodermal cell types, while many clones from below the pigment boundary contain mesodermal cell types. A number of clones give rise to more than one differentiated cell type indicating that the initial steps of mesoderm induction are potentially reversible. Animal hemisphere cells can be induced to form mesoderm by fibroblast growth factor (FGF). Different cell types predominate at different FGF concentrations and the neighbours in this sequence are also the pairs of cell types most usually associated in mixed clones derived from the marginal zone. We propose that the specification of individual cells depends upon both the concentration of inducing factor and on stochastic intracellular events.     

Intermediate filament protein expression and mesoderm formation in the rabbit embryo

Summary This study aims to describe the regulation of vimentin and cytokeratin expression during differentiation of primary mesenchymal cells in the 7 day old rabbit embryo; unusual intermediate filament protein expression patterns have already been found in this species at later embryonic stages. Double-labelling indirect immunofluorescence assays with a panel of monoclonal intermediate filament antibodies are performed on frozen sections and compared with aldehyde-fixed plastic-embedded tissues. The histological part of the study, serving as a basis for the topographical orientation in the immunostained frozen sections, emphasises many similarities between the primitive streak embryos of the rabbit and the chick. The immunohistochemical analysis reveals cytokeratin expression to varying degrees in all germ layers. Vimentin expression, always in combination with cytokeratin expression, is found in a few cells of the ectoderm, endoderm and lateral mesoderm, but not in the primary mesenchymal cells of either the primitive node or the primitive streak. The results are discussed in relation to recent experimental findings on differentiation and morphogenetic processes in the primitive streak embryo. While these complex expression patterns make it seem unlikely that intermediate filament protein subtypes are expressed independently of cellular function during development, no indication can be found for a relation between vimentin expression and the morphogenetic changes thought to be important during mesoderm formation.Supported by the Deutsche Forschungsgemeinschaft (Wa 359-9) and by the Netherlands Cancer Foundation Offprint requests to: C. Viebahn     

Expression of ASK1 during chick and early mouse development

Apoptosis signal-regulating kinase 1 (ASK1) is an important regulator of stress-induced cell death. ASK1 is activated by oxidative stress, TNF and endoplasmatic reticulum stress and activates the JNK- and p38-dependent intracellular death pathways. A number of studies have suggested that ASK1 may also have other roles in addition to its pro-apoptotic activity. Expression of ASK1 during early embryonic development has so far not been analyzed. We have identified and cloned chick ASK1 in a screen for FGF8 inducible genes in chick facial mesenchyme. Here we report the expression of chick ASK1 from the gastrulation stage (HH4) to day 4 of development, its expression in the developing inner organs and limbs, and we compare its expression to the expression of Ask1 during mouse development. Furthermore, we provide evidence that FGF signaling is required for ASK1 expression in chick nasal mesenchyme. In contrast, expression in the mouse nasal region was restricted to the epithelium and was independent of FGF signaling. Our analysis demonstrates that ASK1 has a spatially restricted and temporally dynamic expression pattern in both chick and mouse embryos, which includes conserved as well as species-specific expression domains.     

RNA polymerase during early development in mouse embryo

RNA polymerase activity in mouse embryo homogenates has been measured at various stages of pre-implantation development. The amount of enzyme/embryo appears to increase in the period under consideration. On a per cell basis a decline in the level of polymerase was, however, observed from the 2-cell to the early blastocyst stages.     

Integrin alpha6beta1-laminin interactions regulate early myotome formation in the mouse embryo

We addressed the potential role of cell-laminin interactions during epaxial myotome formation in the mouse embryo. Assembly of the myotomal laminin matrix occurs as epaxial myogenic precursor cells enter the myotome. Most Myf5-positive and myogenin-negative myogenic precursor cells localise near assembled laminin, while myogenin-expressing cells are located either away from this matrix or in areas where it is being assembled. In Myf5(nlacZ/nlacZ) (Myf5-null) embryos, laminin, collagen type IV and perlecan are present extracellularly near myogenic precursor cells, but do not form a basement membrane and cells are not contained in the myotomal compartment. Unlike wild-type myogenic precursor cells, Myf5-null cells do not express the alpha6beta1 integrin, a laminin receptor, suggesting that integrin alpha6beta1-laminin interactions are required for myotomal laminin matrix assembly. Blocking alpha6beta1-laminin binding in cultured wild-type mouse embryo explants resulted in dispersion of Myf5-positive cells, a phenotype also seen in Myf5(nlacZ/nlacZ) embryos. Furthermore, inhibition of alpha6beta1 resulted in an increase in Myf5 protein and ectopic myogenin expression in dermomyotomal cells, suggesting that alpha6beta1-laminin interactions normally repress myogenesis in the dermomyotome. We conclude that Myf5 is required for maintaining alpha6beta1 expression on myogenic precursor cells, and that alpha6beta1 is necessary for myotomal laminin matrix assembly and cell guidance into the myotome. Engagement of laminin by alpha6beta1 also plays a role in maintaining the undifferentiated state of cells in the dermomyotome prior to their entry into the myotome.     

Seasonal variation in cell cycle during early development of the mouse embryo.

Studies of the cell cycle of mouse embryos before implantation were conducted using Giemsa and DAPI stains. The time of embryo recovery did not affect the success rate of cultures during the winter, but embryos cultured during the summer showed the 'two-cell block' phenomenon at the early two-cell stage, 30-37 h after the injection of human chorionic gonadotrophin. There was no significant difference in the number of embryos collected per mouse between summer and winter, but cleavage from the two-cell to the four-cell stage occurred later in the summer than in the winter. Cell cycle of mouse embryos may therefore show seasonal variation.     

Expression of a novel FGF in the Xenopus embryo. A new candidate inducing factor for mesoderm formation and anteroposterior specification.

We have cloned and sequenced a new member of the fibroblast growth factor family from Xenopus laevis embryo cDNA. It is most closely related to both mammalian kFGF (FGF-4) and FGF-6 but as it is not clear whether it is a true homologue of either of these genes we provisionally refer to it as XeFGF (Xenopus embryonic FGF). Two sequences were obtained, differing by 11% in derived amino acid sequence, which probably represent pseudotetraploid variants. Both the sequence and the behaviour of in vitro translated protein indicates that, unlike bFGF (FGF-2), XeFGF is a secreted molecule. Recombinant XeFGF protein has mesoderm-inducing activity with a specific activity similar to bFGF. XeFGF mRNA is expressed maternally and zygotically with a peak during the gastrula stage. Both probe protection and in situ hybridization showed that the zygotic expression is concentrated in the posterior of the body axis and later in the tailbud. Later domains of expression were found near the midbrain/hindbrain boundary and at low levels in the myotomes. Because of its biological properties and expression pattern, XeFGF is a good candidate for an inducing factor with possible roles both in mesoderm induction at the blastula stage and in the formation of the anteroposterior axis at the gastrula stage.     

Expression of the cell surface-associated glycoprotein, fibronectin, in the early mouse embryo.

The expression of the cell surface-associated glycoprotein fibronectin was studied by indirect immunofluorescence in the early stages of mouse embryogenesis. Fibronectin was not detectable in early preimplantation embryos. Trace amounts of the protein were first found between the cells of the inner cell mass of late blastocysts. In implanted early egg cylinders, fibronectin was deposited between the ectoderm and endoderm of the inner cell mass and in the nascent Reichert's membrane. With development, the visceral and the parietal endoderm cells became positive for the protein, but no fibronectin was detected in ectoderm cells. During segregation of mesoderm from ectoderm, fibronectin appeared in mesoderm cells and as a band between the two germ layers. In the developing amnion and chorion, the protein was localized between the ectodermal and mesodermal cell layers. The results indicate that fibronectin is an early differentiation market for the stage of endoderm formation in the inner cell mass of the mouse blastocyst. It is also a marker of mesoderm appearance and seems to be associated with the accumulating extracellular matrix material in the developing embryo.