Morphological and Gene Expression Changes in Cattle Embryos from Hatched Blastocyst to Early Gastrulation Stages after Transfer of In Vitro Produced Embryos

A detailed morphological staging system for cattle embryos at stages following blastocyst hatching and preceding gastrulation is presented here together with spatiotemporal mapping of gene expression for BMP4, BRACHYURY, CERBERUS1 (CER1), CRIPTO, EOMESODERMIN, FURIN and NODAL. Five stages are defined based on distinct developmental events. The first of these is the differentiation of the visceral hypoblast underlying the epiblast, from the parietal hypoblast underlying the mural trophoblast. The second concerns the formation of an asymmetrically positioned, morphologically recognisable region within the visceral hypoblast that is marked by the presence of CER1 and absence of BMP4 expression. We have termed this the anterior visceral hypoblast or AVH. Intra-epiblast cavity formation and the disappearance of the polar trophoblast overlying the epiblast (Rauber’s layer) have been mapped in relation to AVH formation. The third chronological event involves the transition of the epiblast into the embryonic ectoderm with concomitant onset of posterior NODAL, EOMES and BRACHYURY expression. Lastly, gastrulation commences as the posterior medial embryonic ectoderm layer thickens to form the primitive streak and cells ingress between the embryonic ectoderm and hypoblast. At this stage a novel domain of CER1 expression is seen whereas the AVH disappears. Comparison with the mouse reveals that while gene expression patterns at the onset of gastrulation are well conserved, asymmetry establishment, which relies on extraembryonic tissues such as the hypoblast and trophoblast, has diverged in terms of both gene expression and morphology.     

Hypoblast controls mesoderm generation and axial patterning in the gastrulating rabbit embryo

Gastrulation in higher vertebrate species classically commences with the generation of mesoderm cells in the primitive streak by epithelio-mesenchymal transformation of epiblast cells. However, the primitive streak also marks, with its longitudinal orientation in the posterior part of the conceptus, the anterior-posterior (or head-tail) axis of the embryo. Results obtained in chick and mouse suggest that signals secreted by the hypoblast (or visceral endoderm), the extraembryonic tissue covering the epiblast ventrally, antagonise the mesoderm induction cascade in the anterior part of the epiblast and thereby restrict streak development to the posterior pole (and possibly initiate head development anteriorly). In this paper we took advantage of the disc-shape morphology of the rabbit gastrula for defining the expression compartments of the signalling molecules Cerberus and Dickkopf at pre-gastrulation and early gastrulation stages in a mammal other than the mouse. The two molecules are expressed in novel expression compartments in a complementary fashion both in the hypoblast and in the emerging primitive streak. In loss-of-function experiments, carried out in a New-type culturing system, hypoblast was removed prior to culture at defined stages before and at the beginning of gastrulation. The epiblast shows a stage-dependent and topographically restricted susceptibility to express Brachyury, a T-box gene pivotal for mesoderm formation, and to transform into (histologically proven) mesoderm. These results confirm for the mammalian embryo that the anterior-posterior axis of the conceptus is formed first as a molecular prepattern in the hypoblast and then irrevocably fixed, under the control of signals secreted from the hypoblast, by epithelio-mesenchymal transformation (primitive streak formation) in the epiblast.Edited by D. Tautz     

Cell movements during epiboly and gastrulation in zebrafish

Beginning during the late blastula stage in zebrafish, cells located beneath a surface epithelial layer of the blastoderm undergo rearrangements that accompany major changes in shape of the embryo. We describe three distinctive kinds of cell rearrangements. (1) Radial cell intercalations during epiboly mix cells located deeply in the blastoderm among more superficial ones. These rearrangements thoroughly stir the positions of deep cells, as the blastoderm thins and spreads across the yolk cell. (2) Involution at or near the blastoderm margin occurs during gastrulation. This movement folds the blastoderm into two cellular layers, the epiblast and hypoblast, within a ring (the germ ring) around its entire circumference. Involuting cells move anteriorwards in the hypoblast relative to cells that remain in the epiblast; the movement shears the positions of cells that were neighbors before gastrulation. Involuting cells eventually form endoderm and mesoderm, in an anterior-posterior sequence according to the time of involution. The epiblast is equivalent to embryonic ectoderm. (3) Mediolateral cell intercalations in both the epiblast and hypoblast mediate convergence and extension movements towards the dorsal side of the gastrula. By this rearrangement, cells that were initially neighboring one another become dispersed along the anterior-posterior axis of the embryo. Epiboly, involution and convergent extension in zebrafish involve the same kinds of cellular rearrangements as in amphibians, and they occur during comparable stages of embryogenesis.     

Targeted gap junction protein constructs reveal connexin-specific differences in oligomerization

To define further the mechanisms of gap junction protein (connexin (Cx)) oligomerization without pharmacologic disruption, we have examined the transport and assembly of connexin constructs containing C-terminal di-lysine-based endoplasmic reticulum (ER) (HKKSL) or ER-Golgi intermediate compartment (AKKFF) targeting sequences. By immunofluorescence microscopy, Cx43-HKKSL transiently transfected into HeLa cells showed a predominantly ER localization, although Cx43-AKKFF was localized to the perinuclear region of the cell. Sucrose gradient analysis of Triton X-100-solubilized connexins showed that either Cx43-HKKSL or Cx43-AKKFF expressed alone by HeLa cells was maintained as an apparent monomer. In contrast to Cx43-HKKSL, Cx32-HKKSL was maintained in the ER as stable hexamers, consistent with the notion that Cx32 and Cx43 oligomerization occur in distinct intracellular compartments. Furthermore, Cx43-HKKSL and Cx43-AKKFF inhibited trafficking of Cx43 and Cx46 to the plasma membrane. The inhibitory effect was because of the formation of mixed oligomers between Cx43-HKKSL or Cx43-AKKF and wild type Cx43 or Cx46. Taken together, these results suggest that Cx43-HKKSL and Cx43-AKKFF recirculate through compartments where oligomerization occurs and may be maintained as apparent monomers by a putative Cx43-specific quality control mechanism.     

Involvement of fibronectin during epiboly and gastrulation in embryos of the common carp,Cyprinus carpio

Summary The present report firstly describes a pilot study in which, during early development of embryos of the common carp, Cyprinus carpio, the cellular adhesion to fibronectin (FN) was blocked by administration of GRGDS peptide (which binds to the FN-receptor). As this treatment resulted in developmental aberrations, suggesting a functional role for FN, the major part of the work was focussed on the distribution of reactivity of anti-FN antibodies during epiboly and gastrulation. GRGDS treatment had a concentration dependent effect on development. Incubation of embryos in 1.5 mg/ml from the 32-cell stage onwards caused a retardation of epiboly, which did not proceed beyond 60%. The embryos did not show involution, as was confirmed by histological study. These preliminary results suggest that FN is involved in both epiboly and gastrulation of carp embryos. During cleavage, no specific extracellular binding of anti-FN antiserum could be observed. However, binding to a number of cell membranes took place from early epiboly onwards. After the onset of gastrulation, we observed a gradually increasing number of the deepest epiblast cells, showing immunostaining on part of their surface, facing the yolk syncytial layer (YSL) or the involuted cells. During early epiboly, anti-FN binding was restricted to areas in front of the migratory hypoblast cells. Later on, binding was found at the border of hypoblast and epiblast cells. At 100% epiboly, some contact areas of epiblast and hypoblast showed a discontinuous lining of reactivity, whilst other areas appeared devoid of anti-FN binding sites. The results indicate that FN is involved in the migration and guidance of hypoblast cells during gastrulation in carp. Correspondence to: P. Gevers     

Confinement and clearance of OCT4 in the porcine embryo at stereomicroscopically defined stages around gastrulation

In the areas of developmental biology and embryonic stem cell research, reliable molecular markers of pluripotency and early lineage commitment are sparse in large animal species. In this study, we present morphological and immunohistochemical findings on the porcine embryo in the period around gastrulation, days 8-17 postinsemination, introducing a stereomicroscopical staging system in this species. In embryos at the expanding hatched blastocyst stage, OCT4 is confined to the inner cell mass. Following detachment of the hypoblast, and formation of the embryonic disk, this marker of pluripotency was selectively observed in the epiblast. A prominent crescent-shaped thickening at the posterior region of the embryonic disk marked the first polarization within this structure reflecting incipient cell ingression. Following differentiation of the epiblast, clearance of OCT4 from the three germ layers was observed at defined stages, suggesting correlations to lineage specification. In the endoderm, clearance of OCT4 was apparent from early during its formation at the primitive streak stage. The endoderm harbored progenitors of the "fourth germ layer," the primordial germ cells (PGCs), the only cells maintaining expression of OCT4 at the end of gastrulation. In the ectodermal and mesodermal cell lineages, OCT4 became undetectable at the neural groove and somite stage, respectively. As in the mouse, PGCs showed onset of c-kit expression when located in extraembryonal compartments. They appeared to follow the endoderm during extraembryonal allocation and the mesoderm on return to the genital ridge.     

Heart and head defects in mice lacking pairs of connexins

Gene ablation studies in mice have revealed roles for gap junction proteins (connexins) in heart development. Of the 20 connexins in vertebrates, four are expressed in developing heart: connexin37 (Cx37), connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45). Although each cardiac connexin has a different pattern of expression, some heart cells coexpress multiple connexins during cardiac morphogenesis. Since different connexins could have overlapping functions, some developmental phenotypes may only become evident when more than one connexin is ablated. In this study, we interbred Cx40(-/-) and Cx43(-/-) mice to generate mice lacking both Cx40 and Cx43. Cx40(-/-)Cx43(-/-) mice die around embryonic day 12.5 (E12.5), much earlier than either Cx40(-/-) or Cx43(-/-) mice, and they exhibit malformed hearts with ventricles that are abnormally rotated, suggesting a looping defect. Some Cx40(-/-)Cx43(-/-) animals also develop head defects characteristic of exencephaly. In addition, we examined mice lacking both Cx40 and Cx37 and found a high incidence of atrial and ventricular septal defects at birth. These results provide further evidence for the importance of gap junctions in embryonic development. Moreover, ablating different pairs of cardiac connexins results in distinct heart defects, suggesting both common and unique functions for Cx40, Cx43, and Cx37 during cardiac morphogenesis.     

Changes in gap junction distribution and connexin expression pattern during human fetal skin development.

Gap junctions are intercellular channels composed of connexin subunits that mediate cell-cell communication. The functions of gap junctions are believed to be associated with cell proliferation and differentiation and to be important in maintaining tissue homeostasis. We therefore investigated the expression of connexins (Cx)26 and 43, the two major connexins in human epidermis, and examined the formation of gap junctions during human fetal epidermal development. By immunofluorescence, Cx26 expression was observed between 49 and 96 days' estimated gestational age (EGA) but was not present from 108 days' EGA onwards. Conversely, Cx43 expression was observed from 88 days' EGA onwards. Using electron microscopy, the typical structure of gap junctions was observed from 120 days' EGA. The number of gap junctions increased over time and they were more common in the upper layers, within the periderm and intermediate keratinocyte layers rather than the basal layer. Immunoelectron microscopy revealed Cx43 labeling on the gap junction structures after 105 days' EGA. Formation of gap junctions increased as skin developed, suggesting that gap junctions may play an important role in fetal skin development. Furthermore, the changing patterns of connexin expression suggest that Cx26 is important for early fetal epidermal development.     

Effects of diminished expression of connexin43 on gap junction number and size in ventricular myocardium

Gap junction number and size vary widely in cardiac tissues with disparate conduction properties. Little is known about how tissue-specific patterns of intercellular junctions are established and regulated. To elucidate the relationship between gap junction channel protein expression and the structure of gap junctions, we analyzed Cx43 +/- mice, which have a genetic deficiency in expression of the major ventricular gap junction protein, connexin43 (Cx43). Quantitative confocal immunofluorescence microscopy revealed that diminished Cx43 signal in Cx43 +/- mice was due almost entirely to a reduction in the number of individual gap junctions (226 +/- 52 vs. 150 +/- 32 individual gap junctions/field in Cx43 +/+ and +/- ventricles, respectively; P < 0.05). The mean size of an individual gap junction was the same in both groups. Immunofluorescence results were confirmed with electron microscopic morphometry. Thus when connexin expression is diminished, ventricular myocytes become interconnected by a reduced number of large, normally sized gap junctions, rather than a normal number of smaller junctions. Maintenance of large gap junctions may be an adaptive response supporting safe ventricular conduction.     

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.     

Intercellular contact in the unincubated chick embryo

Summary The unincubated chick blastoderm, which consists of a complete upper epithelial layer of one cell thickness (epiblast) and an incomplete lower layer (hypoblast), was examined with the electron microscope in order to define the types of cell contact present. The terminal contacts between the cells of the epiblast invariably involved several focal tight junctions, but only occasionally involved tight junctions. Desmosomes were not observed in these areas, but were encountered in various phases of development in the deeper contact regions between epiblast cells. This deeper region also showed sporadic focal tight junctions and frequent micropapillae. These micropapillae were also common on the surfaces of hypoblast cells. Intercellular spaces between epiblast and hypoblast cells and within the hypoblast were often wide, narrowing to occasional focal tight junctions. Tight junctions and desmosomes were not observed in association with hypoblast cells. Gap junctions were not observed in any region of the embryo.These observations are discussed in relation to the morphogenetic movements occurring in the forming hypoblast and also the influence of this layer on the subsequent development of the embryo. Comparisons are drawn between the contact morphology in the unincubated blastoderm and that in later stages of development.Supported by the Medical Research Council of Canada.     

The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos

At the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell signaling pathways involved in early lineage specification. In mouse, segregation of the primitive endoderm lineage from the pluripotent epiblast lineage depends on FGF/MAP kinase signaling, but it is unknown whether this is conserved between species. Here we examined segregation of the hypoblast and epiblast lineages in bovine and human embryos through modulation of FGF/MAP kinase signaling pathways in cultured embryos. Bovine embryos stimulated with FGF4 and heparin form inner cell masses (ICMs) composed entirely of hypoblast cells and no epiblast cells. Inhibition of MEK in bovine embryos results in ICMs with increased epiblast precursors and decreased hypoblast precursors. The hypoblast precursor population was not fully ablated upon MEK inhibition, indicating that other factors are involved in hypoblast differentiation. Surprisingly, inhibition of FGF signaling upstream of MEK had no effects on epiblast and hypoblast precursor numbers in bovine development, suggesting that GATA6 expression is not dependent on FGF signaling. By contrast, in human embryos, inhibition of MEK did not significantly alter epiblast or hypoblast precursor numbers despite the ability of the MEK inhibitor to potently inhibit ERK phosphorylation in human ES cells. These findings demonstrate intrinsic differences in early mammalian development in the role of the FGF/MAP kinase signaling pathways in governing hypoblast versus epiblast lineage choices.     

Heterocellular gap junctional communication between alveolar epithelial cells

We analyzed the pattern of gap junction protein (connexin) expression in vivo by indirect immunofluorescence. In normal rat lung sections, connexin (Cx)32 was expressed by type II cells, whereas Cx43 was more ubiquitously expressed and Cx46 was expressed by occasional alveolar epithelial cells. In response to bleomycin-induced lung injury, Cx46 was upregulated by alveolar epithelial cells, whereas Cx32 and Cx43 expression were largely unchanged. Given that Cx46 may form gap junction channels with either Cx43 or Cx32, we examined the ability of primary alveolar epithelial cells cultured for 6 days, which express Cx43 and Cx46, to form heterocellular gap junctions with cells expressing other connexins. Day 6 alveolar epithelial cells formed functional gap junctions with other day 6 cells or with HeLa cells transfected with Cx43 (HeLa/Cx43), but they did not communicate with HeLa/Cx32 cells. Furthermore, day 6 alveolar epithelial cells formed functional gap junction channels with freshly isolated type II cells. Taken together, these data are consistent with the notion that type I and type II alveolar epithelial cells communicate through gap junctions compatible with Cx43.     

Appearance of Primordial Germ Cells in Young Chick Blastoderms Cultured in vitro

Appearance of primordial germ cells (PGCs) in young chick blastoderms was investigated by the cultivation of only the epiblast or hypoblast. Presumptive PGCs exist in the epiblast before primitive-streak formation. They translocate gradually to the lower layer during early stages of primitive-streak formation, though substantial number of presumptive PGCs remain in the upper layer. The existing primary hypoblast under the epiblast is dispensable for the further differentiation of the PGCs.     

The hypoblast (visceral endoderm): an evo-devo perspective

When amniotes appeared during evolution, embryos freed themselves from intracellular nutrition; development slowed, the mid-blastula transition was lost and maternal components became less important for polarity. Extra-embryonic tissues emerged to provide nutrition and other innovations. One such tissue, the hypoblast (visceral endoderm in mouse), acquired a role in fixing the body plan: it controls epiblast cell movements leading to primitive streak formation, generating bilateral symmetry. It also transiently induces expression of pre-neural markers in the epiblast, which also contributes to delay streak formation. After gastrulation, the hypoblast might protect prospective forebrain cells from caudalizing signals. These functions separate mesendodermal and neuroectodermal domains by protecting cells against being caught up in the movements of gastrulation.     

Transfer of extracellular matrix components between germ layers in chimaeric chicken-quail blastoderms

Summary A chemical basis for the transmission of signals during gastrulation has been investigated by using chimaeric embryos resulting from the combination of ³H-glucosamine-labelled and unlabelled hypoblast with epiblast taken from chicken and quail embryos at stage 3 of Vakaet (1970). The ability to distinguish chicken from quail cells on the basis of their different nuclear distribution of heterochromatin after Feulgen staining made it possible to determine the origin of the cells in the chimaerae. Tritiated quail hypoblast (after incubation of the embryo in the presence of ³H-glucosamine) was transplanted onto unlabelled chicken blastoderm deprived of its hypoblast. After culture of the chimaera for 5 h, the autoradiographic pattern shows silver grains not only over the graft, but also at the ventral surface of the epiblast of the host. Transfer of label may occur to mesoblast cells, but not between chicken and quail hypoblast cells. Chase experiments exclude the possibility that unprocessed, tritiated glucosamine is transferred. Chemical fixation of the host before transplantation of a labelled quail hypoblast also allows visualization of a transfer of macromolecules from hypoblast to the basement membrane of the epiblast, suggesting that an intervention of the epiblast cells in this process is not necessary. The morphology of the chimaeric embryos, as studied by scanning electron microscopy, suggests a direct deposition of these macromolecules by filopodia of the dorsal surface of the hypoblast. The possibility of diffusion of free macromolecules has been considered and can reasonably be discarded on the basis of several observations. The reverse experiment, in which unlabelled quail hypoblast and possibly some mesoblast have been combined with a tritiated host deprived of its hypoblast, also shows the transfer of label from the host to the cellular surface of the graft. A two-way exchange of glucosamine-containing molecules thus occurs in the blastoderm. It is hypothesized that: (1) low molecular weight compounds, macromolecular material, and/or catabolic products, are exchanged between the different germ layers during gastrulation; (2) the components of the extracellular matrix turn over and are continuously changing; (3) this transfer is a possible mechanism of transmission for developmental or inductive signals during embryonic development. The present results also demonstrate the participation of underlying tissue in the biosynthesis of basement membrane components of an epithelium.     

Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication.

Gap junction communication in some cells has been shown to be inhibited by pp60v-src, a protein tyrosine kinase encoded by the viral oncogene v-src. The gap junction protein connexin43 (Cx43) has been shown to be phosphorylated on serine in the absence of pp60v-src and on both serine and tyrosine in cells expressing pp60v-src. However, it is not known if the effect of v-src expression on communication results directly from tyrosine phosphorylation of the Cx43 or indirectly, for example, by activation of other second-messenger systems. In addition, the effect of v-src expression on communication based on other connexins has not been examined. We have used a functional expression system consisting of paired Xenopus oocytes to examine the effect of v-src expression on the regulation of communication by gap junctions comprised of different connexins. Expression of pp60v-src completely blocked the communication induced by Cx43 but had only a modest effect on communication induced by connexin32 (Cx32). Phosphoamino acid analysis showed that pp60v-src induced tyrosine phosphorylation of Cx43, but not Cx32. A mutation replacing tyrosine 265 of Cx43 with phenylalanine abolished both the inhibition of communication and the tyrosine phosphorylation induced by pp60v-src without affecting the ability of this protein to form gap junctions. These data show that the effect of pp60v-src on gap junctional communication is connexin specific and that the inhibition of Cx43-mediated junctional communication by pp60v-src requires tyrosine phosphorylation of Cx43.