Invasion and migration of a single chick pre-streak stage epiblast cell in vitro: Its implication to the primitive streak formation

To investigate the contribution of the epiblast cell behavior to the primitive streak formation, we examined the motility of a single epiblast cell from pre-streak stage embryo in vitro . On the substratum that was evenly coated with laminin gel, epiblast cells attached well to the gel and one or a few very long and broad cellular processes protruded from their spherical cell bodies; however, they hardly locomoted on it. Unexpectedly, after overnight culture, half of the single cells dissolved the laminin gel beneath them to make well-like holes, and invaded in the holes. On the substratum lined parallel with the fibrous laminin gels supplemented with fibronectin, they locomoted actively in accordance with the alignment. That is, they were subjected to contact guidance. In locomotion they looked like snails, extending one or a few long and broad processes in a forward direction from the spherical cell bodies. However, on the substratum lined with laminin or fibronectin only, they did not locomote actively. Individual chick pre-streak epiblast cells had already been committed to invade, and their migratory nature existed in each cell, even though they were isolated from the epithelial sheet. The implication of these findings on the cellular basis of primitive streak formation will be discussed.     

Cell populations and morphogenetic movements underlying formation of the avian primitive streak and organizer

The cell populations and morphogenetic movements that contribute to the formation of the avian primitive streak and organizer-Hensen's node-are poorly understood. We labeled selected groups of cells with fluorescent dyes and then followed them over time during formation and progression of the primitive streak and formation of Hensen's node. We show that (1) the primitive streak arises from a localized population of epiblast cells spanning the caudal midline of Koller's sickle, with the mid-dorsal cells of the primitive streak arising from the midline of the epiblast overlying Koller's sickle and the deeper and more lateral primitive streak cells arising more laterally within the epiblast overlying the sickle, from an arch subtending about 30 degrees; (2) convergent extension movements of cells in the epiblast overlying Koller's sickle contribute to formation of the initial primitive streak; and (3) Hensen's node is derived from a mixture of cells originating both from the epiblast just rostral to the incipient (stage 2) primitive streak and later from the epiblast just rostral to the elongating (stage 3a/b) primitive streak, as well as from the rostral tip of the progressing streak itself. Collectively, these results provide new information on the formation of the avian primitive streak and organizer, increasing our understanding of these important events of early development of amniotes.     

Differential response of epiblast stem cells to Nodal and Activin signalling: a paradigm of early endoderm development in the embryo

Mouse epiblast stem cells (EpiSCs) display temporal differences in the upregulation of Mixl1 expression during the initial steps of in vitro differentiation, which can be correlated with their propensity for endoderm differentiation. EpiSCs that upregulated Mixl1 rapidly during differentiation responded robustly to both Activin A and Nodal in generating foregut endoderm and precursors of pancreatic and hepatic tissues. By contrast, EpiSCs that delayed Mixl1 upregulation responded less effectively to Nodal and showed an overall suboptimal outcome of directed differentiation. The enhancement in endoderm potency in Mixl1-early cells may be accounted for by a rapid exit from the progenitor state and the efficient response to the induction of differentiation by Nodal. EpiSCs that readily differentiate into the endoderm cells are marked by a distinctive expression fingerprint of transforming growth factor (TGF)-β signalling pathway genes and genes related to the endoderm lineage. Nodal appears to elicit responses that are associated with transition to a mesenchymal phenotype, whereas Activin A promotes gene expression associated with maintenance of an epithelial phenotype. We postulate that the formation of definitive endoderm (DE) in embryoid bodies follows a similar process to germ layer formation from the epiblast, requiring an initial de-epithelialization event and subsequent re-epithelialization. Our results show that priming EpiSCs with the appropriate form of TGF-β signalling at the formative phase of endoderm differentiation impacts on the further progression into mature DE-derived lineages, and that this is influenced by the initial characteristics of the cell population. Our study also highlights that Activin A, which is commonly used as an in vitro surrogate for Nodal in differentiation protocols, does not elicit the same downstream effects as Nodal, and therefore may not effectively mimic events that take place in the mouse embryo.     

A morphogenetic wave in the chick embryo lateral mesoderm generates mesenchymal-epithelial transition through a 3D-rosette intermediate

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5-Bromodeoxyuridine inhibition of the epiblast competence for primitive streak formation in the young chick blastoderm

The competence of stage XIII chick epiblast which under the influence of an inductive hypoblast is directed to form a normal primitive streak, is affected by 5-bromodeoxyuridine (BUdR). The BUdR-treated epiblast forms an atypical primitive streak and no axial mesoderm. However, a nonorganized mesenchymal layer is formed between the epiblast and the hypoblast, and atypical neural tissue in the epiblast. BUdR interferes neither with hypoblast formation nor with its inductivity even when blastoderms are treated with BUdR as early as uterine stage VIII and later.     

Nodal cis-regulatory elements reveal epiblast and primitive endoderm heterogeneity in the peri-implantation mouse embryo

Nodal, a secreted factor known for its conserved functions in cell-fate specification and the establishment of embryonic axes, is also required in mammals to maintain the pluripotency of the epiblast, the tissue that gives rise to all fetal lineages. Although Nodal is expressed as early as E3.5 in the mouse embryo, its regulation and functions at pre- and peri-implantation stages are currently unknown. Sensitive reporter transgenes for two Nodal cis-regulatory regions, the PEE and the ASE, exhibit specific expression profiles before implantation. Mutant and inhibitor studies find them respectively regulated by Wnt/β-catenin signaling and Activin/Nodal signaling, and provide evidence for localized and heterogeneous activities of these pathways in the inner cell mass, the epiblast and the primitive endoderm. These studies also show that Nodal and its prime effector, FoxH1, are not essential to preimplantation Activin/Nodal signaling. Finally, a strong upregulation of the ASE reporter in implanting blastocysts correlates with a downregulation of the pluripotency factor Nanog in the maturing epiblast. This study uncovers conservation in the mouse blastocyst of Wnt/β-catenin and Activin/Nodal-dependent activities known to govern Nodal expression and the establishment of polarity in the blastula of other deuterostomes. Our results indicate that these pathways act early on to initiate distinct cell-specification processes in the ICM derivatives. Our data also suggest that the activity of the Activin/Nodal pathway is dampened by interactions with the molecular machinery of pluripotency until just before implantation, possibly delaying cell-fate decisions in the mouse embryo.     

E-cadherin is required for gastrulation cell movements in zebrafish

E-cadherin is a member of the classical cadherin family and is known to be involved in cell-cell adhesion and the adhesion-dependent morphogenesis of various tissues. We isolated a zebrafish mutant (cdh1(rk3)) that has a mutation in the e-cadherin/cdh1 gene. The mutation rk3 is a hypomorphic allele, and the homozygous mutant embryos displayed variable phenotypes in gastrulation and tissue morphogenesis. The most severely affected embryos displayed epiboly delay, decreased convergence and extension movements, and the dissociation of cells from the embryos, resulting in early embryonic lethality. The less severely affected embryos survived through the pharyngula stage and showed flattened anterior neural tissue, abnormal positioning and morphology of the hatching gland, scattered trigeminal ganglia, and aberrant axon bundles from the trigeminal ganglia. Maternal-zygotic cdh1(rk3) embryos displayed epiboly arrest during gastrulation, in which the enveloping layer (EVL) and the yolk syncytial layer but not the deep cells (DC) completed epiboly. A similar phenotype was observed in embryos that received antisense morpholino oligonucleotides (cdh1MO) against E-cadherin, and in zebrafish epiboly mutants. Complementation analysis with the zebrafish epiboly mutant weg suggested that cdh1(rk3) is allelic to half baked/weg. Immunohistochemistry with an anti-beta-catenin antibody and electron microscopy revealed that adhesion between the DCs and the EVL was mostly disrupted but the adhesion between DCs was relatively unaffected in the MZcdh1(rk3) mutant and cdh1 morphant embryos. These data suggest that E-cadherin-mediated cell adhesion between the DC and EVL plays a role in the epiboly movement in zebrafish.     

Mesoderm-independent regulation of gastrulation movements by the Src tyrosine kinase in Xenopus embryo

In vitro studies have demonstrated the involvement of Src kinases in several aspects of cell scattering, including cell dissociation and motility. We have therefore sought to explore their functions in the context of the whole organism. Loss-of-function microinjection studies indicate that the ubiquitous Src, Fyn, and Yes tyrosine kinases are specifically implicated in Xenopus gastrulation movements. Injection of mRNAs coding for dominant negative forms of the ubiquitous members of the Src family, namely Fyn, Src, and Yes, perturbs gastrulation movements, resulting in the inability to close the blastopore. Injection of mRNA coding for Csk, a natural inhibitor of Src kinase activity, produces the same phenotypic alterations. The ubiquitous Src kinases have redundant functions in gastrulation movements since overexpression of one member of the family can compensate for the inhibition of another. Interfering mutants of the Src family also inhibit activin-induced morphogenetic movements of animal cap explants isolated from injected embryos. In contrast, these mutants do not interfere with mesoderm induction, as inferred from the presence of mesoderm derivatives and from the expression of early mesodermal markers in injected embryos. In addition, Src kinase activity measured by an in vitro kinase assay is elevated in gastrulating embryos and in FGF- and activin-treated animal caps, confirming the implication of Src enzymatic activity during gastrulation. Altogether, our results demonstrate that Src kinases are essential components of the machinery that drives gastrulation movements independent of mesoderm induction and suggest that Src activity is primarily implicated in cellular movements that take place during the process of cell intercalation.     

15-zinc finger protein Bloody Fingers is required for zebrafish morphogenetic movements during neurulation

A novel zebrafish gene bloody fingers (blf) encoding a 478 amino acid protein containing fifteen C(2)H(2) type zinc fingers was identified by expression screening. As determined by in situ hybridization, blf RNA displays strong ubiquitous early zygotic expression, while during late gastrulation and early somitogenesis, blf expression becomes transiently restricted to the posterior dorsal and lateral mesoderm. During later somitogenesis, blf expression appears only in hematopoietic cells. It is completely eliminated in cloche, moonshine but not in vlad tepes (gata1) mutant embryos. Morpholino (MO) knockdown of the Blf protein results in the defects of morphogenetic movements. Blf-MO-injected embryos (morphants) display shortened and widened axial tissues due to defective convergent extension. Unlike other convergent extension mutants, blf morphants display a split neural tube, resulting in a phenotype similar to the human open neural tube defect spina bifida. In addition, dorsal ectodermal cells delaminate in blf morphants during late somitogenesis. We propose a model explaining the role of blf in convergent extension and neurulation. We conclude that blf plays an important role in regulating morphogenetic movements during gastrulation and neurulation while its role in hematopoiesis may be redundant.     

Activation of epiblast gene expression by the hypoblast layer in the prestreak chick embryo

Axis formation is a highly regulated process in vertebrate embryos. In mammals, inductive interactions between an extra-embryonic layer, the visceral endoderm, and the embryonic layer before gastrulation are critical both for anterior neural patterning and normal primitive streak formation. The role(s) of the equivalent extra-embryonic endodermal layer in the chick, the hypoblast, is still less clear, and dramatic effects of hypoblast on embryonic gene expression have yet to be demonstrated. We present evidence that two genes later associated with the gastrula organizer (Gnot-1 and Gnot-2) are induced by hypoblast signals in prestreak embryos. The significance of this induction by hypoblast is discussed in terms of possible hypoblast functions and the regulation of axis formation in the early embryo. Several factors known to be expressed in hypoblast, and retinoic acid, synergistically induce Gnot-1 and Gnot-2 expression in blastoderm cell culture. The presence of retinoic acid in prestreak embryos has not yet been directly demonstrated, but exogenous retinoic acid appears to mimic the effects of hypoblast rotation on primitive streak extension, raising the possibility that retinoid signaling plays some role in the pregastrula embryo.     

Xenopus Tetraspanin-1 regulates gastrulation movements and neural differentiation in the early Xenopus embryo

The tetraspanin family of four-pass transmembrane proteins has been implicated in fundamental biological processes, including cell adhesion, migration, and proliferation. Tetraspanins interact with various transmembrane proteins, establishing a network of large multimolecular complexes that allows specific lateral secondary interactions. Here we report the identification and functional characterization of Xenopus Tetraspanin-1 (xTspan-1). At gastrula and neurula, xTspan-1 is expressed in the dorsal ectoderm and neural plate, respectively, and in the hatching gland, cement gland, and posterior neural tube at tailbud stages. The expression of xTspan-1 in the early embryo is negatively regulated by bone morphogenetic protein (BMP) and stimulated by Notch signals. Microinjection of xTspan-1 mRNA interfered with gastrulation movements and reduced ectodermal cell adhesion in a cadherin-dependent manner. Morpholino knock-down of endogenous xTspan-1 protein revealed a requirement of xTspan-1 for gastrulation movements and primary neurogenesis. Our data suggest that xTspan-1 could act as a molecular link between BMP signalling and the regulation of cellular interactions that are required for gastrulation movements and neural differentiation in the early Xenopus embryo.     

Patterns of protein synthesis in the chick blastula: A comparison of the component areas of the epiblast and the primary hypoblast

The patterns of protein synthesis are examined in the hypoblast and in the areas that comprise the epiblast, that is, the area opaca, the marginal zone, and the central area, during the blastula stage which marks the beginning of the interaction between the epiblast and hypoblast for induction of the primitive streak. The results demonstrate that there are distinct qualitative and quantitative differences in protein patterns in individual areas of blastoderm, the differences being most distinct between the hypoblast and any of the component areas of the epiblast. These differences in patterns of proteins suggest that the component areas of the chick blastula have already diverged to different developmental fates before any apparent morphogenetic differentiation, that is, the appearance of the primitive streak.     

Xoom is required for epibolic movement of animal ectodermal cells in Xenopus laevis gastrulation

Gastrulation is the most dynamic cell movement and initiates the body plan in amphibian development. In contrast to numerous molecular studies on mesodermal induction, the driving force of gastrulation is as yet poorly understood. A novel transmembrane protein, Xoom, was previously reported, which is required for Xenopus gastrulation. In the present study, the role of Xoom during Xenopus gastrulation was further examined in detail. Overexpression and misexpression of Xoom induced overproduction of Xoom protein, but not a changed phenotype. However, Xoom antisense ribonucleic acid (RNA) injection reduced the Xoom protein and caused gastrulation defects without any influence on the involution and translation levels of mesodermal marker genes. Normal migrating activity of dorsal mesodermal cells was recognized in the antisense RNA-injected explant. Morphological examination using artificial exogastrulation showed that convergent extension of mesodermal cells occurred normally, but the ectodermal cell layer significantly shrank in the antisense RNA-injected embryo. Comparison of cell shape among various experimental conditions showed that inhibition of cell spreading occurs specifically in the outer ectodermal layer of the antisense RNA-injected embryo. Cytochemical examination indicated disorganization of F-actin in the ectodermal cells of the antisense RNA-injected embryo. These results suggest that Xoom plays an important role in the epibolic movement of ectodermal cells through some regulation of actin filament organization.     

Wnt3 signaling in the epiblast is required for proper orientation of the anteroposterior axis

The establishment of anteroposterior (AP) polarity in the early mouse epiblast is crucial for the initiation of gastrulation and the subsequent formation of the embryonic (head to tail) axis. The localization of anterior and posterior determining genes to the appropriate region of the embryo is a dynamic process that underlies this early polarity. Several studies indicate that morphological and molecular markers which define the early AP axis are first aligned along the short axis of the elliptical egg cylinder. Subsequently, just prior to the time of primitive streak formation, a conformational change in the embryo realigns these markers with the long axis. We demonstrate that embryos lacking the signaling factor Wnt3 exhibit defects in this axial realignment. In addition, chimeric analyses and conditional removal of Wnt3 activity reveal that Wnt3 expression in the epiblast is required for induction of the primitive streak and mesoderm whereas activity in the posterior visceral endoderm is dispensable.     

Hypoblast cells of chick pre-streak stage embryos invaded basement membrane analogues in vitro: Implications for hypoblast layer formation

In early chick blastodermal morphogenesis, the hypoblast layer is organized beneath the epiblast and induces an axial structure. However, the origin of hypoblast cells and the mechanism of hypoblast layer formation are poorly understood. We hypothesized that the hypoblast layer is formed by an invasive process across the basement membrane of the juxtaposing epiblast, and tested the idea in vitro . Primary and secondary hypoblast cells from embryos at various pre-streak stages were dissociated into single cells and cultured on reconstituted basement membrane gel, laminin gel or fibronectin gel in the culture medium with or without serum for 24–48 h. As a result, we found that after 24 h of serum-supplemented culture, up to 35% of the hypoblast cells dissolved the gel and made holes on it. Similarly, up to 36% of the hypoblast cells showed invasiveness after 48 h in the serum-free culture. Furthermore, it was observed that Koller's sickle cells, which are regarded to be the progenitors of secondary hypoblast cells, penetrated those gels on which they were seeded. The posterior epiblast cells covering Koller's sickle were also invasive. These results suggest that the presumptive primary hypoblast cells that are known to mingle with epiblast cells invade through the basement membrane to form the hypoblast layer. Furthermore, the present results imply that invasion through the basement membrane may be involved in the formation of Koller's sickle, the anlage of secondary hypoblast.     

Significance of bone morphogenetic protein-4 function in the initial myofibrillogenesis of chick cardiogenesis

The heart is the first organ to form and function during vertebrate embryogenesis. Using a secreted protein, noggin, which specifically antagonizes bone morphogenetic protein (BMP)-2 and -4, we examined the role played by BMP during the initial myofibrillogenesis in chick cultured precardiac mesoendoderm (mesoderm + endoderm; ME). Conditioned medium from COS7 cells transfected with Xenopus noggin cDNA inhibited the expression of sarcomeric proteins (such as sarcomeric alpha-actinin, Z-line titin, and sarcomeric myosin), and so myofibrillogenesis was perturbed in cultured stage 4 precardiac ME; however, it did not inhibit the expression of smooth muscle alpha-actin (the first isoform of alpha-actin expressed during cardiogenesis). In cultured stage 5 precardiac ME, noggin did not inhibit either the formation of I-Z-I components or the expression of sarcomeric myosin, but it did inhibit the formation of A-bands. Although BMP4 was required to induce expressions of sarcomeric alpha-actinin, titin, and sarcomeric myosin in cultured stage 6 posterolateral mesoderm (noncardiogenic mesoderm), smooth muscle alpha-actin was expressed without the addition of BMP4. Interestingly, in cultured stage 6 posterolateral mesoderm, BMP2 induced the expressions of sarcomeric alpha-actinin and titin, but not of sarcomeric myosin. These results suggest that (1) BMP4 function lies upstream of the initial formation of I-Z-I components and A-bands separately in a stage-dependent manner, and (2) at least two signaling pathways are involved in the initial cardiac myofibrillogenesis: one is an unknown pathway responsible for the expression of smooth muscle alpha-actin; the other is BMP signaling, which is involved in the expression of sarcomeric alpha-actinin, titin, and sarcomeric myosin.     

Nodal signaling is required for closure of the anterior neural tube in zebrafish

Background  

Nodals are secreted signaling proteins with many roles in vertebrate development. Here, we identify a new role for Nodal signaling in regulating closure of the rostral neural tube of zebrafish.     

Asymmetric Nodal expression in the mouse is governed by the combinatorial activities of two distinct regulatory elements

In all vertebrates, invariant left/right (L/R) positioning and organization of the internal viscera is controlled by a conserved pathway. Nodal, a member of the TGFbeta superfamily is a critical upstream component responsible for initiating L/R axis determination. Asymmetric Nodal expression in the node preceeds and foreshadows morphological L/R asymmetry. Here we address the mechanism of Nodal activation in the left LPM by studying the function of a novel enhancer element, the AIE. We show this element is exclusively active in cells of the left lateral plate mesoderm (LPM) and is not itself responding to Nodal asymmetry. To test the hypothesis that this element may initiate asymmetric Nodal expression in the LPM, we deleted it from the mouse germ line. Mice homozygous for the AIE deletion (Nodal(deltaaie/deltaaie)) show no defects. However, we find that the AIE contributes to regulating the level of asymmetric Nodal activity; analysis of transheterozygous embryos (Nodal(deltaaie/null)) shows reduced Nodal expression in the left LPM associated with a low penetrance of L/R defects. Our findings point to the existence of two independent pathways that control Nodal expression in the left LPM.