Effects of ethanol on the primitive streak stage mouse embryo

Recent studies of mouse models have suggested that malformations associated with the fetal alcohol syndrome (FAS) are caused by the effects of ethanol on early embryos during gastrulation and neurulation. A study of Xenopus laevis embryos showed that exposure of gastrula stage amphibian embryos to ethanol inhibits migration of the mesodermal cells, causes formation of small neural plates, and subsequently causes hypoplastic craniofacial malformations in tadpoles. We now report effects of ethanol on the primitive streak stage mouse embryos. An ethanol solution (25%) was injected intraperitonealy twice into mice of 6.5-7.0 days of pregnancy at a dose of 0.015 ml/gm of body weight. Histological and morphometric examinations of 7.5-day embryos, 20 hr after the second injection, showed that the epiblast layer was disorganized and shrunk with formation of many blebs. In addition, formation of the mesodermal cell layer was retarded in the ethanol-treated embryos, suggesting that exposure of gastrula stage embryos to ethanol causes similar abnormalities in mouse and Xenopus embryos. These results suggest that the inhibition of the morphogenetic movements during gastrulation may be the primary effect of ethanol in causing major craniofacial malformations of FAS.     

Conditional activation of RhoA suppresses the epithelial to mesenchymal transition at the primitive streak during mouse gastrulation

Gastrulation is a pivotal event of mouse early embryogenesis. In telencephalin (TLCN)-Cre mice carrying the Cre recombinase gene inserted into the translational initiation site of the TLCN gene, Cre-mediated recombination took place at the postimplantation stage. To examine the role of RhoA signaling in early embryogenesis, we produced Rho36 mice carrying constitutively active RhoA(G14V) gene inducible by Cre recombinase and crossed with TLCN-Cre mice. In doubly transgenic embryos at the gastrulation stage, there appeared an abnormal bulge of cells protruded from the primitive streak region into the amniotic cavity. The bulged cell mass expressed the epiblast marker gene Oct3 and E-cadherin, but not the primitive streak marker gene T except for the basal portion. These results suggest that the conditional activation of RhoA signaling suppressed the epithelial to mesenchymal transition at the primitive streak during mouse gastrulation.     

Introduction of cell markers into germ layer tissues of the mouse gastrula by whole embryo electroporation

We have optimized the technique of electroporation for introducing genetic markers into cells of the gastrulating mouse embryo to follow cell fates, tissue movement, and lineage differentiation. Using a plate-needle electrode combination and specific route of plasmid delivery, labeling could be targeted to discrete regions of the epiblast or the endoderm of the late gastrula. Among the various types of fluorescent and chromogenic reporter constructs tested, those driven by CMV promoter are efficient and strong expression can be detected as soon as 2-3 h after electroporation. The efficacy of marking cell lineages by CRE-mediated activation of reporters proved to be inefficient for tracking cell lineages due to an obligatory 8-9-h lag from the electroporation of constructs to the expression of reporter. This significant time lag also raises concern of the temporal precision at which tissue- or stage-specific knock-out or activation of genetic activity may be achieved by the Cre-loxP mechanism.     

Analysis of mouse Evx genes: Evx-1 displays graded expression in the primitive streak.

Two mouse genes, Evx-1 and Evx-2, each encoding a homeodomain closely related to that of the Drosophila even-skipped gene were isolated using a PCR-based strategy. The structure and sequence of these genes are described. Mapping studies localized Evx-1 to chromosome 6, near the Hox-1 gene cluster, and Evx-2 to chromosome 2, near the Hox-4 cluster. The evolutionary implications of these linkages are discussed. RNA in situ hybridization analysis of Evx expression in embryos demonstrated a striking pattern of Evx-1 expression during gastrulation, whereas Evx-2 RNA could not be detected at any stage by this technique. Evx-1 RNA is first detected shortly before the onset of gastrulation in a region of ectoderm containing cells that will soon be found in the primitive streak. This localized expression of Evx-1 provides the first molecular evidence for regional differences in the mouse embryonic ectoderm before gastrulation. Throughout gastrulation, Evx-1 expression is limited to cells near and within the streak and that expression is graded, with a posterior-to-anterior decrease in the level of RNA. Based on fate-mapping studies indicating that different types of mesoderm emerge from different regions of the primitive streak and our observation that high levels of expression are localized to the region that will give rise to extraembryonic and ventral mesoderm, we speculate that Evx-1 plays a role in the dorsoventral specification of mesodermal cell fate.     

A model of primitive streak initiation in the chick embryo

Initiation of the primitive streak in avian embryos provides a well-studied example of a pattern-forming event that displays a striking capacity for regulation. The mechanisms underlying the regulative properties are, however, poorly understood and are not easily accounted for by traditional models of pattern formation, such as reaction-diffusion models. In this paper, we propose a new activator-inhibitor model for streak initiation. We show that the model is consistent with experimental observations, both in its pattern-forming properties and in its ability to form these patterns on the correct time-scales for biologically realistic parameter values. A key component of the model is a travelling wave of inhibition. We present a mathematical analysis of the speed of such waves in both diffusive and juxtacrine relay systems. We use the streak initiation model to make testable predictions. By varying parameters of the model, two very different types of patterning can be obtained, suggesting that our model may be applicable to other processes in addition to streak initiation.     

The somitogenetic potential of cells in the primitive streak and the tail bud of the organogenesis-stage mouse embryo.

The developmental potency of cells isolated from the primitive streak and the tail bud of 8.5- to 13.5-day-old mouse embryos was examined by analyzing the pattern of tissue colonization after transplanting these cells to the primitive streak of 8.5-day embryos. Cells derived from these progenitor tissues contributed predominantly to tissues of the paraxial and lateral mesoderm. Cells isolated from older embryos could alter their segmental fate and participated in the formation of anterior somites after transplantation to the primitive streak of 8.5-day host embryo. There was, however, a developmental lag in the recruitment of the transplanted cells to the paraxial mesoderm and this lag increased with the extent of mismatch of developmental ages between donor and host embryos. It is postulated that certain forms of cell-cell or cell-matrix interaction are involved in the specification of segmental units and that there may be age-related variations in the interactive capability of the somitic progenitor cells during development. Tail bud mesenchyme isolated from 13.5-day embryos, in which somite formation will shortly cease, was still capable of somite formation after transplantation to 8.5-day embryos. The cessation of somite formation is therefore likely to result from a change in the tissue environment in the tail bud rather than a loss of cellular somitogenetic potency.     

Epithelial and basement membrane responses to chick embryo primitive streak grafts

Chick embryo primitive streak grafts, placed beneath the epiblast of host embryos, tend to result in the formation of either a neural plate in response to anterior streak grafts, or in de-epithelialization in response to posterior grafts. Ultrastructural and immunocytochemical examination shows that both reactions are preceded by basement membrane disruption and early removal of fibronectin therefrom. This disruption does not occur in response to non-streak grafts. It is suggested that the disruption, evoked by primitive streak cells, is a prerequisite first step, allowing direct graft-epiblast cell contact. This contact elicits a specific cytoskeletal reaction determining the epiblast response.     

Antagonism of Nodal signaling by BMP/Smad5 prevents ectopic primitive streak formation in the mouse amnion

The strength and spatiotemporal activity of Nodal signaling is tightly controlled in early implantation mouse embryos, including by autoregulation and feedback loops, and involves secreted and intracellular antagonists. These control mechanisms, which are established at the extra-embryonic/embryonic interfaces, are essential for anterior-posterior patterning of the epiblast and correct positioning of the primitive streak. Formation of an ectopic primitive streak, or streak expansion, has previously been reported in mutants lacking antagonists that target Nodal signaling. Here, we demonstrate that loss-of-function of a major bone morphogenetic protein (BMP) effector, Smad5, results in formation of an ectopic primitive streak-like structure in mutant amnion accompanied by ectopic Nodal expression. This suggests that BMP/Smad5 signaling contributes to negative regulation of Nodal. In cultured cells, we find that BMP-activated Smad5 antagonizes Nodal signaling by interfering with the Nodal-Smad2/4-Foxh1 autoregulatory pathway through the formation of an unusual BMP4-induced Smad complex containing Smad2 and Smad5. Quantitative expression analysis supports that ectopic Nodal expression in the Smad5 mutant amnion is induced by the Nodal autoregulatory loop and a slow positive-feedback loop. The latter involves BMP4 signaling and also induction of ectopic Wnt3. Ectopic activation of these Nodal feedback loops in the Smad5 mutant amnion results in the eventual formation of an ectopic primitive streak-like structure. We conclude that antagonism of Nodal signaling by BMP/Smad5 signaling prevents primitive streak formation in the amnion of normal mouse embryos.     

Anterior neural plate regionalization in cripto null mutant mouse embryos in the absence of node and primitive streak

The relation between the role of the organizer at the gastrula stage and the activity of earlier signals in the specification, maintenance, and regionalization of the developing brain anlage is still controversial. Mouse embryos homozygous for null mutation in the cripto gene die at about 9.0 days postcoitum (d.p.c.) and fail to gastrulate and to form the node (the primary organizer). Here, we study the presence and the distribution of anterior neural plate molecular domains in cripto null mutants. We demonstrate that, in cripto(-/-) embryos, the main prosencephalic and mesencephalic regions are present and that they assume the correct topological organization. The identity of the anterior neural domains is maintained in mutant embryos at 8.5 d.p.c., as well as in mutant explants dissected at 8.5 d.p.c. and cultured in vitro for 24 h. Our data imply the existence of a stable neural regionalization of anterior character inside the cripto(-/-) embryos, despite the failure in both the gastrulation process and node formation. These results suggest that, in mouse embryos, the specification of the anterior neural identities can be maintained without an absolute requirement for the embryonic mesoderm and the node.     

Correct patterning of the primitive streak requires the anterior visceral endoderm

Anterior-posterior axis specification in the mouse requires signalling from a specialised extra-embryonic tissue called the anterior visceral endoderm (AVE). AVE precursors are induced at the distal tip of the embryo and move to the prospective anterior. Embryological and genetic analysis has demonstrated that the AVE is required for anterior patterning and for correctly positioning the site of primitive streak formation by inhibiting Nodal activity. We have carried out a genetic ablation of the Hex-expressing cells of the AVE (Hex-AVE) by knocking the Diphtheria toxin subunit A into the Hex locus in an inducible manner. Using this model we have identified that, in addition to its requirement in the anterior of the embryo, the Hex-AVE sub-population has a novel role between 5.5 and 6.5dpc in patterning the primitive streak. Embryos lacking the Hex-AVE display delayed initiation of primitive streak formation and miss-patterning of the anterior primitive streak. We demonstrate that in the absence of the Hex-AVE the restriction of Bmp2 expression to the proximal visceral endoderm is also defective and expression of Wnt3 and Nodal is not correctly restricted to the posterior epiblast. These results, coupled with the observation that reducing Nodal signalling in Hex-AVE ablated embryos increases the frequency of phenotypes observed, suggests that these primitive streak patterning defects are due to defective Nodal signalling. Together, our experiments demonstrate that the AVE is not only required for anterior patterning, but also that specific sub-populations of this tissue are required to pattern the posterior of the embryo.     

Formation of the chick primitive streak as studied in computer simulations

We have used a computer simulation system to examine formation of the chick primitive streak and to test the proposal (Wei and Mikawa Development 127 (2000) 87) that oriented cell division could account for primitive streak elongation. We find that this proposal is inadequate to explain elongation of the streak. In contrast, a correctly patterned model streak can be generated if two putative mechanisms are operative. First, a subpopulation of precursor cells that is known to contribute to the streak is assigned a specific, but simple, movement pattern. Second, additional cells within the epiblast are allowed to incorporate into the streak based on near-neighbor relations. In this model, the streak is cast as a steady-state system with continuous recruitment of neighboring epiblast cells, egress of cells into deeper layers and an internal pattern of cell movement. The model accurately portrays elongation and maintenance of a robust streak, changes in the composition of the streak and defects in the streak after experimental manipulation.