Rho kinase activity controls directional cell movements during primitive streak formation in the rabbit embryo.

During animal gastrulation, the specification of the embryonic axes is accompanied by epithelio-mesenchymal transition (EMT), the first major change in cell shape after fertilization. EMT takes place in disparate topographical arrangements, such as the circular blastopore of amphibians, the straight primitive streak of birds and mammals or in intermediate gastrulation forms of other amniotes such as reptiles. Planar cell movements are prime candidates to arrange specific modes of gastrulation but there is no consensus view on their role in different vertebrate classes. Here, we test the impact of interfering with Rho kinase-mediated cell movements on gastrulation topography in blastocysts of the rabbit, which has a flat embryonic disc typical for most mammals. Time-lapse video microscopy, electron microscopy, gene expression and morphometric analyses of the effect of inhibiting ROCK activity showed - besides normal specification of the organizer region - a dose-dependent disruption of primitive streak formation; this disruption resulted in circular, arc-shaped or intermediate forms, reminiscent of those found in amphibians, fishes and reptiles. Our results reveal a crucial role of ROCK-controlled directional cell movements during rabbit primitive streak formation and highlight the possibility that temporal and spatial modulation of cell movements were instrumental for the evolution of gastrulation forms.

Acetylcholine receptors regulate gene expression that is essential for primitive streak formation in murine embryoid bodies.

Muscarinic acetylcholine receptors (mAchRs) are critical components of the cholinergic system, which is the key regulator of both the central and peripheral nervous systems in mammals. Interestingly, several components of the cholinergic system, including mAchRs and choline acetyltransferase (ChAT), have recently been found to be expressed in mouse embryonic stem (ES) cells and human placenta. These results raise the intriguing possibility that mAchRs play physiological roles in the regulation of early embryogenesis. Early embryogenesis can be mimicked in vitro using an ES cell-based culture system in which the cells form a primitive streak-like structure and efficiently develop into mesodermal progenitors. Here we report that chemical inhibitors specifically targeting mAchRs suppressed the expression of genes essential for primitive streak formation, including Wnt3, and thereby blocked mesodermal progenitor differentiation. Interestingly, mAchR inhibitors also reduced the expression of Cyp26a1, an enzyme involved in the catabolism of retinoic acid (RA). RA is an important regulator of Wnt3 signaling. Our study presents evidence indicating that mAchRs influence RA signaling necessary for the induction of the primitive streak. To our knowledge, this is the first report showing that mAchRs have important functions not only in adult mammals but also during early mammalian embryogenesis.

Directed induction of anterior and posterior primitive streak by Wnt from embryonic stem cells cultured in a chemically defined serum-free medium.

Formation of the primitive streak (PS) is the initial specification step that generates all the mesodermal and endodermal tissue lineages during early differentiation. Thus, a therapeutically compatible and efficient method for differentiation of the PS is crucial for regenerative medicine. In this study, we developed chemically defined serum-free culture conditions for the differentiation of embryonic stem (ES) cells into the PS-like cells. Cultures supplemented with Wnt showed induction of expression of a PS marker, the brachyury gene, followed by induction of the anterior PS markers goosecoid and foxa2, a posterior PS marker, evx1, and the endoderm marker sox17. Similar differentiation of PS by Wnt was also observed in human ES cells. Moreover, we revealed that the activation of the Wnt canonical pathway is essential for PS differentiation in mouse ES cells. These results demonstrated that Wnt is an essential and sufficient factor for the induction of the PS-like cells in vitro. These conditions of induction could constitute the initial step in generating therapeutically useful cells of the definitive endoderm lineage, such as hepatocytes and pancreatic endocrine cells, under chemically defined conditions.

Modulation of cell surface architecture in gastrulating chick embryo in response to altered fibroblast growth factor (FGF) signaling.

Gastrulation is a fundamental process that results in formation of the three germ layers in an embryo. It involves highly coordinated cell migration. Cell to cell communication through cell surface and the surrounding molecular environment governs cell migration. In the present work, cell surface features, which are indicative of the migratory status of a cell, of an early gastrulating chick embryo were studied using scanning electron microscopy. The distinct ultrastructural features of cells located in the various regions of the epiblast are described. Differences in the surface features of cells from distinct embryonic regions indicate differences in their migratory capacities. Further, the dynamic nature of these cell surface features by their response to altered fibroblast growth factor (FGF) signaling, experimentally created by using either excess FGF or inhibition of FGF signaling are demonstrated.

Bmp indicator mice reveal dynamic regulation of transcriptional response.

Cellular responses to Bmp ligands are regulated at multiple levels, both extracellularly and intracellularly. Therefore, the presence of these growth factors is not an accurate indicator of Bmp signaling activity. While a common approach to detect Bmp signaling activity is to determine the presence of phosphorylated forms of Smad1, 5 and 8 by immunostaining, this approach is time consuming and not quantitative. In order to provide a simpler readout system to examine the presence of Bmp signaling in developing animals, we developed BRE-gal mouse embryonic stem cells and a transgenic mouse line that specifically respond to Bmp ligand stimulation. Our reporter identifies specific transcriptional responses that are mediated by Smad1 and Smad4 with the Schnurri transcription factor complex binding to a conserved Bmp-Responsive Element (BRE), originally identified among Drosophila, Xenopus and human Bmp targets. Our BRE-gal mES cells specifically respond to Bmp ligands at concentrations as low as 5 ng/ml; and BRE-gal reporter mice, derived from the BRE-gal mES cells, show dynamic activity in many cellular sites, including extraembryonic structures and mammary glands, thereby making this a useful scientific tool.

Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/beta-catenin, Activin/Nodal and BMP signaling.

The canonical Wnt/beta-catenin signaling has remarkably diverse roles in embryonic development, stem cell self-renewal and cancer progression. Here, we show that stabilized expression of beta-catenin perturbed human embryonic stem (hES)-cell self-renewal, such that up to 80% of the hES cells developed into the primitive streak (PS)/mesoderm progenitors, reminiscent of early mammalian embryogenesis. The formation of the PS/mesoderm progenitors essentially depended on the cooperative action of beta-catenin together with Activin/Nodal and BMP signaling pathways. Intriguingly, blockade of BMP signaling completely abolished mesoderm generation, and induced a cell fate change towards the anterior PS progenitors. The PI3-kinase/Akt, but not MAPK, signaling pathway had a crucial role in the anterior PS specification, at least in part, by enhancing beta-catenin stability. In addition, Activin/Nodal and Wnt/beta-catenin signaling synergistically induced the generation and specification of the anterior PS/endoderm. Taken together, our findings clearly demonstrate that the orchestrated balance of Activin/Nodal and BMP signaling defines the cell fate of the nascent PS induced by canonical Wnt/beta-catenin signaling in hES cells.