Activin/Nodal signalling before implantation: setting the stage for embryo patterning.

International audienceActivins and Nodal are members of the transforming growth factor beta (TGF-β) family of growth factors. Their Smad2/3-dependent signalling pathway is well known for its implication in the patterning of the embryo after implantation. Although this pathway is active early on at preimplantation stages, embryonic phenotypes for loss-of-function mutations of prominent components of the pathway are not detected before implantation. It is only fairly recently that an understanding of the role of the Activin/Nodal signalling pathway at these stages has started to emerge, notably from studies detailing how it controls the expression of target genes in embryonic stem cells. We review here what is currently known of the TGF-β-related ligands that determine the activity of Activin/Nodal signalling at preimplantation stages, and recent advances in the elucidation of the Smad2/3-dependent mechanisms underlying developmental progression

A novel Nodal enhancer dependent on pluripotency factors and Smad2/3 signaling conditions a regulatory switch during epiblast maturation

During early development, modulations in the expression of Nodal, a TGFβ family member, determine the specification of embryonic and extra-embryonic cell identities. Nodal has been extensively studied in the mouse, but aspects of its early expression remain unaccounted for. We identified a conserved hotspot for the binding of pluripotency factors at the Nodal locus and called this sequence “highly bound element” (HBE). Luciferase-based assays, the analysis of fluorescent HBE reporter transgenes, and a conditional mutation of HBE allowed us to establish that HBE behaves as an enhancer, is activated ahead of other Nodal enhancers in the epiblast, and is essential to Nodal expression in embryonic stem cells (ESCs) and in the mouse embryo. We also showed that HBE enhancer activity is critically dependent on its interaction with the pluripotency factor Oct4 and on Activin/Nodal signaling. Use of an in vitro model of epiblast maturation, relying on the differentiation of ESCs into epiblast stem cells (EpiSCs), revealed that this process entails a shift in the regulation of Nodal expression from an HBE-driven phase to an ASE-driven phase, ASE being another autoregulatory Nodal enhancer. Deletion of HBE in ESCs or in EpiSCs allowed us to show that HBE, although not necessary for Nodal expression in EpiSCs, is required in differentiating ESCs to activate the differentiation-promoting ASE and therefore controls this regulatory shift. Our findings clarify how early Nodal expression is regulated and suggest how this regulation can promote the specification of extra-embryonic precusors without inducing premature differentiation of epiblast cells. More generally, they open new perspectives on how pluripotency factors achieve their function

Development of a Method for the In Vivo Generation of Allogeneic Hearts in Chimeric Mouse Embryos

Worldwide, there is a great gap between the demand and supply of organs for transplantations. Organs generated from the patients’ cells would not only solve the problem of transplant availability but also overcome the complication of incompatibility and tissue rejection by the host immune system. One of the most promising methods tested for the production of organs in vivo is blastocyst complementation (BC). Regrettably, BC is not suitable for the creation of hearts. We have developed a novel method, induced blastocyst complementation (iBC), to surpass this shortcoming. By applying iBC, we generated chimeric mouse embryos, made up of “host” and “donor” cells. We used a specific cardiac enhancer to drive the expression of the diphtheria toxin gene (dtA) in the “host” cells, so that these cells are depleted from the developing hearts, which now consist of “donor” cells. This is a proof-of-concept study, showing that it is possible to produce allogeneic and ultimately, xenogeneic hearts in chimeric organisms. The ultimate goal is to generate, in the future, human hearts in big animals such as pigs, from the patients’ cells, for transplantations. Such a system would generate transplants in a relatively short amount of time, improving the quality of life for countless patients around the world

Cell communication with the neural plate is required for induction of neural markers by BMP inhibition:evidence for homeogenetic induction and implications for Xenopus animal cap and chick explant assays

In Xenopus, the animal cap is very sensitive to BMP antagonists, which result in neuralization. In chick, however, only cells at the border of the neural plate can be neuralized by BMP inhibition. Here we compare the two systems. BMP antagonists can induce neural plate border markers in both ventral Xenopus epidermis and non-neural chick epiblast. However, BMP antagonism can only neuralize ectodermal cells when the BMP-inhibited cells form a continuous trail connecting them to the neural plate or its border, suggesting that homeogenetic neuralizing factors can only travel between BMP-inhibited cells. Xenopus animal cap explants contain cells fated to contribute to the neural plate border and even to the anterior neural plate, explaining why they are so easily neuralized by BMP-inhibition. Furthermore, chick explants isolated from embryonic epiblast behave like Xenopus animal caps and express border markers. We propose that the animal cap assay in Xenopus and explant assays in the chick are unsuitable for studying instructive signals in neural induction. (C) 2009 Elsevier Inc. All rights reserved

A conserved role for non-neural ectoderm cells in early neural development

During the early steps of head development, ectodermal patterning leads to the emergence of distinct non-neural and neural progenitor cells. The induction of the preplacodal ectoderm and the neural crest depends onwell-studied signalling interactions between the non-neural ectoderm fated to become epidermis and the prospective neural plate. By contrast, the involvement of the non-neural ectoderm in the morphogenetic events leading to the development and patterning of the central nervous systemhas been studied less extensively. Here,we show that the removal of the rostral non-neural ectoderm abutting the prospective neural plate at late gastrulation stage leads, in mouse and chick embryos, to morphological defects in forebrain and craniofacial tissues. In particular, this ablation compromises the development of the telencephalon without affecting that of the diencephalon. Further investigations of ablated mouse embryos established that signalling centres crucial for forebrain regionalization, namely the axial mesendoderm and the anterior neural ridge, form normally. Moreover, changes in cell death or cell proliferation could not explain the specific loss of telencephalic tissue. Finally, we provide evidence that the removal of rostral tissues triggers misregulation of the BMP, WNT and FGF signalling pathways that may affect telencephalon development. This study opens new perspectives on the role of the neural/non-neural interface and reveals its functional relevance across higher vertebrates.status: publishe

HBE is an enhancer active in pluripotent cells.

<p>(A) HBE is a hotspot for the binding of pluripotency factors and Smad3. <i>Nodal</i> regulatory elements are represented by green boxes and <i>Nodal</i> exons by blue boxes. Binding peaks of Nanog, Sox2, Klf4, Oct4, and Smad3 at the <i>Nodal</i> locus in ESCs are represented by black bars that represent either the summit of the peak of ChIP-seq data or its center for ChIP-chip data aligned to UCSC Genome Browser on Mouse Feb. 2006 (NCBI36/mm8) Assembly (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>). (B and C) Luciferase reporter assays for early <i>Nodal</i> enhancers using either a minimal (E1b) or the endogenous promoter (NIS), in ESCs (B), or in EpiSCs (C). Luciferase activities are shown relative to HBE construct. An asterisk indicates significant differences from the control (ctrl) (<i>p</i><0.01).</p

HBE is required for activation of <i>Nodal</i> in the early mouse embryo.

<p>Detection of mCherry (A, B, C, and D) and YFP (A′, B′, C′, and D′) in E4.5 (A–B″) or E6.5 (C–D″) aggregation chimeras generated from <i>Nodal^condHBE-YFP</i> (A–A″ and C–C″) or <i>Nodal^ΔHBE-YFP</i> (B–B″ and D–D″) ES cells and WT embryos. Images are single confocal sections. Cortical actin in blue. <i>n</i> is the number of representative embryos on the total number of embryos analyzed. Scale bar, 25 µm for E4.5 embryos and 50 µm for E6.5 embryos.</p