Spécification des destins cellulaires chez la souris
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Role of cis-regulatory elements in the regulation of preimplantation Nodal expression
Four Nodal cis-regulatory elements have been characterized, only two of which, PEE and ASE, are known to be active in the epiblast prior to the onset of gastrulation (Norris etal., 1999; Adachi et al., 1999). PEE contains two LEF/TCF binding sites and mediates the control of Wnt/β-catenin signalling over Nodal expression (Ben-Haim et al., 2006, Granier et al., 2011). ASE contains two functional FoxH1-Smad2/3 binding motifs and acts as an auto-regulatory element allowing Nodal to amplify its own expression (Norris et al., 2002; Yamamoto et al., 2001). Deletions of PEE or ASE result in phenotypes far less severe than that of Nodal-/- embryos and characterized by later AP or LR patterning defects (Norris et al., 2002; Vincent et al., 2003). This suggests neither is required to initiate Nodal expression.
To study the regulation and functions of Nodal at pre and peri-implantation stages we generated transgenic lines where the expression of fluorescent proteins was placed under the control of PEE or ASE. Expression analysis of the PEE-GFP and ASE-FP transgenes delivered new insights into the activities of the canonical Wnt signalling and Activin/Nodal signalling pathways at pre and peri-implantation stages, and showed for the first time how these activities contribute to the regulation of early Nodal expression (Granier et al., 2011). However, the dynamics and the heterogeneity of the transgenes’expression profiles indicate that other influences shape early Nodal expression. Comparison of the transgenes expression profiles with that of Nodal suggests that cis-regulatory sequences other than ASE and PEE must contribute to Nodal expression in the ICM and the nascent epiblast.
We are currently using bioinformatics, luciferase-based assays, ChIPs, cultured pluripotent stem-cells, transgenesis and homologous recombination to identify novel Nodal regulatory sequences and to investigate their implication in the regulation of the gene.
Regulation of Nodal expression in the mouse
Regulation of preimplantation Activin/Nodal signalling
The Nodal cis-regulatory element ASE contains two functional FoxH1-Smad2,3 binding motifs and acts as an auto-regulatory element allowing Nodal to amplify its own expression (Yamamoto et al., 2001; Norris et al., 2002). The ASE-YFP transgene was validated as a valuable sensor of Activin/Nodal signalling in the early mouse embryo (Granier et al., 2011). One striking observation is the heterogeneity of its expression in the ICM, the nascent epiblast and the primitive endoderm (PrE), tissues where Nodal and other TGFβ ligands are known to be expressed. This suggests that these signals are partially blocked, or that cells in each of these layers do not all have the same ability to respond to them. In this respect, the heterogeneity revealed by the ASE-FP transgene in the peri-implantation epiblast is reminiscent of heterogeneities described among mESCs with respect to the expression of pluripotency markers such as Nanog or Stella, and also of the differences characterized between embryonic stem-cells (ESCs) and epiblast stem-cells (EpiSCs). It may relate to the different maturation status of these cells (Chambers et al., 2007; Hayashi et al., 2008). In particular, it is consistent with the recent hypothesis that there may be in ESCs, a ground state of pluripotency, which exposure to extrinsic signals transforms into a primed state of pluripotency (Ying et al., 2008; Nichols and Smith, 2009).
We are currently investigating the molecular basis for the heterogeneous expression of ASE-YFP, both in cultured pluripotent stem-cells (ESCs and EpiSCs) and in the embryo.
ASE-YFP shows heterogeneous expression in the ICM, the PrE and the epiblast
Specification of anterior cell fates
The role of Nodal had originally been studied with respect to the fate of cells that derive from the posterior epiblast, i. e. cells that go through the primitive streak during gastrulation to form mesoderm and definitive endoderm. Epiblast cells not recruited into the primitive streak will give rise to the ectoderm layer, which later on forms surface ectoderm and neurectoderm. As Nodal mutant embryos fail to form the anterior visceral endoderm (AVE), the organizer and the anterior mesendoderm, the three structures that are supposedly necessary to induce and pattern the anterior region of the embryo, we were interested to investigate if, and how, an anterior identity was specified in the epiblast of these embryos.
Using a panel of specific molecular markers we were able to show that the epiblast of Nodal mutant embryos is precociously anteriorized (Hesx1) and neuralized (Sox1), and even displays expression of neural markers specific of anterior ventral forebrain (Nkx2.1). The first marker of anterior ectoderm, Hesx1, starts to be expressed in the epiblast of Nodal mutant embryos at E5.5, 48h ahead of its normal timing of expression (Camus et al., 2006). The mutant epiblast then follows a temporal sequence of differentiation similar to that of the wildtype forebrain. These observations led to two main conclusions :
- The signals necessary for the specification of anterior and neural identities are present in the mouse embryo well before gastrulation has started.
- The initial function of Nodal in the epiblast is perhaps to inhibit the differentiation of this tissue along the anterior neural pathway.
We are now using the Nodal mutant to identify the signals that are required to elicit anterior neural specification in the mouse embryo.
Premature expression of Hesx1 and Nkx2.1 in Nodal mutant embryos
Onset of expression of Hesx1, Sox1 and Nkx2.1 as detected by real time-RTPCR
on single Nodal-/- embryos as compared to wildtype
Dernière modification 27/07/2011