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Nodal expression starts in the ICM of the E3.5 blastocyst. At E4.0, shortly before implantation, Nodal is detected in the two tissues that derive from the ICM: the epiblast, which will give rise to all fetal lineages, and the primitive endoderm (PrE), an extra-embryonic layer. Nodal expression remains detectable in their post-implantation derivatives up to gastrulation stages, but exhibits complex dynamics, foreshadowing the establishment of the anterior-posterior axis and the formation of the primitive streak, processes which are defective in Nodal mutant embryos. Using fluorescent reporter transgenic mouse lines for the two Nodal enhancer known to be active just after implantation, ASE and PEE, we found that although they could recapitulate some aspects of Nodal expression at preimplantation stages, they could not account for the timing of its onset in the ICM and its presence in nascent preimplantation epiblast cells (Granier et al., 2011). Embryonic stem cells (ESCs) are derived from the nascent preimplantation epiblast and offer a convenient in vitro model of this tissue. They express Nodal and have an active ACTIVIN/NODAL signalling pathway but this is not essential to their maintenance. In contrast, epiblast stem cells (EpiSCs) are derived from the post-implantation epiblast and their capacity to self-renew depends critically on ACTIVIN/NODAL signalling. When exposed to ACTIVIN and FGF, ESCs can be converted into EpiSC-like cells (EpiLCs), a differentiation process described as a transition from a ground state of pluripotency to a formative state of pluripotency. We routinely use this approach to mimic events taking place during what is now called epiblast maturation.
Several studies showed that in ESCs Nodal expression is dependent on pluripotency factors or on ACTIVIN/NODAL signalling itself. Four Nodal cis-regulatory elements were already known. None was controlled by pluripotency factors and only one, ASE, was both dependent on ACTIVIN/NODAL signalling and known to be active before implantation. However, we found that the ASE reporter transgene was not active in ESCs. All of this strongly suggested that this particular aspect of Nodal expression was dependent on cis-regulatory sequences other than that of the Nodal enhancers identified thus far. We aimed to identify these regulatory sequences and to characterize their implication in the regulation of Nodal expression.
Identification and characterization of a novel Nodal enhancer
We identified a conserved hotspot for the binding of pluripotency factors at the Nodal locus, and called this sequence Highly Bound Element (HBE, Fig. 1). 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 preimplantation epiblast and is essential to Nodal expression in ESCs and in the mouse embryo. We also showed that HBE enhancer activity is critically dependent on its interaction with the pluripotency factor OCT4. The study of ESC to EpiLC conversions revealed that epiblast maturation entails a shift in the regulation of Nodal expression from an HBE-driven phase to an ASE-driven phase. Deletion of HBE in ESCs or in EpiLCs allowed us to show that HBE, although not necessary for Nodal expression in EpiLCs, is required in differentiating ESCs to activate the differentiation-promoting ASE, and therefore controls this regulatory shift (Papanayotou et al., 2014). This strongly suggests that a memory of the presence of HBE is deposited at the locus in ESCs, possibly via an epigenetic mechanism, a memory that is later required to allow ASE to drive Nodal expression. We suspect that HBE-bound pluripotency factors and SMAD factors contribute to this process via the recruitment of chromatin remodelers or modifiers. Our results so far suggest that HBE controls the later switch of Nodal regulation to ASE via a specific modification introduced at the level of the promoter and/or at the level of ASE.
Hotspots for the binding of pluripotency factors such as HBE could therefore control gene expression via the influence they exert over the epigenetic status of other regulatory elements. This could be a significant finding as about 4000 such hotspots, also known as multi transcription factors binding loci (MTL), have been found in the genome. They are critical for the acquisition and maintenance of pluripotency, and there are indications that they play a role in the exit of pluripotency and in differentiation.
Transcriptomic and epigenomic comparisons between ESCs, EpiLCs and EpiSCs revealed that a global rearrangement of enhancer chromatin landscape takes place during epiblast maturation, that leads to a shift in enhancer usage (Buecker et al., 2014; Factor et al., 2014; Yang et al., 2014), even for genes that do not see a change in their expression levels, as we showed for Nodal. For such genes, enhancers specifically active in ESCs, which tend to be enriched with DNA binding motifs for SMAD2/3 and SMAD4, are decommissioned once their EpiLC/EpiSC-specific enhancers are activated in differentiating ESCs. All of this suggests that a sudden increase in ACTIVIN concentration induces cells to reach a new equilibrium, which defines the EpiLC/EpiSC state, via the impact this increase has on regulatory elements. Nodal, as a well-characterized target of both the molecular machinery of pluripotency and its own signalling pathway, is an ideal gene model to study the mechanism underlying this process.
Our HBE-related objectives are, 1), to elucidate how HBE controls the enhancer activity of ASE, 2), to characterize the possible implication of HBE in the regulation of genes other than Nodal, the modalities of these interactions and how they may contribute to developmental progression, 3), to characterize the impact of HBE deletion on Nodal expression and function at various stages during embryonic development.
The ability to follow and modify cell behaviour with accurate spatiotemporal resolution is a prerequisite to study morphogenesis in developing organisms. Electroporation, the delivery of exogenous molecules into targeted cell populations through electric permeation of the plasma membrane, has been used with this aim in different model systems. However, current localised electroporation strategies suffer from insufficient reproducibility and mediocre survival when applied to small and delicate organisms such as early post-implantation mouse embryos. We collaborated with a team of physicists to develop a microdevice to locally electroporate embryos with high efficiency and reduced cell damage.
In silico simulations using a simple electrical model of mouse embryos indicated that a dielectric guide-based design would improve on existing alternatives (Fig. 2). Such a device was microfabricated and its capacities tested by targeting the distal visceral endoderm (DVE), a migrating cell population essential for anterior-posterior axis establishment. Transfection was efficiently and reproducibly restricted to fewer than four visceral endoderm cells without compromising cell behaviour and embryo survival. Combining targeted mosaic expression of fluorescent markers with live imaging in transgenic embryos revealed that, like leading DVE cells, non-leading ones send long basal projections and intercalate during their migration. Finally, we showed that the use of our microsystem can be extended to a variety of embryological contexts, from preimplantation stages to organ explants. Hence, we have experimentally validated an approach delivering a tailor-made tool for the study of morphogenesis in the mouse embryo. Furthermore, we have delineated a comprehensive strategy for the development of ad hoc electroporation devices (Mazari et al., 2014).
In the last 10 years a number of studies have established a link between NODAL and tumour progression. We recently started to explore its role in melanoma. Some studies had already established that NODAL is implicated in this cancer (MacAllister et al., 2010 ; Yu et al., 2010) : its most advanced stages (III and IV) were associated with increased NODAL expression, and inhibition of its signalling pathway in aggressive melanoma cell lines reduced their tumorigenicity and promoted reversion to a benign phenotype (Topczewska et al., 2006).
We found that cells from a metastatic cell line, a recognized in vitro model of human melanoma, when cultured at low density in a medium conditioned by the same cells cells cultured at high density, acquire in just 24h a more aggressive phenotype, characterized by a change in cell shape, increased motility and a specific molecular profile, marked by an increase in NODAL expression. CRISPR/Cas9-mediated deletion of NODAL from their genome then showed that the capacity of these cells to express an invasive phenotype is highly dependent on the presence of the gene. Efficient genome editing in this particular melanoma line and the inductive capacity of its conditioned medium provide us with the means to dissect the regulation and key role of NODAL in tumour progression in greater detail than was previously possible. Together with a team of biophysicists, we currently use an array of genetic (genome-editing, RNAi, transcriptome analysis) and biophysical (micropatterned co-cultures of stromal and melanoma cells) approaches to address the following questions:
- How is NODAL expression activated?
- How does NODAL promote motility and invasion?
- How conserved are these mechanisms in other metastatic cell lines?