Inducible stem cell-derived embryos capture mouse morphogenetic events in vitro

The development of mouse embryos can be partially recapitulated by combining embryonic (ES), trophoblast (TS) and extra-embryonic endoderm (XEN) stem cells to generate ETX-embryos. Although ETX-embryos transcriptionally capture the mouse gastrula, their ability to recapitulate complex morphogenic events such as gastrulation is limited, possibly due to the limited potential of XEN cells. To address this, we generated ES cells transiently expressing transcription factor Gata4 that drives the extra-embryonic endoderm fate and combined them together with ES cells and TS cells to generate induced ETX-embryos (iETX-embryos). We show that iETX-embryos establish a robust anterior signalling centre that migrates unilaterally to break embryo symmetry. Furthermore, iETX-embryos gastrulate generating embryonic and extra-embryonic mesoderm, and definitive endoderm. Our findings reveal that replacement of XEN cells with ES cells transiently expressing Gata4 endows iETX-embryos with greater developmental potential, thus enabling the study of the establishment of anterior-posterior patterning and gastrulation in an in vitro system.This work was supported by a European Research Council Grant (RG77946)Wellcome Trust (207415/Z/17/Z), Open Philanthropy, Shurl and Kay Curci, and Weston Havens Foundations grants awarded to M.Z.G.; K.Y.C.L. is supported by the Croucher Foundation and Cambridge Trust. F.H. is supported by a European Research Council Grant (695669) and Wellcome Trust (WT108438/C/15/Z). J.D.J. is supported by the Biotechnology and Biological Sciences Research Council

Inducible stem cell-derived embryos capture mouse morphogenetic events in vitro

The development of mouse embryos can be partially recapitulated by combining embryonic (ES), trophoblast (TS) and extra-embryonic endoderm (XEN) stem cells to generate ETX-embryos. Although ETX-embryos transcriptionally capture the mouse gastrula, their ability to recapitulate complex morphogenic events such as gastrulation is limited, possibly due to the limited potential of XEN cells. To address this, we generated ES cells transiently expressing transcription factor Gata4 that drives the extra-embryonic endoderm fate and combined them together with ES cells and TS cells to generate induced ETX-embryos (iETX-embryos). We show that iETX-embryos establish a robust anterior signalling centre that migrates unilaterally to break embryo symmetry. Furthermore, iETX-embryos gastrulate generating embryonic and extra-embryonic mesoderm, and definitive endoderm. Our findings reveal that replacement of XEN cells with ES cells transiently expressing Gata4 endows iETX-embryos with greater developmental potential, thus enabling the study of the establishment of anterior-posterior patterning and gastrulation in an in vitro system.This work was supported by a European Research Council Grant (RG77946)Wellcome Trust (207415/Z/17/Z), Open Philanthropy, Shurl and Kay Curci, and Weston Havens Foundations grants awarded to M.Z.G.; K.Y.C.L. is supported by the Croucher Foundation and Cambridge Trust. F.H. is supported by a European Research Council Grant (695669) and Wellcome Trust (WT108438/C/15/Z). J.D.J. is supported by the Biotechnology and Biological Sciences Research Council

Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures.

Embryonic stem cells can be incorporated into the developing embryo and its germ line, but, when cultured alone, their ability to generate embryonic structures is restricted. They can interact with trophoblast stem cells to generate structures that break symmetry and specify mesoderm, but their development is limited as the epithelial-mesenchymal transition of gastrulation cannot occur. Here, we describe a system that allows assembly of mouse embryonic, trophoblast and extra-embryonic endoderm stem cells into structures that acquire the embryo's architecture with all distinct embryonic and extra-embryonic compartments. Strikingly, such embryo-like structures develop to undertake the epithelial-mesenchymal transition, leading to mesoderm and then definitive endoderm specification. Spatial transcriptomic analyses demonstrate that these morphological transformations are underpinned by gene expression patterns characteristic of gastrulating embryos. This demonstrates the remarkable ability of three stem cell types to self-assemble in vitro into gastrulating embryo-like structures undertaking spatio-temporal events of the gastrulating mammalian embryo.European Research Council (669198) Wellcome Trust (098287/Z/12/Z)

Elastic properties of proteins: insight on the folding process and evolutionary selection of native structures

We carry out a theoretical study of the vibrational and relaxation properties of naturally-occurring proteins with the purpose of characterizing both the folding and equilibrium thermodynamics. By means of a suitable model we provide a full characterization of the spectrum and eigenmodes of vibration at various temperatures by merely exploiting the knowledge of the protein native structure. It is shown that the rate at which perturbations decay at the folding transition correlates well with experimental folding rates. This validation is carried out on a list of about 30 two-state folders. Furthermore, the qualitative analysis of residues mean square displacements (shown to accurately reproduce crystallographic data) provides a reliable and statistically accurate method to identify crucial folding sites/contacts. This novel strategy is validated against clinical data for HIV-1 Protease. Finally, we compare the spectra and eigenmodes of vibration of natural proteins against randomly-generated compact structures and regular random graphs. The comparison reveals a distinctive enhanced flexibility of natural structures accompanied by slow relaxation times at the folding temperature. The fact that these properties are intimately connected to the presence and assembly of secondary motifs hints at the special criteria adopted by evolution in the selection of viable folds.Comment: Revtex 17 pages, 13 eps figure

Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures

Embryonic stem cells can be incorporated into the developing embryo and its germ line, but, when cultured alone, their ability to generate embryonic structures is restricted. They can interact with trophoblast stem cells to generate structures that break symmetry and specify mesoderm, but their development is limited as the epithelial–mesenchymal transition of gastrulation cannot occur. Here, we describe a system that allows assembly of mouse embryonic, trophoblast and extra-embryonic endoderm stem cells into structures that acquire the embryo’s architecture with all distinct embryonic and extra-embryonic compartments. Strikingly, such embryo-like structures develop to undertake the epithelial–mesenchymal transition, leading to mesoderm and then definitive endoderm specification. Spatial transcriptomic analyses demonstrate that these morphological transformations are underpinned by gene expression patterns characteristic of gastrulating embryos. This demonstrates the remarkable ability of three stem cell types to self-assemble in vitro into gastrulating embryo-like structures undertaking spatio-temporal events of the gastrulating mammalian embryo

Mechanism of Action of Cyclophilin A Explored by Metadynamics Simulations

Trans/cis prolyl isomerisation is involved in several biological processes, including the development of numerous diseases. In the HIV-1 capsid protein (CA), such a process takes place in the uncoating and recruitment of the virion and is catalyzed by cyclophilin A (CypA). Here, we use metadynamics simulations to investigate the isomerization of CA's model substrate HAGPIA in water and in its target protein CypA. Our results allow us to propose a novel mechanistic hypothesis, which is finally consistent with all of the available molecular biology data

Post-transcriptional Regulatory Mechanisms Controlling Development of Murine Cerebral Cortical Precursors

The complex neural circuitry of the mammalian nervous system arises from a small pool of neural precursors that, during development, sequentially gives rise to neurons, astrocytes and oligodendrocytes. Several molecular mechanisms and environmental stimuli regulate the expansion of the neural precursor pool and the subsequent generation of differentiated progeny. In this thesis, I sought to investigate whether post-transcriptional regulation plays a role in the development of mammalian neural precursors. In the first part of this thesis, I show that the double stranded RNA binding protein Staufen2 is part of a repressive complex, with Pumilio2 and DDX1, that prevents early differentiation of neural precursors into neurons by repressing the translation of neurogenic mRNAs such as prox1. In the second part of the thesis I show that Smaug2, a translational repressor, also prevents early generation of neurons by repressing nanos1 mRNA, which encodes another RNA-binding protein. I also show that nanos1 repression occurs by its inclusion in a P-body like granule with 4E-T, another known repressor of mRNA translation. In the last chapter I identify mRNAs associating with Smaug2 and I suggest that Smaug2 may have additional functions that are independent of nanos1 regulation and that unlike nanos1, which is regulated together by Smaug2 and 4E-T, the majority of Smaug2 mRNAs are regulated independently from 4E-T. Together, these studies suggest that several RNA-binding proteins are crucial regulators of cortical development and that they perform this role by forming several, largely independent, repressive RNA-protein complexes. I suggest that these repressive complexes prime neural precursors to generate progeny at the appropriate time by repressing proneurogenic mRNAs until the appropriate developmental cue.Ph.D