Morphogenesis of the early post-implantation mouse embryo

The morphogenetic events that give rise to the early post-implantation mouse embryo (egg cylinder) have not been thoroughly studied and our knowledge is restricted to “snap-shot” descriptions of embryos recovered at different stages of implantation from the mother. A central feature of the egg cylinder is the pro-amniotic cavity, which spans the embryo and participates in formation of the extraembryonic membranes. The major aims of my PhD studies have been to reveal how this cavity is formed (Aim 1) and then how the egg cylinder grows (Aim 2). In order to address how the pro-amniotic cavity forms (Aim 1), I first characterised in detail development of the architecture of the extra-embryonic ectoderm (ExE), which has to be remodelled to permit cavity formation. My findings indicate that the ExE comprises cells in direct contact with a basement membrane and cells that lie deeper in the tissue. The ExE originates in the polar trophectoderm, a monolayer covering the epiblast of the blastocyst, which expands and undergoes invagination to form a slit-like cavity. By carrying out analyses of fixed specimens and live imaging of cultured embryos, I have found that the epiblast and ExE cavity extend towards each other through the formation and resolution of multiple rosette structures. This leads to the fusion of the ExE and epiblast cavities to form the unified pro-amniotic cavity. I show that this process is dependent on signalling cues stemming from the underlying basement membrane that activate the b1-integrin signalling pathway to regulate cell polarity, ExE tissue architecture and rosette formation. In addition to the basement membrane’s role in b1-integrin signalling, it also has physical functions that I characterise in the second part of my study (Aim 2). High resolution imaging revealed that the basement membrane underlying the epiblast is highly perforated during the implantation stages. These perforations are initially evenly distributed and then accumulate asymmetrically at the future posterior part of the embryo, just prior to gastrulation. Finally, I demonstrate that remodelling of the basement membrane requires the expression of matrix metalloproteinases (MMPs) in the epiblast under the control of Nodal. The anterior visceral endoderm inhibits Nodal signalling and hence MMP inhibition in the anterior. I demonstrate that activity of the MMPs and perforations in the basement membrane are essential for embryo growth. The domain of posterior basement membrane perforations persists beyond gastrulation suggesting a potential role for these perforations in primitive streak formation and extension. Together, my studies bring new important insights into the understanding of early mouse embryo morphogenesis.BBSRC Doctoral Training Partnershi

Proposal for an eHealth Based Ecosystem Serving National Healthcare

The European Union (EU)'s keen concern about citizens' health and well-being advancement has been expressed at all levels. It has been understood that at present, these can only be achieved through coordinated actions at the individual member states' level based on EU directives, as well as through promoting and funding R&D and expanding the use of eHealth technologies. Despite the diversities and particularities among member states, common values such as universal access to good quality healthcare, equity, and solidarity have been widely accepted across EU. That demanded the adoption of policies and follow directives, which streamlined actions to bridge healthcare gaps, and facilitate cross-border healthcare. This paper articulates a framework for deriving a national healthcare system, based on interoperable Electronic Health Record (EHR) with safeguarding healthcare quality, enabling quadruple helix (Public, Academia, Industry, NGOs) driven R&D and guided by a patient-centered approach. A methodology to develop an integrated EHR at National level is proposed as a prerequisite for eHealth and put into perspective. Recommendations are given for the steps needed, from the managerial, legal, technical, and financial concerns in developing an open access, patient-centered national healthcare system based on the context and constraints of a country. The example of a small country to apply the proposed methodology is demonstrated. Stakeholders, including citizens, healthcare professionals, academia, and the industry are mobilized, enabled, and incentivized for implementing the methodology. Experiences are aspired to be offered as lessons learned for other countries to adapt on their environment

Embryo size regulates the timing and mechanism of pluripotent tissue morphogenesis.

Mammalian embryogenesis is a paradigm of regulative development as mouse embryos show plasticity in the regulation of cell fate, cell number, and tissue morphogenesis. However, the mechanisms behind embryo plasticity remain largely unknown. Here, we determine how mouse embryos respond to an increase in cell numbers to regulate the timing and mechanism of embryonic morphogenesis, leading to the formation of the pro-amniotic cavity. Using embryos and embryonic stem cell aggregates of different size, we show that while pro-amniotic cavity formation in normal-sized embryos is achieved through basement membrane-induced polarization and exocytosis, cavity formation of increased-size embryos is delayed and achieved through apoptosis of cells that lack contact with the basement membrane. Importantly, blocking apoptosis, both genetically and pharmacologically, alters pro-amniotic cavity formation but does not affect size regulation in enlarged embryos. We conclude that the regulation of embryonic size and morphogenesis, albeit concomitant, have distinct molecular underpinnings

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

Embryo Size Regulates the Timing and Mechanism of Pluripotent Tissue Morphogenesis

Mammalian embryogenesis is a paradigm of regulative development as mouse embryos show plasticity in the regulation of cell fate, cell number, and tissue morphogenesis. However, the mechanisms behind embryo plasticity remain largely unknown. Here, we determine how mouse embryos respond to an increase in cell numbers to regulate the timing and mechanism of embryonic morphogenesis, leading to the formation of the pro-amniotic cavity. Using embryos and embryonic stem cell aggregates of different size, we show that while pro-amniotic cavity formation in normal-sized embryos is achieved through basement membrane-induced polarization and exocytosis, cavity formation of increased-size embryos is delayed and achieved through apoptosis of cells that lack contact with the basement membrane. Importantly, blocking apoptosis, both genetically and pharmacologically, alters pro-amniotic cavity formation but does not affect size regulation in enlarged embryos. We conclude that the regulation of embryonic size and morphogenesis, albeit concomitant, have distinct molecular underpinnings

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

Sequential formation and resolution of multiple rosettes drive embryo remodelling after implantation

The morphogenetic remodelling of embryo architecture after implantation culminates in pro-amniotic cavity formation. Despite its key importance, how this transformation occurs remains unknown. Here, we apply high-resolution imaging of embryos developing in vivo and in vitro, spatial RNA sequencing and 3D trophoblast stem cell models to determine the sequence and mechanisms of these remodelling events. We show that cavitation of the embryonic tissue is followed by folding of extra-embryonic tissue to mediate the formation of a second extra-embryonic cavity. Concomitantly, at the boundary between embryonic and extra-embryonic tissues, a hybrid 3D rosette forms. Resolution of this rosette enables the embryonic cavity to invade the extra-embryonic tissue. Subsequently, β1-integrin signalling mediates the formation of multiple extra-embryonic 3D rosettes. Podocalyxin exocytosis leads to their polarized resolution, permitting the extension of embryonic and extra-embryonic cavities and their fusion into a unified pro-amniotic cavity. These morphogenetic transformations of embryogenesis reveal a previously unappreciated mechanism for lumen expansion and fusion

Sequential formation and resolution of multiple rosettes drive embryo remodelling after implantation

The morphogenetic remodelling of embryo architecture after implantation culminates in pro-amniotic cavity formation. Despite its key importance, how this transformation occurs remains unknown. Here, we apply high-resolution imaging of embryos developing in vivo and in vitro, spatial RNA sequencing and 3D trophoblast stem cell models to determine the sequence and mechanisms of these remodelling events. We show that cavitation of the embryonic tissue is followed by folding of extra-embryonic tissue to mediate the formation of a second extra-embryonic cavity. Concomitantly, at the boundary between embryonic and extra-embryonic tissues, a hybrid 3D rosette forms. Resolution of this rosette enables the embryonic cavity to invade the extra-embryonic tissue. Subsequently, β1-integrin signalling mediates the formation of multiple extra-embryonic 3D rosettes. Podocalyxin exocytosis leads to their polarized resolution, permitting the extension of embryonic and extra-embryonic cavities and their fusion into a unified pro-amniotic cavity. These morphogenetic transformations of embryogenesis reveal a previously unappreciated mechanism for lumen expansion and fusionThe M.Z.G lab is supported by grants from the European Research Council (669198) and the Welcome Trust (098287/Z/12/Z) and the EU Horizon 2020 Marie Sklodowska-Curie actions (ImageInLife,721537). C.K is supported by BBSRC Doctoral training studentship