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Identification and characterisation of novel cell-fate influencing genes in pre-implantation mouse development

Final Report Summary - IDNOVCELFAT2011 (Identification and characterisation of novel cell-fate influencing genes in pre-implantation mouse development)

The four-year ‘Marie Curie Career Integration Grant’ award was intended to facilitate the fellow (Alexander W. Bruce Ph.D) as an independent research group leader within the Department of Molecular Biology, University of South Bohemia (Czech Republic) – herein referred to as the ‘host institute’. Accordingly it also supported an active and high impact research programme, centred on the regulation of cell-lineage formation during the preimplantation stages of mouse embryo development. Accordingly, this ‘Final Report Publishable Summary’ is split between two sections; 1) A summary of the fellow’s successful professional integration, 2) A scientific review of achievements and progress of the supported research.

1) The fellow’s current research team comprises eight people that include himself, a post-doctoral researcher, three Ph.D. students, two bachelors students and a technician; with the most senior Ph.D. student scheduled to defend their thesis in spring 2016 (an additional two bachelors students have successfully transited the laboratory and defended their dissertations in this time). Moreover, the fellow also oversaw a sabbatical research training visit (one year), from a clinical embryologist (with whom contact is maintained). The fellow himself has been fully integrated as a completely independent group leader within the host institute (see website - http://kmb.prf.jcu.cz/en/laboratories/en-bruce-lab) and is currently contracted there until October 2018, although it is mutually planned he will apply for full habilitation during 2016. During the time of this award, the fellow has secured additional funding for his groups independent research programme from an additional four successful grant applications, totalling approximately 240 000 EUR. The host institutes commitment to the fellow is also illustrated by significant capital purchases to support his research programme (e.g. confocal and inverted fluorescence microscopes). Moreover, host institute integration of the fellow is also exemplified by his expansive teaching responsibilities. He has established, teaches, examines and administers two international, English-taught lecture series (each comprising greater than 20 hours of student contact time) in addition to other ad hoc teaching (also outside the host institute). Moreover, he has been centrally active in organising workshops and specialised courses (e.g. advanced microscopy and functional genomics-based) at the host institute, that recruit practical experts in their respective fields to teach technical skills to post-graduate students in the early stages of their research degrees; thus facilitating a transfer and retention of contemporary skills and knowledge at the host institute. It is noteworthy that the fellow has also been an active and engaged participant in host institute’s ‘open days’, designed to educate, inform and entertain the public (in particular high school students) about the active research programmes. The fellow has also effectively integrated into the broader scene of his scientific field. Indeed, during the realisation of this award, he has initiated and maintained 11 different collaborations with different research groups, at the host institute, national and international levels. Additionally, the fellow has been active in dissemination of his group’s research; presenting 5 oral and 3 poster conference presentations and 8 invitational seminars at non-host institutions. He has also been appointed to the editorial board of the relevant journal, Reproductive Biomedicine Online, until December 2018. In summary, the fellow has with the help of this award fully integrated into the host institute and his wider scientific community and is ideally placed to professionally develop his independent research programme and career.

2) After mammalian oocytes/ eggs are fertilised by the sperm, they undergo a series of cell cleavage divisions that ultimately derive the first tissue structure of development, known as the blastocyst. It is this blastocyst, essentially a epithelialized sheet of cells encapsulating an inner-cell-mass (ICM) at one end and a growing fluid filled cavity at the other, that will emerge from its protective proteinaceous shell and implant into the uterus/ womb to continue its development to form supportive extraembryonic tissues, such as the placenta, and the foetus itself. The period leading up to the formation of the blastocyst, known as the preimplantation developmental period (that mimicked during clinical human in vitro fertilisation/ IVF treatments), is characterised not just be increasing cell number but by the emergence of three distinct cell types/ lineages. Two lineages (the outer epithelialized ‘trophectoderm/ TE’ layer and inner ‘primitive endoderm/ PrE’ cells in contact with the cavity) have already begun specialisation/ differentiation, whereas the third lineage (cells deep within the ICM called the ‘epiblast/ EPI’) remain pluripotent and contribute to all the cell types of the subsequent foetus.

The main project aim of this award was to identify and characterise novel genes and mechanisms that impact on the emergence of these three blastocyst lineages, i.e. that effect cell-fate. To do this, the fellow’s group utilised and bioinformatically interrogated information from genetic expression screens of preimplantation stage mouse embryos to identify potential novel cell-fate genes. These genes were then experimentally dysregulated in the context of the developing preimplantation mouse embryo (often only with a discreet number/ clone of the overall total number of cells) using a combination of RNAi/ mRNA-mediated diminution/ over-expression, functional inactivation using small chemical inhibitor compounds or complete genetic ablation. The effect of the interventions upon the normal formation of the blastocyst cell lineages was then compared against controls, to confirm if they indeed act in a cell-fate role.
The fellow’s group has identified and characterised (to varying degrees) multiple genes with novel cell-fate roles in early mouse embryos and as such has published two peer-reviewed manuscripts, has another currently at review, with a further four in the preparation stages (two in the advanced pre submission stage). A resume of results relating to a selection of such genes is given below:

A) The Socs3 gene (a previously characterised immunity-related gene) was shown to regulate the rate at which cells divide, as diminution of expression and genetic ablation led to blastocysts with greater number of cells versus controls. This effect was non-cell autonomous, not restricted to any particular cell lineage and associated with increased production of the pro-inflammatory cytokine Il6 (known to be important in preimplantation embryo development). Understanding what regulates the timing of early mammalian embryo development is important, as compared to other non-mammalian species it is very slow, in order to allow time for the uterus to become receptive to blastocyst implantation – so called ‘viviparity’. Additionally, a role for Socs3 during the latter stages of oogenesis (egg) formation was unexpectedly uncovered, with Socs3 protein found to localise to the meiotic spindle (the apparatus required to faithfully segregate chromosomes, ensuring appropriate genetic dosage in the embryo). Initial genetic diminution and ablation experiments suggest Socs3 acts to stabilise and thus facilitate spindle functioning (this work forming the foundation of a post-doctoral fellowship application, within the host group; especially as chromosomal segregation errors are a major course of female infertility in the developed world).

B) Classically, the emergence of the three blastocyst lineages has been presented as a two step process; firstly, outer TE is separated from inner ICM, secondly, ICM cells segregate to either PrE or EPI. We have demonstrated, with our work on clonal diminution of the Tead4 gene (previously known to have TE role), that the blastocyst lineages emerge in a single ‘integrated’ rather than the classically described ‘two-step’ process. Briefly, the timing of the internalisation of ICM progenitor cells, either in the fourth or fifth rounds of cell cleavage division, biases their ultimate cell-fate. Those cells internalised earlier are more likely to form EPI whereas those coming in later, derived from outer-parental cells that have been subject to a greater amount Tead4 regulation during an additional ~12 hours, favour the PrE. Moreover, we find that Tead4 achieves this PrE bias by potentiating the expression of genes required to form PrE (further integrated using mRNA-Seq based strategies – an aim of this project); thus placing Tead4 at the apex of an integrated mechanism of early embryo cell-fate that leads to TE, PrE and EPI formation. Consequently, these results (and those of other groups) prompt a reappraisal of the ‘text-book’ mechanisms of early embryo cell-fate. Additionally we have demonstrated Tead4 regulates aspects of outer TE cell biology (namely apical-basolateral intra-cellular polarisation and expression/ regulation of Rho-GTPases) required to inhibit mechanisms associated with retention of pluripotency (e.g. Hippo-signalling pathway), further impacting and increasing our mechanistic understanding of this most critical of developmental periods.

C) Additionally, we have uncovered essential roles during preimplantation mouse embryo development for genes of the p38Mapk type; as there are multiple isoforms of this gene, we interrogated its role using specific small chemical compound inhibitors of its catalytic activity. We have discovered inhibition from the 2-cell stage results in embryonic arrest prior to blastocyst formation. Moreover, that the phenotype can be somewhat ameliorated by the exogenous addition of amino-acids and anti-oxidants to the culture embryo media, implying a metabolic defect associated with p38Mapk–inhibition. Indeed, we find that the process of autophagy (when cells break-down internal structures to provide required metabolites) is severely attenuated in p38Mapk-inhibitted embryos. We also find that administration of the inhibitor after initiation of blastocyst formation, circumventing the phenotypic developmental block referred to above, leads to a near complete failure of mature PrE formation (plus some defects in TE cell number) with enhanced EPI cell numbers. The p38-Mapk inhibitor mediated PrE cell-fate blockage is not sensitive to exogenous amino acid addition (unlike the TE phenotype) but is mediated, in part, via a non-classical signalling pathway (related to Tgfβ).These results have the potential to prompt a rethink on the composition of in vitro embryo cultivation media and how it can affect cell-fate and appropriate development (with relevance to human IVF treatments and derivation of therapeutic stem cell types).

In overall summary the award and execution of this grant has undoubtedly supported the fellow’s complete integration into the host institute, the establishment of his independent research/ teaching careers and the foundation of his future research programme.