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Regulation of early embryo development and pluripotency through alternative splicing

Periodic Reporting for period 1 - EDPAS (Regulation of early embryo development and pluripotency through alternative splicing)

Reporting period: 2015-07-01 to 2017-06-30

In mammals, embryo development takes place inside the maternal uterus and the foetus is permanently linked to the mother though the placenta, ensuring a source of nutrients during the whole pregnancy. The early phases of development take place before the embryo attaches or implants into the uterus, during the pre-implantation period. The correct advancement of this period is crucial for the embryo to be able to successfully implant and progress though pregnancy. Indeed, implantation failure remains the limiting step in assisted reproduction as only around 25% of human embryos implant and develop to term. Therefore, understanding how embryo development proceeds prior to implantation is a fundamental question not only in developmental biology but also in the clinic.
Just before implantation, the mammalian embryo is composed of cells that will either form the placenta or that have the ability to turn into all cell types found in the embryo proper. The later are called embryonic stem cells (ESCs) and as the embryo develops they gradually form more specialized cell types that are no longer able to contribute to all body tissues. This process is called cell differentiation. The ability of embryo cells to form any cell type is maintained for a short period of time after implantation, however the characteristics of ESCs change as the embryo implants so they become ready to for differentiation. This change is necessary for development to proceed and what determines it remains incompletely understood. ESCs can be isolated from embryos and cultured in the laboratory under specific conditions. By modifying these conditions we can induce their differentiation to form specific tissues, such as liver, lung or neurons, mimicking what occurs in the embryo. This makes them a very powerful tool in regenerative medicine. However, to be able to use ESCs in a safe and efficient manner a complete understanding of how their differentiation occurs during normal embryo development is necessary.
All processes taking place at early developmental stages are very tightly controlled by instructions encoded in the genes. Each gene codes for a specific protein using an intermediate product called messenger RNA (mRNA), and it is the final protein repertoire of a particular cell which determines its identity and how it behaves within the embryo (for instance if it will detach from its neighbour cells or change shape). Each gene is divided into fragments called exons, and some of the exons can be used or not to form the mRNA that will be translated into the protein. Thus, depending on whether a particular exon is used the resulting protein will be slightly different and can exert different functions within the cell. To fully understand how cell identity and behaviour is regulated we have to take into account which particular fragments (exons) are being used to form the proteins in each cell type.
The main objective of this project was to use data from pre-implantation embryos as well as ESCs cultured in the laboratory, to generate a database that allow us to identify proteins that despite being encoded by the same gene differ in the exons used for their production. Using this information, the second objective was to experimentally test to what extend the usage of a particular exon to form a protein has an impact on the final protein function during development and in stem cells. We have successfully completed both objectives and the results obtained will be ready for publication in the near future.
All the mRNA present in a cell at a specific time point can be isolated and read using a technique called RNA- sequencing, so we can distinguish which proteins are being produced in each cell type. This type of analysis has been done for embryos of different mammalian species, however in all cases researchers focused on investigating proteins produced from different genes not protein variants produced by exon selection from the same genes.
We have developed a method to detect proteins coming from different genes and protein variants encoded in a single gene. We have used it to re-analyse RNA-sequencing data from mouse, human and cow embryos and to generate a very complete database of the whole protein repertoire present at each developmental stage before embryo implantation. We have detected hundreds of protein variants that change in abundance during the course of development and that are likely associated to specific processes, i.e. the initial segregation between cells that will form either the placenta or the foetus. By applying this method to mRNA obtained from ESCs cultured under different conditions, we have also created a catalogue of protein variants associated with the early stages of stem cell differentiation. All this information contributes to the current knowledge on pre-implantation development and stem cell differentiation and has opened up new lines of research in our group.
The generation of genetically modified ESCs has allowed us to experimentally evaluate the function of a subset of protein variants found to have altered exon usage during the course of development and/or stem cell differentiation. Amongst these, we have found a protein which is known to be required for the initiation of differentiation but which conversely has a variant which is required to prevent premature/excess differentiation. We also found a protein with a variant with a novel function involved in stem cell differentiation into the haematopoietic (blood cell) lineage; and a protein with a variant with a role in maintaining proper cell adhesion.
We will make the database publically available so other research groups can use it to progress in the understanding of embryo development. In addition, some of the results obtained in this project have already been presented in national and international scientific meetings and we are currently preparing manuscripts for their publication in specialized scientific journals.
We have developed novel bioinformatics methods to generate for the first time a database of all protein variants present in each cell type during the early stages of mammalian development and stem cell differentiation. So far this data has already allowed us to find variants with a great effect on stem cell differentiation and with a potential role in the development of disease, and represents the stating point for many new projects in our group. Of special relevance is the fact that to date most studies had focused only in the study of how different proteins produced from different genes affect a particular cell type, not taking into account the production of various protein variants from a single gene. In this sense, our results highlight the importance of protein variants in the maintenance of cell identity in the context of early embryo development.
Moreover, upon publication in a scientific journal, our database will be made publically available for all research groups, opening up new research avenues in the developmental biology, assisted reproduction and stem cell fields.
Graphical summary of the project