Lab rotation project description
During the lab Rotations the student will gain experience in three main areas: Rotation 1- Embryology and single cell transcriptomics; Rotation 2- Bioinformatics, Rotation 3- Mathematical modelling.
Rotation 1: During the first rotation the student will learn how to use novel cell sorting techniques for large scale transcriptomic analysis of developing embryos. The student will use 10xGenomics platforms for cDNA library preparations and will also learn how to dissect embryos for these purposes. The student will also carry out quality controls using quantitative PCR and immunhistochemistry.
Rotation 2: During this rotation the student will learn basic principles of bioinformatics analysis of large datasets. Datasets available from public databases will be downloaded, normalized and compared to datasets obtained in our laboratory. R-packages will be used to analyse the datasets. The student will become familiar with most common techniques for gene expression analysis of deep-sequencing data.
Rotation 3: The student will acquire basic skills for creating predictive models of cell specification during gastrulation. The student will develop an individual- based model for the movement of cells through the embryo, considering morphogen gradients and with internal dynamics modelling of the relevant signalling pathways (based upon the literature). These skills will equip the student for the future analysis of the datasets planned for the project.
The aim of this proposal is to address a gap in our knowledge of the regulation of germ layer segregation during gastrulation in non-rodent embryos. This essential event during mammalian development partitions pools of cells in preparation for tissue specification.
Although our primary understanding of gastrulation in mammals comes from studies in mice, many important differences in gene regulation have been reported between rodent and non-rodent species. An example of these differences was recently provided by our research showing molecular differences in the specification of the germ cell lineage, the precursors of sperm and eggs, in pigs and humans compared to mice. These findings represent a paradigm of developmental differences between species and suggest that other germ layers may also be regulated by different gene networks from those described in mice. Furthermore, numerous studies have shown considerable differences in the developmental decisions in pre-implantation development of mice, human and other large mammals, supporting the idea that the mechanisms of lineage specification may also differ in these species. The study of the human embryo during gastrulation (which starts on week 2 of pregnancy) is hindered by the lack of embryological material. Because the early embryology of pigs and humans is largely conserved and we presented evidence of a shared developmental program of the germ cell lineage, we propose that the pig can inform on the developmental mechanisms of early human embryos. Pig and human embryos form a flat embryonic disc, rather than an egg cylinder characteristic of the mouse. Based on the similarities between pig and human embryos, and the gap of our knowledge of how gastrulation is regulated in non-rodent mammals, this project will study how cell differentiate during gastrulation in the pig embryo as a paradigm for embryonic disc forming species.
Hypothesis: It is hypothesized that the regulatory mechanisms of gastrulation in pig and humans are largely conserved, and that the pig embryo can be used as a surrogate system to study early human development.
Objective 1: To delineate the segregation of the three germ layers (ectoderm, mesoderm and endoderm) in the pig embryo using scRNA-Seq.
Objective 2: Perform functional evaluation of the roles of key transcription factors during gastrulation using Crispr/CAS9 gene editing in embryos.
Objective 3: Use mathematical modelling to predict lineage trajectories based on information generated in Objectives 1 and 2.
This research will further our understanding of the distinctive features of lineage segregation during gastrulation in the pig embryo and conserved features with humans. This research will further our knowledge of distinctive features of lineage segregation during gastrulation in the pig embryo and conserved features with humans. The research will also inform on new differentiation methods for the generation of germ layer precursors from hPSC and will impact the efficiency for directed differentiation towards specific cell types.