In this project, we have implemented a robust experimental pipeline delivering the following readouts: i) to quantify the expression levels (transcriptional activity) of specific sets of genes in a spatially resolved manner in intact embryos and ii) to visualise the three-dimensional structure of genomic DNA at the same time. The technical focus was to obtain these readouts in whole embryos, without the need for sectioning or dissociation of cells, and to also maintain a high level of structural sample integrity, allowing us to faithfully interpret associations between DNA structure and gene expression at the nanoscale.
These goals were achieved by i) using a particularly mild version of DNA FISH termed RASER FISH, which is able to preserve chromatin ultrastructure during labelling with optimised FISH probe designs targeting specific genomic regions and associated RNA transcripts ii) using advanced fluorescence microscopy to achieve high-resolution images deep inside the embryo (with resolutions around 100 nm throughout the entire ~500 000 µm^3 embryonic volume) and iii) by developing automated fluidics and microscopy to create a reliable, high throughput workflow capable of measuring structural DNA features of large genomic stretches in multiple embryos. As a proof of concept, this pipeline was applied to study the OCT4 locus, an important lineage decision gene during early embryonic development.
These results were disseminated to a large number of international scientists, at group meetings, EMBL internal seminars as well as multiple international conferences. The methodological approaches developed in this project lay the basis for further studies into the relationships between genomic structure and function with potential applications in better understanding of physiological development, cell differentiation as well as infertility as the result of misregulated development.
