Periodic Reporting for period 1 - EmbryoPAINT (PAINTing the architecture of the totipotency gene network during early mammalian development)
Reporting period: 2020-09-01 to 2022-08-31
While the different cell types found in the human body have very different morphologies and functions, each cell has an identical set of chromosomes with the same genetic sequence and is derived from only a single fertilised egg cell. Therefore, to give rise to different cell types, DNA activity is regulated at the level of its physical structure and by chemical modifications throughout mammalian development. These changes are responsible for the activation or the silencing of different sets of genes and thus for the differentiation of the early embryo into an intricate organism with many different cell types.
In order to study the relationship between these structural changes of DNA and the first cell fate decisions during mammalian development, this project aimed at i) developing a technology that would allow us to interrogate DNA structure at the nanoscale in whole preimplantation mouse embryos and ii) use this technology to gain insights into the structural changes occurring at a set of marker genes which are activated during the first cellular differentiation step in early embryogenesis.
In order to study the relationship between these structural changes of DNA and the first cell fate decisions during mammalian development, this project aimed at i) developing a technology that would allow us to interrogate DNA structure at the nanoscale in whole preimplantation mouse embryos and ii) use this technology to gain insights into the structural changes occurring at a set of marker genes which are activated during the first cellular differentiation step in early embryogenesis.
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.
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.
The technology developed in this project goes beyond the current state of the art in multiple aspects. While conventional tools to study chromatin architecture such as chromosome conformation capture techniques typically suffer from low sampling and therefore resolution at the single cell or single embryo level, our oligoPAINTs technology delivers a readout from most cells within an embryo at high genomic resolution (around 5 kb). Furthermore, we can visualise genome structure in the context of the whole embryo and analyse the transcriptional output from certain genes in the different cells of the embryo at the same time, a multimodal readout not possible with other methods. This allows us to distinguish different cell populations (such as the outer trophectoderm layer from the inner cell mass in blastocysts), their positions and interactions, and generate hypothesis about the structure-function relationship between genome architecture, transcriptional activity and cell differentiation. Lastly, the ability to directly visualise genomic architecture inside its nuclear environment offers the potential to uncover relationships between nuclear architecture, functional compartments and gene positioning and activity in single cells in the developing embryo. These insights will advance our understanding of cellular differentiation and reprogramming, crucial aspects for the field of regenerative medicine, which has a large potential for advancing medical treatments in the future.