Our lab's interests lie in taking quantitative fixed and live-cell approaches to understand cell decision-making processes during early development. We use mouse embryonic stem cells (mESCs) to study these processes in 2D and 3D Gastruloid culture. Gastruloids are aggregates of mESCs generated in non-adherent culture, which are able to effectively recapitulate many of the processes of early mammalian development such as symmetry-breaking, polarisation, gastrulation-like movements and the development of the three embryonic axes. Crucially, Gastruloids are easy to generate, can be experimentally manipulated with ease, and can be used to ask questions which are exceptionally difficult or impossible to address in the embryo.
Using Gastruloids to study axial patterning
The exterior of all vertebrates is bilaterally symmetric, but their interior is highly asymmetrical in both the positioning and the anatomy of internal organs such as the heart, viscera, lungs, gut and brain. The patterning process that leads to this left-right (LR) asymmetry is highly conserved, with the same positional bias in structures such as the heart and visceral organs observed from amphibians to birds and mammals. In humans, failure to correctly specify the asymmetrical placement (situs) and orientation of the different organs leads to a number of pathologies and severe birth defects such as heterotaxia, situs inversus, right/left isomerism, and congenital heart defects. Therefore, in addition to its central biological importance, understanding the origin and regulation of asymmetric patterning has clear biomedical implications.
Despite a vast number of studies into the regulation of LR patterning, the precise mechanisms generating this asymmetry are still subject to debate. Furthermore, the only current methods available to study these events involve the use of forward or reverse genetics in animal models. Although these have been used to great effect, they are difficult to manipulate, expensive and require large numbers of animals to ensure proper statistical power.
We are taking an alternative approach to study the mechanisms of LR asymmetry by using a novel technique called Gasturloids. Gastruloids ('Embryonic Organoids) are 3D aggregates of mouse embryonic stem cells (mESCs) that, over time, develop axial organisation and display gastrulation-like movements that mimic events in embryos. Gastruloids generate all embryonic axes (anteroposterior, dorsoventral, mediolateral), and develop bilateral asymmetry, similar to LR-asymmetry. This patterning is associated with a Node-like structure that is dependent on Nodal signalling, as in the embryo.
This opens up a unique experimental system to study the molecular mechanisms associated with the emergence of LR asymmetry in the mammalian embryo as well as a system for the study of genes and disease states. Such an experimental system allows experiments to be performed which are otherwise difficult to accomplish in embryos and since it is a tissue-culture based assay, it has the added benefit of replacing, reducing and refining the requirement for animal-based studies.
The role of NF-κB in early development
In vivo studies using the mouse embryo has allowed us to dissect many cell-fate decisions that lead to defined cell and tissue identities, however at this stage of development, embryos are difficult to manipulate, expensive, and require large numbers of animals to ensure proper statistical power. Embryonic stem (ES) cells, offer a useful and tractable alternative model to study cell fate decision- making due to their unlimited capacity for self-renewal, and their ability to differentiate into all tissues and lineages of the embryo proper. This approach simplifies the experimental system, and has the potential to provide a more nuanced understanding of how key cell-fate decisions are modulated during early development.
Recent work has suggested that the balance between differentiation and pluripotency in ES cells is modulated by members of the heterodimeric Nuclear Factor kappaB (NF-κB) family. These transcription factors are critical in determining cell fate in a number of contexts (inflammation, immunity etc.), yet its role in early mammalian development is highly controversial, and poorly defined. Furthermore, an important, and oft-overlooked characteristic of NF-κB signalling is its intracellular dynamics. Following activation, its localisation oscillates between the nucleus and cytoplasm, the oscillatory frequency encoding specific transcriptional programs, intricately linking dynamic activity to cell fate. As of yet, only bulk-cell assays or fixed samples have been used to study the role of NF-κB during ES cell multilineage differentiation. Consequently, there has been no attempt to understand whether the dynamics of NF-κB facilitate or control the cell-fate decisions that lead to the exit from pluripotency towards a defined embryonic lineage.
We're taking a combinatorial approach combining bulk-cell biochemical analysis, single-cell immunofluorescence and live-cell imaging to define the role of NF-κB in cell fate decisions during early mammalian lineage commitment.
A 3Rs approach to assess drug toxicity on the mammalian embryo during early pregnancy in vitro using Gastruloids
It is important for pregnant women, or women trying to conceive, to be aware of the risks posed by any medication they may have to take during pregnancy. This is especially important during the 1st trimester, when the embryo is at its most sensitive to developmental defects. The general advice given to women is not to take medication at all during their pregnancy & healthcare professionals are advised to prescribe only when the benefit to the mother outweighs the potential effects on the embryo. This demonstrates the need for adequate information on the effect of current & new drugs during pregnancy, especially when treating certain conditions (e.g. epilepsy, HIV) where withholding treatment might not be prudent. As pregnant women cannot be used to test whether drugs are harmful in pregnancy, we must rely on results from animals during pre-clinical testing, where millions of animals (a large proportion of which are rodents) are used & consequently the tests are highly expensive. Unfortunately, the accuracy of these Developmental & Reproductive Toxicity studies (DARTs) in predicting human developmental toxicity is still unknown & the absence of any effect in animal tests does not necessarily mean a drug is safe to take during pregnancy. Although there are validated in vitro alternatives, none of these provide an integrated approach to look at the early developing embryo.
We have developed a novel 3D tissue-culture protocol (Gastruloids) that uses small numbers of mouse embryonic stem cells (mESCs), grown in suspension. Over time, these gastruloids mimic many of the early developmental events seen during mouse development: symmetry-breaking, polarised gene expression, axis elongation & formation of 3 embryonic axes. Gastruloids are highly tractable, allowing precise experimental manipulation, difficult to do with embryos. This project aims to examine whether gastruloids will substitute mice during DARTs, directly targeting the area of drug development where animals are the predominant model for DARTs.
A multidisciplinary approach to define the dynamics of mammalian midline specification using Gastruloids
BIOTECHNOLOGY & BIOLOGICAL SCIENCE RESEARCH COUNCIL
December 2022 - December 2025
Development of the UK study for the ESA FLUMIAS project: Mitochondrial hydrogen peroxide as a mediator of muscle loss under microgravity and ageing
UK SPACE AGENCY (UK)
July 2022 - December 2024
Characterising the role of NF-𝜅B and metabolism in mouse embryonic stem cells at the transition from pluripotency to differentiation
March 2021 - March 2022
The Establishment of Left-Right Asymmetry in Mammalian Development
NATIONAL CENTRE FOR THE REP, REF AND RED OF ANIMALS USED IN RESEARCH (NC3RS)
March 2019 - February 2021
Project: Role of Wt1 in early development
Using in vitro and in vivo methods to dissect the role of Wt1 in early lineage decisions
Project: NF-kB signalling in early development
Taking quantitative, single-cell approaches to study the role of NF-kappaB signalling in cell fate decisions
Project: NF-kB signalling in early development
External: University of Newcastle
Dissecting the role of NF-kB signalling in early development
External: University of Aberdeen
Using Gastruloids to studying the effect of teratogens on early mammalian development