The Establishment of Left-Right Asymmetry in Mammalian Development

Our external body plan is overtly symmetrical the distribution of organs & tissues throughout our body, is highly asymmetrical: left placement of the heart & the direction of intestine looping. Although the evolutionary reasons for this asymmetrical placement & anatomy of organs are not fully understood, mutations in genes that impact the proper development of L-R Asymmetry leads to a number of pathologies & birth defects demonstrating its central importance during the early embryo development. It is therefore of great clinical importance to have a clear understanding of these mechanisms if one is to understand these disease states.

The Node, a structure that forms during early development is central to the development of laterality. The cells of the Node contain motile cilia, shown to be critical to LR asymmetry: where either their motion generates a flow of extracellular fluid (nodal-flow) create morphogen gradients, carry vesicles of signalling factors or generate leftwards pressure on other, non-motile cilia surrounding the node: the outcome is the asymmetric expression of genes such as Nodal, Lefty2 (its inhibitor) & Pitx2 in the left of the embryo & the establishment of left identity. However, as the exact mechanisms generating LR asymmetry are still unclear and as the proper generation of laterality has clear clinical implications, it is beneficial to garner a better understanding of how LR asymmetry is established.

The only experimental models that exist to study this phenomena involve animals which are costly & difficult to manipulate at these early developmental stages, therefore a more tractable model to study the acquisition of laterality is therefore required. I have recently developed an experimental approach which involves growing mESCs in suspension where they aggregate, following which & after application of appropriate stimulation, they display many of the characteristics of the embryo including polarised gene expression & axial elongation. Significantly the generate a small region of cells (the node-like structure; Nd-LS) which express Nodal, a marker of the node, & they are able to break bilateral symmetry, manifested by one-sided expression of genes. I therefore propose to use this embryonic organoid (Gastruloid) system to study the mechanisms that result in the establishment of LR asymmetry during mammalian development.

Firstly, I will undertake a detailed analysis of the structure & function of the Gastruloid Nd-LS, focusing on its molecular & cellular components, comparing it to the embryo node by quantitatively analysing the expression node-specific fluorescent reporter genes in Gastruloids & measuring the transcription of genes associated with the node by in situ hybridisation chain reaction. Electron microscopy & immunostaining will allow an assessment of the structure & topographical features of the Nd-LS, determining the presence of cilia similar to the Node in vivo. Through a combination of microfluidics, chemical & genetic gain and loss of function experiments, I will be able to precisely apply treatments to the Gastruloids & quantitatively establish which signals are important in the establishment of the Nd-LS and the midline. I will then quantitatively measure & record the dynamics of the LR symmetry-breaking event in real time by generating knock-in lines expressing fluorescent fusion proteins & transcriptional reporters for two genes important in establishing laterality: Nodal & Lefty. Single-cell tracking will allow me to correlate the emergence of asymmetries with reporter expression. Finally, by removing this Nd-LS through microsurgical techniques, I will assess the importance of the Nd-LS on the generation of LR asymmetry following its physical ablation.

I will be able to use the system to gain a significant insight into the mechanisms involved in LR patterning as well as further demonstrating how Gastruloids are an excellent replacement for studying in vivo development.

The Node is a transient structure found at the distal-most tip of the embryo central to the development of Left-Right (LR) asymmetry. Motile cilia generate a net leftward flow of extracellular fluid across the node which is hypothesised to either create morphogen gradients, carry vesicles of signalling factors (e.g. Shh) or generate leftwards pressure on other, non-motile cilia surrounding the node. However, the exact mechanisms generating asymmetry are still unclear. No techniques are available to study these events in detail ex vivo, and in vivo experiments are difficult & expensive to accomplish. I have developed a novel 3D tissue culture technique (Gastruloids) where mouse ES cells aggregated in suspension, display a number of processes similar to the early gastrulating embryo such as symmetry-breaking, polarised gene expression and axial elongation. Interestingly, they also develop a node-like structure (Nd-LS) & bilateral asymmetry. I therefore propose to use Gastruloids to study the acquisition of LR asymmetry, through a combination of live imaging, quantitative cell biology & biomechanics, focusing on the role of the node in this process. I will first undertake a quantitative molecular analysis of the gene expression, analysing the structure & surface topology of the Nd-LS by in situ HCR, fluorescence & electron microscopy to assess the similarity between the Gastruloid Ns-S and the node. Coupling microfluidics, chemical gain or loss of function experiments & biomechanics will define signals that define the placement of the node and midline. I will then quantitatively measure & record the dynamics of the LR symmetry-breaking event by generating knock-in lines expressing Nodal and Lefty fluorescent fusion proteins & transcriptional reporters. Single-cell tracking will allow me to correlate the emergence of asymmetries with reporter expression. Finally, I will assess the importance of the Nd-LS on the generation of LR asymmetry following its physical ablation.

The research that will be undertaken in this project focuses on understanding how laterality is established during mammalian development using a novel tissue-culture method in place of the traditional animal models. This technique uses aggregates of mouse embryonic stem cells (Gastruloids) & permits the study of early developmental events, the dynamics of which are difficult to study in the embryo in real time. The work that will be undertaken in this project will significantly deliver on the NC3R's remit for the Replacement, Reduction & Refinement of animals in research.

Currently, the only methods used to assess LR patterning during development is to use mouse studies, sacrificing a large number of animals & their embryos. Additionally, to generate transgenic lines that allow dynamic visualisation of biological processes require upwards of 100 mice for each line. It is envisaged that a major outcome from this project is in cementing the Gastruloid technique as a excellent in vitro alternative ti study early developmental events, significantly reducing the number of animals used in these experiments with the overall aim for their complete replacement. Through the continued use of the Gastruloid model system, it is likely that it can be used as an initial screen prior to the use of animals for drug screens. As these Gastruloids undergo axial extensions & similar patterning events to the embryo, high throughput screens can be easily established to assess whether potential drug candidates will have detrimental effects early development, reducing the number of animals & embryos that may otherwise suffer will be significantly reduced.

It is envisioned that a better understanding of how interactions between Nodal & its inhibitors Lefty1/2, key proteins during early development, interact to specify distinct anterior & posterior regions within aggregates of mESCs. Using aggregates of mESCs is a novel approach to understand the early development of the embryo, as they mimic a number of important developmental events that are difficult to study in the embryo in real time (symmetry-breaking, axial elongation etc.). From the iterative processes between experimental & mathematical approaches, this project has the potential to generate a robust model that will be able to not only recapitulate the processes involved in early symmetry-breaking events, but may also serve as a generic framework for understanding symmetry-breaking at other stages of development.

The various outcomes & findings from the work undertaken in this project will be published in a timely manner firstly using online preprint servers to allow maximum distribution of the data followed by publication in open access journals. This will ensure that I can disseminate my work & findings not only to subscription paying institutions & their members, but to members of the public who are unlikely to have access to these journals. This will allow the public to see where publically funded money is being spent.

It is envisaged that I will be impacted by the programme of work proposed for this project. Over the course of the project I will be enhancing my skills in molecular biology techniques through extensive use of CRISPR/Cas technology, learning in situ hybridisation chain reaction (HCR) & mastering microsurgical techniques for the dissection of small areas of the Gastruloids: these skills are invaluable for my future work in this field. Through my collaboration with Matthias Lutolf, I will be further developing transferable skills in my ability to work in a multidisciplinary environment as well as being trained in the use of microfluidics & biosynthetic materials. In addition, through the frequent interactions with my collaborators, management of the research programme & dissemination of the findings from my research, I will be developing the skills that are essential for my development as a researcher & a future group leader.