A multidisciplinary approach to define the dynamics of mammalian midline specification using Gastruloids

Mammals are 'bilaterians', meaning their body is divided into a left & right side that are mirror images along the 'head-to-toe' (anteroposterior; AP) axis. This division arises in early development following the formation of the midline, a virtual stripe along the AP axis of the embryo. Structures that form along the midline are important sources of signals that instruct the organisation of the embryo's back-front (dorsoventral; DV) & left-right (medio-lateral; ML) axes. The midline is a crucial reference point for the placement of tissues & organs. An improperly functioning midline can result in congenital malformations, chronic birth defects & heart conditions. Therefore, it is important to understand the biological mechanisms the embryo uses (genetic, chemical, mechanical) to precisely define where & how the midline is laid down, what controls its width, and how midline structures arise from it. Although traditional animal experiments using genetic approaches (remove/enhance gene function) have revealed the signals that specify & pattern the AP axis, less is known regarding the mechanisms (chemical, mechanical;mechanochemical) that direct embryonic cells to form the midline and control its boundaries.
These are fundamental, unresolved issues which this project will resolve by taking an innovative approach, using our advanced, state-of-the-art experimental system called 'gastruloids'. Gastruloids, 3D aggregates of embryonic stem cells, develop the 3 embryonic axes, mimicking many early embryonic patterning events. Importantly, gastruloids develop a midline, offering a tractable means to study the dynamic processes involved in midline specification, formation & morphogenesis. Our approach, coupling gastruloids with quantitative biological techniques and mathematical modelling, allows us to perform experiments that would otherwise be costly, difficult or impossible to do in embryos alone.

Firstly, we will characterise the gastruloid midline prior to & during the expression of multiple midline markers using techniques measuring transcript and protein. We will determine the role chemical signalling plays in midline specification through combination of chemical & genetic loss/gain of function experiments. We will target key signalling pathways at precise time-points, and establish which signals are important for controlling cell allocation and midline boundaries. Live, time-lapse imaging experiments will allow us to quantitatively measure the dynamics of midline specification in response to applied or inhibited chemical signals, allowing us to determine whether there is a correlation between the initial cell location and its final position in the midline.

Secondly, we will precisely perturb the mechanochemical signalling environment in which gastruloids reside by embedding them in a specialised chemically-defined 'gel'. These gels will be formulated with defined properties and composition, making sure the tensile strength & elasticity is kept the same for each formulation so as not to add additional complicating factors. This will allow us to disentangle the role of mechanical or biochemical signals in influencing the formation and placement of the midline. We will correlate the timing, composition & properties of these gels with the specification and placement of midline fates & morphology by live microscopy and the aforementioned quantitative biochemical techniques.

Finally will take a mathematical approach, formulating models to explore the theoretical basis for midline formation. We will infer the genetic mechanisms that could drive the emergence of midline patterning. We will explore alternative mechanisms for midline patterning generating explicit hypotheses & predictions that, critically, will be tested experimentally in gastruloids.
Our multidisciplinary approach will deliver a detailed understanding on how midline tissues are specified in gastruloids & how mechanochemical signalling fine-tunes midline fates.

The evolution of the midline was important for the development & diversification of complex bilaterian organisms organised with respect to a central axis. However the signalling requirements and dynamics governing its formation & morphogenesis are still unclear. We will define how the mammalian embryo specifies and places its midline using a novel experimental system called 'gastruloids' coupled with mathematical modelling, live imaging & quantitative biochemical techniques. Gastruloids, aggregates of embryonic stem cells, develop all three orthogonal embryonic axes in a spatially & temporally coordinated manner, mimicking early embryonic patterning events. Importantly gastruloids develop a midline, as indicated by midline gene expression along the AP axis. Gastruloids therefore allow the study of dynamic processes involved in midline specification, formation & morphogenesis using experimental approaches that would otherwise be costly, difficult or impossible to do in embryos alone.
We will firstly undertake a detailed quantitative characterisation of the gastruloid midline using in situ hybridisation chain reaction (HCR), immunofluorescence (IF) & live imaging, analysing the expression of markers that are confined to the midline. We will define what signalling inputs govern cell allocation to the midline, its gene-expression boundaries & morphogenesis by coupling genetic/chemical LOF/GOF with precise modulation of the mechanochemical environment using peptide hydrogels (PHs) with defined compositions and properties. Midline markers will be analysed with IF and HCR & live imaging will allow us to quantify these dynamics in real time. We will explore the theoretical basis for midline formation, generating explicit hypotheses & predictions that will be tested experimentally in gastruloids. Our multidisciplinary approach will deliver a detailed understanding on how midline tissues are specified in gastruloids & how mechanochemical signalling fine-tunes midline fates.