Investigating Wnt3-mediated symmetry breaking and cell fate in engineered gastruloids

Abstract

Establishment of the anteroposterior (AP) axis in developing mouse embryos is informed by cues from extraembryonic tissues (Arnold & Robertson, 2009; Rivera-Pérez & Magnuson, 2005). In contrast, aggregates of embryonic stem cells (ESCs) termed gastruloids are able to display symmetry breaking and develop an AP axis in the absence of extraembryonic tissues or localised exogenous positional signalling (Baillie-Johnson, van den Brink, Balayo, Turner, & Arias, 2015; Beccari et al., 2018; Turner et al., 2017; Van Den Brink et al., 2014). The exact mechanisms behind these processes remain unclear but depend on feedback in Wnt signalling (Turner et al., 2017), as well as heterogeneity in Wnt activity, which has been shown to precede symmetry breaking (McNamara, Solley, Adamson, Chan, & Toettcher, 2023).

2D culture systems have been used to study heterogeneity and local propagation of Wnt activity and local community effect in other contexts (Martyn, Brivanlou, & Siggia, 2019; Nemashkalo, Ruzo, Heemskerk, & Warmflash, 2017; Yoney, Bai, Brivanlou, & Siggia, 2022). However, it is challenging to study the effects on symmetry breaking and AP axis determination in the absence of 3D architexture and patterning. Chimeric gastruloids (Wehmeyer et al., 2022) generated by co-aggregating ES cell lines with distinct genetic backgrounds offer the opportunity to probe the effects of engineered heterogeneity in Wnt activity in a 3D environment. In combination with cell neighbour- labelling technologies (Malaguti, Lebek, Blin, & Lowell, 2024; Malaguti et al., 2021; Morsut et al., 2016), I attempt to investigate non-cell-autonomous effects of Wnt heterogeneity.

Here I present my work combining these two technologies to test the hypothesis that Wnt3 locally amplifies its own expression to influence the position, identity and local coherence of mesoderm fated cells during symmetry breaking in gastruloids.

I describe the design and engineering of novel tools for expressing tagged but functional Wnt3 in gastruloids, either in a uniform or chimeric manner. Building upon the resulting experimental background, I explore the use of cell contact-labelling technology in a 3D setting to ask how Wnt3 influences multicellular organisation of cell fate decisions.

I demonstrate that synthetic-Notch based cell contact labelling can successfully distinguish between neighbours and non-neighbours of Wnt3-overexpressing experimental or control cells in early gastruloids and discuss the limitations of this approach at later stages. My work probing the characteristics and limitations of this system have broader implications as it showcases areas that challenge synthetic neighbour-labelling technologies in their current form.

In addition, I describe the development of a set of image analysis pipelines for quantitative analysis of gastruloid morphology, cell fate decisions, local coherence of identity and the clustering and positioning of cells of shared identity within homogenous and chimeric gastruloids. In future, these could be adapted for comprehensive high-throughput analysis of self-organisation and morphogenesis in gastruloids or other 3D embryoid models.

Using these tools, I found that gastruloids are able to break symmetry, elongate and generate both mesodermal and neural cell types in the absence of exogenous Wnt activation when an internally tagged version of Wnt3 is constitutively expressed in all cells of the gastruloid. This observation is consistent with the idea that heterogeneity in Wnt expression may not be the only factor driving symmetry breaking. Moreover, consistently forming gastruloids in the absence of global exogenous Wnt activation opens up opportunities for manipulating Wnt activity in a mosaic manner in chimeric gastruloids.

Finally, by generating chimeric gastruloids from labelled and reporter cell lines, I have obtained preliminary evidence that in the absence of global Wnt activation Wnt3 over- expressing cells within chimeric gastruloids may be biased towards contribution to the elongating tip, that Wnt3 may upregulate Wnt3 expression in neighbours in some contexts, and that internal overexpression of Wnt3 may affect the proportion neural and mesodermal descendants.

Alongside these results, I discuss a number of technical challenges in obtaining and interpreting data from these complex experimental systems, and I offer suggestions for improving these systems in future work. The main barriers to interpretation of the collected data stem from variability within population-level gene expression analysis of sorted sub- populations, and from difficulties in single-cell resolution imaging and image analysis. Most of these obstacles could be overcome through single-cell RNA sequencing approaches in future studies.

In summary, I have established a novel set of tools and approaches to aid investigations into the role of Wnt signalling in mediating local cell identity to generate coherent patterning in gastruloids. These methods could be of use for future studies of the prerequisites and disruptors of self-organisation within 3D organoid systems.