Investigating the impact of Wnt pathway modulation from targeted inhibition and over-the-counter medications on gastrulation of an in vitro 3D mouse model of early post-implantation development

Mammalian development is a highly complex and tightly regulated process that developmental biology still seeks to understand. Because embryogenesis occurs within the maternal uterus, stem cell-based in vitro models have been established to study these events without extensive animal-based experimentation. This study aimed to investigate the effects of the canonical Wnt pathway inhibitor XAV939 and the over-the-counter drug ibuprofen on ETiX embryoids, a recently generated 3D mouse embryoid model. XAV939 treatment at 10 μM reduced the expression of the Wnt-associated genes Dkk1 and especially T, while immunofluorescence analysis showed a marked loss of T-positive embryoids after 24 and 48 hours of exposure, indicating impaired gastrulation. These findings confirm that Wnt signaling is required for proper primitive streak formation and further support the validity of ETiX embryoids as a model of early mouse development. In contrast, ibuprofen did not significantly affect ETiX formation efficiency or the expression of most lineage and Wnt-related markers. However, it selectively upregulated Eomes and Snai1 at the highest concentration tested, suggesting a limited transcriptional effect and a potentially low impact on embryoid patterning under the tested conditions. Overall, these findings support the use of ETiX embryoids for studying early mouse development and pharmacological screening.