Unified Transcriptome and Mechanics Map of the Intact Mammalian Preimplantation Embryo In Situ

The development and maintenance of multicellular tissues require that cell states be closely coupled to their local environment, including geometric and mechanical cues. However, studying this coupling in intact tissues has been challenging, because existing measurement technologies cannot simultaneously assess mechanical properties and molecularly defined cell states. To address this gap, we introduce the Unified Transcriptome and Mechanics Map (UTMM), a method for concurrent measurement of the transcriptome and cytoplasmic stiffness (high-frequency intracellular mechanical compliance) within intact 3D multicellular structures. UTMM relies on two innovations: (1) a targeted in situ RNA sequencing approach for intact 3D embryos (3DISS), and (2) a strategy that leverages high-frequency intracellular organelle fluctuations to provide a relative, non-invasive readout of cell-level viscoelastic compliance in situ. We applied UTMM to mouse embryos from the zygote through the morula stage to characterize how RNA expression and intracellular mechanical state becomes coupled during early lineage specification. Our data reveal that, in the early morula, transcriptional and morphological distinctions emerge between trophectoderm and inner cell mass (ICM) lineages, coinciding with a gradual increase in intracellular compliance (softening) across all cells from the 2-cell through morula stages. Furthermore, we observe that early lineage biases associated with distinct mechanical states, reflecting emerging developmental programs. When we osmotically delayed this softening, embryonic progression was impeded, supporting a functional relationship between intracellular material state and developmental timing. Extending this approach to neural stem cells in the adult brain (NSCs), we further show that proliferated NSCs exhibit greater intracellular compliance than quiescent NSCs, NSC state-dependent differences in the mechanical properties and that the analytical framework can resolve biologically associated mechanical differences in a distinct tissue context. Together, these results demonstrate UTMM's ability to bridge molecular and mechanical dynamics in multicellular systems, providing a powerful framework for investigating how molecular state, geometry, and intracellular mechanics become coordinated during multicellular development.