Recording and controlling signaling dynamics during organoid symmetry breaking

The recent emergence of in vitro models of embryonic development and organogenesis has revealed the remarkable potential of stem cells to self-organize morphogenesis and cell fate specification. While great progress has been made towards characterizing these ‘stembryo’ models and validating them against in vivo standards, we have a comparatively less complete understanding of how signaling dynamics orchestrate stem cell self-organization. We address this challenging by developing precision tools for recording and manipulating signaling dynamics in gastruloid models of anterior-posterior axis formation.

First, we implement digital recorders of morphogen signaling using recombinase circuits. Recombinase circuits irreversibly label cells according to their signaling state within a “listening window” defined through addition of the small molecule doxycycline. Clonal mouse embryonic cell lines expressing morphogen recorder circuits can achieve high fidelity labeling with temporal resolution between 1-3 hours. By sampling a Wnt morphogen recorder at different timepoints during gastruloid morphogenesis, we map how dynamics of Wnt signaling encode anterior-posterior positional information. These measurements localize the timepoint of Wnt symmetry breaking to within a 6-hour time window (90 to 96 hrs). Notably, this window occurs before any apparent polarization in either gastruloid morphology or Wnt signaling domains, suggesting a key role for cellular rearrangements in axial morphogenesis.

Second, we are developing optogenetic methods for patterning signaling within gastruloids. Light- and small-molecule dependent genetic circuits enable spatiotemporally precise control of morphogen and/or inhibitor expression. As part of this effort, we are also designing custom instrumentation for patterning light over centimeter-scale samples with 10-20 um resolution, enabling high throughput studies of spatially defined signaling perturbations. We anticipate that these technologies will enable new quantitative descriptions of how morphogen signaling dynamics coordinate stem cell self-organization.