Optogenetic control of morphogen signaling in embryonic stem cells

The processes of cell proliferation, differentiation, migration, and self-organization during early embryonic development are governed by spatially and temporally varying morphogen signals. Such dynamic morphogen patterns drive cell fate specification at the proper location and time. However, current in vitro methods typically do not allow for precise, dynamic, spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics impact cell behavior. In embryonic stem cell (ESC) models for early development, for example, analogous tissue patterns spontaneously emerge but mechanistic insight into this self-organization has been limited. In this dissertation, we combine engineered methods for optogenetic stimulation and biological analysis to photoactivate Wnt/B-catenin signaling and to study morphogen dynamics and self-organization of human pluripotent stem cells./nFirst, we show that optogenetic Wnt/B-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns in human ESCs (hESCs). We expressed a fusion of the plant blue-light photoreceptor Cryptochrome 2 (Cry2) to the Wnt co-receptor LRP6 (the optoWnt system) in hESCs and induce Cry2-LRP6c oligomerization and subsequent canonical Wnt/B-catenin activation using novel engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). Such LAVA devices are a LED-based, programmable illumination system for photostimulation of multi-well plates that can be readily incorporated into the workflow of routine cell culture and allow controlled and quantitative spatiotemporal light patterning. We optimized the LAVA board optical configuration for illumination uniformity and achieve programmable photostimulation of independent wells of 24-well or 96-well culture plates kept in standard 37C tissue culture incubators. Each well can be wirelessly programmed through a graphical user interface (GUI) at user-defined intensities (0 -- 20 uWmm-2, 0.005 uWmm-2 resolution), temporal sequences (10 ms resolution), and spatial patterns (100 um resolution). We demonstrate LAVA board performance by modulating the intensity, timing, and spatial location of canonical Wnt/$\beta$-catenin signaling in hESC cultures using the optoWnt optogenetic system. We show that Wnt pathway activation and hESC differentiation is dose-responsive to light intensity and duration of illumination, and that spatial patterning can be used to simulate the embryonic, spatially polarized presentation of the Wnt ligand./nIn parallel, we engineered a microscope capable of dynamic photostimulation and high-resolution imaging with structured illumination microscopy (SIM). A digital micromirror device (DMD) is used to project user-defined photostimulation sequences onto the sample with diffraction-limited spatial resolution (800 nm). For fluorescence imaging, a series of multi-spot patterns is projected onto the sample to reconstruct a high-resolution SIM image with a 2-fold resolution improvement over the diffraction limit. Such multi-color, user-defined photostimulation and SIM imaging (opto-SIM) allows live-cell timelapse imaging while localizing Wnt/B-catenin photoactivation to specific regions within hESC cultures. Unlike LED-based illumination devices, such as the LAVA board system, the opto-SIM system enables pattern projection at subcellular resolution. Furthermore, projected patterns can be dynamically updated based on feedback from measurements acquired during experiment progression. The opto-SIM system also enables temporal control of signaling at a resolution of 60 Hz, as well as 8-bit greyscale modulation to achieve dose-dependent optoWnt activation. Such flexibility in spatiotemporal patterning gives the unprecedented ability to stimulate stem-cell based model systems, organoids, or embryos with time-varying patterns of morphogen gradients./nLastly, we implement optogenetic control of canonical Wnt signaling to determine whether differential Wnt signaling can model human gastrulation and lead to emergence of organized shape and structure through collective cell rearrangement. We achieve optogenetic control of Wnt signaling in hESCs by illuminating hESC cultures expressing the optoWnt system with LAVA devices. Wnt/B-catenin signaling was activated over a high dynamic range (>500-fold) and drove broad transcriptional changes and mesendoderm differentiation of human ESCs at high efficiency (>95% cells). Furthermore, activating Wnt signaling in subpopulations of ESCs in 2D and 3D cultures induced cell self-organization and morphogenesis reminiscent of human gastrulation, including changes in cell migration and epithelial to mesenchymal transition. We thus developed an hESC model for studying Wnt-mediated morphogenesis in early development. Using this gastrulation model in combination with transcriptomic analysis and single-cell migration studies, we show that Wnt signaling is sufficient for inducing self-organization of cells in an EMT-dependent manner. Our findings thus reveal an instructive role for Wnt in directing cell patterning in this ESC model for early embryogenesis./nIn summary, we developed and utilized engineered methods for spatial and temporal light patterning to photoactivate Wnt signaling in a precise location and at a given time within embryonic stem cell cultures. Such optogenetic activation of morphogen signaling in specific cell subpopulations allows studies of cell-cell interactions and signaling dynamics that regulate early embryonic development.

Advisor: Schaffer, David