Optogenetic control of Wnt signaling for modeling early embryogenic patterning with human pluripotent stem cells

Abstract

20The processes of cell proliferation, differentiation, migration, and self-organization during early 21 embryonic development are governed by dynamic, spatially and temporally varying morphogen 22 signals. Analogous tissue patterns emerge spontaneously in embryonic stem cell (ESC) models 23 for gastrulation, but mechanistic insight into this self-organization is limited by a lack of molecular 24 methods to precisely control morphogen signal dynamics. Here we combine optogenetic 25 stimulation and single-cell imaging approaches to study self-organization of human pluripotent 26 stem cells. Precise control of morphogen signal dynamics, achieved through activation of 27 canonical Wnt/b-catenin signaling over a broad high dynamic range (>500-fold) using an optoWnt 28 optogenetic system, drove broad transcriptional changes and mesendoderm differentiation of 29 human ESCs at high efficiency (>95% cells). Furthermore, activating Wnt signaling in 30 subpopulations of ESCs in 2D and 3D cultures induced cell self-organization and morphogenesis 31 reminiscent of human gastrulation, including changes in cell migration and epithelial to 32 mesenchymal transition. Our findings thus reveal an instructive role for Wnt in directing cell 33 patterning in this ESC model for gastrulation. 34Molecular regulation of embryonic morphogenesis, a process where seemingly identical cells 35 differentiate and organize into spatially defined regions, remains poorly understood in mammalian 36 developmental biology 1 . The emergence of such spatial organization is attributed to cell-intrinsic 37 differences in gene expression 2,3 or extrinsic asymmetries within the cell environment 4,5 . The 38 resulting variability in intracellular signaling leads to changes in cell migration, cell-cell 39 interactions, and/or cell polarity that in turn drive the coordinated organization of specific cell 40 populations 6,7 .41 Self-organization within the embryonic tissue proper, or epiblast, is first evident during 42 gastrulation, where subpopulations of cells reorganize and differentiate along distinct cell lineages 43 to form the three germ layers of the future organism 4 . Initially, asymmetric patterns of molecular 44 signals -morphogens such as Wnt, BMP, Nodal, and FGF -emerge across the morphologically 45 symmetric epiblast of the mouse embryo 4,8-12 . The establishment of such signaling asymmetry is 46 followed by morphological symmetry-breaking, where a subpopulation of posterior epiblast cells 47 undergoes mesendoderm differentiation and an epithelial to mesenchymal transition (EMT), then 48 migrates away from the epithelial epiblast cells in the region of the primitive streak 13,14 . During this 49 dynamic process of gastrulation, the temporal order and location of cell migration through the 50 primitive streak is correlated with downstream cell fate outcome, resulting in organization of the 51 developing cell lineages into defined regions within the embryo 13,15-17 .52 However, while advances in live embryo imaging and transcriptomic analysis hav...

β-catenin-Mediated Wnt Signal Transduction Proceeds Through an Endocytosis-Independent Mechanism

“…Additional lines of evidence in the field support that internalization of the ligand and the receptor complex via endocytosis is not a general requirement for Wnt pathway activation. There are instances in which overexpression or manipulation of Wnt pathway components within the cytoplasm is sufficient to initiate signaling: overexpression of the cytoplasmic Wnt factor Dishevelled (Dvl) or optogenetic oligomerization of the cytoplasmic portion of Lrp6 (35) (36). Second, exposure to Wnt molecules covalently attached to a magnetic bead that therefore cannot be internalized by cells is sufficient for pathway activation in mESCs (37).…”

Engineered illumination devices for optogenetic control of cellular signaling dynamics

“…Also, the intensity range should be sufficient to activate the photosensory protein of interest, which can vary widely from continuous illumination at 1 µWmm -2 (Repina et al 2019) to short pulses at 1,000 – 10,000 µWmm -2 (Yizhar et al 2011). Moreover, the required temporal resolution also depends on the photosensory domain and experimental application: 1-100 ms pulses are typically used for control of neural circuits (Yizhar et al 2011), whereas signal pathway dynamics are controlled on the second to multi-day timescales (Repina et al 2019; Johnson & Toettcher 2018). Furthermore, for spatial resolution requirements, optimal resolution for subcellular stimulation would be 0.2 – 2µm, but such resolutions are infeasible without complex optical systems.…”

“…In particular, a variety of photosensory domains have been discovered, optimized, and repurposed to place intracellular signaling pathways under light control, capabilities that offer the unique advantage of using light patterns to stimulate signaling in a specific location and at a specific time (Repina et al 2017). Within mammalian cells, optogenetic tools that control protein-protein interactions have for example been developed for diverse signaling pathways such as Wnt/β-catenin (Bugaj et al 2013; Repina et al 2019), Ras/Raf/Mek/Erk (Toettcher et al 2011; Johnson & Toettcher 2019; Toettcher et al 2013; Johnson et al 2017), fibroblast growth factor (FGF) (Kainrath et al 2017), and Rho-family GTPase signaling (Levskaya et al 2009; Yazawa et al 2009; Strickland et al 2012; Guntas et al 2015; Bugaj et al 2015; Wang et al 2010). Optogenetic methods have also been recently applied to studies in developmental biology in key model organisms (Johnson & Toettcher 2018).…”

“…For example, in Drosophila and zebrafish embryos, dynamic control of signaling elucidated the spatiotemporal limits of ERK (Johnson et al 2017; Johnson & Toettcher 2019), Nodal (Sako et al 2016), and Bicoid (Huang et al 2017) patterning and how local modulation of cell migration and contractility drives tissue morphogenesis (Izquierdo et al 2018; Guglielmi et al 2015; Čapek et al 2019). Furthermore, in embryonic stem cell models for mammalian development, we have recently achieved optogenetic control of canonical Wnt signaling and elucidated mechanisms of tissue self-organization during mesendoderm differentiation (Bugaj et al 2013; Repina et al 2019).…”

“…Each well can be wirelessly programmed through a graphical user interface (GUI) at user-defined intensities (0 – 20 µWmm -2 , 0.005 µWmm -2 resolution), temporal sequences (10 ms resolution), and spatial patterns (100 µm resolution). We demonstrate LAVA board performance by modulating the intensity, timing, and spatial location of canonical Wnt/β-catenin signaling in human embryonic stem cell (hESC) cultures using the optoWnt optogenetic system (Bugaj et al 2013; Repina et al 2019). 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.…”