Curated Optogenetic Publication Database

Abstract: Morphogen gradients convey essential spatial information during tissue patterning. Although the concentration and timing of morphogen exposure are both crucial, how cells interpret these graded inputs remains challenging to address. We employed an optogenetic system to acutely and reversibly modulate the nuclear concentration of the morphogen Dorsal (DL), homolog of NF-κB, which orchestrates dorsoventral patterning in the Drosophila embryo. By controlling DL nuclear concentration while simultaneously recording target gene outputs in real time, we identified a critical window for DL action that is required to instruct patterning and characterized the resulting effect on spatiotemporal transcription of target genes in terms of timing, coordination and bursting. We found that a transient decrease in nuclear DL levels at nuclear cycle 13 leads to reduced expression of the mesoderm-associated gene snail (sna) and partial derepression of the neurogenic ectoderm-associated target short gastrulation (sog) in ventral regions. Surprisingly, the mispatterning elicited by this transient change in DL was detectable at the level of single-cell transcriptional bursting kinetics, specifically affecting long inter-burst durations. Our approach of using temporally resolved and reversible modulation of a morphogen in vivo, combined with mathematical modeling, establishes a framework for understanding the stimulus-response relationships that govern embryonic patterning.

Abstract: To address the need for methods for tagging and manipulating neuronal ensembles underlying specific behaviors, we present an improved version of FLARE, termed cytoFLARE (cytosol-expressed FLARE). cytoFLARE incorporates cytosolic tethering of a transcription factor and expression of a more sensitive pair of calcium-sensing domains. We show that cytoFLARE captures more calcium- and light-dependent signals in HEK293T cells and higher signal-to-background ratios in neuronal cultures. We further establish cytoFLARE transgenic Drosophila models and apply cytoFLARE to label activated neurons upon sensory or optogenetic stimulation within a defined time window. Notably, through the cytoFLARE-driven expression of optogenetic actuators, we successfully reactivated and inhibited neurons involved in the larval nociceptive system. Our findings demonstrate the characterization and application of time-gated calcium integrators for both recording and manipulating neuronal activity in Drosophila larvae.

Abstract: Morphogen gradients convey essential spatial information during tissue patterning. While both concentration and timing of morphogen exposure are crucial, how cells interpret these graded inputs remains challenging to address. We employed an optogenetic system to acutely and reversibly modulate the nuclear concentration of the morphogen Dorsal (DL), homologue of NF-κB, which orchestrates dorso-ventral patterning in the Drosophila embryo. By controlling DL nuclear concentration while simultaneously recording target gene outputs in real time, we identified a critical window for DL action that is required to instruct patterning, and characterized the resulting effect on spatio-temporal transcription of target genes in terms of timing, coordination, and bursting. We found that a transient decrease in nuclear DL levels at nuclear cycle 13 leads to reduced expression of the mesoderm-associated gene snail (sna) and partial derepression of the neurogenic ectoderm-associated target short gastrulation (sog) in ventral regions. Surprisingly, the mispatterning elicited by this transient change in DL is detectable at the level of single cell transcriptional bursting kinetics, specifically affecting long inter-burst durations. Our approach of using temporally-resolved and reversible modulation of a morphogen in vivo, combined with mathematical modeling, establishes a framework for understanding the stimulus-response relationships that govern embryonic patterning.

Abstract: Transcriptional control is fundamental to cellular function. However, despite knowing that transcription factors can repress or activate specific genes, how these functions are implemented at the molecular level has remained elusive, particularly in the endogenous context of developing animals. Here, we combine optogenetics, single-cell live-imaging, and mathematical modeling to study how a zinc-finger repressor, Knirps, induces switch-like transitions into long-lived quiescent states. Using optogenetics, we demonstrate that repression is rapidly reversible (~1 min) and memoryless. Furthermore, we show that the repressor acts by decreasing the frequency of transcriptional bursts in a manner consistent with an equilibrium binding model. Our results provide a quantitative framework for dissecting the in vivo biochemistry of eukaryotic transcriptional regulation.

Abstract: During animal development, the spatiotemporal properties of molecular events largely determine the biological outcomes. Conventional gene analysis methods lack the spatiotemporal resolution for precise dissection of developmental mechanisms. Although optogenetic tools exist for manipulating designer proteins in cultured cells, few have been successfully applied to endogenous proteins in live animals. Here, we report OptoTrap, a light-inducible clustering system for manipulating endogenous proteins of diverse sizes, subcellular locations, and functions in Drosophila. This system turns on fast, is reversible in minutes or hours, and contains variants optimized for neurons and epithelial cells. By using OptoTrap to disrupt microtubules and inhibit kinesin-1 in neurons, we show that microtubules support the growth of highly dynamic dendrites and that kinesin-1 is required for patterning of low- and high-order dendritic branches in differential spatiotemporal domains. OptoTrap allows for precise manipulation of endogenous proteins in a spatiotemporal manner and thus holds promise for studying developmental mechanisms in a wide range of cell types and developmental stages.

Abstract: Extracellular-signal-regulated kinase (ERK) signaling controls development and homeostasis and is genetically deregulated in human diseases, including neurocognitive disorders and cancers. Although the list of ERK functions is vast and steadily growing, the full spectrum of processes controlled by any specific ERK activation event remains unknown. Here, we show how ERK functions can be systematically identified using targeted perturbations and global readouts of ERK activation. Our experimental model is the Drosophila embryo, where ERK signaling at the embryonic poles has thus far only been associated with the transcriptional patterning of the future larva. Through a combination of live imaging and phosphoproteomics, we demonstrated that ERK activation at the poles is also critical for maintaining the speed and synchrony of embryonic cleavages. The presented approach to interrogating phosphorylation networks identifies a hidden function of a well-studied signaling event and sets the stage for similar studies in other organisms.

Abstract: Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate an optogenetic tool that disrupts Gαq signaling through membrane recruitment of a minimal regulator of G protein signaling (RGS) domain. This approach, Photo-induced Gα Modulator-Inhibition of Gαq (PiGM-Iq), exhibited potent and selective inhibition of Gαq signaling. Using PiGM-Iq we alter the behavior of Caenorhabditis elegans and Drosophila with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq changes axon guidance in cultured dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. Furthermore, by altering the minimal RGS domain, we show that this approach is amenable to Gαi signaling. Our unique and robust optogenetic Gα inhibiting approaches complement existing neurobiological tools and can be used to investigate the functional effects neuromodulators that signal through GPCR and trimeric G proteins.

Abstract: Associative learning enables the adaptive adjustment of behavioral decisions based on acquired, predicted outcomes. The valence of what is learned is influenced not only by the learned stimuli and their temporal relations, but also by prior experiences and internal states. In this study, we used the fruit fly Drosophila melanogaster to demonstrate that neuronal circuits involved in associative olfactory learning undergo restructuring during extended periods of low-caloric food intake. Specifically, we observed a decrease in the connections between specific dopaminergic neurons (DANs) and Kenyon cells at distinct compartments of the mushroom body. This structural synaptic plasticity was contingent upon the presence of allatostatin A receptors in specific DANs and could be mimicked optogenetically by expressing a light-activated adenylate cyclase in exactly these DANs. Importantly, we found that this rearrangement in synaptic connections influenced aversive, punishment-induced olfactory learning but did not impact appetitive, reward-based learning. Whether induced by prolonged low-caloric conditions or optogenetic manipulation of cAMP levels, this synaptic rearrangement resulted in a reduction of aversive associative learning. Consequently, the balance between positive and negative reinforcing signals shifted, diminishing the ability to learn to avoid odor cues signaling negative outcomes. These results exemplify how a neuronal circuit required for learning and memory undergoes structural plasticity dependent on prior experiences of the nutritional value of food.

Abstract: During development, epithelia function as malleable substrates that undergo extensive remodeling to shape developing embryos. Optogenetic control of Rho signaling provides an avenue to investigate the mechanisms of epithelial morphogenesis, but transgenic optogenetic tools can be limited by variability in tool expression levels and deleterious effects of transgenic overexpression on development. Here, we use CRISPR/Cas9 to tag Drosophila RhoGEF2 and Cysts/Dp114RhoGEF with components of the iLID/SspB optogenetic heterodimer, permitting light-dependent control over endogenous protein activities. Using quantitative optogenetic perturbations, we uncover a dose-dependence of tissue furrow depth and bending behavior on RhoGEF recruitment, revealing mechanisms by which developing embryos can shape tissues into particular morphologies. We show that at the onset of gastrulation, furrows formed by cell lateral contraction are oriented and size-constrained by a stiff basal actomyosin layer. Our findings demonstrate the use of quantitative, 3D-patterned perturbations of cell contractility to precisely shape tissue structures and interrogate developmental mechanics.

Abstract: Epithelial furrowing is a fundamental morphogenetic process during gastrulation, neurulation, and body shaping. A furrow often results from a fold that propagates along a line. How fold formation and propagation are controlled and driven is poorly understood. To shed light on this, we study the formation of the cephalic furrow, a fold that runs along the embryo dorsal-ventral axis during Drosophila gastrulation and the developmental role of which is still unknown. We provide evidence of its function and show that epithelial furrowing is initiated by a group of cells. This cellular cluster works as a pacemaker, triggering a bidirectional morphogenetic wave powered by actomyosin contractions and sustained by de novo medial apex-to-apex cell adhesion. The pacemaker's Cartesian position is under the crossed control of the anterior-posterior and dorsal-ventral gene patterning systems. Thus, furrow formation is driven by a mechanical trigger wave that travels under the control of a multidimensional genetic guide.

Abstract: Positional information in development often manifests as stripes of gene expression, but how stripes form remains incompletely understood. Here, we use optogenetics and live-cell biosensors to investigate the posterior brachyenteron (byn) stripe in early Drosophila embryos. This stripe depends on interpretation of an upstream ERK activity gradient and the expression of two target genes, tailless (tll) and huckebein (hkb), that exert antagonistic control over byn. We find that high or low doses of ERK signaling produce transient or sustained byn expression, respectively. Although tll transcription is always rapidly induced, hkb converts graded ERK inputs into a variable time delay. Nuclei thus interpret ERK amplitude through the relative timing of tll and hkb transcription. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop, which is sufficient to explain byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. We further show that 'blurring' of an all-or-none stimulus through intracellular diffusion non-locally produces a byn stripe. Overall, we provide a blueprint for using optogenetics to dissect developmental signal interpretation in space and time.

Abstract: Miro GTPases control mitochondrial morphology, calcium homeostasis, and regulate mitochondrial distribution by mediating their attachment to the kinesin and dynein motor complex. It is not clear, however, how Miro proteins spatially and temporally integrate their function as acute disruption of protein function has not been performed. To address this issue, we have developed an optogenetic loss of function "Split-Miro" allele for precise control of Miro-dependent mitochondrial functions in Drosophila. Rapid optogenetic cleavage of Split-Miro leads to a striking rearrangement of the mitochondrial network, which is mediated by mitochondrial interaction with the microtubules. Unexpectedly, this treatment did not impact the ability of mitochondria to buffer calcium or their association with the endoplasmic reticulum. While Split-Miro overexpression is sufficient to augment mitochondrial motility, sustained photocleavage shows that Split-Miro is surprisingly dispensable to maintain elevated mitochondrial processivity. In adult fly neurons in vivo, Split-Miro photocleavage affects both mitochondrial trafficking and neuronal activity. Furthermore, functional replacement of endogenous Miro with Split-Miro identifies its essential role in the regulation of locomotor activity in adult flies, demonstrating the feasibility of tuning animal behaviour by real-time loss of protein function.

Abstract: Rapid epithelial tissue flows are essential to building and shaping developing embryos. However, the mechanical properties of embryonic epithelial tissues and the factors that control these properties are not well understood. Actomyosin generates contractile tensions and contributes to the mechanical properties of cells and cytoskeletal networks in vitro, but it remains unclear how the levels and patterns of actomyosin activity contribute to embryonic epithelial tissue mechanics in vivo. To dissect the roles of cell-generated tensions in the mechanics of flowing epithelial tissues, we use optogenetic tools to manipulate actomyosin contractility with spatiotemporal precision in the Drosophila germband epithelium, which rapidly flows during body axis elongation. We find that manipulating actomyosin-dependent tensions by either optogenetic activation or deactivation of actomyosin alters the solid-fluid mechanical properties of the germband epithelium, leading to changes in cell rearrangements and tissue-level flows. Optogenetically activating actomyosin leads to increases in the overall level but decreases in the anisotropy of tension in the tissue, whereas optogenetically deactivating actomyosin leads to decreases in both the level and anisotropy of tension compared to in wild-type embryos. We find that optogenetically activating actomyosin results in more solidlike (less fluidlike) tissue properties, which is associated with reduced cell rearrangements and tissue flow compared to in wild-type embryos. Optogenetically deactivating actomyosin also results in more solidlike properties than in wild-type embryos but less solidlike properties compared to optogenetically activating actomyosin. Together, these findings indicate that increasing the overall tension level is associated with more solidlike properties in tissues that are relatively isotropic, whereas high-tension anisotropy fluidizes the tissue. Our results reveal that epithelial tissue flows in developing embryos involve the coordinated actomyosin-dependent regulation of the mechanical properties of tissues and the tensions driving them to flow in order to achieve rapid tissue remodeling.

Abstract: Active Notch signalling is elicited through receptor-ligand interactions that result in release of the Notch intracellular domain (NICD), which translocates into the nucleus. NICD activates transcription at target genes forming a complex with the DNA-binding transcription factor CSL (CBF1/Su(H)/Lag-1) and co-activator Mastermind. Despite this, CSL lacks its own nuclear localisation sequence, and it remains unclear where the tripartite complex is formed. To probe mechanisms involved, we designed an optogenetic approach to control NICD release (OptIC-Notch) and monitored consequences on complex formation and target gene activation. Strikingly we observed that, when uncleaved, OptIC-Notch sequestered CSL in the cytoplasm. Hypothesising that exposure of a juxta membrane ΦWΦP motif is key to sequestration, we masked this motif with a second light sensitive domain in OptIC-Notch{ω}, which was sufficient to prevent CSL sequestration. Furthermore, NICD produced by light-induced cleavage of OptIC-Notch or OptIC-Notch{ω} chaperoned CSL into the nucleus and induced target gene expression, showing efficient light controlled activation. Our results demonstrate that exposure of the ΦWΦP motif leads to CSL recruitment and suggest this can occur in the cytoplasm prior to nuclear entry.

Abstract: Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G-protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate a new optogenetic tool that disrupt Gαq signaling through membrane recruitment of a minimal Regulator of G-protein signaling (RGS) domain. This approach, Photo-induced Modulation of Gα protein – Inhibition of Gαq (PiGM-Iq), exhibited potent and selective inhibition of Gαq signaling. We alter the behavior of C. elegans and Drosophila with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq also changes axon guidance in culture dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. By altering the choice of minimal RGS domain, we also show that this approach is amenable to Gαi signaling.

Abstract: Contractile forces generated by actin and non-muscle myosin II ("actomyosin contractility") are critical for morphological changes of cells and tissues at multiple length scales, such as cell division, cell migration, epithelial folding, and branching morphogenesis. An in-depth understanding of the role of actomyosin contractility in morphogenesis requires approaches that allow the rapid inactivation of actomyosin, which is difficult to achieve using conventional genetic or pharmacological approaches. The presented protocol demonstrates the use of a CRY2-CIBN based optogenetic dimerization system, Opto-Rho1DN, to inhibit actomyosin contractility in Drosophila embryos with precise temporal and spatial controls. In this system, CRY2 is fused to the dominant negative form of Rho1 (Rho1DN), whereas CIBN is anchored to the plasma membrane. Blue light-mediated dimerization of CRY2 and CIBN results in rapid translocation of Rho1DN from the cytoplasm to the plasma membrane, where it inactivates actomyosin by inhibiting endogenous Rho1. In addition, this article presents a detailed protocol for coupling Opto-Rho1DN-mediated inactivation of actomyosin with laser ablation to investigate the role of actomyosin in generating epithelial tension during Drosophila ventral furrow formation. This protocol can be applied to many other morphological processes that involve actomyosin contractility in Drosophila embryos with minimal modifications. Overall, this optogenetic tool is a powerful approach to dissect the function of actomyosin contractility in controlling tissue mechanics during dynamic tissue remodeling.

Abstract: The light-regulated Gal4-UAS system has offered new ways to control cellular activities with precise spatial and temporal resolution in zebrafish and Drosophila. However, the existing optogenetic Gal4-UAS systems suffer from having multiple protein components and a dependence on extraneous light-sensitive cofactors, which increase the technical complexity and limit the portability of these systems. To overcome these limitations, we herein describe the development of a novel optogenetic Gal4-UAS system (ltLightOn) for both zebrafish and Drosophila based on a single light-switchable transactivator, termed GAVPOLT, which dimerizes and binds to gene promoters to activate transgene expression upon blue light illumination. The ltLightOn system is independent of exogenous cofactors and exhibits a more than 2400-fold ON/OFF gene expression ratio, allowing quantitative, spatial, and temporal control of gene expression. We further demonstrate the usefulness of the ltLightOn system in regulating zebrafish embryonic development by controlling the expression of lefty1 by light. We believe that this single-component optogenetic system will be immensely useful in understanding the gene function and behavioral circuits in zebrafish and Drosophila.

Abstract: The control of cell shape during cytokinesis requires a precise regulation of mechanical properties of the cell cortex. Only few studies have addressed the mechanisms underlying the robust production of unequal-sized daughters during asymmetric cell division. Here we report that unequal daughter-cell sizes resulting from asymmetric sensory organ precursor divisions in Drosophila are controlled by the relative amount of cortical branched Actin between the two cell poles. We demonstrate this by mistargeting the machinery for branched Actin dynamics using nanobodies and optogenetics. We can thereby engineer the cell shape with temporal precision and thus the daughter-cell size at different stages of cytokinesis. Most strikingly, inverting cortical Actin asymmetry causes an inversion of daughter-cell sizes. Our findings uncover the physical mechanism by which the sensory organ precursor mother cell controls relative daughter-cell size: polarized cortical Actin modulates the cortical bending rigidity to set the cell surface curvature, stabilize the division and ultimately lead to unequal daughter-cell size.

Abstract: We describe a protocol for optogenetic inhibition of the small GTPase Rho1 (RhoA) in Drosophila embryos, which allows rapid and spatially confined inactivation of Rho1 and Rho1-mediated actomyosin contractility. We provide step-by-step instruction for optogenetic manipulations of Drosophila embryos using confocal and multiphoton imaging systems. This tool is useful for determining the site- and stage-specific function of Rho1 in Drosophila embryos and for studying the immediate tissue response to acute elimination of cellular contractility. For complete details on the use and execution of this protocol, please refer to Guo et al. (2022).1.

Abstract: The development of optogenetic control over signaling pathways has provided a unique opportunity to decode the role of signaling dynamics in cell fate programing. Here I present a protocol for decoding cell fates through systematic interrogation with optogenetics and visualization of signaling with live biosensors. Specifically, this is written for Erk control of cell fates using the optoSOS system in mammalian cells or Drosophila embryos, though it is intended to be adapted to apply generally for several optogenetic tools, pathways, and model systems. This guide focuses on calibrating these tools, tricks of their use, and using them to interrogate features which program cell fates.

Abstract: Morphogenesis, the coordinated execution of developmental programs that shape embryos, raises many fundamental questions at the interface between physics and biology. In particular, how the dynamics of active cytoskeletal processes are coordinated across the surface of entire embryos to generate global cell flows is poorly understood. Two distinct regulatory principles have been identified: genetic programs and dynamic response to mechanical stimuli. Despite progress, disentangling these two contributions remains challenging. Here, we combine in toto light sheet microscopy with genetic and optogenetic perturbations of tissue mechanics to examine theoretically predicted dynamic recruitment of non-muscle myosin II to cell junctions during Drosophila embryogenesis. We find dynamic recruitment has a long-range impact on global myosin configuration, and the rate of junction deformation sets the rate of myosin recruitment. Mathematical modeling and high frequency analysis reveal myosin fluctuations on junctions around a mean value set by mechanical feedback. Our model accounts for the early establishment of the global myosin pattern at 80% fidelity. Taken together our results indicate spatially modulated mechanical feedback as a key regulatory input in the establishment of long-range gradients of cytoskeletal configurations and global tissue flow patterns.

Abstract: Integration of collective cell direction and coordination is believed to ensure collective guidance for efficient movement. Previous studies demonstrated that chemokine receptors PVR and EGFR govern a gradient of Rac1 activity essential for collective guidance of Drosophila border cells, whose mechanistic insight is unknown. By monitoring and manipulating subcellular Rac1 activity, here we reveal two switchable Rac1 pools at border cell protrusions and supracellular cables, two important structures responsible for direction and coordination. Rac1 and Rho1 form a positive feedback loop that guides mechanical coupling at cables to achieve migration coordination. Rac1 cooperates with Cdc42 to control protrusion growth for migration direction, as well as to regulate the protrusion-cable exchange, linking direction and coordination. PVR and EGFR guide correct Rac1 activity distribution at protrusions and cables. Therefore, our studies emphasize the existence of a balance between two Rac1 pools, rather than a Rac1 activity gradient, as an integrator for the direction and coordination of collective cell migration.

Abstract: The ability to drive expression of exogenous genes in different tissues and cell types, under the control of specific enhancers, has been crucial for discovery in biology. While many enhancers drive expression broadly, several genetic tools were developed to obtain access to isolated cell types. Studies of spatially organized neuropiles in the central nervous system of fruit flies have raised the need for a system that targets subsets of cells within a single neuronal type, a feat currently dependent on stochastic flip-out methods. To access the same cells within a given expression pattern consistently across fruit flies, we developed the light-gated expression system LOV-LexA. We combined the bacterial LexA transcription factor with the plant-derived light, oxygen, or voltage photosensitive domain and a fluorescent protein. Exposure to blue light uncages a nuclear localizing signal in the C-terminal of the light, oxygen, or voltage domain and leads to the translocation of LOV-LexA to the nucleus, with the subsequent initiation of transcription. LOV-LexA enables spatial and temporal control of expression of transgenes under LexAop sequences in larval fat body and pupal and adult neurons with blue light. The LOV-LexA tool is ready to use with GAL4 and Split-GAL4 drivers in its current form and constitutes another layer of intersectional genetics that provides light-controlled genetic access to specific cells across flies.

Abstract: The expulsion of dying epithelial cells requires well-orchestrated remodelling steps to maintain tissue sealing. This process, named cell extrusion, has been mostly analysed through the study of actomyosin regulation. Yet, the mechanistic relationship between caspase activation and cell extrusion is still poorly understood. Using the Drosophila pupal notum, a single layer epithelium where extrusions are caspase-dependent, we showed that the initiation of cell extrusion and apical constriction are surprisingly not associated with the modulation of actomyosin concentration and dynamics. Instead, cell apical constriction is initiated by the disassembly of a medio-apical mesh of microtubules which is driven by effector caspases. Importantly, the depletion of microtubules is sufficient to bypass the requirement of caspases for cell extrusion, while microtubule stabilisation strongly impairs cell extrusion. This study shows that microtubules disassembly by caspases is a key rate-limiting step of extrusion, and outlines a more general function of microtubules in epithelial cell shape stabilisation.

Abstract: Proper embryonic development requires directional axes to pattern cells into embryonic structures. In Drosophila, spatially discrete expression of transcription factors determines the anterior to posterior organization of the early embryo, while the Toll and TGFβ signalling pathways determine the early dorsal to ventral pattern. Embryonic MAPK/ERK signaling contributes to both anterior to posterior patterning in the terminal regions and to dorsal to ventral patterning during oogenesis and embryonic stages. Here we describe a novel loss of function mutation in the Raf kinase gene, which leads to loss of ventral cell fates as seen through the loss of the ventral furrow, the absence of Dorsal/NFκB nuclear localization, the absence of mesoderm determinants Twist and Snail, and the expansion of TGFβ. Gene expression analysis showed cells adopting ectodermal fates much like loss of Toll signaling. Our results combine novel mutants, live imaging, optogenetics and transcriptomics to establish a novel role for Raf, that appears to be independent of the MAPK cascade, in embryonic patterning.