Showing 1 - 25 of 66 results
1.
Dimerization activates the Inversin complex in C. elegans.
Abstract:
Genetic, colocalization, and biochemical studies suggest that the ankyrin repeat-containing proteins Inversin (INVS) and ANKS6 function with the NEK8 kinase to control tissue patterning and maintain organ physiology. It is unknown whether these three proteins assemble into a static “Inversin complex” or one that adopts multiple bioactive forms. Through characterization of hyperactive alleles in C. elegans, we discovered that the Inversin complex is activated by dimerization. Genome engineering of an RFP tag onto the nematode homologues of INVS (MLT-4) and NEK8 (NEKL-2) induced a gain-of-function, cyst-like phenotype that was suppressed by monomerization of the fluorescent tag. Stimulated dimerization of MLT-4 or NEKL-2 using optogenetics was sufficient to recapitulate the phenotype of a constitutively active Inversin complex. Further, dimerization of NEKL-2 bypassed a lethal MLT-4 mutant, demonstrating that the dimeric form is required for function. We propose that dynamic switching between at least two functionally distinct states–-an active dimer and an inactive monomer–-gates the output of the Inversin complex.
2.
Endogenous OptoRhoGEFs reveal biophysical principles of epithelial tissue furrowing.
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.
3.
Optogenetic control of Nodal signaling patterns.
Abstract:
A crucial step in early embryogenesis is the establishment of spatial patterns of signaling activity. Tools to perturb morphogen signals with high resolution in space and time can help reveal how embryonic cells decode these signals to make appropriate fate decisions. Here, we present new optogenetic reagents and an experimental pipeline for creating designer Nodal signaling patterns in live zebrafish embryos. Nodal receptors were fused to the light sensitive heterodimerizing pair Cry2/CIB1N, and the Type II receptor was sequestered to the cytosol. The improved optoNodal2 reagents eliminate dark activity and improve response kinetics, without sacrificing dynamic range. We adapted an ultra-widefield microscopy platform for parallel light patterning in up to 36 embryos and demonstrated precise spatial control over Nodal signaling activity and downstream gene expression. Patterned Nodal activation drove precisely controlled internalization of endodermal precursors. Further, we used patterned illumination to generate synthetic signaling patterns in Nodal signaling mutants, rescuing several characteristic developmental defects. This study establishes an experimental toolkit for systematic exploration of Nodal signaling patterns in live embryos.
4.
Epithelial folding through local degradation of an elastic basement membrane plate.
Abstract:
Epithelia are polarised layers of cells that line the outer and inner surfaces of organs. At the basal side, the epithelial cell layer is supported by a basement membrane, which is a thin polymeric layer of self-assembled extracellular matrix (ECM) that tightly adheres to the basal cell surface. Proper shaping of epithelial layers is an important prerequisite for the development of healthy organs during the morphogenesis of an organism. Experimental evidence indicates that local degradation of the basement membrane drives epithelial folding. Here, we present a coarse-grained plate theory model of the basement membrane that assumes force balance between i) cell-transduced active forces and ii) deformation-induced elastic forces. We verify key assumptions of this model through experiments in the Drosophila wing disc epithelium and demonstrate that the model can explain the emergence of outward epithelial folds upon local plate degradation. Our model accounts for local degradation of the basement membrane as a mechanism for the generation of epithelial folds in the absence of epithelial growth.
5.
Temporal dynamics of BMP/Nodal ratio drive tissue-specific gastrulation morphogenesis.
Abstract:
Anteroposterior (AP) elongation of the vertebrate body plan is driven by convergence and extension (C&E) gastrulation movements in both the mesoderm and neuroectoderm, but how or whether molecular regulation of C&E differs between tissues remains an open question. Using a zebrafish explant model of AP axis extension, we show that C&E of the neuroectoderm and mesoderm can be uncoupled ex vivo, and that morphogenesis of individual tissues results from distinct morphogen signaling dynamics. Using precise temporal manipulation of BMP and Nodal signaling, we identify a critical developmental window during which high or low BMP/Nodal ratios induce neuroectoderm- or mesoderm-driven C&E, respectively. Increased BMP activity similarly enhances C&E specifically in the ectoderm of intact zebrafish gastrulae, highlighting the in vivo relevance of our findings. Together, these results demonstrate that temporal dynamics of BMP and Nodal morphogen signaling activate distinct morphogenetic programs governing C&E gastrulation movements within individual tissues.
6.
Optogenetic generation of leader cells reveals a force-velocity relation for collective cell migration.
Abstract:
The front of migratory cellular clusters during development, wound healing and cancer invasion is typically populated with highly protrusive cells that are called leader cells. Leader cells are thought to physically pull and direct their cohort of followers, but how leaders and followers are mechanically organized to migrate collectively remains controversial. One possibility is that the autonomous local action of a leader cell is sufficient to drive migration of the group. Yet another possibility is that a global mechanical organization is required for the group to move cohesively. Here we show that the effectiveness of leader-follower organization is proportional to the asymmetry of traction and tension within the cellular cluster. By combining hydrogel micropatterning and optogenetic activation of Rac1, we locally generate highly protrusive leaders at the edge of minimal cell groups. We find that the induced leader can robustly drag one follower but is generally unable to direct larger groups. By measuring traction forces and tension propagation in groups of increasing size, we establish a quantitative relationship between group velocity and the asymmetry of the traction and tension profiles. We propose a model of the motile cluster as an active polar fluid that explains this force-velocity relationship in terms of asymmetries in the distribution of active tractions. Our results challenge the notion of autonomous leader cells by showing that collective cell migration requires a global mechanical organization within the cluster.
7.
A mechanical wave travels along a genetic guide to drive the formation of an epithelial furrow during Drosophila gastrulation.
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.
8.
Lifelong molecular consequences of high Glucocorticoids exposure during development
Abstract:
Early life stress (ELS) is one of the strongest risk factors for developing psychiatric disorders in humans. As conserved key stress hormones of vertebrates, glucocorticoids (GCs) are thought to play an important role in mediating the effects of ELS exposure in shaping adult phenotypes. In this process, early exposure to high level of GCs may induce molecular changes that alter developmental trajectory of an animal and primes differential adult responses. However, comprehensive characterization of identities of molecules that are targeted by developmental GC exposure is currently lacking. In our study, we describe lifelong molecular consequences of high level of developmental GC exposure using an optogenetic zebrafish model. First, we developed a new double-hit stress model using zebrafish by combining exposure to a high endogenous GC level during development and acute adulthood stress exposure. Our results establish that similar to ELS-exposed humans and rodents, developmental GC exposed zebrafish model shows altered behavior and stress hypersensitivity in adulthood. Second, we generated time-series gene expression profiles of the brains in larvae, in adult, and upon stress exposure to identify molecular alterations induced by high developmental GC exposure at different developmental stages. Third, we identify a set of GC-primed genes that show altered expression upon acute stress exposure only in animals exposed to a high developmental GC. Interestingly, our datasets of GC primed genes are enriched in risk factors identified for human psychiatric disorders. Lastly, we identify potential epigenetic regulatory elements and associated post-transcriptional modifications following high developmental GC exposure. Thus, we present a translationally relevant zebrafish model for studying stress hypersensitivity and alteration of behavior induced by exposure to elevated GC levels during development. Our study provides comprehensive datasets delineating potential molecular targets underlying the impact of developmental high GC exposure on adult responses.
9.
Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo.
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.
10.
Optogenetic control of Wnt signaling models cell-intrinsic embryogenic patterning using 2D human pluripotent stem cell culture.
Abstract:
In embryonic stem cell (ESC) models for early development, spatially and temporally varying patterns of signaling and cell types emerge spontaneously. However, mechanistic insight into this dynamic self-organization is limited by a lack of methods for spatiotemporal control of signaling, and the relevance of signal dynamics and cell-to-cell variability to pattern emergence remains unknown. Here, we combine optogenetic stimulation, imaging, and transcriptomic approaches to study self-organization of human ESCs (hESC) in two-dimensional (2D) culture. Morphogen dynamics were controlled via optogenetic activation of canonical Wnt/β-catenin signaling (optoWnt), which drove broad transcriptional changes and mesendoderm differentiation at high efficiency (>99% cells). When activated within cell subpopulations, optoWnt induced cell self-organization into distinct epithelial and mesenchymal domains, mediated by changes in cell migration, an epithelial to mesenchymal-like transition, and TGF-β signaling. Furthermore, we demonstrate that such optogenetic control of cell subpopulations can be used to uncover signaling feedback mechanisms between neighboring cell types. These findings reveal that cell-to-cell variability in Wnt signaling is sufficient to generate tissue-scale patterning and establish an hESC model system for investigating feedback mechanisms relevant to early human embryogenesis.
11.
Rab8, Rab11, and Rab35 coordinate lumen and cilia formation during zebrafish left-right organizer development.
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Aljiboury, AA
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Ingram, E
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Krishnan, N
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Ononiwu, F
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Pal, D
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Manikas, J
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Taveras, C
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Hall, NA
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Da Silva, J
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Freshour, J
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Hehnly, H
Abstract:
An essential process during Danio rerio's left-right organizer (Kupffer's Vesicle, KV) formation is the formation of a motile cilium by developing KV cells which extends into the KV lumen. Beating of motile cilia within the KV lumen directs fluid flow to establish the embryo's left-right axis. However, the timepoint at which KV cells start to form cilia and how cilia formation is coordinated with KV lumen formation have not been examined. We identified that nascent KV cells form cilia at their centrosomes at random intracellular positions that then move towards a forming apical membrane containing cystic fibrosis transmembrane conductance regulator (CFTR). Using optogenetic clustering approaches, we found that Rab35 positive membranes recruit Rab11 to modulate CFTR delivery to the apical membrane, which is required for lumen opening, and subsequent cilia extension into the lumen. Once the intracellular cilia reach the CFTR positive apical membrane, Arl13b-positive cilia extend and elongate in a Rab8 dependent manner into the forming lumen once the lumen reaches an area of 300 μm2. These studies demonstrate the need to acutely coordinate Rab8, Rab11, and Rab35-mediated membrane trafficking events to ensure appropriate timing in lumen and cilia formation during KV development.
12.
Optogenetic inhibition of Gα signalling alters and regulates circuit functionality and early circuit formation.
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.
13.
Controlling protein stability with SULI, a highly sensitive tag for stabilization upon light induction.
Abstract:
Optogenetics tools for precise temporal and spatial control of protein abundance are valuable in studying diverse complex biological processes. In the present study, we engineer a monomeric tag of stabilization upon light induction (SULI) for yeast and zebrafish based on a single light-oxygen-voltage domain from Neurospora crassa. Proteins of interest fused with SULI are stable upon light illumination but are readily degraded after transfer to dark conditions. SULI shows a high dynamic range and a high tolerance to fusion at different positions of the target protein. Further studies reveal that SULI-mediated degradation occurs through a lysine ubiquitination-independent proteasome pathway. We demonstrate the usefulness of SULI in controlling the cell cycle in yeast and regulating protein stability in zebrafish, respectively. Overall, our data indicate that SULI is a simple and robust tool to quantitatively and spatiotemporally modulate protein levels for biotechnological or biomedical applications.
14.
Optogenetic dissection of transcriptional repression in a multicellular organism.
Abstract:
Transcriptional control is fundamental to cellular function. However, despite knowing that transcription factors can act as repressors or activators, how these functions are implemented at the molecular level has remained elusive. 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 minute) and memoryless. Finally, 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.
15.
Patterned mechanical feedback establishes a global myosin gradient.
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.
16.
Spatiotemporal control of ERK pulse frequency coordinates fate decisions during mammary acinar morphogenesis.
Abstract:
The signaling events controlling proliferation, survival, and apoptosis during mammary epithelial acinar morphogenesis remain poorly characterized. By imaging single-cell ERK activity dynamics in MCF10A acini, we find that these fates depend on the average frequency of non-periodic ERK pulses. High pulse frequency is observed during initial acinus growth, correlating with rapid cell motility and proliferation. Subsequent decrease in motility correlates with lower ERK pulse frequency and quiescence. Later, during lumen formation, coordinated multicellular ERK waves emerge, correlating with high and low ERK pulse frequencies in outer surviving and inner dying cells, respectively. Optogenetic entrainment of ERK pulses causally connects high ERK pulse frequency with inner cell survival. Acini harboring the PIK3CA H1047R mutation display increased ERK pulse frequency and inner cell survival. Thus, fate decisions during acinar morphogenesis are coordinated by different spatiotemporal modalities of ERK pulse frequency.
17.
A non-canonical Raf function is required for dorsal-ventral patterning during Drosophila embryogenesis.
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.
18.
Optogenetic inhibition of actomyosin reveals mechanical bistability of the mesoderm epithelium during Drosophila mesoderm invagination.
Abstract:
Apical constriction driven by actin and non-muscle myosin II (actomyosin) provides a well-conserved mechanism to mediate epithelial folding. It remains unclear how contractile forces near the apical surface of a cell sheet drive out-of-the-plane bending of the sheet and whether myosin contractility is required throughout folding. By optogenetic-mediated acute inhibition of actomyosin, we find that during Drosophila mesoderm invagination, actomyosin contractility is critical to prevent tissue relaxation during the early, 'priming' stage of folding but is dispensable for the actual folding step after the tissue passes through a stereotyped transitional configuration. This binary response suggests that Drosophila mesoderm is mechanically bistable during gastrulation. Computer modeling analysis demonstrates that the binary tissue response to actomyosin inhibition can be recapitulated in the simulated epithelium that undergoes buckling-like deformation jointly mediated by apical constriction in the mesoderm and in-plane compression generated by apicobasal shrinkage of the surrounding ectoderm. Interestingly, comparison between wild-type and snail mutants that fail to specify the mesoderm demonstrates that the lateral ectoderm undergoes apicobasal shrinkage during gastrulation independently of mesoderm invagination. We propose that Drosophila mesoderm invagination is achieved through an interplay between local apical constriction and mechanical bistability of the epithelium that facilitates epithelial buckling.
19.
Spatio-temporal, optogenetic control of gene expression in organoids.
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Legnini, I
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Emmenegger, L
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Zappulo, A
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Wurmus, R
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Martinez, AO
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Jara, CC
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Boltengagen, A
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Hessler, T
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Mastrobuoni, G
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Rybak-Wolf, A
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Kempa, S
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Zinzen, R
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Woehler, A
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Rajewsky, N
Abstract:
Organoids derived from stem cells become increasingly important to study human development and to model disease. However, methods are needed to control and study spatio-temporal patterns of gene expression in organoids. To this aim, we combined optogenetics and gene perturbation technologies to activate or knock-down RNA of target genes, at single-cell resolution and in programmable spatio-temporal patterns. To illustrate the usefulness of our approach, we locally activated Sonic Hedgehog (SHH) signaling in an organoid model for human neurodevelopment. High-resolution spatial transcriptomic and single-cell analyses showed that this local induction was sufficient to generate stereotypically patterned organoids in three dimensions and revealed new insights into SHH’s contribution to gene regulation in neurodevelopment. With this study, we propose optogenetic perturbations in combination with spatial transcriptomics as a powerful technology to reprogram and study cell fates and tissue patterning in organoids.
20.
Mechanical competition alters the cellular interpretation of an endogenous genetic programme.
Abstract:
The intrinsic genetic programme of a cell is not sufficient to explain all of the cell’s activities. External mechanical stimuli are increasingly recognized as determinants of cell behaviour. In the epithelial folding event that constitutes the beginning of gastrulation in Drosophila, the genetic programme of the future mesoderm leads to the establishment of a contractile actomyosin network that triggers apical constriction of cells, and thereby, furrow formation. However, some cells do not constrict but instead stretch, even though they share the same genetic programme as their constricting neighbours. We show here that tissue-wide interactions force these cells to expand even when an otherwise sufficient amount of apical, active actomyosin is present. Models based on contractile forces and linear stress-strain responses are not sufficient to reproduce experimental observations, but simulations in which cells behave as ductile materials with non-linear mechanical properties do. Our models show that this behaviour is a general emergent property of supracellular actomyosin networks, in accordance with our experimental observations of actin reorganisation within stretching cells.
21.
Desensitisation of Notch signalling through dynamic adaptation in the nucleus.
Abstract:
During embryonic development, signalling pathways orchestrate organogenesis by controlling tissue-specific gene expression programmes and differentiation. Although the molecular components of many common developmental signalling systems are known, our current understanding of how signalling inputs are translated into gene expression outputs in real-time is limited. Here we employ optogenetics to control the activation of Notch signalling during Drosophila embryogenesis with minute accuracy and follow target gene expression by quantitative live imaging. Light-induced nuclear translocation of the Notch Intracellular Domain (NICD) causes a rapid activation of target mRNA expression. However, target gene transcription gradually decays over time despite continuous photo-activation and nuclear NICD accumulation, indicating dynamic adaptation to the signalling input. Using mathematical modelling and molecular perturbations, we show that this adaptive transcriptional response fits to known motifs capable of generating near-perfect adaptation and can be best explained by state-dependent inactivation at the target cis-regulatory region. Taken together, our results reveal dynamic nuclear adaptation as a novel mechanism controlling Notch signalling output during tissue differentiation.
22.
Extremely rapid and reversible optogenetic perturbation of nuclear proteins in living embryos.
Abstract:
Many developmental regulators have complex and context-specific roles in different tissues and stages, making the dissection of their function extremely challenging. As regulatory processes often occur within minutes, perturbation methods that match these dynamics are needed. Here, we present the improved light-inducible nuclear export system (iLEXY), an optogenetic loss-of-function approach that triggers translocation of proteins from the nucleus to the cytoplasm. By introducing a series of mutations, we substantially increased LEXY's efficiency and generated variants with different recovery times. iLEXY enables rapid (t1/2 < 30 s), efficient, and reversible nuclear protein depletion in embryos, and is generalizable to proteins of diverse sizes and functions. Applying iLEXY to the Drosophila master regulator Twist, we phenocopy loss-of-function mutants, precisely map the Twist-sensitive embryonic stages, and investigate the effects of timed Twist depletions. Our results demonstrate the power of iLEXY to dissect the function of pleiotropic factors during embryogenesis with unprecedented temporal precision.
23.
Spatiotemporal sensitivity of mesoderm specification to FGFR signalling in the Drosophila embryo.
Abstract:
Development of the Drosophila embryonic mesoderm is controlled through both internal and external inputs to the mesoderm. One such factor is Heartless (Htl), a Fibroblast Growth Factor Receptor (FGFR) expressed in the mesoderm. Although Htl has been extensively studied, the dynamics of its action are poorly understood after the initial phases of mesoderm formation and spreading. To begin to address this challenge, we have developed an optogenetic version of the FGFR Heartless in Drosophila (Opto-htl). Opto-htl enables us to activate the FGFR pathway in selective spatial (~ 35 μm section from one of the lateral sides of the embryo) and temporal domains (ranging from 40 min to 14 h) during embryogenesis. Importantly, the effects can be tuned by the intensity of light-activation, making this approach significantly more flexible than other genetic approaches. We performed controlled perturbations to the FGFR pathway to define the contribution of Htl signalling to the formation of the developing embryonic heart and somatic muscles. We find a direct correlation between Htl signalling dosage and number of Tinman-positive heart cells specified. Opto-htl activation favours the specification of Tinman positive cardioblasts and eliminates Eve-positive DA1 muscles. This effect is seen to increase progressively with increasing light intensity. Therefore, fine tuning of phenotypic responses to varied Htl signalling dosage can be achieved more conveniently than with other genetic approaches. Overall, Opto-htl is a powerful new tool for dissecting the role of FGFR signalling during development.
24.
Temporal integration of inductive cues on the way to gastrulation.
Abstract:
Markers for the endoderm and mesoderm germ layers are commonly expressed together in the early embryo, potentially reflecting cells' ability to explore potential fates before fully committing. It remains unclear when commitment to a single-germ layer is reached and how it is impacted by external signals. Here, we address this important question in Drosophila, a convenient model system in which mesodermal and endodermal fates are associated with distinct cellular movements during gastrulation. Systematically applying endoderm-inducing extracellular signal-regulated kinase (ERK) signals to the ventral medial embryo-which normally only receives a mesoderm-inducing cue-reveals a critical time window during which mesodermal cell movements and gene expression are suppressed by proendoderm signaling. We identify the ERK target gene huckebein (hkb) as the main cause of the ventral furrow suppression and use computational modeling to show that Hkb repression of the mesoderm-associated gene snail is sufficient to account for a broad range of transcriptional and morphogenetic effects. Our approach, pairing precise signaling perturbations with observation of transcriptional dynamics and cell movements, provides a general framework for dissecting the complexities of combinatorial tissue patterning.
25.
Optogenetic Control of the Canonical Wnt Signaling Pathway During Xenopus laevis Embryonic Development.
Abstract:
Optogenetics uses light-inducible protein-protein interactions to precisely control the timing, localization, and intensity of signaling activity. The precise spatial and temporal resolution of this emerging technology has proven extremely attractive to the study of embryonic development, a program faithfully replicated to form the same organism from a single cell. We have previously performed a comparative study for optogenetic activation of receptor tyrosine kinases, where we found that the cytoplasm-to-membrane translocation-based optogenetic systems outperform the membrane-anchored dimerization systems in activating the receptor tyrosine kinase signaling in live Xenopus embryos. Here, we determine if this engineering strategy can be generalized to other signaling pathways involving membrane-bound receptors. As a proof of concept, we demonstrate that the cytoplasm-to-membrane translocation of the low-density lipoprotein receptor-related protein-6 (LRP6), a membrane-bound coreceptor for the canonical Wnt pathway, triggers Wnt activity. Optogenetic activation of LRP6 leads to axis duplication in developing Xenopus embryos, indicating that the cytoplasm-to-membrane translocation of the membrane-bound receptor could be a generalizable strategy for the construction of optogenetic systems.