Optogenetic control of Nodal signaling patterns.

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 creaHng 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.

Temporal dynamics of BMP/Nodal ratio drive tissue-specific gastrulation morphogenesis.

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.

The pattern of nodal morphogen signaling is shaped by co-receptor expression.

Embryos must communicate instructions to their constituent cells over long distances. These instructions are often encoded in the concentration of signals called morphogens. In the textbook view, morphogen molecules diffuse from a localized source to form a concentration gradient, and target cells adopt fates by measuring the local morphogen concentration. However, natural patterning systems often incorporate numerous co-factors and extensive signaling feedback, suggesting that embryos require additional mechanisms to generate signaling patterns. Here, we examine the mechanisms of signaling pattern formation for the mesendoderm inducer Nodal during zebrafish embryogenesis. We find that Nodal signaling activity spans a normal range in the absence of signaling feedback and relay, suggesting that diffusion is sufficient for Nodal gradient formation. We further show that the range of endogenous Nodal ligands is set by the EGF-CFC co-receptor Oep: in the absence of Oep, Nodal activity spreads to form a nearly uniform distribution throughout the embryo. In turn, increasing Oep levels sensitizes cells to Nodal ligands. We recapitulate these experimental results with a computational model in which Oep regulates the diffusive spread of Nodal ligands by setting the rate of capture by target cells. This model predicts, and we confirm in vivo, the surprising observation that a failure to replenish Oep transforms the Nodal signaling gradient into a travelling wave. These results reveal that patterns of Nodal morphogen signaling are shaped by co-receptor-mediated restriction of ligand spread and sensitization of responding cells.

Stochastic antagonism between two proteins governs a bacterial cell fate switch.

Cell fate decision circuits must be variable enough for genetically identical cells to adopt a multitude of fates, yet ensure that these states are distinct, stably maintained, and coordinated with neighboring cells. A long-standing view is that this is achieved by regulatory networks involving self-stabilizing feedback loops that convert small differences into long-lived cell types. We combined regulatory mutants and in vivo reconstitution with theory for stochastic processes to show that the marquee features of a cell fate switch in Bacillus subtilis-discrete states, multigenerational inheritance, and timing of commitments-can instead be explained by simple stochastic competition between two constitutively produced proteins that form an inactive complex. Such antagonistic interactions are commonplace in cells and could provide powerful mechanisms for cell fate determination more broadly.

Nodal patterning without Lefty inhibitory feedback is functional but fragile.

Developmental signaling pathways often activate their own inhibitors. Such inhibitory feedback has been suggested to restrict the spatial and temporal extent of signaling or mitigate signaling fluctuations, but these models are difficult to rigorously test. Here, we determine whether the ability of the mesendoderm inducer Nodal to activate its inhibitor Lefty is required for development. We find that zebrafish lefty mutants exhibit excess Nodal signaling and increased specification of mesendoderm, resulting in embryonic lethality. Strikingly, development can be fully restored without feedback: Lethal patterning defects in lefty mutants can be rescued by ectopic expression of lefty far from its normal expression domain or by spatially and temporally uniform exposure to a Nodal inhibitor drug. While drug-treated mutants are less tolerant of mild perturbations to Nodal signaling levels than wild type embryos, they can develop into healthy adults. These results indicate that patterning without inhibitory feedback is functional but fragile.

Toddler signaling regulates mesodermal cell migration downstream of Nodal signaling.

Toddler/Apela/Elabela is a conserved secreted peptide that regulates mesendoderm development during zebrafish gastrulation. Two non-exclusive models have been proposed to explain Toddler function. The 'specification model' postulates that Toddler signaling enhances Nodal signaling to properly specify endoderm, whereas the 'migration model' posits that Toddler signaling regulates mesendodermal cell migration downstream of Nodal signaling. Here, we test key predictions of both models. We find that in toddler mutants Nodal signaling is initially normal and increasing endoderm specification does not rescue mesendodermal cell migration. Mesodermal cell migration defects in toddler mutants result from a decrease in animal pole-directed migration and are independent of endoderm. Conversely, endodermal cell migration defects are dependent on a Cxcr4a-regulated tether of the endoderm to mesoderm. These results suggest that Toddler signaling regulates mesodermal cell migration downstream of Nodal signaling and indirectly affects endodermal cell migration via Cxcr4a-signaling.

Synchronous long-term oscillations in a synthetic gene circuit.

Synthetically engineered genetic circuits can perform a wide variety of tasks but are generally less accurate than natural systems. Here we revisit the first synthetic genetic oscillator, the repressilator, and modify it using principles from stochastic chemistry in single cells. Specifically, we sought to reduce error propagation and information losses, not by adding control loops, but by simply removing existing features. We show that this modification created highly regular and robust oscillations. Furthermore, some streamlined circuits kept 14 generation periods over a range of growth conditions and kept phase for hundreds of generations in single cells, allowing cells in flasks and colonies to oscillate synchronously without any coupling between them. Our results suggest that even the simplest synthetic genetic networks can achieve a precision that rivals natural systems, and emphasize the importance of noise analyses for circuit design in synthetic biology.

Stochastic activation of a DNA damage response causes cell-to-cell mutation rate variation.

Cells rely on the precise action of proteins that detect and repair DNA damage. However, gene expression noise causes fluctuations in protein abundances that may compromise repair. For the Ada protein in Escherichia coli, which induces its own expression upon repairing DNA alkylation damage, we found that undamaged cells on average produce one Ada molecule per generation. Because production is stochastic, many cells have no Ada molecules and cannot induce the damage response until the first expression event occurs, which sometimes delays the response for generations. This creates a subpopulation of cells with increased mutation rates. Nongenetic variation in protein abundances thus leads to genetic heterogeneity in the population. Our results further suggest that cells balance reliable repair against toxic side effects of abundant DNA repair proteins.

Stochastic Switching of Cell Fate in Microbes.

Microbes transiently differentiate into distinct, specialized cell types to generate functional diversity and cope with changing environmental conditions. Though alternate programs often entail radically different physiological and morphological states, recent single-cell studies have revealed that these crucial decisions are often left to chance. In these cases, the underlying genetic circuits leverage the intrinsic stochasticity of intracellular chemistry to drive transition between states. Understanding how these circuits transform transient gene expression fluctuations into lasting phenotypic programs will require a combination of quantitative modeling and extensive, time-resolved observation of switching events in single cells. In this article, we survey microbial cell fate decisions demonstrated to involve a random element, describe theoretical frameworks for understanding stochastic switching between states, and highlight recent advances in microfluidics that will enable characterization of key dynamic features of these circuits.

Memory and modularity in cell-fate decision making.

Genetically identical cells sharing an environment can display markedly different phenotypes. It is often unclear how much of this variation derives from chance, external signals, or attempts by individual cells to exert autonomous phenotypic programs. By observing thousands of cells for hundreds of consecutive generations under constant conditions, we dissect the stochastic decision between a solitary, motile state and a chained, sessile state in Bacillus subtilis. We show that the motile state is 'memoryless', exhibiting no autonomous control over the time spent in the state. In contrast, the time spent as connected chains of cells is tightly controlled, enforcing coordination among related cells in the multicellular state. We show that the three-protein regulatory circuit governing the decision is modular, as initiation and maintenance of chaining are genetically separable functions. As stimulation of the same initiating pathway triggers biofilm formation, we argue that autonomous timing allows a trial commitment to multicellularity that external signals could extend.