DO YOU KNOW WHERE YOU’RE GOING TO? A QUANTITATIVE FRAMEWORK FOR PREDICTING NUCLEAR MOVEMENT IN EARLY INSECT EMBRYOS.
During early animal embryogenesis, one cell divides to become two, two divide to become four, and before too long the embryo, a complex aggregate of hundreds to millions of cells is formed. Embryonic cells must take on the appropriate fates to ensure that the embryo and adult organism is functional, and acquisition of their fates often relies on cells being in the right place at the right time within the embryo. In the largest group of animals on earth, the insects, the early embryo is first made of thousands of nuclei sharing a common cytoplasm, called a syncytium. These nuclei move to appropriate positions within the egg, then acquire membranes and become true cells, ready to adopt their fate within the embryo. The mechanisms that ensure nuclei end up in the right places are largely unknown. Moreover, it is unclear whether the mechanisms that operate in the well-studied model Drosophila, can explain nuclear behaviour in the eggs of other insects, which can be are orders of magnitude smaller and larger than fruit fly eggs, and have radically different shapes. Here we present a model for nuclear behaviour in embryonic syncytia based on quantitative analysis of cricket embryos, and discuss its possible application to a wide range of eggs and other syncytial systems.
University of Chicago
TOWARD A PREDICTIVE MODEL OF CYTOSKELETAL DYNAMICS
The actin cytoskeleton underlies the ability of cells to move, change their shapes, polarize, and exert mechanical forces on their surroundings. These dynamics are key for a wide range of biological processes, including developmental patterning. Incomplete knowledge of the participating molecules and their states has traditionally presented challenges for developing microscopic models of cytoskeletal dynamics. Experiments that reconstitute biological-like behaviors in assemblies of purified cytoskeletal proteins in vitro now enable developing predictive models. I will describe one such model and its use for understanding contractility and molecular sorting in assemblies of actin filaments, their crosslinkers, and myosin motors.
IMAGING TRANSCRIPTION IN LIVING EMBRYOS : HOW DATA DRIVEN MODELING CAN HELP UNDERSTAND PATTERNING
Morphogen gradients are used by various organisms to establish polarity along embryonic axes or within organs. The extreme robustness of these processes ensures the reproducibility of developmental patterns and the emergence of properly proportioned individuals. To understand the robustness of patterning downstream of the Bicoid gradient in developing fruit fly embryos, we used the MS2 approach, which combines fluorescent labeling of nascent mRNA with live-cell imaging at high spatial and temporal resolution and which allows to monitor transcription in real time in leaving embryos. This showed that despite high nucleus-to-nucleus variability in transcription kinetics and absence of transcription during mitosis, a Bicoid target promoter was able to measure subtle differences in the morphogen concentration and establish a sharp expression boundary in about 3 min. Modeling based on the cooperativity between the 6 known Bicoid binding sites in the target promoter, assuming rate limiting concentrations of the Bicoid transcription factor at the boundary, was able to capture the observed dynamics of pattern establishment but not the steepness of the boundary. Recently, in order to get mechanistic insights into this process, we built synthetic MS2 reporters with various numbers of binding sites for Bicoid and/or for its two partners Zelda and Hunchback. While Bicoid and Zelda act in synergy as sources of information to set-up the position of the boundary along the axis, Hunchback acts independently and targets the downstream steps of the transcription process by increasing the probability for a locus to be expressed. Modeling is currently in progress to accommodate all the experimental data and should help understand when and how robustness in the pattern is achieved.
University of California, Berkeley
TRANSCRIPTION FACTOR-DRIVEN CONTROL OF CHROMATIN ACCESSIBILITY IN EMBRYONIC DEVELOPMENT
An open challenge in developmental biology is to predict gene expression patterns from knowledge of the concentration dynamics of input transcription factors and their binding arrangement on regulatory DNA. While thermodynamic models can predict transcriptional regulation in bacteria, in eukaryotes chromatin accessibility and energy expenditure may call for a different framework. We systematically tested the predictive power of models of DNA accessibility based on the Monod-Wyman-Changeux (MWC) model of allostery, which posits that chromatin fluctuates between accessible and inaccessible states. We dissected the regulatory dynamics of hunchback by the activator Bicoid and the pioneer-like transcription factor Zelda in living Drosophila embryos and showed that no thermodynamic or non-equilibrium MWC model can recapitulate hunchback transcription. In contrast, a model where DNA accessibility is not the result of thermal fluctuations, but where accessibility is catalyzed by these transcription factors, can predict hunchback dynamics. Thus, our theory-experiment dialogue uncovered potential molecular mechanisms of governing transcriptional regulatory dynamics, a key step toward reaching a predictive understanding of developmental decision-making.
SINGLE MOLECULE DYNAMICS AT THE INTERFACE BETWEEN CYTOPLASMIC ORGANELLES
Membrane-bound organelles provide distinct compartments where incompatible biological processes can be separated from one another. However, biochemical reactions in these disparate environments must be coordinated to facilitate homeostasis, particularly in response to environmental changes. Sites of direct contact between the organelles play an important role in this process, serving as signaling hubs and locations of direct transfer for macromolecules. Mechanisms for regulating contact site structure and function remain enigmatic, but it seems likely to involve substantial heterogeneity and plasticity within the system. Numerous specific molecular tethers have been implicated in the process, but the small size and dynamic nature of contact sites have prevented understanding of their spatiotemporal regulation. Here, we use high-speed single molecule imaging to directly observe tethering and release dynamics of putative tethers in individual contact sites between the ER and mitochondria. We demonstrate the existence of structurally regulated subdomains within single contact sites by comparative imaging with 3D electron microscopy of vitreously-frozen samples. We show that distinct tethering molecules have unique patterns of motion, respond differently to nutrient availability, and can actively exclude one another from regions of high density. Thus, changes in the recruitment efficiency of individual tethers can support a change in contact site function without necessitating the breaking of one contact site and formation of another.
The Rockefeller University
MULTICELLULAR DYNAMICS DURING MORPHOGENESIS OF THE VERTEBRATE SKIN
One of the most striking aspects of the emergence of biological form, or morphogenesis, is that the process requires no physical input from an external agent. Unlike Michelangelo’s David, the human body appears to shape itself, absent of a visible master sculptor. Upon closer inspection of a developing embryo, however, one becomes aware that its material provides both raw matter as well as the forces to shape it. This distinct ability comes from the cells that comprise the embryo, which are able to exert forces at the cellular level, but also at larger length scales through aggregation into cellular collectives and tissues. From this perspective, one can ask: how does a homogeneous multicellular field, such as that found in the emerging skin, break symmetry to form the intricate patterns observed in the adult? We are investigating this mechanism through a novel synthetic morphogenesis assay where dynamics of the developing skin are recreated in a minimal ex vivo environment, allowing for more systematic characterization of cellular mechanics. In doing so we aim to uncover fundamental construction rules of periodic pattern formation.
Princeton University and The CUNY Graduate Center
SQUEEZING MORE BITS OUT OF FEWER MOLECULES
Life requires information, but organisms often are starved for bits. In single cells, crucial signals are represented by molecules at very low concentration, so that the arrival of these molecules at their targets is an irreducibly noisy process. Under these conditions there is considerable pressure to make the best possible use of these limited signals. If we take this idea seriously, we can predict, quantitatively, aspects of the signal processing mechanisms that must be present in order for cells to squeeze out enough bits. My colleagues and I have developed these theoretical ideas in dialogue with a series of experiments on the early events in the development of the fruit fly embryo, and the same ideas appear in thinking about coding and computation in the brain. I’ll give a progress report on this work, and point to open problems. Two important themes are that this approach gives us a way of circumventing complicated, highly parameterized models, and that we now can reach a level of precision in theory/experiment comparison that is expected in physics but once seemed impossible in the complex context of living systems.
ANKUR SAXENA &
Illinois - Chicago
Year 02 Pilot Project Investigators
TEMPORALLY-DYNAMIC REGULATORY FEEDBACK YIELDS SPATIALLY-CONSERVED NEIGHBORHOODS OF OLFACTORY NEUROGENESIS
The vertebrate olfactory epithelium is a complex tissue that includes several types of continuously regenerating olfactory sensory neurons (OSNs) and their progenitor cells. While the mature olfactory system has been well-studied, far less is known about the gene regulatory programs that drive timely OSN differentiation during vertebrate embryogenesis. We tracked signaling changes at a single-cell level and perturbed pathways with temporal specificity in live zebrafish embryos to demonstrate that the Notch signaling pathway and transcription factor insm1a operate in a mutually antagonistic feedback loop. This temporally-dynamic feedback controls OSN differentiation by altering proneural gene expression in a defined developmental region and time window. Quantitation of mRNA levels for insm1a and the Notch signaling effector her4 revealed recurring multicellular ‘neighborhoods’ of complementary expression that correlate with olfactory neurogenesis. We are computationally modeling these expression profiles to determine how this highly dynamic intracellular regulatory feedback yields neighborhood-based expression patterns and cell fates. Our findings offer new insights into the delicate balance between stem cells and their neuronal derivatives in vivo and how that balance is tightly regulated to ensure appropriate numbers of differentiated neurons in a rapidly developing sensory organ.
DAVID LUBENSKY &
University of Michigan
Year 02 Pilot Project Investigators
GENERATION OF FATE PATTERNS BY INTERCELLULAR FORCES: THEORY AND A FEW EXPERIMENTS
Studies of embryonic fate patterning often focus on diffusible chemical signals. Intercellular forces’ role in generating embryonic fate patterns is much less thoroughly explored. Recent experiments on stem cell colonies on elastic substrates demonstrated that a key patterning event, neural induction, can occur without exogenous chemical gradients and is affected by mechanical stretching. Inspired by these findings, here we propose a mathematical model of mechanical patterning: cell contractility depends on fate, and in-plane mechanical pressure biases fate. The cells at the colony boundary, more contractile than those at the colony center, generate a fate pattern by transmitting forces to the cells at the colony center. In agreement with previous observations, our model implies that the outer fate domain’s width is independent of colony diameter. Importantly, the model further predicts that the outer fate domain’s width depends non-monotonically on substrate stiffness, which we confirm experimentally. We conclude by discussing alternative explanations for this non-monotonic behavior and model predictions that will allow for a more stringent test of the mechanical origin of the fate patterns.