Session X65: Physics of Development and Stem Cells

The mechanics of epithelial folding

Throughout the lifespan of an organism, tissues are remodeled to shape organs and organisms and to maintain tissue integrity and homeostasis. Apical constriction is a ubiquitous cell shape change of epithelial tissues that promotes epithelia folding and cell/tissue invagination in a variety of contexts. Apical constriction promotes tissue bending by changing the shape of constituent cells from a columnar-shape to a wedge-shape. Drosophilagastrulation is one of the classic examples of apical constriction, where cells constrict to fold the primitive epithelial sheet and internalize cells that will give rise to internal organs. We find that the apical constriction of these cells constrict via repeated contractile pulses of the cytoskeleton, specifically actin filaments and the molecular motor myosin II. Contractility of the cells results in anisotropic tension, which is essential for the proper orientation of the fold. We are investigating mechanisms that pattern contractility across the tissue to ensure robust folding.   

Lineage and state dynamics in stem cell differentiation

High-dimensional single cell measurements allow capturing snapshots of dynamic transitions in biological systems. Over the past few years, measurements such as single cell RNA-Sequencing (RNA-Seq) have come to be routinely used in stem cell biology. However, as single cell measurements necessarily destroy cells, they do not definitively reveal long-term dynamic behaviors. Various theoretical models have been invoked to link static snapshots to dynamics, all necessarily making assumptions. I will describe the assumptions underlying one such model of a Fokker-Planck type, and its limitations. I will then show how the combination of lineage data in single cell transcriptomes allows explicitly testing dynamic inference models on single cell RNA-Seq data. Our results show the successes but also failures of dynamic inference, and provide a test demonstrating the existence of "hidden variables" underlying cellular dynamics as measured by RNA-Seq. The results and methods are applied to the lineage hierarchy of hematopoiesis.   

Precise spatial scaling in the early fly embryo

Animals vary more in size than in proportions; a possible quantitative version of this observation would that organisms exhibit scaling, so that the dimensions of different body segments all vary in proportion to the overall size of the organism. If this were true, pattern formation in biological systems would be qualitatively different from that in the non-biological pattern forming systems that we understand, such as Rayleigh-Benard convection and directional solidification. A relatively pure version of this question is accessible in the early development of insect embryos, where a “blueprint” for the final segmented body plan is visible in striped patterns of gene expression. We measure the positions of these stripes in an ensemble of 100+ embryos from a laboratory strain of Drosophila melanogaster, under controlled conditions. These embryos vary in length by only 4% (rms), yet we can resolve the scaling of stripe positions with length. The resulting 1% accuracy of relative positions is so high as to exclude alternatives, such as combinations of unscaled signals from the two ends of the embryo.   

The Role of Bio-Mechanics in Somite Formation

Early vertebrate segmentation results from the creation of a series of metameric epithelial spheres called somites. While the timing of somite formation is believed to be regulated by a molecular level clock, the formation of somites is a self-organizing mechanical process that has received little attention so far. In this work, we build a 2D computer model of mesenchymal to epithelial transition process to explore the cellular mechanisms that promote somite organization. We focus on the first stage of somite formation, the establishment of the dorsal layer as an anterior propagating wave of cell maturation. First we explore and discuss the separate and combined contributions of cell polarization, adhesion and elongation to the formation of the dorsal layer. Finally, we explore the process of apical constriction and show how the forming layer is segmented into discrete units.   

Limiting-pool mechanism of size control of nucleoli in the C. elegans embryo

Cells contain organelles which maintain a dynamic yet coherent structure, but the mechanisms by which
these assemblies form and stably persist are not well understood. Here, we consider the assembly of
nucleoli in C. elegans embryo as a model-system for questions regarding the mechanisms controlling the
size of self-assembling structures within the cell. Recent experiments suggest that, in C. elegans early
embryo, nucleoli grow until the pool of its building blocks is depleted from the nucleoplasm. This is
known as the ‘limiting-pool’ mechanism.
In order to study experimental signatures of the limiting-pool mechanism, we consider a simple model
of the self-assembly of two spherical structures from a common pool of building blocks. Using theory
and simulations, we analyze the time series of the sizes of the spherical structures and make specific
predictions about their autocorrelation functions. We then compare these theoretical results to
fluorescence data from experiments that measure nucleoli size as a function of time in late stages of C.
elegans embryo development. This approach can be used in other experimental systems to
quantitatively test for the limiting-pool mechanism, and can be extended to other size-control
mechanisms.   

Single cell morphology trajectory analysis reveals different cell states and transition paths in Epithelial-to-Mesenchymal transition

From physics perspective a cell is a nonequilibrium nonlinear dynamical system that evolves over time, and cell phenotype conversion corresponds to transitions between attractors. Quantitative and mechanistic understanding of a phenotypic conversion process is of fundamental significance, but conventional fixed-cell snapshot data miss some key dynamical information. We developed a deep-learning based live cell imaging and analysis procedure to trace single cell trajectories in high-dimensional morphology space. Using TGF-β induced Epithelial-to-Mesenchymal transition (EMT) in human HK2 cells as a model system, we experimentally depicted the quasi-potential landscape of EMT, identified three distinct morphology states that are correlated with different expression levels of EMT regulators and markers. We further reconstructed an extended state network that depicts coupling between cell cycle and EMT, and quantified the transition matrix from single cell data. In many aspects the single cell studies are analogous to the more established single molecule approaches in molecular biophysics, while the transition process under study is complicated by existence of dynamic and static disorders in the system due to coupling to other dynamical processes.   

Structural redundancy in supracellular actomyosin network connections enables robust tissue folding

It is essential for the fate of an organism that key morphogenetic processes occur reproducibly. While much is known about how genetic regulation achieves robustness, less is known about how a tissue mechanically ensures reproducibility. Gastrulation is an indispensable stage of development. Drosophila fruit fly gastrulation is driven by myosin-dependent constriction and this robust large-scale movement requires coordinated forces at the tissue level. Yet, how the cytoskeleton is connected across the constricting tissue to realize shape change is unknown. We demonstrate a high degree of robustness in Drosophila gastrulation whereby ablating many cells doesn’t halt shape change. To understand this robustness, we integrated concepts from graph theory to analyze the connectivity of a supracellular network of mysoin connections that links cells in the tissue. We found that a dense meshwork of tissue scale connections ensures structural redundancy and that even when large numbers of connections are lost some paths across the tissue persists and folding proceeds. We propose that in the same way that redundancy ensures the large-scale function of telecommunication or transportation networks under localized failures, the organization of myosin activation enables robust morphogenesis.   

Diffusion vs. direct transport in the precision of morphogen readout

Morphogen profiles allow cells to determine their position within a developing organism, but there are multiple mechanisms by which these profiles form, and in some cases the mechanism is still not agreed upon. Here we derive fundamental limits to the precision of morphogen concentration sensing for two canonical mechanisms: the diffusion of morphogen through extracellular space and the direct transport of morphogen from source cell to target cell, e.g. via cytonemes. We find that direct transport establishes a morphogen profile without adding extrinsic noise. Despite this advantage, we find that for sufficiently large values of population size and profile length, the diffusion mechanism is many times more precise due to a higher refresh rate of morphogen molecules. We compare our predictions with data from a wide variety of morphogens in developing organisms.
  

Intracellular crowding as mechanoregulator of intestinal organoid growth via modulating Wnt-receptor complex phase transition

Enormous amounts of essential intracellular events are crowdedly packed inside picoliter-sized cellular space. However, the significance of the physical properties of cells remains underappreciated due to a lack of evidence of how they impact cellular functionalities. Here, we show that the degree of intracellular crowding serves as a mechanoregulator of intestinal organoid growth via modulating Wnt/β-catenin signaling. Intracellular crowding varies upon stimulation by different types of extracellular physical and mechanical cues, and leads to a significant enhancement of Wnt/β-catenin signaling by promoting phase transition of LRP6 receptor complex. By enhancing intracellular crowding using either osmotic or mechanical compression, we show that expansion of intestinal organoids was facilitated through elevated Wnt/β-catenin signaling and greater intestinal stem cells (ISCs) self-renewal. Our results provide an entry point for understanding how intracellular crowdedness functions as a mechanotransducer linking extracellular physical cues with intracellular signaling, and potentially facilitate the design of engineering approaches for expansion of stem cells and organoids.   

Population variation in human pre-implantation embryo development, or: how I learned to stop worrying and love IVF data.

The early mammalian embryo is the physicist's dream for studying development. Between fertilization and implantation in the uterus, the mammalian embryo undergoes a global reorganization (compaction) followed by a symmetry-breaking differentiation (blastocyst formation), with no external direction from the mother. While studies on mouse embryos illuminate the mechanisms of development, the genetic homogeneity of lab mice obscures the natural variability in mammalian development. Here, we examine thousands of videos of human embryos recorded during IVF procedures to explore the natural variability in mammalian preimplantation embryo development. We leverage this variability to infer possible regulatory mechanisms in early development. Finally, we will discuss our work on extending non-invasive 3D microscopy to imaging human embryos.   

Biophysical studies of metabolic control in mouse oocytes and embryos

While a great deal is known about pathways and enzymology of carbohydrate metabolism, it is still poorly understood how metabolic fluxes are modulated during development and in response to environmental factors, or degraded in disease. Mounting evidence suggests that defects in metabolism may cause chromosome segregation errors in eggs and embryos, leading to age related infertility in women, but the possible underlying mechanisms remain unclear. We are developing and testing a coarse-grained biophysical model of enzyme engagement of electron carriers, with the goal of extracting metabolic fluxes from fluorescence lifetime imaging microscopy measurements. We are attempting to construct a theory of the control of mitochondrial respiration and cytoplasmic fermentation in mouse oocytes and embryos by using this approach in conjunction with metabolic manipulations. Our preliminary results argue that the fluxes through these pathways can be redirected in response to perturbations, but this is accompanied by large changes in cell biological features, including disassembly of the spindle, which we speculate might underlie the connection between metabolic defects and chromosome segregation errors.