Adhesion-induced cortical flows pattern E-cadherin-mediated cell contacts.

Metazoan development relies on the formation and remodeling of cell-cell contacts. Dynamic reorganization of adhesion receptors and the actomyosin cell cortex in space and time plays a central role in cell-cell contact formation and maturation. Nevertheless, how this process is mechanistically achieved when new contacts are formed remains unclear. Here, by building a biomimetic assay composed of progenitor cells adhering to supported lipid bilayers functionalized with E-cadherin ectodomains, we show that cortical F-actin flows, driven by the depletion of myosin-2 at the cell contact center, mediate the dynamic reorganization of adhesion receptors and cell cortex at the contact. E-cadherin-dependent downregulation of the small GTPase RhoA at the forming contact leads to both a depletion of myosin-2 and a decrease of F-actin at the contact center. At the contact rim, in contrast, myosin-2 becomes enriched by the retraction of bleb-like protrusions, resulting in a cortical tension gradient from the contact rim to its center. This tension gradient, in turn, triggers centrifugal F-actin flows, leading to further accumulation of F-actin at the contact rim and the progressive redistribution of E-cadherin from the contact center to the rim. Eventually, this combination of actomyosin downregulation and flows at the contact determines the characteristic molecular organization, with E-cadherin and F-actin accumulating at the contact rim, where they are needed to mechanically link the contractile cortices of the adhering cells.

Lineage tracing identifies heterogeneous hepatoblast contribution to cell lineages and postembryonic organ growth dynamics.

To meet the physiological demands of the body, organs need to establish a functional tissue architecture and adequate size as the embryo develops to adulthood. In the liver, uni- and bipotent progenitor differentiation into hepatocytes and biliary epithelial cells (BECs), and their relative proportions, comprise the functional architecture. Yet, the contribution of individual liver progenitors at the organ level to both fates, and their specific proportion, is unresolved. Combining mathematical modelling with organ-wide, multispectral FRaeppli-NLS lineage tracing in zebrafish, we demonstrate that a precise BEC-to-hepatocyte ratio is established (i) fast, (ii) solely by heterogeneous lineage decisions from uni- and bipotent progenitors, and (iii) independent of subsequent cell type-specific proliferation. Extending lineage tracing to adulthood determined that embryonic cells undergo spatially heterogeneous three-dimensional growth associated with distinct environments. Strikingly, giant clusters comprising almost half a ventral lobe suggest lobe-specific dominant-like growth behaviours. We show substantial hepatocyte polyploidy in juveniles representing another hallmark of postembryonic liver growth. Our findings uncover heterogeneous progenitor contributions to tissue architecture-defining cell type proportions and postembryonic organ growth as key mechanisms forming the adult liver.

A Guide Toward Multi-scale and Quantitative Branching Analysis in the Mammary Gland.

The mammary gland consists of a bilayered epithelial structure with an extensively branched morphology. The majority of this epithelial tree is laid down during puberty, during which actively proliferating terminal end buds repeatedly elongate and bifurcate to form the basic structure of the ductal tree. Mammary ducts consist of a basal and luminal cell layer with a multitude of identified sub-lineages within both layers. The understanding of how these different cell lineages are cooperatively driving branching morphogenesis is a problem of crossing multiple scales, as this requires information on the macroscopic branched structure of the gland, as well as data on single-cell dynamics driving the morphogenic program. Here we describe a method to combine genetic lineage tracing with whole-gland branching analysis. Quantitative data on the global organ structure can be used to derive a model for mammary gland branching morphogenesis and provide a backbone on which the dynamics of individual cell lineages can be simulated and compared to lineage-tracing approaches. Eventually, these quantitative models and experiments allow to understand the couplings between the macroscopic shape of the mammary gland and the underlying single-cell dynamics driving branching morphogenesis.

DUCT reveals architectural mechanisms contributing to bile duct recovery in a mouse model for Alagille syndrome.

Organ function depends on tissues adopting the correct architecture. However, insights into organ architecture are currently hampered by an absence of standardized quantitative 3D analysis. We aimed to develop a robust technology to visualize, digitalize, and segment the architecture of two tubular systems in 3D: double resin casting micro computed tomography (DUCT). As proof of principle, we applied DUCT to a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice), characterized by intrahepatic bile duct paucity, that can spontaneously generate a biliary system in adulthood. DUCT identified increased central biliary branching and peripheral bile duct tortuosity as two compensatory processes occurring in distinct regions of Jag1Ndr/Ndr liver, leading to full reconstitution of wild-type biliary volume and phenotypic recovery. DUCT is thus a powerful new technology for 3D analysis, which can reveal novel phenotypes and provide a standardized method of defining liver architecture in mouse models.

Many essential parts of the body contain tubes: the liver for example, contains bile ducts and blood vessels. These tubes develop right next to each other, like entwined trees. To do their jobs, these ducts must communicate and collaborate, but they do not always grow properly. For example, babies with Alagille syndrome are born with few or no bile ducts, resulting in serious liver disease. Understanding the architecture of the tubes in their livers could explain why some children with this syndrome improve with time, but many others need a liver transplant. Visualising biological tubes in three dimensions is challenging. One major roadblock is the difficulty in seeing several tubular structures at once. Traditional microscopic imaging of anatomy is in two dimensions, using slices of tissue. This approach shows the cross-sections of tubes, but not how the ducts connect and interact. An alternative is to use micro computed tomography scans, which use X-rays to examine structures in three dimensions. The challenge with this approach is that soft tissues, which tubes in the body are made of, do not show up well on X-ray. One way to solve this is to fill the ducts with X-ray absorbing resins, making a cast of the entire tree structure. The question is, can two closely connected tree structures be distinguished if they are cast at the same time? To address this question, Hankeova, Salplachta et al. developed a technique called double resin casting micro computed tomography, or DUCT for short. The approach involved making casts of tube systems using two types of resin that show up differently under X-rays. The new technique was tested on a mouse model of Alagille syndrome. One resin was injected into the bile ducts, and another into the blood vessels. This allowed Hankeova, Salplachta et al. to reconstruction both trees digitally, revealing their length, volume, branching, and interactions. In healthy mice, the bile ducts were straight with uniform branches, but in mice with Alagille syndrome ducts were wiggly, and had extra branches in the centre of the liver. This new imaging technique could improve the understanding of tube systems in animal models of diseases, both in the liver and in other organs with tubes, such as the lungs or the kidneys. Hankeova, Salplachta et al. also lay a foundation for a deeper understanding of bile duct recovery in Alagille syndrome. In the future, DUCT could help researchers to see how mouse bile ducts change in response to experimental therapies.

Tracing the cellular dynamics of sebaceous gland development in normal and perturbed states.

The sebaceous gland (SG) is an essential component of the skin, and SG dysfunction is debilitating1,2. Yet, the cellular bases for its origin, development and subsequent maintenance remain poorly understood. Here, we apply large-scale quantitative fate mapping to define the patterns of cell fate behaviour during SG development and maintenance. We show that the SG develops from a defined number of lineage-restricted progenitors that undergo a programme of independent and stochastic cell fate decisions. Following an expansion phase, equipotent progenitors transition into a phase of homeostatic turnover, which is correlated with changes in the mechanical properties of the stroma and spatial restrictions on gland size. Expression of the oncogene KrasG12D results in a release from these constraints and unbridled gland expansion. Quantitative clonal fate analysis reveals that, during this phase, the primary effect of the Kras oncogene is to drive a constant fate bias with little effect on cell division rates. These findings provide insight into the developmental programme of the SG, as well as the mechanisms that drive tumour progression and gland dysfunction.

Tracing the origin of adult intestinal stem cells.

Adult intestinal stem cells are located at the bottom of crypts of Lieberkühn, where they express markers such as LGR51,2 and fuel the constant replenishment of the intestinal epithelium1. Although fetal LGR5-expressing cells can give rise to adult intestinal stem cells3,4, it remains unclear whether this population in the patterned epithelium represents unique intestinal stem-cell precursors. Here we show, using unbiased quantitative lineage-tracing approaches, biophysical modelling and intestinal transplantation, that all cells of the mouse intestinal epithelium-irrespective of their location and pattern of LGR5 expression in the fetal gut tube-contribute actively to the adult intestinal stem cell pool. Using 3D imaging, we find that during fetal development the villus undergoes gross remodelling and fission. This brings epithelial cells from the non-proliferative villus into the proliferative intervillus region, which enables them to contribute to the adult stem-cell niche. Our results demonstrate that large-scale remodelling of the intestinal wall and cell-fate specification are closely linked. Moreover, these findings provide a direct link between the observed plasticity and cellular reprogramming of differentiating cells in adult tissues following damage5-9, revealing that stem-cell identity is an induced rather than a hardwired property.

Statistical theory of branching morphogenesis.

Branching morphogenesis remains a subject of abiding interest. Although much is known about the gene regulatory programs and signaling pathways that operate at the cellular scale, it has remained unclear how the macroscopic features of branched organs, including their size, network topology and spatial patterning, are encoded. Lately, it has been proposed that, these features can be explained quantitatively in several organs within a single unifying framework. Based on large-scale organ reconstructions and cell lineage tracing, it has been argued that morphogenesis follows from the collective dynamics of sublineage-restricted self-renewing progenitor cells, localized at ductal tips, that act cooperatively to drive a serial process of ductal elongation and stochastic tip bifurcation. By correlating differentiation or cell cycle exit with proximity to maturing ducts, this dynamic results in the specification of a complex network of defined density and statistical organization. These results suggest that, for several mammalian tissues, branched epithelial structures develop as a self-organized process, reliant upon a strikingly simple, but generic, set of local rules, without recourse to a rigid and deterministic sequence of genetically programmed events. Here, we review the basis of these findings and discuss their implications.

Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland.

Recent lineage tracing studies have revealed that mammary gland homeostasis relies on unipotent stem cells. However, whether and when lineage restriction occurs during embryonic mammary development, and which signals orchestrate cell fate specification, remain unknown. Using a combination of in vivo clonal analysis with whole mount immunofluorescence and mathematical modelling of clonal dynamics, we found that embryonic multipotent mammary cells become lineage-restricted surprisingly early in development, with evidence for unipotency as early as E12.5 and no statistically discernable bipotency after E15.5. To gain insights into the mechanisms governing the switch from multipotency to unipotency, we used gain-of-function Notch1 mice and demonstrated that Notch activation cell autonomously dictates luminal cell fate specification to both embryonic and basally committed mammary cells. These functional studies have important implications for understanding the signals underlying cell plasticity and serve to clarify how reactivation of embryonic programs in adult cells can lead to cancer.

Theory of Epithelial Cell Shape Transitions Induced by Mechanoactive Chemical Gradients.

Cell shape is determined by a balance of intrinsic properties of the cell as well as its mechanochemical environment. Inhomogeneous shape changes underlie many morphogenetic events and involve spatial gradients in active cellular forces induced by complex chemical signaling. Here, we introduce a mechanochemical model based on the notion that cell shape changes may be induced by external diffusible biomolecules that influence cellular contractility (or equivalently, adhesions) in a concentration-dependent manner-and whose spatial profile in turn is affected by cell shape. We map out theoretically the possible interplay between chemical concentration and cellular structure. Besides providing a direct route to spatial gradients in cell shape profiles in tissues, we show that the dependence on cell shape helps create robust mechanochemical gradients.

Multipotent Basal Stem Cells, Maintained in Localized Proximal Niches, Support Directed Long-Ranging Epithelial Flows in Human Prostates.

Sporadic mitochondrial DNA mutations serve as clonal marks providing access to the identity and lineage potential of stem cells within human tissues. By combining quantitative clonal mapping with 3D reconstruction of adult human prostates, we show that multipotent basal stem cells, confined to discrete niches in juxta-urethral ducts, generate bipotent basal progenitors in directed epithelial migration streams. Basal progenitors are then dispersed throughout the entire glandular network, dividing and differentiating to replenish the loss of apoptotic luminal cells. Rare lineage-restricted luminal stem cells, and their progeny, are confined to proximal ducts and provide only minor contribution to epithelial homeostasis. In situ cell capture from clonal maps identified delta homolog 1 (DLK1) enrichment of basal stem cells, which was validated in functional spheroid assays. This study establishes significant insights into niche organization and function of prostate stem and progenitor cells, with implications for disease.

Transmission of cytokinesis forces via E-cadherin dilution and actomyosin flows.

During epithelial cytokinesis, the remodelling of adhesive cell-cell contacts between the dividing cell and its neighbours has profound implications for the integrity, arrangement and morphogenesis of proliferative tissues. In both vertebrates and invertebrates, this remodelling requires the activity of non-muscle myosin II (MyoII) in the interphasic cells neighbouring the dividing cell. However, the mechanisms that coordinate cytokinesis and MyoII activity in the neighbours are unknown. Here we show that in the Drosophila notum epithelium, each cell division is associated with a mechanosensing and transmission event that controls MyoII dynamics in neighbouring cells. We find that the ring pulling forces promote local junction elongation, which results in local E-cadherin dilution at the ingressing adherens junction. In turn, the reduction in E-cadherin concentration and the contractility of the neighbouring cells promote self-organized actomyosin flows, ultimately leading to accumulation of MyoII at the base of the ingressing junction. Although force transduction has been extensively studied in the context of adherens junction reinforcement to stabilize adhesive cell-cell contacts, we propose an alternative mechanosensing mechanism that coordinates actomyosin dynamics between epithelial cells and sustains the remodelling of the adherens junction in response to mechanical forces.

RhoA regulates actin network dynamics during apical surface emergence in multiciliated epithelial cells.

Homeostatic replacement of epithelial cells from basal precursors is a multistep process involving progenitor cell specification, radial intercalation and, finally, apical surface emergence. Recent data demonstrate that actin-based pushing under the control of the formin protein Fmn1 drives apical emergence in nascent multiciliated epithelial cells (MCCs), but little else is known about this actin network or the control of Fmn1. Here, we explore the role of the small GTPase RhoA in MCC apical emergence. Disruption of RhoA function reduced the rate of apical surface expansion and decreased the final size of the apical domain. Analysis of cell shapes suggests that RhoA alters the balance of forces exerted on the MCC apical surface. Finally, quantitative time-lapse imaging and fluorescence recovery after photobleaching studies argue that RhoA works in concert with Fmn1 to control assembly of the specialized apical actin network in MCCs. These data provide new molecular insights into epithelial apical surface assembly and could also shed light on mechanisms of apical lumen formation.

Defining the clonal dynamics leading to mouse skin tumour initiation.

The changes in cell dynamics after oncogenic mutation that lead to the development of tumours are currently unknown. Here, using skin epidermis as a model, we assessed the effect of oncogenic hedgehog signalling in distinct cell populations and their capacity to induce basal cell carcinoma, the most frequent cancer in humans. We found that only stem cells, and not progenitors, initiated tumour formation upon oncogenic hedgehog signalling. This difference was due to the hierarchical organization of tumour growth in oncogene-targeted stem cells, characterized by an increase in symmetric self-renewing divisions and a higher p53-dependent resistance to apoptosis, leading to rapid clonal expansion and progression into invasive tumours. Our work reveals that the capacity of oncogene-targeted cells to induce tumour formation is dependent not only on their long-term survival and expansion, but also on the specific clonal dynamics of the cancer cell of origin.

Emergence of an Apical Epithelial Cell Surface In Vivo.

Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, we combine molecular, pharmacological, and laser-dissection experiments with theoretical modeling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium in vivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging cell and mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis.

Physics of active jamming during collective cellular motion in a monolayer.

Although collective cell motion plays an important role, for example during wound healing, embryogenesis, or cancer progression, the fundamental rules governing this motion are still not well understood, in particular at high cell density. We study here the motion of human bronchial epithelial cells within a monolayer, over long times. We observe that, as the monolayer ages, the cells slow down monotonously, while the velocity correlation length first increases as the cells slow down but eventually decreases at the slowest motions. By comparing experiments, analytic model, and detailed particle-based simulations, we shed light on this biological amorphous solidification process, demonstrating that the observed dynamics can be explained as a consequence of the combined maturation and strengthening of cell-cell and cell-substrate adhesions. Surprisingly, the increase of cell surface density due to proliferation is only secondary in this process. This analysis is confirmed with two other cell types. The very general relations between the mean cell velocity and velocity correlation lengths, which apply for aggregates of self-propelled particles, as well as motile cells, can possibly be used to discriminate between various parameter changes in vivo, from noninvasive microscopy data.

Cortical instability drives periodic supracellular actin pattern formation in epithelial tubes.

An essential question of morphogenesis is how patterns arise without preexisting positional information, as inspired by Turing. In the past few years, cytoskeletal flows in the cell cortex have been identified as a key mechanism of molecular patterning at the subcellular level. Theoretical and in vitro studies have suggested that biological polymers such as actomyosin gels have the property to self-organize, but the applicability of this concept in an in vivo setting remains unclear. Here, we report that the regular spacing pattern of supracellular actin rings in the Drosophila tracheal tubule is governed by a self-organizing principle. We propose a simple biophysical model where pattern formation arises from the interplay of myosin contractility and actin turnover. We validate the hypotheses of the model using photobleaching experiments and report that the formation of actin rings is contractility dependent. Moreover, genetic and pharmacological perturbations of the physical properties of the actomyosin gel modify the spacing of the pattern, as the model predicted. In addition, our model posited a role of cortical friction in stabilizing the spacing pattern of actin rings. Consistently, genetic depletion of apical extracellular matrix caused strikingly dynamic movements of actin rings, mirroring our model prediction of a transition from steady to chaotic actin patterns at low cortical friction. Our results therefore demonstrate quantitatively that a hydrodynamical instability of the actin cortex can trigger regular pattern formation and drive morphogenesis in an in vivo setting.

Theory of epithelial sheet morphology in three dimensions.

Morphogenesis during embryo development requires the coordination of mechanical forces to generate the macroscopic shapes of organs. We propose a minimal theoretical model, based on cell adhesion and actomyosin contractility, which describes the various shapes of epithelial cells and the bending and buckling of epithelial sheets, as well as the relative stability of cellular tubes and spheres. We show that, to understand these processes, a full 3D description of the cells is needed, but that simple scaling laws can still be derived. The morphologies observed in vivo can be understood as stable points of mechanical equations and the transitions between them are either continuous or discontinuous. We then focus on epithelial sheet bending, a ubiquitous morphogenetic process. We calculate the curvature of an epithelium as a function of actin belt tension as well as of cell-cell and and cell-substrate tension. The model allows for a comparison of the relative stabilities of spherical or cylindrical cellular structures (acini or tubes). Finally, we propose a unique type of buckling instability of epithelia, driven by a flattening of individual cell shapes, and discuss experimental tests to verify our predictions.

Alignment of cellular motility forces with tissue flow as a mechanism for efficient wound healing.

Recent experiments have shown that spreading epithelial sheets exhibit a long-range coordination of motility forces that leads to a buildup of tension in the tissue, which may enhance cell division and the speed of wound healing. Furthermore, the edges of these epithelial sheets commonly show finger-like protrusions whereas the bulk often displays spontaneous swirls of motile cells. To explain these experimental observations, we propose a simple flocking-type mechanism, in which cells tend to align their motility forces with their velocity. Implementing this idea in a mechanical tissue simulation, the proposed model gives rise to efficient spreading and can explain the experimentally observed long-range alignment of motility forces in highly disordered patterns, as well as the buildup of tensile stress throughout the tissue. Our model also qualitatively reproduces the dependence of swirl size and swirl velocity on cell density reported in experiments and exhibits an undulation instability at the edge of the spreading tissue commonly observed in vivo. Finally, we study the dependence of colony spreading speed on important physical and biological parameters and derive simple scaling relations that show that coordination of motility forces leads to an improvement of the wound healing process for realistic tissue parameters.

Mechanical instabilities of biological tubes.

We study theoretically the morphologies of biological tubes affected by various pathologies. When epithelial cells grow, the negative tension produced by their division provokes a buckling instability. Several shapes are investigated: varicose, dilated, sinuous, or sausagelike. They are all found in pathologies of tracheal, renal tubes, or arteries. The final shape depends crucially on the mechanical parameters of the tissues: Young's modulus, wall-to-lumen ratio, homeostatic pressure. We argue that since tissues must be in quasistatic mechanical equilibrium, abnormal shapes convey information as to what causes the pathology. We calculate a phase diagram of tubular instabilities which could be a helpful guide for investigating the underlying genetic regulation.

Glass-like dynamics of collective cell migration.

Collective cell migration in tissues occurs throughout embryonic development, during wound healing, and in cancerous tumor invasion, yet most detailed knowledge of cell migration comes from single-cell studies. As single cells migrate, the shape of the cell body fluctuates dramatically through cyclic processes of extension, adhesion, and retraction, accompanied by erratic changes in migration direction. Within confluent cell layers, such subcellular motions must be coupled between neighbors, yet the influence of these subcellular motions on collective migration is not known. Here we study motion within a confluent epithelial cell sheet, simultaneously measuring collective migration and subcellular motions, covering a broad range of length scales, time scales, and cell densities. At large length scales and time scales collective migration slows as cell density rises, yet the fastest cells move in large, multicell groups whose scale grows with increasing cell density. This behavior has an intriguing analogy to dynamic heterogeneities found in particulate systems as they become more crowded and approach a glass transition. In addition we find a diminishing self-diffusivity of short-wavelength motions within the cell layer, and growing peaks in the vibrational density of states associated with cooperative cell-shape fluctuations. Both of these observations are also intriguingly reminiscent of a glass transition. Thus, these results provide a broad and suggestive analogy between cell motion within a confluent layer and the dynamics of supercooled colloidal and molecular fluids approaching a glass transition.