Combinatorial patterns of graded RhoA activation and uniform F-actin depletion promote tissue curvature.

During development, gene expression regulates cell mechanics and shape to sculpt tissues. Epithelial folding proceeds through distinct cell shape changes that occur simultaneously in different regions of a tissue. Here, using quantitative imaging in Drosophila melanogaster, we investigate how patterned cell shape changes promote tissue bending during early embryogenesis. We find that the transcription factors Twist and Snail combinatorially regulate a multicellular pattern of lateral F-actin density that differs from the previously described Myosin-2 gradient. This F-actin pattern correlates with whether cells apically constrict, stretch or maintain their shape. We show that the Myosin-2 gradient and F-actin depletion do not depend on force transmission, suggesting that transcriptional activity is required to create these patterns. The Myosin-2 gradient width results from a gradient in RhoA activation that is refined through the balance between RhoGEF2 and the RhoGAP C-GAP. Our experimental results and simulations of a 3D elastic shell model show that tuning gradient width regulates tissue curvature.

Dynamics of hydraulic and contractile wave-mediated fluid transport during Drosophila oogenesis.

From insects to mice, oocytes develop within cysts alongside nurse-like sister germ cells. Prior to fertilization, the nurse cells' cytoplasmic contents are transported into the oocyte, which grows as its sister cells regress and die. Although critical for fertility, the biological and physical mechanisms underlying this transport process are poorly understood. Here, we combined live imaging of germline cysts, genetic perturbations, and mathematical modeling to investigate the dynamics and mechanisms that enable directional and complete cytoplasmic transport in Drosophila melanogaster egg chambers. We discovered that during "nurse cell (NC) dumping" most cytoplasm is transported into the oocyte independently of changes in myosin-II contractility, with dynamics instead explained by an effective Young-Laplace law, suggesting hydraulic transport induced by baseline cell-surface tension. A minimal flow-network model inspired by the famous two-balloon experiment and motivated by genetic analysis of a myosin mutant correctly predicts the directionality, intercellular pattern, and time scale of transport. Long thought to trigger transport through "squeezing," changes in actomyosin contractility are required only once NC volume has become comparable to nuclear volume, in the form of surface contractile waves that drive NC dumping to completion. Our work thus demonstrates how biological and physical mechanisms cooperate to enable a critical developmental process that, until now, was thought to be mainly biochemically regulated.

Tension, contraction and tissue morphogenesis.

D'Arcy Thompson was a proponent of applying mathematical and physical principles to biological systems, an approach that is becoming increasingly common in developmental biology. Indeed, the recent integration of quantitative experimental data, force measurements and mathematical modeling has changed our understanding of morphogenesis - the shaping of an organism during development. Emerging evidence suggests that the subcellular organization of contractile cytoskeletal networks plays a key role in force generation, while on the tissue level the spatial organization of forces determines the morphogenetic output. Inspired by D'Arcy Thompson's On Growth and Form, we review our current understanding of how biological forms are created and maintained by the generation and organization of contractile forces at the cell and tissue levels. We focus on recent advances in our understanding of how cells actively sculpt tissues and how forces are involved in specific morphogenetic processes.

Actomyosin-based tissue folding requires a multicellular myosin gradient.

Tissue folding promotes three-dimensional (3D) form during development. In many cases, folding is associated with myosin accumulation at the apical surface of epithelial cells, as seen in the vertebrate neural tube and the Drosophila ventral furrow. This type of folding is characterized by constriction of apical cell surfaces, and the resulting cell shape change is thought to cause tissue folding. Here, we use quantitative microscopy to measure the pattern of transcription, signaling, myosin activation and cell shape in the Drosophila mesoderm. We found that cells within the ventral domain accumulate different amounts of active apical non-muscle myosin 2 depending on the distance from the ventral midline. This gradient in active myosin depends on a newly quantified gradient in upstream signaling proteins. A 3D continuum model of the embryo with induced contractility demonstrates that contractility gradients, but not contractility per se, promote changes to surface curvature and folding. As predicted by the model, experimental broadening of the myosin domain in vivo disrupts tissue curvature where myosin is uniform. Our data argue that apical contractility gradients are important for tissue folding.

Apical constriction: themes and variations on a cellular mechanism driving morphogenesis.

Apical constriction is a cell shape change that promotes tissue remodeling in a variety of homeostatic and developmental contexts, including gastrulation in many organisms and neural tube formation in vertebrates. In recent years, progress has been made towards understanding how the distinct cell biological processes that together drive apical constriction are coordinated. These processes include the contraction of actin-myosin networks, which generates force, and the attachment of actin networks to cell-cell junctions, which allows forces to be transmitted between cells. Different cell types regulate contractility and adhesion in unique ways, resulting in apical constriction with varying dynamics and subcellular organizations, as well as a variety of resulting tissue shape changes. Understanding both the common themes and the variations in apical constriction mechanisms promises to provide insight into the mechanics that underlie tissue morphogenesis.

Force transmission in epithelial tissues.

In epithelial tissues, cells constantly generate and transmit forces between each other. Forces generated by the actomyosin cytoskeleton regulate tissue shape and structure and also provide signals that influence cells' decisions to divide, die, or differentiate. Forces are transmitted across epithelia because cells are mechanically linked through junctional complexes, and forces can propagate through the cell cytoplasm. Here, we review some of the molecular mechanisms responsible for force generation, with a specific focus on the actomyosin cortex and adherens junctions. We then discuss evidence for how these mechanisms promote cell shape changes and force transmission in tissues.

Pulsed contractions of an actin-myosin network drive apical constriction.

Apical constriction facilitates epithelial sheet bending and invagination during morphogenesis. Apical constriction is conventionally thought to be driven by the continuous purse-string-like contraction of a circumferential actin and non-muscle myosin-II (myosin) belt underlying adherens junctions. However, it is unclear whether other force-generating mechanisms can drive this process. Here we show, with the use of real-time imaging and quantitative image analysis of Drosophila gastrulation, that the apical constriction of ventral furrow cells is pulsed. Repeated constrictions, which are asynchronous between neighbouring cells, are interrupted by pauses in which the constricted state of the cell apex is maintained. In contrast to the purse-string model, constriction pulses are powered by actin-myosin network contractions that occur at the medial apical cortex and pull discrete adherens junction sites inwards. The transcription factors Twist and Snail differentially regulate pulsed constriction. Expression of snail initiates actin-myosin network contractions, whereas expression of twist stabilizes the constricted state of the cell apex. Our results suggest a new model for apical constriction in which a cortical actin-myosin cytoskeleton functions as a developmentally controlled subcellular ratchet to reduce apical area incrementally.

Volume conservation principle involved in cell lengthening and nucleus movement during tissue morphogenesis.

Tissue morphogenesis is the process in which coordinated movements and shape changes of large numbers of cells form tissues, organs, and the internal body structure. Understanding morphogenetic movements requires precise measurements of whole-cell shape changes over time. Tissue folding and invagination are thought to be facilitated by apical constriction, but the mechanism by which changes near the apical cell surface affect changes along the entire apical-basal axis of the cell remains elusive. Here, we developed Embryo Development Geometry Explorer, an approach for quantifying rapid whole-cell shape changes over time, and we combined it with deep-tissue time-lapse imaging based on fast two-photon microscopy to study Drosophila ventral furrow formation. We found that both the cell lengthening along the apical-basal axis and the movement of the nucleus to the basal side proceeded stepwise and were correlated with apical constriction. Moreover, cell volume lost apically due to constriction largely balanced the volume gained basally by cell lengthening. The volume above the nucleus was conserved during its basal movement. Both apical volume loss and cell lengthening were absent in mutants showing deficits in the contractile cytoskeleton underlying apical constriction. We conclude that a single mechanical mechanism involving volume conservation and apical constriction-induced basal movement of cytoplasm accounts quantitatively for the cell shape changes and the nucleus movement in Drosophila ventral furrow formation. Our study provides a comprehensive quantitative analysis of the fast dynamics of whole-cell shape changes during tissue folding and points to a simplified model for Drosophila gastrulation.

Morphogenesis: Setting the pace of embryo folding.

Tissue folding is a key process for shape generation during embryonic development. A new study reports how a fold in the Drosophila embryo forms by a propagating trigger wave.

Drosophila Fog/Cta and T48 pathways have overlapping and distinct contributions to mesoderm invagination.

The regulation of the cytoskeleton by multiple signaling pathways, sometimes in parallel, is a common principle of morphogenesis. A classic example of regulation by parallel pathways is Drosophila gastrulation, where the inputs from the Folded gastrulation (Fog)/Concertina (Cta) and the T48 pathways induce apical constriction and mesoderm invagination. Whether there are distinct roles for these separate pathways in regulating the complex spatial and temporal patterns of cytoskeletal activity that accompany early embryo development is still poorly understood. We investigated the roles of the Fog/Cta and T48 pathways and found that, by themselves, the Cta and T48 pathways both promote timely mesoderm invagination and apical myosin II accumulation, with Cta being required for timely cell shape change ahead of mitotic cell division. We also identified distinct functions of T48 and Cta in regulating cellularization and the uniformity of the apical myosin II network, respectively. Our results demonstrate that both redundant and distinct functions for the Fog/Cta and T48 pathways exist.

Change in RhoGAP and RhoGEF availability drives transitions in cortical patterning and excitability in Drosophila.

Actin cortex patterning and dynamics are critical for cell shape changes. These dynamics undergo transitions during development, often accompanying changes in collective cell behavior. Although mechanisms have been established for individual cells' dynamic behaviors, the mechanisms and specific molecules that result in developmental transitions in vivo are still poorly understood. Here, we took advantage of two developmental systems in Drosophila melanogaster to identify conditions that altered cortical patterning and dynamics. We identified a Rho guanine nucleotide exchange factor (RhoGEF) and Rho GTPase activating protein (RhoGAP) pair required for actomyosin waves in egg chambers. Specifically, depletion of the RhoGEF, Ect2, or the RhoGAP, RhoGAP15B, disrupted actomyosin wave induction, and both proteins relocalized from the nucleus to the cortex preceding wave formation. Furthermore, we found that overexpression of a different RhoGEF and RhoGAP pair, RhoGEF2 and Cumberland GAP (C-GAP), resulted in actomyosin waves in the early embryo, during which RhoA activation precedes actomyosin assembly by ∼4 s. We found that C-GAP was recruited to actomyosin waves, and disrupting F-actin polymerization altered the spatial organization of both RhoA signaling and the cytoskeleton in waves. In addition, disrupting F-actin dynamics increased wave period and width, consistent with a possible role for F-actin in promoting delayed negative feedback. Overall, we showed a mechanism involved in inducing actomyosin waves that is essential for oocyte development and is general to other cell types, such as epithelial and syncytial cells.

Arp2/3 ATP hydrolysis-catalysed branch dissociation is critical for endocytic force generation.

The Arp2/3 complex, which is crucial for actin-based motility, nucleates actin filaments and organizes them into y-branched networks. The Arp2 subunit has been shown to hydrolyse ATP, but the functional importance of Arp2/3 ATP hydrolysis is not known. Here, we analysed an Arp2 mutant in Saccharomyces cerevisiae that is defective in ATP hydrolysis. Arp2 ATP hydrolysis and Arp2/3-dependent actin nucleation occur almost simultaneously. However, ATP hydrolysis is not required for nucleation. In addition, Arp2 ATP hydrolysis is not required for the release of a WASP-like activator from y-branches. ATP hydrolysis by Arp2, and possibly Arp3, is essential for efficient y-branch dissociation in vitro. In living cells, both Arp2 and Arp3 ATP-hydrolysis mutants exhibit defects in endocytic internalization and actin-network disassembly. Our results suggest a critical feature of dendritic nucleation in which debranching and subsequent actin-filament remodelling and/or depolymerization are important for endocytic vesicle morphogenesis.

EGFR-dependent actomyosin patterning coordinates morphogenetic movements between tissues.

The movements that give rise to the body's structure are powered by cell shape changes and rearrangements that are coordinated at supracellular scales. How such cellular coordination arises and integrates different morphogenetic programs is unclear. Using quantitative imaging, we found a complex pattern of adherens junction (AJ) levels in the ectoderm prior to gastrulation onset in Drosophila. AJ intensity exhibited a double-sided gradient, with peaks at the dorsal midline and ventral neuroectoderm. We show that this dorsal-ventral AJ pattern is regulated by epidermal growth factor (EGF) signaling and that this signal is required for ectoderm cell movement during mesoderm invagination and axis extension. We identify AJ levels and junctional actomyosin as downstream effectors of EGFR signaling. Overall, our study demonstrates a mechanism of coordination between tissue folding and convergent extension that facilitates embryo-wide gastrulation movements.

Live Imaging of Nurse Cell Behavior in Late Stages of Drosophila Oogenesis.

Drosophila oogenesis is a powerful and tractable model for studies of cell and developmental biology due to the multitude of well-characterized events in both germline and somatic cells, the ease of genetic manipulation in fruit flies, and the large number of egg chambers produced by each fly. Recent improvements in live imaging and ex vivo culturing protocols have enabled researchers to conduct more detailed, longer-term studies of egg chamber development, enabling insights into fundamental biological processes. Here, we present a protocol for dissection, culturing, and imaging of late-stage egg chambers to study intercellular and directional cytoplasmic flow during "nurse cell dumping." This critical developmental process towards the latter stages of oogenesis (stages 10b/11) results in rapid growth of the oocyte and shrinkage of the nurse cells and is accompanied by dynamic changes in cell shape. We also describe a procedure to record high-time-resolution movies of the flow of unlabeled cytoplasmic contents within nurse cells and through cytoplasmic bridges in the nurse cell cluster using reflection microscopy, and we describe two ways to analyze data from nurse cell dumping.

Topological packing statistics of living and nonliving matter.

Complex disordered matter is of central importance to a wide range of disciplines, from bacterial colonies and embryonic tissues in biology to foams and granular media in materials science to stellar configurations in astrophysics. Because of the vast differences in composition and scale, comparing structural features across such disparate systems remains challenging. Here, by using the statistical properties of Delaunay tessellations, we introduce a mathematical framework for measuring topological distances between general three-dimensional point clouds. The resulting system-agnostic metric reveals subtle structural differences between bacterial biofilms as well as between zebrafish brain regions, and it recovers temporal ordering of embryonic development. We apply the metric to construct a universal topological atlas encompassing bacterial biofilms, snowflake yeast, plant shoots, zebrafish brain matter, organoids, and embryonic tissues as well as foams, colloidal packings, glassy materials, and stellar configurations. Living systems localize within a bounded island-like region of the atlas, reflecting that biological growth mechanisms result in characteristic topological properties.

Bitesize bundles F-actin and influences actin remodeling in syncytial Drosophila embryo development.

Actin networks undergo rearrangements that influence cell and tissue shape. Actin network assembly and organization is regulated in space and time by a host of actin binding proteins. The Drosophila Synaptotagmin-like protein, Bitesize (Btsz), is known to organize actin at epithelial cell apical junctions in a manner that depends on its interaction with the actin-binding protein, Moesin. Here, we showed that Btsz functions in actin reorganization at earlier, syncytial stages of Drosophila embryo development. Btsz was required for the formation of stable metaphase pseudocleavage furrows that prevented spindle collisions and nuclear fallout prior to cellularization. While previous studies focused on Btsz isoforms containing the Moesin Binding Domain (MBD), we found that isoforms lacking the MBD also function in actin remodeling. Consistent with this, we found that the C-terminal half of BtszB cooperatively binds to and bundles F-actin, suggesting a direct mechanism for Synaptotagmin-like proteins regulating actin organization during animal development.

Change in RhoGAP and RhoGEF availability drives transitions in cortical patterning and excitability in Drosophila.

Actin cortex patterning and dynamics are critical for cell shape changes. These dynamics undergo transitions during development, often accompanying changes in collective cell behavior. While mechanisms have been established for individual cells' dynamic behaviors, mechanisms and specific molecules that result in developmental transitions in vivo are still poorly understood. Here, we took advantage of two developmental systems in Drosophila melanogaster to identify conditions that altered cortical patterning and dynamics. We identified a RhoGEF and RhoGAP pair whose relocalization from nucleus to cortex results in actomyosin waves in egg chambers. Furthermore, we found that overexpression of a different RhoGEF and RhoGAP pair resulted in actomyosin waves in the early embryo, during which RhoA activation precedes actomyosin assembly and RhoGAP recruitment by ~4 seconds. Overall, we showed a mechanism involved in inducing actomyosin waves that is essential for oocyte development and is general to other cell types.

The dynamics and biophysics of shape formation: Common themes in plant and animal morphogenesis.

The emergence of tissue form in multicellular organisms results from the complex interplay between genetics and physics. In both plants and animals, cells must act in concert to pattern their behaviors. Our understanding of the factors sculpting multicellular form has increased dramatically in the past few decades. From this work, common themes have emerged that connect plant and animal morphogenesis-an exciting connection that solidifies our understanding of the developmental basis of multicellular life. In this review, we will discuss the themes and the underlying principles that connect plant and animal morphogenesis, including the coordination of gene expression, signaling, growth, contraction, and mechanical and geometric feedback.

Scaling behaviour and control of nuclear wrinkling.

The cell nucleus is enveloped by a complex membrane, whose wrinkling has been implicated in disease and cellular aging. The biophysical dynamics and spectral evolution of nuclear wrinkling during multicellular development remain poorly understood due to a lack of direct quantitative measurements. Here, we characterize the onset and dynamics of nuclear wrinkling during egg development in the fruit fly when nurse cell nuclei increase in size and display stereotypical wrinkling behavior. A spectral analysis of three-dimensional high-resolution live imaging data from several hundred nuclei reveals a robust asymptotic power-law scaling of angular fluctuations consistent with renormalization and scaling predictions from a nonlinear elastic shell model. We further demonstrate that nuclear wrinkling can be reversed through osmotic shock and suppressed by microtubule disruption, providing tuneable physical and biological control parameters for probing mechanical properties of the nuclear envelope. Our findings advance the biophysical understanding of nuclear membrane fluctuations during early multicellular development.