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.

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.

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.

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.

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.

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.

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.

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.

Morphogenetic forces planar polarize LGN/Pins in the embryonic head during Drosophila gastrulation.

Spindle orientation is often achieved by a complex of Partner of Inscuteable (Pins)/LGN, Mushroom Body Defect (Mud)/Nuclear Mitotic Apparatus (NuMa), Gαi, and Dynein, which interacts with astral microtubules to rotate the spindle. Cortical Pins/LGN recruitment serves as a critical step in this process. Here, we identify Pins-mediated planar cell polarized divisions in several of the mitotic domains of the early Drosophila embryo. We found that neither planar cell polarity pathways nor planar polarized myosin localization determined division orientation; instead, our findings strongly suggest that Pins planar polarity and force generated from mesoderm invagination are important. Disrupting Pins polarity via overexpression of a myristoylated version of Pins caused randomized division angles. We found that disrupting forces through chemical inhibitors, depletion of an adherens junction protein, or blocking mesoderm invagination disrupted Pins planar polarity and spindle orientation. Furthermore, directional ablations that separated mesoderm from mitotic domains disrupted spindle orientation, suggesting that forces transmitted from mesoderm to mitotic domains can polarize Pins and orient division during gastrulation. To our knowledge, this is the first in vivo example where mechanical force has been shown to polarize Pins to mediate division orientation.

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.

The nature of cell division forces in epithelial monolayers.

Epithelial cells undergo striking morphological changes during division to ensure proper segregation of genetic and cytoplasmic materials. These morphological changes occur despite dividing cells being mechanically restricted by neighboring cells, indicating the need for extracellular force generation. Beyond driving cell division itself, forces associated with division have been implicated in tissue-scale processes, including development, tissue growth, migration, and epidermal stratification. While forces generated by mitotic rounding are well understood, forces generated after rounding remain unknown. Here, we identify two distinct stages of division force generation that follow rounding: (1) Protrusive forces along the division axis that drive division elongation, and (2) outward forces that facilitate postdivision spreading. Cytokinetic ring contraction of the dividing cell, but not activity of neighboring cells, generates extracellular forces that propel division elongation and contribute to chromosome segregation. Forces from division elongation are observed in epithelia across many model organisms. Thus, division elongation forces represent a universal mechanism that powers cell division in confining epithelia.

Actin-based force generation and cell adhesion in tissue morphogenesis.

The generation of organismal form - morphogenesis - arises from forces produced at the cellular level. In animal cells, much of this force is produced by the actin cytoskeleton. Here, we review how mechanisms of actin-based force generation are deployed during animal morphogenesis to sculpt organs and organisms. Furthermore, we consider how cytoskeletal forces are coupled through cell adhesions to propagate across tissues, and discuss cases where cytoskeletal force or adhesion is patterned across a tissue to direct shape changes. Together, our review provides a conceptual framework that reflects our current understanding of animal morphogenesis and gives perspectives on future opportunities for study.

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.

Self-organized cytoskeletal alignment during Drosophila mesoderm invagination.

During tissue morphogenesis, mechanical forces are propagated across tissues, resulting in tissue shape changes. These forces in turn can influence cell behaviour, leading to a feedback process that can be described as self-organizing. Here, I discuss cytoskeletal self-organization and point to evidence that suggests its role in directing force during morphogenesis. During Drosophila mesoderm invagination, the shape of the region of cells that initiates constriction creates a mechanical pattern that in turn aligns the cytoskeleton with the axis of greatest resistance to contraction. The wild-type direction of the force controls the shape and orientation of the invaginating mesoderm. Given the ability of the actomyosin cytoskeleton to self-organize, these types of feedback mechanisms are likely to play important roles in a range of different morphogenetic events. This article is part of the discussion meeting issue 'Contemporary morphogenesis'.

Divergent and combinatorial mechanical strategies that promote epithelial folding during morphogenesis.

Folding is an important way by which epithelia are sculpted into three-dimensional shapes during development. Recent studies have integrated quantitative image analysis and physical modeling to uncover contributing mechanisms. It is becoming clear that folding processes often employ multiple mechanisms of force generation. Here, we review divergent modes of epithelial folding. We argue that, in many cases, several mechanisms interact at different time-scales and length-scales to generate a fold. In other cases, very similar folds are generated by different folding mechanisms. How one (or a specific combination of) mechanism(s) might be advantageous in one case versus another is not understood, and future work using quantitative analyses and modeling will be required to determine reasons for distinct modes of folding.

Apical Constriction Reversal upon Mitotic Entry Underlies Different Morphogenetic Outcomes of Cell Division.

During development, coordinated cell shape changes and cell divisions sculpt tissues. While these individual cell behaviors have been extensively studied, how cell shape changes and cell divisions that occur concurrently in epithelia influence tissue shape is less understood. We addressed this question in two contexts of the early Drosophila embryo: premature cell division during mesoderm invagination, and native ectodermal cell divisions with ectopic activation of apical contractility. Using quantitative live-cell imaging, we demonstrated that mitotic entry reverses apical contractility by interfering with medioapical RhoA signaling. While premature mitotic entry inhibits mesoderm invagination, which relies on apical constriction, mitotic entry in an artificially contractile ectoderm induced ectopic tissue invaginations. Ectopic invaginations resulted from medioapical myosin loss in neighboring mitotic cells. This myosin loss enabled nonmitotic cells to apically constrict through mitotic cell stretching. Thus, the spatial pattern of mitotic entry can differentially regulate tissue shape through signal interference between apical contractility and mitosis.

The Physical Mechanisms of Drosophila Gastrulation: Mesoderm and Endoderm Invagination.

A critical juncture in early development is the partitioning of cells that will adopt different fates into three germ layers: the ectoderm, the mesoderm, and the endoderm. This step is achieved through the internalization of specified cells from the outermost surface layer, through a process called gastrulation. In Drosophila, gastrulation is achieved through cell shape changes (i.e., apical constriction) that change tissue curvature and lead to the folding of a surface epithelium. Folding of embryonic tissue results in mesoderm and endoderm invagination, not as individual cells, but as collective tissue units. The tractability of Drosophila as a model system is best exemplified by how much we know about Drosophila gastrulation, from the signals that pattern the embryo to the molecular components that generate force, and how these components are organized to promote cell and tissue shape changes. For mesoderm invagination, graded signaling by the morphogen, Spätzle, sets up a gradient in transcriptional activity that leads to the expression of a secreted ligand (Folded gastrulation) and a transmembrane protein (T48). Together with the GPCR Mist, which is expressed in the mesoderm, and the GPCR Smog, which is expressed uniformly, these signals activate heterotrimeric G-protein and small Rho-family G-protein signaling to promote apical contractility and changes in cell and tissue shape. A notable feature of this signaling pathway is its intricate organization in both space and time. At the cellular level, signaling components and the cytoskeleton exhibit striking polarity, not only along the apical-basal cell axis, but also within the apical domain. Furthermore, gene expression controls a highly choreographed chain of events, the dynamics of which are critical for primordium invagination; it does not simply throw the cytoskeletal "on" switch. Finally, studies of Drosophila gastrulation have provided insight into how global tissue mechanics and movements are intertwined as multiple tissues simultaneously change shape. Overall, these studies have contributed to the view that cells respond to forces that propagate over great distances, demonstrating that cellular decisions, and, ultimately, tissue shape changes, proceed by integrating cues across an entire embryo.

The cellular and molecular mechanisms that establish the mechanics of Drosophila gastrulation.

In this review, we cover advances in the field that have contributed to our mechanistic understanding of how tissues internalize during Drosophila melanogaster gastrulation. The changes in tissue shape and architecture that are associated with mesoderm and endoderm invagination in the early Drosophila embryo are accompanied by cell shape changes which are driven by actomyosin contractility. The activation of signal transduction pathways is patterned by embryonic transcription factors, which define distinct geometries of gene expression and the tissue contractile domains. At the subcellular level, outputs from signaling pathways that activate actomyosin contractility are highly polarized and their behavior is fine-tuned by a balance of both positive and negative regulation. Cells are mechanically linked through adherens junctions, allowing forces that are generated by cells to be integrated across the tissue, ensuring coordinated cell behavior during tissue invagination. The transmission of force between cells also enables mechanical feedback whereby force generation influences both cell and cytoskeletal behavior. Finally, after tissue invagination, mesoderm cells undergo an epithelial-to-mesenchymal transition and cell spreading. We highlight studies that have utilized this model system to uncover fundamental principles at molecular-, cell-, and tissue-levels, which have contributed to our understanding of similar tissue morphogenetic processes across different organisms.

Structural Redundancy in Supracellular Actomyosin Networks Enables Robust Tissue Folding.

Tissue morphogenesis is strikingly robust. Yet, how tissues are sculpted under challenging conditions is unknown. Here, we combined network analysis, experimental perturbations, and computational modeling to determine how network connectivity between hundreds of contractile cells on the ventral side of the Drosophila embryo ensures robust tissue folding. We identified two network properties that mechanically promote robustness. First, redundant supracellular cytoskeletal network paths ensure global connectivity, even with network degradation. By forming many more connections than are required, morphogenesis is not disrupted by local network damage, analogous to the way redundancy guarantees the large-scale function of vasculature and transportation networks. Second, directional stiffening of edges oriented orthogonal to the folding axis promotes furrow formation at lower contractility levels. Structural redundancy and directional network stiffening ensure robust tissue folding with proper orientation.