Cytoplasmic division cycles without the nucleus and mitotic CDK/cyclin complexes.

Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. We demonstrate in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, we present evidence that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. We postulate that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes.

Rigidity percolation uncovers a structural basis for embryonic tissue phase transitions.

Embryo morphogenesis is impacted by dynamic changes in tissue material properties, which have been proposed to occur via processes akin to phase transitions (PTs). Here, we show that rigidity percolation provides a simple and robust theoretical framework to predict material/structural PTs of embryonic tissues from local cell connectivity. By using percolation theory, combined with directly monitoring dynamic changes in tissue rheology and cell contact mechanics, we demonstrate that the zebrafish blastoderm undergoes a genuine rigidity PT, brought about by a small reduction in adhesion-dependent cell connectivity below a critical value. We quantitatively predict and experimentally verify hallmarks of PTs, including power-law exponents and associated discontinuities of macroscopic observables. Finally, we show that this uniform PT depends on blastoderm cells undergoing meta-synchronous divisions causing random and, consequently, uniform changes in cell connectivity. Collectively, our theoretical and experimental findings reveal the structural basis of material PTs in an organismal context.

Mechanics of Anteroposterior Axis Formation in Vertebrates.

The vertebrate anteroposterior axis forms through elongation of multiple tissues during embryogenesis. This process is based on tissue-autonomous mechanisms of force generation and intertissue mechanical coupling whose failure leads to severe developmental anomalies such as body truncation and spina bifida. Similar to other morphogenetic modules, anteroposterior body extension requires both the rearrangement of existing materials-such as cells and extracellular matrix-and the local addition of new materials, i.e., anisotropic growth, through cell proliferation, cell growth, and matrix deposition. Numerous signaling pathways coordinate body axis formation via regulation of cell behavior during tissue rearrangements and/or volumetric growth. From a physical perspective, morphogenesis depends on both cell-generated forces and tissue material properties. As the spatiotemporal variation of these mechanical parameters has recently been explored in the context of vertebrate body elongation, the study of this process is likely to shed light on the cross talk between signaling and mechanics during morphogenesis.

SnapShot: Mechanical Forces in Development II.

The forces shaping an organism are not exclusively produced by actin/myosin II networks. In part II of this SnapShot, we present various alternative mechanisms. In addition to driving morphogenesis, cells use mechanical forces to sense and react to the specific mechanical properties of their environment. Also, we present a selection of experimental tools commonly used in force analysis.

A Small RNA-Catalytic Argonaute Pathway Tunes Germline Transcript Levels to Ensure Embryonic Divisions.

Multiple division cycles without growth are a characteristic feature of early embryogenesis. The female germline loads proteins and RNAs into oocytes to support these divisions, which lack many quality control mechanisms operating in somatic cells undergoing growth. Here, we describe a small RNA-Argonaute pathway that ensures early embryonic divisions in C. elegans by employing catalytic slicing activity to broadly tune, instead of silence, germline gene expression. Misregulation of one target, a kinesin-13 microtubule depolymerase, underlies a major phenotype associated with pathway loss. Tuning of target transcript levels is guided by the density of homologous small RNAs, whose generation must ultimately be related to target sequence. Thus, the tuning action of a small RNA-catalytic Argonaute pathway generates oocytes capable of supporting embryogenesis. We speculate that the specialized nature of germline chromatin led to the emergence of small RNA-catalytic Argonaute pathways in the female germline as a post-transcriptional control layer to optimize oocyte composition.

Cell migration, freshly squeezed.

Migrating cells exhibit distinct motility modes and can switch between modes based on chemical or physical cues. Liu et al. and Ruprecht et al. now describe how confinement and contractility influence motility mode plasticity and instigate a mode termed stable bleb migration in embryonic and tumor cells.

Cortical contractility triggers a stochastic switch to fast amoeboid cell motility.

3D amoeboid cell migration is central to many developmental and disease-related processes such as cancer metastasis. Here, we identify a unique prototypic amoeboid cell migration mode in early zebrafish embryos, termed stable-bleb migration. Stable-bleb cells display an invariant polarized balloon-like shape with exceptional migration speed and persistence. Progenitor cells can be reversibly transformed into stable-bleb cells irrespective of their primary fate and motile characteristics by increasing myosin II activity through biochemical or mechanical stimuli. Using a combination of theory and experiments, we show that, in stable-bleb cells, cortical contractility fluctuations trigger a stochastic switch into amoeboid motility, and a positive feedback between cortical flows and gradients in contractility maintains stable-bleb cell polarization. We further show that rearward cortical flows drive stable-bleb cell migration in various adhesive and non-adhesive environments, unraveling a highly versatile amoeboid migration phenotype.

Zygotic genome activation triggers the DNA replication checkpoint at the midblastula transition.

A conserved feature of the midblastula transition (MBT) is a requirement for a functional DNA replication checkpoint to coordinate cell-cycle remodeling and zygotic genome activation (ZGA). We have investigated what triggers this checkpoint during Drosophila embryogenesis. We find that the magnitude of the checkpoint scales with the quantity of transcriptionally engaged DNA. Measuring RNA polymerase II (Pol II) binding at 20 min intervals over the course of ZGA reveals that the checkpoint coincides with widespread de novo recruitment of Pol II that precedes and does not require a functional checkpoint. This recruitment drives slowing or stalling of DNA replication at transcriptionally engaged loci. Reducing Pol II recruitment in zelda mutants both reduces replication stalling and bypasses the requirement for a functional checkpoint. This suggests a model where the checkpoint functions as a feedback mechanism to remodel the cell cycle in response to nascent ZGA.

Perinuclear Anchoring of H3K9-Methylated Chromatin Stabilizes Induced Cell Fate in C. elegans Embryos.

Interphase chromatin is organized in distinct nuclear sub-compartments, reflecting its degree of compaction and transcriptional status. In Caenorhabditis elegans embryos, H3K9 methylation is necessary to silence and to anchor repeat-rich heterochromatin at the nuclear periphery. In a screen for perinuclear anchors of heterochromatin, we identified a previously uncharacterized C. elegans chromodomain protein, CEC-4. CEC-4 binds preferentially mono-, di-, or tri-methylated H3K9 and localizes at the nuclear envelope independently of H3K9 methylation and nuclear lamin. CEC-4 is necessary for endogenous heterochromatin anchoring, but not for transcriptional repression, in contrast to other known H3K9 methyl-binders in worms, which mediate gene repression but not perinuclear anchoring. When we ectopically induce a muscle differentiation program in embryos, cec-4 mutants fail to commit fully to muscle cell fate. This suggests that perinuclear sequestration of chromatin during development helps restrict cell differentiation programs by stabilizing commitment to a specific cell fate. PAPERCLIP.

Self-organized tissue mechanics underlie embryonic regulation.

Early amniote development is highly self-organized, capable of adapting to interference through local and long-range cell-cell interactions. This process, called embryonic regulation1, has been well illustrated in experiments on avian embryos, in which subdividing the epiblast disk into different parts not only redirects cell fates to eventually form a complete and well-proportioned embryo at its original location, but also leads to the self-organization of additional, fully formed embryos2,3 in the other separated parts. The cellular interactions underlying embryonic self-organization are widely believed to be mediated by molecular signals, yet the identity of such signals is unclear. Here, by analysing intact and mechanically perturbed quail embryos, we show that the mechanical forces that drive embryogenesis self-organize, with contractility locally self-activating and the ensuing tension acting as a long-range inhibitor. This mechanical feedback governs the persistent pattern of tissue flows that shape the embryo4-6 and also steers the concomitant emergence of embryonic territories by modulating gene expression, ensuring the formation of a single embryo under normal conditions, yet allowing the emergence of multiple, well-proportioned embryos after perturbations. Thus, mechanical forces act at the core of embryonic self-organization, shaping both tissues and gene expression to robustly yet plastically canalize early development.

Interplay of cell shape and division orientation promotes robust morphogenesis of developing epithelia.

Epithelial cells acquire functionally important shapes (e.g., squamous, cuboidal, columnar) during development. Here, we combine theory, quantitative imaging, and perturbations to analyze how tissue geometry, cell divisions, and mechanics interact to shape the presumptive enveloping layer (pre-EVL) on the zebrafish embryonic surface. We find that, under geometrical constraints, pre-EVL flattening is regulated by surface cell number changes following differentially oriented cell divisions. The division pattern is, in turn, determined by the cell shape distribution, which forms under geometrical constraints by cell-cell mechanical coupling. An integrated mathematical model of this shape-division feedback loop recapitulates empirical observations. Surprisingly, the model predicts that cell shape is robust to changes of tissue surface area, cell volume, and cell number, which we confirm in vivo. Further simulations and perturbations suggest the parameter linking cell shape and division orientation contributes to epithelial diversity. Together, our work identifies an evolvable design logic that enables robust cell-level regulation of tissue-level development.

De novo inference of systems-level mechanistic models of development from live-imaging-based phenotype analysis.

Elucidation of complex phenotypes for mechanistic insights presents a significant challenge in systems biology. We report a strategy to automatically infer mechanistic models of cell fate differentiation based on live-imaging data. We use cell lineage tracing and combinations of tissue-specific marker expression to assay progenitor cell fate and detect fate changes upon genetic perturbation. Based on the cellular phenotypes, we further construct a model for how fate differentiation progresses in progenitor cells and predict cell-specific gene modules and cell-to-cell signaling events that regulate the series of fate choices. We validate our approach in C. elegans embryogenesis by perturbing 20 genes in over 300 embryos. The result not only recapitulates current knowledge but also provides insights into gene function and regulated fate choice, including an unexpected self-renewal. Our study provides a powerful approach for automated and quantitative interpretation of complex in vivo information.

Feedback control of gene expression variability in the Caenorhabditis elegans Wnt pathway.

Variability in gene expression contributes to phenotypic heterogeneity even in isogenic populations. Here, we used the stereotyped, Wnt signaling-dependent development of the Caenorhabditis elegans Q neuroblast to probe endogenous mechanisms that control gene expression variability. We found that the key Hox gene that orients Q neuroblast migration exhibits increased gene expression variability in mutants in which Wnt pathway activity has been perturbed. Distinct features of the gene expression distributions prompted us on a systematic search for regulatory interactions, revealing a network of interlocked positive and negative feedback loops. Interestingly, positive feedback appeared to cooperate with negative feedback to reduce variability while keeping the Hox gene expression at elevated levels. A minimal model correctly predicts the increased gene expression variability across mutants. Our results highlight the influence of gene network architecture on expression variability and implicate feedback regulation as an effective mechanism to ensure developmental robustness.

Specified neural progenitors sort to form sharp domains after noisy Shh signaling.

Sharply delineated domains of cell types arise in developing tissues under instruction of inductive signal (morphogen) gradients, which specify distinct cell fates at different signal levels. The translation of a morphogen gradient into discrete spatial domains relies on precise signal responses at stable cell positions. However, cells in developing tissues undergoing morphogenesis and proliferation often experience complex movements, which may affect their morphogen exposure, specification, and positioning. How is a clear pattern achieved with cells moving around? Using in toto imaging of the zebrafish neural tube, we analyzed specification patterns and movement trajectories of neural progenitors. We found that specified progenitors of different fates are spatially mixed following heterogeneous Sonic Hedgehog signaling responses. Cell sorting then rearranges them into sharply bordered domains. Ectopically induced motor neuron progenitors also robustly sort to correct locations. Our results reveal that cell sorting acts to correct imprecision of spatial patterning by noisy inductive signals.

Differential proliferation regulates multi-tissue morphogenesis during embryonic axial extension: integrating viscous modeling and experimental approaches.

A major challenge in biology is to understand how mechanical interactions and cellular behavior affect the shapes of tissues and embryo morphology. The extension of the neural tube and paraxial mesoderm, which form the spinal cord and musculoskeletal system, respectively, results in the elongated shape of the vertebrate embryonic body. Despite our understanding of how each of these tissues elongates independently of the others, the morphogenetic consequences of their simultaneous growth and mechanical interactions are still unclear. Our study investigates how differential growth, tissue biophysical properties and mechanical interactions affect embryonic morphogenesis during axial extension using a 2D multi-tissue continuum-based mathematical model. Our model captures the dynamics observed in vivo by time-lapse imaging of bird embryos, and reveals the underestimated influence of differential tissue proliferation rates. We confirmed this prediction in quail embryos by showing that decreasing the rate of cell proliferation in the paraxial mesoderm affects long-term tissue dynamics, and shaping of both the paraxial mesoderm and the neighboring neural tube. Overall, our work provides a new theoretical platform upon which to consider the long-term consequences of tissue differential growth and mechanical interactions on morphogenesis.

Local and global changes in cell density induce reorganisation of 3D packing in a proliferating epithelium.

Tissue morphogenesis is intimately linked to the changes in shape and organisation of individual cells. In curved epithelia, cells can intercalate along their own apicobasal axes, adopting a shape named 'scutoid' that allows energy minimization in the tissue. Although several geometric and biophysical factors have been associated with this 3D reorganisation, the dynamic changes underlying scutoid formation in 3D epithelial packing remain poorly understood. Here, we use live imaging of the sea star embryo coupled with deep learning-based segmentation to dissect the relative contributions of cell density, tissue compaction and cell proliferation on epithelial architecture. We find that tissue compaction, which naturally occurs in the embryo, is necessary for the appearance of scutoids. Physical compression experiments identify cell density as the factor promoting scutoid formation at a global level. Finally, the comparison of the developing embryo with computational models indicates that the increase in the proportion of scutoids is directly associated with cell divisions. Our results suggest that apico-basal intercalations appearing immediately after mitosis may help accommodate the new cells within the tissue. We propose that proliferation in a compact epithelium induces 3D cell rearrangements during development.

Confinement promotes nematic alignment of spindle-shaped cells during Drosophila embryogenesis.

Tissue morphogenesis is often controlled by actomyosin networks pulling on adherens junctions (AJs), but junctional myosin levels vary. At an extreme, the Drosophila embryo amnioserosa forms a horseshoe-shaped strip of aligned, spindle-shaped cells lacking junctional myosin. What are the bases of amnioserosal cell interactions and alignment? Compared with surrounding tissue, we find that amnioserosal AJ continuity has lesser dependence on α-catenin, the mediator of AJ-actomyosin association, and greater dependence on Bazooka/Par-3, a junction-associated scaffold protein. Microtubule bundles also run along amnioserosal AJs and support their long-range curvilinearity. Amnioserosal confinement is apparent from partial overlap of its spindle-shaped cells, its outward bulging from surrounding tissue and from compressive stress detected within the amnioserosa. Genetic manipulations that alter amnioserosal confinement by surrounding tissue also result in amnioserosal cells losing alignment and gaining topological defects characteristic of nematically ordered systems. With Bazooka depletion, confinement by surrounding tissue appears to be relatively normal and amnioserosal cells align despite their AJ fragmentation. Overall, the fully elongated amnioserosa appears to form through tissue-autonomous generation of spindle-shaped cells that nematically align in response to confinement by surrounding tissue.

Mechanical stress combines with planar polarised patterning during metaphase to orient embryonic epithelial cell divisions.

The planar orientation of cell division (OCD) is important for epithelial morphogenesis and homeostasis. Here, we ask how mechanics and antero-posterior (AP) patterning combine to influence the first divisions after gastrulation in the Drosophila embryonic epithelium. We analyse hundreds of cell divisions and show that stress anisotropy, notably from compressive forces, can reorient division directly in metaphase. Stress anisotropy influences the OCD by imposing metaphase cell elongation, despite mitotic rounding, and overrides interphase cell elongation. In strongly elongated cells, the mitotic spindle adapts its length to, and hence its orientation is constrained by, the cell long axis. Alongside mechanical cues, we find a tissue-wide bias of the mitotic spindle orientation towards AP-patterned planar polarised Myosin-II. This spindle bias is lost in an AP-patterning mutant. Thus, a patterning-induced mitotic spindle orientation bias overrides mechanical cues in mildly elongated cells, whereas in strongly elongated cells the spindle is constrained close to the high stress axis.

Two sequential gene expression programs bridged by cell division support long-distance collective cell migration.

The precise assembly of tissues and organs relies on spatiotemporal regulation of gene expression to coordinate the collective behavior of cells. In Drosophila embryos, the midgut musculature is formed through collective migration of caudal visceral mesoderm (CVM) cells, but how gene expression changes as cells migrate is not well understood. Here, we have focused on ten genes expressed in the CVM and the cis-regulatory sequences controlling their expression. Although some genes are continuously expressed, others are expressed only early or late during migration. Late expression relates to cell cycle progression, as driving string/Cdc25 causes earlier division of CVM cells and accelerates the transition to late gene expression. In particular, we found that the cell cycle effector transcription factor E2F1 is a required input for the late gene CG5080. Furthermore, whereas late genes are broadly expressed in all CVM cells, early gene transcripts are polarized to the anterior or posterior ends of the migrating collective. We show this polarization requires transcription factors Snail, Zfh1 and Dorsocross. Collectively, these results identify two sequential gene expression programs bridged by cell division that support long-distance directional migration of CVM cells.

Drosophila Myoblast Fusion: Invasion and Resistance for the Ultimate Union.

Cell-cell fusion is indispensable for creating life and building syncytial tissues and organs. Ever since the discovery of cell-cell fusion, how cells join together to form zygotes and multinucleated syncytia has remained a fundamental question in cell and developmental biology. In the past two decades, Drosophila myoblast fusion has been used as a powerful genetic model to unravel mechanisms underlying cell-cell fusion in vivo. Many evolutionarily conserved fusion-promoting factors have been identified and so has a surprising and conserved cellular mechanism. In this review, we revisit key findings in Drosophila myoblast fusion and highlight the critical roles of cellular invasion and resistance in driving cell membrane fusion.