Notochord segmentation in zebrafish controlled by iterative mechanical signaling.

In bony fishes, patterning of the vertebral column, or spine, is guided by a metameric blueprint established in the notochord sheath. Notochord segmentation begins days after somitogenesis concludes and can occur in its absence. However, somite patterning defects lead to imprecise notochord segmentation, suggesting that these processes are linked. Here, we identify that interactions between the notochord and the axial musculature ensure precise spatiotemporal segmentation of the zebrafish spine. We demonstrate that myoseptum-notochord linkages drive notochord segment initiation by locally deforming the notochord extracellular matrix and recruiting focal adhesion machinery at these contact points. Irregular somite patterning alters this mechanical signaling, causing non-sequential and dysmorphic notochord segmentation, leading to altered spine development. Using a model that captures myoseptum-notochord interactions, we find that a fixed spatial interval is critical for driving sequential segment initiation. Thus, mechanical coupling of axial tissues facilitates spatiotemporal spine patterning.

Axial segmentation by iterative mechanical signaling.

In bony fishes, formation of the vertebral column, or spine, is guided by a metameric blueprint established in the epithelial sheath of the notochord. Generation of the notochord template begins days after somitogenesis and even occurs in the absence of somite segmentation. However, patterning defects in the somites lead to imprecise notochord segmentation, suggesting these processes are linked. Here, we reveal that spatial coordination between the notochord and the axial musculature is necessary to ensure segmentation of the zebrafish spine both in time and space. We find that the connective tissues that anchor the axial skeletal musculature, known as the myosepta in zebrafish, transmit spatial patterning cues necessary to initiate notochord segment formation, a critical pre-patterning step in spine morphogenesis. When an irregular pattern of muscle segments and myosepta interact with the notochord sheath, segments form non-sequentially, initiate at atypical locations, and eventually display altered morphology later in development. We determine that locations of myoseptum-notochord connections are hubs for mechanical signal transmission, which are characterized by localized sites of deformation of the extracellular matrix (ECM) layer encasing the notochord. The notochord sheath responds to the external mechanical changes by locally augmenting focal adhesion machinery to define the initiation site for segmentation. Using a coarse-grained mathematical model that captures the spatial patterns of myoseptum-notochord interactions, we find that a fixed-length scale of external cues is critical for driving sequential segment patterning in the notochord. Together, this work identifies a robust segmentation mechanism that hinges upon mechanical coupling of adjacent tissues to control patterning dynamics.

Minimal vertex model explains how the amnioserosa avoids fluidization during Drosophila dorsal closure.

Dorsal closure is a process that occurs during embryogenesis of Drosophila melanogaster . During dorsal closure, the amnioserosa (AS), a one-cell thick epithelial tissue that fills the dorsal opening, shrinks as the lateral epidermis sheets converge and eventually merge. During this process, the aspect ratio of amnioserosa cells increases markedly. The standard 2-dimensional vertex model, which successfully describes tissue sheet mechanics in multiple contexts, would in this case predict that the tissue should fluidize via cell neighbor changes. Surprisingly, however, the amnioserosa remains an elastic solid with no such events. We here present a minimal extension to the vertex model that explains how the amnioserosa can achieve this unexpected behavior. We show that continuous shrinkage of the preferred cell perimeter and cell perimeter polydispersity lead to the retention of the solid state of the amnioserosa. Our model accurately captures measured cell shape and orientation changes and predicts non-monotonic junction tension that we confirm with laser ablation experiments. Significance Statement: During embryogenesis, cells in tissues can undergo significant shape changes. Many epithelial tissues fluidize, i.e. cells exchange neighbors, when the average cell aspect ratio increases above a threshold value, consistent with the standard vertex model. During dorsal closure in Drosophila melanogaster , however, the amnioserosa tissue remains solid even as the average cell aspect ratio increases well above threshold. We introduce perimeter polydispersity and allow the preferred cell perimeters, usually held fixed in vertex models, to decrease linearly with time as seen experimentally. With these extensions to the standard vertex model, we capture experimental observations quantitatively. Our results demonstrate that vertex models can describe the behavior of the amnioserosa in dorsal closure by allowing normally fixed parameters to vary with time.

Minimal vertex model explains how the amnioserosa avoids fluidization during Drosophila dorsal closure.

Dorsal closure is a process that occurs during embryogenesis of Drosophila melanogaster. During dorsal closure, the amnioserosa (AS), a one-cell thick epithelial tissue that fills the dorsal opening, shrinks as the lateral epidermis sheets converge and eventually merge. During this process, the aspect ratio of amnioserosa cells increases markedly. The standard 2-dimensional vertex model, which successfully describes tissue sheet mechanics in multiple contexts, would in this case predict that the tissue should fluidize via cell neighbor changes. Surprisingly, however, the amnioserosa remains an elastic solid with no such events. We here present a minimal extension to the vertex model that explains how the amnioserosa can achieve this unexpected behavior. We show that continuous shrink-age of the preferred cell perimeter and cell perimeter polydispersity lead to the retention of the solid state of the amnioserosa. Our model accurately captures measured cell shape and orientation changes and predicts non-monotonic junction tension that we confirm with laser ablation experiments.

Wound repair in sea urchin larvae involves pigment cells and blastocoelar cells.

Sea urchin larvae spend weeks to months feeding on plankton prior to metamorphosis. When handled in the laboratory they are easily injured, suggesting that in the plankton they are injured with some frequency. Fortunately, larval wounds are repaired through an efficient wound response with mesenchymal pigment cells and blastocoelar cells assisting as the epithelium closes. An injury to the epithelium leads to an immediate calcium transient that rapidly spreads around the entire larva and is necessary for activating pigment cell migration toward the wound. If calcium transport is blocked, the pigment cells fail to activate and remain in place. When activated, pigment cells initiate directed migration to the wound site from distances of at least 85 â€‹µm. Upon arrival at the wound site they participate in an innate immune response. Blastocoelar cells are recruited to the injury site as well, though the calcium transient is unnecessary for activating these cells. At the wound site, blastocoelar cells participate in several functions including remodeling the skeleton if it protrudes through the epithelium.

DeepProjection: specific and robust projection of curved 2D tissue sheets from 3D microscopy using deep learning.

The efficient extraction of image data from curved tissue sheets embedded in volumetric imaging data remains a serious and unsolved problem in quantitative studies of embryogenesis. Here, we present DeepProjection (DP), a trainable projection algorithm based on deep learning. This algorithm is trained on user-generated training data to locally classify 3D stack content, and to rapidly and robustly predict binary masks containing the target content, e.g. tissue boundaries, while masking highly fluorescent out-of-plane artifacts. A projection of the masked 3D stack then yields background-free 2D images with undistorted fluorescence intensity values. The binary masks can further be applied to other fluorescent channels or to extract local tissue curvature. DP is designed as a first processing step than can be followed, for example, by segmentation to track cell fate. We apply DP to follow the dynamic movements of 2D-tissue sheets during dorsal closure in Drosophila embryos and of the periderm layer in the elongating Danio embryo. DeepProjection is available as a fully documented Python package.

Superresolution microscopy reveals actomyosin dynamics in medioapical arrays.

Arrays of actin filaments (F-actin) near the apical surface of epithelial cells (medioapical arrays) contribute to apical constriction and morphogenesis throughout phylogeny. Here, superresolution approaches (grazing incidence structured illumination, GI-SIM, and lattice light sheet, LLSM) microscopy resolve individual, fluorescently labeled F-actin and bipolar myosin filaments that drive amnioserosa cell shape changes during dorsal closure in Drosophila. In expanded cells, F-actin and myosin form loose, apically domed meshworks at the plasma membrane. The arrays condense as cells contract, drawing the domes into the plane of the junctional belts. As condensation continues, individual filaments are no longer uniformly apparent. As cells expand, arrays of actomyosin are again resolved-some F-actin turnover likely occurs, but a large fraction of existing filaments rearrange. In morphologically isotropic cells, actin filaments are randomly oriented and during contraction are drawn together but remain essentially randomly oriented. In anisotropic cells, largely parallel actin filaments are drawn closer to one another. Our images offer unparalleled resolution of F-actin in embryonic tissue, show that medioapical arrays are tightly apposed to the plasma membrane and are continuous with meshworks of lamellar F-actin. Medioapical arrays thereby constitute modified cell cortex. In concert with other tagged array components, superresolution imaging of live specimens will offer new understanding of cortical architecture and function.

Mutations in Drosophila crinkled/Myosin VIIA disrupt denticle morphogenesis.

Actin filament crosslinking, bundling and molecular motor proteins are necessary for the assembly of epithelial projections such as microvilli, stereocilia, hairs, and bristles. Mutations in such proteins cause defects in the shape, structure, and function of these actin - based protrusions. One protein necessary for stereocilia formation, Myosin VIIA, is an actin - based motor protein conserved throughout phylogeny. In Drosophila melanogaster, severe mutations in the MyoVIIA homolog crinkled (ck) are "semi - lethal" with only a very small percentage of flies surviving to adulthood. Such survivors show morphological defects related to actin bundling in hairs and bristles. To better understand ck/MyoVIIA's function in bundled - actin structures, we used dominant female sterile approaches to analyze the loss of maternal and zygotic (M/Z) ck/MyoVIIA in the morphogenesis of denticles, small actin - based projections on the ventral epidermis of Drosophila embryos. M/Z ck mutants displayed severe defects in denticle morphology - actin filaments initiated in the correct location, but failed to elongate and bundle to form normal projections. Using deletion mutant constructs, we demonstrated that both of the C - terminal MyTH4 and FERM domains are necessary for proper denticle formation. Furthermore, we show that ck/MyoVIIA interacts genetically with dusky - like (dyl), a member of the ZPD family of proteins that links the extracellular matrix to the plasma membrane, and when mutated also disrupts normal denticle formation. Loss of either protein alone does not alter the localization of the other; however, loss of the two proteins together dramatically enhances the defects in denticle shape observed when either protein alone was absent. Our data indicate that ck/MyoVIIA plays a key role in the formation and/or organization of actin filament bundles, which drive proper shape of cellular projections.

Identifying Key Genetic Regions for Cell Sheet Morphogenesis on Chromosome 2L Using a Drosophila Deficiency Screen in Dorsal Closure.

Cell sheet morphogenesis is essential for metazoan development and homeostasis of animal form - it contributes to developmental milestones including gastrulation, neural tube closure, heart and palate formation and to tissue maintenance during wound healing. Dorsal closure, a well-characterized stage in Drosophila embryogenesis and a model for cell sheet morphogenesis, is a remarkably robust process during which coordination of conserved gene expression patterns and signaling cascades regulate the cellular shape changes and movements. New 'dorsal closure genes' continue to be discovered due to advances in imaging and genetics. Here, we extend our previous study of the right arm of the 2nd chromosome to the left arm of the 2nd chromosome using the Bloomington deficiency kit's set of large deletions, which collectively remove 98.9% of the genes on the left arm of chromosome two (2L) to identify 'dorsal closure deficiencies'. We successfully screened 87.2% of the genes and identified diverse dorsal closure defects in embryos homozygous for 49 deficiencies, 27 of which delete no known dorsal closure gene. These homozygous deficiencies cause defects in cell shape, canthus formation and tissue dynamics. Within these deficiencies, we have identified pimples, odd-skipped, paired, and sloppy-paired 1 as dorsal closure genes on 2L that affect lateral epidermal cells. We will continue to identify novel 'dorsal closure genes' with further analysis. These forward genetic screens are expected to identify new processes and pathways that contribute to closure and links between pathways and structures already known to coordinate various aspects of closure.

Contractile protein biochemistry in the Pollard Lab in Baltimore.

We describe our search for the molecular mechanisms of cell motility with personal recollections of bucket biochemistry in Tom Pollards Lab at the Johns Hopkins, circa 1980.

Visualizing Intracellular Organelle and Cytoskeletal Interactions at Nanoscale Resolution on Millisecond Timescales.

In eukaryotic cells, organelles and the cytoskeleton undergo highly dynamic yet organized interactions capable of orchestrating complex cellular functions. Visualizing these interactions requires noninvasive, long-duration imaging of the intracellular environment at high spatiotemporal resolution and low background. To achieve these normally opposing goals, we developed grazing incidence structured illumination microscopy (GI-SIM) that is capable of imaging dynamic events near the basal cell cortex at 97-nm resolution and 266 frames/s over thousands of time points. We employed multi-color GI-SIM to characterize the fast dynamic interactions of diverse organelles and the cytoskeleton, shedding new light on the complex behaviors of these structures. Precise measurements of microtubule growth or shrinkage events helped distinguish among models of microtubule dynamic instability. Analysis of endoplasmic reticulum (ER) interactions with other organelles or microtubules uncovered new ER remodeling mechanisms, such as hitchhiking of the ER on motile organelles. Finally, ER-mitochondria contact sites were found to promote both mitochondrial fission and fusion.

Unified biophysical mechanism for cell-shape oscillations and cell ingression.

We describe a mechanochemical and percolation cascade that augments myosin's regulatory network to tune cytoskeletal forces. Actomyosin forces collectively generate cytoskeletal forces during cell oscillations and ingression, which we quantify by elastic percolation of the internally driven, cross-linked actin network. Contractile units can produce relatively large, oscillatory forces that disrupt crosslinks to reduce cytoskeletal forces. A (reverse) Hopf bifurcation switches contractile units to produce smaller, steady forces that enhance crosslinking and consequently boost cytoskeletal forces to promote ingression. We describe cell-shape changes and cell ingression in terms of intercellular force imbalances along common cell junctions.

Identifying Genetic Players in Cell Sheet Morphogenesis Using a Drosophila Deficiency Screen for Genes on Chromosome 2R Involved in Dorsal Closure.

Cell sheet morphogenesis characterizes key developmental transitions and homeostasis, in vertebrates and throughout phylogeny, including gastrulation, neural tube formation and wound healing. Dorsal closure, a process during Drosophila embryogenesis, has emerged as a model for cell sheet morphogenesis. ∼140 genes are currently known to affect dorsal closure and new genes are identified each year. Many of these genes were identified in screens that resulted in arrested development. Dorsal closure is remarkably robust and many questions regarding the molecular mechanisms involved in this complex biological process remain. Thus, it is important to identify all genes that contribute to the kinematics and dynamics of closure. Here, we used a set of large deletions (deficiencies), which collectively remove 98.5% of the genes on the right arm of Drosophila melanogaster's 2nd chromosome to identify "dorsal closure deficiencies". Through two crosses, we unambiguously identified embryos homozygous for each deficiency and time-lapse imaged them for the duration of closure. Images were analyzed for defects in cell shapes and tissue movements. Embryos homozygous for 47 deficiencies have notable, diverse defects in closure, demonstrating that a number of discrete processes comprise closure and are susceptible to mutational disruption. Further analysis of these deficiencies will lead to the identification of at least 30 novel "dorsal closure genes". We expect that many of these novel genes will identify links to pathways and structures already known to coordinate various aspects of closure. We also expect to identify new processes and pathways that contribute to closure.

Cell Sheet Morphogenesis: Dorsal Closure in Drosophila melanogaster as a Model System.

Dorsal closure is a key process during Drosophila morphogenesis that models cell sheet movements in chordates, including neural tube closure, palate formation, and wound healing. Closure occurs midway through embryogenesis and entails circumferential elongation of lateral epidermal cell sheets that close a dorsal hole filled with amnioserosa cells. Signaling pathways regulate the function of cellular structures and processes, including Actomyosin and microtubule cytoskeletons, cell-cell/cell-matrix adhesion complexes, and endocytosis/vesicle trafficking. These orchestrate complex shape changes and movements that entail interactions between five distinct cell types. Genetic and laser perturbation studies establish that closure is robust, resilient, and the consequence of redundancy that contributes to four distinct biophysical processes: contraction of the amnioserosa, contraction of supracellular Actomyosin cables, elongation (stretching?) of the lateral epidermis, and zipping together of two converging cell sheets. What triggers closure and what the emergent properties are that give rise to its extraordinary resilience and fidelity remain key, extant questions.

Tension Creates an Endoreplication Wavefront that Leads Regeneration of Epicardial Tissue.

Mechanisms that control cell-cycle dynamics during tissue regeneration require elucidation. Here we find in zebrafish that regeneration of the epicardium, the mesothelial covering of the heart, is mediated by two phenotypically distinct epicardial cell subpopulations. These include a front of large, multinucleate leader cells, trailed by follower cells that divide to produce small, mononucleate daughters. By using live imaging of cell-cycle dynamics, we show that leader cells form by spatiotemporally regulated endoreplication, caused primarily by cytokinesis failure. Leader cells display greater velocities and mechanical tension within the epicardial tissue sheet, and experimentally induced tension anisotropy stimulates ectopic endoreplication. Unbalancing epicardial cell-cycle dynamics with chemical modulators indicated autonomous regenerative capacity in both leader and follower cells, with leaders displaying an enhanced capacity for surface coverage. Our findings provide evidence that mechanical tension can regulate cell-cycle dynamics in regenerating tissue, stratifying the source cell features to improve repair.

Quantifying dorsal closure in three dimensions.

Dorsal closure is an essential stage of Drosophila embryogenesis and is a powerful model system for morphogenesis, wound healing, and tissue biomechanics. During closure, two flanks of lateral epidermis close an eye-shaped dorsal opening that is filled with amnioserosa. The two flanks of lateral epidermis are zipped together at each canthus ("corner" of the eye). Actomyosin-rich purse strings are localized at each of the two leading edges of lateral epidermis ("lids" of the eye). Here we report that each purse string indents the dorsal surface at each leading edge. The amnioserosa tissue bulges outward during the early-to-mid stages of closure to form a remarkably smooth, asymmetric dome indicative of an isotropic and uniform surface tension. Internal pressure of the embryo and tissue elastic properties help to shape the dorsal surface.

MRCK-1 Drives Apical Constriction in C. elegans by Linking Developmental Patterning to Force Generation.

Apical constriction is a change in cell shape that drives key morphogenetic events including gastrulation and neural tube formation. Apical force-producing actomyosin networks drive apical constriction by contracting while connected to cell-cell junctions. The mechanisms by which developmental patterning regulates these actomyosin networks and associated junctions with spatial precision are not fully understood. Here we identify a myosin light-chain kinase MRCK-1 as a key regulator of C. elegans gastrulation that integrates spatial and developmental patterning information. We show that MRCK-1 is required for activation of contractile actomyosin dynamics and elevated cortical tension in the apical cell cortex of endoderm precursor cells. MRCK-1 is apically localized by active Cdc42 at the external, cell-cell contact-free surfaces of apically constricting cells, downstream of cell fate determination mechanisms. We establish that the junctional components α-catenin, ß-catenin, and cadherin become highly enriched at the apical junctions of apically constricting cells and that MRCK-1 and myosin activity are required in vivo for this enrichment. Taken together, our results define mechanisms that position a myosin activator to a specific cell surface where it both locally increases cortical tension and locally enriches junctional components to facilitate apical constriction. These results reveal crucial links that can tie spatial information to local force generation to drive morphogenesis.

Remodeling Tissue Interfaces and the Thermodynamics of Zipping during Dorsal Closure in Drosophila.

Dorsal closure during Drosophila embryogenesis is an important model system for investigating the biomechanics of morphogenesis. During closure, two flanks of lateral epidermis (with actomyosin-rich purse strings near each leading edge) close an eye-shaped opening that is filled with amnioserosa. At each canthus (corner of the eye) a zipping process remodels the tissue interfaces between the leading edges of the lateral epidermis and the amnioserosa. We investigated zipping dynamics and found that apposing leading edge cells come together at their apical ends and then square off basally to form a lateral junction. Meanwhile, the purse strings act as contractile elastic rods bent toward the embryo interior near each canthus. We propose that a canthus-localized force contributes to both bending the ends of the purse strings and the formation of lateral junctions. We developed a thermodynamic model for zipping based on three-dimensional remodeling of the tissue interfaces and the reaction dynamics of adhesion molecules in junctions and elsewhere, which we applied to zipping during unperturbed wild-type closure and to laser or genetically perturbed closure. We identified two processes that can contribute to the zipping mechanism, consistent with experiments, distinguished by whether amnioserosa dynamics do or do not augment canthus adhesion dynamics.

Moving Inward: Establishing the Mammalian Inner Cell Mass.

Early in mammalian development, a few cells move to the center of the embryo to establish the inner cell mass-the early precursor of the fetus. In this issue of Developmental Cell, Samarage et al. (2015) shed light on how these cells move inward.

Epithelial morphogenesis: apoptotic forces drive cell shape changes.

Studying how cells produce and transmit forces that drive morphogenesis is critical to understanding organismal development. A new paper by Monier et al. (2015) identifies an apicobasal actomyosin cable that characterizes apoptotic cells and contributes force(s) for cell sheet bending.