Left-right symmetry of zebrafish embryos requires somite surface tension.

The body axis of vertebrate embryos is periodically segmented into bilaterally symmetric pairs of somites1,2. The anteroposterior length of somites, their position and left-right symmetry are thought to be molecularly determined before somite morphogenesis3,4. Here we show that, in zebrafish embryos, initial somite anteroposterior lengths and positions are imprecise and, consequently, many somite pairs form left-right asymmetrically. Notably, these imprecisions are not left unchecked and we find that anteroposterior lengths adjust within an hour after somite formation, thereby increasing morphological symmetry. We find that anteroposterior length adjustments result entirely from changes in somite shape without change in somite volume, with changes in anteroposterior length being compensated by corresponding changes in mediolateral length. The anteroposterior adjustment mechanism is facilitated by somite surface tension, which we show by comparing in vivo experiments and in vitro single-somite explant cultures using a mechanical model. Length adjustment is inhibited by perturbation of molecules involved in surface tension, such as integrin and fibronectin. By contrast, the adjustment mechanism is unaffected by perturbations to the segmentation clock, therefore revealing a distinct process that influences morphological segment lengths. We propose that tissue surface tension provides a general mechanism to adjust shapes and ensure precision and symmetry of tissues in developing embryos.

An emerging role for tissue plasticity in developmental precision.

Reproducible tissue morphology is a fundamental feature of embryonic development. To ensure such robustness during tissue morphogenesis, inherent noise in biological processes must be buffered. While redundant genes, parallel signaling pathways and intricate network topologies are known to reduce noise, over the last few years, mechanical properties of tissues have been shown to play a vital role. Here, taking the example of somite shape changes, I will discuss how tissues are highly plastic in their ability to change shapes leading to increased precision and reproducibility.

Highly-Efficient Guiding of Motile Microtubules on Non-Topographical Motor Patterns.

Molecular motors, highly efficient biological nanomachines, hold the potential to be employed for a wide range of nanotechnological applications. Toward this end, kinesin, dynein, or myosin motor proteins are commonly surface-immobilized within engineered environments in order to transport cargo attached to cytoskeletal filaments. Being able to flexibly control the direction of filament motion, and in particular on planar, non-topographical surfaces, has, however, remained challenging. Here, we demonstrate the applicability of a UV-laser-based ablation technique to programmably generate highly localized patterns of functional kinesin-1 motors with different shapes and sizes on PLL-g-PEG-coated polystyrene surfaces. Straight and curved motor tracks with widths of less than 500 nm could be generated in a highly reproducible manner and proved to reliably guide gliding microtubules. Though dependent on track curvature, the characteristic travel lengths of the microtubules on the tracks significantly exceeded earlier predictions. Moreover, we experimentally verified the performance of complex kinesin-1 patterns, recently designed by evolutionary algorithms for controlling the global directionality of microtubule motion on large-area substrates.

Asymmetric Flows in the Intercellular Membrane during Cytokinesis.

Eukaryotic cells undergo shape changes during their division and growth. This involves flow of material both in the cell membrane and in the cytoskeletal layer beneath the membrane. Such flows result in redistribution of phospholipid at the cell surface and actomyosin in the cortex. Here we focus on the growth of the intercellular surface during cell division in a Caenorhabditis elegans embryo. The growth of this surface leads to the formation of a double-layer of separating membranes between the two daughter cells. The division plane typically has a circular periphery and the growth starts from the periphery as a membrane invagination, which grows radially inward like the shutter of a camera. The growth is typically not concentric, in the sense that the closing internal ring is located off-center. Cytoskeletal proteins anillin and septin have been found to be responsible for initiating and maintaining the asymmetry of ring closure but the role of possible asymmetry in the material flow into the growing membrane has not been investigated yet. Motivated by experimental evidence of such flow asymmetry, here we explore the patterns of internal ring closure in the growing membrane in response to asymmetric boundary fluxes. We highlight the importance of the flow asymmetry by showing that many of the asymmetric growth patterns observed experimentally can be reproduced by our model, which incorporates the viscous nature of the membrane and contractility of the associated cortex.

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos.

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

Emergence of a left-right symmetric body plan in vertebrate embryos.

External bilateral symmetry is a prevalent feature in vertebrates, which emerges during early embryonic development. To begin with, vertebrate embryos are largely radially symmetric before transitioning to bilaterally symmetry, after which, morphogenesis of various bilateral tissues (e.g somites, otic vesicle, limb bud), and structures (e.g palate, jaw) ensue. While a significant amount of work has probed the mechanisms behind symmetry breaking in the left-right axis leading to asymmetric positioning of internal organs, little is known about how bilateral tissues emerge at the same time with the same shape and size and at the same position on the two sides of the embryo. By discussing emergence of symmetry in many bilateral tissues and structures across vertebrate model systems, we highlight that understanding symmetry establishment is largely an open field, which will provide deep insights into fundamental problems in developmental biology for decades to come.

Atypical Protein Kinase C Promotes its own Asymmetric Localisation by Phosphorylating Cdc42 in Polarising Cells.

Atypical protein kinase C (aPKC) is a major regulator of cell polarity. Acting in conjunction with Par6, Par3 and the small GTPase Cdc42, aPKC becomes asymmetrically localised and drives the polarisation of cells. aPKC activity is crucial for its own asymmetric localisation, suggesting a hitherto unknown feedback mechanism contributing to polarisation. Here we show in C. elegans zygotes that the feedback relies on CDC-42 phosphorylation at serine 71 by aPKC, which in turn results in aPKC dissociation from CDC-42. The dissociated aPKC then associates with PAR-3 clusters, which are transported anteriorly by actomyosin-based cortical flow. Moreover, the turnover of aPKC-mediated CDC-42 phosphorylation regulates the organisation of the actomyosin cortex that drives aPKC asymmetry. Given the widespread role of aPKC and Cdc42 in cell polarity, this form of self-regulation of aPKC may be vital for the robust polarisation of many cell types.

Preparation of Single-Somite Explants from Zebrafish Embryos.

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular prepatterns determine the initial length-scale of somites, mechanical processes have been implicated in setting their final size and shape. To better understand the intrinsic material properties of somites, a method is developed to culture single-somite explant from zebrafish embryos. Single somites are isolated by first removing the skin of embryos, followed by yolk removal and sequential excision of neighboring tissues. Using transgenic embryos, the distribution of various sub-cellular structures can be observed by fluorescent time-lapse microscopy. Dynamics of explanted somites can be followed for several hours, thus providing an experimental framework for studying tissue-scale shape changes at single-cell resolution. This approach enables direct mechanical manipulation of somites, allowing for dissection of the material properties of the tissue. Finally, the technique outlined here can be readily extended for explanting other tissues such as the notochord, neural plate, and lateral plate mesoderm.

Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma.

The mesothelium lines body cavities and surrounds internal organs, widely contributing to homeostasis and regeneration. Mesothelium disruptions cause visceral anomalies and mesothelioma tumors. Nonetheless, the embryonic emergence of mesothelia remains incompletely understood. Here, we track mesothelial origins in the lateral plate mesoderm (LPM) using zebrafish. Single-cell transcriptomics uncovers a post-gastrulation gene expression signature centered on hand2 in distinct LPM progenitor cells. We map mesothelial progenitors to lateral-most, hand2-expressing LPM and confirm conservation in mouse. Time-lapse imaging of zebrafish hand2 reporter embryos captures mesothelium formation including pericardium, visceral, and parietal peritoneum. We find primordial germ cells migrate with the forming mesothelium as ventral migration boundary. Functionally, hand2 loss disrupts mesothelium formation with reduced progenitor cells and perturbed migration. In mouse and human mesothelioma, we document expression of LPM-associated transcription factors including Hand2, suggesting re-initiation of a developmental program. Our data connects mesothelium development to Hand2, expanding our understanding of mesothelial pathologies.

Morphogenetic degeneracies in the actomyosin cortex.

One of the great challenges in biology is to understand the mechanisms by which morphogenetic processes arise from molecular activities. We investigated this problem in the context of actomyosin-based cortical flow in C. elegans zygotes, where large-scale flows emerge from the collective action of actomyosin filaments and actin binding proteins (ABPs). Large-scale flow dynamics can be captured by active gel theory by considering force balances and conservation laws in the actomyosin cortex. However, which molecular activities contribute to flow dynamics and large-scale physical properties such as viscosity and active torque is largely unknown. By performing a candidate RNAi screen of ABPs and actomyosin regulators we demonstrate that perturbing distinct molecular processes can lead to similar flow phenotypes. This is indicative for a 'morphogenetic degeneracy' where multiple molecular processes contribute to the same large-scale physical property. We speculate that morphogenetic degeneracies contribute to the robustness of bulk biological matter in development.

The Sweetness of Embryonic Elongation and Differentiation.

Metabolic pathways play a vital yet poorly understood role in embryogenesis. In this issue of Developmental Cell, Bulusu et al. (2017) and Oginuma et al. (2017) provide insights into the intricate relationship between metabolism and morphogenesis, showing that glycolysis facilitates body elongation and balances neural and mesodermal differentiation.

Controlling contractile instabilities in the actomyosin cortex.

The actomyosin cell cortex is an active contractile material for driving cell- and tissue morphogenesis. The cortex has a tendency to form a pattern of myosin foci, which is a signature of potentially unstable behavior. How a system that is prone to such instabilities can rveliably drive morphogenesis remains an outstanding question. Here, we report that in the Caenorhabditis elegans zygote, feedback between active RhoA and myosin induces a contractile instability in the cortex. We discover that an independent RhoA pacemaking oscillator controls this instability, generating a pulsatory pattern of myosin foci and preventing the collapse of cortical material into a few dynamic contracting regions. Our work reveals how contractile instabilities that are natural to occur in mechanically active media can be biochemically controlled to robustly drive morphogenetic events.

Actomyosin-driven left-right asymmetry: from molecular torques to chiral self organization.

Chirality or mirror asymmetry is a common theme in biology found in organismal body plans, tissue patterns and even in individual cells. In many cases the emergence of chirality is driven by actin cytoskeletal dynamics. Although it is well established that the actin cytoskeleton generates rotational forces at the molecular level, we are only beginning to understand how this can result in chiral behavior of the entire actin network in vivo. In this review, we will give an overview of actin driven chiralities across different length scales known until today. Moreover, we evaluate recent quantitative models demonstrating that chiral symmetry breaking of cells can be achieved by properly aligning molecular-scale torque generation processes in the actomyosin cytoskeleton.

Active torque generation by the actomyosin cell cortex drives left-right symmetry breaking.

Many developmental processes break left-right (LR) symmetry with a consistent handedness. LR asymmetry emerges early in development, and in many species the primary determinant of this asymmetry has been linked to the cytoskeleton. However, the nature of the underlying chirally asymmetric cytoskeletal processes has remained elusive. In this study, we combine thin-film active chiral fluid theory with experimental analysis of the C. elegans embryo to show that the actomyosin cortex generates active chiral torques to facilitate chiral symmetry breaking. Active torques drive chiral counter-rotating cortical flow in the zygote, depend on myosin activity, and can be altered through mild changes in Rho signaling. Notably, they also execute the chiral skew event at the 4-cell stage to establish the C. elegans LR body axis. Taken together, our results uncover a novel, large-scale physical activity of the actomyosin cytoskeleton that provides a fundamental mechanism for chiral morphogenesis in development.

Systematic genetic interaction screens uncover cell polarity regulators and functional redundancy.

Although single-gene loss-of-function analyses can identify components of particular processes, important molecules are missed owing to the robustness of biological systems. Here we show that large-scale RNAi screening for suppression interactions with functionally related mutants greatly expands the repertoire of genes known to act in a shared process and reveals a new layer of functional relationships. We performed RNAi screens for 17 Caenorhabditis elegans cell polarity mutants, generating the most comprehensive polarity network in a metazoan, connecting 184 genes. Of these, 72% were not previously linked to cell polarity and 80% have human homologues. We experimentally confirmed functional roles predicted by the network and characterized through biophysical analyses eight myosin regulators. In addition, we discovered functional redundancy between two unknown polarity genes. Similar systematic genetic interaction screens for other biological processes will help uncover the inventory of relevant genes and their patterns of interactions.