Guy B. Blanchard

Tissue tectonics: morphogenetic strain rates, cell shape change and intercalation

The dynamic reshaping of tissues during morphogenesis results from a combination of individual cell behaviors and collective cell rearrangements. However, a comprehensive framework to unambiguously measure and link cell behavior to tissue morphogenesis is lacking. Here we introduce such a kinematic framework, bridging cell and tissue behaviors at an intermediate, mesoscopic, level of cell clusters or domains. By measuring domain deformation in terms of the relative motion of cell positions and the evolution of their shapes, we characterized the basic invariant quantities that measure fundamental classes of cell behavior, namely tensorial rates of cell shape change and cell intercalation. In doing so we introduce an explicit definition of cell intercalation as a continuous process. We mapped strain rates spatiotemporally in three models of tissue morphogenesis, gaining insight into morphogenetic mechanisms. Our quantitative approach has broad relevance for the precise characterization and comparison of morphogenetic phenotypes.

Cytoskeletal dynamics and supracellular organisation of cell shape fluctuations during dorsal closure

Fluctuations in the shape of amnioserosa (AS) cells during Drosophila dorsal closure (DC) provide an ideal system with which to understand contractile epithelia, both in terms of the cellular mechanisms and how tissue behaviour emerges from the activity of individual cells. Using quantitative image analysis we show that apical shape fluctuations are driven by the medial cytoskeleton, with periodic foci of contractile myosin and actin travelling across cell apices. Shape changes were mostly anisotropic and neighbouring cells were often, but transiently, organised into strings with parallel deformations. During the early stages of DC, shape fluctuations with long cycle lengths produced no net tissue contraction. Cycle lengths shortened with the onset of net tissue contraction, followed by a damping of fluctuation amplitude. Eventually, fluctuations became undetectable as AS cells contracted rapidly. These transitions were accompanied by an increase in apical myosin, both at cell-cell junctions and medially, the latter ultimately forming a coherent, but still dynamic, sheet across cells. Mutants with increased myosin activity or actin polymerisation exhibited precocious cell contraction through changes in the subcellular localisation of myosin. thickveins mutant embryos, which exhibited defects in the actin cable at the leading edge, showed similar timings of fluctuation damping to the wild type, suggesting that damping is an autonomous property of the AS. Our results suggest that cell shape fluctuations are a property of cells with low and increasing levels of apical myosin, and that medial and junctional myosin populations combine to contract AS cell apices and drive DC.

Cell shape changes indicate a role for extrinsic tensile forces in Drosophila germ-band extension.

Drosophila germ-band extension (GBE) is an example of the convergence and extension movements that elongate and narrow embryonic tissues. To understand the collective cell behaviours underlying tissue morphogenesis, we have continuously quantified cell intercalation and cell shape change during GBE. We show that the fast, early phase of GBE depends on cell shape change in addition to cell intercalation. In antero-posterior patterning mutants such as those for the gap gene Krüppel, defective polarized cell intercalation is compensated for by an increase in antero-posterior cell elongation, such that the initial rate of extension remains the same. Spatio-temporal patterns of cell behaviours indicate that an antero-posterior tensile force deforms the germ band, causing the cells to change shape passively. The rate of antero-posterior cell elongation is reduced in twist mutant embryos, which lack mesoderm. We propose that cell shape change contributing to germ-band extension is a passive response to mechanical forces caused by the invaginating mesoderm.

Mechanical control of global cell behaviour during dorsal closure in Drosophila

Halfway through embryonic development, the epidermis of Drosophila exhibits a gap at the dorsal side covered by an extraembryonic epithelium, the amnioserosa (AS). Dorsal closure (DC) is the process whereby interactions between the two epithelia establish epidermal continuity. Although genetic and biomechanical analysis have identified the AS as a force-generating tissue, we do not know how individual cell behaviours are transformed into tissue movements. To approach this question we have applied a novel image-analysis method to measure strain rates in local domains of cells and performed a kinematic analysis of DC. Our study reveals spatial and temporal differences in the rate of apical constriction of AS cells. We find a slow phase of DC, during which apical contraction of cells at the posterior end predominates, and a subsequent fast phase, during which all the cells engage in the contraction, which correlates with the zippering process. There is a radial gradient of AS apical contraction, with marginal cells contracting earlier than more centrally located cells. We have applied this analysis to the study of mutant situations and associated a particular genotype with quantitative and reproducible changes in the rate of cell contraction and hence in the overall rate of the process. Our mutant analysis reveals the contribution of mechanical elements to the rate and pattern of DC.

A dynamic fate map of the forebrain shows how vertebrate eyes form and explains two causes of cyclopia

Mechanisms for shaping and folding sheets of cells during development are poorly understood. An example is the complex reorganisation of the forebrain neural plate during neurulation, which must fold a sheet into a tube while evaginating two eyes from a single contiguous domain within the neural plate. We, for the first time, track these cell rearrangements to show that forebrain morphogenesis differs significantly from prior hypotheses. We postulate a new model for forebrain neurulation and demonstrate how mutations affecting two signalling pathways can generate cyclopic phenotypes by disrupting normal cell movements or introducing new erroneous behaviours.

Dynamics of actomyosin contractile activity during epithelial morphogenesis

In the past few years, advances in microscopy and quantitative image analysis have lead to a completely new understanding of the processes underlying the cell shape changes and cell rearrangements that drive tissue morphogenesis. In a handful of tissues so far, though the number will surely increase rapidly, it has been shown that cell behaviour is not continuous but proceeds in pulses driven by the contractile activity of dynamic cortical actomyosin networks. The patterns and dynamics of temporary subcellular contractile foci, driven by local increases in actin and myosin, are remarkably similar in disparate tissues. Cells in all tissues display a similar range of intervals between contractions, with increasing frequencies associated with stronger tissue morphogenesis. Contractile foci appear to flow within cells with speeds that are consistent across tissues. We highlight the difference between contractile tension and stiffness, the latter being a requirement for any ratchet mechanism that stabilises contraction to produce effective tissue morphogenesis. At least two different types of ratchet mechanism are discussed, with the stiffness conferred either by a more stable actomyosin population at cell-cell junctions or through cortical actomyosin forming a quasi-stable supra-cellular network. Pulsatile contractions, polarized cell organization and various stiffening ratchet mechanisms combine to provide a rich variety of options for robust epithelial tissue remodelling.

Division of labour and seasonality in the ant Leptothorax albipennis: worker corpulence and its influence on behaviour.

We address the organization of workers in social insect societies. We distinguish between changes in behavioural role over the nurse to forager role sequence, which may depend on changes in physiology, and potentially more rapid changes of task within role. We investigated the association between role and nutrient status in the ant Leptothorax albipennis. Worker lipid stores were quantified using a new body size-controlled method, and were related to worker behaviour. Worker lipid stores were evenly distributed amongst colony members at the end of winter, splitting rapidly into two distinct modes (replete nurses and lean foragers) in spring. The proportion of lean foragers increased throughout spring and summer, until most colonies contained only workers of this type. Callow workers then eclosed with intermediate lipid stores. We developed a computer vision system that tracks all nest ants to extract detailed behaviour of individuals of known lipid stores. Lipid storage was negatively correlated with a workers foraging propensity, and with measures of spatial occupation in the nest and of activity. Different colonies showed a similar quantitative correlation between lipid stores and behavioural role, suggesting that lipid stores were not only correlated with the relative organization of individuals within each nest, but may also have influenced their absolute role. We reviewed the literature and found evidence that nutrient status influences role predisposition in social insect workers. We conclude that the distribution of worker roles may be linked to the balance between foraging income and energetic consumption within the colony directly via worker nutrient status. Copyright 2000 The Association for the Study of Animal Behaviour.

Integrative approaches to morphogenesis: Lessons from dorsal closure

Although developmental biology has been dominated by the genetic analysis of embryonic development, in recent years genetic tools have been combined with new approaches such as imaging of live processes, automated and quantitative image analysis, mechanical perturbation and mathematical modeling, to study the principles underlying the formation of organisms. Here we focus on recent work carried out on Dorsal Closure, a morphogenetic process during Drosophila embryogenesis, to illustrate how this multidisciplinary approach is yielding new and unexpected insights into how cells organize themselves through the activity of their molecular components to give rise to the stereotyped and macroscopic movements observed during development. genesis 49:522–533, 2011.

Mechanical Coupling between Endoderm Invagination and Axis Extension in Drosophila.

How genetic programs generate cell-intrinsic forces to shape embryos is actively studied, but less so how tissue-scale physical forces impact morphogenesis. Here we address the role of the latter during axis extension, using Drosophila germband extension (GBE) as a model. We found previously that cells elongate in the anteroposterior (AP) axis in the extending germband, suggesting that an extrinsic tensile force contributed to body axis extension. Here we further characterized the AP cell elongation patterns during GBE, by tracking cells and quantifying their apical cell deformation over time. AP cell elongation forms a gradient culminating at the posterior of the embryo, consistent with an AP-oriented tensile force propagating from there. To identify the morphogenetic movements that could be the source of this extrinsic force, we mapped gastrulation movements temporally using light sheet microscopy to image whole Drosophila embryos. We found that both mesoderm and endoderm invaginations are synchronous with the onset of GBE. The AP cell elongation gradient remains when mesoderm invagination is blocked but is abolished in the absence of endoderm invagination. This suggested that endoderm invagination is the source of the tensile force. We next looked for evidence of this force in a simplified system without polarized cell intercalation, in acellular embryos. Using Particle Image Velocimetry, we identify posteriorwards Myosin II flows towards the presumptive posterior endoderm, which still undergoes apical constriction in acellular embryos as in wildtype. We probed this posterior region using laser ablation and showed that tension is increased in the AP orientation, compared to dorsoventral orientation or to either orientations more anteriorly in the embryo. We propose that apical constriction leading to endoderm invagination is the source of the extrinsic force contributing to germband extension. This highlights the importance of physical interactions between tissues during morphogenesis.

A Dynamic Microtubule Cytoskeleton Directs Medial Actomyosin Function during Tube Formation

Summary The cytoskeleton is a major determinant of cell-shape changes that drive the formation of complex tissues during development. Important roles for actomyosin during tissue morphogenesis have been identified, but the role of the microtubule cytoskeleton is less clear. Here, we show that during tubulogenesis of the salivary glands in the fly embryo, the microtubule cytoskeleton undergoes major rearrangements, including a 90° change in alignment relative to the apicobasal axis, loss of centrosomal attachment, and apical stabilization. Disruption of the microtubule cytoskeleton leads to failure of apical constriction in placodal cells fated to invaginate. We show that this failure is due to loss of an apical medial actomyosin network whose pulsatile behavior in wild-type embryos drives the apical constriction of the cells. The medial actomyosin network interacts with the minus ends of acentrosomal microtubule bundles through the cytolinker protein Shot, and disruption of Shot also impairs apical constriction.