The wing imaginal disc

The Drosophila wing imaginal disc is a tissue of undifferentiated cells that are precursors of the wing and most of the notum of the adult fly. The wing disc first forms during embryogenesis from a cluster of ∼30 cells located in the second thoracic segment, which invaginate to form a sac-like structure. They undergo extensive proliferation during larval stages to form a mature larval wing disc of ∼35,000 cells. During this time, distinct cell fates are assigned to different regions, and the wing disc develops a complex morphology. Finally, during pupal stages the wing disc undergoes morphogenetic processes and then differentiates to form the adult wing and notum. While the bulk of the wing disc comprises epithelial cells, it also includes neurons and glia, and is associated with tracheal cells and muscle precursor cells. The relative simplicity and accessibility of the wing disc, combined with the wealth of genetic tools available in Drosophila, have combined to make it a premier system for identifying genes and deciphering systems that play crucial roles in animal development. Studies in wing imaginal discs have made key contributions to many areas of biology, including tissue patterning, signal transduction, growth control, regeneration, planar cell polarity, morphogenesis, and tissue mechanics.     

Establishment of global patterns of planar polarity during growth of the Drosophila wing epithelium

Epithelial tissues develop planar polarity that is reflected in the global alignment of hairs and cilia with respect to the tissue axes. The planar cell polarity (PCP) proteins form asymmetric and polarized domains across epithelial junctions that are aligned locally between cells and orient these external structures. Although feedback mechanisms can polarize PCP proteins intracellularly and locally align polarity between cells, how global PCP patterns are specified is not understood. It has been proposed that the graded distribution of a biasing factor could guide long-range PCP. However, we recently identified epithelial morphogenesis as a mechanism that can reorganize global PCP patterns; in the Drosophila pupal wing, oriented cell divisions and rearrangements reorient PCP from a margin-oriented pattern to one that points distally. Here, we use quantitative image analysis to study how PCP patterns first emerge in the wing. PCP appears during larval growth and is spatially oriented through the activities of three organizer regions that control disc growth and patterning. Flattening morphogen gradients emanating from these regions does not reduce intracellular polarity but distorts growth and alters specific features of the PCP pattern. Thus, PCP may be guided by morphogenesis rather than morphogen gradients.     

Growth and patterning are evolutionarily dissociated in the vestigial wing discs of workers of the red imported fire ant, Solenopsis invicta

Over the last decade, it has become clear that organismal form is largely determined by developmental and evolutionary changes in the growth and pattern formation of tissues. Yet, there is little known about how these two integrated processes respond to environmental cues or how they evolve relative to one another. Here, we present the discovery of vestigial wing imaginal discs in worker larvae of the red imported fire ant, Solenopsis invicta. These vestigial wing discs are present in all worker larvae, which is uncommon for a species with a large worker size distribution. Furthermore, the growth trajectory of these vestigial discs is distinct from all of the ant species examined to date because they grow at a rate slower than the leg discs. We predicted that the growth trajectory of the vestigial wing discs would be mirrored by evolutionary changes in their patterning. We tested this prediction by examining the expression of three patterning genes, extradenticle, ultrabithorax, and engrailed, known to underlie the wing polyphenism in ants. Surprisingly, the expression patterns of these three genes in the vestigial wing discs was the same as those found in ant species with different worker size distributions and wing disc growth than fire ants. We conclude that growth and patterning are evolutionarily dissociated in the vestigial wing discs of S. invicta because patterning in these discs is conserved, whereas their growth trajectories are not. The evolutionary dissociation of growth and patterning may be an important feature of gene networks that underlie polyphenic traits.     

BMP morphogen gradients in flies

Bone morphogenetic proteins (BMPs) act as morphogens to control patterning and growth in a variety of developing tissues in different species. How BMP morphogen gradients are established and interpreted in the target tissues has been extensively studied in Drosophila melanogaster. In Drosophila, Decapentaplegic (Dpp), a homologue of vertebrate BMP2/4, acts as a morphogen to control dorsal–ventral patterning of the early embryo and anterior–posterior patterning and growth of the wing imaginal disc. Despite intensive efforts over the last twenty years, how the Dpp morphogen gradient in the wing imaginal disc forms remains controversial, while gradient formation in the early embryo is well understood. In this review, we first focus on the current models of Dpp morphogen gradient formation in these two tissues, and then discuss new strategies using genome engineering and nanobodies to tackle open questions.     

Mechanical forces shaping the development of the inner ear

The inner ear is one of the most complex structures in the mammalian body. Embedded within it are the hearing and balance sensory organs that contain arrays of hair cells that serve as sensors of sound and acceleration. Within the sensory organs, these hair cells are prototypically arranged in regular mosaic patterns. The development of such complex, yet precise, patterns require the coordination of differentiation, growth, and morphogenesis, both at the tissue and cellular scales. In recent years, there is accumulating evidence that mechanical forces at the tissue, the cellular, and the subcellular scales coordinate the development and organization of this remarkable organ. Here, we review recent works that reveal how such mechanical forces shape the inner ear, control its size, and establish regular cellular patterns. The insights learned from studying how mechanical forces drive the inner ear development are relevant for many other developmental systems in which precise cellular patterns are essential for their function.     

The Mechanical Role of Microtubules in Tissue Remodeling

During morphogenesis, tissues undergo extensive remodeling to get their final shape. Such precise sculpting requires the application of forces generated within cells by the cytoskeleton and transmission of these forces through adhesion molecules within and between neighboring cells. Within individual cells, microtubules together with actomyosin filaments and intermediate filaments form the composite cytoskeleton that controls cell mechanics during tissue rearrangements. While studies have established the importance of actin-based mechanical forces that are coupled via intercellular junctions, relatively little is known about the contribution of other cytoskeletal components such as microtubules to cell mechanics during morphogenesis. In this review the focus is on recent findings, highlighting the direct mechanical role of microtubules beyond its well-established role in trafficking and signaling during tissue formation.     

神经元轴突生长的力生物学

神经系统作为一个复杂的体系,在其发育过程中轴突需要延伸较长的距离才能与下一级神经元或靶细胞形成突触。在这个复杂的移动过程中,神经元轴突在空间分布上形成了精确有序的结构。过去认为这种有序结构的形成主要由形态发生素的化学浓度梯度来指导,而最近的研究发现力学因素对调控轴突的延伸速度与方向发挥着重要的作用。因此,轴突的延伸本质上是一个力化学耦合过程。本文将结合自己过去的工作论述力学因素对轴突延伸的调控机制及相关的信号转导。这一领域的研究将为认识对神经系统疾病的发生以及神经再生提供重要的参考。     

Mechanical loading history and skeletal biology

A comprehensive theory which relates tissue mechanical stresses to many features of skeletal morphogenesis, growth, regeneration, maintenance and degeneration is reviewed. The theory considers the repeated or intermittent mechanical forces which constitute the loading history on the chondro-osseous skeleton. The results of numerous mechanical stress analyses indicate that the local tissue stress history plays a major role in controlling connective tissue biology. The strong influence of mechanical energy in ontogenesis implies a comparably strong influence in phylogenesis. The fact that the mechanical stress histories in skeletal tissues are directly related to the force of gravity suggests that the life forms that have evolved on Earth are closely tied to our gravitational field.     

The structural and mechanical complexity of cell-growth control

Tight control of cell proliferation is required to ensure normal tissue patterning and prevent cancer formation. The analysis of cultured cells has led to an explosion in our understanding of the molecules that trigger growth and mediate cell-cycle progression. However, the mechanism by which the local growth differentials that drive morphogenesis are established and maintained still remains unknown. Here we review recent work that reveals the importance of cell binding to the extracellular matrix, and associated changes in cell shape and cytoskeletal tension, to the spatial control of cell-cycle progression. These findings change the paradigm of cell-growth control, by placing our understanding of molecular signalling cascades in the context of the structural and mechanical complexity of living tissues.     

Pinching and pushing: fold formation in the Drosophila dorsal epidermis

Epithelial folding is a fundamental morphogenetic process that shapes planar epithelial sheets into complex three-dimensional structures. Multiple mechanisms can generate epithelial folds, including apical constriction, which acts locally at the cellular level, differential growth on the tissue scale, or buckling because of compression from neighboring tissues. Here, we investigate the formation of dorsally located epithelial folds at segment boundaries during the late stages of Drosophila embryogenesis. We found that the fold formation at the segment boundaries occurs through the juxtaposition of two key morphogenetic processes: local apical constriction and tissue-level compressive forces from posterior segments. Further, we found that epidermal spreading and fold formation are accompanied by spatiotemporal pulses of Hedgehog (Hh) signaling. A computational model that incorporates the local forces generated from the differential tensions of the apical, basal, and lateral sides of the cell and active forces generated within the whole tissue recapitulates the overall fold formation process in wild-type and Hh overexpression conditions. In sum, this work demonstrates how epithelial folding depends on multiple, separable physical mechanisms to generate the final morphology of the dorsal epidermis. This work illustrates the modularity of morphogenetic unit operations that occur during epithelial morphogenesis.     

Drosophila dumpy is a gigantic extracellular protein required to maintain tension at epidermal-cuticle attachment sites

BACKGROUND: Growth and morphogenesis during development depend both on patterning genes, which assign positional information, and on genes that regulate mechanical forces. The dumpy gene of the fruit fly Drosophila melanogaster is an example of the latter class, with mutant phenotypes affecting size and shape of the limbs, thoracic cuticle, trachea and mouthparts. RESULTS: The genetically complex dumpy locus was found to span over 100 kb and encode a gigantic 2.5 MDa extracellular matrix protein. Dumpy represents an extreme form of modular protein evolution, containing 308 epidermal growth factor (EGF) modules, interspersed with a new module class, DPY, and terminating in a crosslinking zona pellucida domain and membrane anchor sequence. We determined the three-dimensional structure of the DPY module by nuclear magnetic resonance (NMR) spectroscopy and found that it forms a disulphide-stabilised beta sheet motif, capable of linking end-to-end with EGF modules to form a fibre. Consistent with its cuticle phenotypes, dumpy is expressed at several sites of cuticle-epidermal cell attachment, including the trachea and the muscle tendon cells, which mediate anchorage of the muscles to the cuticle. CONCLUSIONS: The dumpy gene encodes a gigantic extracellular molecule that we predict to be a membrane-anchored fibre of almost a micrometer in length. Insertion and crosslinking of this fibre within the cuticle may provide a strong anchor for the underlying tissue, allowing it to maintain mechanical tension at sites under stress. This would explain its contribution to tissue morphogenesis through its regulation of mechanical properties.     

Mechanical influences on morphogenesis of the knee joint revealed through morphological, molecular and computational analysis of immobilised embryos

Very little is known about the regulation of morphogenesis in synovial joints. Mechanical forces generated from muscle contractions are required for normal development of several aspects of normal skeletogenesis. Here we show that biophysical stimuli generated by muscle contractions impact multiple events during chick knee joint morphogenesis influencing differential growth of the skeletal rudiment epiphyses and patterning of the emerging tissues in the joint interzone. Immobilisation of chick embryos was achieved through treatment with the neuromuscular blocking agent Decamethonium Bromide. The effects on development of the knee joint were examined using a combination of computational modelling to predict alterations in biophysical stimuli, detailed morphometric analysis of 3D digital representations, cell proliferation assays and in situ hybridisation to examine the expression of a selected panel of genes known to regulate joint development. This work revealed the precise changes to shape, particularly in the distal femur, that occur in an altered mechanical environment, corresponding to predicted changes in the spatial and dynamic patterns of mechanical stimuli and region specific changes in cell proliferation rates. In addition, we show altered patterning of the emerging tissues of the joint interzone with the loss of clearly defined and organised cell territories revealed by loss of characteristic interzone gene expression and abnormal expression of cartilage markers. This work shows that local dynamic patterns of biophysical stimuli generated from muscle contractions in the embryo act as a source of positional information guiding patterning and morphogenesis of the developing knee joint.     

Dpp signaling activity requires Pentagone to scale with tissue size in the growing Drosophila wing imaginal disc

The wing of the fruit fly, Drosophila melanogaster, with its simple, two-dimensional structure, is a model organ well suited for a systems biology approach. The wing arises from an epithelial sac referred to as the wing imaginal disc, which undergoes a phase of massive growth and concomitant patterning during larval stages. The Decapentaplegic (Dpp) morphogen plays a central role in wing formation with its ability to co-coordinately regulate patterning and growth. Here, we asked whether the Dpp signaling activity scales, i.e. expands proportionally, with the growing wing imaginal disc. Using new methods for spatial and temporal quantification of Dpp activity and its scaling properties, we found that the Dpp response scales with the size of the growing tissue. Notably, scaling is not perfect at all positions in the field and the scaling of target gene domains is ensured specifically where they define vein positions. We also found that the target gene domains are not defined at constant concentration thresholds of the downstream Dpp activity gradients P-Mad and Brinker. Most interestingly, Pentagone, an important secreted feedback regulator of the pathway, plays a central role in scaling and acts as an expander of the Dpp gradient during disc growth.     

Differential proliferation rates generate patterns of mechanical tension that orient tissue growth

Orientation of cell divisions is a key mechanism of tissue morphogenesis. In the growing Drosophila wing imaginal disc epithelium, most of the cell divisions in the central wing pouch are oriented along the proximal–distal (P–D) axis by the Dachsous‐Fat‐Dachs planar polarity pathway. However, cells at the periphery of the wing pouch instead tend to orient their divisions perpendicular to the P–D axis despite strong Dachs polarization. Here, we show that these circumferential divisions are oriented by circumferential mechanical forces that influence cell shapes and thus orient the mitotic spindle. We propose that this circumferential pattern of force is not generated locally by polarized constriction of individual epithelial cells. Instead, these forces emerge as a global tension pattern that appears to originate from differential rates of cell proliferation within the wing pouch. Accordingly, we show that localized overgrowth is sufficient to induce neighbouring cell stretching and reorientation of cell division. Our results suggest that patterned rates of cell proliferation can influence tissue mechanics and thus determine the orientation of cell divisions and tissue shape.     

Integrating force-sensing and signaling pathways in a model for the regulation of wing imaginal disc size

The regulation of organ size constitutes a major unsolved question in developmental biology. The wing imaginal disc of Drosophila serves as a widely used model system to study this question. Several mechanisms have been proposed to have an impact on final size, but they are either contradicted by experimental data or they cannot explain a number of key experimental observations and may thus be missing crucial elements. We have modeled a regulatory network that integrates the experimentally confirmed molecular interactions underlying other available models. Furthermore, the network includes hypothetical interactions between mechanical forces and specific growth regulators, leading to a size regulation mechanism that conceptually combines elements of existing models, and can be understood in terms of a compression gradient model. According to this model, compression increases in the center of the disc during growth. Growth stops once compression levels in the disc center reach a certain threshold and the compression gradient drops below a certain level in the rest of the disc. Our model can account for growth termination as well as for the paradoxical observation that growth occurs uniformly in the presence of a growth factor gradient and non-uniformly in the presence of a uniform growth factor distribution. Furthermore, it can account for other experimental observations that argue either in favor or against other models. The model also makes specific predictions about the distribution of cell shape and size in the developing disc, which we were able to confirm experimentally.     

Effect of non-uniform thickness of samples in stress relaxation tests under unconfined compression of samples of articular discs

A precise information of the biomechanical properties of soft tissues is required to develop a suitable simulation model, with which the distribution of stress and strain in the complex structures can be estimated. Many soft tissues have been mechanically characterized by stress relaxation tests under unconfined or confined compression. In general, full-thickness samples are extracted to reduce the damage in the tissue as much as possible. However, it is not guaranteed that these samples have a uniform thickness or, in other words, planar parallel faces. In particular, in the articular disc of the temporomandibular joint, many studies can be found testing full-thickness samples for which that thickness is known to be non-uniform, while making the assumption of uniaxial stress state to extract the mechanical properties from those tests. That inaccuracy may have a strong influence in some cases and needs a profound revision. The main goal of this work is to quantify the error committed in that assumption and the influence of the variation of thickness on that error in a particular test: stress relaxation tests under unconfined compression. Based on this error and defining an allowable tolerance, a criterion is established to reject samples depending on their aspect ratio.     

Reciprocal roles for bowl and lines in specifying the peripodial epithelium and the disc proper of the Drosophila wing primordium

Central to embryonic development is the generation of molecular asymmetries across fields of undifferentiated cells. The Drosophila wing imaginal disc provides a powerful system with which to understand how such asymmetries are generated and how they contribute to formation of a complex structure. Early in development, the wing primordium is subdivided into a thin layer of peripodial epithelium (PE) and an apposing thickened layer of pseudostratified columnar epithelium (CE), known as the disc proper (DP). The DP gives rise to the wing blade, hinge and dorsal mesothorax, whereas the PE makes only a minor contribution to the ventral hinge and pleura. The mechanisms that generate this major asymmetry and its contribution to wing development are poorly understood. The Lines protein destabilizes the nuclear protein Bowl in ectodermal structures. Here, we show that Bowl accumulates in the PE from early stages of wing development and is absent from the DP. Broad inhibition of Bowl in the PE resulted in the replacement of the PE with a mirror image duplication of the DP. The failure to generate the PE severely compromised wing growth and the formation of the notum. Conversely, the activation of bowl in the DP (by removal or inhibition of lines function) resulted in the transformation of the DP into PE. Thus, we provide evidence that bowl and lines act as a binary switch to subdivide the wing primordium into PE and DP, and assign crucial roles for this asymmetry in wing growth and patterning.     

Mechanical Control of Organ Size in the Development of the Drosophila Wing Disc

Control of cessation of growth in developing organs has recently been proposed to be influenced by mechanical forces acting on the tissue due to its growth. In particular, it was proposed that stretching of the tissue leads to an increase in cell proliferation. Using the model system of the Drosophila wing imaginal disc, we directly stretch the tissue finding a significant increase in cell proliferation, thus confirming this hypothesis. In addition, we characterize the growth over the entire growth period of the wing disc finding a correlation between the apical cell area and cell proliferation rate.PACS numbers: 87.19.lx, 87.18.Nq, 87.80.Ek, 87.17.Ee, 87.85.Xd