Hydrodynamic stress and phenotypic plasticity of the zebrafish regenerating fin.

Understanding how extrinsic factors modulate genetically encoded information to produce a specific phenotype is of prime scientific interest. In particular, the feedback mechanism between abiotic forces and locomotory organs during morphogenesis to achieve efficient movement is a highly relevant example of such modulation. The study of this developmental process can provide unique insights on the transduction of cues at the interface between physics and biology. Here, we take advantage of the natural ability of adult zebrafish to regenerate their amputated fins to assess its morphogenic plasticity upon external modulations. Using a variety of surgical and chemical treatments, we could induce phenotypic responses to the structure of the fin. Through the ablation of specific rays in regenerating caudal fins, we generated artificially narrowed appendages in which the fin cleft depth and the positioning of rays bifurcations were perturbed compared with normal regenerates. To dissect the role of mechanotransduction in this process, we investigated the patterns of hydrodynamic forces acting on the surface of a zebrafish fin during regeneration by using particle tracking velocimetry on a range of biomimetic hydrofoils. This experimental approach enabled us to quantitatively compare hydrodynamic stress distributions over flapping fins of varying sizes and shapes. As a result, viscous shear stress acting on the distal margin of regenerating fins and the resulting internal tension are proposed as suitable signals for guiding the regulation of ray growth dynamics and branching pattern. Our findings suggest that mechanical forces are involved in the fine-tuning of the locomotory organ during fin morphogenesis.

Correction: Long-term C. elegans immobilization enables high resolution developmental studies in vivo.

Correction for 'Long-term C. elegans immobilization enables high resolution developmental studies in vivo' by Simon Berger et al., Lab Chip, 2018, 18, 1359-1368.

Long-term C. elegans immobilization enables high resolution developmental studies in vivo.

Live-imaging of C. elegans is essential for the study of conserved cellular pathways (e.g. EGFR/Wnt signaling) and morphogenesis in vivo. However, the usefulness of live imaging as a research tool has been severely limited by the need to immobilize worms prior to and during imaging. Conventionally, immobilization is achieved by employing both physical and chemical interventions. These are known to significantly affect many physiological processes, and thus limit our understanding of dynamic developmental processes. Herein we present a novel, easy-to-use microfluidic platform for the long-term immobilization of viable, normally developing C. elegans, compatible with image acquisition at high resolution, thereby overcoming the limitations associated with conventional worm immobilization. The capabilities of the platform are demonstrated through the continuous assessment of anchor cell (AC) invasion and distal tip cell (DTC) migration in larval C. elegans and germ cell apoptosis in adult C. elegans in vivo for the first time.

In vivo quantification of mechanical properties of caudal fins in adult zebrafish.

The caudal fins of adult zebrafish are supported by multiple bony rays that are laterally interconnected by soft interray tissue. Little is known about the fin's mechanical properties that influence bending in response to hydrodynamic forces during swimming. Here, we developed an experimental setup to measure the elastic properties of caudal fins in vivo by applying micro-Newton forces to obtain bending stiffness and a tensional modulus. We detected overall bending moments of 1.5×10-9-4×10-9 N m2 along the proximal-distal axis of the appendage showing a non-monotonous pattern that was not due to the geometry of the fin itself. Surgical disruption of the interray tissues along the proximal-distal axis revealed no significant changes to the overall bending stiffness, which we confirmed by determining a tensional modulus of the interray tissue. Thus, the biophysical values suggest that the flexibility of the fin during its hydrodynamic performance predominantly relies on the mechanical properties of the rays.

Endoglin controls blood vessel diameter through endothelial cell shape changes in response to haemodynamic cues.

The hierarchical organization of properly sized blood vessels ensures the correct distribution of blood to all organs of the body, and is controlled via haemodynamic cues. In current concepts, an endothelium-dependent shear stress set point causes blood vessel enlargement in response to higher flow rates, while lower flow would lead to blood vessel narrowing, thereby establishing homeostasis. We show that during zebrafish embryonic development increases in flow, after an initial expansion of blood vessel diameters, eventually lead to vessel contraction. This is mediated via endothelial cell shape changes. We identify the transforming growth factor beta co-receptor endoglin as an important player in this process. Endoglin mutant cells and blood vessels continue to enlarge in response to flow increases, thus exacerbating pre-existing embryonic arterial-venous shunts. Together, our data suggest that cell shape changes in response to biophysical cues act as an underlying principle allowing for the ordered patterning of tubular organs.

MorphoGraphX: A platform for quantifying morphogenesis in 4D.

Morphogenesis emerges from complex multiscale interactions between genetic and mechanical processes. To understand these processes, the evolution of cell shape, proliferation and gene expression must be quantified. This quantification is usually performed either in full 3D, which is computationally expensive and technically challenging, or on 2D planar projections, which introduces geometrical artifacts on highly curved organs. Here we present MorphoGraphX ( www.MorphoGraphX.org), a software that bridges this gap by working directly with curved surface images extracted from 3D data. In addition to traditional 3D image analysis, we have developed algorithms to operate on curved surfaces, such as cell segmentation, lineage tracking and fluorescence signal quantification. The software's modular design makes it easy to include existing libraries, or to implement new algorithms. Cell geometries extracted with MorphoGraphX can be exported and used as templates for simulation models, providing a powerful platform to investigate the interactions between shape, genes and growth.

The Triple-Repeat Protein Anakonda Controls Epithelial Tricellular Junction Formation in Drosophila.

In epithelia, specialized tricellular junctions (TCJs) mediate cell contacts at three-cell vertices. TCJs are fundamental to epithelial biology and disease, but only a few TCJ components are known, and how they assemble at tricellular vertices is not understood. Here we describe a transmembrane protein, Anakonda (Aka), which localizes to TCJs and is essential for the formation of tricellular, but not bicellular, junctions in Drosophila. Loss of Aka causes epithelial barrier defects associated with irregular TCJ structure and geometry, suggesting that Aka organizes cell corners. Aka is necessary and sufficient for accumulation of Gliotactin at TCJs, suggesting that Aka initiates TCJ assembly by recruiting other proteins to tricellular vertices. Aka's extracellular domain has an unusual tripartite repeat structure that may mediate self-assembly, directed by the geometry of tricellular vertices. Conversely, Aka's cytoplasmic tail is dispensable for TCJ localization. Thus, extracellular interactions, rather than TCJ-directed intracellular transport, appear to mediate TCJ assembly.

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.

In-vivo imaging of the Drosophila wing imaginal disc over time: novel insights on growth and boundary formation.

In developmental biology, the sequence of gene induction and pattern formation is best studied over time as an organism develops. However, in the model system of Drosophila larvae this oftentimes proves difficult due to limitations in imaging capabilities. Using the larval wing imaginal disc, we show that both overall growth, as well as the creation of patterns such as the distinction between the anterior(A) and posterior(P) compartments and the dorsal(D) and ventral(V) compartments can be studied directly by imaging the wing disc as it develops inside a larva. Imaged larvae develop normally, as can be seen by the overall growth curve of the wing disc. Yet, the fact that we can follow the development of individual discs through time provides the opportunity to simultaneously assess individual variability. We for instance find that growth rates can vary greatly over time. In addition, we observe that mechanical forces act on the wing disc within the larva at times when there is an increase in growth rates. Moreover, we observe that A/P boundary formation follows the established sequence and a smooth boundary is present from the first larval instar on. The division of the wing disc into a dorsal and a ventral compartment, on the other hand, develops quite differently. Contrary to expectation, the specification of the dorsal compartment starts with only one or two cells in the second larval instar and a smooth boundary is not formed until the third larval instar.

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.

Cell-sorting at the A/P boundary in the Drosophila wing primordium: a computational model to consolidate observed non-local effects of Hh signaling.

Non-intermingling, adjacent populations of cells define compartment boundaries; such boundaries are often essential for the positioning and the maintenance of tissue-organizers during growth. In the developing wing primordium of Drosophila melanogaster, signaling by the secreted protein Hedgehog (Hh) is required for compartment boundary maintenance. However, the precise mechanism of Hh input remains poorly understood. Here, we combine experimental observations of perturbed Hh signaling with computer simulations of cellular behavior, and connect physical properties of cells to their Hh signaling status. We find that experimental disruption of Hh signaling has observable effects on cell sorting surprisingly far from the compartment boundary, which is in contrast to a previous model that confines Hh influence to the compartment boundary itself. We have recapitulated our experimental observations by simulations of Hh diffusion and transduction coupled to mechanical tension along cell-to-cell contact surfaces. Intriguingly, the best results were obtained under the assumption that Hh signaling cannot alter the overall tension force of the cell, but will merely re-distribute it locally inside the cell, relative to the signaling status of neighboring cells. Our results suggest a scenario in which homotypic interactions of a putative Hh target molecule at the cell surface are converted into a mechanical force. Such a scenario could explain why the mechanical output of Hh signaling appears to be confined to the compartment boundary, despite the longer range of the Hh molecule itself. Our study is the first to couple a cellular vertex model describing mechanical properties of cells in a growing tissue, to an explicit model of an entire signaling pathway, including a freely diffusible component. We discuss potential applications and challenges of such an approach.

Exploring the effects of mechanical feedback on epithelial topology.

Apical cell surfaces in metazoan epithelia, such as the wing disc of Drosophila, resemble polygons with different numbers of neighboring cells. The distribution of these polygon numbers has been shown to be conserved. Revealing the mechanisms that lead to this topology might yield insights into how the structural integrity of epithelial tissues is maintained. It has previously been proposed that cell division alone, or cell division in combination with cell rearrangements, is sufficient to explain the observed epithelial topology. Here, we extend this work by including an analysis of the clustering and the polygon distribution of mitotic cells. In addition, we study possible effects of cellular growth regulation by mechanical forces, as such regulation has been proposed to be involved in wing disc size regulation. We formulated several theoretical scenarios that differ with respect to whether cell rearrangements are allowed and whether cellular growth rates are dependent on mechanical stress. We then compared these scenarios with experimental data on the polygon distribution of the entire cell population, that of mitotic cells, as well as with data on mitotic clustering. Surprisingly, we observed considerably less clustering in our experiments than has been reported previously. Only scenarios that include mechanical-stress-dependent growth rates are in agreement with the experimental data. Interestingly, simulations of these scenarios showed a large decrease in rearrangements and elimination of cells. Thus, a possible growth regulation by mechanical force could have a function in releasing the mechanical stress that evolves when all cells have similar growth rates.

Determination of mechanical stress distribution in Drosophila wing discs using photoelasticity.

Morphogenesis, the process by which all complex biological structures are formed, is driven by an intricate interplay between genes, growth, as well as intra- and intercellular forces. While the expression of different genes changes the mechanical properties and shapes of cells, growth exerts forces in response to which tissues, organs and more complex structures are shaped. This is exemplified by a number of recent findings for instance in meristem formation in Arabidopsis and tracheal tube formation in Drosophila. However, growth not only generates forces, mechanical forces can also have an effect on growth rates, as is seen in mammalian tissues or bone growth. In fact, mechanical forces can influence the expression levels of patterning genes, allowing control of morphogenesis via mechanical feedback. In order to study the connections between mechanical stress, growth control and morphogenesis, information about the distribution of stress in a tissue is invaluable. Here, we applied stress-birefringence to the wing imaginal disc of Drosophila melanogaster, a commonly used model system for organ growth and patterning, in order to assess the stress distribution present in this tissue. For this purpose, stress-related differences in retardance are measured using a custom-built optical set-up. Applying this method, we found that the stresses are inhomogeneously distributed in the wing disc, with maximum compression in the centre of the wing pouch. This compression increases with wing disc size, showing that mechanical forces vary with the age of the tissue. These results are discussed in light of recent models proposing mechanical regulation of wing disc growth.

Model for the regulation of size in the wing imaginal disc of Drosophila.

For animal development it is necessary that organs stop growing after they reach a certain size. However, it is still largely unknown how this termination of growth is regulated. The wing imaginal disc of Drosophila serves as a commonly used model system to study the regulation of growth. Paradoxically, it has been observed that growth occurs uniformly throughout the disc, even though Decapentaplegic (Dpp), a key inducer of growth, forms a gradient. Here, we present a model for the control of growth in the wing imaginal disc, which can account for the uniform occurrence and termination of growth. A central feature of the model is that net growth is not only regulated by growth factors, but by mechanical forces as well. According to the model, growth factors like Dpp induce growth in the center of the disc, which subsequently causes a tangential stretching of surrounding peripheral regions. Above a certain threshold, this stretching stimulates growth in these peripheral regions. Since the stretching is not completely compensated for by the induced growth, the peripheral regions will compress the center of the disc, leading to an inhibition of growth in the center. The larger the disc, the stronger this compression becomes and hence the stronger the inhibiting effect. Growth ceases when the growth factors can no longer overcome this inhibition. With numerical simulations we show that the model indeed yields uniform growth. Furthermore, the model can also account for other experimental data on growth in the wing disc.

Comment on "Precise domain specification in the developing Drosophila embryo".

In a recent paper, Houchmandzadeh [Phys. Rev. E 72, 061920 (2005)] introduce a correlated bigradient model in order to explain the robust scaling of the boundary of hunchback (hb) expression in the early Drosophila embryo. In particular, they stress that recent experiments by Lucchetta [Nature (London) 434, 1134 (2005)], where embryos whose anterior and posterior halves develop at different temperatures still show excellent precision in the hb boundary, are in good agreement with such a model. We would like to show here that this conclusion is unwarranted. This is because the experiments of Lucchetta were done at different temperatures from those studied in the model. Since in other temperature combinations the model does not produce precise boundaries and there are no systematic trends in these deviations, a comparison to the experiment is not possible. Furthermore, we would like to point out that any correlated bigradient model should also take into account the fluctuations of the bicoid profile within an embryo. When forming correlated bigradients of experimental profiles from an online library, we observe that these intraembryo variations destroy robustness of the hb boundary even in the wild-type situation.

Model for the robust establishment of precise proportions in the early Drosophila embryo.

During embryonic development, a spatial pattern is formed in which proportions are established precisely. As an early pattern formation step in Drosophila embryos, an anterior-posterior gradient of Bicoid (Bcd) induces hunchback (hb) expression (Nature 337 (1989) 138; Nature 332 (1988) 281). In contrast to the Bcd gradient, the Hb profile includes information about the scale of the embryo. Furthermore, the resulting hb expression pattern shows a much lower embryo-to-embryo variability than the Bcd gradient (Nature 415 (2002) 798). An additional graded posterior repressing activity could theoretically account for the observed scaling. However, we show that such a model cannot produce the observed precision in the Hb boundary, such that a fundamentally different mechanism must be at work. We describe and simulate a model that can account for the observed precise generation of the scaled Hb profile in a highly robust manner. The proposed mechanism includes Staufen (Stau), an RNA binding protein that appears essential to precision scaling (Nature 415 (2002) 798). In the model, Stau is released from both ends of the embryo and relocalizes hb RNA by increasing its mobility. This leads to an effective transport of hb away from the respective Stau sources. The balance between these opposing effects then gives rise to scaling and precision. Considering the biological importance of robust precision scaling and the simplicity of the model, the same principle may be employed more often during development.

Digital learning material for student-directed model building in molecular biologyS.

The building of models to explain data and make predictions constitutes an important goal in molecular biology research. To give students the opportunity to practice such model building, two digital cases had previously been developed in which students are guided to build a model step by step. In this article, the development and initial evaluation of a third digital case is described. It concerns the selection of bristles during Drosophila development. To mimic a real research situation in a more realistic way, students are given much more freedom while building their models and can thus follow their own model-building approach. At the same time, however, students are provided with a sufficient amount of support to ensure that they can build their models without the requirement of intensive supervision.