Tinri Aegerter-Wilmsen

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.

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 softwares 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. DOI: http://dx.doi.org/10.7554/eLife.05864.001

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.

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.

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.

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

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.

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. Akas extracellular domain has an unusual tripartite repeat structure that may mediate self-assembly, directed by the geometry of tricellular vertices. Conversely, Akas cytoplasmic tail is dispensable for TCJ localization. Thus, extracellular interactions, rather than TCJ-directed intracellular transport, appear to mediate TCJ assembly.

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.

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

ABSTRACT 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 fins 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. Summary: The quantitative in vivo determination of the zebrafish caudal fins main constituents (bony rays and interray tissue) shows that flexibility is dominated by the elastic properties of the bony rays, whereas the elastic properties of the interray tissue co-define the fins complex 3D deformation during swimming and will also be needed as a crucial input for hydrodynamic simulations.