Wing Epithelium Research Articles

Deep learning for rapid analysis of cell divisions in vivo during epithelial morphogenesis and repair.

Cell division is fundamental to all healthy tissue growth, as well as being rate-limiting in the tissue repair response to wounding and during cancer progression. However, the role that cell divisions play in tissue growth is a collective one, requiring the integration of many individual cell division events. It is particularly difficult to accurately detect and quantify multiple features of large numbers of cell divisions (including their spatio-temporal synchronicity and orientation) over extended periods of time. It would thus be advantageous to perform such analyses in an automated fashion, which can naturally be enabled using deep learning. Hence, we develop a pipeline of deep learning models that accurately identify dividing cells in time-lapse movies of epithelial tissues in vivo. Our pipeline also determines their axis of division orientation, as well as their shape changes before and after division. This strategy enables us to analyse the dynamic profile of cell divisions within the Drosophila pupal wing epithelium, both as it undergoes developmental morphogenesis and as it repairs following laser wounding. We show that the division axis is biased according to lines of tissue tension and that wounding triggers a synchronised (but not oriented) burst of cell divisions back from the leading edge.

Deep learning for rapid analysis of cell divisions in vivo during epithelial morphogenesis and repair

Cell division is fundamental to all healthy tissue growth, as well as being rate-limiting in the tissue repair response to wounding and during cancer progression. However, the role that cell divisions play in tissue growth is a collective one, requiring the integration of many individual cell division events. It is particularly difficult to accurately detect and quantify multiple features of large numbers of cell divisions (including their spatio-temporal synchronicity and orientation) over extended periods of time. It would thus be advantageous to perform such analyses in an automated fashion, which can naturally be enabled using deep learning. Hence, we develop a pipeline of deep learning models that accurately identify dividing cells in time-lapse movies of epithelial tissues in vivo. Our pipeline also determines their axis of division orientation, as well as their shape changes before and after division. This strategy enables us to analyse the dynamic profile of cell divisions within the Drosophila pupal wing epithelium, both as it undergoes developmental morphogenesis and as it repairs following laser wounding. We show that the division axis is biased according to lines of tissue tension and that wounding triggers a synchronised (but not oriented) burst of cell divisions back from the leading edge.

Dissociation of Drosophila Evi-Wg Complex Occurs Post Apical Internalization in the Maturing Acidic Endosomes.

Signaling pathways activated by secreted Wnt ligands play an essential role in tissue development and the progression of diseases, like cancer. Secretion of the lipid-modified Wnt proteins is tightly regulated by a repertoire of intracellular factors. For instance, a membrane protein, Evi, interacts with the Wnt ligand in the ER, and it is essential for its further trafficking and release in the extracellular space. After dissociating from the Wnt, the Wnt-unbound Evi is recycled back to the ER via Golgi. However, where in this trafficking path Wnt proteins dissociate from Evi remains unclear. Here, we have used the Drosophila wing epithelium to trace the route of the Evi-Wg (Wnt homolog) complex leading up to their separation. In these polarized cells, Wg is first trafficked to the apical surface; however, the secretion of Wg is believed to occurs post-internalization via recycling. Our results show that the Evi-Wg complex is internalized from the apical surface and transported to the retromer-positive endosomes. Furthermore, using antibodies that specifically label the Wnt-unbound Evi, we show that Evi and Wg separation occurs post-internalization in the acidic endosomes. These results refine our understanding of the polarized trafficking of Wg and highlight the importance of Wg endocytosis in its secondary secretion.

Deep learning reveals a damage signalling hierarchy that coordinates different cell behaviours driving wound re-epithelialisation.

One of the key tissue movements driving closure of a wound is re-epithelialisation. Earlier wound healing studies describe the dynamic cell behaviours that contribute to wound re-epithelialisation, including cell division, cell shape changes and cell migration, as well as the signals that might regulate these cell behaviours. Here, we have used a series of deep learning tools to quantify the contributions of each of these cell behaviours from movies of repairing wounds in the Drosophila pupal wing epithelium. We test how each is altered after knockdown of the conserved wound repair signals Ca2+ and JNK, as well as after ablation of macrophages that supply growth factor signals believed to orchestrate aspects of the repair process. Our genetic perturbation experiments provide quantifiable insights regarding how these wound signals impact cell behaviours. We find that Ca2+ signalling is a master regulator required for all contributing cell behaviours; JNK signalling primarily drives cell shape changes and divisions, whereas signals from macrophages largely regulate cell migration and proliferation. Our studies show deep learning to be a valuable tool for unravelling complex signalling hierarchies underlying tissue repair.

Evolution and development of Drosophila melanogaster under different thermal conditions affected cell sizes and sensitivity to paralyzing hypoxia

Environmental gradients cause evolutionary and developmental changes in the cellular composition of organisms, but the physiological consequences of these effects are not well understood. Here, we studied experimental populations of Drosophila melanogaster that had evolved in one of three selective regimes: constant 16 °C, constant 25 °C, or intergenerational shifts between 16 °C and 25 °C. Genotypes from each population were reared at three developmental temperatures (16 °C, 20.5 °C, and 25 °C). As adults, we measured thorax length and cell sizes in the Malpighian tubules and wing epithelia of flies from each combination of evolutionary and developmental temperatures. We also exposed flies from these treatments to a short period of nearly complete oxygen deprivation to measure hypoxia tolerance. For genotypes from any selective regime, development at a higher temperature resulted in smaller flies with smaller cells, regardless of the tissue. At every developmental temperature, genotypes from the warm selective regime had smaller bodies and smaller wing cells but had larger tubule cells than did genotypes from the cold selective regime. Genotypes from the fluctuating selective regime were similar in size to those from the cold selective regime, but their cells of either tissue were the smallest among the three regimes. Evolutionary and developmental treatments interactively affected a fly’s sensitivity to short-term paralyzing hypoxia. Genotypes from the cold selective regime were less sensitive to hypoxia after developing at a higher temperature. Genotypes from the other selective regimes were more sensitive to hypoxia after developing at a higher temperature. Our results show that thermal conditions can trigger evolutionary and developmental shifts in cell size, coupled with changes in body size and hypoxia tolerance. These patterns suggest links between the cellular composition of the body, levels of hypoxia within cells, and the energetic cost of tissue maintenance. However, the patterns can be only partially explained by existing theories about the role of cell size in tissue oxygenation and metabolic performance.

Nuclear position and local acetyl-CoA production regulate chromatin state

Histone acetylation regulates gene expression, cell function and cell fate1. Here we study the pattern of histone acetylation in the epithelial tissue of the Drosophila wing disc. H3K18ac, H4K8ac and total lysine acetylation are increased in the outer rim of the disc. This acetylation pattern is controlled by nuclear position, whereby nuclei continuously move from apical to basal locations within the epithelium and exhibit high levels of H3K18ac when they are in proximity to the tissue surface. These surface nuclei have increased levels of acetyl-CoA synthase, which generates the acetyl-CoA for histone acetylation. The carbon source for histone acetylation in the rim is fatty acid β-oxidation, which is also increased in the rim. Inhibition of fatty acid β-oxidation causes H3K18ac levels to decrease in the genomic proximity of genes involved in disc development. In summary, there is a physical mark of the outer rim of the wing and other imaginal epithelia in Drosophila that affects gene expression.

Maintenance of proteostasis by Drosophila Rer1 is essential for competitive cell survival and Myc-driven overgrowth.

Defects in protein homeostasis can induce proteotoxic stress, affecting cellular fitness and, consequently, overall tissue health. In various growing tissues, cell competition based mechanisms facilitate detection and elimination of these compromised, often referred to as 'loser', cells by the healthier neighbors. The precise connection between proteotoxic stress and competitive cell survival remains largely elusive. Here, we reveal the function of an endoplasmic reticulum (ER) and Golgi localized protein Rer1 in the regulation of protein homeostasis in the developing Drosophila wing epithelium. Our results show that loss of Rer1 leads to proteotoxic stress and PERK-mediated phosphorylation of eukaryotic initiation factor 2α. Clonal analysis showed that rer1 mutant cells are identified as losers and eliminated through cell competition. Interestingly, we find that Rer1 levels are upregulated upon Myc-overexpression that causes overgrowth, albeit under high proteotoxic stress. Our results suggest that increased levels of Rer1 provide cytoprotection to Myc-overexpressing cells by alleviating the proteotoxic stress and thereby supporting Myc-driven overgrowth. In summary, these observations demonstrate that Rer1 acts as a novel regulator of proteostasis in Drosophila and reveal its role in competitive cell survival.

Osiris17 is essential for stable integrin localization and function during insect wing epithelia remodeling

The dynamic adhesion between cells and their extracellular matrix is essential for the development and function of organs. During insect wing development, two epithelial sheets contact each other at their basal sites through the interaction of βPS integrins with the extracellular matrix. We report that Osiris17 contributes to the maintenance of βPS integrins localization and function in developing wing of Drosophila and locust. In flies with reduced Osiris17 expression the epithelia sheets fail to maintain the integrity of basal cytoplasmic junctional bridges and basal adhesion. In contrast to the continuous basal integrin localization in control wings, this localization is disrupted during late stages of wing development in Osiris17 depleted flies. In addition, the subcellular localization revealed that Osiris17 co-localizes with the endosomal markers Rab5 and Rab11. This observation suggests an involvement of Osiris17 in endosomal recycling of integrins. Indeed, Osiris17 depletion reduced the numbers of Rab5 and Rab11 positive endosomes. Moreover, overexpression of Osiris17 increased co-localization of Rab5 and βPS integrins and partially rescued the detachment phenotype in flies with reduced βPS integrins. Taken together, our data suggest that Osiris17 is an endosome related protein that contributes to epithelial remodeling and morphogenesis by assisting basal integrins localization in insects.

Programmed disassembly of a microtubule-based membrane protrusion network coordinates 3D epithelial morphogenesis in Drosophila.

Comprehensive analysis of cellular dynamics during the process of morphogenesis is fundamental to understanding the principles of animal development. Despite recent advancements in light microscopy, how successive cell shape changes lead to complex three-dimensional tissue morphogenesis is still largely unresolved. Using in vivo live imaging of Drosophila wing development, we have studied unique cellular structures comprising a microtubule-based membrane protrusion network. This network, which we name here the Interplanar Amida Network (IPAN), links the two wing epithelium leaflets. Initially, the IPAN sustains cell-cell contacts between the two layers of the wing epithelium through basal protrusions. Subsequent disassembly of the IPAN involves loss of these contacts, with concomitant degeneration of aligned microtubules. These processes are both autonomously and non-autonomously required for mitosis, leading to coordinated tissue proliferation between two wing epithelia. Our findings further reveal that a microtubule organization switch from non-centrosomal to centrosomal microtubule-organizing centers (MTOCs) at the G2/M transition leads to disassembly of non-centrosomal microtubule-derived IPAN protrusions. These findings exemplify how cell shape change-mediated loss of inter-tissue contacts results in 3D tissue morphogenesis.

α-Spectrin regulates cell shape changes during disassembly of microtubule-driven protrusions in Drosophila wings.

The dynamics of microtubule-mediated protrusions, termed Interplanar Amida Network (IPAN) in Drosophila pupal wing, involve cell shape changes. The molecular mechanisms underlying these processes are yet to be fully understood. This study delineates the stages of cell shape alterations during the disassembly of microtubule protrusions and underscores the pivotal role of α-Spectrin in driving these changes by regulating both the microtubule and actomyosin networks. Our findings also demonstrate that α-Spectrin is required for the apical relaxation of wing epithelia during protrusion disassembly, indicating its substantial contribution to the robustness of 3D tissue morphogenesis.

Frizzled2 receives WntA signaling during butterfly wing pattern formation.

Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes. Although the secreted ligand WntA has been shown to instruct the color pattern formation in butterflies, its mode of reception remains elusive. Butterfly genomes encode four homologs of the Frizzled-family of Wnt receptors. Here, we show that CRISPR mosaic knockouts of frizzled2 (fz2) phenocopy the color pattern effects of WntA loss of function in multiple nymphalids. Whereas WntA mosaic clones result in intermediate patterns of reduced size, fz2 clones are cell-autonomous, consistent with a morphogen function. Shifts in expression of WntA and fz2 in WntA crispant pupae show that they are under positive and negative feedback, respectively. Fz1 is required for Wnt-independent planar cell polarity in the wing epithelium. Fz3 and Fz4 show phenotypes consistent with Wnt competitive-antagonist functions in vein formation (Fz3 and Fz4), wing margin specification (Fz3), and color patterning in the Discalis and Marginal Band Systems (Fz4). Overall, these data show that the WntA/Frizzled2 morphogen-receptor pair forms a signaling axis that instructs butterfly color patterning and shed light on the functional diversity of insect Frizzled receptors.

Live Detection of Intracellular Chitin in Butterfly Wing Epithelial Cells In Vivo Using Fluorescent Brightener 28: Implications for the Development of Scales and Color Patterns.

Chitin is the major component of the extracellular cuticle and plays multiple roles in insects. In butterflies, chitin builds wing scales for structural colors. Here, we show that intracellular chitin in live cells can be detected in vivo with fluorescent brightener 28 (FB28), focusing on wing epithelial cells of the small lycaenid butterfly Zizeeria maha immediately after pupation. A relatively small number of cells at the apical surface of the epithelium were strongly FB28-positive in the cytosol and seemed to have extensive ER-Golgi networks, which may be specialized chitin-secreting cells. Some cells had FB28-positive tadpole-tail-like or rod-like structures relative to the nucleus. We detected FB28-positive hexagonal intracellular objects and their associated structures extending toward the apical end of the cell, which may be developing scale bases and shafts. We also observed FB28-positive fibrous intracellular structures extending toward the basal end. Many cells were FB28-negative in the cytosol, which contained FB28-positive dots or discs. The present data are crucial to understanding the differentiation of the butterfly wing epithelium, including scale formation and color pattern determination. The use of FB28 in probing intracellular chitin in live cells may be applicable to other insect systems.

Spatiotemporal remodeling of extracellular matrix orients epithelial sheet folding.

Biological systems are inherently noisy; however, they produce highly stereotyped tissue morphology. Drosophila pupal wings show a highly stereotypic folding through uniform expansion and subsequent buckling of wing epithelium within a surrounding cuticle sac. The folding pattern produced by buckling is generally stochastic; it is thus unclear how buckling leads to stereotypic tissue folding of the wings. We found that the extracellular matrix (ECM) protein, Dumpy, guides the position and direction of buckling-induced folds. Dumpy anchors the wing epithelium to the overlying cuticle at specific tissue positions. Tissue-wide alterations of Dumpy deposition and degradation yielded different buckling patterns. In summary, we propose that spatiotemporal ECM remodeling shapes stereotyped tissue folding through dynamic interactions between the epithelium and its external structures.

Cell death and wing reduction during the metamorphosis of sex-specific flightless morphs in winter geometrid moths.

Winter geometrid moths show striking sexual dimorphism by having female-specific flightless morphs. The evolutionary grades of wing reduction in winter geometrid moths vary and range from having short wings, vestigial wings, to being wingless. Although the ontogenetic processes underlying the development of the wingless or short-wing morphs in Lepidoptera has been well studied, the mechanisms underlying the development of vestigial wing morphs in winter geometrid moths during metamorphosis are poorly understood. In the winter geometrid moth Sebastosema bubonaria Warren, 1896, the males have functional wings, but the females have vestigial wings. Here, we studied the ontogenetic processes affecting wing reduction in the winter geometrid moth S. bubonaria using light microscopy and transmission electron microscopy, and compared the ontogenetic process of wing reduction in this species with that in another species of the wingless-female winter moth that we investigated previously. Our results showed that, in the vestigial-wing morphs, the loss of pupal wing epithelium was terminated in the middle of the wing degeneration process, whereas in the wingless morph, the pupal wing epithelium disappeared almost completely and the final appearance of the wings differed slightly among flightless morphs. We propose that the extent and location of cell death in the pupal wing play an important role in the various patterns of reduced wings that are observed in flightless moths.

Deciphering the roles of cell shape and Fat and Dachsous planar polarity in arranging the Drosophila apical microtubule network through quantitative image analysis.

In epithelial cells, planar polarization of subapical microtubule networks is thought to be important for both breaking cellular symmetry and maintaining the resulting cellular polarity. Studies in the Drosophila pupal wing and other tissues have suggested two alternative mechanisms for specifying network polarity. On one hand, mechanical strain and/or cell shape have been implicated as key determinants; on the other hand, the Fat-Dachsous planar polarity pathway has been suggested to be the primary polarizing cue. Using quantitative image analysis in the pupal wing, we reassess these models. We found that cell shape was a strong predictor of microtubule organization in the developing wing epithelium. Conversely, Fat-Dachsous polarity cues do not play any direct role in the organization of the subapical microtubule network, despite being able to weakly recruit the microtubule minus-end capping protein Patronin to cell boundaries. We conclude that any effect of Fat-Dachsous on microtubule polarity is likely to be indirect, via their known ability to regulate cell shape.

Attachment and detachment of cortical myosin regulates cell junction exchange during cell rearrangement in the Drosophila wing epithelium

Epithelial cells remodel cell adhesion and change their neighbors to shape a tissue. This cellular rearrangement proceeds in three steps: the shrinkage of a junction, exchange of junctions, and elongation of the newly generated junction. Herein, by combining live imaging and physical modeling, we showed that the formation of myosin-II (myo-II) cables around the cell vertices underlies the exchange of junctions in the Drosophila wing epithelium. The local and transient detachment of myo-II from the cell cortex is regulated by the LIM domain-containing protein Jub and the tricellular septate junction protein M6. Moreover, we found that M6 shifts to the adherens junction plane on jub RNAi and that Jub is persistently retained at reconnecting junctions in m6 RNAi cells. This interplay between Jub and M6 can depend on the junction length and thereby couples the detachment of cortical myo-II cables and the shrinkage/elongation of the junction during cell rearrangement. Furthermore, we developed a mechanical model based on the wetting theory and clarified how the physical properties of myo-II cables are integrated with the junction geometry to induce the transition between the attached and detached states and support the unidirectionality of cell rearrangement. Collectively, this study elucidates the orchestration of geometry, mechanics, and signaling for exchanging junctions.

Early cellular development induced by ecdysteroid in sex-specific wing degeneration of the wingless female winter moth.

The winter moth, Nyssiodes lefuarius, exhibits striking sexual dimorphism in wing form; males have functional wings of normal size, whereas females lack wings. We previously found that the steroid hormone 20-hydroxyecdysone (20E) triggered massive programmed cell death (PCD) only in the female pupal wing epithelium; however, when and how early sexual trait development of the pupal wings is initiated during pupal-adult metamorphosis remains obscure. To clarify the detailed morphological changes and mechanisms underlying early sexual trait development and cell death, we examined the effects of 20E on early ultrastructural and histological changes in the pupal wing epithelium of both sexes. Before the onset of adult differentiation, no morphological differences were observed in the epithelial cells of both sexes at an ultrastructural level. When 5.4µg of 20E was injected into pupae of both sexes at 15days after the onset of pupation, retraction of the wing epithelium from the pupal cuticle was initiated at day 2 after 20E injection in both sexes. Although overt degeneration of wing tissue was not still obvious, apoptotic body-like structures and auto-phagosomes were visible at day 3 after 20E injection in females, whereas development of scale precursor cells started on day 4 after injection in males. Our results suggest that (1) the injection of 20E induced sexually dimorphic changes in the pattern of organelle distribution in wing epithelial cells, and (2) abnormally shaped mitochondria in the cytoplasm of the female wing epithelium might be involved in the PCD that occurs during wing tissue degeneration.

Inferring the flow properties of epithelial tissues from their geometry

Amorphous materials exhibit complex material properties with strongly nonlinear behaviors. Below a yield stress they behave as plastic solids, while they start to yield above a critical stress Σc. A key quantity controlling plasticity which is, however, hard to measure is the density P(x) of weak spots, where x is the additional stress required for local plastic failure. In the thermodynamic limit P(x) ∼ x θ is singular at x = 0 in the solid phase below the yield stress Σc. This singularity is related to the presence of system spanning avalanches of plastic events. Here we address the question if the density of weak spots and the flow properties of a material can be determined from the geometry of an amorphous structure alone. We show that a vertex model for cell packings in tissues exhibits the phenomenology of plastic amorphous systems. As the yield stress is approached from above, the strain rate vanishes and the avalanches size S and their duration τ diverge. We then show that in general, in materials where the energy functional depends on topology, the value x is proportional to the length L of a bond that vanishes in a plastic event. For this class of models P(x) is therefore readily measurable from geometry alone. Applying this approach to a quantification of the cell packing geometry in the developing wing epithelium of the fruit fly, we find that in this tissue P(L) exhibits a power law with exponents similar to those found numerically for a vertex model in its solid phase. This suggests that this tissue exhibits plasticity and non-linear material properties that emerge from collective cell behaviors and that these material properties govern developmental processes. Our approach based on the relation between topology and energetics suggests a new route to outstanding questions associated with the yielding transition.

Morphological and histological examination of short-wing formation in the winter moth Protalcis concinnata (Insecta: Lepidoptera, Geometridae).

Winter geometrid moths exhibit sexual dimorphism in wing length and female-specific flightlessness. Female-specific flightlessness in insects is an interesting phenomenon in terms of sexual dimorphism and reproductive biology. In the winter geometrid moth, Protalcis concinnata (Wileman), adult females have short wings and adult males have fully developed wings. Although the developmental process for wing reduction in Lepidoptera is well studied, little is known about the morphology and the developmental pattern of short-winged flightless morphs in Lepidoptera. To clarify the precise mechanisms and developmental processes that produce short-winged morphs, we performed morphological and histological investigations of adult and pupal wing development in the winter geometrid moth P. concinnata. Our findings showed that (a) wing development in both sexes is similar until larval-pupal metamorphosis, (b) the shape of the sexually dimorphic wings is determined by the position of the bordering lacuna (BL), (c) the BL is positioned farther inward in females than in males, and (d) after the short pupal diapause period, the female pupal wing epithelium degenerates to approximately two-thirds its original size due to cell death. We propose that this developmental pattern is a previously unrecognized process among flightless Lepidoptera.

Ykt6-dependent endosomal recycling is required for Wnt secretion in the Drosophila wing epithelium.

ABSTRACTMorphogens are important signalling molecules for tissue development and their secretion requires tight regulation. In the wing imaginal disc of flies, the morphogen Wnt/Wingless is apically presented by the secreting cell and re-internalized before final long-range secretion. Why Wnt molecules undergo these trafficking steps and the nature of the regulatory control within the endosomal compartment remain unclear. Here, we have investigated how Wnts are sorted at the level of endosomes by the versatile v-SNARE Ykt6. Using in vivo genetics, proximity-dependent proteomics and in vitro biochemical analyses, we show that most Ykt6 is present in the cytosol, but can be recruited to de-acidified compartments and recycle Wnts to the plasma membrane via Rab4-positive recycling endosomes. Thus, we propose a molecular mechanism by which producing cells integrate and leverage endocytosis and recycling via Ykt6 to coordinate extracellular Wnt levels.