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Differentiation of the Drosophila compound eye from the eye imaginal disc is a progressive process: columns of cells successively differentiate in a posterior to anterior sequence, clusters of cells form at regularly spaced intervals within each column, and individual photoreceptors differentiate in a defined order within each cluster. The progress...
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... the anterior-posterior axis, this organization arises because signals from the photoreceptor axons induce the formation of their target cells. Hh, which drives the propagation of the MF within the eye disc, is also transported along the photoreceptor axons and released to induce the final division of lamina precursor cells (LPCs) and their expression of the differentiation marker Dachshund (Dac) (Huang and Kunes, 1996) (Figure 5). Interestingly, this axonal transport requires a targeting signal that lies within the C-terminal protease domain of Hh ( Chu et al., 2006), although LPCs respond to the N-terminal secreted domain through the canonical Hh signaling pathway . ...
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The Drosophila Hedgehog receptor functions to regulate the essential downstream pathway component, Smoothened, and to limit the range of signaling by sequestering Hedgehog protein signal within imaginal disc epithelium. Hedgehog receptor function requires both Patched and Ihog activity, the latter interchangeably encoded by interference hedgehog (i...
Precise planar cell polarity (PCP) is critical for the development of multiple organ systems in animals. A group of core-PCP proteins are recognized to play crucial roles in convergent extension and other PCP-related processes in mammals. However, the functions of another group of PCP-regulating proteins, the PCP-effector proteins, are yet to be fu...
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... In addition to Dpp (represented by the action of Rn) also Hh has a contribution to the differentiation of G cells [16,24,33], which is represented by the action of the Hh-producing R cells. Finally, the final differentiation, or transition from Rn into R, can be expressed as being proportional to Rn, indicating that Rn differentiates into R cells with a constant pace, simplifying a complex process of successive cell induction leading to the formation of the ommatidial cell types [34]. However, eye size does not depend on this latter process, as the number of ommatidia and, therefore, the size of the eye, is directly related to the number of G cells that are recruited as Rn cells. ...
Biological processes are intrinsically noisy, and yet, the result of development—like the species-specific size and shape of organs—is usually remarkably precise. This precision suggests the existence of mechanisms of feedback control that ensure that deviations from a target size are minimized. Still, we have very limited understanding of how these mechanisms operate. Here, we investigate the problem of organ size precision using the Drosophila eye. The size of the adult eye depends on the rates at which eye progenitor cells grow and differentiate. We first find that the progenitor net growth rate results from the balance between their proliferation and apoptosis, with this latter contributing to determining both final eye size and its variability. In turn, apoptosis of progenitor cells is hampered by Dpp, a BMP2/4 signaling molecule transiently produced by early differentiating retinal cells. Our genetic and computational experiments show how the status of retinal differentiation is communicated to progenitors through the differentiation-dependent production of Dpp, which, by adjusting the rate of apoptosis, exerts a feedback control over the net growth of progenitors to reduce final eye size variability.
... To perform cell tracking, we used cultured eye imaginal disc explants ( Figure 1b). The adult fly eye develops from a neuro-epithelium that progressively differentiates during the late third instar as a differentiation front, known as the morphogenetic furrow (MF), sweeps through the eye epithelium from posterior to anterior such that cells anterior to the MF are proliferative and nondifferentiated whereas cells posterior to the MF are differentiated, mostly post-mitotic and adopt characteristic cell shapes to form a highly ordered epithelium [16]. Upon heat-induced expression of the FLP, stochastic recombination of the FRT stop resulted in the expression of the nlsHalo protein. ...
Multiscale analysis of morphogenesis requires to follow and measure in real-time the in vivo behaviour of large numbers of individual cells over long period of time. Despite recent progress, the large-scale automated tracking of cells in developing embryos and tissues remains a challenge. Here we describe a genetic tool for the random and sparse labelling of individual cells in developing Drosophila tissues. This tool is based on the conditional expression of a nuclear HaloTag protein that can be fluorescently labelled upon the irreversible binding of a cell permeable synthetic ligand. While the slow maturation of genetically encoded fluorescent renders the tracking of individual cells difficult in rapidly dividing tissues, nuclear HaloTag proteins allowed for rapid labelling of individual cells in cultured imaginal discs. To study cell shape changes, we also produced an HaloTag version of the actin-bound protein LifeAct. Since sparse labelling facilitates cell tracking, nuclear HaloTag reporters will be useful for the single-cell analysis of fate dynamics in Drosophila tissues cultured ex vivo.
... The Drosophila eye develops from an epithelial imaginal disc during larval stages, which is initially composed of identical pluripotent precursor cells ahead of the morphogenetic furrow (MF) (upper left panel). As the MF sweeps across the disc from posterior to anterior, preclusters of differentiating cells start to emerge which will then develop into mature ommatidia (Cagan & Ready, 1989;Roignant & Treisman, 2009;Tomlinson & Ready, 1987). During differentiation and maturation, these clusters rotate 90°in opposite directions in the dorsal and ventral halves of the eye to establish the final mirror-symmetric pattern of ommatidia across the D/V midline ( Jenny, 2010;Mlodzik, 1999). ...
... During larval stages, the eye develops from an epithelial imaginal disc, which is initially composed of identical pluripotent precursor cells (Fig. 2). As a wave of cell proliferation and differentiation (called morphogenetic furrow, MF) travels across the disc from posterior to anterior, it leaves in its wake preclusters of differentiating cells that will mature into ommatidia (Cagan & Ready, 1989;Roignant & Treisman, 2009;Tomlinson & Ready, 1987). During differentiation and maturation, these clusters rotate 90°in opposite directions in the dorsal and ventral halves of the eye to establish the mirror-symmetric pattern of adult ommatidia across the D/V midline (Fig. 2) ( Jenny, 2010;Mlodzik, 1999). ...
The molecular complexes underlying planar cell polarity (PCP) were first identified in Drosophila through analysis of mutant phenotypes in the adult cuticle and the orientation of associated polarized protrusions such as wing hairs and sensory bristles. The same molecules are conserved in vertebrates and are required for the localization of polarized protrusions such as primary or sensory cilia and the orientation of hair follicles. Not only is PCP signaling required to align cellular structures across a tissue, it is also required to coordinate movement during embryonic development and adult homeostasis. PCP signaling allows cells to interpret positional cues within a tissue to move in the appropriate direction and to coordinate this movement with their neighbors. In this review we outline the molecular basis of the core Wnt-Frizzled/PCP pathway, and describe how this signaling orchestrates collective motility in Drosophila and vertebrates. Here we cover the paradigms of ommatidial rotation and border cell migration in Drosophila, and convergent extension in vertebrates. The downstream cell biological processes that underlie polarized motility include cytoskeletal reorganization, and adherens junctional and extracellular matrix remodeling. We discuss the contributions of these processes in the respective cell motility contexts. Finally, we address examples of individual cell motility guided by PCP factors during nervous system development and in cancer disease contexts.
... We expressed ERK-KTR under the control of Tubulin (Tub)-Gal4 and performed immunofluorescence against doubly phosphorylated ERK (dpERK) to detect endogenous, active ERK. In third instar larval eye-antennal imaginal discs, ERK activity is required for differentiation of photoreceptors (Roignant and Treisman, 2009). A band of apical constriction, called the morphogenetic furrow, progressively sweeps across the eye disc from posterior to anterior. ...
Extracellular signal-regulated kinase (ERK) lies downstream of a core signalling cascade that controls all aspects of development and adult homeostasis. Recent developments have led to new tools to image and manipulate the pathway. However, visualising ERK activity in vivo with high temporal resolution remains a challenge in Drosophila. We adapted a kinase translocation reporter (KTR) for use in Drosophila, which shuttles out of the nucleus when phosphorylated by ERK. We show that ERK-KTR faithfully reports endogenous ERK signalling activity in developing and adult tissues, and that it responds to genetic perturbations upstream of ERK. Using ERK-KTR in time-lapse imaging, we made two novel observations: firstly, sustained hyperactivation of ERK by expression of dominant-active epidermal growth factor receptor raised the overall level but did not alter the kinetics of ERK activity; secondly, the direction of migration of retinal basal glia correlated with their ERK activity levels, suggesting an explanation for the heterogeneity in ERK activity observed in fixed tissue. Our results show that KTR technology can be applied in Drosophila to monitor ERK activity in real-time and suggest that this modular tool can be further adapted to study other kinases.
This article has an associated First Person interview with the first author of the paper.
... In other systems, morphogenesis is driven by an individual wave traversing the tissue. For example, in the formation of bird feathers and Drosophila ommatidia, periodic spatial structures are specified following a traveling wavefront of gene expression that traverses the entire tissue (9,63,134). In micropatterned cultures of human embryonic stem cells that mimic the formation of the primitive streak, a wave of Wnt signaling has been shown to control the epithelial-to-mesenchymal transition (EMT) (99). ...
Embryonic development hinges on effective coordination of molecular events across space and time. Waves have recently emerged as constituting an ubiquitous mechanism that ensures rapid spreading of regulatory signals across embryos, as well as reliable control of their patterning, namely, for the emergence of body plan structures. In this article, we review a selection of recent quantitative work on signaling waves and present an overview of the theory of waves. Our aim is to provide a succinct yet comprehensive guiding reference for the theoretical frameworks by which signaling waves can arise in embryos. We start, then, from reaction–diffusion systems, both static and time dependent; move to excitable dynamics; and conclude with systems of coupled oscillators. We link these theoretical models to molecular mechanisms recently elucidated for the control of mitotic waves in early embryos, patterning of the vertebrate body axis, micropattern cultures, and bone regeneration. Our goal is to inspire experimental work that will advance theory in development and connect its predictions to quantitative biological observations.
Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Here, we consider a layer called disc proper, which is a single layer columnar epithelium [1]. During third instar larva development, it is transformed into an ordered structure of future photoreceptors via gene interactions and morphological transformations [2,3]. The photoreceptor specification proceeds in the posterior-to-anterior direction through the movement of an epithelial invagination (morphogenetic furrow, MF) along the disc (Fig. 1). ...
... As described above, eye disk differentiation is accompanied by the changes in mutual arrangement and shape of the cells. To analyze the mechanisms of photoreceptor specification, it is important: (1) to define cell boundaries and cell shape; (2) to determine the levels of gene expression in each cell with a high precision. All image processing procedures are implemented in the ProStack program developed by the authors [6]. ...
... Hh diffuses in the epithelial tissue up to and including the constricted cells in the morphogenetic furrow [46]. The propagation of the MF is mediated by Decapentaplegic and regulated by Hemothorax, Hairy and extra macrochaetae [46,[54][55][56]. In the model herein, a simplified gene regulatory network was used, see [57] for a model with a detailed treatment of the gene regulatory network. ...
Epithelial tissues constitute an exotic type of active matter with non-linear properties reminiscent of amorphous materials. In the context of a proliferating epithelium, modeled by the quasistatic vertex model, we identify novel discrete tissue scale rearrangements, i.e. cellular rearrangement avalanches, which are a form of collective cell movement. During the avalanches, the vast majority of cells retain their neighbors, and the resulting cellular trajectories are radial in the periphery, a vortex in the core. After the onset of these avalanches, the epithelial area grows discontinuously. The avalanches are found to be stochastic, and their strength is correlated with the density of cells in the tissue. Overall, avalanches redistribute accumulated local spatial pressure along the tissue. Furthermore, the distribution of avalanche magnitudes is found to obey a power law, with an exponent consistent with sheer induced avalanches in amorphous materials. To understand the role of avalanches in organ development, we simulate epithelial growth of the Drosophila eye disc during the third instar using a computational model, which includes both chemical and mechanistic signaling. During the third instar, the morphogenetic furrow (MF), a ~10 cell wide wave of apical area constriction propagates through the epithelium. These simulations are used to understand the details of the growth process, the effect of the MF on the growth dynamics on the tissue scale, and to make predictions for experimental observations. The avalanches are found to depend on the strength of the apical constriction of cells in the MF, with a stronger apical constriction leading to less frequent and more pronounced avalanches. The results herein highlight the dependence of simulated tissue growth dynamics on relaxation timescales, and serve as a guide for in vitro experiments.
... We expressed ERK-KTR under the control of Tubulin (Tub)-Gal4 and performed immunofluorescence against doubly phosphorylated ERK (dpERK) to detect endogenous, active ERK. In third instar larval eyeantennal imaginal discs, ERK activity is required for differentiation of photoreceptors (Roignant and Treisman, 2009). A band of apical constriction, called the morphogenetic furrow, progressively sweeps across the eye disc from posterior to anterior. ...
Extracellular Signal-Regulated Kinase (ERK) lies downstream of a core signalling cascade that controls all aspects of development and adult homeostasis. Recent developments have led to new tools to image and manipulate the pathway. However, visualising ERK activity in vivo with high temporal resolution remains a challenge in Drosophila. We adapted a kinase translocation reporter (KTR) for use in Drosophila, which shuttles out of the nucleus when phosphorylated by ERK. We show that ERK-KTR faithfully reports endogenous ERK signalling activity in developing and adult tissues, and that it responds to genetic perturbations upstream of ERK. Using ERK-KTR in time-lapse imaging, we made two novel observations: firstly, sustained hyperactivation of ERK by expression of dominant-active Epidermal Growth Factor Receptor raised the overall level but did not alter the kinetics of ERK activity; secondly, heterogeneity in ERK activity in retinal basal glia correlated with the direction of migration of individual cells. Our results show that KTR technology can be applied in Drosophila to monitor ERK activity in real-time and suggest that this modular tool can be further adapted to study other kinases.
Summary Statement
We describe a reporter to study the dynamics of ERK signalling in Drosophila, use it to measure signalling in individual cells over time, and monitor development.
... The early role of photoreceptor identity during development has been extensively studied. R1-R6s develop in three sequential pairs during eye development: R2/R5, then R3/R4 and last R1/R6 [12][13][14]. The R3/R4 pair is particularly important in breaking symmetry, including the 90˚rotation of R-cell clusters in the developing eye disc and the asymmetric trapezoidal arrangement of the adult ommatidia [15,16]. ...
A fascinating question in neuroscience is how ensembles of neurons, originating from different locations, extend to the proper place and by the right time to create precise circuits. Here, we investigate this question in the Drosophila visual system, where photoreceptors re-sort in the lamina to form the crystalline-like neural superposition circuit. The repeated nature of this circuit allowed us to establish a data-driven, standardized coordinate system for quantitative comparison of sparsely perturbed growth cones within and across specimens. Using this common frame of reference, we investigated the extension of the R3 and R4 photoreceptors, which is the only pair of symmetrically arranged photoreceptors with asymmetric target choices. Specifically, we found that extension speeds of the R3 and R4 growth cones are inherent to their cell identities. The ability to parameterize local regularity in tissue organization facilitated the characterization of ensemble cellular behaviors and dissection of mechanisms governing neural circuit formation.
... Movie 5). The eye disc is highly dynamic with multiple developmental events compressed in a strong temporal gradient (Roignant and Treisman, 2009, Tsachaki and Sprecher, 2012, Wolff and Ready, 1991. Accordingly, we focused on Ca 2+ activities in the latter half of pupal development, when pattern formation is complete (Cagan and Ready, 1989) suggesting that the construction of the entire IOC wave network took place within this time. ...
Actomyosin contraction shapes the Drosophila eye's panoramic view. The convex curvature of the retinal epithelium, organized in ∼800 close-packed ommatidia, depends upon a fourfold condensation of the retinal floor mediated by contraction of actin stress fibers in the endfeet of interommatidial cells (IOCs). How these tensile forces are coordinated is not known. Here, we discover a previously unobserved phenomenon: Ca2+ waves regularly propagate across the IOC network in pupal and adult eyes. Genetic evidence demonstrates that IOC waves are independent of phototransduction, but require the inositol 1,4,5-triphosphate receptor (IP3R), suggesting that these waves are mediated by Ca2+ releases from endoplasmic reticulum stores. Removal of IP3R disrupts stress fibers in IOC endfeet and increases the basal retinal surface by ∼40%, linking IOC waves to facilitation of stress fiber contraction and floor morphogenesis. Furthermore, IP3R loss disrupts the organization of a collagen IV network underneath the IOC endfeet, implicating the extracellular matrix and its interaction with stress fibers in eye morphogenesis. We propose that coordinated cytosolic Ca2+ increases in IOC waves promote stress fiber contractions, ensuring an organized application of the planar tensile forces that condense the retinal floor.
This article has an associated ‘The people behind the papers’ interview.
