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SUMMARYPost-developmental organ resizing improves organismal fitness under constantly changing nutrient environments. Although stem cell abundance is a fundamental determinant of adaptive resizing, our understanding of its underlying mechanisms remains primarily limited to the regulation of stem cell division. Here we demonstrate that nutrient fluctuation induces dedifferentiation in theDrosophilaadult midgut to drive adaptive intestinal growth. From lineage tracing and single-cell RNA-sequencing, we identify a subpopulation of enteroendocrine cells (EEs) that convert into functional intestinal stem cells (ISCs) in response to dietary glucose and amino acids by activating the JAK-STAT pathway. Genetic ablation of EE-derived ISCs severely impairs ISC expansion and midgut growth despite the retention of resident ISCs, andin silicomodeling further indicates that EE dedifferentiation enables efficient increase in the midgut cell number while maintaining epithelial cell composition. Our findings uncover a physiologically-induced dedifferentiation that ensures ISC expansion during adaptive organ growth in concert with nutrient conditions.

Signal transduction by the Toll-like receptors (TLRs) is conserved and essential for innate immunity in metazoans. The founding member of the TLR family

Estimation of dynamic change of crossbridge formation in living cardiomyocytes is expected to provide crucial information for elucidating cardiomyopathy mechanisms, efficacy of an intervention, and other parameters. Here, we developed an assay system to dynamically measure second harmonic generation (SHG) polarization in pulsating cardiomyocyte and proved that the SHG anisotropy derived from myosin filaments in disease-model cardiomyocytes depended on their crossbridge status, providing an evaluation method for myosin force generation. Experiments utilizing an inheritable mutation that induces excessive myosin-actin interactions revealed that the correlation between sarcomere length and SHG anisotropy represents crossbridge formation ratio during pulsation. Furthermore, the present method found that ultraviolet irradiation induced an increased population of attached crossbridges that lost force-generating ability upon myocardial differentiation, causing acquired dysfunction. Taking an advantage of infrared two-photon excitation in SHG microscopy, myocardial dysfunction could be intravitally evaluated in aDrosophiladisease model. Thus, along with the establishment of the methodology, we successfully demonstrated the applicability and effectiveness of the present method to evaluate the actomyosin activity of a drug or genetic defect on living cardiomyocytes.

The epithelium is one of the best studied tissues for morphogenesis, pattern formation, cell polarity, cell division, cell competition, tumorigenesis, and metastatic behaviors. However, it has been challenging to analyze real-time cell interactions or cell dynamics within the epithelia under physiological conditions. The Drosophila pupal abdominal epidermis is a model system that allows to combine long-term real-time imaging under physiological conditions with the use of powerful Drosophila genetics tools. The abdominal epidermis displays a wide range of stereotypical characteristics of the epithelia and cellular behaviors including cell division, cell death, cell rearrangement, apical constriction, and apicobasal/planar polarity, making this tissue a first choice for the study of epithelial morphogenesis and relevant phenomena. In this chapter, I describe the staging and mounting of pupae and the live imaging of the abdominal epidermis. Moreover, methods to combine live imaging with mosaic analysis or drug injection will be presented. The long-term live imaging of the pupal abdominal epidermis is straightforward and opens up the possibility to analyze cell dynamics during epithelial morphogenesis at an unprecedented resolution.

Maintaining lineage restriction boundaries in proliferating tissues is vital to animal development. A long-standing thermodynamics theory, the differential adhesion hypothesis, attributes cell sorting phenomena to differentially expressed adhesion molecules. However, the contribution of the differential adhesion system during tissue morphogenesis has been unsubstantiated despite substantial theoretical support. Here, we report that Toll-1, a transmembrane receptor protein, acts as a differentially expressed adhesion molecule that straightens the fluctuating anteroposterior compartment boundary in the abdominal epidermal epithelium of the Drosophila pupa. Toll-1 is expressed across the entire posterior compartment under the control of the selector gene engrailed and displays a sharp expression boundary that coincides with the compartment boundary. Toll-1 corrects local distortions of the boundary in the absence of cable-like Myosin II enrichment along the boundary. The reinforced adhesion of homotypic cell contacts, together with pulsed cell contraction, achieves a biased vertex sliding action by resisting the separation of homotypic cell contacts in boundary cells. This work reveals a self-organizing system that integrates a differential adhesion system with pulsed contraction of cells to maintain lineage restriction boundaries., The differential adhesion hypothesis is proposed to play a role during epithelial tissue morphogenesis but it has remained unclear. Here, the authors identify the Toll-1 receptor as a differentially expressed adhesion molecule that maintains lineage restriction boundaries in the Drosophila epidermal epithelium.

The complex shapes of animal bodies are constructed through a sequence of simple physical interactions of constituent cells. Mechanical forces generated by cellular activities, such as division, death, shape change and rearrangement, drive tissue morphogenesis. By confining assembly or disassembly of actomyosin networks within the three-dimensional space of the cell, cells can localize forces to induce tissue deformation. Tissue-scale morphogenesis emerges from a collective behavior of cells that coordinates the force generation in space and time. Thus, the molecular mechanisms that govern the temporal and spatial regulation of forces in individual cells are elemental to organogenesis, and the tissue-scale coordination of forces generated by individual cells is key to determining the final shape of organs.

Epithelial tissues undergo cell turnover both during development and for homeostatic maintenance. Cells no longer needed are quickly removed without compromising barrier function of the tissue. During metamorphosis, insects undergo developmentally programed tissue remodeling. However, the mechanisms that regulate this rapid tissue remodeling are not precisely understood. Here, we show that the temporal dynamics of endocytosis modulate physiological cell properties to potentiate larval epidermal cells for cell elimination. Endocytic activity gradually reduces as tissue remodeling progresses. This reduced endocytic activity accelerates cell elimination through the regulation of Myosin II subcellular reorganization, junctional E-cadherin levels, and caspase activation. Whereas the increased Myosin II dynamics accelerates cell elimination, E-cadherin rather plays a protective role against cell elimination. Reduced E-cadherin is involved in the amplification of caspase activation by forming a positive feedback loop with caspase. These findings reveal the role of endocytosis in preventing cell elimination and in the cell property switching initiated by the temporal dynamics of endocytic activity to achieve rapid cell elimination during tissue remodeling.

Epithelial tissues undergo cell turnover both during development and for homeostatic maintenance. Cells that are no longer needed are quickly removed without compromising the barrier function of the tissue. During metamorphosis, insects undergo developmentally programmed tissue remodeling. However, the mechanisms that regulate this rapid tissue remodeling are not precisely understood. Here, we show that the temporal dynamics of endocytosis modulate physiological cell properties to prime larval epidermal cells for cell elimination. Endocytic activity gradually reduces as tissue remodeling progresses. This reduced endocytic activity accelerates cell elimination through the regulation of Myosin II subcellular reorganization, junctional E-cadherin levels, and caspase activation. Whereas the increased Myosin II dynamics accelerates cell elimination, E-cadherin plays a protective role against cell elimination. Reduced E-cadherin is involved in the amplification of caspase activation by forming a positive-feedback loop with caspase. These findings reveal the role of endocytosis in preventing cell elimination and in the cell-property switching initiated by the temporal dynamics of endocytic activity to achieve rapid cell elimination during tissue remodeling.

Tissue organization requires the interplay between biochemical signaling and cellular force generation. The formation of straight boundaries separating cells with different fates into compartments is important for growth and patterning during tissue development. In the developing Drosophila wing disc, maintenance of the straight anteroposterior (AP) compartment boundary involves a local increase in mechanical tension at cell bonds along the boundary. The biochemical signals that regulate mechanical tension along the AP boundary, however, remain unknown. Here, we show that a local difference in Hedgehog signal transduction activity between anterior and posterior cells is necessary and sufficient to increase mechanical tension along the AP boundary. This difference in Hedgehog signal transduction is also required to bias cell rearrangements during cell intercalations to keep the characteristic straight shape of the AP boundary. Moreover, severing cell bonds along the AP boundary does not reduce tension at neighboring bonds, implying that active mechanical tension is upregulated, cell bond by cell bond. Finally, differences in the expression of the homeodomain-containing protein Engrailed also contribute to the straight shape of the AP boundary, independently of Hedgehog signal transduction and without modulating cell bond tension. Our data reveal a novel link between local differences in Hedgehog signal transduction and a local increase in active mechanical tension of cell bonds that biases junctional rearrangements. The large-scale shape of the AP boundary thus emerges from biochemical signals inducing patterns of active tension on cell bonds.

During animal development groups of cells with similar fates and functions often stay together and separate from cells with different fates. An example for this cellular behavior is the formation of compartments, groups of cells with similar fates that are separated by sharp boundaries from neighboring groups of cells. Compartments play important roles during patterning by serving as units of growth and gene expression. Boundaries between compartments are associated with organizers that secrete signaling molecules instructing growth and differentiation throughout the tissue. The straight shape of the boundary between compartments is important for maintaining the position and shape of the organizer and thus for precise patterning. The straight shape of compartment boundaries, however, is challenged by cell divisions and cell intercalations that take place in many developing tissues. Early work established a role for selector genes and signaling pathways in setting up and keeping boundaries straight. Recent work in Drosophila has now begun to further unravel the physical and cellular mechanisms that maintain compartment boundaries. Key to the separation of compartments is a local increase of actomyosin-dependent mechanical tension at cell junctions along the boundary. Increased mechanical tension acts as a barrier to cell mixing during cell division and influences cell rearrangements during cell intercalations along the compartment boundary in a way that the straight shape of the boundary is maintained. An important question for the future is how the signaling pathways that maintain the straight shape of compartment boundaries control mechanical tension along these boundaries. WIREs Dev Biol 2015, 4:407–417. doi: 10.1002/wdev.178 For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article.

During the initial stage of tumor progression, oncogenic cells spread despite spatial confinement imposed by surrounding normal tissue. This spread of oncogenic cells (winners) is thought to be governed by selective killing of surrounding normal cells (losers) through a phenomenon called "cell competition" (i.e., supercompetition). Although the mechanisms underlying loser elimination are increasingly apparent, it is not clear how winner cells selectively occupy the space made available following loser apoptosis. Here, we combined live imaging analyses of two different oncogenic clones (Yki/YAP activation and Ras activation) in the Drosophila epithelium with computer simulation of tissue mechanics to elucidate such a mechanism. Contrary to the previous expectation that cell volume loss after apoptosis of loser cells was simply compensated for by the faster proliferation of winner cells, we found that the lost volume was compensated for by rapid cell expansion of winners. Mechanistically, the rapid winner-dominated cell expansion was driven by apoptosis-induced epithelial junction remodeling, which causes re-connection of local cellular connectivity (cell topology) in a manner that selectively increases winner apical surface area. In silico experiments further confirmed that repetition of loser elimination accelerates tissue-scale winner expansion through topological changes over time. Our proposed mechanism for linking loser death and winner expansion provides a new perspective on how tissue homeostasis disruption can initiate from an oncogenic mutation.

Cell populations in multicellular organisms show genetic and non-genetic heterogeneity, even in undifferentiated tissues of multipotent cells during development and tumorigenesis. The heterogeneity causes difference of mechanical properties, such as, cell bond tension or adhesion, at the cell–cell interface, which determine the shape of clonal population boundaries via cell sorting or mixing. The boundary shape could alter the degree of cell–cell contacts and thus influence the physiological consequences of sorting or mixing at the boundary (e.g., tumor suppression or progression), suggesting that the cell mechanics could help clarify the physiology of heterogeneous tissues. While precise inference of mechanical tension loaded at each cell–cell contacts has been extensively developed, there has been little progress on how to distinguish the population-boundary geometry and identify the cause of geometry in heterogeneous tissues. We developed a pipeline by combining multivariate analysis of clone shape with tissue mechanical simulations. We examined clones with four different genotypes within Drosophila wing imaginal discs: wild-type, tartan (trn) overexpression, hibris (hbs) overexpression, and Eph RNAi. Although the clones were previously known to exhibit smoothed or convoluted morphologies, their mechanical properties were unknown. By applying a multivariate analysis to multiple criteria used to quantify the clone shapes based on individual cell shapes, we found the optimal criteria to distinguish not only among the four genotypes, but also non-genetic heterogeneity from genetic one. The efficient segregation of clone shape enabled us to quantitatively compare experimental data with tissue mechanical simulations. As a result, we identified the mechanical basis contributed to clone shape of distinct genotypes. The present pipeline will promote the understanding of the functions of mechanical interactions in heterogeneous tissue in a non-invasive manner.

Epithelia are tissues that regulate exchanges with the environment. They are very dynamic and can acquire virtually any shape; at the cellular level, they are composed of cells tightly connected by junctions. Most often epithelia are amenable to live imaging; however, the large number of cells composing an epithelium and the absence of informatics tools dedicated to epithelial analysis largely prevented tissue scale studies. Here we present Tissue Analyzer, a free tool that can be used to segment and analyze epithelial cells and monitor tissue dynamics.

The subdivision of proliferating tissues into groups of non-intermingling sets of cells, termed compartments, is a common process of animal development. Signaling between adjacent compartments induces the local expression of morphogens that pattern the surrounding tissue. Sharp and straight boundaries between compartments stabilize the source of such morphogens during tissue growth and, thus, are of crucial importance for pattern formation. Signaling pathways required to maintain compartment boundaries have been identified, yet the physical mechanisms that maintain compartment boundaries remained elusive. Recent data now show that a local increase in actomyosin-based mechanical tension on cell bonds is vital for maintaining compartment boundaries in Drosophila.

Neural stem cells called neuroblasts (NBs) generate a variety of neuronal and glial cells in the central nervous system of the Drosophilaembryo. These NBs, few in number, are selected from a field of neuroepithelial(NE) cells. In the optic lobe of the third instar larva, all NE cells of the outer optic anlage (OOA) develop into either NBs that generate the medulla neurons or lamina neuron precursors of the adult visual system. The number of lamina and medulla neurons must be precisely regulated because photoreceptor neurons project their axons directly to corresponding lamina or medulla neurons. Here, we show that expression of the proneural protein Lethal of scute [L(1)sc] signals the transition of NE cells to NBs in the OOA. L(1)sc expression is transient, progressing in a synchronized and ordered `proneural wave' that sweeps toward more lateral NEs. l(1)sc expression is sufficient to induce NBs and is necessary for timely onset of NB differentiation. Thus, proneural wave precedes and induces transition of NE cells to NBs. Unpaired (Upd), the ligand for the JAK/STAT signaling pathway,is expressed in the most lateral NE cells. JAK/STAT signaling negatively regulates proneural wave progression and controls the number of NBs in the optic lobe. Our findings suggest that NBs might be balanced with the number of lamina neurons by JAK/STAT regulation of proneural wave progression, thereby providing the developmental basis for the formation of a precise topographic map in the visual center.

Epithelia are tissues that regulate exchanges with the environment. They are very dynamic and can acquire virtually any shape; at the cellular level, they are composed of cells tightly connected by junctions. Most often epithelia are amenable to live imaging; however, the large number of cells composing an epithelium and the absence of informatics tools dedicated to epithelial analysis largely prevented tissue scale studies. Here we present Tissue Analyzer, a free tool that can be used to segment and analyze epithelial cells and monitor tissue dynamics.

Photoreceptor cell axons (R axons) innervate optic ganglia in the Drosophila brain through the tubular optic stalk. This structure consists of surface glia (SG) and forms independently of R axon projection. In a screen for genes involved in optic stalk formation, we identified Fak56D encoding a Drosophila homolog of mammalian focal adhesion kinase (FAK). FAK is a main component of the focal adhesion signaling that regulates various cellular events, including cell migration and morphology. We show that Fak56D mutation causes severe disruption of the optic stalk structure. These phenotypes were completely rescued by Fak56D transgene expression in the SG cells but not in photoreceptor cells. Moreover, Fak56D genetically interacts with myospheroid, which encodes an integrin β subunit. In addition,we found that CdGAPr is also required for optic stalk formation and genetically interacts with Fak56D. CdGAPr encodes a GTPase-activating domain that is homologous to that of mammalian CdGAP, which functions in focal adhesion signaling. Hence the optic stalk is a simple monolayered structure that can serve as an ideal system for studying glial cell morphogenesis and the developmental role(s) of focal adhesion signaling.

During animal development groups of cells with similar fates and functions often stay together and separate from cells with different fates. An example for this cellular behavior is the formation of compartments, groups of cells with similar fates that are separated by sharp boundaries from neighboring groups of cells. Compartments play important roles during patterning by serving as units of growth and gene expression. Boundaries between compartments are associated with organizers that secrete signaling molecules instructing growth and differentiation throughout the tissue. The straight shape of the boundary between compartments is important for maintaining the position and shape of the organizer and thus for precise patterning. The straight shape of compartment boundaries, however, is challenged by cell divisions and cell intercalations that take place in many developing tissues. Early work established a role for selector genes and signaling pathways in setting up and keeping boundaries straight. Recent work in Drosophila has now begun to further unravel the physical and cellular mechanisms that maintain compartment boundaries. Key to the separation of compartments is a local increase of actomyosin-dependent mechanical tension at cell junctions along the boundary. Increased mechanical tension acts as a barrier to cell mixing during cell division and influences cell rearrangements during cell intercalations along the compartment boundary in a way that the straight shape of the boundary is maintained. An important question for the future is how the signaling pathways that maintain the straight shape of compartment boundaries control mechanical tension along these boundaries.

During neurogenesis in the medulla of the Drosophila optic lobe, neuroepithelial cells are programmed to differentiate into neuroblasts at the medial edge of the developing optic lobe. The wave of differentiation progresses synchronously in a row of cells from medial to the lateral regions of the optic lobe, sweeping across the entire neuroepithelial sheet; it is preceded by the transient expression of the proneural gene lethal of scute [l(1)sc] and is thus called the proneural wave. We found that the epidermal growth factor receptor (EGFR) signaling pathway promotes proneural wave progression. EGFR signaling is activated in neuroepithelial cells and induces l(1)sc expression. EGFR activation is regulated by transient expression of Rhomboid (Rho), which is required for the maturation of the EGF ligand Spitz. Rho expression is also regulated by the EGFR signal. The transient and spatially restricted expression of Rho generates sequential activation of EGFR signaling and assures the directional progression of the differentiation wave. This study also provides new insights into the role of Notch signaling. Expression of the Notch ligand Delta is induced by EGFR, and Notch signaling prolongs the proneural state. Notch signaling activity is downregulated by its own feedback mechanism that permits cells at proneural states to subsequently develop into neuroblasts. Thus, coordinated sequential action of the EGFR and Notch signaling pathways causes the proneural wave to progress and induce neuroblast formation in a precisely ordered manner.

Topographic maps, which maintain the spatial order of neurons in the order of their axonal connections, are found in many parts of the nervous system. Here, we focus on the communication between retinal axons and their postsynaptic partners, lamina neurons, in the first ganglion of the Drosophila visual system, as a model for the formation of topographic maps. Post-mitotic lamina precursor cells differentiate upon receiving Hedgehog signals delivered through newly arriving retinal axons and, before maturing to extend neurites, extend short processes toward retinal axons to create the lamina column. The lamina column provides the cellular basis for establishing stereotypic synapses between retinal axons and lamina neurons. In this study, we identified two cell-adhesion molecules: Hibris, which is expressed in post-mitotic lamina precursor cells; and Roughest, which is expressed on retinal axons. Both proteins belong to the nephrin/NEPH1 family. We provide evidence that recognition between post-mitotic lamina precursor cells and retinal axons is mediated by interactions between Hibris and Roughest. These findings revealed mechanisms by which axons of presynaptic neurons deliver signals to induce the development of postsynaptic partners at the target area. Postsynaptic partners then recognize the presynaptic axons to make ensembles, thus establishing a topographic map along the anterior/posterior axis.

Summary Subdividing proliferating tissues into compartments is an evolutionarily conserved strategy of animal development [1–6]. Signals across boundaries between compartments can result in local expression of secreted proteins organizing growth and patterning of tissues [1–6]. Sharp and straight interfaces between compartments are crucial for stabilizing the position of such organizers and therefore for precise implementation of body plans. Maintaining boundaries in proliferating tissues requires mechanisms to counteract cell rearrangements caused by cell division; however, the nature of such mechanisms remains unclear. Here we quantitatively analyzed cell morphology and the response to the laser ablation of cell bonds in the vicinity of the anteroposterior compartment boundary in developing Drosophila wings. We found that mechanical tension is approximately 2.5-fold increased on cell bonds along this compartment boundary as compared to the remaining tissue. Cell bond tension is decreased in the presence of Y-27632 [7], an inhibitor of Rho-kinase whose main effector is Myosin II [8]. Simulations using a vertex model [9] demonstrate that a 2.5-fold increase in local cell bond tension suffices to guide the rearrangement of cells after cell division to maintain compartment boundaries. Our results provide a physical mechanism in which the local increase in Myosin II-dependent cell bond tension directs cell sorting at compartment boundaries.

During development of the Drosophila visual center, photoreceptor cells extend their axons (R axons) to the lamina ganglion layer, and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal Hedgehog (Hh). This inductive mechanism helps to establish an orderly arrangement of connections between the R axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding R axons. We found that the bHLH-PAS transcription factor Single-minded (Sim) is induced by Hh in the lamina neurons and is required for the association of lamina neurons with R axons. In sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with R axons. As a result, lamina neurons are set aside from R axons. The data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks.