Osiris gene family defines the cuticle nanopatterns of Drosophila.

Nanostructures of pores and protrusions in the insect cuticle modify molecular permeability and surface wetting and help insects sense various environmental cues. However, the cellular mechanisms that modify cuticle nanostructures are poorly understood. Here, we elucidate how insect-specific Osiris family genes are expressed in various cuticle-secreting cells in the Drosophila head during the early stages of cuticle secretion and cover nearly the entire surface of the head epidermis. Furthermore, we demonstrate how each sense organ cell with various cuticular nanostructures expressed a unique combination of Osiris genes. Osiris gene mutations cause various cuticle defects in the corneal nipples and pores of the chemosensory sensilla. Thus, our study emphasizes on the importance of Osiris genes for elucidating cuticle nanopatterning in insects.

Publishing in the Open Access and Open Science era.

Our research activities would be better served if they were communicated in a manner that is openly accessible to the public and all researchers. The research we share is often limited to representative data included in research papers-science would be much more efficient if all reproducible research data were shared alongside detailed methods and protocols, in the paradigm called Open Science. On the other hand, one primary function of research journals is to select manuscripts of good quality, verify the authenticity of the data and its impact, and deliver to the appropriate audience for critical evaluation and verification. In the current paradigm, where publication in a subset of journals is intimately linked to research evaluation, a hypercompetitive "market" has emerged where authors compete to access a limited number of top-tier journals, leading to high rejection rates. Competition among publishers and scientific journals for market dominance resulted in an increase in both the number of journals and the cost of publishing and accessing scientific papers. Here we summarize the current problems and potential solutions from the development of AI technology discussed in the seminar at the 46th Annual Meeting of the Molecular Biology Society of Japan.

Emergence of periodic circumferential actin cables from the anisotropic fusion of actin nanoclusters during tubulogenesis.

The periodic circumferential cytoskeleton supports various tubular tissues. Radial expansion of the tube lumen causes anisotropic tensile stress, which can be exploited as a geometric cue. However, the molecular machinery linking anisotropy to robust circumferential patterning is poorly understood. Here, we aim to reveal the emergent process of circumferential actin cable formation in a Drosophila tracheal tube. During luminal expansion, sporadic actin nanoclusters emerge and exhibit circumferentially biased motion and fusion. RNAi screening reveals the formin family protein, DAAM, as an essential component responding to tissue anisotropy, and non-muscle myosin II as a component required for nanocluster fusion. An agent-based model simulation suggests that crosslinkers play a crucial role in nanocluster formation and cluster-to-cable transition occurs in response to mechanical anisotropy. Altogether, we propose that an actin nanocluster is an organizational unit that responds to stress in the cortical membrane and builds a higher-order cable structure.

Epidermal growth factor receptor signaling protects epithelia from morphogenetic instability and tissue damage in Drosophila.

Dying cells in the epithelia communicate with neighboring cells to initiate coordinated cell removal to maintain epithelial integrity. Naturally occurring apoptotic cells are mostly extruded basally and engulfed by macrophages. Here, we have investigated the role of Epidermal growth factor (EGF) receptor (EGFR) signaling in the maintenance of epithelial homeostasis. In Drosophila embryos, epithelial tissues undergoing groove formation preferentially enhanced extracellular signal-regulated kinase (ERK) signaling. In EGFR mutant embryos at stage 11, sporadic apical cell extrusion in the head initiates a cascade of apical extrusions of apoptotic and non-apoptotic cells that sweeps the entire ventral body wall. Here, we show that this process is apoptosis dependent, and clustered apoptosis, groove formation, and wounding sensitize EGFR mutant epithelia to initiate massive tissue disintegration. We further show that tissue detachment from the vitelline membrane, which frequently occurs during morphogenetic processes, is a key trigger for the EGFR mutant phenotype. These findings indicate that, in addition to cell survival, EGFR plays a role in maintaining epithelial integrity, which is essential for protecting tissues from transient instability caused by morphogenetic movement and damage.

Transcriptomic exploration of the Coleopteran wings reveals insight into the evolution of novel structures associated with the beetle elytron.

The acquisition of novel traits is central to organismal evolution, yet the molecular mechanisms underlying this process are elusive. The beetle forewings (elytra) are evolutionarily modified to serve as a protective shield, providing a unique opportunity to study these mechanisms. In the past, the orthologs of genes within the wing gene network from Drosophila studies served as the starting point when studying the evolution of elytra (candidate genes). Although effective, candidate gene lists are finite and only explore genes conserved across species. To go beyond candidate genes, we used RNA sequencing and explored the wing transcriptomes of two Coleopteran species, the red flour beetle (Tribolium castaneum) and the Japanese stag beetle (Dorcus hopei). Our analysis revealed sets of genes enriched in Tribolium elytra (57 genes) and genes unique to the hindwings, which possess more "typical" insect wing morphologies (29 genes). Over a third of the hindwing-enriched genes were "candidate genes" whose functions were previously analyzed in Tribolium, demonstrating the robustness of our sequencing. Although the overlap was limited, transcriptomic comparison between the beetle species found a common set of genes, including key wing genes, enriched in either elytra or hindwings. Our RNA interference analysis for elytron-enriched genes in Tribolium uncovered novel genes with roles in forming various aspects of morphology that are unique to elytra, such as pigmentation, hardening, sensory development, and vein formation. Our analyses deepen our understanding of how gene network evolution facilitated the emergence of the elytron, a unique structure critical to the evolutionary success of beetles.

Mechano-chemical enforcement of tendon apical ECM into nano-filaments during Drosophila flight muscle development.

Contractile tension is critical for musculoskeletal system development and maintenance. In insects, the muscular force is transmitted to the exoskeleton through the tendon cells and tendon apical extracellular matrix (ECM). In Drosophila, we found tendon cells secrete Dumpy (Dpy), a zona pellucida domain (ZPD) protein, to form the force-resistant filaments in the exuvial space, anchoring the tendon cells to the pupal cuticle. We showed that Dpy undergoes filamentous conversion in response to the tension increment during indirect flight muscle development. We also found another ZPD protein Quasimodo (Qsm) protects the notum epidermis from collapsing under the muscle tension by enhancing the tensile strength of Dpy filaments. Qsm is co-transported with Dpy in the intracellular vesicles and diffuses into the exuvial space after secretion. Tissue-specific qsm expression rescued the qsm mutant phenotypes in distant tissues, suggesting Qsm can function in a long-range, non-cell-autonomous manner. In the cell culture assay, Qsm interacts with Dpy-ZPD and promotes secretion and polymerization of Dpy-ZPD. The roles of Qsm underlies the positive feedback mechanism of force-dependent organization of Dpy filaments, providing new insights into apical ECM remodeling through the unconventional interaction of ZPD proteins.

polished rice mediates ecdysone-dependent control of Drosophila embryonic organogenesis.

In many animals, progression of developmental stages is temporally controlled by steroid hormones. In Drosophila, the level of ecdysone titer oscillates and developmental stage transitions, such as larval molting and metamorphosis, are induced at each of ecdysone peaks. Ecdysone titer also peaks at the stage of mid-embryogenesis and the embryonic ecdysone is necessary for morphogenesis of several organs, although the regulatory mechanisms of embryonic organogenesis dependent on ecdysone signaling are still open questions. In this study, we find that absence or interruption of embryonic ecdysone signaling caused multiple defects in the tracheal system, including decrease in luminal protein deposition, uneven dilation of the dorsal trunk and loss of terminal branches. We also reveal that an ecdysone-inducible gene polished rice (pri) is essential for tip cell fate decision in dorsal branches. As over-expression of pri can restore the defects caused by disturbance of ecdysone biosynthesis, pri functions as one of the major mediators of embryonic ecdysone signal in tracheogenesis. These results demonstrate that ecdysone and its downstream target pri play essential roles in tracheal development by modulating cell fate decision.

ERK signaling dynamics in the morphogenesis and homeostasis of Drosophila.

Receptor tyrosine kinases (RTK) are transmembrane kinases that receive signals for intercellular communication to help organize body plan and sustain tissue homeostasis. These signals converge into the major signaling module of ERK, which transduces signals to the cytoplasm and nucleus. How this module responds to multiple RTK signals, and specifies unique outcomes in each cell, is still poorly understood. Recent technological advances in the quantitative imaging of ERK activity and its manipulation have yielded significant information on the cellular logic behind ERK activation and its readout in the context of Drosophila development. While in the pregastrulation stage, ERK plays a decisive on/off switch; its role changes to modulatory functions of morphogenesis and tissue quality control in the late embryonic stages.

Two-step regulation of trachealess ensures tight coupling of cell fate with morphogenesis in the Drosophila trachea.

During organogenesis, inductive signals cause cell differentiation and morphogenesis. However, how these phenomena are coordinated to form functional organs is poorly understood. Here, we show that cell differentiation of the Drosophila trachea is sequentially determined in two steps and that the second step is synchronous with the invagination of the epithelial sheet. The master gene trachealess is dispensable for the initiation of invagination, while it is essential for maintaining the invaginated structure, suggesting that tracheal morphogenesis and differentiation are separately induced. trachealess expression starts in bipotential tracheal/epidermal placode cells. After invagination, its expression is maintained in the invaginated cells but is extinguished in the remaining sheet cells. A trachealess cis-regulatory module that shows both tracheal enhancer activity and silencer activity in the surface epidermal sheet was identified. We propose that the coupling of trachealess expression with the invaginated structure ensures that only invaginated cells canalize robustly into the tracheal fate.

The plus-tip tracking and microtubule stabilizing activities of Javelin-like regulate microtubule organization and cell polarity.

Cell polarity is essential for building cell asymmetry in all eukaryotic cells. Drosophila oocyte and bristle development require the newly characterized Spn-F protein complex, which includes Spn-F, IKKε, and Javelin-like (Jvl), to establish polarity. Jvl is a novel microtubule (MT)-associated protein; however, the mechanism by which it regulates MT organization is still unknown. We found that overexpression of Jvl stabilizes MTs and that jvl is needed for stable MT arrangement at the bristle tip and organization of the dynamic MT throughout the bristle shaft. At low levels of expression in cultured cells, Jvl behaved as a microtubule plus-end tracking protein. We demonstrated that Jvl physically interacts with the highly conserved MT end-binding protein 1 (EB1) using yeast two-hybrid and GST pull-down assays. This interaction is, however, dispensable for Jvl function in oocyte and bristle development. In addition, using a MT-binding assay, we saw that Jvl-C terminus directly binds to MTs. We also revealed that oocyte developmental arrest caused by Jvl overexpression in the germline can be rescued by mutations in its partners, spn-F and ikkε, suggesting that complex formation with Spn-F and IKKε is required for Jvl function in vivo. In summary, our results show that the microtubule plus-end tracking and stabilizing activities of Jvl are central for controlling cell polarity of oocytes and bristles.

Nanopore Formation in the Cuticle of an Insect Olfactory Sensillum.

Nanometer-level patterned surface structures form the basis of biological functions, including superhydrophobicity, structural coloration, and light absorption [1-3]. In insects, the cuticle overlying the olfactory sensilla has multiple small (50- to 200-nm diameter) pores [4-8], which are supposed to function as a filter that admits odorant molecules, while preventing the entry of larger airborne particles and limiting water loss. However, the cellular processes underlying the patterning of extracellular matrices into functional nano-structures remain unknown. Here, we show that cuticular nanopores in Drosophila olfactory sensilla originate from a curved ultrathin film that is formed in the outermost envelope layer of the cuticle and secreted from specialized protrusions in the plasma membrane of the hair forming (trichogen) cell. The envelope curvature coincides with plasma membrane undulations associated with endocytic structures. The gore-tex/Osiris23 gene encodes an endosomal protein that is essential for envelope curvature, nanopore formation, and odor receptivity and is expressed specifically in developing olfactory trichogen cells. The 24-member Osiris gene family is expressed in cuticle-secreting cells and is found only in insect genomes. These results reveal an essential requirement for nanopores for odor reception and identify Osiris genes as a platform for investigating the evolution of surface nano-fabrication in insects.

Automated FRET quantification shows distinct subcellular ERK activation kinetics in response to graded EGFR signaling in Drosophila.

Threshold responses to an activity gradient allow a single signaling pathway to yield multiple outcomes. Extracellular signal-regulated kinase (ERK) is one such signal, which couples receptor tyrosine kinase signaling with multiple cellular responses in various developmental processes. Recent advances in the development of fluorescent biosensors for live imaging have enabled the signaling activities accompanying embryonic development to be monitored in real time. Here, we used an automated computational program to quantify the signals of a fluorescence resonance energy transfer (FRET) reporter for activated ERK, and we used this system to monitor the spatio-temporal dynamics of ERK during neuroectoderm patterning in Drosophila embryos. We found that the cytoplasmic and nuclear ERK activity gradients show distinct kinetics in response to epidermal growth factor receptor activation. The ERK activation patterns implied that the cytoplasmic ERK activity is modulated into a threshold response in the nucleus.

Trynity controls epidermal barrier function and respiratory tube maturation in Drosophila by modulating apical extracellular matrix nano-patterning.

The outer surface of insects is covered by the cuticle, which is derived from the apical extracellular matrix (aECM). The aECM is secreted by epidermal cells during embryogenesis. The aECM exhibits large variations in structure, function, and constituent molecules, reflecting the enormous diversity in insect appearances. To investigate the molecular principles of aECM organization and function, here we studied the role of a conserved aECM protein, the ZP domain protein Trynity, in Drosophila melanogaster. We first identified trynity as an essential gene for epidermal barrier function. trynity mutation caused disintegration of the outermost envelope layer of the cuticle, resulting in small-molecule leakage and in growth and molting defects. In addition, the tracheal tubules of trynity mutants showed defects in pore-like structures of the cuticle, and the mutant tracheal cells failed to absorb luminal proteins and liquid. Our findings indicated that trynity plays essential roles in organizing nano-level structures in the envelope layer of the cuticle that both restrict molecular trafficking through the epidermis and promote the massive absorption pulse in the trachea.

A Switch-like Activation Relay of EGFR-ERK Signaling Regulates a Wave of Cellular Contractility for Epithelial Invagination.

The dynamics of extracellular signal-regulated kinase (ERK) signaling underlies its versatile functions in cell differentiation, cell proliferation, and cell motility. Classical studies in Drosophila established that a gradient of epidermal growth factor receptor (EGFR)-ERK signaling is essential for these cellular responses. However, we challenge this view by the real-time monitoring of ERK activation; we show that a switch-like ERK activation is essential for the invagination movement of the Drosophila tracheal placode. This switch-like ERK activation stems from the positive feedback regulation of the EGFR-ERK signaling and a resultant relay of EGFR-ERK signaling among tracheal cells. A key transcription factor Trachealess (Trh) permissively regulates the iteration of the relay, and the ERK activation becomes graded in trh mutant. A mathematical model based on these observations and a molecular link between ERK activation dynamics and myosin shows that the relay mechanism efficiently promotes epithelial invagination while the gradient mechanism does not.

Development and Function of the Drosophila Tracheal System.

The tracheal system of insects is a network of epithelial tubules that functions as a respiratory organ to supply oxygen to various target organs. Target-derived signaling inputs regulate stereotyped modes of cell specification, branching morphogenesis, and collective cell migration in the embryonic stage. In the postembryonic stages, the same set of signaling pathways controls highly plastic regulation of size increase and pattern elaboration during larval stages, and cell proliferation and reprograming during metamorphosis. Tracheal tube morphogenesis is also regulated by physicochemical interaction of the cell and apical extracellular matrix to regulate optimal geometry suitable for air flow. The trachea system senses both the external oxygen level and the metabolic activity of internal organs, and helps organismal adaptation to changes in environmental oxygen level. Cellular and molecular mechanisms underlying the high plasticity of tracheal development and physiology uncovered through research on Drosophila are discussed.

Shape and geometry control of the Drosophila tracheal tubule.

For efficient respiration, tubular airways must be constructed with an optimal diameter and length for the dimensions of the body. In Drosophila, the growth of embryonic tracheal tubules proceeds in two dimensions, by axial elongation and diameter expansion. The growth forces in each dimension are controlled by distinct genetic programs and cellular mechanisms. Recent studies reveal that the apical cortex and the apical extracellular matrix filling the luminal space are essential for the generation, balancing, and equilibrium of these growth forces. We here discuss the mechanical properties and architecture of the apical cortex and extracellular matrix, and their crucial roles in the tissue-level coordination of tubule shape and geometry.

Escargot controls the sequential specification of two tracheal tip cell types by suppressing FGF signaling in Drosophila.

Extrinsic branching factors promote the elongation and migration of tubular organs. In the Drosophila tracheal system, Branchless (Drosophila FGF) stimulates the branching program by specifying tip cells that acquire motility and lead branch migration to a specific destination. Tip cells have two alternative cell fates: the terminal cell (TC), which produces long cytoplasmic extensions with intracellular lumen, and the fusion cell (FC), which mediates branch connections to form tubular networks. How Branchless controls this specification of cells with distinct shapes and behaviors is unknown. Here we report that this cell type diversification involves the modulation of FGF signaling by the zinc-finger protein Escargot (Esg), which is expressed in the FC and is essential for its specification. The dorsal branch begins elongation with a pair of tip cells with high FGF signaling. When the branch tip reaches its final destination, one of the tip cells becomes an FC and expresses Esg. FCs and TCs differ in their response to FGF: TCs are attracted by FGF, whereas FCs are repelled. Esg suppresses ERK signaling in FCs to control this differential migratory behavior.

IKKε inhibits PKC to promote Fascin-dependent actin bundling.

Signaling molecules have pleiotropic functions and are activated by various extracellular stimuli. Protein kinase C (PKC) is activated by diverse receptors, and its dysregulation is associated with diseases including cancer. However, how the undesired activation of PKC is prevented during development remains poorly understood. We have previously shown that a protein kinase, IKKε, is active at the growing bristle tip and regulates actin bundle organization during Drosophila bristle morphogenesis. Here, we demonstrate that IKKε regulates the actin bundle localization of a dynamic actin cross-linker, Fascin. IKKε inhibits PKC, thereby protecting Fascin from inhibitory phosphorylation. Excess PKC activation is responsible for the actin bundle defects in IKKε-deficient bristles, whereas PKC is dispensable for bristle morphogenesis in wild-type bristles, indicating that PKC is repressed by IKKε in wild-type bristle cells. These results suggest that IKKε prevents excess activation of PKC during bristle morphogenesis.