Augmin complex activity finetunes dendrite morphology through non-centrosomal microtubule nucleation in vivo.

During development, neurons achieve a stereotyped neuron type-specific morphology, which relies on dynamic support by microtubules (MTs). An important player is the augmin complex (hereafter augmin), which binds to existing MT filaments and recruits the γ-tubulin ring complex (γ-TuRC), to form branched MTs. In cultured neurons, augmin is important for neurite formation. However, little is known about the role of augmin during neurite formation in vivo. Here, we have revisited the role of mammalian augmin in culture and then turned towards the class four Drosophila dendritic arborization (c4da) neurons. We show that MT density is maintained through augmin in cooperation with the γ-TuRC in vivo. Mutant c4da neurons show a reduction of newly emerging higher-order dendritic branches and in turn also a reduced number of their characteristic space-filling higher-order branchlets. Taken together, our data reveal a cooperative function for augmin with the γ-TuRC in forming enough MTs needed for the appropriate differentiation of morphologically complex dendrites in vivo.

Dynamic de novo adipose tissue development during metamorphosis in Drosophila melanogaster.

Adipose tissue is a central organ for controlling systemic metabolism both in invertebrates and vertebrates. Here, we have investigated the developmental processes of the adult-type fat body (AFB) in Drosophila. We have established genetic tools that allow visualization and genetic manipulations of cells in the AFB lineage from early in metamorphosis. We identified precursor cells that give rise to the AFB and delineated dynamic cellular behaviors underlying AFB formation. These precursor cells displayed polarized cell shapes and oriented motility, with emigration from the thorax and subsequent dispersal to the abdomen and head. After the migration period, these cells adhered to each other, assembling into the AFB with a sheet-like architecture. Continuous cell proliferation occurred during and after the large-scale migration to achieve appropriate fat tissue mass. Homotypic cell fusion after the sheet formation contributed to the establishment of multinucleated cells in the AFB. We also examined candidate gene functions, and our results argue that ecdysone signaling and the transcription factor Serpent support adult fat body organogenesis.

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.

E-cadherin-dependent coordinated epithelial rotation on a two-dimensional discoidal pattern.

In vivo, cells collectively migrate in a variety of developmental and pathological contexts. Coordinated epithelial rotation represents a unique type of collective cell migrations, which has been modeled in vitro under spatially confined conditions. Although it is known that the coordinated rotation depends on intercellular interactions, the contribution of E-cadherin, a major cell-cell adhesion molecule, has not been directly addressed on two-dimensional (2D) confined substrates. Here, using well-controlled fibronectin-coated surfaces, we tracked and compared the migratory behaviors of MDCK cells expressing or lacking E-cadherin. We observed that wild-type MDCK II cells exhibited persistent and coordinated rotations on discoidal patterns, while E-cadherin knockout cells migrated in a less coordinated manner without large-scale rotation. Our comparison of the collective dynamics between these two cell types revealed a series of changes in migratory behavior caused by the loss of E-cadherin, including a decreased global migration speed, less regularity in quantified coordination, and increased average density of topological defects. Taken together, these data demonstrate that spontaneous initiation of collective epithelial rotations depends on E-cadherin under 2D discoidal confinements.

Refinement of a technique for collecting and evaluating the osmolality of haemolymph from Drosophila larvae.

Ex vivo physiological experiments using small insect models such as Drosophila larvae have become increasingly useful to address fundamental biological questions. To perform such experiments, various artificial saline solutions have been developed, but their osmolality varies significantly from one to the next. Such a variation of osmolality stems, in part, from the difficulty of determining the true value of haemolymph osmolality in Drosophila larvae. Thus, there is a pressing need to refine protocols for collecting and measuring the osmolality of the larval haemolymph. Two major obstacles are thought to impede the accurate analysis of haemolymph collected from small insects: melanin formation and gut-derived contamination. Here, we greatly refined existing haemolymph collection methods, evaluated the purity of the collected haemolymph under melanin-free conditions, and concluded that the true value of haemolymph osmolality is close to 306.0 mOsm kg-1 in Drosophila larvae.

Image-based parameter inference for epithelial mechanics.

Measuring mechanical parameters in tissues, such as the elastic modulus of cell-cell junctions, is essential to decipher the mechanical control of morphogenesis. However, their in vivo measurement is technically challenging. Here, we formulated an image-based statistical approach to estimate the mechanical parameters of epithelial cells. Candidate mechanical models are constructed based on force-cell shape correlations obtained from image data. Substitution of the model functions into force-balance equations at the cell vertex leads to an equation with respect to the parameters of the model, by which one can estimate the parameter values using a least-squares method. A test using synthetic data confirmed the accuracy of parameter estimation and model selection. By applying this method to Drosophila epithelial tissues, we found that the magnitude and orientation of feedback between the junction tension and shrinkage, which are determined by the spring constant of the junction, were correlated with the elevation of tension and myosin-II on shrinking junctions during cell rearrangement. Further, this method clarified how alterations in tissue polarity and stretching affect the anisotropy in tension parameters. Thus, our method provides a novel approach to uncovering the mechanisms governing epithelial morphogenesis.

Shortened lifespan induced by a high-glucose diet is associated with intestinal immune dysfunction in Drosophila sechellia.

Organisms can generally be divided into two nutritional groups: generalists that consume various types of food and specialists that consume specific types of food. However, it remains unclear how specialists adapt to only limited nutritional conditions in nature. In this study, we addressed this question by focusing on Drosophila fruit flies. The generalist Drosophila melanogaster can consume a wide variety of foods that contain high glucose levels. In contrast, the specialist Drosophila sechellia consumes only the Indian mulberry, known as noni (Morinda citrifolia), which contains relatively little glucose. We showed that the lifespan of D. sechellia was significantly shortened under a high-glucose diet, but this effect was not observed for D. melanogaster. In D. sechellia, a high-glucose diet induced disorganization of the gut epithelia and visceral muscles, which was associated with abnormal digestion and constipation. RNA-sequencing analysis revealed that many immune-responsive genes were suppressed in the gut of D. sechellia fed a high-glucose diet compared with those fed a control diet. Consistent with this difference in the expression of immune-responsive genes, high glucose-induced phenotypes were restored by the addition of tetracycline or scopoletin, a major nutritional component of noni, each of which suppresses gut bacterial growth. We propose that, in D. sechellia, a high-glucose diet impairs gut immune function, which leads to a change in gut microbiota, disorganization of the gut epithelial structure and a shortened lifespan.

Divergence of Drosophila species: Longevity and reproduction under different nutrient balances.

How nutrition impacts growth, reproduction and longevity is complex because relationships between these life events are difficult to disentangle. As a first step in sorting out these processes, we carried out a comparative analysis of related species of Drosophila with distinct feeding habits. In particular, we examined life spans and egg laying of two generalists and three specialists on diets with distinct protein-to-carbohydrate ratios. In contrast to the generalist D. melanogaster, adult males of two specialists, D. sechellia and D. elegans, lived longer on a protein-rich diet. These results and our previous studies collectively show that the diet to which larvae of each specialist species have adapted ensures a longer life span of adult males of that same species. We also found a species-specific sexual dimorphism of life span in the above two specialists regardless of the diets, which was in sharp contrast to D. melanogaster. In D. melanogaster, males lived longer than females, whereas females of D. sechellia and D. elegans were longer-lived than males, and those specialist females were exceedingly low in egg production, relative to the other species. We discuss our findings from perspectives of mechanisms, including a possible contribution of egg production to life span.

DeTerm: Software for automatic detection of neuronal dendritic branch terminals via an artificial neural network.

Dendrites of neurons receive and process synaptic or sensory inputs. The Drosophila class IV dendritic arborization (da) neuron is an established model system to explore molecular mechanisms of dendrite morphogenesis. The total number of dendritic branch terminals is one of the frequently employed parameters to characterize dendritic arborization complexity of class IV neurons. This parameter gives a useful phenotypic readout of arborization during neurogenesis, and it is typically determined by laborious manual analyses of numerous images. Ideally, an automated analysis would greatly reduce the workload; however, it is challenging to automatically discriminate dendritic branch terminals from signals of surrounding tissues in whole-mount live larvae. Here, we describe our newly developed software, called DeTerm, which automatically recognizes and quantifies dendrite branch terminals via an artificial neural network. Once we input an image file of a neuronal dendritic arbor and its region of interest information, DeTerm is capable of labeling terminals of larval class IV neurons with high precision, and it also provides positional data of individual terminals. We further show that DeTerm is applicable to other types of neurons, including mouse cerebellar Purkinje cells. DeTerm is freely available on the web and was successfully tested on Mac, Windows and Linux.

Map7/7D1 and Dvl form a feedback loop that facilitates microtubule remodeling and Wnt5a signaling.

The Wnt signaling pathway can be grouped into two classes, the ß-catenin-dependent and ß-catenin-independent pathways. Wnt5a signaling through a ß-catenin-independent pathway promotes microtubule (MT) remodeling during cell-substrate adhesion, cell migration, and planar cell polarity formation. Although Wnt5a signaling and MT remodeling are known to form an interdependent regulatory loop, the underlying mechanism remains unknown. Here we show that in HeLa cells, the paralogous MT-associated proteins Map7 and Map7D1 (Map7/7D1) form an interdependent regulatory loop with Disheveled, the critical signal transducer in Wnt signaling. Map7/7D1 bind to Disheveled, direct its cortical localization, and facilitate the cortical targeting of MT plus-ends in response to Wnt5a signaling. Wnt5a signaling also promotes Map7/7D1 movement toward MT plus-ends, and depletion of the Kinesin-1 member Kif5b abolishes the Map7/7D1 dynamics and Disheveled localization. Furthermore, Disheveled stabilizes Map7/7D1. Intriguingly, Map7/7D1 and its Drosophila ortholog, Ensconsin show planar-polarized distribution in both mouse and fly epithelia, and Ensconsin influences proper localization of Drosophila Disheveled in pupal wing cells. These results suggest that the role of Map7/7D1/Ensconsin in Disheveled localization is evolutionarily conserved.

The seven-pass transmembrane cadherin Flamingo controls dendritic self-avoidance via its binding to a LIM domain protein, Espinas, in Drosophila sensory neurons.

Members of the Flamingo cadherin family are required in a number of different in vivo contexts of neural development. Even so, molecular identities downstream from the family have been poorly understood. Here we show that a LIM domain protein, Espinas (Esn), binds to an intracellular juxtamembrane domain of Flamingo (Fmi), and that this Fmi-Esn interplay elicits repulsion between dendritic branches of Drosophila sensory neurons. In wild-type larvae, branches of the same class IV dendritic arborization neuron achieve efficient coverage of its two-dimensional receptive field with minimum overlap with each other. However, this self-avoidance was disrupted in a fmi hypomorphic mutant, in an esn knockout homozygote, and in the fmi/esn trans-heterozygote. A functional fusion protein, Fmi:3eGFP, was localized at most of the branch tips, and in a heterologous system, assembly of Esn at cell contact sites required its LIM domain and Fmi. We further show that genes controlling epithelial planar cell polarity (PCP), such as Van Gogh (Vang) and RhoA, are also necessary for the self-avoidance, and that fmi genetically interacts with these loci. On the basis of these and other results, we propose that the Fmi-Esn complex, together with the PCP regulators and the Tricornered (Trc) signaling pathway, executes the repulsive interaction between isoneuronal dendritic branches.

Nutrient-dependent increased dendritic arborization of somatosensory neurons.

Suboptimal nutrition imposes developmental constraints on infant animals, which marshal adaptive responses to eventually become mature adults. Such responses are mounted at multiple levels from systemic to cellular. At the cellular level, the underlying mechanisms of cell proliferation control have been intensively studied. However, less is known about how growth of postmitotic and morphologically complex cells, such as neurons, is controlled by nutritional status. We address this question using Class I and Class IV dendritic arborization neurons in Drosophila larvae. Class IV neurons have been shown to sense nociceptive thermal, mechanical and light stimuli, whereas Class I neurons are proprioceptors. We reared larvae on diets with different protein and carbohydrate content throughout larval stages and examined how morphologies of Class I or Class IV neurons were affected. Dendritic arbors of Class IV neurons became more complex when larvae were reared on a low-yeast diet, which contains lower amounts of amino acids and other ingredients, compared to a high-yeast diet. In contrast, such low-yeast-dependent hyperarborization was not seen in Class I neurons. The physiological and metabolic implications of the hyperarborization phenotype are discussed in relation to a recent hypothesis that Class IV neurons sense protein-deficient stress and to our characterization of how the dietary yeast contents impacted larval metabolism.

Celsr1 is required for the generation of polarity at multiple levels of the mouse oviduct.

The oviduct is an important organ in reproduction where fertilization occurs, and through which the fertilized eggs are carried to the uterus in mammals. This organ is highly polarized, where the epithelium forms longitudinal folds along the ovary-uterus axis, and the epithelial multicilia beat towards the uterus to transport the ovulated ova. Here, we analyzed the postnatal development of mouse oviduct and report that multilevel polarities of the oviduct are regulated by a planar cell polarity (PCP) gene, Celsr1. In the epithelium, Celsr1 is concentrated in the specific cellular boundaries perpendicular to the ovary-uterus axis from postnatal day 2. We found a new feature of cellular polarity in the oviduct - the apical surface of epithelial cells is elongated along the ovary-uterus axis. In Celsr1-deficient mice, the ciliary motion is not orchestrated along the ovary-uterus axis and the transport ability of beating cilia is impaired. Epithelial cells show less elongation and randomized orientation, and epithelial folds show randomized directionality and ectopic branches in the mutant. Our mosaic analysis suggests that the geometry of epithelial cells is primarily regulated by Celsr1 and as a consequence the epithelial folds are aligned. Taken together, we reveal the characteristics of the multilevel polarity formation processes in the mouse oviduct epithelium and suggest a novel function of the PCP pathway for proper tissue morphogenesis.

Mechanical Regulation of Three-Dimensional Epithelial Fold Pattern Formation in the Mouse Oviduct.

Epithelia exhibit various three-dimensional morphologies linked to organ function in animals. However, the mechanisms of three-dimensional morphogenesis remain elusive. The luminal epithelium of the mouse oviduct forms well-aligned straight folds along the longitudinal direction of the tubes. Disruption of the Celsr1 gene, a planar cell polarity-related gene, causes ectopically branched folds. Here, we evaluated the mechanical contributions of the epithelium to the fold pattern formation. In the mutant oviduct, the epithelium was more intricate along the longitudinal direction than in the wild-type, suggesting a higher ratio of the longitudinal length of the epithelial layer to that of the surrounding smooth muscle (SM) layer (L-Epi/SM ratio). Our mathematical modeling and computational simulations suggested that the L-Epi/SM ratio could explain the differences in fold branching between the two genotypes. Longitudinal epithelial tensions were increased in well-aligned folds compared with those in disorganized folds both in the simulations and in experimental estimations. Artificially increasing the epithelial tensions suppressed the branching in simulations, suggesting that the epithelial tensions can regulate fold patterning. The epithelial tensions could be explained by the combination of line tensions along the epithelial cell-cell boundaries with the polarized cell arrays observed in vivo. These results suggest that the fold pattern is associated with the polarized cell array through the longitudinal epithelial tension. Further simulations indicated that the L-Epi/SM ratio could contribute to fold pattern diversity, suggesting that the L-Epi/SM ratio is a critical parameter in the fold patterning in tubular organs.

Age-dependent deterioration of locomotion in Drosophila melanogaster deficient in the homologue of amyotrophic lateral sclerosis 2.

Recessive mutations in the amyotrophic lateral sclerosis 2 (ALS2) gene have been linked to juvenile-onset ALS2. Although one of the molecular functions of the ALS2 protein is clearly the activation of Rab5, the mechanisms underlying the selective dysfunction and degeneration of motor neurons in vivo remain to be fully understood. Here, we focused on the ALS2 homologue of Drosophila melanogaster, isolated two independent deletions, and systematically compared phenotypes of the mutants with those of animals in which Rab5 function in identified neurons was abrogated. In the dALS2 mutant flies, we found that the stereotypic axonal and dendritic morphologies of neurons shared some features with those in Rab5-deficient flies, but the dALS2 mutant phenotypes were much milder. We also found that the abrogation of Rab5 function in motor neurons strongly depressed the locomotion activity of adults, resembling the behavior of aged dALS2 mutants. Importantly, this age-dependent locomotion deficit of dALS2 mutants was restored to normal by expressing the dALS2 transgene in a wide range of tissues. This finding provided a platform where we could potentially identify particular cell types responsible for the phenotype by tissue-specific rescue experiments. We discuss our results and the future usage of the dALS2 mutant as a new ALS model.

A novel planar polarity gene pepsinogen-like regulates wingless expression in a posttranscriptional manner.

BACKGROUND: Planar cell polarity (PCP) originally referred to the coordination of global organ axes and individual cell polarity within the plane of the epithelium. More recently, it has been accepted that pertinent PCP regulators play essential roles not only in epithelial sheets, but also in various rearranging cells. RESULTS: We identified pepsinogen-like (pcl) as a new planar polarity gene, using Drosophila wing epidermis as a model. Pcl protein is predicted to belong to a family of aspartic proteases. When pcl mutant clones were observed in pupal wings, PCP was disturbed in both mutant and wild-type cells that were juxtaposed to the clone border. We examined levels of known PCP proteins in wing imaginal discs. The amount of the seven-pass transmembrane cadherin Flamingo (Fmi), one of the PCP "core group" members, was significantly decreased in mutant clones, whereas neither the amount of nor the polarized localization of Dachsous (Ds) at cell boundaries was affected. In addition to the PCP phenotype, the pcl mutation caused loss of wing margins. Intriguingly, this was most likely due to a dramatic decrease in the level of Wingless (Wg) protein, but not due to a decrease in the level of wg transcripts. CONCLUSIONS: Our results raise the possibility that Pcl regulates Wg expression post-transcriptionally, and PCP, by proteolytic cleavages.

Snakeskin, a membrane protein associated with smooth septate junctions, is required for intestinal barrier function in Drosophila.

Septate junctions (SJs) are the membrane specializations observed between epithelial cells in invertebrates. SJs play a crucial role in epithelial barrier function by restricting the free diffusion of solutes through the intercellular space. In arthropod species, two morphologically different types of SJs have been described: pleated septate junctions (pSJs) and smooth septate junctions (sSJs), which are specific to ectodermal and endodermal epithelia, respectively. In contrast to the recent identification of pSJ-related proteins, the molecular constituents of sSJs are mostly unknown. Here, we report the discovery of a new sSJ-specific membrane protein, designated 'Snakeskin' (Ssk). Ssk is highly concentrated in sSJs in the Drosophila midgut and Malpighian tubules. Lack of Ssk expression is embryonically lethal in Drosophila and results in defective sSJ formation accompanied by abnormal morphology of midgut epithelial cells. We also show that the barrier function of the midgut to a fluorescent tracer is impaired in ssk-knockdown larvae. These results suggest that Ssk is required for the intestinal barrier function in Drosophila.

In vivo fluorescent adenosine 5'-triphosphate (ATP) imaging of Drosophila melanogaster and Caenorhabditis elegans by using a genetically encoded fluorescent ATP biosensor optimized for low temperatures.

Adenosine 5'-triphosphate (ATP) is the major energy currency of all living organisms. Despite its important functions, the spatiotemporal dynamics of ATP levels inside living multicellular organisms is unclear. In this study, we modified the genetically encoded Förster resonance energy transfer (FRET)-based ATP biosensor ATeam to optimize its affinity at low temperatures. This new biosensor, AT1.03NL, detected ATP changes inside Drosophila S2 cells more sensitively than the original biosensor did, at 25 °C. By expressing AT1.03NL in Drosophila melanogaster and Caenorhabditis elegans, we succeeded in imaging the in vivo ATP dynamics of these model animals at single-cell resolution.

High-resolution in vivo imaging of regenerating dendrites of Drosophila sensory neurons during metamorphosis: local filopodial degeneration and heterotypic dendrite-dendrite contacts.

Neuronal circuits that are formed in early development are reorganized at later developmental stages to support a wide range of adult behaviors. At Drosophila pupal stages, one example of this reorganization is dendritic remodeling of multidendritic neurons, which is accomplished by pruning and subsequent regeneration of branches in environments quite distinct from those in larval life. Here, we used long-term in vivo time-lapse recordings at high spatiotemporal resolution and analyzed the dynamics of two adjacent cell types that remodel dendritic arbors, which eventually innervate the lateral plate of the adult abdomen. These neurons initially exhibited dynamic extension, withdrawal and local degeneration of filopodia that sprouted from all along the length of regenerating branches. At a midpupal stage, branches extending from the two cell types started fasciculating with each other, which prompted us to test the hypothesis that this heterotypic contact may serve as a guiding scaffold for shaping dendritic arbors. Unexpectedly, our cell ablation study gave only marginal effects on the branch length and the arbor shape. This result suggests that the arbor morphology of the adult neurons in this study can be specified mostly in the absence of the dendrite-dendrite contact.

Cohesin controls planar cell polarity by regulating the level of the seven-pass transmembrane cadherin Flamingo.

Planar cell polarity (PCP) refers to the coordination of global organ axes and individual cell polarity in vertebrate and invertebrate epithelia. Mechanisms of PCP have been best studied in the Drosophila wing, in which each epidermal cell produces a single wing hair at the distal cell edge, and this spatial specification is mediated by redistribution of the core group proteins, including the seven-pass transmembrane cadherin Flamingo/Starry night (Fmi/Stan), to selective plasma membrane domains. Through genetic screening, we found that a mutation of the SMC3 gene caused dramatic misspecification of wing hair positions. SMC3 protein is one subunit of the cohesin complex, which regulates sister chromatid cohesion and also plays a role in transcriptional control of gene expression. In the SMC3 mutant cells, Fmi appeared to be upregulated by a posttranscriptional mechanism(s), and this elevation of Fmi was at least one cause of the PCP defect. In addition to the PCP phenotype, the loss of the cohesin function affected wing morphogenesis at multiple levels: one malformation was loss of the wing margin, and this was most likely a result of downregulation of the homeodomain protein Cut. At the cellular level, apical cell size and hexagonal packing were affected in the mutant wing. Dysfunction of cohesin in humans results in Cornelia de Lange syndrome (CdLS), which is characterized by various developmental abnormalities and mental retardation. Our analysis of cohesin in epithelia may provide new insight into cellular and molecular mechanisms of CdLS.