midline represses Dpp signaling and target gene expression in Drosophila ventral leg development

Ventral leg patterning in Drosophila is controlled by the expression of the redundant T-box Transcription factors midline (mid) and H15. Here we show that mid represses the Dpp-activated gene Daughters against decapentaplegic (Dad) through a consensus TBE site in the minimal enhancer, Dad13. Mutating the Dad13 DNA sequence results in an increased and broadening of Dad expression. We further demonstrate that the engrailed-homology-1 domain of Mid is critical for regulating the levels of phospho-Mad, a transducer of Dpp-signaling. However, we find that mid does not affect all Dpp-target genes as we demonstrate that brinker (brk) expression is unresponsive to mid. This study further illuminates the interplay between mechanisms involved in determination of cellular fate and the varied roles of mid.Summary statementVentral patterning is controlled in part by the T-box Transcription factor midline blocking Dpp signaling and Dpp-activated genes, though midline does not affect the Dpp-repressed gene brk.

A new animal model signifying a decisive bridge between innate immunity and the pathognomonic morphological characteristics of Type 1 Diabetes

Available animal models for Type 1 Diabetes (T1D) show limited similarities with the human disease and have no predictive value in screening for effective intervention therapies. Heat-inactivated bacteria instilled in the ductal compartment of the pancreas in healthy rats rapidly cause periductal inflammation and accumulation of mainly granulocytes and monocytes in the exocrine pancreas and in the peri-islet area mimicking the acute pancreatic inflammation in subjects with recent onset T1D. After three weeks, the triggered exocrine inflammation had vanished and pancreases showed normal morphology. However, a distinct accumulation of both CD4+ and CD8+ T cells within and adjacent to affected islets was found in one third of the rats, mimicking the pathognomonic insulitis in T1D. As in human T1D, the insulitis affected a fraction of all islets and was observed only in certain lobes of the pancreases. The presented animal model for T1D will allow detailed mechanistic studies to unravel a previously unknown interplay between bacteria-activated innate immunity and an acquired cellular immunity forming the immunopathological events described in humans at different stages of T1D.Summary StatementThe results presented signify a previously unknown decisive bridge between innate immunity and formation of the pathognomonic immunopathological events described in subjects with recent onset T1D.

Mitochondrial antiviral-signalling protein is a client of the BAG6 protein quality control complex

ABSTRACTThe heterotrimeric BAG6 complex coordinates the direct handover of newly synthesised tail-anchored (TA) membrane proteins from an SGTA-bound preloading complex to the endoplasmic reticulum (ER) delivery component TRC40. In contrast, defective precursors, including aberrant TA proteins, form a stable complex with this cytosolic protein quality control factor, enabling such clients to be either productively re-routed or selectively degraded. We identify the mitochondrial TA protein MAVS (mitochondrial antiviral-signalling protein) as an endogenous client of both SGTA and the BAG6 complex. Our data suggest that the BAG6 complex binds to a cytosolic pool of MAVS before its misinsertion into the ER membrane, from where it can subsequently be removed via ATP13A1-mediated dislocation. This BAG6- associated fraction of MAVS is dynamic and responds to the activation of an innate immune response, suggesting that BAG6 may modulate the pool of MAVS that is available for coordinating the cellular response to viral infection.SUMMARY STATEMENTMitochondrial antiviral-signalling (MAVS) protein is a favoured client of the cytosolic BAG6 complex. We discuss how this dynamic interaction may modulate MAVS biogenesis at signalling membranes.

Responses to Temperatures of Different Drosophila Species

Temperature is a critical environmental variable that affects the distribution, survival, and reproduction of most animals. Although temperature receptors have been identified in different animals, how these receptors respond to temperatures is largely unknown. Here we use modified single-fly thermotactic assays to analyze movements and temperature preferences of nine Drosophila species. The ability/inclination to move varies among these species and at different temperatures. Importantly, different species prefer various ranges of temperatures. While wild-type D. melanogaster flies avoid the warm temperature in the warm avoidance assay and the cool temperature in the cool avoidance assay, D. bipectinata and D. yakuba avoid neither warm nor cool temperatures and D. biarmipes and D. mojavensis do not avoid the warm temperature in the warm avoidance assay. These results demonstrate that Drosophila species have different mobilities and temperature preferences, thereby benefiting the research on molecular mechanisms of temperature responsiveness.Summary statementThe ability to move and the preference for temperatures vary among fly species when flies are exposed to steep temperature gradients.

Fibroblast-derived HGF integrates muscle and nerve development during morphogenesis of the mammalian diaphragm

The diaphragm is a domed muscle between the thorax and abdomen essential for breathing in mammals. Diaphragm development requires the coordinated development of muscle, connective tissue, and nerve, which are derived from different embryonic sources. Defects in diaphragm development cause the common and often lethal birth defect, Congenital Diaphragmatic Hernias (CDH). HGF/MET signaling is required for diaphragm muscularization, but the source of HGF and the specific functions of this pathway in muscle progenitors or potentially the phrenic nerve have not been explicitly tested. Using conditional mutagenesis and pharmacological inhibition of MET, we demonstrate that the pleuroperitoneal folds (PPFs), transient embryonic structures that give rise to the connective tissue, are the source of HGF critical for diaphragm muscularization and phrenic nerve primary branching. HGF not only is required for recruitment of muscle progenitors to the diaphragm, but is continuously required for maintenance and motility of the pool of progenitors to enable full muscularization. Thus, the connective tissue fibroblasts and HGF coordinately regulate diaphragm muscularization and innervation. Defects in PPF-derived HGF result in muscleless regions that are susceptible to CDH.Summary StatementFibroblast-derived HGF signals to Met+ muscle progenitors and nerve to control the expansion of diaphragm muscle and primary branching of phrenic nerve axons - structures critical for breathing in mammals.

Using TrackMate to Analyze Drosophila Larval and Adult Locomotion

Drosophila adult and larvae exhibit sophisticated behaviors that are widely used in development, synaptic transmission, sensory physiology, and learning and memory research. Many of these behaviors depend on locomotion, the ability of an animal to move. However, the statistical analysis of locomotion is not trivial. Here we use an open-source Fiji plugin TrackMate to track the locomotion of Drosophila adults and larvae. We build optimal experimental setups to rapidly process recordings by Fiji and analyze by TrackMate. We also provide tips for analyzing non-optimal recordings. TrackMate extracts the X and Y positions of an animal on each frame of an image sequence or a video. This information allows for generating moving trajectories, calculating moving distances, and determining preference indices in two-choice assays. Notably, this free-cost analysis method does not require programming skills.Summary statementThis study uses an open-source Fiji plugin TrackMate to computationally analyze Drosophila adult and larval behavioral assays, which does not require programming skills.

Admp regulates tail bending by controlling the intercalation of the ventral epidermis through myosin phosphorylation

Chordate tailbud embryos have similar morphological features, including a bending tail. A recent study revealed that the actomyosin of the notochord changes the contractility and drive tail bending of the early Ciona tailbud embryo. Yet, the upstream regulator of tail bending remains unknown. In this study, we find that Admp regulates tail bending of Ciona mid-tailbud embryos. Anti-pSmad antibody signal was detected at the ventral midline tail epidermis. Admp knock-down embryo completely inhibited the ventral tail bending and reduced the number of the triangular-shaped cells, which has the apical accumulation of the myosin phosphorylation and inhibited specifically the cell-cell intercalation of the ventral epidermis. The degree of myosin phosphorylation of the ventral cells and tail bending were correlated. Finally, the laser cutter experiments demonstrated the myosin-phosphorylation-dependent tension of the ventral midline epidermis during tail bending. We conclude that Admp is an upstream regulator of the tail bending by controlling myosin phosphorylation and its localization of ventral epidermal cells. These data reveal a new aspect of the function of the Admp that might be evolutionarily conserved in bilaterian animals.Summary StatementAdmp is an upstream regulator of the bending of the tail in the tailbud embryo regulating tissue polarity of the ventral midline epidermis by phosphorylation of myosin.

Myosin-1b interacts with UNC45A and controls intestinal epithelial morphogenesis

Vesicle trafficking and the establishment of apico-basal polarity are essential processes in epithelium morphogenesis. Myosin-1b, an actin-motor able to bind membranes, regulates membrane shaping and vesicle trafficking. Here, we investigate Myosin-1b function in gut morphogenesis and congenital disorders using cell line and zebrafish larvae as well as patient biopsies. In a 3D Caco-2 cyst model, lumen formation is impaired in absence of Myosin-1b. In zebrafish, both Morpholino knock-down and genetic mutation of myo1b result in intestinal bulb epithelium folding defects associated with vesicle accumulation, reminiscent of a villous atrophy phenotype. We show that Myosin-1b interacts with the chaperone UNC45A, genetic deletion of which also results in gut folding defects in zebrafish. Loss of function mutations in UNC45A have been reported in complex hereditary syndromes, notably exhibiting intestinal disorders associated with villous atrophy. In UNC45A-depleted cells and in patient biopsies, Myosin-1b protein level is strikingly decreased. The appearance of Myosin-1b aggregates upon proteasome inhibition in cells points at a degradation mechanism of misfolded Myosin-1b in the absence of its chaperone. In conclusion, Myosin-1b plays an unexpected role in the development of the intestinal epithelium folds or villi downstream UNC45A, establishing its role in the gut defects reported in UNC45A patients.Summary statementMyosin-1b is important for intestinal epithelium folding during zebrafish development and participates in the villous atrophy clinical manifestation downstream UNC45A loss of function.

Experimental Evolution of a Pheromone Signal

ABSTRACTSexual signals are important in speciation, but understanding their evolution is complex as these signals are often composed of multiple, genetically interdependent components. To understand how signals evolve, we thus need to consider selection responses in multiple components and account for the genetic correlations among components. One intriguing possibility is that selection changes genetic correlations between components under selection and those not under selection, reducing evolutionary constraints. However, this hypothesis remains largely untested empirically. In this study, we investigate the evolutionary response of the multi-component female sex pheromone blend of the moth Heliothis subflexa to 10 generations of artificial selection. We observed a selection response of about 3/4s of a phenotypic standard deviation in the components under selection. Interestingly, other pheromone components that are biochemically and genetically linked to the components under selection did not change. We found that this component-specific selection response was likely facilitated due to reduced genetic coupling between the component under selection and the components not under selection, resulting from changes in genetic covariances within the 10 generations of selection. Our findings provide rare empirical support for an intriguing mechanism by which a sexual signal can respond to selection without possible constraints from indirect selection responses.SUMMARY STATEMENTThis study reveals a mechanism that can facilitate evolution of sex pheromone components under selection independent of other components of the sex pheromone that are not under selection.

Entry to and exit from diapause arrest in Caenorhabditis elegans are both regulated by a steroid hormone pathway

Diapause arrest in animals such as Caenorhabditis elegans is tightly regulated so that animals make appropriate developmental decisions amidst environmental challenges. Fully understanding diapause requires mechanistic insight of both entry and exit from the arrested state. While a steroid hormone pathway regulates the entry decision into Caenorhabditis elegans dauer diapause, its role in the exit decision is less clear. A complication to understanding steroid hormonal regulation of dauer has been the peculiar fact that steroid hormone mutants such as daf-9 form partial dauers under normal growth conditions. Here, we corroborate previous findings that daf-9 mutants remain capable of forming full dauers under unfavorable growth conditions, and we establish that the daf-9 partial dauer state is likely a partially exited dauer that has initiated but cannot complete the dauer exit decision. We show that the steroid hormone pathway is both necessary for and promotes complete dauer exit, and that the spatiotemporal dynamics of steroid hormone regulation during dauer exit resembles that of dauer entry. Overall, dauer entry and dauer exit are distinct developmental decisions that are both controlled by steroid hormone signaling.Summary StatementIn animals such as Caenorhabditis elegans, a steroid hormone pathway controls both the entry and exit decisions into and out of the developmentally arrested dauer state in response to environmental signaling.