Long-range formation of the Bicoid gradient requires multiple dynamic modes that spatially vary across the embryo.

Morphogen gradients provide essential positional information to gene networks through their spatially heterogeneous distribution, yet how they form is still hotly contested, with multiple models proposed for different systems. Here, we focus on the transcription factor Bicoid (Bcd), a morphogen that forms an exponential gradient across the anterior-posterior (AP) axis of the early Drosophila embryo. Using fluorescence correlation spectroscopy we find there are spatial differences in Bcd diffusivity along the AP axis, with Bcd diffusing more rapidly in the posterior. We establish that such spatially varying differences in Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole. In the nucleus, we demonstrate that Bcd dynamics are impacted by binding to DNA. Addition of the Bcd homeodomain to eGFP::NLS qualitatively replicates the Bcd concentration profile, suggesting this domain regulates Bcd dynamics. Our results reveal how a long-range gradient can form while retaining a steep profile through much of its range.

Deep learning reduces data requirements and allows real-time measurements in imaging FCS.

Imaging fluorescence correlation spectroscopy (FCS) is a powerful tool to extract information on molecular mobilities, actions, and interactions in live cells, tissues, and organisms. Nevertheless, several limitations restrict its applicability. First, FCS is data hungry, requiring 50,000 frames at 1-ms time resolution to obtain accurate parameter estimates. Second, the data size makes evaluation slow. Third, as FCS evaluation is model dependent, data evaluation is significantly slowed unless analytic models are available. Here, we introduce two convolutional neural networks-FCSNet and ImFCSNet-for correlation and intensity trace analysis, respectively. FCSNet robustly predicts parameters in 2D and 3D live samples. ImFCSNet reduces the amount of data required for accurate parameter retrieval by at least one order of magnitude and makes correct estimates even in moderately defocused samples. Both convolutional neural networks are trained on simulated data, are model agnostic, and allow autonomous, real-time evaluation of imaging FCS measurements.

Mechanics of epidermal morphogenesis in the Drosophila pupa.

Adult epidermal development in Drosophila showcases a striking balance between en masse spreading of the developing adult precursor tissues and retraction of the degenerating larval epidermis. The adult precursor tissues, driven by both intrinsic plasticity and extrinsic mechanical cues, shape the segments of the adult epidermis and appendages. Here, we review the tissue architectural changes that occur during epidermal morphogenesis in the Drosophila pupa, with a particular emphasis on the underlying mechanical principles. We highlight recent developments in our understanding of adult epidermal morphogenesis. We further discuss the forces that drive these morphogenetic events and finally outline open questions and challenges.

NMR Spectroscopy-based Metabolomics of Drosophila Model of Huntington's Disease Suggests Altered Cell Energetics.

Huntington's disease (HD) is a neurodegenerative disorder induced by aggregation of the pathological form of Huntingtin protein that has expanded polyglutamine (polyQ) repeats. In the Drosophila model, for instance, expression of transgenes with polyQ repeats induces HD-like pathologies, progressively correlating with the increasing lengths of these repeats. Previous studies on both animal models and clinical samples have revealed metabolite imbalances during HD progression. To further explore the physiological processes linked to metabolite imbalances during HD, we have investigated the 1D 1H NMR spectroscopy-based metabolomics profile of Drosophila HD model. Using multivariate analysis (PCA and PLS-DA) of metabolites obtained from methanolic extracts of fly heads displaying retinal deformations due to polyQ overexpression, we show that the metabolite imbalance during HD is likely to affect cell energetics. Six out of the 35 metabolites analyzed, namely, nicotinamide adenine dinucleotide (NAD), lactate, pyruvate, succinate, sarcosine, and acetoin, displayed segregation with progressive severity of HD. Specifically, HD progression was seen to be associated with reduction in NAD and increase in lactate-to-pyruvate ratio. Furthermore, comparative analysis of fly HD metabolome with those of mouse HD model and HD human patients revealed comparable metabolite imbalances, suggesting altered cellular energy homeostasis. These findings thus raise the possibility of therapeutic interventions for HD via modulation of cellular energetics.

Proximate larval epidermal cell layer generates forces for Pupal thorax closure in Drosophila.

During tissue closures, such as embryonic dorsal closure in Drosophila melanogaster, a proximate extra-embryonic layer, amnioserosa, generates forces that drive migration of the flanking lateral embryonic epidermis, thereby zip-shutting the embryo. Arguably, this paradigm of tissue closure is also recapitulated in mammalian wound healing wherein proximate fibroblasts transform into contractile myofibroblasts, develop cell junctions, and form a tissue layer de novo: contraction of the latter then aids in wound closure. Given this parallelism between disparate exemplars, we posit a general principle of tissue closure via proximate cell layer-generated forces. Here, we have tested this hypothesis in pupal thorax closure wherein 2 halves of the presumptive adult thorax of Drosophila, the contralateral heminotal epithelia, migrate over an underlying larval epidermal cell layer. We show that the proximate larval epidermal cell layer promotes thorax closure by its active contraction, orchestrated by its elaborate actomyosin network-driven epithelial cell dynamics, cell delamination, and death-the latter being prefigured by the activation of caspases. Larval epidermal cell dynamics generate contraction forces, which when relayed to the flanking heminota-via their mutual integrin-based adhesions-mediate thorax closure. Compromising any of these contraction force-generating mechanisms in the larval epidermal cell layer slows down heminotal migration, while loss of its relay to the flanking heminota abrogates the thorax closure altogether. Mathematical modeling further reconciles the biophysical underpinning of this emergent mechanism of thorax closure. Revealing mechanism of thorax closure apart, these findings show conservation of an essential principle of a proximate cell layer-driven tissue closure.

Heterophilic cell-cell adhesion of atypical cadherins Fat and Dachsous regulate epithelial cell size dynamics during Drosophila thorax morphogenesis.

Spatiotemporal changes in epithelial cell sizes-or epithelial cell size dynamics (ECD)-during morphogenesis entail interplay between two opposing forces: cell contraction via actomyosin cytoskeleton and cell expansion via cell-cell adhesion. Cell-cell adhesion-based ECD, however, has not yet been clearly demonstrated. For instance, changing levels of homophilic E-cadherin-based cell-cell adhesion induce cell sorting, but not ECD. Here we show that cell-expansive forces of heterophilic cell-cell adhesion regulate ECD: higher cell-cell adhesion results in cell size enlargement. Thus, ECD during morphogenesis in the heminotal epithelia of Drosophila pupae leading to thorax closure corresponds with spatiotemporal gradients of two heterophilic atypical cadherins-Fat (Ft) and Dachsous (Ds)-and the levels of Ft-Ds heterodimers formed concomitantly. Our mathematical modeling and genetic tests validate this mechanism of dynamic heterophilic cell-cell adhesion-based regulation of ECD. Conservation of these atypical cadherins suggests a wider prevalence of heterophilic cell-cell adhesion-based ECD regulation during animal morphogenesis.

Discovery of Arginine Ethyl Ester as Polyglutamine Aggregation Inhibitor: Conformational Transitioning of Huntingtin N-Terminus Augments Aggregation Suppression.

Huntington's disease (HD) is a genetic disorder caused by a CAG expansion mutation in the huntingtin gene leading to polyglutamine (polyQ) expansion in the N-terminal part of huntingtin (Httex1). Expanded polyQ, through a complex aggregation pathway, forms aggregates in neurons and presents a potential therapeutic target. Here we show Httex1 aggregation suppression by arginine and arginine ethyl ester (AEE) in vitro, as well as in yeast and mammalian cell models of HD, bearing expanded polyQ. These molecules also rescue locomotion dysfunction in HD Drosophila model. Both molecules alter the hydrogen bonding network of polyQ to enhance its aqueous solubility and delay aggregation. AEE shows direct binding with the NT17 part of Httex1 to induce structural changes to impart an enhanced inhibitory effect. This study provides a platform for the development of better arginine based therapeutic molecules against polyQ-rich Httex1 aggregation.