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Memory guides behavior across widely varying environments and must therefore be both sufficiently specific and general. A memory too specific will be useless in even a slightly different environment, while an overly general memory may lead to suboptimal choices. Animals successfully learn to both distinguish between very similar stimuli and generalize across cues. Rather than forming memories that strike a balance between specificity and generality,Drosophilacan flexibly categorize a given stimulus into different groups depending on the options available. We asked how this flexibility manifests itself in the well-characterized learning and memory pathways of the fruit fly. We show that flexible categorization in neuronal activity as well as behavior depends on the order and identity of the perceived stimuli. Our results identify the neural correlates of flexible stimulus-categorization in the fruit fly.Impact StatementFlies can optimally recall a memory with high specificity by comparing options close in time, or default to generalization when they cannot.

Representing direction in the fly A population of cells called compass neurons represents a fruitfly's heading direction. Kim et al. used imaging and optogenetics in behaving flies to elucidate the functional architecture of the underlying neuronal network. They observed local excitation and global inhibition between the compass neurons. The features of the network were best explained by a ring attractor network model. Until now, this hypothesized network structure has been difficult to demonstrate in a real brain. Science , this issue p. 849

Many animals maintain an internal representation of their heading as they move through their surroundings. Such a compass representation was recently discovered in a neural population in the Drosophila melanogaster central complex, a brain region implicated in spatial navigation. Here, we use two-photon calcium imaging and electrophysiology in head-fixed walking flies to identify a different neural population that conjunctively encodes heading and angular velocity, and is excited selectively by turns in either the clockwise or counterclockwise direction. We show how these mirror-symmetric turn responses combine with the neurons’ connectivity to the compass neurons to create an elegant mechanism for updating the fly’s heading representation when the animal turns in darkness. This mechanism, which employs recurrent loops with an angular shift, bears a resemblance to those proposed in theoretical models for rodent head direction cells. Our results provide a striking example of structure matching function for a broadly relevant computation. DOI: http://dx.doi.org/10.7554/eLife.23496.001

International audience; INTRODUCTION: Spatial patterning in developing multicellular organisms relies on positional cues and cell-cell interactions. Stereotyped sensory organ arrangements in Drosophila are commonly attributed to a prepattern that defines regions of neural competence. Notch-mediated interactions then isolate sensory organ precursor (SOP) cells from among the competent cells. In support of this view, prepattern factors direct the expression of proneural factors in discrete clusters and determine the location of large bristles on the dorsal thorax. However, no such prepattern is known to establish the proneural stripes that give rise to finer-bristle rows.RATIONALE: By analogy with reaction-diffusion systems, we wondered whether Notch-mediated cell-cell interactions might organize a pattern of proneural stripes. To explore a possible role for Notch in proneural patterning, we generated fluorescent reporters for the proneural factors Achaete and Scute, the ligand Delta, and the Notch early-response factor E(spl)m3-HLH, which antagonizes proneural activity. We observed expression of these reporters in live and fixed samples throughout early pupal development. In parallel, we developed a mathematical model for Notch-mediated patterning. In this abstract model, the dynamics of a cell is expressed in terms of just two variables, for the state of the cell and the level of signal it receives. The model incorporates a series of plausible assumptions that govern its patterning behavior: Cells, which adopt the SOP fate in the absence of signal and the alternative, epidermal fate under high enough signal, exhibit a bistable response under intermediate signal levels. Inhibitory signaling from a cell varies nonlinearly with cell state and reaches beyond immediate neighbors.RESULTS: Before the onset of proneural gene expression, a bimodal gradient of Delta expression drove Notch activity; as a result of cis-inhibition, Notch was activated only in regions of intermediate Delta levels. This defined an inhibitory template for a first series of three proneural stripes. The spatial pattern of Notch activity dynamically changed as these first proneural stripes emerged, forming a negative template for a second group of intercalating stripes. Eventually, each stripe resolved into a row of isolated SOPs through Notch signaling. Thus, Notch mediated both proneural stripe patterning and SOP selection via a self-organized process. Simulations of the model, with a time-dependent inhibitory gradient describing the proneural-independent signaling seen in vivo, recapitulated the sequential emergence and resolution of proneural stripes. In both model and experiments, mutual inhibition drove a gradual refinement of the proneural group, concomitant with the buildup of proneural activity. Cells on the sides of the stripes were excluded first, such that stripes narrowed before isolated SOPs emerged. In terms of the model, cell-intrinsic bistability allowed cells with higher proneural activity, at the center of the stripes, to evade levels of inhibition that were sufficient to exclude cells on the sides. Nonlinear signaling allowed a smooth transition from weak mutual inhibition within an extended proneural group to strong lateral inhibition from emerging SOPs, ensuring that attrition of the proneural group proceeds until only isolated cells remain. Finally, the model correctly predicted the outcome of perturbation experiments affecting the pattern of Notch activity, the level and activity of Delta, and the range of signaling.CONCLUSION:Our results show that self-organized Notch signaling can establish stripe and dot patterns on a tissue-wide scale. A transient spatial bias, mediated by an initial gradient of Delta, is transduced by cell-cell interactions to produce a finer pattern of proneural stripes and bristle rows. Input from extrinsic positional cues and self-organization, sometimes considered competing paradigms for fate patterning, combine during bristle development and operate through the same signal. Self-organized Notch dynamics may provide a flexible substrate to generate diverse patterns in response to varying inputs.

Ring attractors are a class of recurrent networks hypothesized to underlie the representation of heading direction. Such network structures, schematized as a ring of neurons whose connectivity depends on their heading preferences, can sustain a bump-like activity pattern whose location can be updated by continuous shifts along either turn direction. We recently reported that a population of fly neurons represents the animal's heading via bump-like activity dynamics. We combined two-photon calcium imaging in head-fixed flying flies with optogenetics to overwrite the existing population representation with an artificial one, which was then maintained by the circuit with naturalistic dynamics. A network with local excitation and global inhibition enforces this unique and persistent heading representation. Ring attractor networks have long been invoked in theoretical work; our study provides physiological evidence of their existence and functional architecture.

The emergence of spatial patterns in developing multicellular organisms relies on positional cues and cell-cell communication.

The acquisition of different fates by cells that are initially in the same state is central to development. Here, we investigate the possible structures of bistable genetic networks that can allow two identical cells to acquire different fates through cell-cell interactions. Cell-autonomous bistable networks have been previously sampled using an evolutionary algorithm. We extend this evolutionary procedure to take into account interactions between cells. We obtain a variety of simple bistable networks that we classify into major subtypes. Some have long been proposed in the context of lateral inhibition through the Notch-Delta pathway, some have been more recently considered and others appear to be new and based on mechanisms not previously considered. The results highlight the role of posttranscriptional interactions and particularly of protein complexation and sequestration, which can replace cooperativity in transcriptional interactions. Some bistable networks are entirely based on posttranscriptional interactions and the simplest of these is found to lead, upon a single parameter change, to oscillations in the two cells with opposite phases. We provide qualitative explanations as well as mathematical analyses of the dynamical behaviors of various created networks. The results should help to identify and understand genetic structures implicated in cell-cell interactions and differentiation.

Cell fate determination depends in part on the establishment of specific transcriptional programs of gene expression. These programs result from the interpretation of the genomic cis-regulatory information by sequence-specific factors. Decoding this information in sequenced genomes is an important issue. Here, we developed statistical analysis tools to computationally identify the cis-regulatory elements that control gene expression in a set of coregulated genes. Starting with a small number of validated and/or predicted cis-regulatory modules (CRMs) in a reference species as a training set, but with no a priori knowledge of the factors acting in trans , we computationally predicted transcription factor binding sites (TFBSs) and genomic CRMs underlying coregulation. This method was applied to the gene expression program active in Drosophila melanogaster sensory organ precursor cells (SOPs), a specific type of neural progenitor cells. Mutational analysis showed that four, including one newly characterized, out of the five top-ranked families of predicted TFBSs were required for SOP-specific gene expression. Additionaly, 19 out of the 29 top-ranked predicted CRMs directed gene expression in neural progenitor cells, i.e., SOPs or larval brain neuroblasts, with a notable fraction active in SOPs (11/29). We further identified the lola gene as the target of two SOP-specific CRMs and found that the lola gene contributed to SOP specification. The statistics and phylogeny-based tools described here can be more generally applied to identify the cis-regulatory elements of specific gene regulatory networks in any family of related species with sequenced genomes.

A fundamental and unresolved problem in animal development is the question of how a growing tissue knows when it has achieved its correct final size. A widely held view suggests that this process is controlled by morphogen gradients, which adapt to tissue size and become flatter as tissue grows, leading eventually to growth arrest. Here, we present evidence that the decapentaplegic (Dpp) morphogen distribution in the developing Drosophila wing imaginal disk does not adapt to disk size. We measure the distribution of a functional Dpp-GFP transgene and the Dpp signal transduced by phospho-Mad and show that the characteristic length scale of the Dpp profile remains approximately constant during growth. This finding suggests an alternative scenario of size determination, where disk size is determined relative to the fixed morphogen distribution by a certain threshold level of morphogen required for growth. We propose that when disk boundary reaches the threshold the arrest of cell proliferation throughout the disk is induced by mechanical stress in the tissue. Mechanical stress is expected to arise from the nonuniformity of morphogen distribution that drives growth. This stress, through a negative feedback on growth, can compensate for the nonuniformity of morphogen, achieving uniform growth with the rate that vanishes when disk boundary reaches the threshold. The mechanism is demonstrated through computer simulations of a tissue growth model that identifies the key assumptions and testable predictions. This analysis provides an alternative hypothesis for the size determination process. Novel experimental approaches will be needed to test this model.

Summary During development, cell-fate diversity can result from the unequal segregation of fate determinants at mitosis [1]. Polarization of the mother cell is essential for asymmetric cell division (ACD). It often involves the formation of a cortical domain containing the PAR complex proteins Par3, Par6, and atypical protein kinase C (aPKC) [1–5]. In the fly notum, sensory organ precursor cells (SOPs) divide asymmetrically within the plane of the epithelium and along the body axis to generate two distinct cells [6–12]. Fate asymmetry depends on the asymmetric localization of the PAR complex. In the absence of planar cell polarity (PCP), SOPs divide with a random planar orientation but still asymmetrically, showing that PCP is dispensable for PAR asymmetry at mitosis [6, 13–15]. To study when and how the PAR complex localizes asymmetrically, we have used a quantitative imaging approach to measure the planar polarization of the proteins Bazooka (Baz, fly Par3), Par6, and aPKC in living pupae. By using imaging of functional GFP-tagged proteins with image processing and computational modeling, we find that Baz, Par6, and aPKC become planar polarized prior to mitosis in a manner independent of the AuroraA kinase and that PCP is required for the planar polarization of Baz, Par6, and aPKC during interphase. This indicates that a "mitosis rescue" mechanism establishes asymmetry at mitosis in PCP mutants. This study therefore identifies PCP as the initial symmetry-breaking signal for the planar polarization of PAR proteins in asymmetrically dividing SOPs.

International audience; Cis-regulatory modules (CRMs) and motifs play a central role in tissue and condition-specific gene expression. Here we present Imogene, an ensemble of statistical tools that we have developed to facilitate their identification and implemented in a publicly available software. Starting from a small training set of mammalian or fly CRMs that drive similar gene expression profiles, Imogene determines de novo cis-regulatory motifs that underlie this co-expression. It can then predict on a genome-wide scale other CRMs with a regulatory potential similar to the training set. Imogene bypasses the need of large datasets for statistical analyses by making central use of the information provided by the se-quenced genomes of multiple species, based on the developed statistical tools and explicit models for transcription factor binding site evolution. We test Imogene on characterized tissue-specific mouse developmental CRMs. Its ability to identify CRMs with the same specificity based on its de novo created motifs is comparable to that of previously evaluated 'motif-blind' methods. We further show, both in flies and in mammals, that Imogene de novo generated motifs are sufficient to discriminate CRMs related to different developmental programs. Notably, purely relying on sequence data, Imogene performs as well in this discrimination task as a previously reported learning algorithm based on Chromatin Immunopre-cipitation (ChIP) data for multiple transcription factors at multiple developmental stages.

Organismal development is under genetic control. Ultimately, mechanical forces shape embryos. If we want to understand the precise regulation of size and shape in animals, we must dissect how forces are distributed in developing tissues, and how they drive cell behavior to shape organs. This has not been addressed fully in the context of growing tissues. As cells grow and divide, they exert a pressure on their neighbors. How these local stresses add up or dissipate as the tissue grows is an unanswered question. We address this issue in the growing wing imaginal disc of Drosophila larvae, the precursor of the adult wing. We used a quantitative approach to analyze the strains and stresses of cells of the wing pouch, and found a global pattern of stress whereby cells in the periphery of the tissue are mechanically stretched and cells in the center are compressed. This pattern has important consequences on cell shape in the wing pouch: cells respond to it by polarizing their acto-myosin cortex, and aligning their divisions with the main axis of cell stretch, thereby polarizing tissue growth. Ectopic perturbations of tissue growth by the Hippo signaling pathway reorganize this pattern in a non-autonomous manner, suggesting a synergy between tissue mechanics and growth control during wing disc morphogenesis.

International audience; Background: Developmental programs are implemented by regulatory interactions between Transcription Factors (TFs) and their target genes, which remain poorly understood. While recent studies have focused on regulatory cascades of TFs that govern early development, little is known about how the ultimate effectors of cell differentiation are selected and controlled. We addressed this question during late Drosophila embryogenesis, when the finely tuned expression of the TF Ovo/Shavenbaby (Svb) triggers the morphological differentiation of epidermal trichomes.Results: We defined a sizeable set of genes downstream of Svb and used in vivo assays to delineate 14 enhancers driving their specific expression in trichome cells. Coupling computational modeling to functional dissection, we investigated the regulatory logic of these enhancers. Extending the repertoire of epidermal effectors using genome-wide approaches showed that the regulatory models learned from this first sample are representative of the whole set of trichome enhancers. These enhancers harbor remarkable features with respect to their functional architectures, including a weak or non-existent clustering of Svb binding sites. The in vivo function of each site relies on its intimate context, notably the flanking nucleotides. Two additional cis-regulatory motifs, present in a broad diversity of composition and positioning among trichome enhancers, critically contribute to enhancer activity.Conclusions: Our results show that Svb directly regulates a large set of terminal effectors of the remodeling of epidermal cells. Further, these data reveal that trichome formation is underpinned by unexpectedly diverse modes of regulation, providing fresh insights into the functional architecture of enhancers governing a terminal differentiation program.

Notch receptors in a given cell are activated by cell surface ligands in neighbouring cells but can also be inhibited by the ligands present within the same cell. This process is known as cis-inhibition of Notch. Additionally, reciprocal cis-inhibition of the ligands by Notch has also been observed, albeit to a limited extent. Here, we review the mechanisms, functional relevance and potential implications of these cis-inhibitory interactions for Notch-mediated fate decisions.