Although different animal species often exhibit extensive variation in many behaviors, typically scientists examine one or a small number of behaviors in any single study. Here, we propose a new framework to simultaneously study the evolution of many behaviors. We measured the behavioral repertoire of individuals from six species of fruit flies using unsupervised techniques and identified all stereotyped movements exhibited by each species. We then fit a Generalized Linear Mixed Model to estimate the intra- and inter-species behavioral covariances, and, by using the known phylogenetic relationships among species, we estimated the (unobserved) behaviors exhibited by ancestral species. We found that much of intra-specific behavioral variation has a similar covariance structure to previously described long-time scale variation in an individual's behavior, suggesting that much of the measured variation between individuals of a single species in our assay reflects differences in the status of neural networks, rather than genetic or developmental differences between individuals. We then propose a method to identify groups of behaviors that appear to have evolved in a correlated manner, illustrating how sets of behaviors, rather than individual behaviors, likely evolved. Our approach provides a new framework for identifying co-evolving behaviors and may provide new opportunities to study the mechanistic basis of behavioral evolution.

Regions of genomic DNA called enhancers encode binding sites for transcription factor proteins. Binding of activators and repressors increase and reduce transcription, respectively, but it is not understood how combinations of activators and repressors generate precise patterns of transcription during development. Here, we explore this problem using a fully synthetic transcriptional platform in Drosophila consisting of engineered transcription factor gradients and artificial enhancers. We found that binding sites for a transcription factor that makes DNA accessible are required together with binding sites for transcriptional activators to produce a functional enhancer. Only in this context can changes in the number of activator binding sites mediate quantitative control of transcription. Using an engineered transcriptional repressor gradient, we demonstrate that overlapping repressor and activator binding sites provide more robust repression and sharper expression boundaries than non-overlapping sites. This may explain why this common motif is observed in many developmental enhancers.

Topographic maps, the systematic spatial ordering of neurons by response tuning, are common across species. In Drosophila, the lobula columnar (LC) neuron types project from the optic lobe to the central brain, where each forms a glomerulus in a distinct position. However, the advantages of this glomerular arrangement are unclear. Here, we examine the functional and spatial relationships of 10 glomeruli using single-neuron calcium imaging. We discover novel detectors for objects smaller than the lens resolution (LC18) and for complex line motion (LC25). We find that glomeruli are spatially clustered by selectivity for looming versus drifting object motion and ordered by size tuning to form a topographic visual feature map. Furthermore, connectome analysis shows that downstream neurons integrate from sparse subsets of possible glomeruli combinations, which are biased for glomeruli encoding similar features. LC neurons are thus an explicit example of distinct feature detectors topographically organized to facilitate downstream circuit integration.

We report the larval CNS expression patterns for 6,650 GAL4 lines based on cis-regulatory regions (CRMs) from the Drosophila genome. Adult CNS expression patterns were previously reported for this collection, thereby providing a unique resource for determining the origins of adult cells. An illustrative example reveals the origin of the astrocyte-like glia of the ventral CNS. Besides larval neurons and glia, the larval CNS contains scattered lineages of immature, adult-specific neurons. Comparison of lineage expression within this large collection of CRMs provides insight into the codes used for designating neuronal types. The CRMs encode both dense and sparse patterns of lineage expression. There is little correlation between brain and thoracic lineages in patterns of sparse expression, but expression in the two regions is highly correlated in the dense mode. The optic lobes, by comparison, appear to use a different set of genetic instructions in their development.

We established a collection of 7,000 transgenic lines of Drosophila melanogaster. Expression of GAL4 in each line is controlled by a different, defined fragment of genomic DNA that serves as a transcriptional enhancer. We used confocal microscopy of dissected nervous systems to determine the expression patterns driven by each fragment in the adult brain and ventral nerve cord. We present image data on 6,650 lines. Using both manual and machine-assisted annotation, we describe the expression patterns in the most useful lines. We illustrate the utility of these data for identifying novel neuronal cell types, revealing brain asymmetry, and describing the nature and extent of neuronal shape stereotypy. The GAL4 lines allow expression of exogenous genes in distinct, small subsets of the adult nervous system. The set of DNA fragments, each driving a documented expression pattern, will facilitate the generation of additional constructs for manipulating neuronal function. synapse was substantially elevated, at the endocytic zone there was no enhanced polymerization activity. We conclude that actin subserves spatially diverse, independently regulated processes throughout spines. Perisynaptic actin forms a uniquely dynamic structure well suited for direct, active regulation of the synapse.

For the overall strategy and methods used to produce the GAL4 lines:
Pfeiffer, B.D., Jenett, A., Hammonds, A.S., Ngo, T.T., Misra, S., Murphy, C., Scully, A., Carlson, J.W., Wan, K.H., Laverty, T.R., Mungall, C., Svirskas, R., Kadonaga, J.T., Doe, C.Q., Eisen, M.B., Celniker, S.E., Rubin, G.M. (2008). Tools for neuroanatomy and neurogenetics in Drosophila. Proc. Natl. Acad. Sci. USA 105, 9715-9720. http://www.pnas.org/content/105/28/9715.full.pdf+html synapse was substantially elevated, at the endocytic zone there was no enhanced polymerization activity. We conclude that actin subserves spatially diverse, independently regulated processes throughout spines. Perisynaptic actin forms a uniquely dynamic structure well suited for direct, active regulation of the synapse.

For data on expression in the embryo:
Manning, L., Purice, M.D., Roberts, J., Pollard, J.L., Bennett, A.L., Kroll, J.R., Dyukareva, A.V., Doan, P.N., Lupton, J.R., Strader, M.E., Tanner, S., Bauer, D., Wilbur, A., Tran, K.D., Laverty, T.R., Pearson, J.C., Crews, S.T., Rubin, G.M., and Doe, C.Q. (2012) Annotated embryonic CNS expression patterns of 5000 GMR GAL4 lines: a resource for manipulating gene expression and analyzing cis-regulatory motifs. Cell Reports (2012) Doi: 10.1016/j.celrep.2012.09.009 http://www.cell.com/cell-reports/fulltext/S2211-1247(12)00290-2 synapse was substantially elevated, at the endocytic zone there was no enhanced polymerization activity. We conclude that actin subserves spatially diverse, independently regulated processes throughout spines. Perisynaptic actin forms a uniquely dynamic structure well suited for direct, active regulation of the synapse.

For data on expression in imaginal discs:
Jory, A., Estella, C., Giorgianni, M.W., Slattery, M., Laverty, T.R., Rubin, G.M., and Mann, R.S. (2012) A survey of 6300 genomic fragments for cis-regulatory activity in the imaginal discs of Drosophila melanogaster. Cell Reports (2012) Doi: 10.1016/j.celrep.2012.09.010 http://www.cell.com/cell-reports/fulltext/S2211-1247(12)00291-4 synapse was substantially elevated, at the endocytic zone there was no enhanced polymerization activity. We conclude that actin subserves spatially diverse, independently regulated processes throughout spines. Perisynaptic actin forms a uniquely dynamic structure well suited for direct, active regulation of the synapse.

For data on expression in the larval nervous system:
Li, H.-H., Kroll, J.R., Lennox, S., Ogundeyi, O., Jeter, J., Depasquale, G., and Truman, J.W. (2013) A GAL4 driver resource for developmental and behavioral studies on the larval CNS of Drosophila. Cell Reports (submitted).

Fruit flies recognize hundreds of ecologically relevant odors and respond appropriately to them. The complexity, redundancy and interconnectedness of the olfactory machinery complicate efforts to pinpoint the functional contributions of any component neuron or receptor to behavior. Some contributions can only be elucidated in flies that carry multiple mutations and transgenes, but the production of such flies is currently labor-intensive and time-consuming. Here, we describe a set of transgenic flies that express the GAL80 in specific olfactory sensory neurons (). The GAL80s effectively and specifically subtract the activities of GAL4-driven transgenes that impart anatomical and physiological phenotypes. can allow researchers to efficiently activate only one or a few types of functional neurons in an otherwise nonfunctional olfactory background. Such experiments will improve our understanding of the mechanistic connections between odorant inputs and behavioral outputs at the resolution of only a few functional neurons.

We engineered electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with “positive-going” fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transformed the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further applied this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters.

Pushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. We recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the Janelia Fluor (JF) series of dyes. We refined and extended this strategy, finding that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chemical properties of rhodamine dyes with unprecedented precision. This strategy allowed us to establish principles for fine-tuning the properties of fluorophores and to develop a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red. Our results demonstrate the versatility of these new dyes in cells, tissues and animals.

Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

Fluorescence microscopy relies on dyes that absorb and then emit photons. In addition to fluorescence, fluorophores can undergo photochemical processes that decrease quantum yield or result in spectral shifts and irreversible photobleaching. Chemical strategies that suppress these undesirable pathways—thereby increasing the brightness and photostability of fluorophores—are crucial for advancing the frontier of bioimaging. Here, we describe a general method to improve small-molecule fluorophores by incorporating deuterium into the alkylamino auxochromes of rhodamines and other dyes. This strategy increases fluorescence quantum yield, inhibits photochemically induced spectral shifts, and slows irreparable photobleaching, yielding next-generation labels with improved performance in cellular imaging experiments.