The fan-shaped body is the largest substructure of the central complex in Drosophila melanogaster. Two groups of large-field neurons that innervate the fan-shaped body, viz., F1 and F5 neurons, have recently been found to be involved in visual pattern memory for "contour orientation" and "elevation" in a rut-dependent manner. The F5 neurons have been found to be responsible for the parameter "elevation" in a for-dependent manner. We have shown here that the F1 neuron also affects visual memory for "contour orientation" in a for-dependent way. With the help of Gal4/UAS and FLP-out techniques, we have characterized the morphological features of these two groups of neurons at single neuron resolution. We have observed that F1 or F5 neurons are groups of isomorphic individual neurons. Single F1 neurons have three main arborization regions: one in the first layer of the fan-shaped body, one in the ventral body, and another in the inferior medial protocerebrum. Single F5 neurons have two arborization regions: one in the fifth layer of the fan-shaped body and the other in the superior medial protocerebrum. The polarity of the F1 and F5 neurons has been studied with the Syt-GFP marker. Our results indicate the existence of presynaptic sites of both F1 and F5 neurons located in the fan-shaped body and postsynaptic sites outside of the fan-shaped body.

Morphology evolves often through changes in developmental genes, but the causal mutations, and their effects, remain largely unknown. The evolution of naked cuticle on larvae of Drosophila sechellia resulted from changes in five transcriptional enhancers of shavenbaby (svb), a transcript of the ovo locus that encodes a transcription factor that governs morphogenesis of microtrichiae, hereafter called ’trichomes’. Here we show that the function of one of these enhancers evolved through multiple single-nucleotide substitutions that altered both the timing and level of svb expression. The consequences of these nucleotide substitutions on larval morphology were quantified with a novel functional assay. We found that each substitution had a relatively small phenotypic effect, and that many nucleotide changes account for this large morphological difference. In addition, we observed that the substitutions had non-additive effects. These data provide unprecedented resolution of the phenotypic effects of substitutions and show how individual nucleotide changes in a transcriptional enhancer have caused morphological evolution.

One central, and yet unsolved, question in evolutionary biology is the relationship between the genetic variants segregating within species and the causes of morphological differences between species. The classic neo-darwinian view postulates that species differences result from the accumulation of small-effect changes at multiple loci. However, many examples support the possible role of larger abrupt changes in the expression of developmental genes in morphological evolution. Although this evidence might be considered a challenge to a neo-darwinian micromutationist view of evolution, there are currently few examples of the actual genes causing morphological differences between species. Here we examine the genetic basis of a trichome pattern difference between Drosophila species, previously shown to result from the evolution of a single gene, shavenbaby (svb), probably through cis-regulatory changes. We first identified three distinct svb enhancers from D. melanogaster driving reporter gene expression in partly overlapping patterns that together recapitulate endogenous svb expression. All three homologous enhancers from D. sechellia drive expression in modified patterns, in a direction consistent with the evolved svb expression pattern. To test the influence of these enhancers on the actual phenotypic difference, we conducted interspecific genetic mapping at a resolution sufficient to recover multiple intragenic recombinants. This functional analysis revealed that independent genetic regions upstream of svb that overlap the three identified enhancers are collectively required to generate the D. sechellia trichome pattern. Our results demonstrate that the accumulation of multiple small-effect changes at a single locus underlies the evolution of a morphological difference between species. These data support the view that alleles of large effect that distinguish species may sometimes reflect the accumulation of multiple mutations of small effect at select genes.

We describe a genetic mosaic system in Drosophila, in which a dominant repressor of a cell marker is placed in trans to a mutant gene of interest. Mitotic recombination events between homologous chromosomes generate homozygous mutant cells, which are exclusively labeled due to loss of the repressor. Using this system, we are able to visualize axonal projections and dendritic elaboration in large neuroblast clones and single neuron clones with a membrane-targeted GFP marker. This new method allows for the study of gene functions in neuroblast proliferation, axon guidance, and dendritic elaboration in the complex central nervous system. As an example, we show that the short stop gene is required in mushroom body neurons for the extension and guidance of their axons.

To coordinate cellular physiology, eukaryotic cells rely on the inter-organelle transfer of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondria contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signaling molecules, lipids, and metabolites3. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle4,5. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation6,7, a clear understanding of their nanoscale structure and regulation is still lacking. Here, we combine 3D electron microscopy with high-speed molecular tracking of a model organelle tether, VAPB, to map the structure and diffusion landscape of ERMCSs. From EM reconstructions, we identified subdomains within the contact site where ER membranes dramatically deform to match local mitochondrial curvature. In parallel live cell experiments, we observed that the VAPB tethers that mediate this interface were not immobile, but rather highly dynamic, entering and leaving the site in seconds. These subdomains enlarged during nutrient stress, indicating ERMCSs can readily remodel under different physiological conditions. An ALS-associated mutation in VAPB altered the normal fluidity of contact sites, likely perturbing effective communication across the contact site and preventing remodeling. These results establish high speed single molecule imaging as a new tool for mapping the structure of contact site interfaces and suggest that the diffusion landscape of VAPB is a crucial component of ERMCS homeostasis.

To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.

Functional imaging in behaving animals is essential to understanding brain function. However, artifacts resulting from animal motion, including locomotion, can severely corrupt functional measurements. To dampen tissue motion, we designed a new optical window with minimal optical aberrations. Using the newly developed high-speed continuous volumetric imaging system based on an optical phase-locked ultrasound lens, we quantified motion of the cerebral cortex and hippocampal surface during two-photon functional imaging in behaving mice. We find that the out-of-plane motion is generally greater than the axial dimension of the point-spread-function during mouse locomotion, which indicates that high-speed continuous volumetric imaging is necessary to minimize motion artifacts.

Many animals utilize acoustic signals-or songs-to attract mates. During courtship, Drosophila melanogaster males vibrate a wing to produce trains of pulses and extended tone, called pulse and sine song, respectively. Courtship songs in the genus Drosophila are exceedingly diverse, and different song features appear to have evolved independently of each other. How the nervous system allows such diversity to evolve is not understood. Here, we identify a wing muscle in D. melanogaster (hg1) that is uniquely male-enlarged. The hg1 motoneuron and the sexually dimorphic development of the hg1 muscle are required specifically for the sine component of the male song. In contrast, the motoneuron innervating a sexually monomorphic wing muscle, ps1, is required specifically for a feature of pulse song. Thus, individual wing motor pathways can control separate aspects of courtship song and may provide a "modular" anatomical substrate for the evolution of diverse songs.

Following considerable progress on the molecular and cellular basis of taste perception in fly sensory neurons, the time is now ripe to explore how taste information, integrated with hunger and satiety, undergo a sensorimotor transformation to lead to the motor actions of feeding behavior. I examine what is known of feeding circuitry in adult flies from more than 250 years of work in larger flies and from newer work in Drosophila. I review the anatomy of the proboscis, its muscles and their functions (where known), its motor neurons, interneurons known to receive taste inputs, interneurons that diverge from taste circuitry to provide information to other circuits, interneurons from other circuits that converge on feeding circuits, proprioceptors that influence the motor control of feeding, and sites of integration of hunger and satiety on feeding circuits. In spite of the several neuron types now known, a connected pathway from taste inputs to feeding motor outputs has yet to be found. We are on the threshold of an era where these individual components will be assembled into circuits, revealing how nervous system architecture leads to the control of behavior.

Many forms of human and animal behavior involve head movements. A new study reveals the neural code for three-dimensional head movements in the midbrain of freely moving mice.