The Drosophila melanogaster olfactory system is one of the most intensively studied parts of the nervous system in any animal. Composed of ~60 independent olfactory neuron classes, with several associated hygrosensory and thermosensory pathways, it has been subject to diverse types of experimental analyses. However, synthesizing the available data is limited by the incompleteness and inconsistent nomenclature found in the literature. In this work, we first “complete” the peripheral sensory map through the identification of a previously uncharacterized antennal sensory neuron population expressing Or46aB, and the definition of an exceptional “hybrid” olfactory neuron class comprising functional Or and Ir receptors. Second, we survey developmental, anatomical, connectomic, functional and evolutionary studies to generate an integrated dataset of these sensory neuron pathways – and associated visualizations – creating an unprecedented comprehensive resource. Third, we illustrate the utility of the dataset to reveal relationships between different organizational properties of this sensory system, and the new questions these stimulate. These examples emphasize the power of this resource to promote further understanding of the construction, function and evolution of these neural circuits.
Sensory neurons must be reproducibly specified to permit accurate neural representation of external signals but also able to change during evolution. We studied this paradox in the Drosophila olfactory system by establishing a single-cell transcriptomic atlas of all developing antennal sensory lineages, including latent neural populations that normally undergo programmed cell death (PCD). This atlas reveals that transcriptional control is robust, but imperfect, in defining selective sensory receptor expression. A second layer of precision is afforded by the intersection of expression of functionally-interacting receptor subunits. A third layer is defined by stereotyped PCD patterning, which masks promiscuous receptor expression in neurons fated to die and removes “empty” neurons lacking receptors. Like receptor choice, PCD is under lineage-specific transcriptional control; promiscuity in this regulation leads to previously-unappreciated heterogeneity in neuronal numbers. Thus functional precision in the mature olfactory system belies developmental noise that might facilitate the evolution of sensory pathways.
Transcription does not occur diffusely throughout the nucleus but is concentrated in specific areas. Areas of accumulated transcriptional machinery have been called clusters, hubs, or condensates, while transcriptionally active areas have been referred to as transcription factories or transcription bodies. Despite the widespread occurrence of transcription bodies, it has been difficult to study their assembly, function, and effect on gene expression. This review highlights the advantages of developmental model systems such as zebrafish and fruit fly embryos, in addressing these questions. We focus on three important discoveries that were made in embryos. (i) It had previously been suggested that, in transcription bodies, the different steps of the transcription process are organized in space. We explore how work in embryos has revealed that they can also be organized in time. In this case, transcription bodies mature from transcription factor clusters to elongating transcription bodies. This type of organization has important implications for transcription body function. (ii) The relevance of clustering for in vivo gene regulation has benefited greatly from studies in embryos. We discuss examples in which transcription bodies regulate developmental gene expression by compensating for low transcription factor concentrations and low-affinity enhancers. Finally, (iii) while accumulations of transcriptional machinery can facilitate transcription locally, work in embryos showed that transcription bodies can also sequester the transcriptional machinery, modulating the availability for activity at other sites. In brief, the reviewed literature highlights the properties of developmental model organisms that make them powerful systems for uncovering the form and function of transcription bodies.
Neuronal phenotypic traits such as morphology, connectivity and function are dictated, to a large extent, by a specific combination of differentially expressed genes. Clusters of neurons in transcriptomic space correspond to distinct cell types and in some cases-for example, Caenorhabditis elegans neurons1 and retinal ganglion cells2-4-have been shown to share morphology and function. The zebrafish optic tectum is composed of a spatial array of neurons that transforms visual inputs into motor outputs. Although the visuotopic map is continuous, subregions of the tectum are functionally specialized5,6. Here, to uncover the cell-type architecture of the tectum, we transcriptionally profiled its neurons, revealing more than 60 cell types that are organized in distinct anatomical layers. We measured the visual responses of thousands of tectal neurons by two-photon calcium imaging and matched them with their transcriptional profiles. Furthermore, we characterized the morphologies of transcriptionally identified neurons using specific transgenic lines. Notably, we found that neurons that are transcriptionally similar can diverge in shape, connectivity and visual responses. Incorporating the spatial coordinates of neurons within the tectal volume revealed functionally and morphologically defined anatomical subclusters within individual transcriptomic clusters. Our findings demonstrate that extrinsic, position-dependent factors expand the phenotypic repertoire of genetically similar neurons.
Lukas Breitzler (Group Larsch) writes about research from colleagues in the Benton lab.
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On the third floor of the Genopode, many doors open to fruit flies. A first-time visitor might walk past rows of incubators and mistake them for the usual industrial-sized refrigerators and freezers that line the hallways of the CIG. But behind their doors are hundreds of plastic tubes teeming with numberless small fruit flies. Another door opens to a room with two large utility shelves stacked with more boxes of the same tubes. Foam plugs prevent their inhabitants from escaping, while food at the bottom allows them to feed ad libitum. At first glance, the flies look identical. But handwritten notes on labelling tape indicate that the tubes house several different species and genetic strains. Unassuming as they initially appear, these flies are central to the Benton lab’s efforts to answer a fundamental question in biology: how does behavior evolve?
“As a geneticist, I am interested in relating gene changes to neural circuits and behavior,” says Richard Benton, principal investigator of the Benton lab. Richard occupies one of the corner offices on the floor with a view of Lac Léman. Next to his desk, a table holds more boxes of tubes filled with flies. Richard explains that in the past 10 to 20 years, many labs have made impressive advances explaining how genes and neural circuits influence behavior in Drosophila melanogaster—the most commonly studied species of fruit fly. Researchers typically manipulate genes to alter neuronal processes and observe their impact on behavior. “We’re taking a slightly different approach, which is looking at natural variation in behavior to learn how circuits function and how they’re built,” says Richard. Animal genomes contain tens of thousands of genes, some of which influence behaviors. Pinpointing a single gene’s role often involves a fair amount of educated guesswork; not only do you have to pick a gene, but you also have to know what behavior to look for. Richard’s group prefers to approach this problem by working backward: identify differences in behavior first, then investigate the “nuts and bolts of what’s changing genetically.” That’s precisely the approach the lab took in their latest study, published in Nature, which revealed how changes in gene regulation enabled Drosophila to adapt their behavior to varying day lengths. A key aspect to this approach was examining the behavior of not only D. melanogaster but also that of closely related Drosophila species.
One of these species will be familiar to those who have joined the Benton lab’s progress reports: Drosophila sechellia; a close cousin to D. melanogaster. The two species are so closely related, in fact, that they are capable of producing hybrid offspring. But despite their genetic similarities, their habitats are strikingly different. D. sechellia feeds and lays eggs exclusively on Noni fruit, a plant toxic to other Drosophila species. (The fruit also emits a horrendous odor once it reaches a certain stage of ripeness, earning it the nickname ‘vomit fruit’ in regions where it grows. The Benton lab keeps a small, airtight incubator filled with them.) Additionally, while D. melanogaster can be found virtually all over the world—including in northern latitudes, where they experience highly variable day lengths throughout the year—D. sechellia live exclusively on the Seychelles islands near the equator, where the sun rises and sets at nearly the same time year-round. Previously, the group detailed some of the genetic differences that enabled D. sechellia’s specialization to noni fruit. However, whether the pronounced differences in seasonal daylight drove evolutionary adaptation had never been investigated—until Michael Shahandeh, the recent study’s first author, decided to take a look.
Indeed Michael and collaborators conducted experiments demonstrating that D. sechellia barely adjusted their behavior to longer photoperiods. The flies were most active around the same time every day, regardless of whether days were 12 or 20 hours long. D. Melanogaster on the other hand adjusted their activity without problem. Importantly, other species closely related to D. sechellia also adjusted their behavior without problem, suggesting that D. sechellia lost its ability to regulate daily rhythms through evolution as it adapted to a stable environment.
With a behavioral phenotype in hand, the difficulty now lay in identifying the genetic mechanism. Fortunately, the team had decades of research on the circadian biology of D. melanogaster to draw on. This allowed them to identify a list of candidate genes to test.
To find the gene relevant for regulating daily activity, “Michael devised a clever approach,” says Richard. By taking D. melanogaster strains with mutations for the different candidate genes and interbreeding them with D. sechellia, the lab created hybrid offspring that carried one gene copy from each species. In most cases the D. sechellia allele compensated for the missing D. melanogaster gene and the hybrids behaved normally. But when one specific gene—called Pdf—was disrupted, the sechellia allele failed to make up for the loss; the hybrids lost their ability to shift activity under longer days. This indicated that Pdf, which encodes a neuropeptide that transmits signals generated by core molecular timekeeping mechanisms in Drosophila, regulated behavioral plasticity.
Follow-up experiments found that Pdf expression was weaker and less variable in neurons of D. sechellia compared to D. melanogaster and pinpointed those differences to changes in the gene’s regulatory region. However, the lab needed to establish that those differences in gene regulation actually caused the behavior they observed. Using genetic engineering, they therefore cloned the Pdf regulatory sequences of both species and inserted them into mutant flies lacking their own functional Pdf regulatory sequence. And indeed, flies with the D. sechellia sequence showed reduced plasticity compared to flies with the melanogaster sequence. This final experiment completed the picture, showing how a subtle gene regulatory change could alter neuronal function and, ultimately, the way the flies behave.
It is exceedingly difficult to understand how evolution links genetic changes to their effects on neurons and behavior. The Benton lab achieves this by asking the right questions in the right system. “It’s really Drosophila as a whole and Drosophila sechellia as a specialized species. And it pays off to have an ecological framework,” says Richard. Genetic mechanisms can shape behavior through subtle and unexpected pathways. The Benton lab’s latest study is one of several examples from their research demonstrating how this framework not only deepens our understanding of evolution, but also uncovers mechanisms that might otherwise remain hidden.
Dysfunctions in autophagy, a cellular mechanism for breaking down components within lysosomes, often lead to neurodegeneration. The specific mechanisms underlying neuronal vulnerability due to autophagy dysfunction remain elusive. Here we show that autophagy contributes to cerebellar Purkinje cell (PC) survival by safeguarding their glycolytic activity. Outside the conventional housekeeping role, autophagy is also involved in the ATG5-mediated regulation of glucose transporter 2 (GLUT2) levels during cerebellar maturation. Autophagy-deficient PCs exhibit GLUT2 accumulation on the plasma membrane, along with increased glucose uptake and alterations in glycolysis. We identify lysophosphatidic acid and serine as glycolytic intermediates that trigger PC death and demonstrate that the deletion of GLUT2 in ATG5-deficient mice mitigates PC neurodegeneration and rescues their ataxic gait. Taken together, this work reveals a mechanism for regulating GLUT2 levels in neurons and provides insights into the neuroprotective role of autophagy by controlling glucose homeostasis in the brain.
