Recognition of environmental cues is essential for the survival of all organisms. Transcriptional changes occur to enable the generation and function of the neural circuits underlying sensory perception. To gain insight into these changes, we generated single-cell transcriptomes of olfactory- (ORNs), thermo-, and hygro-sensory neurons at an early developmental and adult stage using single-cell and single-nucleus RNA sequencing. We discovered that ORNs maintain expression of the same olfactory receptors across development. Using receptor expression and computational approaches, we matched transcriptomic clusters corresponding to anatomically and physiologically defined neuron types across multiple developmental stages. We found that cell-type-specific transcriptomes partly reflected axon trajectory choices in development and sensory modality in adults. We uncovered stage-specific genes that could regulate the wiring and sensory responses of distinct ORN types. Collectively, our data reveal transcriptomic features of sensory neuron biology and provide a resource for future studies of their development and physiology.

The regulatory mechanisms by which neurons coordinate their physiology and connectivity are not well understood. The Drosophila olfactory receptor neurons (ORNs) provide an excellent system to investigate this question. Each ORN type expresses a unique olfactory receptor, or a combination thereof, and sends their axons to a stereotyped glomerulus. Using single-cell RNA sequencing, we identified 33 transcriptomic clusters for ORNs and mapped 20 to their glomerular types, demonstrating that transcriptomic clusters correspond well with anatomically and physiologically defined ORN types. Each ORN type expresses hundreds of transcription factors. Transcriptome-instructed genetic analyses revealed that (1) one broadly expressed transcription factor (Acj6) only regulates olfactory receptor expression in one ORN type and only wiring specificity in another type, (2) one type-restricted transcription factor (Forkhead) only regulates receptor expression, and (3) another type-restricted transcription factor (Unplugged) regulates both events. Thus, ORNs utilize diverse strategies and complex regulatory networks to coordinate their physiology and connectivity.

The precise assembly of a neural circuit involves many consecutive steps. The conflict between a limited number of wiring molecules and the complexity of the neural network impels each molecule to execute multiple functions at different steps. Here, we examined the cell-type specific distribution of endogenous levels of axon guidance receptor Plexin B (PlexB) in the developing antennal lobe, the first olfactory processing center in . We found that different classes of olfactory receptor neurons (ORNs) express PlexB at different levels in two wiring steps - axonal trajectory choice and subsequent target selection. In line with its temporally distinct patterns, the proper levels of PlexB control both steps in succession. Genetic interactions further revealed that the effect of high-level PlexB is antagonized by its canonical partner Sema2b. Thus, PlexB plays a multifaceted role in instructing the assembly of the olfactory circuit through temporally-regulated expression patterns and expression level-dependent effects.

Neurons undergo substantial morphological and functional changes during development to form precise synaptic connections and acquire specific physiological properties. What are the underlying transcriptomic bases? Here, we obtained the single-cell transcriptomes of olfactory projection neurons (PNs) at four developmental stages. We decoded the identity of 21 transcriptomic clusters corresponding to 20 PN types and developed methods to match transcriptomic clusters representing the same PN type across development. We discovered that PN transcriptomes reflect unique biological processes unfolding at each stage-neurite growth and pruning during metamorphosis at an early pupal stage; peaked transcriptomic diversity during olfactory circuit assembly at mid-pupal stages; and neuronal signaling in adults. At early developmental stages, PN types with adjacent birth order share similar transcriptomes. Together, our work reveals principles of cellular diversity during brain development and provides a resource for future studies of neural development in PNs and other neuronal types.

Transcription factors specify the fate and connectivity of developing neurons. We investigate how a lineage-specific transcription factor, Acj6, controls the precise dendrite targeting of Drosophila olfactory projection neurons (PNs) by regulating the expression of cell-surface proteins. Quantitative cell-surface proteomic profiling of wild-type and acj6 mutant PNs in intact developing brains, and a proteome-informed genetic screen identified PN surface proteins that execute Acj6-regulated wiring decisions. These include canonical cell adhesion molecules and proteins previously not associated with wiring, such as Piezo, whose mechanosensitive ion channel activity is dispensable for its function in PN dendrite targeting. Comprehensive genetic analyses revealed that Acj6 employs unique sets of cell-surface proteins in different PN types for dendrite targeting. Combined expression of Acj6 wiring executors rescued acj6 mutant phenotypes with higher efficacy and breadth than expression of individual executors. Thus, Acj6 controls wiring specificity of different neuron types by specifying distinct combinatorial expression of cell-surface executors.

Drosophila larval locomotion, which entails rhythmic body contractions, is controlled by sensory feedback from proprioceptors. The molecular mechanisms mediating this feedback are little understood. By using genetic knock-in and immunostaining, we found that the Drosophila melanogaster transmembrane channel-like (tmc) gene is expressed in the larval class I and class II dendritic arborization (da) neurons and bipolar dendrite (bd) neurons, both of which are known to provide sensory feedback for larval locomotion. Larvae with knockdown or loss of tmc function displayed reduced crawling speeds, increased head cast frequencies, and enhanced backward locomotion. Expressing Drosophila TMC or mammalian TMC1 and/or TMC2 in the tmc-positive neurons rescued these mutant phenotypes. Bending of the larval body activated the tmc-positive neurons, and in tmc mutants this bending response was impaired. This implicates TMC's roles in Drosophila proprioception and the sensory control of larval locomotion. It also provides evidence for a functional conservation between Drosophila and mammalian TMCs.

Our understanding of the mechanisms of neural circuit assembly is far from complete. Identification of wiring molecules with novel mechanisms of action will provide insights into how complex and heterogeneous neural circuits assemble during development. In the olfactory system, 50 classes of olfactory receptor neurons (ORNs) make precise synaptic connections with 50 classes of partner projection neurons (PNs). Here, we performed an RNA interference screen for cell surface molecules and identified the leucine-rich repeat-containing transmembrane protein known as Fish-lips (Fili) as a novel wiring molecule in the assembly of the olfactory circuit. Fili contributes to the precise axon and dendrite targeting of a small subset of ORN and PN classes, respectively. Cell-type-specific expression and genetic analyses suggest that Fili sends a transsynaptic repulsive signal to neurites of nonpartner classes that prevents their targeting to inappropriate glomeruli in the antennal lobe.