Temporal evolution of single-cell transcriptomes ofDrosophilaolfactory projection neurons

Abstract

Neurons undergo substantial morphological and functional changes during development to form precise synaptic connections and acquire specific physiological features. What are the underlying transcriptomic bases? Here, we obtained the single-cell transcriptomes of Drosophila 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.

Single-cell transcriptomes of developing and adult olfactory receptor neurons in Drosophila

“…Many of them were members of protein families implicated in circuit assembly, such as Beaten Path (beat), Down Syndrome Cell Adhesion Molecule (Dscam), Fasciclin (Fas), Netrin (Net), Roundabout (robo), Defective Proboscis Extension Response (Dpr), and Semaphorin (Sema; Figure 8—figure supplements 1 – 3 ; Kolodkin and Tessier-Lavigne, 2011 ; Sanes and Zipursky, 2020 ), suggesting that these differentially expressed CSMs could regulate ORN wiring. Similar to other scRNA-seq studies of developing neurons ( Kurmangaliyev et al, 2020 ; Özel et al, 2021 ; Xie et al, 2021 ), many CSMs exhibited graded and/or broad expression in multiple neuron types. Semaphorin 2B ( Sema-2b ) and Teneurin-a ( Ten-a ), which display restricted expression at the protein level ( Hong et al, 2012 ; Joo et al, 2013 ), were broadly expressed at the mRNA level ( Figure 8—figure supplement 1 ), indicating post-transcriptional regulation of some of these CSMs.…”

“…By profiling Drosophila ORNs at multiple stages, we have generated a rich dataset that can be used as a foundation to reveal cell-type-specific regulators of neuron differentiation, axon targeting, synapse formation, and sensory function. In conclusion, this work, together our accompanying paper profiling PNs at multiple stages ( Xie et al, 2021 ), should spur novel and important biological discoveries in the fields of sensory system development and function.…”

“…Next, we sought to map 24 hr APF ORNs to their respective glomerular types ( Figure 4A ). In the companion manuscript that analyzed single-cell transcriptomes of PNs ( Xie et al, 2021 ), we demonstrated that using differentially expressed genes from the stage with maximal transcriptomic diversity between different cell types for dimensionality reduction achieved the best clustering results. For the three stages of ORNs that we profiled, we found that transcriptomes at 42 hr APF exhibited highest diversity ( Figure 4B ).…”

“…We used pan-neuronal nSyb-GAL4 to label adult ORNs. To evaluate if this technique was broadly applicable to fly neurons, we also sequenced adult PN nuclei using VT033006-GAL4 to label the majority of PN types ( Tirian and Dickson, 2017 ; see the companion manuscript Xie et al, 2021 for details) and compared them with stage-matched cells labeled with GH146-GAL4 ( Figure 2A ).…”

“…Finally, to aid in identification of molecules important for synaptic partner matching, we queried differentially expressed genes in six synaptic partner ORNs and PNs (from the companion manuscript, Xie et al, 2021 ) at 42 hr APF and 48 hr APF, respectively. From this gene set, we identified 203 differentially expressed CSMs ( Figure 8—figure supplements 5 , 6 ).…”