Claude Desplan

Glial cells are essential for proper nervous system development and function. To understand glial development and function, we comprehensively annotated the glial cells from two large single-cell mRNA-sequencing (scRNA-seq) atlases of the developing Drosophila visual system. This allowed us to identify all glial cell types from larval to adult stages and follow their developmental trajectories to understand how the diversity of glial types is generated during development. We show that whereas most glial types, such as chiasm glia, gradually change their transcriptome as they mature during development, neuropil glia that transcriptionally appear as a single-cell class in larvae, splits into ensheathing (EG) and astrocyte-like (ALG) glia types during pupal stages. We have experimentally validated these developmental trajectories and identified the genetic markers expressed through the differentiation between EG and ALG classes. Unexpectedly, our analysis of scRNA-seq datasets allowed us to discover that the transcriptome of glial cell bodies can be distinguished from that of their processes. We have identified that processes are enriched for distinct mRNAs that were validated in vivo. This work provides the most detailed transcriptomic analysis of optic lobe glia during development and helps explain the expansion of glial diversity observed in the adult visual system. We also present an innovative computational approach to identify mRNA species that are differentially localized to cell bodies or cellular processes.

Combinations of terminal selector transcription factors (tsTFs) are thought to establish and maintain the unique identities of the numerous cell types found in nervous systems. However, it remains largely unclear how tsTF combinations are specified during development and how they then coordinate the type-specific differentiation programs of each neuron. To investigate these regulatory mechanisms, we performed simultaneous single-cell RNA and ATAC (assay for transposase-accessible chromatin) sequencing on the Drosophila optic lobes at four stages of their development and identified over 250 distinct cell types. We characterized the common cis-regulatory features of neuronal enhancers and performed comprehensive inference of gene regulatory networks across cell types and stages. Our results reveal cell type– and stage-specific enhancers of many neuronal genes and the cooperative actions of tsTFs, pan-neuronal and ecdysone-responsive TFs on these enhancers. We show that the same effector genes are often regulated by different tsTF combinations acting through distinct enhancers in different neurons. During neurogenesis, tsTF codes are established within a brief critical period in newborn neurons, often through cell type–specific enhancers that are not accessible in their progenitors. Accordingly, when neuroblast temporal patterning TFs are reutilized as tsTFs in neurons, they are regulated independently through separate enhancers. Therefore, neuronal identity specification and differentiation is a multistep regulatory program, wherein the same TFs enact distinct regulatory codes at different steps and across cell types.

Spatial patterning generates neuronal diversity by compartmentalizing progenitors into domains with distinct molecular identities. However, these patterning cues are often transient in neurogenic domains, raising the question of how spatial information can be preserved and how rigidly it constrains neuronal fates. Here we show that a single-locus chromatin memory in the medulla of the Drosophila optic lobe faithfully preserves spatial identity while conferring the flexibility for specific neuronal fates to bypass spatial restriction. Medulla progenitors are partitioned into three spatial domains marked by Vsx1, Optix and Bifid. Domain-resolved single-cell multiome profiling reveals that progenies from different neuroepithelial domains are nearly indistinguishable for both transcriptome and chromatin accessibility, although persistent domain-specific accessibility in Vsx and Bi progenies is restricted to a single corresponding spatial-factor locus, Vsx1 /2 or Bifid , respectively. These same factors are absent when neuroepithelial cells are converted to neural stem cells, but they are re-expressed in postmitotic neurons to execute domain-specific fates. Because this bookmarking is so restricted, specific classes of neurons can bypass spatial restriction by overriding only this single-locus chromatin state, adopting equivalent fates regardless of spatial origin. Other neurons ignore domain boundaries by expressing Vsx1/2 through a program independent of their domain of origin. PRC2-mediated silencing restricts Vsx1 re-expression to its home domain, while temporal identity and Notch signaling in newborn neurons determine which neurons engage or bypass the spatial program. Thus, single-locus chromatin memory preserves spatial information without making it an obligatory determinant of every neuronal fate.

Social interactions establish reproductive hierarchies and regulate division of labor in eusocial insect colonies. In the ant Harpegnathos saltator , queen loss induces a subset of workers to transition into reproductive pseudo-queens, known as gamergates, through a ritualized antennal-dueling tournament that can last for more than a month. Gamergate transition is associated with sustained dueling engagement, yet the interaction networks and social dynamics that shape reproductive hierarchy formation remain unclear. Here, we combine long-term automated tracking with behavioral classifiers to systematically quantify social interactions, including antennal dueling and two distinct biting behaviors: aggressive grooming and mandible locking. We find that antennal dueling is organized into two activity states, an engaged state and a disengaged state, and that persistent maintenance of the engaged state, rather than transient entry into it, predicts successful transition to gamergate fate. Early access to abundant food shapes dueling activity and biases ants toward entry into the engaged state. Moreover, engaged-state individuals exhibit a strong assortative preference for dueling with one another and cluster spatially within the colony. We further show that mandible locking precedes transitions out of the engaged state, whereas aggressive grooming reinforces the disengaged state, together consistent with a role in stabilizing worker fate. Our findings reveal how structured social interactions organize reproductive hierarchy formation by regulating stable behavioral states associated with caste fate.

In the medulla of the Drosophila optic lobe, the identity of each neuronal type is specified in progenitors and newborn neurons via the integration of temporal, spatial and Notch-driven patterning mechanisms. This identity is maintained in differentiating and adult neurons by the continuous expression of neuronal type-specific combinations of transcription factors called terminal selectors, which are thought to control all neuronal type-specific features. How the patterning mechanisms establish terminal selector expression is unknown. Here we have used single-cell mRNA sequencing to characterize the spatial origins of medulla neurons. Combined with our previous characterization of their temporal and Notch origins, this allowed us to identify correlations between patterning information, terminal selector expression and neuronal features. Our results suggest that different subsets of the patterning information accessible to a given neuronal type control the expression of each of its terminal selectors and modules of terminal features, including neurotransmitter identity.

Optimal neural processing relies on precise tissue patterning across diverse cell types. Here, we show that spontaneous calcium waves arise among non-neuronal support cells in the developing Drosophila eye to drive retinal morphogenesis. These waves are initiated by Cad96Ca receptor tyrosine kinase signaling, triggering phospholipase C–γ–mediated calcium release from the endoplasmic reticulum. A cell type–specific “innexin code” coordinates wave propagation through a defined gap junction network among non-neuronal retinal cells, excluding photoreceptors. Wave intensity scales with ommatidial size, triggering stronger myosin II–driven apical contraction at interommatidial boundaries in larger ommatidia. This size-dependent mechanism compensates for early boundary irregularities, ensuring uniform ommatidial packing that is critical for precise optical architecture. Our findings reveal how synchronized calcium signaling among non-neuronal cells orchestrates tissue patterning in the developing nervous system.

To ensure specificity, sensory neurons must select and express a single receptor from often vast gene families, adhering to the rule of ‘one receptor per neuron’. For example, each olfactory sensory neuron in mammals expresses only one odorant receptor (Or) gene^1,2. In Drosophila, which has about 60 Or genes, this selection is deterministic³. By contrast, mice face the challenge of choosing one Or gene from over 1,000 options⁴. They solve this through a complex system of stochastic choices^5,6,7,8,9. Ants also possess many Or genes, most of which are organized into tandem arrays similar to those in mammals, but their regulatory mechanisms have evolved independently. Here we show that, in the ant Harpegnathos saltator, each olfactory sensory neuron activates a single promoter within an Or gene array, producing a mature capped and polyadenylated mRNA. While the promoters of downstream genes in the array are inactive, all downstream genes are nonetheless transcribed due to transcriptional readthrough from the active promoter, probably caused by inefficient RNA polymerase II termination. This readthrough appears to suppress downstream promoters through transcriptional interference, resulting in aberrant non-capped transcripts that are not translated, ensuring that only the active gene is expressed. Simultaneously, long antisense transcription originating from the chosen Or promoter covers upstream genes, presumably silencing them. Ants therefore appear to have evolved a unique transcriptional-interference-based mechanism to express a single OR protein from an array of Or genes with functionally similar promoters.

Production, signaling, and degradation of the inhibitory neurotransmitter GABA requires tight regulation for proper nervous system function, yet the gene regulatory mechanisms underlying this control remain poorly understood. Here, we identify a post-transcriptional mechanism that coordinates expression of the GABA synthetase Gad1 with its vesicular transporter VGAT in Drosophila . While Gad1 is transcribed specifically in GABAergic neurons, VGAT mRNA is transcribed broadly across the nervous system, but is selectively translated in GABAergic neurons. We show that this specificity depends on CG14989, a putative long non-coding RNA located adjacent to the Gad1 gene locus that shares its GABAergic expression pattern. CG14989 RNA contains sequences complementary to three predicted miR-7 sites in the VGAT 3′UTR, and ectopic expression experiments revealed that it is sufficient for VGAT translation in non-GABAergic neurons, consistent with antagonism of miR-7-mediated repression. Furthermore, complementary regions to CG14989 are present in multiple other genes related to metabolism and signaling of GABA, suggesting a broader role for this mechanism. Together, our findings suggest that CG14989 functions as a regulatory hub that coordinates the molecular identity of GABAergic neurons.

Combinations of terminal selector transcription factors (tsTFs) are thought to establish and maintain the unique identities of the numerous cell types found in nervous systems. However, it remains largely unclear how tsTF combinations are specified during development, and how they then coordinate the type-specific differentiation programs of each neuron. To investigate these regulatory mechanisms, we performed simultaneous single-cell RNA and ATAC sequencing on the Drosophila optic lobes at four stages of their development and identified over 250 distinct cell types. We characterized the common cis-regulatory features of neuronal enhancers and performed comprehensive inference of gene regulatory networks across cell types and stages. Our results reveal cell-type and stage-specific enhancers of many neuronal genes and the cooperative actions of tsTFs, pan-neuronal and ecdysone-responsive TFs on these enhancers. We show that the same effector genes are often regulated by different tsTF combinations acting through distinct enhancers in different neurons. During neurogenesis, tsTF codes are established within a brief critical period in newborn neurons, often through cell-type-specific enhancers that are not accessible in their progenitors. Accordingly, when neuroblast temporal patterning TFs are re-utilized as tsTFs in neurons, they are regulated independently through separate enhancers. Therefore, neuronal identity specification and differentiation is a multi-step regulatory program, wherein the same TFs enact distinct regulatory codes at different steps and across cell types.