Biology subjects

Chen, Y.-C. D.

Publications and source records attributed to Chen, Y.-C. D..

6 recordsLinked to original sources

bioRxiv2025

Deciphering the combinatorial expression pattern and genetic regulatory mechanisms of Beats and Sides in the olfactory circuits of Drosophila

Over the past decades, many critical molecular players have been uncovered to control distinct steps in olfactory circuit assembly in Drosophila. Among these, multi-member gene families of cell surface proteins are of interest because they can act as neuron-specific identification/recognition tags in combinations and contribute to circuit assembly in complex brains through their heterophilic or homophilic interactions. Recently, a multi-protein interactome has been described between the Beat and Side families of IgSF proteins. Here, we use the publicly available single-cell RNA-seq datasets and newly generated gene trap transgenic driver lines to probe the in vivo spatial expression pattern of the beat/side gene families in odorant receptor neurons (ORNs) and their synaptic target projection neurons (PNs). Our results revealed that each ORN and its synaptic target PN class expresses a class-specific combination of beat/side genes, hierarchically regulated by lineage-specific genetic programs. Though ORNs or PNs from closer lineages tend to possess more similar beat/side profiles, we also found many examples of divergence from this pattern among closely related ORNs and closely related PNs. To explore whether the class-specific combination of beats/sides defines ORN-PN matching specificity, we perturbed presynaptic beat-IIa and postsynaptic side-IV in two ORN-PN partners. However, disruption of Beat-IIa-Side-IV interaction did not produce any significant mistargeting in these two examined glomeruli. Though without affecting general glomerular targeting, knockdown of side in ORNs leads to the reduction of synaptic development. Interestingly, we found conserved expression patterns of beat/side orthologs across ORNs in ants and mosquitoes, indicating the shared regulatory strategies specifying the expression of these duplicated paralogs in insect evolution. Overall, this comprehensive analysis of expression patterns lays a foundation for in-depth functional investigations into how Beat/Side combinatorial expression contributes to the olfactory circuit assembly.

neuroscience↗

bioRxiv2025

High-resolution comparative single-cell transcriptomics of doublesex-expressing neurons reveals evolutionary conservation and diversity of sexual circuits in Drosophila

Understanding how the cellular and molecular composition of neural circuits change during evolution is essential for deciphering how behavior evolves. Male sexual behaviors in Drosophila species are remarkably diverse, and the underlying sexual circuits are specified by sex determination genes. Here, we employed single-cell transcriptomics to systematically characterize and compare neuron cell types that express the sex determination gene doublesex in adult males across Drosophila species with divergent sexual behaviors. High-resolution profiling to delineate cellular diversity revealed a largely conserved set of cell types across four species, with minimal evolutionary gain or loss and potentially more prevalent changes in cell type abundance. In-depth comparisons between D. melanogaster and D. yakuba showed that transcriptomic conservation and differentially expressed genes between species are highly cell-type-specific, suggesting that cell types evolve as highly independent units. We identified widespread species differences in gene expression, particularly in the neuromodulatory signaling pathway, while preserving a conserved circuit layout in sexual identity and neurotransmitter properties. We further generated a female dataset in D. melanogaster to define sex differences in cell types and examine species divergence in relation to sex differences. Finally, we reported marker gene combinations that uniquely define each cell type, providing a foundational resource that enables the design of cell -type-specific genetic reagents. Overall, our study provides fundamental insights into the cellular diversity of sexual circuits and how evolution shapes cell types and gene expression in behavioral adaptations.

evolutionary biology↗

bioRxiv2025

Decoding neuronal wiring by joint inference of cell identity and synaptic connectivity

Animal behaviors are executed by motor neurons (MNs), which receive information from complex pre-motor neuron (preMN) circuits and output commands to muscles. How motor circuits are established during development remains an important unsolved problem in neuroscience. Here we focus on the development of the motor circuits that control the movements of the adult legs in Drosophila melanogaster. After generating single-cell RNA sequencing (scRNAseq) datasets for leg MNs at multiple time points, we describe the time course of gene expression for multiple gene families. This analysis reveals that transcription factors (TFs) and cell adhesion molecules (CAMs) appear to drive the molecular diversity between individual MNs. In parallel, we introduce ConnectionMiner, a novel computational tool that integrates scRNAseq data with electron microscopy-derived connectomes. ConnectionMiner probabilistically refines ambiguous cell type annotations by leveraging neural wiring patterns, and, in turn, it identifies combinatorial gene expression signatures that correlate with synaptic connectivity strength. Applied to the Drosophila leg motor system, ConnectionMiner yields a comprehensive transcriptional annotation of both MNs and preMNs and uncovers candidate effector gene combinations that likely orchestrate the assembly of neural circuits from preMNs to MNs and ultimately to muscles.

neuroscience↗

bioRxiv2025

Temporal and Notch identity determine layer targeting and synapse location of medulla neurons

How specification mechanisms that generate neural diversity translate into specific neuronal targeting, connectivity, and function in the adult brain is not understood. In the medulla region of the Drosophila optic lobe, neural progenitors generate different neurons in a fixed order by sequentially expressing a series of temporal transcription factors as they age. Then, Notch signaling in intermediate progenitors further diversifies neuronal progeny. Using the Electron Microscopy reconstruction of the adult fly brain, we matched the temporal and Notch identity of medulla neurons with their adult targeting features across the different optic lobe neuropils at synapse resolution, and found that these specification mechanisms are significantly associated with the depth of neuropil targeting in the adult brain, for both local interneurons and projection neurons. We show that this temporal identity-dependent targeting of projection neurons unfolds early in development and is genetically determined. Notably, synapse location in the adult brain of putative medulla neurons can be used to predict their temporal origin. Moreover, synapses of neuronal types from different connectome-predicted functional subsystems are compartmentalized in the different neuropils, suggesting a link between synaptic stratification and specific visual functions. Finally, we show that candidate terminal selector TFs are significantly associated with a specific depth bias in the optic lobe neuropils, providing putative molecular determinants of this targeting. Together, we show that temporal identity and Notch status of medulla neurons can predict their neuropil synapse location, linking their developmental patterning with adult circuit architecture.

developmental biology↗

bioRxiv2023

Biased cell adhesion organizes a circuit for visual motion integration

Layer specific computations in the brain rely on neuronal processes establishing synaptic connections with specific partners in distinct laminae. In the Drosophila lobula plate neuropile, the axons of the four subtypes of T4 and T5 visual motion direction-selective neurons segregate into four layers, based on their directional preference, and form synapses with distinct subsets of postsynaptic neurons. Four bi-stratified inhibitory lobula plate intrinsic cells exhibit a consistent synaptic pattern, receiving excitatory T4/T5 inputs in one layer, and conveying inhibitory signals to an adjacent layer. This layered arrangement establishes motion opponency. Here, we identify layer-specific expression of different receptor-ligand pairs belonging to the Beat and Side families of Cell Adhesion Molecules (CAMs) between T4/T5 neurons and their postsynaptic partners. Genetic analysis reveals that Beat/Side mediated interactions are required to restrict T4/T5 axonal innervation to a single layer. We propose that Beat/Side contribute to synaptic specificity by biasing adhesion between synaptic partners before synaptogenesis.

developmental biology↗

bioRxiv2023

Using single-cell RNA sequencing to generate cell-type-specific split-GAL4 reagents throughout development

Cell-type-specific tools facilitate the identification and functional characterization of distinct cell types, which underly the complexity of neuronal circuits. A large collection of existing genetic tools in Drosophila relies on enhancer activity to label different subsets of cells. These enhancer-based GAL4 lines often fail to show a predicable expression pattern to reflect the expression of nearby gene(s), partly due to an incomplete capture of the full gene regulatory elements. While genetic intersectional technique such as the split-GAL4 system further improve cell-type-specificity, it requires significant time and resource to generate and screen through combinations of enhancer expression patterns. In addition, since existing enhancer-based split-GAL4 lines that show cell-type-specific labeling in adult are not necessarily active nor specific in early development, there is a relative lack of tools for the study of neural development. Here, we use an existing single-cell RNA sequencing (scRNAseq) dataset to select gene pairs and provide an efficient pipeline to generate cell-type-specific split-GAL4 lines based on the native genetic regulatory elements. These gene-specific split-GAL4 lines can be generated from a large collection of coding intronic MiMIC/CRIMIC lines either by embryo injection or in vivo cassette swapping crosses and/or CRISPR knock-in at the N or C terminal of the gene. We use the developing Drosophila visual system as a model to demonstrate the high prediction power of scRNAseq-guided gene specific split-GAL4 lines in targeting known cell types. The toolkit allows efficient cluster annotation in scRNAseq datasets but also the identification of novel cell types. Lastly, the gene-specific split-GAL4 lines are broadly applicable to Drosophila tissues. Our work opens new avenues for generating cell-type-specific tools for the targeted manipulation of distinct cell types throughout development and represents a valuable resource to the fly research community. Significance StatementUnderstanding the functional role of individual cell types in the nervous systems has remained a major challenge for neuroscience researchers, partly due to incomplete identification and characterization of underlying cell types. To study the development of individual cell types and their functional roles in health and disease, experimental access to a specific cell type is often a prerequisite. Here, we establish an experimental pipeline to generate gene-specific split-GAL4 guided by single-cell RNA sequencing datasets. These lines show high accuracy for labeling targeted cell types from early developmental stages to adulthood and can be applied to any tissues in Drosophila. The collection of gene-speicifc-split-GAL4 will provide a valuable resource to the entire fly research community.

developmental biology↗