Biology subjects

Clowney, E. J.

Publications and source records attributed to Clowney, E. J..

11 recordsLinked to original sources

bioRxiv2026

Fruitless produces diverse neuronal sex differences by editing lineage-programmed chromatin landscapes

A major question in metazoan development is how a limited set of DNA-binding transcription factors generate the much larger number of cell fates and states in the organism. In vivo, individual transcription factors are joined by varied sets of collaborating or competing proteins across cellular contexts. Brains provide particularly diverse transcriptional milieus across thousands of neuron types, where each cell is a phenotypic readout of intrinsic transcriptional regulation over developmental time. Todays methods allow interrogation of individual cells, transforming the diversity of neurons in the brain from an experimental hindrance to a benefit in interrogation of combinatorial transcription factor action. Here, we use the BTB domain transcription factor Fruitless as a model to determine how a single factor produces cell-type specific regulatory effects, in this case sexual dimorphisms. In Drosophila melanogaster, Fruitless is transcribed in a precise neuronal repertoire (irrespective of sex) but translated into protein only in male cells. By performing joint scRNA/ATACseq on fruitless neurons from both sexes (i.e. with and without Fru protein) we determine the transcriptional mechanisms by which Fruitless produces cell-type specific regulatory effects. We demonstrate that Fruitlesss actions are constrained by the chromatin landscape produced by cell lineage. Within lineage-specific landscapes, type-specific transcriptional dimorphisms arise from varying "Fru power" across cell types, which emerges as a combination of Fruitless dose, DNA binding domain isoforms, and the motif repertoire of individual enhancers. Together, these mechanisms allow a single factor to produce multifaceted transcriptional effects that subtly alter individual neuron types and produce sex-specific circuit functions.

molecular biology↗

bioRxiv2026

Human neurodevelopmental genes housed in massive, ancient gene deserts

Mammalian genomes contain a remarkable proportion of non-coding sequence relative to protein-coding sequence, including megabase-scale gene deserts. The origin of gene deserts and their function have long been mysteries, and most studies probing these questions have focused on specific deserts linked to transcription factors, like SOX9 and MYC. Here, we describe 21 human "lonely genes" located within some of the largest deserts in the genome, making them exceptionally isolated from other genes in linear sequence space. Together, these regions comprise roughly 3.4% of the human genome. Despite minimal sequence conservation, we find that the sizes and locations of these gene deserts are conserved, and they likely originated at the root of the vertebrate tree. In contrast to the deserts near transcription factors that have been the primary focus to date, we find that most genes housed within these massive deserts, as well as genes on the edges of deserts, are cell adhesion molecules. Additionally, many of the genes associated with these deserts are dysregulated in datasets involving chromatin modifiers linked to neurodevelopmental disorders. Using DNA FISH in mouse olfactory epithelium, we find that lonely genes appose the nuclear lamina. We propose that these ancient regions may play a structural role in the nucleus, and that this structure makes the genes within them uniquely difficult to express. While specific chromatin modifiers are able to access lonely genes, this dependence creates a regulatory vulnerability in brain development.

evolutionary biology↗

bioRxiv2026

Modular presynaptic assemblages scale to postsynaptic partner number

Behavioral diversification can arise through, and is constrained by, evolutionary and inter-individual differences in neural circuit development. Moreover, alteration of focal neural parameters changes the environment in which cells connect into circuits. In the mushroom body, an associative learning center of arthropods, the number of principal Kenyon cells varies widely across species and among individuals. How such variation is developmentally accommodated by projection neurons, which provide sensory input to Kenyon cells, is not understood. In Drosophila melanogaster, we previously demonstrated that projection neurons scale their presynaptic bouton number to Kenyon cell population size. Here, we identify the developmental mechanisms underlying this input flexibility. Boutons arise from projection neuron axonal collaterals; we find that a PNs collateral number is subtype-specific and serves as the substrate through which bouton number scales to Kenyon cell population size. Independent of projection neuron identity or Kenyon cell number, individual collaterals most often produce just one bouton, suggesting collaterals are modular cell biological bouton units. Developing projection neurons initially overproduce nascent collaterals in the early pupa. The set of nascent collaterals that mature and eventually bear boutons is conditional on Kenyon cell number, thereby executing scaling. Finally, early boutons bear filopodia that frequently contact neighboring PN processes, suggesting that bouton-bouton interactions contribute to shaping these structures.

neuroscience↗

bioRxiv2025

Hierarchical diversification of instinctual behavior neurons by lineage, birth order, and sex

Brain regions that regulate motivated behaviors, including the vertebrate hypothalamus and arthropod cerebrum, house bespoke neural circuits dedicated to perceptual and internal regulation of many behavioral states1,2. These circuits are built to purpose from complex sets of cell types whose patterning has been challenging to elucidate. Here, we developed methods in Drosophila melanogaster to embed well-studied neurons that regulate mating in the transcriptional contexts of the neuronal lineages that generate them3-5. By comparing transcription within and between lineages, we identified a large set of transcription factors expressed in complex combinations that delineate cerebral hemilineages - classes of postmitotic neurons born from the same stem cell and sharing Notch status6,7. Hemilineages comprise the major anatomic classes in the cerebrum8-10 and these transcription factors are required to generate their gross features. We show that subtypes of the same hemilineage can provide a common computational module to circuits regulating different drives, and identify an orthogonal set of transcription factors that stratify hemilineage subtypes of differing birth order. Our findings suggest that distinct sets of transcription factors operate in a hierarchical system to build, diversify, and sexually differentiate lineally-related neurons that compose motivated behaviors circuits. By linking developmental patterning to separable transcriptional axes that produce gross versus fine aspects of information flow, we provide a logical framework for cerebral control of diverse drives.

neuroscience↗

bioRxiv2024

Spatial constraints and cell surface molecule depletion structure a randomly connected learning circuit

The brain can represent almost limitless objects to "categorize an unlabeled world" (Edelman, 1989). This feat is supported by expansion layer circuit architectures, in which neurons carrying information about discrete sensory channels make combinatorial connections onto much larger postsynaptic populations. Combinatorial connections in expansion layers are modeled as randomized sets. The extent to which randomized wiring exists in vivo is debated, and how combinatorial connectivity patterns are generated during development is not understood. Non- deterministic wiring algorithms could program such connectivity using minimal genomic information. Here, we investigate anatomic and transcriptional patterns and perturb partner availability to ask how Kenyon cells, the expansion layer neurons of the insect mushroom body, obtain combinatorial input from olfactory projection neurons. Olfactory projection neurons form their presynaptic outputs in an orderly, predictable, and biased fashion. We find that Kenyon cells accept spatially co-located but molecularly heterogeneous inputs from this orderly map, and ask how Kenyon cell surface molecule expression impacts partner choice. Cell surface immunoglobulins are broadly depleted in Kenyon cells, and we propose that this allows them to form connections with molecularly heterogeneous partners. This model can explain how developmentally identical neurons acquire diverse wiring identities.

neuroscience↗

bioRxiv2023

Diversity of visual inputs to Kenyon cells of the Drosophila mushroom body

The arthropod mushroom body is well-studied as an expansion layer that represents olfactory stimuli and links them to contingent events. However, 8% of mushroom body Kenyon cells in Drosophila melanogaster receive predominantly visual input, and their tuning and function are poorly understood. Here, we use the FlyWire adult whole-brain connectome to identify inputs to visual Kenyon cells. The types of visual neurons we identify are similar across hemispheres and connectomes with certain inputs highly overrepresented. Many visual projection neurons presynaptic to Kenyon cells receive input from large swathes of visual space, while local visual interneurons, providing smaller fractions of input, receive more spatially restricted signals that may be tuned to specific features of the visual scene. Like olfactory Kenyon cells, visual Kenyon cells receive sparse inputs from different combinations of visual channels, including inputs from multiple optic lobe neuropils. The sets of inputs to individual visual Kenyon cells are consistent with random sampling of available inputs. These connectivity patterns suggest that visual coding in the mushroom body, like olfactory coding, is sparse, distributed, and combinatorial. However, the expansion coding properties appear different, with a specific repertoire of visual inputs projecting onto a relatively small number of visual Kenyon cells.

neuroscience↗

bioRxiv2023

Clock-dependent chromatin accessibility rhythms regulate circadian transcription

Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility over the circadian cycle. We observed significant circadian oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible throughout the circadian cycle, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons. Significance StatementChromatin organization plays a critical role in gene regulation in development and in disease. In this study, we discovered robust circadian oscillations in the chromatin accessibility of regulatory elements of clock-regulated genes in Drosophila clock neurons, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. We found enrichment of E-box motifs in genes that exhibited enhanced accessibility at dusk, and enrichment of VRI/PDP1-box motifs in genes that exhibited higher accessibility at dawn. Moreover, the complete loss of chromatin accessibility rhythms in per01 mutants highlights the essential role of the Period protein in driving chromatin compaction during the repression phase. This study highlights the significance of chromatin organization in the generation of [~]24-hour circadian rhythms.

neuroscience↗

bioRxiv2023

Hacking brain development to test models of sensory coding

Animals can discriminate myriad sensory stimuli but can also generalize from learned experience. You can probably distinguish the favorite teas of your colleagues while still recognizing that all tea pales in comparison to coffee. Tradeoffs between detection, discrimination, and generalization are inherent at every layer of sensory processing. During development, specific quantitative parameters are wired into perceptual circuits and set the playing field on which plasticity mechanisms play out. A primary goal of systems neuroscience is to understand how material properties of a circuit define the logical operations-- computations--that it makes, and what good these computations are for survival. A cardinal method in biology--and the mechanism of evolution--is to change a unit or variable within a system and ask how this affects organismal function. Here, we make use of our knowledge of developmental wiring mechanisms to modify hard-wired circuit parameters in the Drosophila melanogaster mushroom body and assess the functional and behavioral consequences. By altering the number of expansion layer neurons (Kenyon cells) and their dendritic complexity, we find that input number, but not cell number, tunes odor selectivity. Simple odor discrimination performance is maintained when Kenyon cell number is reduced and augmented by Kenyon cell expansion.

neuroscience↗

bioRxiv2022

A common genomic architecture for interacting with the external world

The radiation of mammals at the extinction of the dinosaurs produced a plethora of new forms--as diverse as bats, dolphins, and elephants--in only 10-20 million years. Behind the scenes, adaptation to new niches is accompanied by extensive innovation in large families of genes that allow animals to contact the environment, including chemosensors, xenobiotic enzymes, and immune and barrier proteins. Genes in these "outward-looking" families are allelically diverse among humans and exhibit tissue-specific and sometimes stochastic expression. Here, we show that outward-looking genes are clustered in tandem arrays, enriched in AT-biased isochores, and lack CpG islands in their promoters. Models of mammalian genome evolution have not incorporated the sharply different functions and transcriptional patterns of genes in AT-versus GC-biased regions. To examine the relationship between gene family expansion, sequence content, and functional diversification, we use population genetic data and comparative analysis. First, we find that AT bias can emerge with gene family expansion in cis. Second, human genes in AT-biased isochores or with GC-poor promoters experience relatively low rates of de novo point mutation today but are enriched for functional variants. Finally, we find that isochores containing gene clusters exhibit low rates of recombination. We hypothesize that the depletion of GC bases in outward-facing gene clusters results from tolerance of sequence variation and low recombination. In turn, high AT content exerts a profound effect on their chromatin organization and transcriptional regulation.

evolutionary biology↗

bioRxiv2020

Fruitless decommissions regulatory elements to implement cell-type-specific neuronal masculinization

In the fruit fly Drosophila melanogaster, male-specific splicing and translation of the Fruitless transcription factor (FruM) alters the presence, anatomy, and/or connectivity of >60 types of central brain neurons that interconnect to generate male-typical behaviors. While the indispensable function of FruM in sex-specific behavior has been understood for decades, the molecular mechanisms underlying its activity remain unknown. Here, we take a genome-wide, brain-wide approach to identifying regulatory elements whose activity depends on the presence of FruM. We identify 436 high-confidence genomic regions differentially accessible in male fruitless neurons, validate candidate regions as bona-fide, differentially regulated enhancers, and describe the particular cell types in which these enhancers are active. We find that individual enhancers are not activated universally but are dedicated to specific fru+ cell types. Aside from fru itself, genes are not dedicated to or common across the fru circuit; rather, FruM appears to masculinize each cell type differently, by tweaking expression of the same effector genes used in other circuits. Finally, we find FruM motifs enriched among regulatory elements that are open in the female but closed in the male. Together, these results suggest that FruM acts cell-type-specifically to decommission regulatory elements in male fruitless neurons.

neuroscience↗

bioRxiv2019

Presynaptic developmental plasticity allows robust sparse wiring of the Drosophila mushroom body

In order to represent complex stimuli, principle neurons of associative learning regions receive combinatorial sensory inputs. Density of combinatorial innervation is theorized to determine the number of distinct stimuli that can be represented and distinguished from one another, with sparse innervation thought to optimize the complexity of representations in networks of limited size. How the convergence of combinatorial inputs to principle neurons of associative brain regions is established during development is unknown. Here, we explore the developmental patterning of sparse olfactory inputs to Kenyon cells of the Drosophila melanogaster mushroom body. By manipulating the ratio between pre- and post-synaptic cells, we find that postsynaptic Kenyon cells set convergence ratio: Kenyon cells produce fixed distributions of dendritic claws while presynaptic processes are plastic. Moreover, we show that sparse odor responses are preserved in mushroom bodies with reduced cellular repertoires, suggesting that developmental specification of convergence ratio allows functional robustness.

neuroscience↗