Nuclear aggregation of olfactory receptor genes governs their monogenic expression.

Gene positioning and regulation of nuclear architecture are thought to influence gene expression. Here, we show that, in mouse olfactory neurons, silent olfactory receptor (OR) genes from different chromosomes converge in a small number of heterochromatic foci. These foci are OR exclusive and form in a cell-type-specific and differentiation-dependent manner. The aggregation of OR genes is developmentally synchronous with the downregulation of lamin b receptor (LBR) and can be reversed by ectopic expression of LBR in mature olfactory neurons. LBR-induced reorganization of nuclear architecture and disruption of OR aggregates perturbs the singularity of OR transcription and disrupts the targeting specificity of the olfactory neurons. Our observations propose spatial sequestering of heterochromatinized OR family members as a basis of monogenic and monoallelic gene expression.

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 at dawn and dusk over the circadian cycle. We observed significant 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 at both dawn and dusk, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase at dawn. 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.

An epigenetic signature for monoallelic olfactory receptor expression.

Constitutive heterochromatin is traditionally viewed as the static form of heterochromatin that silences pericentromeric and telomeric repeats in a cell cycle- and differentiation-independent manner. Here, we show that, in the mouse olfactory epithelium, olfactory receptor (OR) genes are marked in a highly dynamic fashion with the molecular hallmarks of constitutive heterochromatin, H3K9me3 and H4K20me3. The cell type and developmentally dependent deposition of these marks along the OR clusters are, most likely, reversed during the process of OR choice to allow for monogenic and monoallelic OR expression. In contrast to the current view of OR choice, our data suggest that OR silencing takes place before OR expression, indicating that it is not the product of an OR-elicited feedback signal. Our findings suggest that chromatin-mediated silencing lays a molecular foundation upon which singular and stochastic selection for gene expression can be applied.

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.

Emergence and influence of sequence bias in evolutionarily malleable, mammalian tandem arrays.

BACKGROUND: 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. RESULTS: Here, we show that these tandem arrays of outward-looking genes occupy AT-biased isochores and comprise the "tissue-specific" gene class that 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 allelic diversity, we use population genetic data and comparative analysis. First, we find that AT bias can emerge during evolutionary expansion of gene families 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 non-synonymous variants. Finally, we find that isochores containing gene clusters exhibit low rates of recombination. CONCLUSIONS: Our analyses suggest that tolerance of non-synonymous variation and low recombination are two forces that have produced the depletion of GC bases in outward-facing gene arrays. In turn, high AT content exerts a profound effect on their chromatin organization and transcriptional regulation.

Untangling the wires: development of sparse, distributed connectivity in the mushroom body calyx.

Appropriate perception and representation of sensory stimuli pose an everyday challenge to the brain. In order to represent the wide and unpredictable array of environmental stimuli, principle neurons of associative learning regions receive sparse, combinatorial sensory inputs. Despite the broad role of such networks in sensory neural circuits, the developmental mechanisms underlying their emergence are not well understood. As mammalian sensory coding regions are numerically complex and lack the accessibility of simpler invertebrate systems, we chose to focus this review on the numerically simpler, yet functionally similar, Drosophila mushroom body calyx. We bring together current knowledge about the cellular and molecular mechanisms orchestrating calyx development, in addition to drawing insights from literature regarding construction of sparse wiring in the mammalian cerebellum. From this, we formulate hypotheses to guide our future understanding of the development of this critical perceptual center.

Coding exons function as tissue-specific enhancers of nearby genes.

Enhancers are essential gene regulatory elements whose alteration can lead to morphological differences between species, developmental abnormalities, and human disease. Current strategies to identify enhancers focus primarily on noncoding sequences and tend to exclude protein coding sequences. Here, we analyzed 25 available ChIP-seq data sets that identify enhancers in an unbiased manner (H3K4me1, H3K27ac, and EP300) for peaks that overlap exons. We find that, on average, 7% of all ChIP-seq peaks overlap coding exons (after excluding for peaks that overlap with first exons). By using mouse and zebrafish enhancer assays, we demonstrate that several of these exonic enhancer (eExons) candidates can function as enhancers of their neighboring genes and that the exonic sequence is necessary for enhancer activity. Using ChIP, 3C, and DNA FISH, we further show that one of these exonic limb enhancers, Dync1i1 exon 15, has active enhancer marks and physically interacts with Dlx5/6 promoter regions 900 kb away. In addition, its removal by chromosomal abnormalities in humans could cause split hand and foot malformation 1 (SHFM1), a disorder associated with DLX5/6. These results demonstrate that DNA sequences can have a dual function, operating as coding exons in one tissue and enhancers of nearby gene(s) in another tissue, suggesting that phenotypes resulting from coding mutations could be caused not only by protein alteration but also by disrupting the regulation of another gene.

High-throughput mapping of the promoters of the mouse olfactory receptor genes reveals a new type of mammalian promoter and provides insight into olfactory receptor gene regulation.

The olfactory receptor (OR) genes are the largest mammalian gene family and are expressed in a monogenic and monoallelic fashion in olfactory neurons. Using a high-throughput approach, we mapped the transcription start sites of 1085 of the 1400 murine OR genes and performed computational analysis that revealed potential transcription factor binding sites shared by the majority of these promoters. Our analysis produced a hierarchical model for OR promoter recognition in which unusually high AT content, a unique epigenetic signature, and a stereotypically positioned O/E site distinguish OR promoters from the rest of the murine promoters. Our computations revealed an intriguing correlation between promoter AT content and evolutionary plasticity, as the most AT-rich promoters regulate rapidly evolving gene families. Within the AT-rich promoter category the position of the TATA-box does not correlate with the transcription start site. Instead, a spike in GC composition might define the exact location of the TSS, introducing the concept of "genomic contrast" in transcriptional regulation. Finally, our experiments show that genomic neighborhood rather than promoter sequence correlates with the probability of different OR genes to be expressed in the same olfactory cell.

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

The arthropod mushroom body is well-studied as an expansion layer representing olfactory stimuli and linking them to contingent events. However, 8% of mushroom body Kenyon cells in Drosophila melanogaster receive predominantly visual input, and their function remains unclear. Here, we identify inputs to visual Kenyon cells using the FlyWire adult whole-brain connectome. Input repertoires are similar across hemispheres and connectomes with certain inputs highly overrepresented. Many visual neurons presynaptic to Kenyon cells have large receptive fields, while interneuron inputs receive spatially restricted signals that may be tuned to specific visual features. Individual visual Kenyon cells randomly sample sparse inputs from combinations of visual channels, including multiple optic lobe neuropils. These connectivity patterns suggest that visual coding in the mushroom body, like olfactory coding, is sparse, distributed, and combinatorial. However, the specific input repertoire to the smaller population of visual Kenyon cells suggests a constrained encoding of visual stimuli.

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.

Let's talk about sex: Mechanisms of neural sexual differentiation in Bilateria.

In multicellular organisms, sexed gonads have evolved that facilitate release of sperm versus eggs, and bilaterian animals purposefully combine their gametes via mating behaviors. Distinct neural circuits have evolved that control these physically different mating events for animals producing eggs from ovaries versus sperm from testis. In this review, we will describe the developmental mechanisms that sexually differentiate neural circuits across three major clades of bilaterian animals-Ecdysozoa, Deuterosomia, and Lophotrochozoa. While many of the mechanisms inducing somatic and neuronal sex differentiation across these diverse organisms are clade-specific rather than evolutionarily conserved, we develop a common framework for considering the developmental logic of these events and the types of neuronal differences that produce sex-differentiated behaviors. This article is categorized under: Congenital Diseases > Stem Cells and Development Neurological Diseases > Stem Cells and Development.

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.

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.

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.

Input density tunes Kenyon cell sensory responses in the Drosophila mushroom body.

The ability to discriminate sensory stimuli with overlapping features is thought to arise in brain structures called expansion layers, where neurons carrying information about sensory features make combinatorial connections onto a much larger set of cells. For 50 years, expansion coding has been a prime topic of theoretical neuroscience, which seeks to explain how quantitative parameters of the expansion circuit influence sensory sensitivity, discrimination, and generalization. Here, we investigate the developmental events that produce the quantitative parameters of the arthropod expansion layer, called the mushroom body. Using Drosophila melanogaster as a model, we employ genetic and chemical tools to engineer changes to circuit development. These allow us to produce living animals with hypothesis-driven variations on natural expansion layer wiring parameters. We then test the functional and behavioral consequences. By altering the number of expansion layer neurons (Kenyon cells) and their dendritic complexity, we find that input density, but not cell number, tunes neuronal odor selectivity. Simple odor discrimination behavior is maintained when the Kenyon cell number is reduced and augmented by Kenyon cell number expansion. Animals with increased input density to each Kenyon cell show increased overlap in Kenyon cell odor responses and become worse at odor discrimination tasks.

May the Odds Be Ever in Your Favor: Non-deterministic Mechanisms Diversifying Cell Surface Molecule Expression.

How does the information in the genome program the functions of the wide variety of cells in the body? While the development of biological organisms appears to follow an explicit set of genomic instructions to generate the same outcome each time, many biological mechanisms harness molecular noise to produce variable outcomes. Non-deterministic variation is frequently observed in the diversification of cell surface molecules that give cells their functional properties, and is observed across eukaryotic clades, from single-celled protozoans to mammals. This is particularly evident in immune systems, where random recombination produces millions of antibodies from only a few genes; in nervous systems, where stochastic mechanisms vary the sensory receptors and synaptic matching molecules produced by different neurons; and in microbial antigenic variation. These systems employ overlapping molecular strategies including allelic exclusion, gene silencing by constitutive heterochromatin, targeted double-strand breaks, and competition for limiting enhancers. Here, we describe and compare five stochastic molecular mechanisms that produce variety in pathogen coat proteins and in the cell surface receptors of animal immune and neuronal cells, with an emphasis on the utility of non-deterministic variation.

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.

Despite having a limited number of senses, animals can perceive a huge range of sensations. One possible explanation is that the brain combines several stimuli to make each specific sensation. The olfactory learning system in the fruit fly Drosophila melanogaster is in a part of the brain called the mushroom body. It allows fruit flies to associate a specific smell with a reward (e.g. food) or a punishment (e.g. poison) and behave accordingly. Two groups of neurons process stimuli from sensory receptors in the mushroom body: olfactory projection neurons carry information from the receptors and pass it on to neurons called Kenyon cells. The system relies on Kenyon cells receiving the combined input of multiple olfactory projection neurons, and therefore information from multiple receptors. The number of inputs each Kenyon cell receives is thought to determine the number of sensations that can be told apart, and thus, the number of signals that can be used for learning. While many mechanisms dictating the complexity of a neuron's shape have been described, the logic behind how two populations of neurons become connected to combine several inputs into a single sensation has not been addressed. A better understanding of how these connections are established during development can help explain how the brain processes information, and the D. melanogaster mushroom body is a good system to address these questions. Elkahlah, Rogow et al. manipulated the number of olfactory projection neurons and Kenyon cells in the mushroom body of fruit flies during development. They found that despite there being a varying number of cells, the number of connections into a post-synaptic cell remained the same. This indicates that the logic behind the combinations of inputs required for a sensation depends on the Kenyon cell, while olfactory projection neurons can adapt during their development to suit these input demands. Thus, if there are fewer Kenyon cells, the olfactory projection neurons will each provide connections to fewer cells to compensate, and if there are fewer olfactory projection neurons, each of them will input into more Kenyon cells. To show that the developing mushroom body could indeed adapt to different numbers of olfactory projection neurons and Kenyon cells, the modified flies were tested for olfactory perception: their responses to odor were largely normal. These results underline the robustness of neuronal circuits. During development, the mushroom body can compensate for missing or extra neurons by modifying the numbers of connections between two groups of neurons, thus allowing the olfactory system to work normally. This robustness may also predispose the system to evolutionary change, since it allows the system to continue working as it changes. These findings are relevant to any area of the brain where neurons rely on combined input from many sources.

Comparative Development of the Ant Chemosensory System.

The insect antennal lobe (AL) contains the first synapses of the olfactory system, where olfactory sensory neurons (OSNs) contact second-order projection neurons (PNs). In Drosophila melanogaster, OSNs expressing specific receptor genes send stereotyped projections to one or two of about 50 morphologically defined glomeruli [1-3]. The mechanisms for this precise matching between OSNs and PNs have been studied extensively in D. melanogaster, where development is deterministic and independent of neural activity [4-6]. However, a number of insect lineages, most notably the ants, have receptor gene repertoires many times larger than D. melanogaster and exhibit more structurally complex antennal lobes [7-12]. Moreover, perturbation of OSN function via knockout of the odorant receptor (OR) co-receptor, Orco, results in drastic AL reductions in ants [13, 14], but not in Drosophila [15]. Here, we characterize AL development in the clonal raider ant, Ooceraea biroi. We find that, unlike in Drosophila, ORs and Orco are expressed before the onset of glomerulus formation, and Orco protein is trafficked to developing axon terminals, raising the possibility that ORs play a role during ant AL development. Additionally, ablating ant antennae at the onset of pupation results in AL defects that recapitulate the Orco mutant phenotype. Thus, early loss of functional OSN innervation reveals latent structure in the AL that develops independently of peripheral input, suggesting that the AL is initially pre-patterned and later refined in an OSN-dependent manner. This two-step process might increase developmental flexibility and thereby facilitate the rapid evolution and expansion of the ant chemosensory system.

Soft X-Ray Tomography Reveals Gradual Chromatin Compaction and Reorganization during Neurogenesis In Vivo.

The realization that nuclear distribution of DNA, RNA, and proteins differs between cell types and developmental stages suggests that nuclear organization serves regulatory functions. Understanding the logic of nuclear architecture and how it contributes to differentiation and cell fate commitment remains challenging. Here, we use soft X-ray tomography (SXT) to image chromatin organization, distribution, and biophysical properties during neurogenesis in vivo. Our analyses reveal that chromatin with similar biophysical properties forms an elaborate connected network throughout the entire nucleus. Although this interconnectivity is present in every developmental stage, differentiation proceeds with concomitant increase in chromatin compaction and re-distribution of condensed chromatin toward the nuclear core. HP1ß, but not nucleosome spacing or phasing, regulates chromatin rearrangements because it governs both the compaction of chromatin and its interactions with the nuclear envelope. Our experiments introduce SXT as a powerful imaging technology for nuclear architecture.

Multimodal Chemosensory Circuits Controlling Male Courtship in Drosophila.

Throughout the animal kingdom, internal states generate long-lasting and self-perpetuating chains of behavior. In Drosophila, males instinctively pursue females with a lengthy and elaborate courtship ritual triggered by activation of sexually dimorphic P1 interneurons. Gustatory pheromones are thought to activate P1 neurons but the circuit mechanisms that dictate their sensory responses to gate entry into courtship remain unknown. Here, we use circuit mapping and in vivo functional imaging techniques to trace gustatory and olfactory pheromone circuits to their point of convergence onto P1 neurons and reveal how their combined input underlies selective tuning to appropriate sexual partners. We identify inhibition, even in response to courtship-promoting pheromones, as a key circuit element that tunes and tempers P1 neuron activity. Our results suggest a circuit mechanism in which balanced excitation and inhibition underlie discrimination of prospective mates and stringently regulate the transition to courtship in Drosophila.