Opposing, spatially-determined epigenetic forces impose restrictions on stochastic olfactory receptor choice.

Olfactory receptor (OR) choice represents an example of genetically hardwired stochasticity, where every olfactory neuron expresses one out of ~2000 OR alleles in the mouse genome in a probabilistic, yet stereotypic fashion. Here, we propose that topographic restrictions in OR expression are established in neuronal progenitors by two opposing forces: polygenic transcription and genomic silencing, both of which are influenced by dorsoventral gradients of transcription factors NFIA, B, and X. Polygenic transcription of OR genes may define spatially constrained OR repertoires, among which one OR allele is selected for singular expression later in development. Heterochromatin assembly and genomic compartmentalization of OR alleles also vary across the axes of the olfactory epithelium and may preferentially eliminate ectopically expressed ORs with more dorsal expression destinations from this 'privileged' repertoire. Our experiments identify early transcription as a potential 'epigenetic' contributor to future developmental patterning and reveal how two spatially responsive probabilistic processes may act in concert to establish deterministic, precise, and reproducible territories of stochastic gene expression.

retro-Tango enables versatile retrograde circuit tracing in Drosophila.

Transsynaptic tracing methods are crucial tools in studying neural circuits. Although a couple of anterograde tracing methods and a targeted retrograde tool have been developed in Drosophila melanogaster, there is still need for an unbiased, user-friendly, and flexible retrograde tracing system. Here, we describe retro-Tango, a method for transsynaptic, retrograde circuit tracing and manipulation in Drosophila. In this genetically encoded system, a ligand-receptor interaction at the synapse triggers an intracellular signaling cascade that results in reporter gene expression in presynaptic neurons. Importantly, panneuronal expression of the elements of the cascade renders this method versatile, enabling its use not only to test hypotheses but also to generate them. We validate retro-Tango in various circuits and benchmark it by comparing our findings with the electron microscopy reconstruction of the Drosophila hemibrain. Our experiments establish retro-Tango as a key method for circuit tracing in neuroscience research.

Transsynaptic labeling and transcriptional control of zebrafish neural circuits.

Deciphering the connectome, the ensemble of synaptic connections that underlie brain function is a central goal of neuroscience research. The trans-Tango genetic approach, initially developed for anterograde transsynaptic tracing in Drosophila, can be used to map connections between presynaptic and postsynaptic partners and to drive gene expression in target neurons. Here, we describe the successful adaptation of trans-Tango to visualize neural connections in a living vertebrate nervous system, that of the zebrafish. Connections were validated between synaptic partners in the larval retina and brain. Results were corroborated by functional experiments in which optogenetic activation of retinal ganglion cells elicited responses in neurons of the optic tectum, as measured by trans-Tango-dependent expression of a genetically encoded calcium indicator. Transsynaptic signaling through trans-Tango reveals predicted as well as previously undescribed synaptic connections, providing a valuable in vivo tool to monitor and interrogate neural circuits over time.

Opposing, spatially-determined epigenetic forces impose restrictions on stochastic olfactory receptor choice.

Olfactory receptor (OR) choice represents an example of genetically hardwired stochasticity, where every olfactory neuron expresses one out of ~2000 OR alleles in a probabilistic, yet stereotypic fashion. Here, we propose that topographic restrictions in OR expression are established in neuronal progenitors by two opposing forces: polygenic transcription and genomic silencing, both of which are influenced by dorsoventral gradients of transcription factors NFIA, B, and X. Polygenic transcription of OR genes may define spatially constrained OR repertoires, among which one OR allele is selected for singular expression later in development. Heterochromatin assembly and genomic compartmentalization of OR alleles also vary across the axes of the olfactory epithelium and may preferentially eliminate ectopically expressed ORs with more dorsal expression destinations from this "privileged" repertoire. Our experiments identify early transcription as a potential "epigenetic" contributor to future developmental patterning and reveal how two spatially responsive probabilistic processes may act in concert to establish deterministic, precise, and reproducible territories of stochastic gene expression.

Circuit analysis reveals a neural pathway for light avoidance in Drosophila larvae.

Understanding how neural circuits underlie behaviour is challenging even in the connectome era because it requires a combination of anatomical and functional analyses. This is exemplified in the circuit underlying the light avoidance behaviour displayed by Drosophila melanogaster larvae. While this behaviour is robust and the nervous system relatively simple, the circuit is only partially delineated with some contradictions among studies. Here, we devise trans-Tango MkII, an offshoot of the transsynaptic circuit tracing tool trans-Tango, and implement it in anatomical tracing together with functional analysis. We use neuronal inhibition to test necessity of particular neuronal types in light avoidance and selective neuronal activation to examine sufficiency in rescuing light avoidance deficiencies exhibited by photoreceptor mutants. Our studies reveal a four-order circuit for light avoidance connecting the light-detecting photoreceptors with a pair of neuroendocrine cells via two types of clock neurons. This approach can be readily expanded to studying other circuits.

Complex representation of taste quality by second-order gustatory neurons in Drosophila.

Sweet and bitter compounds excite different sensory cells and drive opposing behaviors. However, it remains unclear how sweet and bitter tastes are represented by the neural circuits linking sensation to behavior. To investigate this question in Drosophila, we devised trans-Tango(activity), a strategy for calcium imaging of second-order gustatory projection neurons based on trans-Tango, a genetic transsynaptic tracing technique. We found spatial overlap between the projection neuron populations activated by sweet and bitter tastants. The spatial representation of bitter tastants in the projection neurons was consistent, while that of sweet tastants was heterogeneous. Furthermore, we discovered that bitter tastants evoke responses in the gustatory receptor neurons and projection neurons upon both stimulus onset and offset and that bitter offset and sweet onset excite overlapping second-order projections. These findings demonstrate an unexpected complexity in the representation of sweet and bitter tastants by second-order neurons of the gustatory circuit.

Coordination of two enhancers drives expression of olfactory trace amine-associated receptors.

Olfactory sensory neurons (OSNs) are functionally defined by their expression of a unique odorant receptor (OR). Mechanisms underlying singular OR expression are well studied, and involve a massive cross-chromosomal enhancer interaction network. Trace amine-associated receptors (TAARs) form a distinct family of olfactory receptors, and here we find that mechanisms regulating Taar gene choice display many unique features. The epigenetic signature of Taar genes in TAAR OSNs is different from that in OR OSNs. We further identify that two TAAR enhancers conserved across placental mammals are absolutely required for expression of the entire Taar gene repertoire. Deletion of either enhancer dramatically decreases the expression probabilities of different Taar genes, while deletion of both enhancers completely eliminates the TAAR OSN populations. In addition, both of the enhancers are sufficient to drive transgene expression in the partially overlapped TAAR OSNs. We also show that the TAAR enhancers operate in cis to regulate Taar gene expression. Our findings reveal a coordinated control of Taar gene choice in OSNs by two remote enhancers, and provide an excellent model to study molecular mechanisms underlying formation of an olfactory subsystem.

Transsynaptic mapping of Drosophila mushroom body output neurons.

The mushroom body (MB) is a well-characterized associative memory structure within the Drosophila brain. Analyzing MB connectivity using multiple approaches is critical for understanding the functional implications of this structure. Using the genetic anterograde transsynaptic tracing tool, trans-Tango, we identified divergent projections across the brain and convergent downstream targets of the MB output neurons (MBONs). Our analysis revealed at least three separate targets that receive convergent input from MBONs: other MBONs, the fan-shaped body (FSB), and the lateral accessory lobe (LAL). We describe, both anatomically and functionally, a multilayer circuit in which inhibitory and excitatory MBONs converge on the same genetic subset of FSB and LAL neurons. This circuit architecture enables the brain to update and integrate information with previous experience before executing appropriate behavioral responses. Our use of trans-Tango provides a genetically accessible anatomical framework for investigating the functional relevance of components within these complex and interconnected circuits.

Circuits that encode and guide alcohol-associated preference.

A powerful feature of adaptive memory is its inherent flexibility. Alcohol and other addictive substances can remold neural circuits important for memory to reduce this flexibility. However, the mechanism through which pertinent circuits are selected and shaped remains unclear. We show that circuits required for alcohol-associated preference shift from population level dopaminergic activation to select dopamine neurons that predict behavioral choice in Drosophila melanogaster. During memory expression, subsets of dopamine neurons directly and indirectly modulate the activity of interconnected glutamatergic and cholinergic mushroom body output neurons (MBON). Transsynaptic tracing of neurons important for memory expression revealed a convergent center of memory consolidation within the mushroom body (MB) implicated in arousal, and a structure outside the MB implicated in integration of naïve and learned responses. These findings provide a circuit framework through which dopamine neuronal activation shifts from reward delivery to cue onset, and provide insight into the maladaptive nature of memory.

Transsynaptic Mapping of Second-Order Taste Neurons in Flies by trans-Tango.

Mapping neural circuits across defined synapses is essential for understanding brain function. Here we describe trans-Tango, a technique for anterograde transsynaptic circuit tracing and manipulation. At the core of trans-Tango is a synthetic signaling pathway that is introduced into all neurons in the animal. This pathway converts receptor activation at the cell surface into reporter expression through site-specific proteolysis. Specific labeling is achieved by presenting a tethered ligand at the synapses of genetically defined neurons, thereby activating the pathway in their postsynaptic partners and providing genetic access to these neurons. We first validated trans-Tango in the Drosophila olfactory system and then implemented it in the gustatory system, where projections beyond the first-order receptor neurons are not fully characterized. We identified putative second-order neurons within the sweet circuit that include projection neurons targeting known neuromodulation centers in the brain. These experiments establish trans-Tango as a flexible platform for transsynaptic circuit analysis.

Identifying functional connections of the inner photoreceptors in Drosophila using Tango-Trace.

In Drosophila, the four inner photoreceptor neurons exhibit overlapping but distinct spectral sensitivities and mediate behaviors that reflect spectral preference. We developed a genetic strategy, Tango-Trace, that has permitted the identification of the connections of the four chromatic photoreceptors. Each of the four stochastically distributed chromatic photoreceptor subtypes make distinct connections in the medulla with four different TmY cells. Moreover, each class of TmY cells forms a retinotopic map in both the medulla and the lobula complex, generating four overlapping topographic maps that could carry different color information. Thus, the four inner photoreceptors transmit spectral information through distinct channels that may converge in both the medulla and lobula complex. These projections could provide an anatomic basis for color vision and may relay information about color to motion sensitive areas. Moreover, the Tango-Trace strategy we used may be applied more generally to identify neural circuits in the fly brain.

A critical period defined by axon-targeting mechanisms in the murine olfactory bulb.

The olfactory system remains plastic throughout life because of continuous neurogenesis of sensory neurons in the nose and inhibitory interneurons in the olfactory bulb. Here, we reveal that transgenic expression of an odorant receptor has non-cell autonomous effects on axons expressing this receptor from the endogenous gene. Perinatal expression of transgenic odorant receptor causes rerouting of like axons to new glomeruli, whereas expression after the sensory map is established does not lead to rerouting. Further, chemical ablation of the map after rerouting does not restore the normal map, even when the transgenic receptor is no longer expressed. Our results reveal that glomeruli are designated as targets for sensory neurons expressing specific odorant receptors during a critical period in the formation of the olfactory sensory map.

Alteration of genic 5-hydroxymethylcytosine patterning in olfactory neurons correlates with changes in gene expression and cell identity.

The modified DNA base 5-hydroxymethylcytosine (5hmC) is enriched in neurons where it may contribute to gene regulation and cellular identity. To determine how 5hmC influences gene expression in an in vivo neuronal population, we assessed the patterning and function of the base along the developmental lineage of the main olfactory epithelium-from multipotent stem cells through neuronal progenitors to mature olfactory sensory neurons (mOSNs). We find that 5hmC increases over gene bodies during mOSN development with substantial patterning occuring between the progenitor and mOSN stages. Although gene-body 5hmC levels correlate with gene expression in all three developmental cell types, this association is particularly pronounced within mOSNs. Overexpression of Tet3 in mOSNs markedly alters gene-body 5hmC levels and gene expression in a manner consistent with a positive role for 5hmC in transcription. Moreover, Tet3 overexpression disrupts olfactory receptor expression and the targeting of axons to the olfactory bulb, key molecular and anatomical features of the olfactory system. Our results suggest a physiologically significant role for gene-body 5hmC in transcriptional facilitation and the maintenance of cellular identity independent of its function as an intermediate to demethylation.

An epigenetic trap stabilizes singular olfactory receptor expression.

The molecular mechanisms regulating olfactory receptor (OR) expression in the mammalian nose are not yet understood. Here, we identify the transient expression of histone demethylase LSD1 and the OR-dependent expression of adenylyl cyclase 3 (Adcy3) as requirements for initiation and stabilization of OR expression. As a transcriptional coactivator, LSD1 is necessary for desilencing and initiating OR transcription, but as a transcriptional corepressor, it is incompatible with maintenance of OR expression, and its downregulation is imperative for stable OR choice. Adcy3, a sensor of OR expression and a transmitter of an OR-elicited feedback, mediates the downregulation of LSD1 and promotes the differentiation of olfactory sensory neurons (OSNs). This novel, three-node signaling cascade locks the epigenetic state of the chosen OR, stabilizes its singular expression, and prevents the transcriptional activation of additional OR alleles for the life of the neuron.

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.

Aß alters the connectivity of olfactory neurons in the absence of amyloid plaques in vivo.

The amyloid beta peptide aggregates into amyloid plaques at presymptomatic stages of Alzheimer's disease, but the temporal relationship between plaque formation and neuronal dysfunction is poorly understood. Here we demonstrate that the connectivity of the peripheral olfactory neural circuit is perturbed in mice overexpressing human APPsw (Swedish mutation) before the onset of plaques. Expression of human APPsw exclusively in olfactory sensory neurons also perturbs connectivity with associated reductions in odour-evoked gene expression and olfactory acuity. By contrast, olfactory sensory neuron axons project correctly in mice overexpressing wild-type human amyloid precursor protein throughout the brain and in mice overexpressing M671V human APP, a missense mutation that reduces amyloid beta production, exclusively in olfactory sensory neurons. Furthermore, expression of Aß40 or Aß42 solely in the olfactory epithelium disrupts the olfactory sensory neuron axon targeting. Our data indicate that altering the structural connectivity and function of highly plastic neural circuits is one of the pleiotropic actions of soluble human amyloid beta.

Neurons expressing trace amine-associated receptors project to discrete glomeruli and constitute an olfactory subsystem.

Some chemoreceptors of the trace amine-associated receptor (TAAR) family detect innately aversive odors and are proposed to activate hardwired olfactory circuits. However, the wiring of TAAR neurons, the regulatory mechanisms of Taar gene choice, and the subcellular localization of TAAR proteins remain unknown. Here, we reveal similarities between neurons expressing TAARs and odorant receptors (ORs), but also unexpected differences. Like ORs, TAARs seem to be monoallelically expressed and localized both in cilia, the site of odor detection, and in axons, where they may participate in guidance. TAAR neurons project to discrete glomeruli predominantly localized to a confined bulb region. Taar expression involves different regulatory logic than OR expression, as neurons choosing a Taar5 knockout allele frequently express a second Taar without silencing the deleted allele. Moreover, the epigenetic signature of OR gene choice is absent from Taar genes. The unique molecular and anatomical features of the TAAR neurons suggest that they constitute a distinct olfactory subsystem.

Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing.

Behavior cannot be predicted from a "connectome" because the brain contains a chemical "map" of neuromodulation superimposed upon its synaptic connectivity map. Neuromodulation changes how neural circuits process information in different states, such as hunger or arousal. Here we describe a genetically based method to map, in an unbiased and brain-wide manner, sites of neuromodulation under different conditions in the Drosophila brain. This method, and genetic perturbations, reveal that the well-known effect of hunger to enhance behavioral sensitivity to sugar is mediated, at least in part, by the release of dopamine onto primary gustatory sensory neurons, which enhances sugar-evoked calcium influx. These data reinforce the concept that sensory neurons constitute an important locus for state-dependent gain control of behavior and introduce a methodology that can be extended to other neuromodulators and model organisms.

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.

The genetic design of signaling cascades to record receptor activation.

We have developed an experimental strategy to monitor protein interactions in a cell with a high degree of selectivity and sensitivity. A transcription factor is tethered to a membrane-bound receptor with a linker that contains a cleavage site for a specific protease. Activation of the receptor recruits a signaling protein fused to the protease that then cleaves and releases the transcription factor to activate reporter genes in the nucleus. This strategy converts a transient interaction into a stable and amplifiable reporter gene signal to record the activation of a receptor without interference from endogenous signaling pathways. We have developed this assay for three classes of receptors: G protein-coupled receptors, receptor tyrosine kinases, and steroid hormone receptors. Finally, we use the assay to identify a ligand for the orphan receptor GPR1, suggesting a role for this receptor in the regulation of inflammation.