Rapid threat assessment in the Drosophila thermosensory system.

Neurons that participate in sensory processing often display "ON" responses, i.e., fire transiently at the onset of a stimulus. ON transients are widespread, perhaps universal to sensory coding, yet their function is not always well-understood. Here, we show that ON responses in the Drosophila thermosensory system extrapolate the trajectory of temperature change, priming escape behavior if unsafe thermal conditions are imminent. First, we show that second-order thermosensory projection neurons (TPN-IIIs) and their Lateral Horn targets (TLHONs), display ON responses to thermal stimuli, independent of direction of change (heating or cooling) and of absolute temperature. Instead, they track the rate of temperature change, with TLHONs firing exclusively to rapid changes (>0.2 °C/s). Next, we use connectomics to track TLHONs' output to descending neurons that control walking and escape, and modeling and genetic silencing to demonstrate how ON transients can flexibly amplify aversive responses to small thermal change. Our results suggest that, across sensory systems, ON transients may represent a general mechanism to systematically anticipate and respond to salient or dangerous conditions.

A thermometer circuit for hot temperature adjusts Drosophila behavior to persistent heat.

Small poikilotherms such as the fruit fly Drosophila depend on absolute temperature measurements to identify external conditions that are above (hot) or below (cold) their preferred range and to react accordingly. Hot and cold temperatures have a different impact on fly activity and sleep, but the circuits and mechanisms that adjust behavior to specific thermal conditions are not well understood. Here, we use patch-clamp electrophysiology to show that internal thermosensory neurons located within the fly head capsule (the AC neurons1) function as a thermometer active in the hot range. ACs exhibit sustained firing rates that scale with absolute temperature-but only for temperatures above the fly's preferred ∼25°C (i.e., "hot" temperature). We identify ACs in the fly brain connectome and demonstrate that they target a single class of circadian neurons, the LPNs.2 LPNs receive excitatory drive from ACs and respond robustly to hot stimuli, but their responses do not exclusively rely on ACs. Instead, LPNs receive independent drive from thermosensory neurons of the fly antenna via a new class of second-order projection neurons (TPN-IV). Finally, we show that silencing LPNs blocks the restructuring of daytime "siesta" sleep, which normally occurs in response to persistent heat. Our previous work described a distinct thermometer circuit for cold temperature.3 Together, the results demonstrate that the fly nervous system separately encodes and relays absolute hot and cold temperature information, show how patterns of sleep and activity can be adapted to specific temperature conditions, and illustrate how persistent drive from sensory pathways can impact behavior on extended temporal scales.

Robustness and plasticity in Drosophila heat avoidance.

Simple innate behavior is often described as hard-wired and largely inflexible. Here, we show that the avoidance of hot temperature, a simple innate behavior, contains unexpected plasticity in Drosophila. First, we demonstrate that hot receptor neurons of the antenna and their molecular heat sensor, Gr28B.d, are essential for flies to produce escape turns away from heat. High-resolution fly tracking combined with a 3D simulation of the thermal environment shows that, in steep thermal gradients, the direction of escape turns is determined by minute temperature differences between the antennae (0.1°-1 °C). In parallel, live calcium imaging confirms that such small stimuli reliably activate both peripheral thermosensory neurons and central circuits. Next, based on our measurements, we evolve a fly/vehicle model with two symmetrical sensors and motors (a "Braitenberg vehicle") which closely approximates basic fly thermotaxis. Critical differences between real flies and the hard-wired vehicle reveal that fly heat avoidance involves decision-making, relies on rapid learning, and is robust to new conditions, features generally associated with more complex behavior.

The irritant receptor TRPA1 mediates the mosquito repellent effect of catnip.

Catnip (Nepeta cataria) is a common garden herb well known for its euphoric and hallucinogenic effects on domestic cats,1-3 for its medicinal properties,4,5 as well as for its powerful repellent action on insects.6,7 Catnip extracts have been proposed as a natural alternative to synthetic insect repellents, such as N,N-diethyl-3-methylbenzamide (DEET),8,9 but how catnip triggers aversion in insects is not known. Here, we show that, both in Drosophila melanogaster flies and Aedes aegypti mosquitoes, the major mediator of catnip repellency is the widely conserved chemical irritant receptor TRPA1. In vitro, both catnip extract and its active ingredient nepetalactone can directly activate fly and mosquito TRPA1. In vivo, D. melanogaster and Ae. aegypti TRPA1 mutants are no longer repelled by catnip and nepetalactone. Interestingly, our data show that some, but not all, fly and mosquito TRPA1 variants are catnip targets. Moreover, unlike the broad TRPA1 agonist allyl isothiocyanate (AITC) (an active ingredient of tear gas and wasabi), catnip does not activate human TRPA1. Our results support the use of catnip and nepetalactone as insect-selective irritants and suggest that, despite TRPA1's broad conservation, insect TRPA1 can be targeted for the development of safe repellents.

A Circuit Encoding Absolute Cold Temperature in Drosophila.

Animals react to environmental changes over timescales ranging from seconds to days and weeks. An important question is how sensory stimuli are parsed into neural signals operating over such diverse temporal scales. Here, we uncover a specialized circuit, from sensory neurons to higher brain centers, that processes information about long-lasting, absolute cold temperature in Drosophila. We identify second-order thermosensory projection neurons (TPN-IIs) exhibiting sustained firing that scales with absolute temperature. Strikingly, this activity only appears below the species-specific, preferred temperature for D. melanogaster (∼25°C). We trace the inputs and outputs of TPN-IIs and find that they are embedded in a cold "thermometer" circuit that provides powerful and persistent inhibition to brain centers involved in regulating sleep and activity. Our results demonstrate that the fly nervous system selectively encodes and relays absolute temperature information and illustrate a sensory mechanism that allows animals to adapt behavior specifically to cold conditions on the timescale of hours to days.

µChIP-Seq for Genome-Wide Mapping of In Vivo TF-DNA Interactions in Arabidopsis Root Protoplasts.

Chromatin immunoprecipitation (ChIP) is a widely used method to map the position of DNA-binding proteins such as histones and transcription factors (TFs) upon their interaction with particular regions of the genome. To examine the genomic distribution of a TF in specific cell types in response to a change in nitrogen concentration, we developed a micro-ChIP (µChIP) protocol that requires only ~5000 Arabidopsis cells transiently expressing the Arabidopsis TF Basic Leucine Zipper 1 (bZIP1) fused to the glucocorticoid receptor (GR) domain that mediates nuclear import in the presence of dexamethasone. The DNA fragments obtained from the immunoprecipitation of bZIP1-DNA complexes were analyzed by next-generation sequencing (ChIP-seq), which helped uncover genome-wide associations between a bZIP1 and its targets in plant cells upon fluctuations in nitrogen availability.

Activation of planarian TRPA1 by reactive oxygen species reveals a conserved mechanism for animal nociception.

All animals must detect noxious stimuli to initiate protective behavior, but the evolutionary origin of nociceptive systems is not well understood. Here we show that noxious heat and irritant chemicals elicit robust escape behaviors in the planarian Schmidtea mediterranea and that the conserved ion channel TRPA1 is required for these responses. TRPA1-mutant Drosophila flies are also defective in noxious-heat responses. We find that either planarian or human TRPA1 can restore noxious-heat avoidance to TRPA1-mutant Drosophila, although neither is directly activated by heat. Instead, our data suggest that TRPA1 activation is mediated by H2O2 and reactive oxygen species, early markers of tissue damage rapidly produced as a result of heat exposure. Together, our data reveal a core function for TRPA1 in noxious heat transduction, demonstrate its conservation from planarians to humans, and imply that animal nociceptive systems may share a common ancestry, tracing back to a progenitor that lived more than 500 million years ago.

Early Integration of Temperature and Humidity Stimuli in the Drosophila Brain.

The Drosophila antenna contains receptor neurons for mechanical, olfactory, thermal, and humidity stimuli. Neurons expressing the ionotropic receptor IR40a have been implicated in the selection of an appropriate humidity range [1, 2], but although previous work indicates that insect hygroreceptors may be made up by a "triad" of neurons (with a dry-, a cold-, and a humid-air-responding cell [3]), IR40a expression included only cold- and dry-air cells. Here, we report the identification of the humid-responding neuron that completes the hygrosensory triad in the Drosophila antenna. This cell type expresses the Ir68a gene, and Ir68a mutation perturbs humidity preference. Next, we follow the projections of Ir68a neurons to the brain and show that they form a distinct glomerulus in the posterior antennal lobe (PAL). In the PAL, a simple sensory map represents related features of the external environment with adjacent "hot," "cold," "dry," and "humid" glomeruli-an organization that allows for both unique and combinatorial sampling by central relay neurons. Indeed, flies avoided dry heat more robustly than humid heat, and this modulation was abolished by silencing of dry-air receptors. Consistently, at least one projection neuron type received direct synaptic input from both temperature and dry-air glomeruli. Our results further our understanding of humidity sensing in the Drosophila antenna, uncover a neuronal substrate for early sensory integration of temperature and humidity in the brain, and illustrate the logic of how ethologically relevant combinations of sensory cues can be processed together to produce adaptive behavioral responses.

The Dehydratase ADT3 Affects ROS Homeostasis and Cotyledon Development.

During the transition from seed to seedling, emerging embryos strategically balance available resources between building up defenses against environmental threats and initiating the developmental program that promotes the switch to autotrophy. We present evidence of a critical role for the phenylalanine (Phe) biosynthetic activity of AROGENATE DEHYDRATASE3 (ADT3) in coordinating reactive oxygen species (ROS) homeostasis and cotyledon development in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. We show that ADT3 is expressed in the cotyledon and shoot apical meristem, mainly in the cytosol, and that the epidermis of adt3 cotyledons contains higher levels of ROS Genome-wide proteomics of the adt3 mutant revealed a general down-regulation of plastidic proteins and ROS-scavenging enzymes, corroborating the hypothesis that the ADT3 supply of Phe is required to control ROS concentration and distribution to protect cellular components. In addition, loss of ADT3 disrupts cotyledon epidermal patterning by affecting the number and expansion of pavement cells and stomata cell fate specification; we also observed severe alterations in mesophyll cells, which lack oil bodies and normal plastids. Interestingly, up-regulation of the pathway leading to cuticle production is accompanied by an abnormal cuticle structure and/or deposition in the adt3 mutant. Such impairment results in an increase in cell permeability and provides a link to understand the cell defects in the adt3 cotyledon epidermis. We suggest an additional role of Phe in supplying nutrients to the young seedling.

"Hit-and-Run" leaves its mark: catalyst transcription factors and chromatin modification.

Understanding how transcription factor (TF) binding is related to gene regulation is a moving target. We recently uncovered genome-wide evidence for a "Hit-and-Run" model of transcription. In this model, a master TF "hits" a target promoter to initiate a rapid response to a signal. As the "hit" is transient, the model invokes recruitment of partner TFs to sustain transcription over time. Following the "run", the master TF "hits" other targets to propagate the response genome-wide. As such, a TF may act as a "catalyst" to mount a broad and acute response in cells that first sense the signal, while the recruited TF partners promote long-term adaptive behavior in the whole organism. This "Hit-and-Run" model likely has broad relevance, as TF perturbation studies across eukaryotes show small overlaps between TF-regulated and TF-bound genes, implicating transient TF-target binding. Here, we explore this "Hit-and-Run" model to suggest molecular mechanisms and its biological relevance.

Hit-and-run transcriptional control by bZIP1 mediates rapid nutrient signaling in Arabidopsis.

The dynamic nature of gene regulatory networks allows cells to rapidly respond to environmental change. However, the underlying temporal connections are missed, even in kinetic studies, as transcription factor (TF) binding within at least one time point is required to identify primary targets. The TF-regulated but unbound genes are dismissed as secondary targets. Instead, we report that these genes comprise transient TF-target interactions most relevant to rapid signal transduction. We temporally perturbed a master TF (Basic Leucine Zipper 1, bZIP1) and the nitrogen (N) signal it transduces and integrated TF regulation and binding data from the same cell samples. Our enabling approach could identify primary TF targets based solely on gene regulation, in the absence of TF binding. We uncovered three classes of primary TF targets: (i) poised (TF-bound but not TF-regulated), (ii) stable (TF-bound and TF-regulated), and (iii) transient (TF-regulated but not TF-bound), the largest class. Unexpectedly, the transient bZIP1 targets are uniquely relevant to rapid N signaling in planta, enriched in dynamic N-responsive genes, and regulated by TF and N signal interactions. These transient targets include early N responders nitrate transporter 2.1 and NIN-like protein 3, bound by bZIP1 at 1-5 min, but not at later time points following TF perturbation. Moreover, promoters of these transient targets are uniquely enriched with cis-regulatory motifs coinherited with bZIP1 binding sites, suggesting a recruitment role for bZIP1. This transient mode of TF action supports a classic, but forgotten, "hit-and-run" transcription model, which enables a "catalyst TF" to activate a large set of targets within minutes of signal perturbation.

Arabidopsis thaliana TERMINAL FLOWER2 is involved in light-controlled signalling during seedling photomorphogenesis.

Plants respond to changes in the environment by altering their growth pattern. Light is one of the most important environmental cues and affects plants throughout the life cycle. It is perceived by photoreceptors such as phytochromes that absorb light of red and far-red wavelengths and control, for example, seedling de-etiolation, chlorophyll biosynthesis and shade avoidance response. We report that the terminal flower2 (tfl2) mutant, carrying a mutation in the Arabidopsis thaliana HETEROCHROMATIN PROTEIN1 homolog, functions in negative regulation of phytochrome dependent light signalling. tfl2 shows defects in both hypocotyl elongation and shade avoidance response. Double mutant analysis indicates that mutants of the red/far-red light absorbing phytochrome family of plant photoreceptors, phyA and phyB, are epistatic to tfl2 in far-red and red light, respectively. An overlap between genes regulated by light and by auxin has earlier been reported and, in tfl2 plants light-dependent auxin-regulated genes are misexpressed. Further, we show that TFL2 binds to IAA5 and IAA19 suggesting that TFL2 might be involved in regulation of phytochrome-mediated light responses through auxin action.

A functional genomics approach reveals CHE as a component of the Arabidopsis circadian clock.

Transcriptional feedback loops constitute the molecular circuitry of the plant circadian clock. In Arabidopsis, a core loop is established between CCA1 and TOC1. Although CCA1 directly represses TOC1, the TOC1 protein has no DNA binding domains, which suggests that it cannot directly regulate CCA1. We established a functional genomic strategy that led to the identification of CHE, a TCP transcription factor that binds specifically to the CCA1 promoter. CHE is a clock component partially redundant with LHY in the repression of CCA1. The expression of CHE is regulated by CCA1, thus adding a CCA1/CHE feedback loop to the Arabidopsis circadian network. Because CHE and TOC1 interact, and CHE binds to the CCA1 promoter, a molecular linkage between TOC1 and CCA1 gene regulation is established.

Dictyostelium Dock180-related RacGEFs regulate the actin cytoskeleton during cell motility.

Cell motility of amoeboid cells is mediated by localized F-actin polymerization that drives the extension of membrane protrusions to promote forward movements. We show that deletion of either of two members of the Dictyostelium Dock180 family of RacGEFs, DockA and DockD, causes decreased speed of chemotaxing cells. The phenotype is enhanced in the double mutant and expression of DockA or DockD complements the reduced speed of randomly moving DockD null cells' phenotype, suggesting that DockA and DockD are likely to act redundantly and to have similar functions in regulating cell movement. In this regard, we find that overexpressing DockD causes increased cell speed by enhancing F-actin polymerization at the sites of pseudopod extension. DockD localizes to the cell cortex upon chemoattractant stimulation and at the leading edge of migrating cells and this localization is dependent on PI3K activity, suggesting that DockD might be part of the pathway that links PtdIns(3,4,5)P(3) production to F-actin polymerization. Using a proteomic approach, we found that DdELMO1 is associated with DockD and that Rac1A and RacC are possible in vivo DockD substrates. In conclusion, our work provides a further understanding of how cell motility is controlled and provides evidence that the molecular mechanism underlying Dock180-related protein function is evolutionarily conserved.

PRR3 Is a vascular regulator of TOC1 stability in the Arabidopsis circadian clock.

The pseudoresponse regulators (PRRs) participate in the progression of the circadian clock in Arabidopsis thaliana. The founding member of the family, TIMING OF CAB EXPRESSION1 (TOC1), is an essential component of the transcriptional network that constitutes the core mechanism of the circadian oscillator. Recent data suggest a role in circadian regulation for all five members of the PRR family; however, the molecular function of TOC1 or any other PRRs remains unknown. In this work, we present evidence for the involvement of PRR3 in the regulation of TOC1 protein stability. PRR3 was temporally coexpressed with TOC1 under different photoperiods, yet its tissue expression was only partially overlapping with that of TOC1, as PRR3 appeared restricted to the vasculature. Decreased expression of PRR3 resulted in reduced levels of TOC1 protein, while overexpression of PRR3 caused an increase in the levels of TOC1, all without affecting the amount of TOC1 transcript. PRR3 was able to bind to TOC1 in yeast and in plants and to perturb TOC1 interaction with ZEITLUPE (ZTL), which targets TOC1 for proteasome-dependent degradation. Together, our results indicate that PRR3 might function to modulate TOC1 stability by hindering ZTL-dependent TOC1 degradation, suggesting the existence of local regulators of clock activity and adding to the growing importance of posttranslational regulation in the design of circadian timing mechanisms in plants.

The pleiotropic mutation dar1 affects plant architecture in Arabidopsis thaliana.

Shoot architecture is shaped upon the organogenic activity of the shoot apical meristem (SAM). Such an activity relies on the balance between the maintenance of a population of undifferentiated cells in the centre of the SAM and the recruitment of organ founder cells at the periphery. A novel mutation in Arabidopsis thaliana, distorted architecture1 (dar1), is characterised by disturbed phyllotaxy of the inflorescence and consumption of the apical meristem late in development. SEM and light microscopy analyses of the dar1 SAM reveal an abnormal partitioning of meristematic domains, and mutations known to affect the SAM structure and function were found to interact with dar1. Moreover, the mutant shows an alteration of the root apical meristem (RAM) structure. Those observations support the hypothesis that DAR1 has a role in meristem maintenance and it is required for the normal development of Arabidopsis inflorescence during plant life.