Summary Environmental humidity influences the fitness and geographic distribution of all animals [1]. Insects in particular use humidity cues to navigate the environment, and previous work suggests the existence of specific sensory mechanisms to detect favorable humidity ranges [2–5]. Yet, the molecular and cellular basis of humidity sensing (hygrosensation) remains poorly understood. Here, we describe genes and neurons necessary for hygrosensation in the vinegar fly Drosophila melanogaster. We find that members of the Drosophila genus display species-specific humidity preferences related to conditions in their native habitats. Using a simple behavioral assay, we find that the ionotropic receptors IR40a, IR93a and IR25a are all required for humidity preference in D. melanogaster. Yet, while IR40a is selectively required for hygrosensory responses, IR93a and IR25a mediate both humidity and temperature preference. Consistent with this, the expression of IR93a and IR25a includes thermosensory neurons of the arista. In contrast, IR40a is excluded from the arista, but expressed (and required) in specialized neurons innervating pore-less sensilla of the sacculus, a unique invagination of the third antennal segment. Indeed, calcium imaging shows that IR40a neurons directly respond to changes in humidity, and IR40a knockdown or IR93a mutation reduced their responses to stimuli. Taken together, our results suggest that the preference for a specific humidity range depends on specialized sacculus neurons, and that the processing of environmental humidity can happen largely in parallel to that of temperature.

Determining the pattern of activity of individual connections within a neural circuit could provide insights into the computational processes that underlie brain function. Here, we develop new strategies to label active synapses by trans-synaptic fluorescence complementation in Drosophila. First, we demonstrate that a synaptobrevin-GRASP chimera functions as a powerful activity-dependent marker for synapses in vivo. Next, we create cyan and yellow variants, achieving activity-dependent, multi-colour fluorescence reconstitution across synapses (X-RASP). Our system allows for the first time retrospective labelling of synapses (rather than whole neurons) based on their activity, in multiple colours, in the same animal. As individual synapses often act as computational units in the brain, our method will promote the design of experiments that are not possible using existing techniques. Moreover, our strategies are easily adaptable to circuit mapping in any genetic system.

Summary 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 while 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 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

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 flies (Drosophila) are also defective in noxious heat responses. Unexpectedly, we find that either planarian or human TRPA1 can restore noxious heat avoidance to TRPA1 mutant Drosophila, even though neither is directly activated by heat. Instead, our data suggest that TRPA1 activation is mediated by H2O2/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.

bNeuronally coexpressed ELAV/Hu proteins comprise a family of highly related RNA binding proteins which bind to very similar cognate sequences. How this redundancy is linked to in vivo function and how gene-specific regulation is achieved have not been clear. Analysis of mutants in Drosophila ELAV/Hu family proteins ELAV, FNE, and RBP9 and of genetic interactions among them indicates that they have mostly independent roles in neuronal development and function but have converging roles in the regulation of synaptic plasticity. Conversely, ELAV, FNE, RBP9, and human HuR bind ELAV target RNA in vitro with similar affinities. Likewise, all can regulate alternative splicing of ELAV target genes in nonneuronal wing disc cells and substitute for ELAV in eye development upon artificially increased expression; they can also substantially restore ELAV's biological functions when expressed under the control of the elav gene. Furthermore, ELAV-related Sex-lethal can regulate ELAV targets, and ELAV/Hu proteins can interfere with sexual differentiation. An ancient relationship to Sex-lethal is revealed by gonadal expression of RBP9, providing a maternal fail-safe for dosage compensation. Our results indicate that highly related ELAV/Hu RNA binding proteins select targets for mRNA processing through alteration of their expression levels and subcellular localization but only minimally by altered RNA binding specificity. RNA binding proteins (RBPs) are key regulators of gene expression. Through regulation of alternative splicing and polyadenylation, they expand the proteome and control spatiotemporal expression by affecting mRNA transport, turnover, localization, and translatability (1, 2). In the brain, alternative mRNA processing is particularly abundant and substantially contributes to the complexity of this organ (3, 4). Many RBPs comprise highly related gene families, but they seem to discriminate only marginally between short cognate binding sequences (5). Although redundancy can be evolutionarily stable over extended periods of time (6), it is not clear if highly related RBPs act redundantly in vivo, regulating mostly the same genes in the same biological processes, or if they have diverged such that they regulate genes involved in different biological processes. Detailed analysis of the functions of highly related RBPs in animal models is required to decipher the underlying mechanisms of how highly related RBPs achieve target specificity.ELAV (embryonic-lethal abnormal visual system)/Hu proteins comprise a family of RBPs broadly coexpressed in the nervous system and widely used neuronal markers (7,8). ELAV/Hu proteins are prototype RBPs which harbor three highly conserved RNA recognition motifs (RRMs), whereby the first two RRMs are arranged in tandem and the third RRM is separated by a less conserved hinge region. Humans have four ELAV/Hu protein-encoding genes (HuB, HuC, HuD, and HuR), while Drosophila has three (elav, fne, and Rbp9), which derive from a common ancestor but have duplicated independently in vertebrates ...