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The advent of CRISPR/Cas9-mediated genome editing has expanded the range of animals amenable to targeted genetic analysis. This has accelerated research in animals not traditionally studied using molecular genetics. However, studying genes essential for reproduction or survival in such animals remains challenging, as they lack the tools that aid genetic analysis in traditional genetic model organisms. We recently introduced the use of distinguishably marked knock-in pairs (DMKPs) as a strategy for rapid and reliable genotyping in such species. Here we show that DMKPs also facilitate the maintenance and study of mutations that cannot be maintained in a homozygous state, a group which includes recessive lethal and sterile mutations. Using DMKPs, we disrupt the zero population growth locus in Drosophila melanogaster and in the dengue vector mosquito Aedes aegypti. In both species, DMKPs enable the maintenance of zero population growth mutant strains and the reliable recovery of zero population growth mutant animals. Male and female gonad development is disrupted in fly and mosquito zero population growth mutants, rendering both sexes sterile. In Ae. aegypti, zero population growth mutant males remain capable of inducing a mating refractory period in wild-type females and of competing with wild-type males for mates, properties compatible with zero population growth serving as a target in mosquito population suppression strategies. DMKP is readily generalizable to other species amenable to CRISPR/Cas9-mediated gene targeting, and should facilitate the study of sterile and lethal mutations in multiple organisms not traditionally studied using molecular genetics.

Mosquito-borne diseases sicken >500,000,000 people annually, killing >500,0001. Mosquito host-seeking is guided by multiple host-associated cues, which combine to drive blood feeding in a manner that remains poorly understood2,3. While heat is a powerful mosquito attractant, recent studies indicate that disruption of heat seeking has little impact on host detection by the malaria vector Anopheles gambiae4, suggesting other cues act alongside heat in the complex sensory environment of a human host. Here we show mosquitoes require Ir93a (an Ionotropic Receptor5) to maintain attraction to a human host and feed on warmed blood. Using Ir93a, we uncover the previously uncharacterized mosquito hygrosensory system, and show Ir93a is required for humidity detection by humidity sensors (hygrosensors) as well as temperature detection by thermosensors, and for attraction to each stimulus. These findings indicate that hygrosensation and thermosensation function in parallel, driving host proximity detection in response to the overlapping heat and humidity gradients humans produce6,7. These host cue sensors appear to have arisen by co-opting existing sensors of physical cues rather than de novo, as Ir93a-dependent thermo- and hygro-sensors support physiological homeostasis in non-blood-feeding insects8–11. While Ir93a is conserved among arthropods, reliance on heat and humidity evolved independently in multiple blood-feeding lineages, suggesting multiple, vector-specific implementations of this common host-seeking strategy.

Flexible integration of warming and cooling pathways underlies thermal homeostasis in larval Drosophila., Body temperature homeostasis is essential and reliant upon the integration of outputs from multiple classes of cooling- and warming-responsive cells. The computations that integrate these outputs are not understood. Here, we discover a set of warming cells (WCs) and show that the outputs of these WCs combine with previously described cooling cells (CCs) in a cross-inhibition computation to drive thermal homeostasis in larval Drosophila. WCs and CCs detect temperature changes using overlapping combinations of ionotropic receptors: Ir68a, Ir93a, and Ir25a for WCs and Ir21a, Ir93a, and Ir25a for CCs. WCs mediate avoidance to warming while cross-inhibiting avoidance to cooling, and CCs mediate avoidance to cooling while cross-inhibiting avoidance to warming. Ambient temperature–dependent regulation of the strength of WC- and CC-mediated cross-inhibition keeps larvae near their homeostatic set point. Using neurophysiology, quantitative behavioral analysis, and connectomics, we demonstrate how flexible integration between warming and cooling pathways can orchestrate homeostatic thermoregulation.

Dietary magnesium (MgThe proverbial saying ‘you are what you eat’ perfectly summarizes the concept that our diet can influence both our mental and physical health. We know that foods that are good for the heart, such as nuts, oily fish and berries, are also good for the brain. We know too that vitamins and minerals are essential for overall good health. But is there any evidence that increasing your intake of specific vitamins or minerals could help boost your brain power? While it might sound almost too good to be true, there is some evidence that this is the case for at least one mineral, magnesium. Studies in rodents have shown that adding magnesium supplements to food improves how well the animals perform on memory tasks. Both young and old animals benefit from additional magnesium. Even elderly rodents with a condition similar to Alzheimer’s disease show less memory loss when given magnesium supplements. But what about other species? Wu et al. now show that magnesium supplements also boost memory performance in fruit flies. One group of flies was fed with standard cornmeal for several days, while the other group received cornmeal supplemented with magnesium. Both groups were then trained to associate an odor with a food reward. Flies that had received the extra magnesium showed better memory for the odor when tested 24 hours after training. Wu et al. show that magnesium improves memory in the flies via a different mechanism to that reported previously for rodents. In rodents, magnesium increased levels of a receptor protein for a brain chemical called glutamate. In fruit flies, by contrast, the memory boost depended on a protein that transports magnesium out of neurons. Mutant flies that lacked this transporter showed memory impairments. Unlike normal flies, those without the transporter showed no memory improvement after eating magnesium-enriched food. The results suggest that the transporter may help adjust magnesium levels inside brain cells in response to neural activity. Humans produce four variants of this magnesium transporter, each encoded by a different gene. One of these transporters has already been implicated in brain development. The findings of Wu et al. suggest that the transporters may also act in the adult brain to influence cognition. Further studies are needed to test whether targeting the magnesium transporter could ultimately hold promise for treating memory impairments.

Body temperature homeostasis is an essential function that relies upon the integration of the outputs from multiple classes of cooling- and warming-responsive cells. The computations that integrate these diverse outputs to control body temperature are not understood. Here we discover a new set of Warming Cells (WCs), and show that the outputs of these WCs and previously described Cooling Cells (CCs1) are combined in a cross-inhibition computation to drive thermal homeostasis in larval Drosophila. We find that WCs and CCs are opponent sensors that operate in synchrony above, below, and near the homeostatic set-point, with WCs consistently activated by warming and inhibited by cooling, and CCs the converse. Molecularly, these opponent sensors rely on overlapping combinations of Ionotropic Receptors to detect temperature changes: Ir68a, Ir93a, and Ir25a for WCs; Ir21a, Ir93a, and Ir25a for CCs. Using a combination of optogenetics, sensory receptor mutants, and quantitative behavioral analysis, we find that the larva uses flexible cross-inhibition of WC and CC outputs to locate and stay near the homeostatic set-point. Balanced cross-inhibition near the set-point suppresses any directed movement along temperature gradients. Above the set-point, WCs mediate avoidance to warming while cross-inhibiting avoidance to cooling. Below the set-point, CCs mediate avoidance to cooling while cross-inhibiting avoidance to warming. Our results demonstrate how flexible cross-inhibition between warming and cooling pathways can orchestrate homeostatic thermoregulation.

Summary Animals exhibit innate and learned preferences for temperature and humidity—conditions critical for their survival and reproduction. Leveraging a whole-brain electron microscopy volume, we studied the adult Drosophila melanogaster circuitry associated with antennal thermo- and hygrosensory neurons. We have identified two new target glomeruli in the antennal lobe, in addition to the five known ones, and the ventroposterior projection neurons (VP PNs) that relay thermo- and hygrosensory information to higher brain centers, including the mushroom body and lateral horn, seats of learned and innate behavior. We present the first connectome of a thermo- and hygrosensory neuropil, the lateral accessory calyx (lACA), by reconstructing neurons downstream of heating- and cooling-responsive VP PNs. A few mushroom body-intrinsic neurons solely receive thermosensory input from the lACA, while most receive additional olfactory and thermo- and/or hygrosensory PN inputs. Furthermore, several classes of lACA-associated neurons form a local network with outputs to other brain neuropils, suggesting that the lACA serves as a hub for thermo- and hygrosensory circuitry. For example, DN1a neurons link thermosensory PNs in the lACA to the circadian clock via the accessory medulla. Finally, we survey strongly connected downstream partners of VP PNs across the protocerebrum; these include a descending neuron targeted by dry-responsive VP PNs, meaning that just two synapses might separate hygrosensory inputs from motor circuits. These data provide a comprehensive first- and second-order layer analysis of Drosophila thermo- and hygrosensory systems and an initial survey of third-order neurons that could directly modulate behavior., Graphical Abstract, Highlights • Two novel thermo- and/or hygrosensory glomeruli in the fly antennal lobe • First complete set of thermosensory and hygrosensory projection neurons • First connectome for a thermo- and hygrosensory neuropil • Third-order thermo- and hygrosensory neurons, including link to circadian clock, Marin et al. use connectomics and genetics for comprehensive identification of temperature and humidity sensory neurons in the Drosophila brain. They reconstruct all projections to higher brain areas and select higher-order targets, including the mushroom body lateral accessory calyx, linking thermosensation to memory and the circadian clock.

SUMMARYAnimals exhibit innate and learned preferences for temperature and humidity – conditions critical for their survival and reproduction. Here, we leveraged a whole adult brain electron microscopy volume to study the circuitry associated with antennal thermosensory and hygrosensory neurons, which target specific ventroposterior (VP) glomeruli in theDrosophila melanogasterantennal lobe. We have identified two new VP glomeruli, in addition to the five known ones, and the projection neurons (VP PNs) that relay VP information to higher brain centres, including the mushroom body and lateral horn, seats of learned and innate olfactory behaviours, respectively. Focussing on the mushroom body lateral accessory calyx (lACA), a known thermosensory neuropil, we present a comprehensive connectome by reconstructing neurons downstream of heating- and cooling-responsive VP PNs. We find that a few lACA-associated mushroom body intrinsic neurons (Kenyon cells) solely receive thermosensory inputs, while most receive additional olfactory and thermo- or hygrosensory PN inputs in the main calyx. Unexpectedly, we find several classes of lACA-associated neurons that form a local network with outputs to other brain neuropils, suggesting that the lACA serves as a general hub for thermosensory circuitry. For example, we find DN1 pacemaker neurons that link the lACA to the accessory medulla, likely mediating temperature-based entrainment of the circadian clock. Finally, we survey strongly connected downstream partners of VP PNs across the protocerebrum; these include a descending neuron that receives input mainly from dry-responsive VP PNs, meaning that just two synapses might separate hygrosensory inputs from motor neurons in the nerve cord. (249)HIGHLIGHTSTwo novel thermo/hygrosensory glomeruli in the fly antennal lobeFirst complete set of thermosensory and hygrosensory projection neuronsFirst connectome for a thermosensory centre, the lateral accessory calyxNovel third order neurons, including a link to the circadian clock

Heat seeking is cool Mosquitoes seek hosts using several cues, one of which is body heat. Greppi et al. hypothesized that cooling-activated receptors could be used for locating mammalian hosts if they were rewired downstream for repulsion responses (see the Perspective by Lazzari). A gene family conserved in insects and known to be responsible for sensing changes in temperature in fruit flies was the starting point. Genome-wide analyses and labeled CRISPR-Cas9 mutants allowed visualization of the receptor in neurons of Anopheles gambiae mosquitoes' antennae and assessment of adult female mosquitoes with a disrupted copy of the receptor. This ancestral insect temperature regulatory system has been repurposed for host-finding by malaria mosquitoes. Science , this issue p. 681 ; see also p. 628

How metazoan mechanotransduction channels sense mechanical stimuli is not well understood. NOMPC channel in the transient receptor potential (TRP) family, a mechanotransduction channel for Drosophila touch sensation and hearing, contains 29 Ankyrin repeats (ARs) that associate with microtubules. These ARs have been postulated to act as a tether that conveys force to the channel. Here, we report that these N-terminal ARs form a cytoplasmic domain essential for NOMPC mechanogating in vitro, mechanosensitivity of touch receptor neurons in vivo, and touch-induced behaviors of Drosophila larvae. Duplicating the ARs elongates the filaments that tether NOMPC to microtubules in mechanosensory neurons. Moreover, microtubule association is required for NOMPC mechanogating. Importantly, transferring the NOMPC ARs to mechano-insensitive voltage-gated potassium channels confers mechanosensitivity to the chimeric channels. These experiments strongly support a tether mechanism of mechanogating for the NOMPC channel, providing insights regarding the basis of mechanosensitivity of mechanotransduction channels.

After previously discovering that the ion channel TRPA1 is used as an internal temperature sensor in Drosophila to control the slow response of flies to shallow thermal gradients, the authors show here that the rapid response of flies to steep warming gradients relies on a different protein, GR28B, providing the first example of a thermosensory role for a gustatory receptor. Flies use the TRPA1 ion channel as an internal temperature sensor to slowly adjust their response to shallow thermal gradients. Now Paul Garrity and colleagues show that the rapid response of flies exposed to steep warmth gradients does not require TRPA1, but instead relies on the gustatory receptor GR28B(D), acting in peripheral thermosensing cells. Gustatory receptors have been implicated in taste, olfaction and host-seeking by disease-vector insects, but have not previously been linked with thermosensation. Behavioural responses to temperature are critical for survival, and animals from insects to humans show strong preferences for specific temperatures1,2. Preferred temperature selection promotes avoidance of adverse thermal environments in the short term and maintenance of optimal body temperatures over the long term1,2, but its molecular and cellular basis is largely unknown. Recent studies have generated conflicting views of thermal preference in Drosophila, attributing importance to either internal3 or peripheral4 warmth sensors. Here we reconcile these views by showing that thermal preference is not a singular response, but involves multiple systems relevant in different contexts. We found previously that the transient receptor potential channel TRPA1 acts internally to control the slowly developing preference response of flies exposed to a shallow thermal gradient3. We now find that the rapid response of flies exposed to a steep warmth gradient does not require TRPA1; rather, the gustatory receptor GR28B(D) drives this behaviour through peripheral thermosensors. Gustatory receptors are a large gene family, widely studied in insect gustation and olfaction, and are implicated in host-seeking by insect disease vectors5,6,7, but have not previously been implicated in thermosensation. At the molecular level, GR28B(D) misexpression confers thermosensitivity upon diverse cell types, suggesting that it is a warmth sensor. These data reveal a new type of thermosensory molecule and uncover a functional distinction between peripheral and internal warmth sensors in this tiny ectotherm reminiscent of thermoregulatory systems in larger, endothermic animals2. The use of multiple, distinct molecules to respond to a given temperature, as observed here, may facilitate independent tuning of an animal’s distinct thermosensory responses.

Ionotropic Receptors (IRs) are a large subfamily of variant ionotropic glutamate receptors present across Protostomia. While these receptors are most extensively studied for their roles in chemosensory detection, recent work has implicated two family members, IR21a and IR25a, in thermosensation in Drosophila. Here we characterize one of the most evolutionarily deeply conserved receptors, IR93a, and show that it is co-expressed and functions with IR21a and IR25a to mediate physiological and behavioral responses to cool temperatures. IR93a is also co-expressed with IR25a and a distinct receptor, IR40a, in a discrete population of sensory neurons in the sacculus, a multi-chambered pocket within the antenna. We demonstrate that this combination of receptors is required for neuronal responses to dry air and behavioral discrimination of humidity differences. Our results identify IR93a as a common component of molecularly and cellularly distinct IR pathways important for thermosensation and hygrosensation in insects. DOI: http://dx.doi.org/10.7554/eLife.17879.001

Ionotropic Receptors (IRs) are a large subfamily of variant ionotropic glutamate receptors present across Protostomia. While these receptors are most extensively studied for their roles in chemosensory detection in insects, recent work has implicated two family members, IR21a and IR25a, in thermosensation in Drosophila. Here we characterize one of the most deeply conserved receptors, IR93a, and show that it is co-expressed and functions with IR21a and IR25a to mediate physiological and behavioral responses to cool temperatures. IR93a is also co-expressed with IR25a and a distinct receptor, IR40a, in a discrete population of sensory neurons in the sacculus, a multi-chambered pocket within the antenna. We demonstrate that this combination of receptors is important for neuronal responses to dry air and behavioral discrimination of humidity differences. Our results identify IR93a as a common component of molecularly and cellularly distinct IR pathways underlying thermosensation and hygrosensation in insects.

Our bodies are constantly bombarded by a diversity of environmental stimuli, such as touch, taste, sound, smell, light, etc. To detect and process this broad array of signals, nature has evolved a variety of cellular sensory mechanisms and pathways that interface with the environment and transmit neural signals back to the CNS where they are translated into behavioral decisions. Transient Response Potential (TRP) cation channels were first identified in invertebrates (i.e., Drosophila) and represent a sizeable receptor/channel family in mammals, consisting of 28 individual members grouped into subclasses denoted TRPC, TRPV, TRPM, TRPML, TRPP and TRPA (for a recent review, see ref. 1). Although originally described as ion channels, we now know that many members of the TRP family also function as receptors for a range of stimuli, including temperature, pH, chemical compounds and membrane voltage. In fact, several TRP isoforms display multimodal sensitivity, meaning that they can respond to more than one stimulus. For example, TRPV1, or the capcaisin receptor, displays both thermal and chemical sensitivity, and the two stimuli may act synergistically to increase channel activity. Physiologically, TRP family members are expressed in a variety of sensory afferent nerves that feed environmental information to the CNS, and also in smaller C-type afferent fibers responsible for peripheral pain sensation and transmission. Therapeutically, manipulation of TRP channel activity may represent an effective strategy to treat peripheral pain associated with inflammation and chronic tissue injury.

Environmental temperature strongly affects physiology of ectotherms. Small ectotherms, like Drosophila, cannot endogenously regulate body temperature so must rely on behavior to maintain body temperature within a physiologically permissive range. Here we review what is known about Drosophila thermal preference. Work on thermal behavior in this group is particularly exciting because it provides the opportunity to connect genes to neuromolecular mechanisms to behavior to fitness in the wild.

The ability to sense and respond to subtle variations in environmental temperature is critical for animal survival. Animals avoid temperatures that are too cold or too warm and seek out temperatures favorable for their survival. At the molecular level, members of the transient receptor potential (TRP) family of cation channels contribute to thermosensory behaviors in animals from flies to humans. In Drosophila melanogaster larvae, avoidance of excessively warm temperatures is known to require the TRP protein dTRPA1. Whether larval avoidance of excessively cool temperatures also requires TRP channel function, and whether warm and cool avoidance use the same or distinct TRP channels has been unknown. Here we identify two TRP channels required for cool avoidance, TRPL and TRP. Although TRPL and TRP have previously characterized roles in phototransduction, their function in cool avoidance appears to be distinct, as neither photoreceptor neurons nor the phototransduction regulators NORPA and INAF are required for cool avoidance. TRPL and TRP are required for cool avoidance; however they are dispensable for warm avoidance. Furthermore, cold-activated neurons in the larvae are required for cool but not warm avoidance. Conversely, dTRPA1 is essential for warm avoidance, but not cool avoidance. Taken together, these data demonstrate that warm and cool avoidance in the Drosophila larva involves distinct TRP channels and circuits.

Ptpmeg is a cytoplasmic tyrosine phosphatase containing FERM and PDZ domains. Drosophila Ptpmeg and its vertebrate homologs PTPN3 and PTPN4 are expressed in the nervous system, but their developmental functions have been unknown. We found that ptpmeg is involved in neuronal circuit formation in the Drosophila central brain, regulating both the establishment and the stabilization of axonal projection patterns. In ptpmeg mutants, mushroom body (MB) axon branches are elaborated normally, but the projection patterns in many hemispheres become progressively abnormal as the animals reach adulthood. The two branches of MB alpha/beta neurons are affected by ptpmeg in different ways; ptpmeg activity inhibits alpha lobe branch retraction while preventing beta lobe branch overextension. The phosphatase activity of Ptpmeg is essential for both alpha and beta lobe formation, but the FERM domain is required only for preventing alpha lobe retraction, suggesting that Ptpmeg has distinct roles in regulating the formation of alpha and beta lobes. ptpmeg is also important for the formation of the ellipsoid body (EB), where it influences the pathfinding of EB axons. ptpmeg function in neurons is sufficient to support normal wiring of both the EB and MB. However, ptpmeg does not act in either MB or EB neurons, implicating ptpmeg in the regulation of cell-cell signaling events that control the behavior of these axons.

Animals rely on highly sensitive thermoreceptors to seek out optimal temperatures, but the molecular mechanisms of thermosensing are not well understood. The Dorsal Organ Cool Cells (DOCCs) of the Drosophila larva are a set of exceptionally thermosensitive neurons critical for larval cool avoidance. Here, we show that DOCC cool-sensing is mediated by Ionotropic Receptors (IRs), a family of sensory receptors widely studied in invertebrate chemical sensing. We find that two IRs, IR21a and IR25a, are required to mediate DOCC responses to cooling and are required for cool avoidance behavior. Furthermore, we find that ectopic expression of IR21a can confer cool-responsiveness in an Ir25a-dependent manner, suggesting an instructive role for IR21a in thermosensing. Together, these data show that IR family receptors can function together to mediate thermosensation of exquisite sensitivity. DOI: http://dx.doi.org/10.7554/eLife.13254.001, eLife digest Animals need to be able to sense temperatures for a number of reasons. For example, this ability allows animals to avoid conditions that are either too hot or too cold, and to maintain an optimal body temperature. Most animals detect temperature via nerve cells called thermoreceptors. These sensors are often extremely sensitive and some can even detect changes in temperature of just a few thousandths of a degree per second. However, it is not clear how thermoreceptors detect temperature with such sensitivity, and many of the key molecules involved in this ability are unknown. In 2015, researchers discovered a class of highly sensitive nerve cells that allow fruit fly larvae to navigate away from unfavorably cool temperatures. Now, Ni, Klein et al. – who include some of the researchers involved in the 2015 work – have determined that these nerves use a combination of two receptors to detect cooling. Unexpectedly, these two receptors – Ionotropic Receptors called IR21a and IR25a – had previously been implicated in the detection of chemicals rather than temperature. IR25a was well-known to combine with other related receptors to detect an array of tastes and smells, while IR21a was thought to act in a similar way but had not been associated with detecting any specific chemicals. These findings demonstrate that the combination of IR21a and IR25a detects temperature instead. Together, these findings reveal a new molecular mechanism that underlies an animal’s ability to sense temperature. These findings also raise the possibility that other “orphan” Ionotropic Receptors, which have not been shown to detect any specific chemicals, might actually contribute to sensing temperature instead. Further work will explore this possibility and attempt to uncover precisely how IR21a and IR25a work to detect cool temperatures. DOI: http://dx.doi.org/10.7554/eLife.13254.002

Summary Ambient light affects multiple physiological functions and behaviors, such as circadian rhythms, sleep-wake activities, and development, from flies to mammals [1–6]. Mammals exhibit a higher body temperature when exposed to acute light compared to when they are exposed to the dark, but the underlying mechanisms are largely unknown [7–10]. The body temperature of small ectotherms, such as Drosophila , relies on the temperature of their surrounding environment, and these animals exhibit a robust temperature preference behavior [11–13]. Here, we demonstrate that Drosophila prefer a ∼1° higher temperature when exposed to acute light rather than the dark. This acute light response, light-dependent temperature preference (LDTP), was observed regardless of the time of day, suggesting that LDTP is regulated separately from the circadian clock. However, screening of eye and circadian clock mutants suggests that the circadian clock neurons posterior dorsal neurons 1 (DN1 p s) and Pigment-Dispersing Factor Receptor (PDFR) play a role in LDTP. To further investigate the role of DN1 p s in LDTP, PDFR in DN1 p s was knocked down, resulting in an abnormal LDTP. The phenotype of the pdfr mutant was rescued sufficiently by expressing PDFR in DN1 p s, indicating that PDFR in DN1 p s is responsible for LDTP. These results suggest that light positively influences temperature preference via the circadian clock neurons, DN1 p s, which may result from the integration of light and temperature information. Given that both Drosophila and mammals respond to acute light by increasing their body temperature, the effect of acute light on temperature regulation may be conserved evolutionarily between flies and humans.

Brain morphogenesis depends on the maintenance of boundaries between populations of non-intermingling cells. We used molecular markers to characterize a boundary within the optic lobe of the Drosophila brain and found that Slit and the Robo family of receptors, well-known regulators of axon guidance and neuronal migration, inhibit the mixing of adjacent cell populations in the developing optic lobe. Our data suggest that Slit is needed in the lamina to prevent inappropriate invasion of Robo-expressing neurons from the lobula cortex. We show that Slit protein surrounds lamina glia, while the distal cell neurons in the lobula cortex express all three Drosophila Robos. We examine the function of these proteins in the visual system by isolating a novel allele of slit that preferentially disrupts visual system expression of Slit and by creating transgenic RNA interference flies to inhibit the function of each Drosophila Robo in a tissue-specific fashion. We find that loss of Slit or simultaneous knockdown of Robo, Robo2 and Robo3 causes distal cell neurons to invade the lamina,resulting in cell mixing across the lamina/lobula cortex boundary. This boundary disruption appears to lead to alterations in patterns of axon navigation in the visual system. We propose that Slit and Robo-family proteins act to maintain the distinct cellular composition of the lamina and the lobula cortex.

Cell death plays an essential role in development, and the removal of cell corpses presents an important challenge for the developing organism. Macrophages are largely responsible for the clearance of cell corpses in Drosophila melanogaster and mammalian systems. We have examined the developmental requirement for macrophages in Drosophila and find that macrophage function is essential for central nervous system (CNS)morphogenesis. We generate and analyze mutations in the Pvr locus,which encodes a receptor tyrosine kinase of the PDGF/VEGF family that is required for hemocyte migration. We find that loss of Pvr function causes the mispositioning of glia within the CNS and the disruption of the CNS axon scaffold. We further find that inhibition of hemocyte development or of Croquemort, a receptor required for macrophage-mediated corpse engulfment,causes similar CNS defects. These data indicate that macrophage-mediated clearance of cell corpses is required for proper morphogenesis of the Drosophila CNS.

The neuronal wiring of the Drosophila melanogaster visual system is constructed through an intricate series of cell–cell interactions. Recent studies have identified some of the gene regulatory and cytoskeletal signaling pathways responsible for the layer-specific targeting of Drosophila photoreceptor axons. Target selection decisions of the R1–R6 subset of photoreceptor axons have been found to be influenced by the nuclear factors Brakeless and Runt, and target selection decisions of the R7 subset of axons have been found to require the cell-surface proteins Ptp69d, Lar and N-cadherin. A role for the visual system glia in orienting photoreceptor axon outgrowth and target selection has also been uncovered.

Acetic acid produces an irritating sensation that can be attributed to activation of nociceptors within the trigeminal ganglion that innervate the nasal or oral cavities. These sensory neurons sense a diverse array of noxious agents in the environment, allowing animals to actively avoid tissue damage. Although receptor mechanisms have been identified for many noxious chemicals, the mechanisms by which animals detect weak acids, such as acetic acid, are less well understood. Weak acids are only partially dissociated at neutral pH and, as such, some can cross the cell membrane, acidifying the cell cytosol. The nociceptor ion channel TRPA1 is activated by CO(2), through gating of the channel by intracellular protons, making it a candidate to more generally mediate sensory responses to weak acids. To test this possibility, we measured responses to weak acids from heterologously expressed TRPA1 channels and trigeminal neurons with patch clamp recording and Ca(2+) microfluorometry. Our results show that heterologously expressed TRPA1 currents can be induced by a series of weak organic acids, including acetic, propionic, formic, and lactic acid, but not by strong acids. Notably, the degree of channel activation was predicted by the degree of intracellular acidification produced by each acid, suggesting that intracellular protons are the proximate stimulus that gates the channel. Responses to weak acids produced a Ca(2+)-independent inactivation that precluded further activation by weak acids or reactive chemicals, whereas preactivation by reactive electrophiles sensitized TRPA1 channels to weak acids. Importantly, responses of trigeminal neurons to weak acids were highly overrepresented in the subpopulation of TRPA1-expressing neurons and were severely reduced in neurons from TRPA1 knockout mice. We conclude that TRPA1 is a general sensor for weak acids that produce intracellular acidification and suggest that it functions within the pain pathway to mediate sensitivity to cellular acidosis.

Complex animal behaviors are built from dynamical relationships between sensory inputs, neuronal activity, and motor outputs in patterns with strategic value. Connecting these patterns illuminates how nervous systems compute behavior. Here, we study Drosophila larva navigation up temperature gradients toward preferred temperatures (positive thermotaxis). By tracking the movements of animals responding to fixed spatial temperature gradients or random temperature fluctuations, we calculate the sensitivity and dynamics of the conversion of thermosensory inputs into motor responses. We discover three thermosensory neurons in each dorsal organ ganglion (DOG) that are required for positive thermotaxis. Random optogenetic stimulation of the DOG thermosensory neurons evokes behavioral patterns that mimic the response to temperature variations. In vivo calcium and voltage imaging reveals that the DOG thermosensory neurons exhibit activity patterns with sensitivity and dynamics matched to the behavioral response. Temporal processing of temperature variations carried out by the DOG thermosensory neurons emerges in distinct motor responses during thermotaxis.

Animals use thermosensory systems to achieve optimal temperatures for growth and reproduction and to avoid damaging extremes. Thermoregulation is particularly challenging for small animals like the fruit fly Drosophila melanogaster, whose body temperature rapidly changes in response to environmental temperature fluctuation. Recent work has uncovered some of the key molecules mediating fly thermosensation, including the Transient Receptor Potential (TRP) channels TRPA1 and Painless, and the Gustatory Receptor Gr28b, an unanticipated thermosensory regulator normally associated with a different sensory modality. There is also evidence the Drosophila phototransduction cascade may have some role in thermosensory responses. Together, the fly's diverse thermosensory molecules act in an array of functionally distinct thermosensory neurons to drive a suite of complex, and often exceptionally thermosensitive, behaviors.

From honeybee foraging to bird migration, the orientation of animals in their environments is vital for survival and provides opportunities for studying the neural mechanisms that underlie the perception and processing of sensory information. Temperature is a ubiquitous environmental variable that

Mutations in the Drosophila gene dreadlocks ( dock ) disrupt photoreceptor cell (R cell) axon guidance and targeting. Genetic mosaic analysis and cell-type-specific expression of dock transgenes demonstrate dock is required in R cells for proper innervation. Dock protein contains one SH2 and three SH3 domains, implicating it in tyrosine kinase signaling, and is highly related to the human proto-oncogene Nck . Dock expression is detected in R cell growth cones in the target region. We propose Dock transmits signals in the growth cone in response to guidance and targeting cues. These findings provide an important step for dissection of signaling pathways regulating growth cone motility.

Imaging a fluorophore in a living tissue presents several unique problems. The fluorescence from the labeled cell(s) may be weak, the labeled cells may be buried deep within tissue and the presence of a fluorophore may render the cells photo-sensitive. Two-photon laser-scanning microscopy (TPLSM) offers several advantages in meeting these challenges. We show that TPLSM provides greater sensitivity, better resolution and less photo-bleaching, as compared to confocal laser-scanning microscopy. The dramatically reduced photo-bleaching makes it possible to image cells continuously for long periods of time. Therefore, TPLSM allows a safer and higher-resolution means of imaging living cells labeled with a variety of fluorophores, including green fluorescent protein.

In recent years, interest has grown in the ability to manipulate, in a temporally precise fashion, the electrical activity of specific neurons embedded within densely wired brain circuits, in order to reveal how specific neurons subserve behaviors and neural computations, and to open up new horizons on the clinical treatment of brain disorders. Technologies that enable temporally precise control of electrical activity of specific neurons, and not these neurons ’ neighbors – whose cell bodies or processes might be just tens to hundreds of nanometers away – must involve two components. First, they require as a trigger a transient pulse of energy that supports the temporal precision of the control. Second, they require a molecular sensitizer that can be expressed in specific neurons and which renders those neurons specifically responsive to the triggering energy delivered. Optogenetic tools, such as microbial opsins, can be used to activate or silence neural activity with brief pulses of light. Thermogenetic tools, such as thermosensitive TRP channels, can be used to drive neural activity downstream of increases or decreases in temperature. We here discuss the principles underlying the operation of these two recently developed, but widely used, toolboxes, as well as the directions being taken in the use and improvement of these toolboxes.

When placed on a temperature gradient, a Drosophila larva navigates away from excessive cold or heat by regulating the size, frequency, and direction of reorientation maneuvers between successive periods of forward movement. Forward movement is driven by peristalsis waves that travel from tail to head. During each reorientation maneuver, the larva pauses and sweeps its head from side to side until it picks a new direction for forward movement. Here, we characterized the motor programs that underlie the initiation, execution, and completion of reorientation maneuvers by measuring body segment dynamics of freely moving larvae with fluorescent muscle fibers as they were exposed to temporal changes in temperature. We find that reorientation maneuvers are characterized by highly stereotyped spatiotemporal patterns of segment dynamics. Reorientation maneuvers are initiated with head sweeping movement driven by asymmetric contraction of a portion of anterior body segments. The larva attains a new direction for forward movement after head sweeping movement by using peristalsis waves that gradually push posterior body segments out of alignment with the tail (i.e., the previous direction of forward movement) into alignment with the head. Thus, reorientation maneuvers during thermotaxis are carried out by two alternating motor programs: (1) peristalsis for driving forward movement and (2) asymmetric contraction of anterior body segments for driving head sweeping movement.

The ability to avoid noxious extremes of hot and cold is critical for survival and depends on thermal nociception. The TRPV subset of transient receptor potential (TRP) channels is heat activated and proposed to be responsible for heat detection in vertebrates and fruit flies. To gain insight into the genetic and neural basis of thermal nociception, we developed assays that quantify noxious heat avoidance in the nematode Caenorhabditis elegans and used them to investigate the genetic basis of this behavior. First, we screened mutants for 18 TRP channel genes (including all TRPV orthologs) and found only minor defects in heat avoidance in single and selected double and triple mutants, indicating that other genes are involved. Next, we compared two wild isolates of C. elegans that diverge in their threshold for heat avoidance and linked this phenotypic variation to a polymorphism in the neuropeptide receptor gene npr-1. Further analysis revealed that loss of either the NPR-1 receptor or its ligand, FLP-21, increases the threshold for heat avoidance. Cell-specific rescue of npr-1 implicates the interneuron RMG in the circuit regulating heat avoidance. This neuropeptide signaling pathway operates independently of the TRPV genes, osm-9 and ocr-2, since mutants lacking npr-1 and both TRPV channels had more severe defects in heat avoidance than mutants lacking only npr-1 or both osm-9 and ocr-2. Our results show that TRPV channels and the FLP-21/NPR-1 neuropeptide signaling pathway determine the threshold for heat avoidance in C. elegans.

Like other ectotherms, the roundworm Caenorhabditis elegans and the fruit fly Drosophila melanogaster rely on behavioral strategies to stabilize their body temperature. Both animals use specialized sensory neurons to detect small changes in temperature, and the activity of these thermosensors governs the neural circuits that control migration and accumulation at preferred temperatures. Despite these similarities, the underlying molecular, neuronal, and computational mechanisms responsible for thermotaxis are distinct in these organisms. Here, we discuss the role of thermosensation in the development and survival of C. elegans and Drosophila, and review the behavioral strategies, neuronal circuits, and molecular networks responsible for thermotaxis behavior.

A mechanistic understanding of animal navigation requires quantitative assessment of the sensorimotor strategies used during navigation and quantitative assessment of how these strategies are regulated by cellular sensors. Here, we examine thermotactic behavior of theDrosophila melanogasterlarva using a tracking microscope to study individual larval movements on defined temperature gradients. We discover that larval thermotaxis involves a larger repertoire of strategies than navigation in smaller organisms such as motile bacteria andCaenorhabditis elegans. Beyond regulating run length (i.e., biasing a random walk), theDrosophila melanogasterlarva also regulates the size and direction of turns to achieve and maintain favorable orientations. Thus, the sharp turns in a larva's trajectory represent decision points for selecting new directions of forward movement. The larva uses the same strategies to move up temperature gradients during positive thermotaxis and to move down temperature gradients during negative thermotaxis. Disrupting positive thermotaxis by inactivating cold-sensitive neurons in the larva's terminal organ weakens all regulation of turning decisions, suggesting that information from one set of temperature sensors is used to regulate all aspects of turning decisions. TheDrosophila melanogasterlarva performs thermotaxis by biasing stochastic turning decisions on the basis of temporal variations in thermosensory input, thereby augmenting the likelihood of heading toward favorable temperatures at all times.

Environmental temperature strongly affects physiology of ectotherms. Small ectotherms, like Drosophila, cannot endogenously regulate body temperature so must rely on behavior to maintain body temperature within a physiologically permissive range. Here we review what is known about Drosophila thermal preference. Work on thermal behavior in this group is particularly exciting because it provides the opportunity to connect genes to neuromolecular mechanisms to behavior to fitness in the wild.

In recent years, a number of tools have become available for remotely activating neural circuits in Drosophila. Despite widespread and growing use, very little work has been done to characterize exactly how these tools affect activity in identified fly neurons. Using the GAL4-UAS system, we expressed blue light-gated Channelrhodopsin-2 (ChR2) and a mutated form of ChR2 (H134R-ChR2) in motor and sensory neurons of the Drosophila third-instar locomotor circuit. Neurons expressing H134R-ChR2 show enhanced responses to blue light pulses and less spike frequency adaptation than neurons expressing ChR2. Although H134R-ChR2 was more effective at manipulating behavior than ChR2, the behavioral consequences of firing rate adaptation were different in sensory and motor neurons. For comparison, we examined the effects of ectopic expression of the warmth-activated cation channel Drosophila TRPA1 (dTRPA1). When dTRPA1 was expressed in larval motor neurons, heat ramps from 21 to 27 degrees C evoked tonic spiking at approximately 25 degrees C that showed little adaptation over many minutes. dTRPA1 activation had stronger and longer-lasting effects on behavior than ChR2 variants. These results suggest that dTRPA1 may be particularly useful for researchers interested in activating fly neural circuits over long time scales. Overall, this work suggests that understanding the cellular effects of these genetic tools and their temporal dynamics is important for the design and interpretation of behavioral experiments.

Animals from flies to humans are able to distinguish subtle gradations in temperature and show strong temperature preferences. Animals move to environments of optimal temperature and some manipulate the temperature of their surroundings, as humans do using clothing and shelter. Despite the ubiquitous influence of environmental temperature on animal behaviour, the neural circuits and strategies through which animals select a preferred temperature remain largely unknown. Here we identify a small set of warmth-activated anterior cell (AC) neurons located in the Drosophila brain, the function of which is critical for preferred temperature selection. AC neuron activation occurs just above the fly's preferred temperature and depends on dTrpA1, an ion channel that functions as a molecular sensor of warmth. Flies that selectively express dTrpA1 in the AC neurons select normal temperatures, whereas flies in which dTrpA1 function is reduced or eliminated choose warmer temperatures. This internal warmth-sensing pathway promotes avoidance of slightly elevated temperatures and acts together with a distinct pathway for cold avoidance to set the fly's preferred temperature. Thus, flies select a preferred temperature by using a thermal sensing pathway tuned to trigger avoidance of temperatures that deviate even slightly from the preferred temperature. This provides a potentially general strategy for robustly selecting a narrow temperature range optimal for survival.

We have found that the mouse metallothionein-I (MT-I) gene promoter functions in an unusual, compound manner. It directs both TATA-dependent and TATA-independent modes of transcription in vivo. The TATA-dependent message is initiated at the previously characterized +1 transcription start site and is the predominant species in most tissues. In many cell types it is metal inducible. The TATA-independent initiation sites are distributed over the 160 bp upstream of the previously characterized +1 start site, and the RNA products are present in all tissues examined. Only in testis, however, do the TATA-independent transcripts predominate, accumulating to highest levels in pachytene-stage meiotic cells and early spermatids. Unlike the TATA-dependent +1 transcript, these RNAs are not induced by metal, even in cultured cells in which the +1 species is induced. Transfection studies of site-directed mutants show that destruction of the TATA element drastically alters the ratio of the two RNA classes in cells in which the +1 transcripts normally dominates. In TATA-minus mutants, the TATA-independent RNAs become the most prevalent, although they remain refractory to metal induction. Thus, the MT-I promoter utilizes two different types of core promoter function within a single cell population. The two different types of core promoter respond very differently to environmental stimuli, and the choice between them appears to be regulated in a tissue-specific fashion.

BEACH proteins, an evolutionarily conserved family characterized by the presence of a BEACH (Beige and Chediak-Higashi) domain, have been implicated in membrane trafficking, but how they interact with the membrane trafficking machinery is unknown. Here we show that the Drosophila BEACH protein Bchs (Blue cheese) acts during development as an antagonist of Rab11, a small GTPase involved in vesicle trafficking. We find that reduction in, or loss of, bchs function restores viability and normal bristle development in animals with reduced rab11 function, while reductions in rab11 function exacerbate defects caused by bchs overexpression in the eye. Consistent with a role for Bchs in modulating Rab11-dependent trafficking, Bchs protein is associated with vesicles and extensively colocalized with Rab11 at the neuromuscular junction (NMJ). At the NMJ, we find that rab11 is important for synaptic morphogenesis, as reductions in rab11 function cause increases in bouton density and branching. These defects are also suppressed by loss of bchs . Taken together, these data identify Bchs as an antagonist of Rab11 during development and uncover a role for these regulators of vesicle trafficking in synaptic morphogenesis. This raises the interesting possibility that Bchs and other BEACH proteins may regulate vesicle traffic via interactions with Rab GTPases.

Thermotaxis is important for animal survival, but the molecular identities of temperature sensors controlling this behavior have not been determined. We demonstrate dTRPA1, a heat-activated Transient Receptor Potential (TRP) family ion channel, is essential for thermotaxis in Drosophila. dTrpA1 knockdown eliminates avoidance of elevated temperatures along a thermal gradient. We observe dTRPA1 expression in cells without previously ascribed roles in thermosensation and implicate dTRPA1-expressing neurons in mediating thermotaxis. Our data suggest that thermotaxis relies upon neurons and molecules distinct from those required for high-temperature nociception. We propose dTRPA1 may control thermotaxis by sensing environmental temperature.