Drosophila tissue and organ development: Peripheral nervous system

The Interactive Fly

Genes involved in tissue and organ development

Peripheral Nervous System

What is the peripheral nervous system?
Identification and function of thermosensory neurons in Drosophila larvae

A newly identified type of attachment cell is critical for normal patterning of chordotonal neurons
Integration of complex larval chemosensory organs into the adult nervous system of Drosophila
Genetic programs activated by proneural proteins in the developing Drosophila PNS
Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology
doublesex functions early and late in gustatory sense organ development
Sound response mediated by the TRP channels NOMPC, NANCHUNG, and INACTIVE in chordotonal organs of Drosophila larvae
Transmembrane channel-like (tmc) gene regulates Drosophila larval locomotion
The role of PPK26 in Drosophila larval mechanical nociception
Nociceptor-enriched genes required for normal thermal nociception
Coordination and fine motor control depend on Drosophila TRPγ
Neuroendocrine control of Drosophila larval light preference
A gustatory receptor paralogue controls rapid warmth avoidance in Drosophila
The insulin receptor is required for the development of the Drosophila peripheral nervous system
The adhesion GPCR Latrophilin/CIRL shapes mechanosensation
ROS-mediated activation of Drosophila larval nociceptor neurons by UVC irradiation
Kinematic responses to changes in walking orientation and gravitational load in Drosophila melanogaster
WHAMY is a novel actin polymerase promoting myoblast fusion, macrophage cell motility and sensory organ development
G2-phase arrest prevents bristle progenitor self-renewal and synchronizes cell divisions with cell fate differentiation
The microtubule-based cytoskeleton is a component of a mechanical signaling pathway in fly campaniform receptors

Diagram of the sensory cells of an embryonic or larval abdominal hemisegment (A1-A7)

Orgogozo, V. and Grueber, W. B: FlyPNS, a database of the Drosophila embryonic and larval peripheral nervous system

Genes of the peripheral nervous system

What is the peripheral nervous system?

The peripheral nervous system consists of sensory neurons. There are two types. Type I neurons innervate the sensory organs to which they are related by lineage. Each of these sensory organs is thought to be derived from a single ectodermal precursor (sensory organ precursor or SOP) which gives rise to one or several monodendritic neurons and several support cells. Type I sensory organs have been classified into two major groups: first, mechano- or chemosensory organs that have external sensory structures in the cuticle such as bristles, campaniform, and basiconical sensilla (external sensory organs), and second, chordotonal organs that are internally located stretch receptors. In addition the larval PNS also contains numerous type II neurons with multiple dendrites. These neurons, with one exception, do not seem to be associated with support cells. Multiple dendrite neurons are thought to function as stretch or touch receptors. Multiple dendritic neurons are derived from three sources, one group from external sensory organ lineages, a second set from chordotonal neurons and a third set is unrelated to sensory organs (Brewster, 1995).

For information about the development of the antennal olfactory sense organs see Olfactory Receptors.

Organs of the peripheral nervous system

Identification and function of thermosensory neurons in Drosophila larvae

Although the ability to sense temperature is critical for many organisms, the underlying mechanisms are poorly understood. Using the calcium reporter yellow cameleon 2.1 and electrophysiological recordings, thermosensitive neurons were identified and their physiologic responses were examined in Drosophila larvae. In the head, terminal sensory organ neurons show increased activity in response to cooling by ~1°C, heating reduces their basal activity, and different units show distinct response patterns. Neither cooling nor heating affects dorsal organ neurons. Body wall neurons show a variety of distinct response patterns to both heating and cooling; the diverse thermal responses are strikingly similar to those described in mammals. These data establish a functional map of thermoresponsive neurons in Drosophila larvae and provide a foundation for understanding mechanisms of thermoreception in both insects and mammals (Liu, 2003).

To identify neurons responding to changes in temperature, an optical approach using yellow cameleon 2.1 (YC2.1), an engineered, calmodulin-based, Ca2+-sensitive protein, was used. Its two fluorophores, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), comprise a fluorescence resonance energy transfer (FRET)-capable pair; a conformational change in the protein causes FRET to increase when the Ca2+ concentration rises. Cameleon fluorescence has been used to measure intracellular (or cytosolic) Ca2+ concentration, [Ca2+]i, in vitro and in vivo (in C. elegans). Transgenic Drosophila larvae were developed that express cameleon in their neurons, and FRET was assayed to monitor activity in the peripheral neurons as the temperature was changed. The FRET measurements, plus electrophysiologic and behavioral assays, indicate that the terminal organ is a thermosensitive structure that responds to cool temperatures. Some body wall neurons also showed FRET changes with temperature shifts, and in contrast to the terminal organ, they responded to warm temperatures (Liu, 2003).

The pan-neuronal promoter elav was used to drive expression of the YC2.1 variant of cameleon with the Gal4-UAS system. Heterozygous transgenic larvae (elav-Gal4/+;UAS-YC2.1/+) showed fluorescence signal in all neurons. For example, cameleon fluorescence was distributed in neurons of the lateral pentascolopidial chordotonal organs in a diffuse pattern in the cell bodies and neurites, but not in the cell nuclei. Similar results were obtained in two other lines. To increase fluorescence intensity, homozygous lines (elav-Gal4;UAS-YC2.1) were generated. They produced a more intense fluorescence signal than did the heterozygous line; therefore, homozygous larvae were used for optical recording (Liu, 2003).

The dorsal and terminal organs are the major sensory structures of the larval head. Each organ contains more than 30 bipolar neurons with large dendrites extending to the tip of a dome-like structure where pores open to the environment. Cameleon was expressed in the larval head in terminal and dorsal organ neurons. Terminal and dorsal organ YFP/CFP ratio images were measured at 18, 10 and 40°C. When the temperature fell, the terminal organ YFP/CFP ratio and the calculated [Ca2+]i increased, and when the temperature rose, terminal organ [Ca2+]i fell. In contrast to the terminal organ, temperature variations had little effect on [Ca2+]i in the dorsal organ, salivary gland or trachea (Liu, 2003).

At 18°C, the fluorescence ratio from terminal organ neurons fluctuates spontaneously; this was reflected in the variance of the fluorescence ratio (sigma2). These fluctuations are not an artifact, since the fluorescence ratio in the adjacent dorsal organ and in the salivary gland and trachea did not fluctuate, and the sigma2 was much lower. Cooling increases and heating decreases terminal organ sigma2, but has little effect on sigma2 in the other cell types. These findings suggest that terminal organ neurons have substantial spontaneous activity, even at room temperature, and the large [Ca2+]i fluctuations raise the possibility that some neurons might show oscillating activity (Liu, 2003).

Although the data do not allow distinguishing whether the changes in [Ca2+]i are upstream or downstream of action potential firing, these results indicate that the larval terminal organ contains thermosensory neurons that respond to cooling. Their response profile resembles the behavior of the most commonly observed mammalian cold receptors. The lack of thermoreceptor activity in dorsal organ neurons provides an important control showing the specificity of temperature sensing by the terminal organ: both terminal and dorsal organs contain sensory neurons, they lie adjacent to each other in the larval head; they are exposed to the same temperature stimuli, and cameleon fluorescence from both organs can be examined in the same image. These data also indicate that the cameleon protein itself does not respond to temperature changes (Liu, 2003).

To further test the hypothesis that terminal organ neurons detect cool temperatures and to obtain an independent assessment of the response of thermosensitive neurons, a glass electrode was inserted into early third-instar larval terminal or dorsal organs and extracellular recordings were obtained. Dorsal organ recordings showed no activity at room temperature or any response to warming or cooling. Terminal organ neurons, however, showed spontaneous activity at room temperature. This difference between terminal and dorsal organ basal activity is consistent with the difference in sigma2 in the optical measurements. Depending on the electrode position, recordings were obtained with one or two units. Thirty-six such recordings were analyzed (Liu, 2003).

When larvae are cooled by placing a cold metal block in their vicinity, terminal organ neurons respond in one of three ways. A type-I response is an increased firing frequency that adapts during the time course of the stimulus. This response occurred in 20 of 36 recordings. A type-II response, which occurred in 3 of 36 recordings, showed cold-induced oscillatory activity. This oscillating electrical activity may explain, in part, the increase in FRET sigma2 observed on cooling the terminal organ. In contrast to the type-I response, the onset of the type-II response is slow, and after the cold stimulus is removed, oscillations persist for some time before returning to baseline. A type-III response involves a transient cold-induced reduction in activity; it occurred in 13 recordings (Liu, 2003).

When a warm block was substituted for the cold one, spontaneous activity fell with all three types of response. For example, in recordings showing a type-I response, a warm stimulus reduced activity from 16.7 +/- 2.4 spikes/s to 7.4 +/- 2.9 spikes/s. After removing the warm block, neuronal activity recovered to baseline within 5 s (Liu, 2003).

On exposure to the cold stimulus, temperature will fall, and the closer the stimulus to the terminal organ, the more rapid the fall. In type-I cells, faster cooling elicits a greater increase in neuronal activity, the maximal increase in activity occurs 1 s later, at a time when that terminal organ temperature was calculated to have fallen 0.30°C. With the cold block at position 1, the maximal increase in spike rate occurs at 16 s when a 0.43°C decline was calculated to have occured. At the intermediate position 2, a fall of 0.45°C at 7 s was calculated when activity was maximal. Thus, the response to a cold stimulus seems to be a function of the rate of cooling and the change in temperature (Liu, 2003).

To change temperature by another means, a Peltier-based device was used to cool the solution bathing the larval body. Recordings were studied that showed a type-I response and lowered temperature 0.1-1°C from room temperature. This cooling increased spike frequency by 20%. Thus, similar results were obtained with different methods of lowering temperature and with independent means of measuring the response (Liu, 2003).

Because cooling stimulates terminal organ activity, it was hypothesized that disrupting terminal organ function would blunt the behavioral response to a reduced temperature. Earlier work showed that the GH86 promoter drives expression in the terminal organ; it also drives expression in the dorsal organ, epidermis, enocytes and pharyngeal muscle. The GH86-Gal4 promoter was used to drive a UAS-tetanus toxin light chain transgene (UAS-TNT-C), which specifically degrades synaptobrevin and thereby blocks neurotransmitter release. Because both the GH86-Gal4 and UAS-TNT-C transgenes are located on the X chromosome, only female F1 larvae that contained both transgenes were studied. The GH86 x TNT-C cross showed a reduced preference for 18°C compared with 11°C. These data suggest that larvae use terminal organ thermosensors to sense cool temperatures. Larval terminal organs contain more than 30 neurons and also likely respond to other sensory stimuli. For example, some terminal organ neurons may be involved in the response to low concentrations of salt (Liu, 2003).

To test thermosensitivity at another site, neurons in lateral body segments 5-8 were examined. At this location, there was less interference from fluorescence of the central nervous system and salivary glands than in segments 1-4. Depending on the location of the neurons, it was possible to measure fluorescence from some individual neurons. In other cases, clusters of neurons were studied because single neurons could not be reproducibly identified. However, temperature-dependent changes in the clusters likely arise from single neurons because the fluorescence changes often occur at a single spot within the cluster (Liu, 2003).

Multidendritic neurons show the greatest response to temperature changes, and the amplitude of [Ca2+]i response varies for different neurons and clusters. Different neurons also show distinct thermosensory responses. For example, for neurons in clusters 2 and 3, and neuron ddaB, cooling reduces and heating elevates [Ca2+]i. This response is the opposite of that in the terminal organ. The neurons in cluster 1 and lch5 increase [Ca2+]i when the temperature is either raised or lowered from the preferred temperature of 18°C. The distinct response patterns observed suggest that different neurons carry specific information to the central nervous system (Liu, 2003).

These data provide a functional map of thermosensitive neurons in Drosophila larvae. Neurons with different temperature responses were to be anatomically segregated. Moreover, within different regions there was a striking diversity in the behavior of thermosensitive neurons (Liu, 2003).

Terminal organ cold sensors show activity at room temperature, and this activity increases with cooling and falls with heating. Thus, these thermosensors are poised to respond whenever temperature changes, even slightly. Moreover, terminal organ function is apparently required for a normal response to cool temperature because expressing the tetanus toxin light chain in the terminal organ blunts the behavioral preference for 18°C versus 11°C. The calculations of the temperature shifts induced by a cold block and the measurements of bath temperature indicate that the terminal organ responds to changes of ~1°C. Thus, these data explain how larvae respond to a change in temperature, but how they respond to absolute temperatures remains unclear. The answer probably lies in central integration of output from the complex mixture of thermosensory neurons. In this regard, thermoreceptors with oscillating discharges may be particularly important to sensing absolute temperatures. It has also been suggested that the substantial complexity of thermoreceptive cell types may increase the sensitivity of the system(Liu, 2003).

The data suggest a striking similarity between thermoreceptor physiology in Drosophila and mammals. In both organisms, different types of neurons encode the response to cold and heat stimuli. It was found that in Drosophila, the most common type of terminal organ cold receptive neurons show a characteristic response to cold and heat; they spontaneously discharge at room temperature, cooling reduces the frequency of nerve impulses, and warming decreases activity. This type of cold receptor neuron is also very common in mammals. Additionally, an oscillatory response to cooling was found in 3 of 36 larval terminal organ neurons. The preliminary observations suggest that oscillatory activity may be more common if temperature is reduced more rapidly and to a greater extent. Interestingly, there are several reports of oscillatory activity in mammalian thermoreceptors. Whether this type of activity results from coordinated effects of temperature on multiple channels or on a single type of channel is unknown. Cluster 1 and Ich5 neurons in Drosophila increase activity on both warming and cooling. Mammals also contain these so-called 'paradoxical' temperature receptors. It will be interesting to learn whether this activity is generated by two different temperature-responsive ion channel receptors that are both expressed in a single neuron. Finally, some Drosophila neurons (multidendritic neurons ddaB and clusters 2 and 3) increase their activity during heating and reduce activity during cooling. This pattern of activity also exists in many mammalian warm receptors (Liu, 2003).

These findings reveal a diverse pattern of thermosensory response in larval neurons and provide new insight into the physiology of temperature sensing in Drosophila. Moreover, the results demonstrate common thermosensory response patterns between distantly related animal species. Given the potential relationship between temperature sensing and pain, this work may provide a basis for additional insight into nociception. Thus, these studies help pave the way toward a better understanding of the molecular mechanisms of thermoreception in both insects and mammals (Liu, 2003).

A newly identified type of attachment cell is critical for normal patterning of chordotonal neurons

This work describes unknown aspects of chordotonal organ (ChO) morphogenesis revealed in post-embryonic stages through the use of new fluorescently labeled markers. Towards the end of embryogenesis a hitherto unnoticed phase of cell migration commences in which the cap cells of the ventral ChOs elongate and migrate towards their prospective attachment sites. This migration and consequent cell attachment generates a continuous zigzag line of proprioceptors, stretching from the ventral midline to a dorsolateral position in each abdominal segment. The observation that the cap cell of the ventral-most ChO attaches to a large tendon cell near the midline provides the first evidence for a direct physical connection between the contractile and proprioceptive systems in Drosophila. This analysis has also provided an answer to a longstanding enigma that is what anchors the neurons of the ligamentless ventral ChOs on their axonal side. A new type of ChO attachment cell was identified that binds to the scolopale cells of these organs, thus behaving like a ligament cell, but on the other hand exhibits all the typical features of a ChO attachment cell and is critical for the correct anchoring of these organs (Halachmi, 2016).

Integration of complex larval chemosensory organs into the adult nervous system of Drosophila

The sense organs of adult Drosophila, and holometabolous insects in general, derive essentially from imaginal discs and hence are adult specific. Experimental evidence presented in this study, however, suggests a different developmental design for the three largely gustatory sense organs located along the pharynx. In a comprehensive cellular analysis, it is shown that the posteriormost of the three organs derives directly from a similar larval organ and that the two other organs arise by splitting of a second larval organ. Interestingly, these two larval organs persist despite extensive reorganization of the pharynx. Thus, most of the neurons of the three adult organs are surviving larval neurons. However, the anterior organ includes some sensilla that are generated during pupal stages. Also, apoptosis is observed in a third larval pharyngeal organ. Hence, the experimental data show for the first time the integration of complex, fully differentiated larval sense organs into the nervous system of the adult fly and demonstrate the embryonic origin of their neurons. Moreover, they identify metamorphosis of this sensory system as a complex process involving neuronal persistence, generation of additional neurons and neuronal death. The conclusions are based on combined analysis of reporter expression from P[GAL4] driver lines, horseradish peroxidase injections into blastoderm stage embryos, cell labeling via heat-shock-induced flip-out in the embryo, bromodeoxyuridine birth dating and staining for programmed cell death. They challenge the general view that sense organs are replaced during metamorphosis (Gendre, 2003).

The external gustatory sensilla of the Drosophila larva appear to follow the general holometabolan fate: they degenerate during metamorphosis and are replaced by adult-specific sensilla that derive from the labial imaginal disc. This study examines whether this rule also applies to the internal gustatory system that is located along the pharyngeal tube. Interestingly, the adult pharynx derives essentially from small, densely packed imaginal cells that comprise the clypeolabral bud, which is closely associated with the larval pharyngeal skeleton. Does this imply that adult pharyngeal sensilla are born during metamorphosis, like their external counterparts, or do the anatomical similarities of certain larval and adult pharyngeal organs rather suggest persistence of sensilla through metamorphosis (Gendre, 2003)?

The data prove that most of the neurons of the three major adult pharyngeal sense organs are persisting larval neurons that were born in the embryo. This is unlike other adult sensory neurons, nearly all of which derive from imaginal discs. This interpretation relies on two independent experimental approaches for demonstrating embryonic birth dates (the use of the embryonic lineage tracer HRP and cell labeling by FLPout at late embryonic stages, a novel use of this technique). The experimental data are supported by anatomical observations showing: (1) an almost identical organization of the larval posterior pharyngeal sense organ (pps) and the adult dorsal cibarial sense organ (dcso); (2) the presence of the pps and dorsal pharyngeal sense organ (dps) sensilla continuously through metamorphosis; (3) an uninterrupted expression of the P[GAL4] lines used in these two organs, and (4) the persistence of dendrites and axons in all surviving neurons (Gendre, 2003).

HRP injected at the syncytial blastoderm stage becomes incorporated into every cell upon cellularization. During subsequent development, the marker remains at high levels in cells that divide only a few times but becomes diluted in cells that undergo repeated divisions. Consequently, labeling in the adult is expected in many neurons of the central nervous system known to be persisting larval neurons (e.g. optic lobe pioneers) but should be absent from tissues derived from imaginal discs. This corresponds to what was observed and leads to the postulation of an embryonic origin for the elements containing high HRP levels in adult pharyngeal sense organs (Gendre, 2003).

This interpretation is supported by the FLPout experiments performed at late embryonic stages with the neuron-specific MJ94 line. In adults deriving from this treatment, exclusively single labeled neurons were detected in sensillum 7 of the labral sense organ (lso), containing eight neurons, and in the five multiply innervated sensilla of the vcso and dcso. Although the cell lineage of these sensilla was not studied, they are probably homologous to other multineuronal terminal-pore gustatory sensilla, which derive from a common sensory mother cell. Indeed, apart from its eight neurons, sensillum 7 of the lso corresponds to a typical insect gustatory sensillum in terms of fine structural and cellular organization, containing no more than three accessory cells. Hence, the single labeled neurons in this sensillum and in all sensilla of the vcso and dcso must have been postmitotic during FLPout. This agrees with the observation that formation of head nerves is complete by embryonic stage 15 (Gendre, 2003).

Could these neurons have remained immature during larval life, differentiating only during metamorphosis, similar to subsets of postmitotic cells in the larval central nervous system CNS? This is thought to be rather unlikely because it would require either the entire sensillum or subsets of neurons in multineuronal sensilla to remain immature. Moreover, there is no indication for immature neurons from tracing their development with the marker line mCD8-GFP. Thus, it is suggested that all the neurons of the dcso and vcso, and sensillum 7 of the lso derive from mature, functional larval neurons. Also, continuous reporter expression through metamorphosis suggests that one of the mononeural lso sensilla (perhaps sensillum 3) might be another persisting larval sensillum (Gendre, 2003).

The fact that the pps and dps persist through metamorphosis is remarkable given the origin of the adult labrum and cibarium from imaginal cells of the clypeolabral bud. Massive labeling of pharyngeal epithelial cells was observed after early pupal BrdU application. Moreover, the pharyngeal cuticle is shed and regenerates, a process that includes the cuticular part of the sensilla in question. Perhaps the birth of additional accessory cells during metamorphosis (e.g. in the dcso or vcso, containing exclusively persisting neurons) is related to this modification. Formation of new cuticular structures is also known from persisting external sensilla during larval molts, but the survival of pharyngeal sensilla during the extensive remodeling of the pharynx remains stunning. The morphogenetic movements observed in the sensory system certainly reflect these dramatic changes (Gendre, 2003). Why is the larval pharyngeal sensory apparatus largely conserved through metamorphosis? Small subsets of neurons associated with leg imaginal discs or with abdominal segments persist through metamorphosis. In the fly Phormia, such leg-disc-associated neurons remain immature, implying that they are non-functional. Laser ablation studies suggest that persisting neurons might help adult afferents to navigate from the imaginal discs to their central targets. Whether they become truly integrated in the adult nervous system or die after reaching adulthood (having completed their pathway role) remains to be shown (Gendre, 2003).

The data demonstrate for the first time experimentally the integration of larval sensory neurons into the adult nervous system of Drosophila. Particularly striking and novel is the fact that entire, fully differentiated larval sense organs become incorporated. Also, this is the first observation of metamorphic survival in the chemosensory system (Gendre, 2003).

Concerning the persisting neurons of the lso, a pathway function for the newly developing afferents toward and inside the central nervous system is certainly possible. However, the integration of the surviving pharyngeal neurons into the adult sensory system invites other interpretations. For example, these neurons and/or their central projections might be particularly precious, allowing, for example, the persistence of specific feeding-associated gustatory tasks through metamorphosis. As an alternative explanation, survival might be due to reasons of economy, a principle that governs the metamorphosis of the nervous system. Although neuronal reorganization is indispensable owing to the changing demands of larval and adult life, it is kept at a minimum, as shown by the survival of most larval interneurons and motor neurons. Sophisticated adult sense organs, however, might be easier to build de novo than by the transformation of simple larval organs, explaining the almost complete replacement of the larval sensory system. Why pharyngeal sense organs do not follow this general rule might relate to their largely conserved function at the two stages of life (analyzing the quality of ingested food of similar composition). The presence of larva-specific and adult-specific sensilla, however, suggests the existence of stage-specific gustatory tasks (Gendre, 2003).

Genetic programs activated by proneural proteins in the developing Drosophila PNS

Neurogenesis depends on a family of proneural transcriptional activator proteins, but the 'proneural' function of these factors is poorly understood, in part because the ensemble of genes they activate, directly or indirectly, has not been identified systematically. A direct approach to this problem has been undertaken in Drosophila. Fluorescence-activated cell sorting was used to recover a purified population of the cells that comprise the 'proneural clusters' from which sensory organ precursors of the peripheral nervous system (PNS) arise. Whole-genome microarray analysis and in situ hybridization was then used to identify and verify a set of genes that are preferentially expressed in proneural cluster cells. Genes in this set encode proteins with a diverse array of implied functions, and loss-of-function analysis of two candidate genes shows that they are indeed required for normal PNS development. Bioinformatic and reporter gene studies further illuminate the cis-regulatory codes that direct expression in proneural clusters (Reeves, 2005).

The PNC cells that express the proneural genes achaete (ac) and scute (sc) comprise only a small fraction of the wing imaginal disc of the late third-instar Drosophila larva. It is anticipated that this might frustrate attempts to characterize PNC-specific gene expression in unfractionated wing discs (e.g., by comparison of wild-type and ac-sc mutant tissue). Accordingly, PNC cells were purified by using fluorescence-activated cell sorting (FACS). As a PNC-specific marker, a GFP reporter was chosen representing the Bearded family gene E(spl)m4. m4 is strongly and specifically expressed in PNCs, and a cis-regulatory module has been identified sufficient to recapitulate this activity. Wing imaginal discs were dissected from late third-instar larvae carrying the m4-GFP transgene and dissociated in trypsin-EDTA; cells with fluorescence greater than that of w1118 control cells (GFP-positive cells) and cells with fluorescence comparable to the control (GFP-negative cells) were recovered separately by FACS (Reeves, 2005).

Transcripts from several genes known to be expressed in domains of the wing disc outside of PNCs (en, hh, and twi) were found to be greatly depleted in the GFP-positive cell population. These negative controls provide further evidence of successful separation of PNC cells from other disc cells (Reeves, 2005).

Since the microarray data clearly associates expression of known genes preferentially with the expected cell populations, 43 candidates not known to be expressed in wing imaginal discs were chosen for further analysis. Candidate genes for which cDNA clones were available from the Drosophila Gene Collection were favored. The selected genes exhibit a wide variety of GFP+/GFP- expression ratios in the microarray data, and their products have a broad spectrum of predicted functions (Reeves, 2005).

In situ hybridization was employed as a secondary screening method, both to verify that candidate genes selected from these microarray data are expressed in wing imaginal discs, and to determine their specific patterns of transcript accumulation. The wing disc expression patterns observed can be sorted into three major classes: PNC patterns, SOP patterns, and overlapping patterns. Five of the 43 selected candidate genes exhibit a complete PNC pattern of expression, while 3 other candidates are expressed in subsets of PNCs; phyl is expressed in the SOP and in a subset of non-SOP cells in each PNC. An unexpected 18 candidates are expressed in the presumptive SOP cells of the wing disc. Fourteen of these SOP genes are expressed in a complete pattern of SOPs, whereas the remaining four are expressed either late in SOP development or in subsets of SOPs. The existence of the latter group suggests that the cell sorting strategy made it possible to identify genes that are expressed preferentially in just a few cells of the wing disc. Overall, 27 (63%) of the tested candidates were found to display PNC- or SOP-specific expression patterns. This is likely to be an underestimate of the true success rate of the microarray analysis, since 23 genes known to be expressed in these patterns are not included in the statistic, though they were reidentified in the screen (Reeves, 2005).

In addition to those expressed specifically in PNCs and SOPs, a small group of candidate genes was found that is expressed in patterns that overlap PNCs but appears to be distinct from them. Detection of this class of genes is an important confirmation of the efficacy and unbiased nature of the experimental approach (Reeves, 2005).

Patterned expression of the proneural genes ac and sc defines the PNCs for most external sensory bristles in adult Drosophila, and ac-sc function is required for PNC and SOP gene expression, as well as for specification of the SOP cell fate. Fifteen of the genes identified by the combined cell sorting/microarray approach also require proneural gene function for their expression. In an ac sc proneural mutant background, transcript accumulation from members of both the PNC (CG11798, CG32434/loner, edl, PFE) and SOP (CG3227, CG30492, CG32150, CG32392, Men, qua) classes is lost from PNCs that require ac-sc function. This result is further evidence that the approach has identified bona fide PNC genes, and it demonstrates that expression of these ten genes is, directly or indirectly, downstream of the bHLH activators encoded by ac and sc. The data further show that the PNC-specific imaginal disc expression of the previously studied genes mira, phyl, rho, Spn43Aa, and Traf1 is likewise downstream of proneural gene function (Reeves, 2005).

The identification of sets of genes comprising the genetic programs deployed in PNCs and SOPs by the action of proneural proteins offers a powerful opportunity to investigate the regulatory organization of these programs. Specifically, it was of interest to find out (1) which genes are directly activated by proneural regulators, and which indirectly, and (2) the nature of the cis-regulatory sequences and their cognate transcription factors that distinguish PNC- versus SOP-specific target gene expression. This analysis was initiated by examining potential regulatory sequences of several of the genes that have been identified for the presence of conserved, high-affinity proneural protein binding sites of the form RCAGSTG. The initial approach was to ask whether evolutionarily conserved clusters of these binding sites identify cis-regulatory modules of the appropriate specificity. To date, this strategy has proven very successful. Genomic DNA fragments bearing proneural protein binding site clusters associated with CG11798, edl, Traf1, CG32434/loner, and rho confer PNC-specific activity on a heterologous promoter, while similar modules from CG32150, mira, and PFE drive SOP-specific expression. In three cases, double labeling with the SOP marker anti-Hindsight (Hnt) reveals that PNC-specific expression of the reporter gene includes the SOP as well as the non-SOP cells. Mutation of the proneural protein binding sites in four of the enhancer-bearing fragments severely reduces (CG11798) or abolishes (CG32150, edl, Traf1) reporter gene expression in PNCs/SOPs. Such results indicate that these genes are indeed direct targets of activation by proneural proteins in vivo (Reeves, 2005).

Holometabolous insects like Drosophila carry out two major phases of PNS neurogenesis, one in embryogenesis to form the larval PNS, and a second in the late larval and early pupal stages to construct the adult PNS. Many known genes participate in both phases. Accordingly, it was of interest to determine whether genes identified as being expressed in imaginal disc PNCs or SOPs are also expressed in the developing larval PNS. In situ hybridization reveals that, among others, the PNC genes CG11798 and CG32434/loner and the SOP genes CG3227, CG32150, and CG32392 are indeed expressed in embryonic PNCs and SOPs, respectively (Reeves, 2005).

To determine whether this combined cell sorting/microarray/in situ hybridization approach had indeed identified gene functions required for proper PNS development, loss-of-function alleles of two loci, CG11798 and CG3227, were generated. These were chosen because (1) transcript accumulation from both genes was detected in the primordia of both the larval and adult PNSs; (2) both genes encode proteins with conserved domains; and (3) mobilizable P element transposon insertions were available adjacent to these genes (Reeves, 2005).

CG11798 is predicted to encode a probable transcription factor with four zinc finger domains. Loss-of-function alleles of the gene were generated by mobilizing KG03781, a P element located immediately downstream. A precise excision of the P transposon and two partial deletions of the CG11798 coding region were recovered and characterized by sequencing. Deletions 19E and 34E are both homozygous lethal during early larval stages, and both confer clear defects in the development of the larval PNS. 19E causes the loss or misplacement of sensory neurons marked by mAb 22C10 and sensory organ accessory cells marked by anti-Prospero (αPros). Deletion 34E confers an even more severe PNS phenotype and removes or misplaces many more 22C10-positive and Pros-positive sensory organ cells in each hemisegment. The difference in the severity of the 19E and 34E mutant phenotypes may be due to the fact that the latter deletes a larger portion of the CG11798 coding region, including the codons for the four zinc fingers. As a control genotype, use was made of the precise excision (PE) derivative of the KG03781 transposon insertion. No PNS mutant phenotype was detected in homozygous PE embryos, demonstrating that the defects observed in the 19E and 34E deletion homozygotes do not result from a second-site mutation on the original KG03781 chromosome. The results of complementation tests led to the conclusion that CG11798 corresponds to the previously described charlatan (chn) locus (Reeves, 2005).

To generate loss-of-function alleles of CG3227, the P element transposon KG07404, inserted just upstream of the gene, was mobilized. Imprecise excision created two deletions, 23B and 23I. Homozygosity for either results in nearly complete lethality before adulthood. Mosaic adult flies carrying FLP/FRT-generated mutant clones exhibit a severe PNS defect in which most mechanosensory bristles within the clonal territory not only lack shaft structures but also bear multiple socket structures, suggestive of shaft-to-socket cell fate transformations. The major defects observed in sensory structures in both the larval and adult PNSs prompted giving CG3227 the new name insensitive (insv) (Reeves, 2005).

insv is predicted to encode a protein containing a conserved C-terminal domain of unknown function called DUF1172. DUF1172 was first recognized in the vertebrate NAC1 proteins, transcription factors that also contain BTB/POZ protein-protein interaction domains. Alignment of arthropod and vertebrate DUF1172s reveals that the domain is large (approximately 125 amino acids) and contains a highly conserved central region of alternating polar/charged residues and nonpolar residues. This is the first described loss-of-function phenotype for a gene encoding a DUF1172 domain protein (Reeves, 2005).

Several known or potential components of signaling pathways were uncovered in this analysis as exhibiting either PNC- or SOP-specific expression. These include genes encoding a putative G protein-coupled receptor (CG31660), a receptor tyrosine kinase (Ror), a regulator of G protein signaling (loco), and a modulator of Ets protein activity (edl). Earlier studies have linked both G protein function and Ras/MAPK signaling to the development of Drosophila sensory bristles, but much remains to be learned about their roles in this process. These findings suggest functions in PNS development for both known and previously uncharacterized signaling pathway components (Reeves, 2005).

Perhaps surprisingly, the data indicate the PNS-specific expression in imaginal discs of several genes predicted to encode metabolic enzymes, including a uridine phosphorylase (CG6330), a maleylacetoacetate isomerase (CG9363), and a malate dehydrogenase (Men). Exceptional metabolic requirements or signaling activities in developing sensory organs may underlie these observations (Reeves, 2005).

Loss-of-function analysis of two genes identified by the cell sorting/microarray/in situ hybridization approach, one expressed in PNCs (CG11798/chn) and one in SOPs (CG3227), confirms that they are indeed required for normal PNS development in Drosophila. Deletion mutations of CG3227 (insensitive) cause severe defects in the specification and differentiation of sensory organ cells in the adult PNS, as assayed in mosaic clones. Particularly prevalent is an apparent transformation of the shaft cell to the fate of its sister, the socket cell; this is the same phenotype conferred by loss-of-function mutations in N pathway antagonists such as Hairless and numb. The definition of a loss-of-function phenotype for a DUF1172 gene should prove valuable in investigating the in vivo function of this uncharacterized protein domain (Reeves, 2005).

Certain SOP-specific genes, exemplified by sens and phyl, are required for the execution of the SOP fate itself. insv, by contrast, represents a distinct class of SOP gene, required not for the fate of this cell, but for the specification and/or differentiation of one or more of its progeny. Thus, SOP-specific (or, more generally, precursor-specific) gene expression can serve the same function as maternal gene expression -- providing gene products essential to the development of descendants. It is anticipated that a number of the SOP genes identified will prove to act similarly (Reeves, 2005).

The function of proneural bHLH proteins in Drosophila PNS development is complex, since they not only activate in SOPs genes that promote the neural precursor cell fate (e.g., ac and sc themselves, sens and phyl); they also activate in non-SOPs genes involved in inhibiting this fate (e.g., genes of the Enhancer of split Complex). The nature of the cis-regulatory 'codes' (combinations of transcription factor binding sites) that distinguish the PNC versus SOP expression specificities is of particular interest. One code has been identified for the expression of N-responsive genes in the non-SOP cells of the PNC that consists of binding sites for the proneural proteins plus sites for the N-activated transcription factor Suppressor of Hairless (Su(H)). Importantly, none of the PNC modules identified in this study includes a conserved high-affinity Su(H) site, yet at least three of them do mediate direct transcriptional activation by the proneural proteins. Moreover, the expression driven by these new PNC modules includes the SOP, whereas the 'Su(H) plus proneural' code directs expression that excludes it. These findings indicate the existence of at least one novel code for PNC expression, and of a heretofore hypothetical class of genes -- ones that are directly regulated by the proneural proteins in PNCs/SOPs but are evidently not activated in response to N-mediated lateral inhibitory signaling, perhaps because they are not involved in the inhibitory process (Reeves, 2005).

The proneural genes were first identified by their function in the ectoderm in specifying neural cell fates, and they have been studied almost exclusively in that context in both vertebrates and invertebrates. However, it has become clear that these genes function as well in the other two germ layers. The Drosophila proneural gene lethal of scute (l'sc) is required to specify the fates of muscle progenitor cells in the embryonic mesoderm, and the same gene (and probably also sc) is required for the adult midgut precursor (AMP) cell fate in the embryonic endoderm. In both of these nonectodermal settings, a striking parallel with neurogenesis is seen in the manner in which proneural genes function in close association with the N pathway to select individual precursor cells. In the mesoderm, l'sc is deployed in 'pro-muscle clusters' from which single muscle progenitors emerge by N-mediated 'lateral inhibition'; in the endoderm, where proneural gene expression is initially uniform, AMPs are spaced apart from each other by N signaling in a manner very reminiscent of the spacing of microchaete bristles on the adult thorax. The mouse proneural protein Atoh1 (Math1) has been shown to be required for the specification of nonneural secretory cell precursors in the intestinal epithelium. Thus, proneural transcription factors are not dedicated specifiers of neural cell fates; rather, they appear to be very effective in first conferring on a group of cells the potential to adopt a particular cell fate and then promoting the selection of an individual committed progenitor from within that group. This suggests the existence of a 'core' set of genes that function downstream of the proneural proteins in all such contexts, with other sets of genes contributing to context-dependent (e.g., germ layer-specific) programs. Further investigation of the genes identified in this study should permit a test of this intriguing hypothesis (Reeves, 2005).

Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology

Neurons establish diverse dendritic morphologies during development, and a major challenge is to understand how these distinct developmental programs might relate to, and influence, neuronal function. Drosophila dendritic arborization (da) sensory neurons display class-specific dendritic morphology with extensive coverage of the body wall. To begin to build a basis for linking dendrite structure and function in this genetic system, da neuron axon projections were analyzed in embryonic and larval stages. It was found that multiple parameters of axon morphology, including dorsoventral position, midline crossing and collateral branching, correlate with dendritic morphological class. A class-specific medial-lateral layering of axons in the central nervous system formed during embryonic development was identified; this layering allows different classes of da neurons to develop differential connectivity to second-order neurons. The effect of Robo family members on class-specific axon lamination was examined, and a forward genetic approach has also been taken to identify new genes involved in axon and dendrite development. For the latter, the third chromosome was screened at high resolution in vivo for mutations that affect class IV da neuron morphology. Several known loci, as well as putative novel mutations, were identified that contribute to sensory dendrite and/or axon patterning. This collection of mutants, together with anatomical data on dendrites and axons, should begin to permit studies of dendrite diversity in a combined developmental and functional context, and also provide a foundation for understanding shared and distinct mechanisms that control axon and dendrite morphology (Grueber, 2007).

Drosophila dendritic arborization (da) neurons have been segregated into four classes (classes I-IV) that reflect arbor complexity, arbor size and the length of terminal branches. The cell bodies are distributed in ventral, ventral', lateral and dorsal clusters between the epidermis and muscles, spreading dendrites across the body wall, and axons to the ventral nerve cord. It was reasoned that if morphological classes correspond to at least partially distinct sensory systems, then their axons may have divergent morphologies and target non-overlapping regions of the ventral nerve cord, where information will be relayed to second-order neurons. Previous studies have found that most da neurons arborize together in a common fascicle in the ventral CNS, with a subset, including at least some class I da neurons, projecting to more dorsal neuropil. In light of studies showing distinct morphological types of da neurons, mosaic analysis with a repressible cell marker (MARCM) was used to examine the morphology of da neuron dendrites and axons in third instar larvae. As a MARCM driver Gal4109(2)80 was used; this labels all multidendritic (md) sensory neurons, including those belonging to the da subgroup. Owing to the sparse labeling of central neurons, the 109(2)80 driver combined with MARCM allowed resolution of axon morphology of individual neurons (Grueber, 2007).

Data was collected from wild-type da neuron clones in segments A2-A7 to identify their axon projections in the CNS. Different da classes showed distinctive types of central projections. Class I neurons were unique in their projection to the dorsal neuropil. Class II axons showed collateral branches (branches exiting from the main axonal trunk, although the timing of their emergence has not been determined) that were not observed in class III and IV neurons. The class I neuron vpda also showed such a branch. Class IV neurons projected axon branches across the midline, but these were only rarely observed for the other classes. Dorsal and ventral' class IV axons crossed the midline, but axons from the ventral neuron did not. Each class IV neuron also showed a large accumulation of branches medial to the commissural/longitudinal branch bifurcation. The class III terminals extended in an anteroposterior (AP) orientation and were relatively unbranched, showing neither the collateral branches observed in class II neurons, nor contralateral projections observed among class IV neurons (Grueber, 2007).

The axons of class I and class IV neurons also showed evidence of somatotopic arrangements in the CNS. The trajectory of class I neurons in the CNS mirrored the polarity of their dendrites in the periphery. Dorsal class I neurons have distinct polarity with respect to the AP body axis: dendrites of ddaD extend anteriorly and dendrites of ddaE extend posteriorly. Likewise, it was found that the ddaD axons extended anteriorly in the CNS, whereas the ddaE axons extended posteriorly. Among the class IV neurons, only neurons positioned in the dorsal and ventral regions of the body wall, but not the lateral region, extended axons across the midline, fitting with principles of somatotopy established for body wall bristle neurons. These data together demonstrate that da neuron classes have distinguishing axon terminals, and that neurons in the same class show evidence of somatotopic organization (Grueber, 2007).

The position of sensory axons defines the population of possible second-order targets and thus contributes strongly to sensory information processing in the CNS. Axons of tactile receptors typically project to ventral areas of the neuropil, whereas strain-sensing or proprioceptive neurons usually project to more dorsal regions. Fasciclin II-labeled axon tracts provide a frame of reference for assessing dorsoventral (DV) position in the CNS. The DV positions of axons was studied in 42 ventral nerve cords (VNCs) using MARCM, and 18 VNCs using the FLP-out system. Both techniques revealed that each class I neuron extended axons to dorsal regions of the neuropil, terminating just lateral to the dorsomedial (DM) fascicle. The position of class I axons was therefore indistinguishable at this level of resolution from the position of the dbd terminal arbor, implying that information from class I neurons and the putative stretch-sensing dbd neuron might be processed similarly in the CNS. Class II, III and IV axons targeted the ventral CNS without obvious class-specific dorsoventral lamination of terminal position. The positions of the class II collateral branches were somewhat variable, either terminating on the ventrolateral (VL) fascicle, or slightly lateral to VL (vdaA often had a more lateral termination). These data together provided anatomical support for distinct functions among different da neurons, fitting with their distinct dendritic arbor morphologies. Class II, III and IV axons project similarly to known tactile afferents, while class I neurons have projections like known proprioceptive or strain-sensing neurons (Grueber, 2007).

Whether the terminal positions of the ventral-projecting class II, III and IV neurons could be further distinguished by their position was examined. Short pickpocket (ppk) enhancer sequences can drive gene expression strongly in all class IV neurons and weakly in class III neurons. Viewing all class IV neurons together revealed that they crossed the midline in a single fascicle, that the stereotyped branching at the commissural-longitudinal junction overlapped for all neurons, and that longitudinal projections were not always tightly fasciculated. In ppk-eGFP and ppk-Gal4, UAS-CD8::GFP animals, a strongly labeled set of medial axons and a weakly labeled, slightly more lateral, layer of terminals were observed. It is suspected that the weakly labeled axons were class III axons, which may form a layer next to class IV axons. To test this idea, ppk-Gal4 was introduced into the FLP-out mosaic system. The relative locations were observed of all class III axons except ddaF (whose axon was labeled too weakly) and it was found that their major longitudinal projections terminated immediately lateral to the scaffold of class IV axons (Grueber, 2007).

The ppk reporter lines alone do not label the class II axons, and thus did not allow determination of whether all da classes form a laminar organization or only the class III and IV neurons. However, examination of FLP-out clones produced with Gal4109(2)80, with or without ppk-eGFP to label class IV neurons, permitted labeling of different axon groups. It was found that class II neurons with a significant longitudinal projection formed a third layer of sensory axons that was lateral to both class III and class IV axons, with class II collateral branches terminating in a distinct, even more lateral, position (Grueber, 2007).

The FLP-out data was confirmed by mapping the relationships of individual pairs of sensory afferents using the MARCM technique. Within hemisegments, or in adjacent hemisegments, having two or more da neuron clones axons were organized (medial>lateral) class IV>class III>class II. Laminar patterning was independent of peripheral cell body position. These data together indicate the presence of a laminar arrangement of somatosensory axons in the Drosophila CNS. These data also suggest that somatosensory information carried by different classes of da neurons might be distinguished by sensory axon connectivity to second-order targets (Grueber, 2007).

The above FLP-out and MARCM data were collected from third instar larval stages, so when during development layering of the different classes of axons could be observed was examined. To achieve live two-color discrimination of different neuronal classes in embryonic and early larval stages transgenic flies were generated expressing a photoconvertible fluorescent protein, Kaede, and expression was placed under the control of Gal4109(2)80 in the presence of ppk-eGFP. The Kaede protein was converted from green to red fluorescence using a 10-30 second UV pulse and the position of all da axons was examined relative to ppk-eGFP-labeled class IV axons. As early as the sensory axon scaffold could be visualized (stage 17), class IV axons occupied a medialmost layer with respect to other classes. These data indicate that a laminar pattern develops at least by late embryonic stages and is maintained without qualitative change in larvae (Grueber, 2007).

Much of the knowledge about somatotopic maps in insect mechanosensory systems derives from studies of bristle afferents with peripheral receptive fields that approximate a point source. Drosophila da neurons have largely overlapping peripheral sensory fields and may, as a group, respond to several distinct stimuli. How is information from this predominant body wall sensory system represented in the CNS and what might this organization reveal about the possible functions of da neurons? Neurons with different dendritic branching morphologies target distinct regions of the CNS, supporting the existence of a modality map of da neuron axons. Evidence is provided for nested somatotopic mapping in class I and class IV da neurons. Individual class I neurons extend their dendritic and axonal arbors in the same preferred direction along the AP body axis. Class IV axons project across the midline according to the cell body position along the DV axis of the body wall, with dorsal and ventral cells, but not more lateral cells, crossing the midline. It is possible that class II and III neurons also project in a somatotopic pattern that was not uncovered by this analysis. Thus, da neuron connectivity appears to incorporate both class and position-specific components, with some apparent correlations with dendritic field orientation (Grueber, 2007).

In the context of sensory processing, these data suggest distinct functions for different morphological classes of da neurons. The class II-IV neurons target a ventral region of the neuropil; thus information from these neurons might be processed similarly to ventral-projecting tactile sensory bristle neurons. Within this ventral region, the class-specific laminar pattern could allow differential connectivity with second-order interneurons. Additionally, class II neurons, with their collateral branches, might provide information to distinct central circuits. The class I neurons targeted a more dorsal region of the neuropil, which is generally a characteristic of proprioceptive afferents in insects. Indeed, a class of da neurons in Manduca has been shown to target dorsal neuropil, and to respond to stretch of the cuticle. Many insect proprioceptors, including chordotonal organs and the bipolar dendrite neurons, have dendrites oriented in a preferential direction relative to the body axis. Notably, the primary dendrites of each class I neuron are oriented dorsally and secondary dendrites are oriented anteriorly and posteriorly. This arrangement could allow larvae to compare distension along major body axes. While the anatomical arrangement of their axons suggests distinct functions for different da neurons, dissecting these different functions will ultimately require behavioral and physiological studies (Grueber, 2007).

A notable feature of the mapping of da sensory afferents is their predominant organization into class-correlated mediolateral layers, with class IV neurons in a medial layer, class III neurons intermediate, and class II neurons most lateral. These layers do not correspond to the medial, intermediate and lateral fascicles that have their position specified by a Robo combinatorial code; thus novel molecular cues may contribute to this laminar organization. Indeed, it has been postulated that the Robo code provides information about the broad zone that a growth cone targets, while a complementary fasciculation code fine-tunes pathway choice within that zone. It is conceivable that Robo proteins could participate in specifying the lateral position of collateral branches, since Robo3 overexpression in individual neurons induced ectopic branching from the axon shaft. Although no cell-type-specific expression of Robo3 was detected in neurons that normally show such branching, it is notable that Robo3 has been implicated in cell-type-specific patterning decisions of PNS axons, and that Slit2 has been proposed as a positive regulator of collateral branching of dorsal root ganglion sensory axons (Grueber, 2007).

The mechanisms for targeting of somatosensory afferents should act to properly position axons of different classes relative to one another. Several alternative mechanisms could contribute to this positioning. One potentially important component of axon sorting could involve interactions between homotypic or heterotypic axons. Heterotypic axons could repel each other to sort to discrete bundles. Likewise, homotypic axons could adhere to one another to ensure that they terminate together. Olfactory receptor neuron axons engage in extensive hierarchical interactions to establish precise targeting in olfactory glomeruli, and dendrites of da neurons engage in class-selective interactions during development to ensure their proper spacing. It is therefore conceivable that intraclass and interclass interactions could participate in the sorting of somatosensory axons. Axons from different classes could also project to specific layers that are prepatterned by the processes of target interneurons or by other axons. Finally, the projection of da axons to different layers could conceivably reflect a temporal order of sensory axon arrival in the CNS, similar to the three-way correlation between mediolateral axon position, physiological function and time of differentiation among Drosophila wing campaniform sensilla. Among the ventral cluster da neurons, a group with differentiation that has been examined in the greatest detail, the class II neurons appear to be the first-born, followed by class III neurons and class IV neurons. These data suggest a possible correlation between birth order and axon position in the neuropil, although additional early markers of da neurons and further high-resolution imaging studies are required to further test this scenario (Grueber, 2007).

The molecular basis for insect sensory neuron differentiation, as well as anatomical studies of somatosensory axon mapping and VNC circuitry, have been subjects of considerable study, and principles are emerging that link the two areas into molecular models of connectivity and synaptic specificity. Among embryonic sensory neurons in Drosophila, there is a three-way correlation between soma position, proneural transcription factor expression and axon projection pattern, suggesting that these transcription factors may endow aspects of modality-specific axonal projections. Such a link was recently established between chordotonal-organ-specific expression of the atonal proneural gene and expression of the Robo3 axon guidance molecule in these same organs. Misexpression experiments with atonal, robo3 and comm suggest a model whereby Atonal activates expression of Robo3, which in turn specifies mediolateral positioning in chordotonal versus bipolar dendrite-type axon projections. These studies provide an important basis for understanding the establishment of sensory circuitry in the VNC (Grueber, 2007).

To begin to address the molecular basis of axon and dendrite patterning using the anatomical framework established for the da neurons, a forward genetic approach, which has proven a successful means to identify regulators of neuronal morphogenesis, was undertaken. A strength of this screen was the ability to simultaneously assess phenotypes in dendrites and axons at the level of single identifiable neurons. Numerous complementation groups were identified that affect axon patterning, including several mutations with a molecular nature as yet unknown. These mutations should allow identification of new genes involved in axon morphogenesis and place these into the context of their effects on somatosensory axon patterning and circuitry. Given that many mutations affecting dendrite morphogenesis have been identified, it is expected that studies of the mutations identified from the screen will also allow addressing of the similarities and distinctions between axon and dendrite development (Grueber, 2007).

doublesex functions early and late in gustatory sense organ development

Somatic sexual dimorphisms outside of the nervous system in Drosophila melanogaster are largely controlled by the male- and female-specific Doublesex transcription factors (DSXM and DSXF, respectively). The DSX proteins must act at the right times and places in development to regulate the diverse array of genes that sculpt male and female characteristics across a variety of tissues. To explore how cellular and developmental contexts integrate with doublesex (dsx) gene function, this study focused on the sexually dimorphic number of gustatory sense organs (GSOs) in the foreleg. Tha DSXM and DSXF were shown to promote and repress GSO formation, respectively, and their relative contribution to this dimorphism varies along the proximodistal axis of the foreleg. The results suggest that the DSX proteins impact specification of the gustatory sensory organ precursors (SOPs). DSXF then acts later in the foreleg to regulate gustatory receptor neuron axon guidance. These results suggest that the foreleg provides a unique opportunity for examining the context-dependent functions of DSX (Mellert, 2012).

dsx regulates the sexually dimorphic number of GSOs across all tarsal segments of the foreleg: DSXM promotes and DSXF represses the development of certain GSOs. The effects of this regulation are apparent by 8 h APF, when the GSOs are first identified, and the spatiotemporal pattern of DSX implies that dsx determines the number of gustatory SOPs. dsx exhibits a surprising degree of context sensitivity: the relative importance of DSXM and DSXF varies along the proximodistal axis of the foreleg and, during the course of GSO development, DSXF progresses from regulating cell fate to regulating axon guidance (Mellert, 2012).

Given that dsx controls the formation of the other sexually dimorphic cuticular structures of the fly, as well as the number of GSOs in segment T1 of the foreleg, it was anticipated that dsx would regulate the sex-specific GSO numbers in segments T2–T4 of the foreleg. However, the manner in which this regulation is achieved across the tarsal segments was surprising. Although each of the T1–T4 foreleg tarsal segments produces more GSOs in males than in females, in two segments this difference is achieved by promoting formation of several GSOs in males (via the action of DSXM), in one segment it is achieved by repressing the formation of several GSOs in females (via the action of DSXF), and in another segment, both DSXM and DSXF act to regulate GSO number. This is more complicated than the simpler a priori expectation that the function of dsx would be the same across the T1–T4 foreleg segments (Mellert, 2012).

That DSXM and DSXF can be utilized differentially has been previously established. In the fat body, female-specific expression of Yp1 and Yp2 depends on up-regulation by DSXF in females and down-regulation by DSXM in males. Thus, in dsx null flies, both sexes express these genes at equivalent levels. Similarly, DSXF activates and DSXM represses expression at the bric-a-brac locus to generate sex-specific pigmentation in the abdominal epithelium. In these two cases, both DSX proteins contribute to regulation of a single trait, similar to the regulation of GSO number in T2. In contrast, desatF is activated by DSXF in oenocytes to produce female-specific pheromones without influence from DSXM. This single isoform-mediated regulation bears similarity to the regulation of foreleg GSOs in T1, T3 and T4. Whereas the previous studies found that DSXM or DSXF were differentially utilized to sculpt sexually dimorphic traits arising from developmentally distinct tissues, this study found that these transcription factors can be differentially utilized across a single developmental field–the epithelium of the foreleg disc. Moreover, the differential roles of DSXM and DSXF in different tarsal segments suggest that each segment may have independently evolved a molecular mechanism for integrating sexual and proximodistal axis information within the foreleg disc to produce more GSOs in the male (Mellert, 2012).

Attempts were made to determine when the function of dsx impacts neurogenesis to generate the numbers of GSOs. Although the details of foreleg GSO development have not been specifically reported, studies of the mechanosensory macrochaete lineages of the notum provide a basic framework for the multi-step process of sensory organ neurogenesis. The initiating event is patterned expression of the proneural genes ac and sc, which imparts the potential to produce SOPs to specific clusters of epithelial cells across the disc epithelium. Subsequent cell-cell interactions within the cluster typically specify a single SOP. The nascent SOP must then sustain its fate and undergo a series of stereotyped cell divisions to produce all of the cells of the sensory organ. Any of the molecular processes that underlie these stages could be influenced by the functions of dsx (Mellert, 2012).

Of interest was the broad distribution of the DSX proteins across the T1–T4 foreleg disc epithelium before and at 0 h APF, a time when the gustatory SOPs are specified. Because the number of DSX-positive cells far exceeded the number of gustatory SOPs necessary to give rise to the GSOs, it was inferred that dsx is acting prior to or during SOP formation. This is consistent with the frequent colocalization of DSX with AC in proneural clusters, which suggests that dsx might act within these cells to determine whether the SOP fate is promoted in males or repressed in females. The broad distribution of the DSX proteins could ensure that sexual information is available for integration with positional information across the foreleg disc epithelium to guide sexually dimorphic development (Mellert, 2012).

In contrast to T2–T4, DSX was not apparent in the foreleg epithelium of T5, which produces a sexually monomorphic GSO number. Thus, the presence of DSX in the epithelium correlates with the adult sexual dimorphism in GSO number, consistent with the notion that dsx is expressed at the right time and place to impact SOP selection in the foreleg. However, at 0 h APF DSX was observed in two nascent sensory organs expressing ase-lacZ. It is speculated that these sensory organs correspond to the GSOs containing GRNs that express pickpocket 25 (ppk25), which is enriched in males and required for their normal response to female pheromones. Thus, the presence of DSX in the nascent GSOs may forecast sexually dimorphic gene expression in the adult GSO (Mellert, 2012).

In addition to specifying foreleg GSO numbers, a temporally distinct function was observed for dsx in the GRNs. During pupal development, GRN axons project proximally along the leg nerves and into the VNC, and here the behavior of the axon depends on the activities of FRUM or DSXF. In males, FRUM promotes crossing of the VNC midline by the axons, but in females, DSXF represses this behavior. This dual regulation causes GRN axons to project across the VNC midline only in males. Two competing hypotheses have been proposed to explain the action of DSXF: 1) DSXF directly affects axon guidance in differentiating GRNs; or 2) only male-specific GRNs are competent to cross the VNC midline and DSXF indirectly affects midline crossing by repressing formation of the male-specific GSOs. Having shown that post-mitotic expression of DSXF in FruM-expressing GRNs subsequent to the establishment of GSO number prevents midline crossing, the second hypothesis is now rejected. Moreover, the early sexual information that impacts GSO number does not irreversibly determine sex-specific development of the GRNs as they continue to be sensitive to the action of DSXF (and presumably FRUM). Because dsx and fru are classically thought of as acting in parallel, it was intriguing to find both genes regulating the same phenotype in a common set of GRNs. Determining whether they coregulate a common set of target genes or independently regulate distinct targets will be of great interest (Mellert, 2012).

Because DSXM and DSXF differentially impact GSO numbers in different tarsal segments, and DSXF regulates the later process of axon guidance, the identity of the genes directly regulated by dsx during foreleg development likely changes with spatiotemporal context. Although it is currently unknown which genes are directly regulated by dsx in the foreleg epithelium or the GSO lineage, the available data on in vivo DSX binding sites may reveal genes that are known to be involved in peripheral neurogenesis or axon guidance. The challenge will then be to determine if such candidates exhibit sexually dimorphic expression in the different tarsal segments at different developmental time points. In this way, development of the foreleg GSOs presents a unique opportunity for investigating how dsx function is integrated with spatiotemporal context across a changing developmental landscape (Mellert, 2012).

Sound response mediated by the TRP channels NOMPC, NANCHUNG, and INACTIVE in chordotonal organs of Drosophila larvae

Mechanical stimuli, including tactile and sound signals, convey a variety of information important for animals to navigate the environment and avoid predators. Recent studies have revealed that Drosophila larvae can sense harsh or gentle touch with dendritic arborization (da) neurons in the body wall and can detect vibration with chordotonal organs (Cho). Whether they can also detect and respond to vibration or sound from their predators remains an open question. This study reports that larvae respond to sound of wasps and yellow jackets, as well as to pure tones of frequencies that are represented in such natural sounds, with startle and burrowing behaviors. The larval response to sound/vibration requires Cho neurons and, to a lesser extent, class IV da neurons. Calcium imaging and electrophysiological experiments reveal that Cho neurons, but not class IV da neurons, are excited by natural sounds or pure tones, with tuning curves and intensity dependence appropriate for the behavioral responses. Furthermore, this study implicates the transient receptor potential (TRP) channels NOMPC, NANCHUNG, and INACTIVE, but not the dmPIEZO channel, in the mechanotransduction and/or signal amplification for the detection of sound by the larval Cho neurons. These findings indicate that larval Cho, like their counterparts in the adult fly, use some of the same mechanotransduction channels to detect sound waves and mediate the sensation akin to hearing in Drosophila larvae, allowing them to respond to the appearance of predators or other environmental cues at a distance with behaviors crucial for survival (Zhang, 2013).

The ability to sense mechanical stimuli that indicate potential harm is important for survival. Drosophila larvae use their mechanosensory neurons to sense the mechanical pain caused by a predator attack. The da neurons on the body wall are capable of sensing gentle and harsh touch, allowing larvae to move away from harm. Their survival could be further enhanced if larvae could detect signals such as sound from predators at a distance. The results show that Drosophila larvae exhibit startle behavior in response to certain frequencies of sound, including the sound from predators such as wasps and yellow jackets. This startle behavior and ensuing escape or avoidance behavior may increase a larva's chance of survival. Interestingly, Drosophila larvae are highly sensitive to low-frequency sounds but not to high-frequency sounds, unlike some other insects that can detect high-frequency sounds including ultrasonic sounds. This diversity in hearing might reflect evolutionary adaptation to different predators for organisms ranging from insects to bats, and might entail interspecies differences at both structural and molecular levels (Zhang, 2013).

Although both Cho neurons and class IV da neurons are involved in the sound-triggered startle response, only Cho neurons are sensitive to sound. Class IV da neurons may have modulatory effects on the neural circuits activated by the Cho neuronal response to sound -- a likely scenario, considering that class IV da neurons mediate avoidance behaviors to several noxious stimuli. The startle response and avoidance of sound also may depend on this neural circuit for avoidance behaviors. Alternatively, class IV da neurons may contribute to the behavioral response through their involvement in peristalsis (Zhang, 2013).

Several TRP channels have been implicated in hearing and touch sensation in Drosophila, although the roles of these channels in mechanotransduction may differ in different sensory neurons. For example, NOMPC is critical for touch sensation but IAV and NAN are not, whereas IAV and NAN are important for adult hearing. With respect to larval Cho neurons, it appears that IAV and NAN are required for sound transduction, whereas NOMPC function is important, but not essential, for the detection of loud sound. A possible model is one in which NOMPC serves as one of the primary sensors for sound and enhances the movement of the Cho neuronal cilium to activate IAV and NAN, which may be able to sense loud sound on their own in the absence of NOMPC. An alternative model has been suggested for the adult Johnston organs, which may use IAV and NAN rather that NOMPC as the primary sensor (Zhang, 2013).

Given that the cytoplasmic calcium indicator G-CaMP5 might not be localized to the small structure within the tip of the cilium, the Ca2+ imaging method in these experiments might not be sufficiently sensitive to detect Ca2+ influx at the site of mechanotransduction. Thus, the absence of a Ca2+ signal in Cho neurons might be attributed to the lack of downstream amplification. dmPIEZO, one of the first mechanotransduction channels identified for mechanical nociception in Drosophila larvae, appears to have no involvement in hearing, suggesting that larvae make use of different channels for different modalities of mechanosensation (Zhang, 2013).

Recent microarray studies have identified hundreds of genes implicated in the hearing of adult flies. Many of these genes also have been implicated in other sensory modalities besides hearing. A major challenge is the difficulty of recording from a single neuron in the adult antenna. The larval Cho neurons are accessible to electrophysiological recording at single-cell resolution. Moreover, the entire structure of a Cho neuron can be imaged simultaneously in vivo. In conjunction with the extensive genetic resources available, larval Cho neurons lend themselves to mechanistic studies of mechanotransduction for hearing in Drosophila (Zhang, 2013).

Transmembrane channel-like (tmc) gene regulates Drosophila larval locomotion

Drosophila larval locomotion, which entails rhythmic body contractions, is controlled by sensory feedback from proprioceptors. The molecular mechanisms mediating this feedback are little understood. By using genetic knock-in and immunostaining, this study found that the Drosophila melanogaster transmembrane channel-like (tmc) gene is expressed in the larval class I and class II dendritic arborization (da) neurons and bipolar dendrite (bd) neurons, both of which are known to provide sensory feedback for larval locomotion. Larvae with knockdown or loss of tmc function displayed reduced crawling speeds, increased head cast frequencies, and enhanced backward locomotion. Expressing Drosophila TMC or mammalian TMC1 and/or TMC2 in the tmc-positive neurons rescued these mutant phenotypes. Bending of the larval body activated the tmc-positive neurons, and in tmc mutants this bending response was impaired. This implicates TMC's roles in Drosophila proprioception and the sensory control of larval locomotion. It also provides evidence for a functional conservation between Drosophila and mammalian TMCs (Guo, 2016).

The role of PPK26 in Drosophila larval mechanical nociception

In Drosophila larvae, the class IV dendritic arborization (da) neurons are polymodal nociceptors. This study shows that ppk26 (CG8546) plays an important role in mechanical nociception in class IV da neurons. Immunohistochemical and functional results demonstrate that ppk26 is specifically expressed in class IV da neurons. Larvae with mutant ppk26 showed severe behavioral defects in a mechanical nociception behavioral test but responded to noxious heat stimuli comparably to wild-type larvae. In addition, functional studies suggest that ppk26 and ppk (also called ppk1 or pickpocket) function in the same pathway, whereas Piezo functions in a parallel pathway. Consistent with these functional results, Ppk and Ppk26 are interdependent on each other for their cell surface localization. This work indicates that Ppk26 and Ppk might form heteromeric DEG/ENaC channels that are essential for mechanotransduction in class IV da neurons (Guo, 2014).

The similar cellular functions and behavioral outputs of class IV da neurons in fruit flies and polymodal nociceptors in C. elegans and mammals suggest that they may share similar molecular mechanisms. The current findings confirmed that the DEG/ ENaC channels PPK26 and PPK participate in mechanical nociception in Drosophila larvae. In addition, DEG/ENaC channels are required for response to harsh touch in C. elegans. The role of mammalian DEG/ENaC channels in mechanical nociception remains unclear, because no consistent behavioral defects have been observed in genetic knockout mice, and need to be further clarified. The genetic interaction experiments and surface expression experiments of this study indicated that PPK26 and PPK might translocate to the plasma membrane with each other after they interact and they might form heteromeric channels. The relationship between acid-sensing ion channels (ASICs) in dorsal root ganglion neurons should also be examined to clarify whether, like PPK and PPK26, they are interdependent for their surface expression and whether they form heteromeric channels or work redundantly in mechanical nociception. Unlike ppk and ppk26, this study found that piezo and ppk26 work in parallel pathways. It is reasonable to wonder why mechanical nociception involves two pathways: the ppk pathway and piezo pathway. One possibility is that the two different pathways might respond to forces of different intensity. However, the results of the current behavioral experiments do not support this idea, because mutation of either pathway caused behavioral defects in response to a wide range of forces. Another possibility, is that the sensations of noxious mechanical stimuli are so vital that animals have evolved two redundant pathways to increase survival and provide an evolutionary advantage (Guo, 2014).

There is still no clear evidence that mechanogated ion currents can be detected when DEG/ENaC channels are ectopically expressed in heterogeneous systems. PPK and PPK26 were ectopically coexpressed in human embryonic kidney 293T and Drosophila S2 cells, but unfortunately, no obvious mechanosensitive ion currents were recorded from these cells. In situ whole-cell patch recording was performed on larval class IV da neurons, but no mechanosensitive ion currents were detected in response to mechanical displacements toward the dendritic area of up to 100 mM. It is possible that they are pore-forming subunits but that they need much larger mechanical forces to activate than that was given, because nociceptors are usually activated with a high threshold. PPK has also been reported to be activated by acid, similar to mammalian ASICs. It is possible that DEG/ENaC channels might be activated indirectly by acids in mechanical nociception. The gating mechanism of DEG/ENaC channels in mechanotransduction is still not fully understood, and more thorough studies are needed to address this question (Guo, 2014).

Nociceptor-enriched genes required for normal thermal nociception

This study describe a targeted reverse genetic screen for thermal nociception genes in Drosophila larvae. Using laser capture microdissection and microarray analyses of nociceptive and non-nociceptive neurons, 275 nociceptor-enriched genes were identified. A side-by-side comparison was carried out of the normalized hybridization intensity between class IV and class I neurons for all Affymetrix probe sets, and the selected genes showed a greater than 2-fold higher expression in class IV neurons in comparison to class I neurons. The function of the enriched genes were tested with nociceptor-specific RNAi and thermal nociception assays. Tissue-specific RNAi targeted against 14 genes caused insensitive thermal nociception while targeting of 22 genes caused hypersensitive thermal nociception. Previously uncategorized genes were named for heat resistance (i.e., boilerman (CG12681), fire dancer (CG8968), oven mitt (CG31976), trivet (CG12870), thawb (CG14608), and bunker gear (CG8297)) or heat sensitivity (firelighter (CG14946), black match (CG12524), eucalyptus (CG12269), primacord (CG15704), jet fuel (CG12858), detonator (CG14446), gasoline (CG6018), smoke alarm (CG13988), and jetboil (CG4398)). Insensitive nociception phenotypes were often associated with severely reduced branching of nociceptor neurites and hyperbranched dendrites were seen in two of the hypersensitive cases. Many genes that were identified are conserved in mammals (Honjo, 2016).

Coordination and fine motor control depend on Drosophila TRPγ

Motor coordination is broadly divided into gross and fine motor control, both of which depend on proprioceptive organs. However, the channels that function specifically in fine motor control are unknown. This study shows that mutations in trpγ disrupt fine motor control while leaving gross motor proficiency intact. The mutants are unable to coordinate precise leg movements during walking, and are ineffective in traversing large gaps due to an inability in making subtle postural adaptations that are requisite for this task. TRPγ is expressed in proprioceptive organs, and is required in both neurons and glia for gap crossing. TRPγ was expressed in vitro, and its activity was found to be promoted by membrane stretch. A mutation eliminating the Na+/Ca+ exchanger suppresses the gap-crossing phenotype of trpγ flies. These findings indicate that TRPγ contributes to fine motor control through mechanical activation in proprioceptive organs, thereby promoting Ca+ influx, which is required for function (Akitake, 2015).

Even the most basic tasks, such as acquiring food, locating safe places to rest, avoiding and defending against enemies, and mating requires motile animals to navigate through their environment by moving multiple body parts in a highly coordinated manner. To move fluidly, both vertebrate and invertebrate animals employ complex mechanosensory organs that are designed to gather and interpret feedback information about their movement in real time through an array of specialized receptors and neural networks. These proprioceptive sensory systems provide animals with continuously updated maps of their body positions that are critical for balance and locomotion (Akitake, 2015).

Proprioception is mediated at the cellular level, by stretch-sensitive cells located in muscles, ligaments and joints, which are activated by mechanical forces. In humans, damage to proprioceptive afferents results in a variety of movement disorders such as spasticity, impaired load sensitivity, and altered gait. Proprioceptive dysfunction is also a clinical feature of diseases that affect the nervous system such as Parkinson's disease (Akitake, 2015).

The worm, C. elegans, and the fruit fly, Drosophila melanogaster, have served as animal models for characterizing proprioception. Both of these organisms display highly stereotypic locomotion, which has facilitated the identification of neurons and ion channels that function in proprioception. In flies, proprioceptive neurons are located in specialized sensory structures-mechanosensory bristles, campaniform sensilla, and chordotonal organs. Several invertebrate members of the transient receptor potential (TRP) family of cation channels localize to proprioceptive cells and contribute to sensing bodily movements during locomotion. These include the C. elegans and Drosophila TRPN channels, TRP-4 and NOMPC, respectively, which are required for worms and fly larvae to make gross postural changes during locomotion. Most NompC mutant animals die during the pupal stage. The few mutant animals that survive to adulthood exhibit severe locomotion defects and uncoordinated movement of body parts, indicative of defects in gross motor control. Mutations disrupting the Drosophila TRPV channels, Inactive (Iav) and Nanchung (Nan) also result in severe locomotor defects (Akitake, 2015).

A key question is whether ion channels exist that specifically function in fine motor control. In flies, a defect in fine motor control would not eliminate behaviours that rely principally on gross movements of the body and appendages, such as negative geotaxis, or crossing small gaps. However, loss of fine motor control would be expected to impair performance when the flies are faced with highly challenging tasks, such as traversing wide gaps, which rely on coordinating a repertoire of fine movements, including subtle changes in body angles and leg positions (Akitake, 2015).

The Drosophila genome encodes 13 TRPs, 12 of which have been subjected to genetic analyses. The recurring theme is that these channels are essential for sensory physiology. However, the function of one Drosophila TRP channel, TRPγ, is not known. TRPγ is a TRPC channel, and is most related to the founding TRP channel. This study demonstrates that TRPγ is localized to neurons and glia that comprise the femoral chordotonal organs (FCOs). trpγ null mutant flies were generated and were found to be distinct from the nan and iav mutants in that they displayed much greater levels of negative geotaxis and were proficient in crossing small gaps. However, once the gaps became challenging but were still surmountable for most wild-type flies, the trpγ mutants were unable to make the fine postural adaptations required for negotiating these gaps. Thus, this phenotype sharply contrasted with the loss of other TRP channels that impact on proprioception, as TRPγ was uniquely required to promote this highly coordinated motor control. These data demonstrate that fine motor control is not mediated exclusively through the same repertoire of cation channels that function in gross motor control (Akitake, 2015).

In humans, motor control is categorized into two major types-gross motor skills, required for large body movements such as sitting upright or waving an arm, and fine motor skills, necessary for small precise movements such as picking up and manipulating objects. In Drosophila, gross motor coordination is essential for the large rhythmic movements of the body and limbs during general locomotion (walking), while fine motor coordination is critical for making small changes in the angles and positions of the body and appendages to complete difficult tasks, such as righting or navigating gaps in a terrain (Akitake, 2015).

Three Drosophila TRP channels are expressed in proprioceptive organs (Iav, Nan and NOMPC) and elimination of any of these proteins have severe effects on locomotion and gravity sensation. Owing to the major locomotor deficits resulting from the loss of any of these channels, fine motor control is also profoundly affected (Akitake, 2015).

Before the current study, it was unclear whether there existed cation channels that contribute exclusively to highly coordinated movements of the body and appendages. In principle, it was possible that the two types of motor coordination depended on the same repertoire of channels (for example, Iav and Nan), and that null mutations would strongly impair all types of coordinated movements, while hypomorphic mutations would affect fine motor control only (Akitake, 2015).

This study found that TRPγ was required exclusively for fine motor behaviours. In high-frame rate video analysis, both wild-type and trpγ flies traversed the catwalk at a relatively fixed maximum speed, although the trpγ mutants walked more slowly and displayed decreased precision in their leg placements. However, the trpγ mutants used consistently shortened steps, which differed from the abnormally long and highly variable step lengths exhibited by nan mutants11 (Akitake, 2015).

The impairments in fine motor control exhibited by trpγ flies compromised their ability to cross challenging gap sizes. While the mutant animals negotiated gaps of up to 3.0 mm as well as wild type, they were ineffective in traversing larger gaps. This defect was not due to smaller flies, since the lengths of the trpγ bodies were similar to wild-type animals. It is proposed that the impairment in gap-crossing arises from the inability of the mutants to precisely sense their body position and make the fine postural adjustments required to complete the task. Indeed, the trpγ flies were unable to increase their body angles towards the horizontal position, even as they made successive leg-over-head sweeps. Consequently, they could not fully extend the reach of their front legs to bridge the gap. In sharp contrast, nan and iav mutants were not able to effectively cross even short gaps that virtually all trpγ and wild-type flies were able to negotiate (Akitake, 2015).

The majority of work on Drosophila proprioceptive organs has focused on the contribution of the mechanosensory neurons to motor control. Unexpectedly, this study found that TRPγ was expressed and functioned in both neurons and in glial support cells, called scolopale cells. However, the requirement for TRPγ in neurons appeared to be more significant. RNAi knockdown of trpγ in neurons induced a gap-crossing deficit nearly as severe as the null mutations, while RNAi knockdown of trpγ in scolopale cells, caused a more modest effect. Moreover, the gap-crossing impairment exhibited by trpγ was rescued to a greater extent after re-introducing the wild-type transgene in neurons than in scolopale cells. When the effects of the trpγ mutation on the leg motor circuit were assayed, using EMGs, the deficit in sensitivity was rescued only after expressing the wild-type transgene in neurons. Nevertheless, TRPγ has a dual role in both neurons and scolopale cells, and this is an additional feature that distinguishes TRPγ from TRPs that function in gross motor control (Akitake, 2015).

In neurons, the spatial distribution of TRPγ is different from that of Iav, Nan and NOMPC, consistent with the distinct roles of these channels in promoting fine and gross motor control, respectively. In contrast to the cilia-restricted localizations of Iav, Nan, and NOMPC, detected TRPγ was detected throughout the neuronal cell bodies and dendrites (Akitake, 2015).

It is proposed that TRPγ functions in chordotonal neurons to sense joint movements needed for fine motor control. In support of this proposal, TRPγ was activated directly by membrane stretch in vitro, and expressed in the dendrites. Furthermore, the TRPγ-dependent Ca2+-influx contributes to function, since the severity of the behavioural phenotype was suppressed by eliminating the Na+/Ca2+-exchanger, CalX (Akitake, 2015).

An additional question concerns the potential role of TRPγ in the scolopale cells. This study found that the extensive vacuole network of the mutant scolopale cells in the FCO was reduced in size compared with the wild-type. Various mechanosensors contribute to maintaining vacuolar structures in cells and growing evidence suggests that TRP channels play critical roles in regulating cell size and shape. This raises the possibility that TRPγ plays a similar role in these support cells, which in turn helps maintain the structural stability of the mechanosensory organs (Akitake, 2015).

In summary, this study employed high-frame rate video microscopy of fly locomotive behaviours to identify a requirement for a mechanosensitive TRP channel, TRPγ, for fine motor control. The demonstration that a Drosophila channel functions specifically to promote precise body movements raises the question as to whether there exist mechanosensitive Ca2+-permeable channels in mammals that are uniquely required for fine motor control (Akitake, 2015).

Neuroendocrine control of Drosophila larval light preference

Animal development is coupled with innate behaviors that maximize chances of survival. This study shows that the prothoracicotropic hormone (PTTH), a neuropeptide that controls the developmental transition from juvenile stage to sexual maturation, also regulates light avoidance in Drosophila melanogaster larvae. PTTH, through its receptor Torso, acts on two light sensors (the Bolwig's organ and the peripheral class IV dendritic arborization neurons) to regulate light avoidance. PTTH was found to concomitantly promote steroidogenesis and light avoidance at the end of larval stage, driving animals toward a darker environment to initiate the immobile maturation phase. Thus, PTTH controls the decisions of when and where animals undergo metamorphosis, optimizing conditions for adult development (Yamanaka, 2013)

Animal development is associated with multiple primitive, innate behaviors, allowing inexperienced juveniles to choose an environment that maximizes their survival fitness before the transition to adulthood. In insects, this transition is timed by a peak of ecdysone production induced by the prothoracicotropic hormone (PTTH). In the larval brain of Drosophila, PTTH is produced by two pairs of neurosecretory cells projecting their axons onto the prothoracic gland (PG), where ecdysone is produced. Transition to adulthood is associated with drastic changes in larval behavior: Feeding larvae remain buried in the food, whereas wandering larvae (at the end of larval development) crawl out and find a spot where they immobilize and pupariate. Mechanisms allowing proper coordination of these behavioral changes with the developmental program remain elusive (Yamanaka, 2013)

Two pairs of neurons in the central brain were recently reported to control larval light avoidance. Using specific antibodies to PTTH, this study established that these neurons labeled by the NP0394-Gal4 and NP0423-Gal4 lines correspond to the PTTH-expressing neurons. Moreover, silencing the ptth gene by using NP0423-Gal4 or a ubiquitous driver (tub-Gal4) impaired light avoidance, indicating that PTTH itself controls this behavior. PTTH activates Torso, a receptor tyrosine kinase whose knockdown in the PG prevents ecdysone production and induces a developmental delay. In contrast, knocking down torso in the PG did not cause any change in light avoidance, indicating that the role of PTTH in ecdysteroidogenesis is functionally distinct from its role in light avoidance behavior (Yamanaka, 2013)

Because in Drosophila the PTTH-producing neurons only innervate the PG, it was reasoned that PTTH is secreted into the hemolymph and reaches the cells or organs involved in light avoidance. Consistent with this, inactivation of PTTH-expressing neurons affects light avoidance with 8 to 10 hours delay, arguing against PTTH neurons projecting directly on their target cells to control light avoidance. PTTH peptide is present in the PTTH-expressing neurons throughout larval development and shows a marked increase before wandering, correlating with the rapid increase of ecdysteroidogenesis at this stage. Using an enzyme-linked immunosorbent assay (ELISA), it was found that PTTH is readily detected in the hemolymph with a fluctuation pattern similar to that of its accumulation in the PTTH-expressing neurons. Furthermore, hemolymph PTTH levels were significantly decreased upon RNA interference (RNAi)–mediated knockdown of ptth in the PTTH-expressing neurons, suggesting that in addition to the paracrine control of ecdysteroidogenesis in the PG, PTTH also carries endocrine function (Yamanaka, 2013)

Pan-neuronal knockdown of torso (elav>torso-RNAiGD) recapitulates the loss of light avoidance observed upon torso ubiquitous knockdown (tub>torso-RNAiGD), suggesting that PTTH acts on neuronal cells to control light avoidance. The potential role of torso was specifically tested in two neuronal populations previously identified as light sensors in Drosophila larvae: (1) the Bolwig's organ (BO) and (2) the class IV dendritic arborization (da) neurons tiling the larval body wall. An enhancer trap analysis of torso, as well as in situ hybridization using a torso antisense probe, confirmed torso expression in class IV da neurons. In parallel, torso transcripts were detected by means of quantitative reverse transcription polymerase chain reaction in larval anterior tips containing the BO, and their levels were efficiently knocked down by using the BO-specific drivers Kr5.1-Gal4 and Rh5-Gal4, demonstrating torso expression in the BO. The knockdown of torso in the BO (Kr5.1>torso-RNAiGD and GMR>torso RNAiGD) or in the class IV da neurons (ppk>torso-RNAiGD) abolished larval light avoidance (motoneurons serve as a negative control: OK6>torso-RNAiGD). Knocking down torso in both neuronal populations (ppk>, GMR>torso-RNAiGD) mimicked the effect observed with the BO driver or class IV da neuron driver alone. A similar loss of light avoidance was observed when these neurons were separately inactivated by expressing the hyperpolarizing channel Kir2.1 (GMR>Kir2.1 and ppk>Kir2.1), suggesting that both of these light sensors are necessary for light avoidance behavior. Down-regulation of PTTH/Torso signaling did not lead to any neuronal morphology or locomotion defect, further indicating its direct effect on light sensing. The knockdown of torso in class IV da neurons or in the BO had no effect on the pupariation timing. Taken together, these results indicate that PTTH/Torso signaling is required for light avoidance behavior in two distinct populations of light-sensing neurons and that this function is separate from its role in controlling developmental progression (Yamanaka, 2013)

Drosophila light-sensing cells use photosensitive opsins that upon exposure to light, activate transient receptor potential (TRP) cation channels, thus depolarizing the membrane and triggering neural activation. Although the BO and class IV da neurons use different photosensitive molecules and TRP channels, one can assume that PTTH/Torso signaling regulates the phototransduction pathway through a similar mechanism in both types of neurons. Immunohistochemical detection of Rh5, the opsin involved in light avoidance behavior in the BO, showed no difference in protein level in torso mutant background. PTTH/Torso signaling knockdown did not change the expression level of Gr28b, a gustatory receptor family gene that plays an opsin-like role in class IV da neurons. These results strongly suggest that PTTH affects signaling components downstream of the photoreceptors (Yamanaka, 2013)

The neural activity of the light sensors was investigated using the calcium indicator GCaMP3 for live calcium imaging. torso mutant class IV da neurons showed a 25% reduction of their response to light as compared with that of control. This was accompanied by a loss of light avoidance, indicating that such partial reduction of the GCaMP3 signal corresponds to a reduction of neural activity strong enough to exert a behavioral effect. Indeed, blocking the firing of class IV da neurons by using TrpA1-RNAi caused a similar 25% reduction of the GCaMP3 signal and behavioral effect. This suggests that in da neurons, PTTH/Torso signaling exerts its action upstream of TrpA1 channel activation. Accordingly, a strong genetic interaction was observed between torso and TrpA1 mutants for light preference. A genetic interaction between torso and Rh5 mutants was also detected, further supporting that PTTH/Torso signaling affects a step in phototransduction between the photoreceptor molecule and the TRP channel. Collectively, these data are consistent with the notion that PTTH/Torso signaling acts to facilitate TRP activation downstream of photoreceptor-dependent light sensing (Yamanaka, 2013)

A previous study suggested that larval photophobic behavior diminishes at the end of larval development, perhaps facilitating larval food exit and entry into the wandering phase. The present finding and the increase of PTTH at the beginning of the wandering stage appear to contradict such a hypothesis. Indeed, a sustained larval light avoidance mediated by PTTH was detected that persisted through the wandering stage. These results imply that wandering behavior is triggered by a signal distinct from light preference. Consistent with this notion, the timing of wandering initiation in ppk>torso-RNAiGD or Kr5.1>torso-RNAiGD larvae was found comparable with that of control animals, despite the fact that these animals are not photophobic (Yamanaka, 2013)

As found in other insects, wandering is either directly or indirectly triggered by PTTH- induced ecdysone production. Therefore, concomitant PTTH-mediated photophobicity could ensure that wandering larvae maintain a dark preference for pupariation site, providing better protection from predators and dehydration during the immobile pupal stage. To test this hypothesis, a light/dark preference assay was developed for pupariation. When exposed to a light/dark choice, larvae indeed showed a strong preference to pupariate in the dark. This behavior was abolished either by inactivating PTTH-expressing neurons (ptth>Kir2.1), by silencing ptth in the PTTH-expressing neurons (NP0423>ptth-RNAi, dicer2), or by introducing a torso mutant background (torso[e00150]/[1]). Dark site preference for pupariation was observed in Drosophila populations collected in the wild, confirming that this innate behavior was selected in a natural environment (Yamanaka, 2013)

This work illustrates the use of a single biochemical messenger, PTTH, for the concomitant control of two major functions during larval development. PTTH establishes a neuroendocrine link between distinct neuronal components previously shown to be involved in light avoidance. In contrast to previous interpretations but consistent with another study, this study showed that wandering is independent of light preference and that PTTH maintains a strong light avoidance response through to the time of pupariation. High levels of circulating PTTH during the wandering stage could reinforce the robustness of light avoidance, which might otherwise be compromised by active roaming. This eventually promotes larvae to pupariate in the dark, a trait potentially beneficial for ecological selection. PTTH is thus at the core of a neuroendocrine network, promoting developmental progression and appropriate innate behavioral decisions to optimize fitness and survival (Yamanaka, 2013)

A gustatory receptor paralogue controls rapid warmth avoidance in Drosophila

Behavioural responses to temperature are critical for survival, and animals from insects to humans show strong preferences for specific temperatures. Preferred temperature selection promotes avoidance of adverse thermal environments in the short term and maintenance of optimal body temperatures over the long term, but its molecular and cellular basis is largely unknown. Recent studies have generated conflicting views of thermal preference in Drosophila, attributing importance to either internal or peripheral warmth sensors. This study reconciles these views by showing that thermal preference is not a singular response, but involves multiple systems relevant in different contexts. Previously it was found that the transient receptor potential channel TRPA1 acts internally to control the slowly developing preference response of flies exposed to a shallow thermal gradient. This study now finds that the rapid response of flies exposed to a steep warmth gradient does not require TRPA1; rather, the gustatory receptor GR28B(D), one of five splice variants of Gustatory receptor 28b), 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 vectors, 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 animals. The use of multiple, distinct molecules to respond to a given temperature, as observed in this study, may facilitate independent tuning of an animal's distinct thermosensory responses (Ni, 2013).

In Drosophila two sets of warmth-sensing neurons (activated above ~25°C) have been proposed to control thermal preference: the anterior cell (AC) neurons, located inside the head, and the hot cell (HC) neuron, located peripherally in the arista. However, different studies suggest conflicting cellular and molecular mechanisms for thermal preference control. At the cellular level, primary importance has been attributed to either internal warmth sensors. At the molecular level, the internal AC neurons sense warmth via TrpA1, which encodes a warmth-activated transient receptor potential (TRP) channel, whereas the peripheral HC neurons seem to be TrpA1-independent. To clarify the mechanisms of thermal preference, this study sought to discover the molecular basis of HC neuron function (Ni, 2013).

The arista contains six neurons: three warmth-responsive HC neurons (which can be labelled using cell-specific Gal4 expression in the HC-GAL4 strain) and three cool-responsive (cold cell; CC) neurons (labelled in the CC-GAL4 strain). Three unidentified cells in the arista have been reported to express Gr28b.d-GAL4, a transgene in which promoter sequences upstream of the gustatory receptor GR28B(D) control Gal4 expression. This study found that these Gr28b.d-GAL4-expressing cells resembled thermoreceptors, with cell bodies near the arista base and thin processes in the shaft. To determine the thermoreceptor subset labelled, Gr28b.d-GAL4 was combined with each thermoreceptor-specific Gal4. Gr28b.d-GAL4 plus HC-GAL4 labelled three neurons, whereas Gr28b.d-GAL4 plus CC-GAL4 labelled six neurons, indicating that Gr28b.d-GAL4 is expressed in the HC neurons. Although in situ hybridization was unsuccessful (common for gustatory receptors, GR28B(D) transcripts were robustly detected in dissected antennae/aristae from wild-type, but not Gr28b mutant, animals by reverse transcriptase PCR (RT–PCR), demonstrating expression in this tissue (Ni, 2013).

Gustatory receptors are a large family of seven transmembrane proteins present in invertebrates, with 68 members in Drosophila melanogaster. Insects also contain multiple gustatory receptor-related odorant receptors (62 in D. melanogaster. Gustatory receptors and odorant receptors form a gene family distinct from, and apparently unrelated to, the G-protein-coupled receptor superfamily. Gustatory receptors and odorant receptors have been studied extensively as chemoreceptors for sweet and bitter tastants, food odours, carbon dioxide and other chemicals, but have not previously been implicated in thermosensation. This study examined gustatory receptor involvement in thermosensation using a two-temperature choice assay, exposing flies for 1 min to a steep thermal gradient (initially >5°C per cm) created using tubes of ~25.5 and ~31.0°C air (a preferred and an increased-but-innocuous temperature, respectively) separated by 1 cm. Flies normally prefer the cooler tube, a behaviour termed 'rapid negative thermotaxis'. This and a previous study showed that inhibiting HC neurons by cell-specific expression of tetanus toxin light chain (TNT), a vesicle release inhibitor, using HC-GAL4 strongly reduced such behaviour). In agreement with the importance of HC neurons, and in addition to previous studies, third antennal segment/arista removal strongly reduced this behaviour, whereas ablating other tissues expressing HC-GAL4 and Gr28b.d-GAL4 did not. By contrast, inhibiting AC neurons by TNT expression using TrpA1GAL4, a Gal4 knock-in at the TrpA1 locus, had no effect. (This manipulation disrupted a previously reported AC-dependent thermosensory behaviour) These data indicate that rapid negative thermotaxis depends on the peripheral HC warmth sensors (Ni, 2013).

To probe the molecular basis of rapid negative thermotaxis, its dependence on TrpA1, which is required for AC neuron warmth-sensing, was examined. Consistent with the TrpA1-independence of HC neuron thermosensitivity, a strong loss-of-function TrpA1 mutation did not affect this behaviour. By contrast, strong loss-of-function mutations in the gene encoding GR28B(D) eliminated the response; Gr28b mutants distributed nearly equally between ~25.5°C and ~31.0°C. The defect was specific: excising the transposon in the Gr28bMi allele restored thermotaxis, and both a Gr28b-containing genomic transgene and Gr28b(D) complementary DNA expression rescued the mutant. Attempts were made rescue by expressing cDNAs for the other Drosophila GR28 family members (four other Gr28b isoforms and Gr28a) under Gr28b.d-GAL4 control. Although a negative result could reflect a failure to be properly expressed, only Gr28b(E) yielded significant rescue. However, endogenous Gr28b(E) transcripts were not detected in the antenna/arista, consistent with a previous analysis indicating that GR28B(E) is not expressed there. Together, these data demonstrate that rapid negative thermotaxis depends not on TrpA1, but on Gr28b, consonant with the specific dependence of this behaviour on HC neuron function. Notably, cell-specific GR28B(D) expression using HC-GAL4 strongly rescued the Gr28b mutant, indicating that GR28B(D) function in the HC thermosensors is sufficient to restore rapid negative thermotaxis (Ni, 2013).

To test whether GR28B(D) might act as a thermosensor, whether it conferred warmth-sensitivity when ectopically expressed was examined. Unlike controls, flies broadly expressing GR28B(D) under Actin5C-GAL4 control were incapacitated when heated to 37°C for 3 min, recovering when returned to 23°C. This dramatic effect suggested that GR28B(D) might promote warmth-responsive neuronal activation. It has been shown previously that ectopic expression of the warmth-activated cation channel TRPA1(B), a product of Drosophila TrpA1, renders fly chemosensors warmth-responsive. Like TRPA1(B), chemosensor expression of GR28B(D) (using Gr5a-GAL4) conferred robust warmth-responsiveness. The behavioural consequences of such GR28B(D) expression were examined. When chemically activated, sweet-responsive chemosensors promote proboscis extension. When GR28B(D) was expressed in these cells, strong proboscis extension was elicited by warming to ~32°C. This ability to confer warmth-responsiveness is consistent with GR28B(D) acting as a warmth sensor (Ni, 2013).

Whether GR28B(D) requires sensory neuron-specific cofactors was examined in the neuromuscular system. Unlike controls, motor neurons expressing GR28B(D) (using OK371-GAL4) triggered warmth-responsive excitatory junction potentials at the neuromuscular junction. Thus, GR28B(D)-mediated warmth-responsiveness does not require sensory neuron-specific cofactors. The threshold for GR28B(D)-dependent muscle stimulation was 26.0± 0.3°C, just above TRPA1(B)’s ~25°C threshold in this system, indicating that both molecules mediate responses to innocuous warming (Ni, 2013).

To quantify the thermosensitivity of GR28B(D)-dependent responses, currents were monitored using whole-cell patch-clamp electrophysiology. Unlike controls, voltage-clamped motor neurons expressing GR28B(D) exhibited warmth-responsive inward currents. The response's temperature coefficient (Q10, fold change in current per 10°C change) was calculated by Arrhenius analysis. GR28B(D)-dependent currents were highly thermosensitive (Q10 of 25 ± 5, similar to mammalian neurons expressing thermosensitive TRP channels. Substituting N-methyl-d-glucamine (NMDG)+ for Na+ in the extracellular solution eliminated heat-responsiveness, consistent with cation channel activation (Ni, 2013).

The potential dependence of GR28B(D) on neuron-specific cofactors was tested in muscle. Although control muscles voltage-clamped at −60 mV exhibited modest warmth-responsive outward currents, muscles expressing GR28B(D) (using Mhc-GAL4) exhibited robust warmth-responsive inward currents. The ability of GR28B(D) to confer warmth sensitivity across diverse cell types supports the hypothesis that GR28B(D) acts as a molecular thermoreceptor. It further suggests GR28B(D) as a new class of tool for thermogenetic neuronal activation, adding to the TRP-based toolbox currently used in Drosophila (Ni, 2013).

Although GR28B(D) resembles TRPA1(B) in conferring warmth-sensitivity, these two proteins have distinct functions in the fly, with only Gr28b controlling rapid negative thermotaxis. TrpA1 was found previously to control the slowly developing thermal preference response of flies on a shallow, broad thermal gradient (~0.5°C per cm, 18-32°C). The contribution of Gr28b to this long-term body temperature selection behaviour was tested. As reported previously, TrpA1 mutants selected unusually warm temperatures after 30 min on the gradient, with many accumulating at ≥28°C. By contrast, strong loss-of-function Gr28b mutants behaved indistinguishably from wild type. This neatly distinguishes Gr28b and TrpA1, with the former controlling rapid negative thermotaxis and the latter long-term body temperature selection (Ni, 2013).

These findings reconcile previously disparate views of Drosophila thermosensation by demonstrating that thermal preference is not a singular behaviour, but involves multiple systems relevant in different contexts. It suggests a model in which Gr28b, acting peripherally, controls rapid responses to ambient temperature jumps, whereas TrpA1, acting internally, controls responses to sustained temperature increases reaching the core. In the arista, Gr28b could experience ambient temperature fluctuations in advance of core changes, eliciting rapid avoidance. Such behaviour could be critical for a tiny animal in which ambient and core temperatures equalize rapidly. The dispensability of Gr28b for responses on the shallow gradient could relate to observations in other insects where peripheral thermoreceptors respond more to temperature fluctuations than absolute values. The fly's reliance on distinct sensors for distinct aspects of thermal preference is reminiscent of complex thermosensory systems of larger, endothermic animals. In the fly, these warmth-responsive pathways potentially converge in the brain, where both sets of sensors innervate overlapping regions (Ni, 2013).

Finally, whether Gr28b and TrpA1 were uniquely suited to their roles in the fly was tested. Although TrpA1 was normally not required for rapid negative thermotaxis, when expressed in the arista using Gr28b.d-GAL4, TRPA1(B) significantly rescued the Gr28b mutant defect. (As expected, a less thermosensitive TrpA1 isoform, TRPA1(A), did not rescue the defect). Conversely, although Gr28b was not normally required for slowly developing thermal preference on the shallow gradient, GR28B(D) expression under TrpA1GAL4 control significantly rescued the TrpA1 mutant defect. Thus, when their expression is manipulated appropriately, GR28B(D) and TRPA1(B) can act in the same cells and support the same behaviours, indicating fundamental functional similarities (Ni, 2013).

Although studied extensively, the mechanisms of gustatory receptor action are not fully resolved. Gustatory receptors have been reported to act as cation channels and via G-proteins. Whether GR28B(D) acts by either mechanism remains unknown. Although attempts to study GR28B(D) in heterologous cells (including Xenopus laevis oocytes and HEK cells) were unsuccessful, the ability of GR28B(D) to confer warmth-responsiveness upon diverse cell types argues against a requirement for cell-type-specific cofactors in the fly. Gr28b has been implicated in responses to strong illumination (Xiang, 2010). This seems to be unrelated to GR28B(D)-dependent thermosensation, as Gr28b-dependent photosensors are unresponsive to innocuous warming (Xiang, 2010) and appear to express other Gr28b isoforms. GR28B(D)-expressing muscles were not light-responsive (Ni, 2013).

Previous studies have demonstrated the importance of TRP channels in Drosophila thermosensation, stimulating interest in their potential involvement in warmth-dependent host-seeking by insect disease vectors. This work raises the possibility that gustatory receptors, including GR28 receptors in disease vectors such as tsetse flies and mosquitoes, regulate thermosensation more broadly. GR28B(D) adds to a growing list of highly thermosensitive membrane proteins including not only TRPs, but the mammalian ANO1 chloride channel. The presence of exceptional thermosensitivity in diverse proteins may facilitate temperature-responsive modulation of diverse physiological responses. Furthermore, using multiple molecules to mediate behavioural responses to similar temperatures may facilitate independent tuning of distinct thermosensory responses (Ni, 2013).

The insulin receptor is required for the development of the Drosophila peripheral nervous system

The Insulin Receptor (InR) in Drosophila presents features conserved in its mammalian counterparts. InR is required for growth; it is expressed in the central and embryonic nervous system and modulates the time of differentiation of the eye photoreceptor without altering cell fate. This study shows that the InR is required for the formation of the peripheral nervous system during larval development and more particularly for the formation of sensory organ precursors (SOPs) on the fly notum and scutellum. SOPs arise in the proneural cluster that expresses high levels of the proneural proteins Achaete (Ac) and Scute (Sc). The other cells will become epidermis due to lateral inhibition induced by the Notch (N) receptor signal that prevents its neighbors from adopting a neural fate. In addition, misexpression of the InR or of other components of the pathway (PTEN, Akt, FOXO) induces the development of an abnormal number of macrochaetes, which are Drosophila mechanoreceptors. These data suggest that InR regulates the neural genes ac, sc and sens. The FOXO transcription factor, which becomes localized in the cytoplasm upon insulin uptake, displays strong genetic interaction with the InR and is involved in Ac regulation. The genetic interactions between the epidermal growth factor receptor (EGFR), Ras and InR/FOXO suggest that these proteins cooperate to induce neural gene expression. Moreover, InR/FOXO is probably involved in the lateral inhibition process, since genetic interactions with N are highly significant. These results show that the InR can alter cell fate, independently of its function in cell growth and proliferation (Dutrieux, 2013).

A model is proposed in which the InR receptor plays a role in the development of the peripheral nervous system mainly through FOXO cell localization independently of its role in proliferation and apoptosis. The role of the InR/FOXO pathway appears early in PNS development before SOP formation. The use of different mutants involved in growth indicates that the TOR pathway does not play a major role in the phenotypes observed. The results using genetic and molecular methods strongly suggest that InR/FOXO controls the level of proneuronal genes such as ac, sc and Sens early in PNS development. This explains the interaction observed with N55e11 (Dutrieux, 2013).

Several arguments indicate that the phenotypes observed when InR is overexpressed are not due, at least for the most part, to proliferation, growth or lack of apoptosis. First using anti-PH3 staining that allows to visualize mitotic cells, no extra mitoses are observed in the clusterOverexpression of genes such as dE2F1, or dacapo did not lead to a significant increase or decrease in the number of macrochaetes. In addition co-expression of these genes with InR indicates no interaction. Moreover, the effects of InR and FOXO when overexpressed on respectively the increase and the decrease in cell number, could be estimated by the number of Ac-positive cells in the DC and SC clusters. No significant differences were observed between the control and the overexpressed strain (either InR or FOXO) in the number of cells positive for Ac. If the possibility that proliferation is somehow involved in cluster size cannot be discarded, it does not account for the effects observed since the ratio of Sens-positive cells when InR is overexpressed over the control strain is much higher than the ratio of Ac-positive cells. A similar role for FOXO in apoptosis could also be discarded on the same basis. No clear interactions were observed between FOXO and genes involved in inhibition of apoptosis like diap1 (Dutrieux, 2013).

Along the same line it has been shown that the InR/TOR pathway plays a role in controlling the time of neural differentiation. This has been observed in photoreceptor formation but also in the chordotonal organs of the leg that develop on the same basis as thoracic bristles. The dynamic formation of the SOPs, particularly after a block of InR signaling was undertaken. No differences were observed before the end third larval instar in the test and in the overexpressed strain. Only an increase in the number of positive Sens stained cells are observed in the sca>InR strain (Dutrieux, 2013).

Using Pros staining that marks pIIb cells, this study shows that staining appears in the late third instar larvae at the level of DC SOPs in sca>InR; this is not observed in the control strain. In addition in sca>FOXO RNAi wing discs it also leads to Pros staining. This indicates that the time of differentiation is advanced in the InR strain through the absence of nuclear FOXO. However it was verified that in very early third instar larvae the first scutellar SOP appears at the same time in the control and in the overexpressed strains and that no differences were observed in mid third instar (Dutrieux, 2013).

In addition the observations show that the increase in the number of macrochaetes in sca>InR is independent of the TOR pathway since none of the members induces a similar phenotype as does InR or interacts either with InR or FOXO in this process. However, some interactions were observed with raptor and Rheb that could be the consequence for the latter of its role in PIIa and PIIb formation regulating N (Dutrieux, 2013).

Are InR and FOXO acting on the same target in SOP formation? Several arguments are in favor of this possibility. First underexpression experiments (InR clones, InR RNAi or FOXO RNAi overexpression and FOXO homozygotes and even heterozygotes,) induce exactly opposite phenotypes. This is also true for overexpression experiments with InR and hFOXO3a-TM. Moreover overexpression of both transgenes leads to an intermediate phenotype, very different from the control phenotype. Finally, overexpression of InR in a heterozygote FOXO mutant background leads to an increase in the number of macrochaetes compared to InR alone. FOXO null flies are fully viable and do not usually display any phenotype. However an increase in the number of pDC and aSC macrochaetes is observed in some FOXO homozygotes and even heterozygotes that are nor observed in the control strain. This could indicate that FOXO function is in part dispensable. Even if the InR/FOXO double heterozygote is completely normal, the double null mutant InR/FOXO shows either an excess or a lack of macrochaetes, that is in favor of the hypothesis that InR acts through FOXO. FOXO null clones do not display any phenotype comparable to FOXO RNAi overexpression. However overexpression of InR in a FOXO null clone leads to stronger phenotypes than overexpression of InR alone in a clone. Yet, it cannot be excluded that part of the InR overexpression phenotype is not due to the absence of FOXO or its cytoplasmic retention (Dutrieux, 2013).

The absence of FOXO, using FOXO RNAi, or its retention in the cytoplasm by InR or Akt overexpression produces the same neurogenic phenotypes that are exactly the opposite when nuclear hFOXO3a-TM is overexpressed. In addition overexpression of both hFOXO3a-TM and InR leads to a decrease in the number of highly positive Ac and Sens expressing cells compared to overexpression of InR alone. Finally, overexpression of FOXO RNAi in dpp regulatory sequences, induces Ac expression. All these results should be explained by the same molecular process. One possibility would be that InR/FOXO regulates one or several neural genes involved in cluster formation and maintenance. The results are in favor of the hypothesis that genes of the Ac/Sc complex could be regulated by InR. Either InR via nuclear FOXO represses the Ac/Sc pathway, or FOXO activates a repressor of the pathway (Dutrieux, 2013).

Since it has been well established that InR induces cell proliferation, it remains possible that these functions could affect the size of the proneural clusters when the genes are overexpressed. However, when the number of the Ac-positive cells in the DC and SC clusters in the different genotypes was estimated, it was not significantly different (Dutrieux, 2013).

Several relevant arguments exist suggesting that InR is necessary for SOP formation and regulation of neural gene expression. (1) The phenotype of the overexpression experiments either with InR or with InR RNAi suggests that InR perturbs the normal pattern of singling out a cell in the proneural cluster that will become an SOP. The fact that the sensitive period occurs in the late second/beginning third instar is in accordance with this hypothesis. The phenotype of the InR null clones comfort this hypothesis. (2) When InR is overexpressed the level of Ac is significantly higher. This is confirmed by the IMARIS technique that estimated that in this genotype, the number of cells with the highest scores (106 and 107 units) is larger than in the control strain. These 'highly Ac-positive cells' seem to also be Sens positive cells indicating a correlation between the two events. (3) In sca>InR the level of Sens, measured by the IMARIS technique is higher than in the test raising the possibility that InR regulates several neural genes independently. However another possibility would be that this high Sens expression level would be indirectly due to the induction by InR of a Sens-positive regulator such as sc. (4) Several sc enhancers are regulated by InR, the sc promoter, and the SRV and DC enhancers. As sc is auto-regulated through its different enhancers, it is difficult to evaluate if a specific enhancer is involved although the effect on the 3.8 kb sc promoter is the most striking. For FOXO the absence of FOXO using the FOXO RNAi strain shows that Ac is induced. The double expression of InR and hFOXO3a-TM produces an intermediate phenotype and decreases the effects of InR, on Ac and Sens expression. The results using the sc enhancers when hFOXO3a-TM is overexpressed showed that only a decrease in the expression of the SRV enhancer is observed. However, the phenotypes observed in sca>hFOXO3a-TM agree with the hypothesis of repression of ac and sc by hFOXO3a-TM. As expected, overexpression of FOXO RNAi induces sc-lacZ enhancer. (5) Overexpression of both InR and sc leads to a significant increase in the effect of a single transgene. This indicates that both transgenes have a common target; one of them could be sc itself. An opposite effect is observed with constitutively active hFOXO3a-TM. This favors the model whereby InR and FOXO act in opposite ways on the sc target in SOP formation. (6) Highly significant genetic interactions are observed between sc and InR, and sc and FOXO. (7) Another gene charlatan (chn) which is both upstream and downstream of sc, strongly interacts genetically with InR (Dutrieux, 2013).

Lateral inhibition is determined by the activity of the N receptor. When N is mutated, cell fate changes and extra macrochaete singling appear. Using the N deletion (N55e11) to test possible genetic interaction with InR and with FOXO in heterozygote females, interaction was observed with the InR RNAi strain. Moreover strong interaction is observed with InR overexpression. This indicates that InR impairs lateral inhibition and cooperates with N in this process. In parallel, as for Inr overexpression, the absence of nuclear FOXO either using FOXO25 homozygotes (or even heterozygotes) or FOXO RNAi overexpression induces an increase in the neurogenic phenotype. With this latter strain, tufted microchaetes were observed, indicating that FOXO could also act later in development. Overexpression of hFOXO3a-TM displays highly significant interaction with N55e11 as the neurogenic phenotype is increased compared to overexpression in a wild type background. However, overexpression of InR RNAi in a N55e11 heterozygote background leads to a significant increase but only at the level of aSC, raising the possibility of a local interaction or appearing at a specific time for the different clusters (Dutrieux, 2013).

Moreover the fact that there is no differences when Suppressor of Hairless (Su(H)) which transduces the N pathway, is expressed with or without the InR, indicates that lateral inhibition is not affected. In addition in the InR strain, Sens stained cells were clearly individualized and separated from one another. These results clearly indicate that InR and FOXO act with N on the choice of the cell that will become an SOP (Dutrieux, 2013).

EGFR has also been implicated in macrochaete development. Indeed EGFR mutants and EGFR null clones display macrochaete phenotypes. This could be explained since in EGFR hypomorphic mutants the level of Sc is reduced in some clusters and increased in others suggesting a different requirement of EGFR for the different SOPs. If RasV12 was overexpressed with an ubiquitous driver, sc was ectopically expressed. Thus, Ac/Sc induction by Ras overrules lateral inhibition due to N. Moreover N downregulation enhances EGFR signaling. A model has been established of antagonist interaction between EGFR and N in which Ac/Sc activates both pathways that in turn act on the same SOP specific enhancers (Dutrieux, 2013).

Moreover, the InR/TOR pathway regulates the expression of some of the components of the EGFR signaling pathway such as argos, rhomboid and pointed. The results suggest that both the InR and the EGFR/Ras pathways induce sc in a synergic manner and this further overrules the lateral inhibition mechanism induced by N. The fact that overexpression of RasV12 in an InR null heterozygote background significantly lowers the phenotype observed with RasV12 only, is in agreement with this hypothesis. The interactions observed with the EGFR RNAi strain seem to be FOXO independent (Dutrieux, 2013).

Taken together these results show that InR and several components of the pathway such as PTEN, Akt and FOXO are involved in PNS development independently of their role in growth, proliferation and delay in the time of neural differentiation. The function of InR in PNS development seems to be independent of TOR/4E-BP. FOXO cytoplasmic retention either by InR activation or by the use of FOXO RNAi produces opposite phenotypes suggesting that nuclear FOXO could be a repressor of PNS development. These results using antibody staining and reporters of sc enhancers indicate that InR targets are the neural genes ac, sc and sens. However, as most of these neural genes display a complex co-regulation, it is difficult to demonstrate whether or not sc is the primary target of the pathway. A strong interaction is observed between the EGFR/Ras pathways and InR suggesting that both could act together to induce neural gene expression and this would explain the strong interaction observed between InR/FOXO and N (Dutrieux, 2013).

The adhesion GPCR Latrophilin/CIRL shapes mechanosensation

G-protein-coupled receptors (GPCRs) are typically regarded as chemosensors that control cellular states in response to soluble extracellular cues. However, the modality of stimuli recognized through adhesion GPCR (aGPCR), the second largest class of the GPCR superfamily, is unresolved. This study study characterizes the Drosophila aGPCR Latrophilin/dCirl, a prototype member of this enigmatic receptor class. dCirl is shown to shapes the perception of tactile, proprioceptive, and auditory stimuli through chordotonal neurons, the principal mechanosensors of Drosophila. dCirl sensitizes these neurons for the detection of mechanical stimulation by amplifying their input-output function. These results indicate that aGPCR may generally process and modulate the perception of mechanical signals, linking these important stimuli to the sensory canon of the GPCR superfamily (Scholz, 2015).

Because of the nature of their activating agents, G-protein-coupled receptors (GPCRs) are established sensors of chemical compounds. The concept that GPCRs may also be fit to detect and transduce physical modalities, i.e., mechanical stimulation, has received minor support thus far. In vitro observations showed that, in addition to classical soluble agonists, mechanical impact such as stretch, osmolarity, and plasma membrane viscosity may alter the metabotropic activity of individual class A GPCR. However, the ratio and relationship between chemical and mechanical sensitivity and the physiological role of the latter remain unclear (Scholz, 2015).

Genetic studies have indicated that adhesion GPCRs (aGPCRs), a large GPCR class with more than 30 mammalian members, are essential components in developmental processes. Human mutations in aGPCR loci are notoriously linked to pathological conditions emanating from dysfunction of these underlying mechanisms, including disorders of the nervous and cardiovascular systems, and neoplasias of all major tissues. However, as the identity of aGPCR stimuli is unclear, it has proven difficult to comprehend how a GPCRs exert physiological control during these processes (Scholz, 2015).

Latrophilins constitute a prototype aGPCR subfamily because of their long evolutionary history. Latrophilins are present in invertebrate and vertebrate animals, and their receptor architecture has remained highly conserved across this large phylogenetic distance. The mammalian Latrophilin 1 homolog was identified through its capacity to bind the black widow spider venom component α-latrotoxin, which induces a surge of vesicular release from synaptic terminals and neuroendocrine cells through formation of membrane pores. Latrophilin 1/ADGRL1 was suggested to partake in presynaptic calcium homeostasis by interacting with a teneurin ligand and in trans-cellular adhesion through interaction with neurexins 1b and 2b. Further, engagement of Latrophilin 3/ADGRL3 with FLRT proteins may contribute to synapse development. The role of Latrophilins in the nervous system thus appears complex (Scholz, 2015).

This study has used a genomic engineering approach to remove and modify the Latrophilin locus dCirl, the only Latrophilin homolog of Drosophila melanogaster. dCirl was shown to be required in chordotonal neurons for adequate sensitivity to gentle touch, sound, and proprioceptive feedback during larval locomotion. This indicates an unexpected role of the aGPCR Latrophilin in the recognition of mechanosensory stimuli and provides a unique in vivo demonstration of a GPCR in mechanoception (Scholz, 2015).

This analysis provides multiple lines of evidence to support that Latrophilin/dCirl, one of only two aGPCRs in the fly, is a critical regulator of mechanosensation through chordotonal neurons in Drosophila larvae. Larval chordotonal organs respond to tactile stimuli arising through gentle touch, mechanical deformation of the larval body wall and musculature during the locomotion cycle, and vibrational cues elicited through sound. First, this study determined that registration of all these mechanical qualities is reduced in the absence of dCirl, based on behavioral assays. Then it was established that behavioral defects can be rescued by re-expression of dCirl in chordotonal neurons, one of several cell types with endogenous dCirl expression. Mechanically stimulated lch5 neurons lacking dCirl were shown to responded with action currents at approximately half the control rate across a broad spectrum of stimulation frequencies, providing direct functional evidence for a role of dCirl in chordotonal dendrites, the site of mechanotransduction and receptor potential generation, or somata, where action potentials are likely initiated. Further, the ability of chordotonal neurons to generate mechanical responses relative to their background spike activity appears to be modulated by dCirl (Scholz, 2015).

Combining dCirl KO with strong hypomorphs of trp homologs, ion channels that are directly responsible for the conversion of mechanical stimulation into electrical signals within chordotonal neurons, implied that dCirl operates upstream of them. Intriguingly, removing dCirl from nompC or nan mutant backgrounds resulted in inverse outcomes, i.e., decreased and increased crawling distances, respectively. This suggests that dCirl enhances nompC activity while curtailing nan function. These experiments demonstrate that dCirl genetically interacts with essential elements of the mechanotransduction machinery in chordotonal cilia. Additional studies showed that the dCirl promoter contains a RFX/Fd3F transcription factor signature that implicates dCirl in the mechanosensitive specialization of sensory cilia. On the basis of these results, it is proposed that dCirl partakes in the process of mechanotransduction or spike initiation and transmission to promote sensory encoding (Scholz, 2015).

The classical model of GPCR activation has become the archetypical example for cellular perception of external signals. It comprises soluble ligands that bind to the extracellular portions of a cognate receptor, whereby receptor conformation is stabilized in a state that stimulates metabotropic effectors. Thus, GPCRs are primarily regarded as chemosensors due to the nature of their activating agents. The concept that GPCRs may also be fit to detect and transduce physical modalities, i.e., mechanical stimulation, has received little support thus far (Scholz, 2015).

aGPCRs display an exceptional property among the GPCR superfamily in that they recognize cellular or matricellular ligands. To date, only one ligand, Type 4 collagen, has proved adequate to induce intracellular signaling, whereas for the vast majority of ligand-aGPCR interactions this proof either failed or is lacking). This implies that sole ligand recognition is generally not sufficient to induce a metabotropic response of aGPCRs. Thus, in addition to ligand engagement, the current results suggest that mechanical load is a co-requirement to trigger the activity of dCIRL, a prototypical aGPCR homolog (Scholz, 2015).

Recent findings place aGPCRs in the context of mechanically governed cellular functions, but how mechanical perception through aGPCR activity impinges on cell responses has not yet been established. In addition, the molecular structure of aGPCR is marked by the presence of a GPCR auto-proteolysis inducing (GAIN) domain, which plays a paramount role in signaling scenarios for aGPCRs. This domain type is also present in PKD-1/Polycystin-1-like proteins, which are required to sense osmotic stress and fluid flow in different cell types and are thus considered bona fide mechanosensors. In addition, studies on EGF-TM7-, BAI-, and GPR56-type aGPCRs further showed that proteolytic processing and loss of NTF may figure prominently in activation of the receptors' metabotropic signaling output and that mechanical forces exerted through receptor-ligand contact are required for receptor internalization (Scholz, 2015 and references therein).

dCirl is not the only aGPCR associated with mechanosensation. Celsr1 is required during planar cell polarity establishment of neurons of the inner ear sensory epithelium. Similarly, the very large G-protein-coupled receptor 1 (VLGR1) exerts an ill-defined developmental role in cochlear inner and outer hair cells, where the receptor connects the ankle regions of neighboring stereocilia. In addition, VLGR1 forms fibrous links between ciliary and apical inner segment membranes in photoreceptors. Both cell types are affected in a type of Usher syndrome, a congenital combination of deafness and progressive retinitis pigmentosa in humans, which is caused by loss of VLGR1 function. Although present evidence derived from studies of constitutively inactive alleles suggests a requirement for Celsr1 and VLGR1 aGPCR for sensory neuron development, their putative physiological roles after completion of tissue differ-entiation have remained unclear and should be of great interest (Scholz, 2015 and references therein).

In the current model on dCirl function, aGPCR activity, adjusted by mechanical challenge, modulates the molecular machinery gating mechanotransduction currents or the subsequent initiation of action potentials and ensures that mechanical signals are encoded distinctly from the background activity of the sen-sory organ. Thereby, dCirl shapes amplitude and kinetics of the sensory neuronal response. Linking adequate physiological receptor stimulation to downstream pathways and cell function is an essential next step to grasp the significance of aGPCR function and the consequences of their malfunction in human conditions. The versatility of the dCirl model now provides an unprecedented opportunity to study the mechanosensory properties of an exemplary aGPCR and to uncover features that might prove of general relevance for the function and regulation of the entire aGPCR class (Scholz, 2015).

ROS-mediated activation of Drosophila larval nociceptor neurons by UVC irradiation

The complex Drosophila larval peripheral nervous system, capable of monitoring sensory input from the external environment, includes a family of multiple dendritic (md) neurons with extensive dendritic arbors tiling the inner surface of the larval body wall. The class IV multiple dendritic (mdIV) neurons are the most complex with dendritic nerve endings forming direct intimate contacts with epithelial cells of the larval body wall. Functioning as polymodal mechanonociceptors with the ability to respond to both noxious mechanical stimulation and noxious heat, the mdIV neurons are also activated by nanomolar levels of the endogenous reactive oxygen species (ROS), H2O2. Although often associated with tissue damage related to oxidative stress, endogenous ROS have also been shown to function as signaling molecules at lower concentrations. The overall role of ROS in sensory signaling is poorly understood but the acutely sensitive response of mdIV neurons to ROS-mediated activation is consistent with a routine role in the regulation of mdIV neuronal activity. Larvae respond to short wavelength ultraviolet (UVC) light with an immediate and visual system-independent writhing and twisting of the body previously described as a nociceptive response. Molecular and cellular mechanisms mediating this response and potential relationships with ROS generation are not well understood. This study has used the UVC-induced writhing response as a model for investigation of the proposed link between endogenous ROS production and mdIV neuron function in the larval body wall. Transgenic inactivation of mdIV neurons caused a strong suppression of UVC-induced writhing behavior consistent with a key role for the mdIV neurons as mediators of the behavioral response. Direct imaging of ROS-activated fluorescence showed that UVC irradiation caused a significant increase in endogenous ROS levels in the larval body wall and transgenic overexpression of antioxidant enzymes strongly suppressed the UVC-induced writhing response. Direct electrophysiological recordings demonstrated that UVC irradiation also increased neuronal activity of the mdIV neurons. Therefore, results obtained using UVC irradiation to induce ROS generation provide evidence that UVC-induced writhing behavior is mediated by endogenous production of ROS capable of activating mdIV mechanonociceptors in the larval body wall (Kim, 2014).

Kinematic responses to changes in walking orientation and gravitational load in Drosophila melanogaster

Walking behavior is context-dependent, resulting from the integration of internal and external influences by specialized motor and pre-motor centers. Neuronal programs must be sufficiently flexible to the locomotive challenges inherent in different environments. Although insect studies have contributed substantially to the identification of the components and rules that determine locomotion, understanding of how multi-jointed walking insects respond to changes in walking orientation and direction and strength of the gravitational force is still not understood. In order to answer these questions the kinematic properties of untethered Drosophila was measured with high temporal and spatial resolution during inverted and vertical walking. In addition, the kinematic responses to increases in gravitational load were measured. Animals were found to be capable of shifting their step, spatial and inter-leg parameters in order to cope with more challenging walking conditions. For example, flies walking in an inverted orientation decreased the duration of their swing phase leading to increased contact with the substrate and, as a result, greater stability. It was also found that when flies carry additional weight, thereby increasing their gravitational load, some changes in step parameters vary over time, providing evidence for adaptation. However, above a threshold that is between 1 and 2 times their body weight flies display locomotion parameters that suggest they are no longer capable of walking in a coordinated manner. Finally, it was found that functional chordotonal organs are required for flies to cope with additional weight, as animals deficient in these proprioceptors display increased sensitivity to load bearing as well as other locomotive defects (Mendes, 2014: PubMed).

WHAMY is a novel actin polymerase promoting myoblast fusion, macrophage cell motility and sensory organ development

Wiskott-Aldrich syndrome proteins (WASP) are nucleation promoting factors (NPF) that differentially control the Arp2/3 complex. In Drosophila, three different family members, SCAR/WAVE, WASP and WASH, have been analyzed so far. This study characterizes WHAMY, the fourth Drosophila WASP family member. whamy originated from a wasp gene duplication and underwent a sub-neofunctionalization. Unlike WASP, WHAMY specifically interacts with activated Rac1 through its two CRIB domains that are sufficient for targeting WHAMY to lamellipodial and filopodial tips. Biochemical analyses showed that WHAMY promotes exceptionally fast actin filament elongation, while it does not activate the Arp2/3 complex. Loss- and gain-of function studies revealed an important function of WHAMY in membrane protrusions and cell migration in macrophages. Genetic data further imply synergistic functions between WHAMY and WASP during morphogenesis. Double mutants are late-embryonic lethal and show severe defects in myoblast fusion. Trans-heterozygous mutant animals show strongly increased defects in sensory cell fate specification. Thus, WHAMY is a novel actin polymerase with an initial partitioning of ancestral WASP functions in development and subsequent acquisition of a new function in cell motility during evolution (Brinkmann, 2015).

The actin cytoskeleton plays a central role in a number of different cellular functions, such as cell shape changes, cell motility and membrane trafficking. Members of the Wiskott-Aldrich syndrome protein (WASP) family are conserved nucleation-promoting factors (NPF) that activate the Arp2/3 complex, a major actin nucleator in eukaryotic cells. In mammals, the WASP protein family consists of eight different members: the two Wiskott-Aldrich syndrome proteins WASP and N-WASP (also known as WAS and WASL, respectively), the related WASP family Verprolin homologous proteins WAVE1-WAVE3 (also known as SCAR1-SCAR3 and WASF1-WASF3, the Wiskott-Aldrich syndrome protein and SCAR homolog WASH (also known as WASH1), and the WHAMM and JMY proteins. WASP proteins share a conserved C-terminal Arp2/3-complex-activating WCA module. This module consists of either one or multiple actin-monomer-binding WH2 (W) domains, a central domain (C) and an acidic (A) domain, which mediate Arp2/3 binding. Apart from the catalytic WCA module, WASP proteins often share a proline-rich region and a basic region, which bind SH3-domain containing proteins and acidic phosphoinositides, respectively. WASP proteins are regulated by similar molecular principles. Under resting conditions NPFs are primarily inactive and become activated upon binding of the Rho GTPases Cdc42 and Rac1. Additionally, a variety of factors further modulate proper activation and recruitment of WASP proteins (Brinkmann, 2015).

In Drosophila, only three WASP subfamily members have been described, namely WAVE, WASP and WASH (also known as CG13176). Insects like Drosophila have subsequently lost a WHAMM/JMY gene, although the common ancestor first arose in invertebrates. Genetic studies indicate that WAVE and WASP are the central activators of the Arp2/3 complex, differentially regulating most aspects of Arp2/3 function in Drosophila. These studies highlight distinct, but also overlapping cellular requirements of WAVE and WASP during development. WAVE function is in particular essential for cell shape and morphogenetic cell movements during development. By contrast, WASP function is needed for cell fate specification of sensory organ precursors (SOPs) and spermatid Both, WASP and WAVE are required for myoblast fusion (Brinkmann, 2015).

Loss of maternal and zygotic WASP results in late-embryonic lethality due to strong defects in cell fate decisions of neuronal cell lineages and myoblast fusion defects. Remarkably, animals lacking zygotic WASP function survive until early adulthood. Thus, maternally provided WASP protein is sufficient for proper embryonic and larval development. Mutant wasp flies show no strong morphological defects except a partial loss of sensory bristles. Loss of zygotic Arp2/3 function results in a similar, albeit stronger, neurogenic phenotype suggesting an involvement of additional factors in Arp2/3-dependent SOP development (Rajan et al., 2009). The loss of sensory bristles in wasp and arp2/3 mutants phenocopies Notch loss-of-function and is caused by a pIIa-to-pIIb cell fate transformation. This results in an excess of neurons at the expense of bristle sheath, shaft and socket cells. Recent work further suggests that the WASP-Arp2/3 pathway rather plays an important role in the trafficking of Delta-positive vesicles from the basal area to the apical cortex of the signal-sending pIIb cell (Brinkmann, 2015).

Remarkably, rescue experiments have implied that established activators of WASP, such as Cdc42 or phosphatidylinositol 4,5-bisphosphate (PIP2), are not required for WASP function, neither for the myoblast fusion process nor for SOP development. The identity of an independent activator that might act cooperatively to control Arp2/3 function in these contexts is unknown. This study presents a functional analysis of WHAMY, a new WASP-like protein that regulates cell motility of Drosophila blood cells but also synergizes with WASP during embryonic muscle formation and cell fate specification of adult SOPs (Brinkmann, 2015).

The identification of all WASP family homologs in all sequenced organisms allows a detailed phylogenetic analysis of the origin of diverse subfamilies evolving differential cellular functions. WASP proteins are multi-domain proteins. They share functions that are encoded by similar domains at the C-termini, whereas different N-terminal domains mainly define their diverse cellular processes. Gene duplication and domain shuffling are two important mechanisms driving novel and increasingly complex developmental programs during evolution. It is thought that this boost in domain shuffling is responsible for the apparent disconnection between greatly increased phenotypic complexity and a relatively small difference in gene number between humans and Drosophila (Brinkmann, 2015).

The whamy gene is an excellent example for how gene duplication and subsequent domain shuffling can create new gene functions after initial gene duplication. It arose through a duplication of wasp at the base of the genus Drosophila. Although the encoded protein has evolved a new function in cell motility, it also functions synergistically with WASP in muscle formation and sensory organ development. In the latter, WHAMY can even partially substitute for WASP, indicating that it has kept functionality following the duplication. This duality is reflected in the sequence of the WHAMY CRIB domains. As there is an overlap in function with WASP, selective pressure has been reduced since the duplication, leading to the observed increase of evolutionary rate. Following the duplication of the CRIB domains within WHAMY, a similar trend can be found. Whereas one domain has kept the function of binding to Cdc42-GTP, the other has lost the ability to interact. This is reflected in domain-specific conserved substitutions. The duplication of the wasp gene and subsequent subneofunctionalization of whamy might have occurred at the same time as the loss of a true WHAMM/JMY ancestor during insect evolution (Veltman, 2010). Like Drosophila WHAMY, the common ancestor of WHAMM/JMY proteins in invertebrates also lacks the characteristic C-terminal tryptophan residue in their VCA domains that is crucial for Arp2/3 binding and activation (Veltman, 2010). This further implies a primary Arp2/3-independent function of the common ancestor of invertebrate WHAMM/JMY proteins (Brinkmann, 2015).

WHAMY shows no Arp2/3-activating nucleation promoting factor (NPF) activity in vitro. However, different from WASP, WHAMY itself is able to promote fast elongation of linear actin filaments from actin-rich clusters. With respect to its activity, WHAMY resembles the WH2-domain containing Ena/VASP polymerases that actively drive processive actin-filament elongation and promote assembly of both lamellipodial and filopodia actin networks. Notably, Ena/VASP proteins are tetramers, and their oligomerization is mandatory to allow for polymerase activity in experiments in solution, as used in this study. Since fast filament elongation was exclusively observed from WHAMY clusters in total internal reflection fluorescence (TIRF) experiments, and consistent with the size exclusion chromatography experiments, it is proposed that WHAMY requires oligomerization to acquire actin polymerase activity. Concerning previously analyzed proteins of the WASP family, the filament elongation activity of WHAMY is therefore rather unique, and when compared to other fast actin polymerases, only the Drosophila formin Diaphanous achieves comparable high elongation activity in vitro. As evidenced from the pyrene data, the activity of WHAMY can further be increased by Rac1 (Brinkmann, 2015).

Rac1 seems to act on both the activity and the localization of WHAMY at lamellipodial tips. Both of the two CRIB domains of WHAMY bind equally to activated Rac1, and only loss of both CRIB domains abolishes Rac1 binding and the localization to the leading edge. Therefore, it currently remains unclear why WHAMY contains two CRIB domains and whether they differentially mediate distinct cellular functions. They might contribute to a local clustering of WHAMY and Rac1 at the leading edge. The most prominent Rac1 effector represents the WAVE regulatory complex (WRC) that drives Arp2/3-mediated branched actin nucleation. Rac1 directly binds and activates the WRC by allosterically releasing the bound Arp2/3-activating WCA domain of WAVE. Overexpression of WHAMY leads to a strong induction of filopodia, presumably due to the filament elongation activity of WHAMY. Additionally, competition between WHAMY and the WRC for Rac1 could disturb the balance between nucleation and elongation activity, and therefore might contribute to the observed overexpression phenotype. Different from WHAMY, WRC function is essential for lamellipodia formation and cell migration in most eukaryotic cells. By contrast, loss of WHAMY function does not impair lamellipodia formation but rather regulates cell spreading and contributes to cell motility (Brinkmann, 2015).

WHAMY does not compete but rather functions together with WASP in Drosophila morphogenesis. Previous studies have revealed that the major established activators of WASP, such as Cdc42 and PIP2, are not required for the function of WASP in sensory organ development or myoblast fusion. This observation already suggests that additional components, such as WHAMY, might act together with WASP in sensory organ development and myoblast fusion. Consistent with this, further reduction of whamy function in wasp mutants was found to phenocopy loss of arp2/3 function, resulting in an excess of neurons and a near absence of bristle sheath, shaft and socket cells. Rescue data further indicate that WHAMY can partially substitute for WASP function. Thus, WHAMY cooperates with WASP rather than acting redundantly in sensory organ development. Based on TIRF microscopy data, it is suggested that WHAMY might potentially generate mother filaments in close vicinity of Arp2/3 complex facilitating Arp2/3-mediated actin assembly (Brinkmann, 2015).

How might WHAMY and WASP act on actin dynamics during sensory organ development? Recent work suggests that the WASP-Arp2/3 pathway is not involved in Notch receptor endocytosis or its processing in the signal-receiving cell (pIIa) but rather plays an important role in the trafficking of Delta-positive vesicles from the basal area to the apical cortex of the signal-sending pIIb cell. This model also implies that recycled Notch ligands such as Delta and Serrate are active at apical junctions with actin-rich structures induced by WASP and the Arp2/3 complex, which in turn activate apical Notch receptor in pIIa. In vivo, WHAMY localizes at dynamic vesicles during sensory organ precursor formation and, together with WASP, becomes strongly enriched at apical junctions shortly after SOP division. Thus, a scenario is proposed in which WASP and WHAMY might act either on the assembly of actin-rich structures or directly promote apical trafficking of Delta through Rab11-recycling endosomes (Brinkmann, 2015).

A dynamic reorganization of the actin cytoskeleton into distinct cellular structures is also necessary to ensure successful myogenesis. Filopodial protrusions are crucial for the attachment of FCMs to the founder cell and growing myotube, and for the initiation of the fusion process. The recognition and adhesion of myoblasts depends on members of the immunoglobulin superfamily (IgSF) that are expressed specifically in myoblasts in a ring-like structure. The interaction of these proteins leads to the formation of a cell communication structure, which has been termed fusion-restricted myogenic adhesive structure (FuRMAS) or podosome-like structure. The cytodomains of the IgSFs trigger the activation of WAVE in founder cells, and of WAVE and WASP in FCMs. In FCMs, WAVE- and WASP-mediated Arp2/3 activation results in the formation of a dense F-actin focus that accumulates at the interface of adhering myoblasts. Electron microscopy studies have revealed that WASP is required for the formation of fusion pores at apposing myoblasts during embryonic and indirect flight muscle development. These fusion pores expand until full cytoplasmic continuity is achieved, and WASP has implicated to be required for fusion pore expansion. It has been discussed that WASP is required for the removal of membrane residuals during membrane vesiculation. WHAMY might contribute to this process, but the detailed mechanistic contribution of WHAMY in fusion pore formation needs to be addressed in future studies by ultrastructural analyses (Brinkmann, 2015).

G2-phase arrest prevents bristle progenitor self-renewal and synchronizes cell divisions with cell fate differentiation

Developmentally regulated cell cycle arrest is a fundamental feature of neurogenesis, whose significance is poorly understood. During Drosophila sensory organ (SO) development, primary progenitor (pI) cells arrest in G2-phase for precisely defined periods. Upon re-entering the cell cycle in response to developmental signals, these G2-arrested precursor cells divide and generate specialized neuronal and non-neuronal cells. To study how G2-phase arrest affects SO lineage specification, pI-cells were forced to divide prematurely. This produced SO with normal neuronal lineages but supernumerary non-neuronal cell types. The reason was that prematurely dividing pI-cells generated a secondary pI-cell that produced a complete SO and an external precursor cell that underwent amplification divisions producing supernumerary non-neural cells. This means that pI-cells are capable to undergo self-renewal before transit to a terminal mode of division. Regulation of G2-phase arrest therefore serves a dual role in SO development: preventing progenitor self-renewal and synchronizing cell division with developmental signals. Cell cycle arrest in G2-phase therefore temporally coordinates the precursor cells proliferation potential with terminal cell fate determination to ensure formation of organs with a normal set of sensory cells (Ayeni, 2016).

The microtubule-based cytoskeleton is a component of a mechanical signaling pathway in fly campaniform receptors

In mechanoreceptors, mechanical stimulation by external forces leads to the rapid opening of transduction channels followed by an electrical response. Despite intensive studies in various model systems, the molecular pathway by which forces are transmitted to the transduction channels remains elusive. In fly campaniform mechanoreceptors, the mechanotransduction channels are gated by compressive forces conveyed via two rows of microtubules that are hypothesized to be mechanically reinforced by an intervening electron-dense material (EDM). This hypothesis was tested by studying a mutant fly in which the EDM was nearly absent, whereas the other ultrastructural elements in the mechanosensitive organelle were still present at 50% (or greater) of normal levels. The mechanosensory response in this mutant was reduced by 90% and the sensitivity by at least 80%. To test whether loss of the EDM could lead to such a reduction in response, a mechanical analysis was performed, and the loss of the EDM was expected to greatly decrease the overall rigidity, leading to a marked reduction in the gating force conveyed to the channel. It is argued that this reduction in force, rather than the reduction in the number of transduction channels, is primarily responsible for the nearly complete loss of mechanosensory response observed in the mutant fly. Based on these experiments and analysis, it is concluded that the microtubule-based cytoskeleton (i.e., microtubules and EDM) is an essential component of the mechanical signaling pathway in fly campaniform mechanoreceptor (Liang, 2014).


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Genes expressed in the PNS

Genes involved in organ development

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