InteractiveFly: GeneBrief

pickpocket : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - pickpocket

Synonyms - Ppk1, dmdNaC1

Cytological map position -

Function - channel

Keywords - peripheral nervous system, locomotion, mechanosensory signal transduction of proprioceptive sensory information

Symbol - ppk

FlyBase ID: FBgn0020258

Genetic map position -

Classification - amiloride-sensitive sodium channel

Cellular location - surface transmembrane



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Koseki, N., Mori, S., Suzuki, S., Tonooka, Y., Kosugi, S., Miyakawa, H. and Morimoto, T. (2016). Individual differences in sensory responses influence decision making by Drosophila melanogaster larvae on exposure to contradictory cues. J Neurogenet: 1-39. PubMed ID: 27309770
Summary:
Animals make decisions on behavioral choice by evaluating internal and external signals. Individuals often make decisions in different ways, but the underlying neural mechanisms are not well understood. This study describes a system for observing the behavior of individual Drosophila melanogaster larvae simultaneously presented with contradictory signals, in this case attractive (yeast paste) and aversive (NaCl) signals. Olfaction was used to detect the yeast paste, whereas the ENaC/Pickpocket channel was important for NaCl detection. Wild-type (Canton-S) larvae fall into two decision making groups: one group decided to approach the yeast paste by overcoming the aversive signal, whereas the other group decided to forgo the yeast paste because of the aversive signal. These findings indicate that different endogenous sensitivities to NaCl contribute to make differences between two groups and that diverse decision making steps occur in individual animals.
BIOLOGICAL OVERVIEW

Coordination of rhythmic locomotion depends upon a precisely balanced interplay between central and peripheral control mechanisms. Although poorly understood, peripheral proprioceptive mechanosensory input is thought to provide information about body position for moment-to-moment modifications of central mechanisms mediating rhythmic motor output. Pickpocket1 (PPK1) is a Drosophila subunit of the epithelial sodium channel (ENaC) family displaying limited expression in multiple dendritic (md) sensory neurons tiling the larval body wall and a small number of bipolar neurons in the upper brain (Adams, 1998). ppk1 null mutant larvae have normal external touch sensation and md neuron morphology but display striking alterations in crawling behavior. Loss of PPK1 function causes an increase in crawling speed and an unusual straight path with decreased stops and turns relative to wild-type. This enhanced locomotion results from sustained peristaltic contraction wave cycling at higher frequency with a significant decrease in pause period between contraction cycles. The mutant phenotype is rescued by a wild-type PPK1 transgene and duplicated by expressing a ppk1RNAi transgene or a dominant-negative PPK1 isoform. These results demonstrate that the Ppk1 channel plays an essential role in controlling rhythmic locomotion by providing mechanosensory signal transduction of proprioceptive sensory information (Ainsley, 2003).

The DEG/ENaC superfamily includes proteins involved in mechanotransduction, proprioception, neurotransmission, and fluid and electrolyte homeostasis. Members of this superfamily are united by similarities in their amino acid sequences and in some cases by their function. The first identified protein and the largest number of family members are from Caenorhabditis elegans; these include MEC-4, MEC-10, DEG-1, UNC-105, UNC-8, and DEL-1. Elegant genetic studies in that organism have suggested a role for DEG/ ENaC proteins in mechanotransduction. MEC-4 and MEC-10 are expressed in mechanosensory neurons and are required for normal sensitivity to touch. In addition, UNC-105 and UNC-8 are expressed in muscle and motor neurons, respectively, and are required for coordinated movement. Three lines of evidence suggest that C. elegans family members may be ion channels. (1) Other family members have been shown to be ion channels. (2) Residues in the transmembrane sequence of a related DEG/ ENaC channel, alphaENaC, can be substituted for residues in M2 of MEC-4 without loss of function in the worm and vice versa. (3) Specific mutations in several C. elegans family members cause a phenotype marked by neuronal swelling; this may suggest a loss of cell volume control, perhaps caused by unregulated opening of an ion channel. Nevertheless, these C. elegans, proteins have not been shown experimentally to be channels either in vivo or in heterologous expression systems (Adams, 1998 and references therein).

Subunits of the vertebrate epithelial Na+ channel (ENaC) subfamily form ion channels that mediate Na+ absorption across the apical membrane of epithelia. Studies of ENaC have served to define several functional properties of the family. (1) ENaC generates Na+ currents that are reversibly blocked by the diuretic amiloride. Although Na+ selectivity is not a general feature, all DEG/ENaC proteins shown to be ion channels are amiloride-sensitive and conduct cations. (2) ENaC functions as a multimeric complex of three subunits, alpha-, ß-, and γENaC. In Xenopus oocytes, expression of alphaENaC, but not ß- or γENaC, generates a small amiloride-sensitive Na+ current. However, when ß- and γENaC are coexpressed with alphaENaC, much larger Na+ currents are produced. This finding illustrates (3) -- some members can form ion channels when expressed alone, whereas others are ion channel subunits that do not function by themselves in oocytes. (4) Biochemical studies of alphaENaC reveal a membrane topology consisting of two transmembrane domains (M1 and M2), cytoplasmic NH2 and COOH termini, and a large extracellular region containing cysteine-rich domains. Other family members are thought to have a similar molecular organization (Adams, 1998 and references therein).

In addition to the C. elegans proteins, three other neuronal family members have been identified. FaNaCh is a neuronal channel activated by the neuropeptide FMRFamide. Brain Na+ channel 1 (BNC1, also named MDEG and BNaC1) is widely expressed in human brain. Although its physiologic function is unknown, its channel activity can be enhanced by mutation of a residue associated with neuronal swelling in C. elegans DEG/ENaC proteins. A channel very similar to BNC1, BNaC2 (also named ASIC), is expressed in brain and dorsal root ganglia, and is activated by extracellular protons. These observations demonstrate a fifth feature of the family; in contrast to ENaC, neuronal DEG/ENaC channels may open only in the presence of specific stimuli or an activating mutation (Adams, 1998 and references therein).

The Drosophila embryonic and larval peripheral nervous system (PNS) is composed of segmentally repeated neuronal clusters within the body wall (designated d, l, v', and v), each containing a defined set of sensory neurons responsible for the innervation of different sensory structures. Ciliated organs such as the adult external sensory bristles and the larval chordotonal organs, thought to sense external touch and cuticle deformation, are innervated by bipolar type I sensory neurons. Type II multiple dendritic (md) neurons within the PNS, also referred to as dendritic arborization (da) neurons, extend dendritic processes to uniformly tile the internal epithelial surface. md neurons have been proposed to play a proprioceptive mechanosensory function to coordinate muscle contractions for larval movement. However, recent reports have implicated md neurons as possible thermosensory and/or nociceptive neurons. Distinct differences in morphology between subsets of md neurons suggest that subtypes may serve different sensory functions (Ainsley, 2003).

pickpocket1 (ppk1), encoding a member of the degenerin/epithelial sodium channel(DEG/ENaC) family, is expressed in a single md neuron within each of the d, v', and v PNS clusters (Adams, 1998). DEG/ENaC proteins were first characterized based upon genetic studies in the nematode that sought touch-insensitive mutants (Lingueglia, 1995). These proteins have subsequently been implicated as central components of a heteromultimeric mechanotransduction channel (Goodman, 2003). DEG/ENaC family members may also function as peptide neurotransmitter receptors, as salt taste sensors, or pH sensors with a possible role in synaptic plasticity. Therefore, the role of PPK1 protein in Drosophila neurons cannot necessarily be predicted based upon protein structure alone (Ainsley, 2003 and references therein).

In a pattern identical to the endogenous ppk1 gene, a ppk1.9-GAL4 transgene was expressed in a single md neuron within each of the d, v', and v PNS clusters as well as in four bipolar neurons in each of the upper brain lobes. ppk1 is expressed in the class IV md neurons ddaC, v'ada, and vdaB, displaying a characteristically uniform extension of processes to tile the internal surface of the epithelium between segmental boundaries (see Grueber, 2000 for a portrayal of these neurons). Extensive dendritic branches approach but do not cross the anterior and posterior segmental boundaries and appear to never overlap. Each PNS neuronal cluster contains multiple md neurons, only one of which expresses PPK1. Each md neuron cell body produces a single axon extending toward the ventral nerve cord with extensive axonal branching at the midline of the ventral nerve cord. Expression of nuclear-localized lacZ controlled by ppk1.9GAL4 reveals no detectable ppk1 gene expression in neuronal cell bodies of the ventral nerve cord (Ainsley, 2003).

ppk1 is located at position 35B1 just upstream of the alcohol dehydrogenase (Adh) gene. The small genomic overlap (44 kb) between two existing deficiency breakpoints, Df(2L)b88h49 and Df(2L)A400, precisely removes ppk1 to create a null genotype in transheterozygous flies (Df/Df). ppk1 null mutant animals (Df/Df) are viable as larvae and adults with no morphological or behavioral abnormalities easily detected during routine culture (Ainsley, 2003).

No defects in md neuron morphology were detected in ppk1 mutant larvae carrying ppk1.9GAL4 and UAS-DsRed, suggesting that PPK1 is not required for basic developmental steps. ppk1 mutant larvae were also indistinguishable from wild-type when tested using previously published paradigms for external touch with an eyelash or single hair and for their response to four different odorants in traditional olfactory taxis assays. md neurons have been implicated as nociceptors based upon the loss-of-function phenotype for the painless gene encoding a TRP-like channel protein. This phenotype is manifested as a putative pain response displayed as larval twisting and rolling when a hot (>38°C) probe is placed nearby. ppk1 mutant larvae were indistinguishable from wild-type in their ability to respond to a hot probe, suggesting that ppk1 does not participate in the same signal transduction pathway defined by the painless mutation (Ainsley, 2003).

ppk1 mutants were examined for larval locomotion by testing for differences in larval wandering behavior. When placed on an agarose sheet in the absence of food, wild-type third instar larvae display a characteristic pattern of wandering behavior, in which short bursts of forward movement (3-10 forward contraction waves) are separated by stops and repeated side-to-side head probes followed normally by a change in direction. Larval contour trails were created using digital larval motion analysis and scored for total contour trail area as a useful and accurate indirect measure of speed and stops. In addition, larval centroid trails were used to more precisely quantitate a number of specific parameters describing larval locomotion pattern (Ainsley, 2003).

ppk1 mutants move with a decreased number of stops or turns, resulting in an extended straight or slightly arching contour trail and a total contour trail area nearly twice that of wild-type. This enhanced locomotion pattern can be returned to wild-type values by providing wild-type PPK1 from a UAS-PPK1 transgene driven by ppk1.9GAL4. As an independent means of disrupting PPK1 activity, a ppk1RNAi transgene was created and expressed in transgenic flies. ppk1RNAi larvae display the same increase in contour trail area in a dosage-dependent manner. The moderate mutant phenotype caused by a single copy of ppk1RNAi can be shifted to a level comparable to the Df/Df null genotype by adding an additional copy of the transposon or by reducing endogenous gene dosage using a ppk1 deficiency chromosome. The UAS-PPK1[E145X] transgene encoding a truncated form of PPK1 containing only the first transmembrane domain was used to express a dominant-negative PPK1 isoform. UAS-PPK1[E145X] expression duplicated the significant increase in total contour trail area previously observed in ppk1 null mutants (Ainsley, 2003).

Additional behavioral parameters were determined using computer-generated larval centroid trails. Maximum translocation from the starting centroid was drastically increased in ppk1 mutants to a value of 66.99 mm (± 5.9) relative to only 29.93 mm (± 3.6) in wild-type. Fraction of time spent stopped was decreased from 0.41 (± 0.046) in wild-type to only .097 (± 0.017) in ppk1 mutants. Changes in direction of forward motion (>20°) was found to be decreased to 2.92 turns (± 0.61) in ppk1 mutants relative to 6.85 turns (± 0.76) in wild-type. Each of these values was returned to wild-type levels by providing wild-type PPK1 controlled by ppk1.9GAL4. Crawling speed during a linear locomotion burst of 5 or more frames was increased to 55.76 mm/min (± 1.1) in ppk1 mutants compared to 41.97 mm/min (± 1.7) in wild-type, and the duration of linear locomotion bursts without stops, only 6.7 s (± 0.45) in wild-type, was increased to 41.4 s (± 4.1) in ppk1 mutants. This value was returned to 8.24 s (± 0.48) in rescued larvae (Ainsley, 2003).

Larval contraction wave cycles during sustained linear locomotion can be divided into two parts consisting of the actual progression of the telescoping peristaltic wave of contraction from the posterior to anterior ends of the larva, separated by a brief pause prior to initiation of the next wave of contraction. Using high-resolution digital video, wild-type larvae were demonstrated to complete a full contraction wave cycle every 1.94 s, but ppk1 mutants complete the cycle in only 1.11 s, resulting in an increase in linear speed. This is accomplished by more rapid progression of the contraction wave from posterior to anterior and by a significant decrease in the length of the pause period. Wild-type larvae pause an average of 1.06 s between contraction waves while ppk1 mutants pause only 0.43 s (Ainsley, 2003).

ppk1 mutants crawl with significantly fewer stops, side-to-side head probes, and changes in direction and also cycle peristaltic contraction waves at a higher rate, causing increased linear speed. These characteristics could be mechanistically separable or could result from disruption of the same PPK1-mediated function. Overall neural control of rhythmic locomotion such as swimming, walking, or crawling is a complex ensemble performance with contributions from both central neurons responsible for rhythmic motor output and peripheral mechanosensory neurons providing moment-to-moment proprioceptive input concerning relative body position. The presence of PPK1 in peripheral md neurons is consistent with a role in mechanosensory signal transduction of proprioceptive sensory information from individual larval segments. However, rhythmic motor output driving locomotion originates in neural networks known as central pattern generators (CPG) present in the spinal cord or the analogous ventral nerve cord. The absence of cellular PPK1 expression in the ventral nerve cord suggests that PPK1 does not participate directly in function of individual CPGs. Ample axonal projections of md neurons to the ventral nerve cord imply that PPK1 may function in the regulatory control of motor output from the nerve cord (Ainsley, 2003).

Regulation of CPG motor output also extends from higher brain centers. Mutations disrupting the central complex, a large median neuropil in the insect brain, result in impaired walking in adults and defective crawling behavior in larvae. These defects are invariably displayed as a decrease in locomotion and do not resemble the enhanced locomotion observed in ppk1 mutants. Limited expression of PPK1 in only a subset of neurons may indicate that it is involved in only a small component of the overall regulatory mechanism (Ainsley, 2003).

The exact physiological functions of PPK1 and other DEG/ENaC proteins are not well understood and could contribute to neuronal excitability in a wide variety of direct and indirect ways. This new genetic model of DEG/ENaC function in the Drosophila system should serve as a valuable tool for further studies. Using the unusual enhanced locomotion phenotype and the restricted ppk1 expression pattern, future work should be able to gather useful information concerning motor control and the specific function of DEG/ENaC proteins in both peripheral and central neurons (Ainsley, 2003).


REGULATION

Control of multidendritic neuron differentiation in Drosophila: the role of Collier

Proper sampling of sensory inputs critically depends upon neuron morphogenesis and expression of sensory channels. The highly stereotyped organisation of the Drosophila peripheral nervous system (PNS) provides a model to study neuronal determination and morphogenesis. This study reports that Collier/Knot (Col/Kn), the Drosophila member of the COE family of transcription factors, is transiently expressed in the subset of multidendritic arborisation (da) sensory neurons that display an highly branched dendritic arborisation, class IV neurons. When lacking Col activity, class IV da neurons are formed but display a reduced dendrite arborisation. Col control on dendrite branching is distinct from that exerted by Cut, another transcription factor expressed in class IV neurons and necessary for proper dendrite morphogenesis. Col is also required for the class IV da-specific expression of pickpocket (ppk), which encodes a degenerin/epithelial sodium channel subunit required for larval locomotion. Characterisation of the col upstream region identified a 9-kb cis-regulatory region driving col expression in all class IV md neurons, even though these originate from two types of sensory precursor cells. Altogether, these findings indicate that col is required in at least two distinct programs that control the morphological and sensory specificity of Drosophila md neurons (Crozatier, 2008).

Each individual cell of the Drosophila embryonic PNS has been described, providing a unique model system to investigate the mechanisms of coding of neural identity. This study shows that expression of the COE transcription factor Collier/Knot is specific to a subset of md neurons, the class IV neurons. Col is required both for the specific expression of ppk, a gene encoding a subunit of a sodium-gated channel involved in locomotion and aspects of the elaborate dendritic branching pattern of class IV md neurons. It should be noted that an independent study of col function in md neurons came to very similar conclusions (Hattori, 2007: Crozatier, 2008).

Systematic screens for transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites have revealed that a number of distinct transcription factors are involved in different aspects of dendrite extension, lateral branching and arborisation as well as in restriction of the dendritic coverage. However, to date the expression pattern of very few of these transcription factors is known during embryonic and post embryonic development. Col is the only known transcription factor whose expression is restricted to a subtype of md neurons. One particularly intriguing feature is that the three class IV md neurons found per abdominal hemisegment originate from at least two different types of lineages, the md-es (v'ada) or md-solo lineages (ddaC and vdaB), pointing to the existence of a convergence process towards the same neuronal phenotype. Since col remains transcribed in these three neurons in embryos mutant for col, it suggests that they are specified independently of col activity. The regulation of Col expression and the function in class IV md neurons provide a unique paradigm to study how the transcriptional control of md specification operates in independent lineages (Crozatier, 2008).

col transcription is first observed in the pII cells at the origin of class IV md neurons, indicating that it becomes restricted to these neurons following asymmetric division of the pIIb cell. Two or three Col-positive cells in place of each dorsal md neuron were often detected in embryos mutant for sanpodo, which encodes a four-pass transmembrane protein which interacts with the Notch receptor. Conversely, md-specific col transcription is often lost in embryos mutant for numb (numb1, a null allele), which down regulates Notch signalling in one of the pIIb daughter cells. It is therefore concluded that col transcription is repressed by Notch signalling. Previous studies on col requirement for the formation of the DA3 embryonic muscle showed that col transcription in the sibling DO5 muscle lineage is also repressed by Notch. It thus appears that col repression by Notch is used in several independent cell lineage decisions. Whether the same effectors of the Notch pathway are involved both in the md neuronal and DA3 muscle lineages remains to be determined. One intriguing feature of col transcription in post-mitotic md neurons is that it is only transient. This could be due to a direct control by maternal and/or early zygotic transcription activators that dilute away during embryogenesis. Alternatively, it could involve the progressive accumulation in md neurons of a repressor able to shut down col transcription. Preliminary dissection of the Drosophila col upstream region mapped the cis-regulatory information necessary for col transcription in md neurons to a fragment located between − 9 and − 5 kbp upstream of the transcription start site (Dubois, 2007). Expression of the reporter gene was not detected, however, in the neurons innervating the dh2 and vp5 es organs, indicating that Col expression in md neurons and es neurons is under the control of separate cis-elements. Whether (partly) different cis-elements are also involved in md-solo, versus md-es col activation remains an open question. An identical pattern of Col expression in Class IV md neurons is observed in Drosophila virilis, indicating that the transcriptional regulatory network controlling this expression has been conserved between these two Drosophila species. The − 9- to − 5-kbp fragment of col upstream DNA contains many sequences, between 20 and 80 bp in length, which are identical between D. melanogaster and D. virilis and represent as many potential regulatory sequences. Further dissection of this fragment should allow identifying which combinations of transcription factors are involved in the specific activation and temporal restriction of col transcription in class IV md neurons (Crozatier, 2008).

Activation of ppk transcription in class IV md neurons requires Col activity. Unlike Col, ppk remains expressed in these neurons until the end of larval development, showing that once activated, ppk expression is maintained independently of Col. This suggests the occurrence of a relay mechanism. The several hours delay between Col accumulation and ppk activation, in both normal embryos and upon pan-neuronal expression of Col, also indicates that the ability of Col to activate ppk depends upon another md-specific factor. Whether this factor(s) is itself a target of Col only activated in md neurons or an md-specific factor whose activity is potentiated by Col remains to be investigated. Finally, whether and how the delay and maintenance mechanisms are coupled is also unknown, at present. The transient character of Col expression does not favour a feed-forward mechanism such as that proposed for the specification of two lineage-related neurons in the CNS, where Col and its downstream targets act together to activate lineage-specific neuropeptides. The cis-regulatory region driving ppk expression in class IV md neurons has been identified. Parallel studies on col and ppk cis-regulation should now allow deciphering more precisely the transcriptional control of class IV md neuron differentiation (Crozatier, 2008).

In addition to ppk expression, class IV md neurons differ from the other md neurons by the length and degree of arborisation of their dendrite network. This dendritic arborisation is unchanged in ppk mutant larvae, showing that ppk expression and the dendritic network of class IV md neurons are specified by two independent programs. Formation of the primary branches of the md dendrite network starts to form at the end of embryogenesis and continues to elongate during larval development. At the same time, a more elaborate pattern of secondary arbors develops. The recent identification of no less than 76 transcription factors influencing (class I, in that case) dendrite formation suggests that the formation and maintenance of the dendritic network is regulated at many different, likely successive levels. One of these factors is Cut, which regulates distinct dendrite branching patterns in different md classes in a dose-dependent manner. It is therefore proposed that Col plays a dual function in implementing the class IV md neuron identity. According to this model, at least one unidentified class IV md neuron-specific TF is required for activating Col expression and regulating the level of Cut (Cutmedium) expression. On one side, Col cooperates with other md-specific TFs to activate ppk transcription specifically in class IV md neurons, independent of Cut. On the other, Col and Cutmedium are involved in establishing the secondary complex dendrite network, typical of these neurons that develops in larvae. Since col transcription is not maintained beyond embryogenesis, its role in secondary dendrite branching and maintaining ppk transcription in larvae must be indirect and likely involves (an) intermediate TF(s). Systematic identification of col and cut targets in embryonic class IV md neurons should allow to better understand their respective roles. col involvement in two parallel regulatory networks (or dual function) links two salient features of class IV md neurons, an extended dendritic field and ppk expression. Dendrites act as information-integrating centers as they receive sensory or synaptic inputs. How expression of the Na+ channel subunit Ppk and extended dendrite arborisation are physiologically linked is a next question (Crozatier, 2008).

Biochemical Characterization and Protein Interactions

The hypothesis that Ppk might form an ion channel was tested by expressing it in Xenopus oocytes and measuring whole cell current with the two-electrode voltage-clamp. Expression of Ppk alone failed to generate basal currents larger than those of uninjected control oocytes. In this respect, Ppk resembles several other DEG/ENaC proteins, such as MEC-4 and MEC-10 and ß- and γENaC subunits, that do not produce current when expressed in Xenopus oocytes. These observations suggested that activation of Ppk might require a specific, unknown ligand, or coexpression with other DEG/ ENaC proteins (Adams, 1998).

Although Ppk does not produce currents when expressed alone, its structural similarity to DEG/ENaC channels suggested it was likely to participate in the formation of channels. To test this possibility, Ppk was coexpressed with a closely related DEG/ENaC protein, BNC1. The rationale for this experiment was based on the observation that channels can be composed of multiple ENaC subunits, even though some of the subunits do not form channels by themselves. A BNC1 mutant, BNC1G430V, was used that contains an activating mutation and produces much larger whole cell currents than wild-type BNC1. Expression of Ppk with BNC1G430V generated amiloride-sensitive currents that were much smaller than currents in oocytes expressing BNC1G430V with control proteins. An interaction between BNC1 and Ppk was also detected biochemically. Ppk coimmunoprecipitates with BNC1. These data do not explain how association of Ppk disrupts the function of BNC1; it could alter gating, conduction, or delivery to the cell surface. Nevertheless, the biochemical and functional association of Ppk with BNC1 suggests that Ppk may be an ion channel subunit that is dependent upon another DEG/ENaC protein for its channel function (Adams, 1998).

fragile X-related regulates the mRNA level of Pickpocket1

Fragile X syndrome is caused by loss-of-function mutations in the fragile X mental retardation 1 (FMR1) gene. How FMR1 affects the function of the central and peripheral nervous systems is still unclear. FMR1 is an RNA binding protein that associates with a small percentage of total mRNAs in vivo. It remains largely unknown what proteins encoded by mRNAs in the FMR1-messenger ribonuclear protein (mRNP) complex are most relevant to the affected physiological processes. Loss-of-function mutations in the Drosophila fragile X-related (dfmr1) gene, which is highly homologous to the human fmr1 gene, decrease the duration and percentage of time that crawling larvae spend on linear locomotion. Overexpression of DFMR1 in multiple dendritic (MD) sensory neurons increases the time percentage and duration of linear locomotion; this phenotype is similar to that caused by reduced expression of the MD neuron subtype-specific degenerin/epithelial sodium channel (DEG/ENaC) family protein Pickpocket1 (PPK1). Genetic analyses indicate that PPK1 is a key component downstream of DFMR1 in controlling the crawling behavior of Drosophila larvae. DFMR1 and ppk1 mRNA are present in the same mRNP complex in vivo and can directly bind to each other in vitro. DFMR1 downregulates the level of ppk1 mRNA in vivo, and this regulatory process also involves Argonaute2 (Ago2), a key component in the RNA interference pathway. These studies identify ppk1 mRNA as a physiologically relevant in vivo target of DFMR1. The finding that the level of ppk1 mRNA is regulated by DFMR1 and Ago2 reveals a genetic pathway that controls sensory input-modulated locomotion behavior (Xu, 2004).
To study subtle behavioral defects caused by dfmr1 mutations, the crawling behavior of wild-type and mutant larvae were examined at the wandering stage with DIAS software in an environment devoid of cues for chemotaxis and phototaxis; because larvae were placed on a flat surface, as verified by a level, there were presumably no cues for geotaxis. The larval crawling behavior is relatively simple and stereotyped and can be separated into two phases: linear locomotion and nonlocomotive turning. As shown by analysis of the centroid paths of crawling larvae, wild-type and dfmr14 mutant larvae have different crawling patterns. Analysis at higher magnification showed that the wild-type larvae had longer linear paths and made fewer turns than dfmr14 mutants (Xu, 2004).

To quantify differences between wild-type and dfmr14 mutant larvae, the direction change was determined, defined as the absolute value of the difference in direction from one frame to the next. Computer analysis of crawling routes indicated that, during 1.5 min of recording (150 data points), a representative dfmr14 mutant larva had 17 data points with direction changes larger than 60°, and a representative wild-type larva had only eight such points (Xu, 2004).

Because the number of direction changes varied substantially among larvae of a given genotype, a large number of larvae were recorded and analyzed to quantify the difference at the population level. The average direction change for a larva is the mean value of all the data points. More dfmr14/dfmr14 mutants than wild-type larvae exhibited an average direction change greater than 20°. dfmr14/dmr14 mutant larvae showed a greater average direction change than wild-type larvae. No significant differences were seen between dfmr14/+ heterozygous larvae and wild-type larvae. To confirm that the abnormal crawling behavior was caused by dfmr1 mutations, mutant larvae were examined that had a combination of different dfmr1 loss-of-function alleles that were independently generated from the genetic screen. Larvae with different dfmr1 alleles all exhibited similar crawling behaviors. In addition, the alteration in direction change could be rescued by introducing a genomic DNA fragment containing the wild-type dfmr1 gene, indicating that dfmr1 was indeed responsible for the observed phenotype in mutant larvae (Xu, 2004).

To further characterize the alterations in the crawling pattern of dfmr1 mutant larvae, 'linear locomotion' was defined as the time period during which at least five consecutive frames showed a direction change that was smaller than 20°. Wild-type larvae spent a greater percentage of time on linear locomotion than dfmr14 mutant larvae. Mutant larvae with different dfmr1 alleles showed a phenotype similar to that of dfmr14 mutant larvae. In addition, the average duration of linear locomotion of dfmr14 mutant larvae was shorter than that of wild-type larvae. Both alterations were rescued by the introduction of a genomic DNA fragment containing the wild-type dfmr1 gene. These findings indicate that dfmr1 mutations significantly alter the crawling pattern of wandering larvae (Xu, 2004).

DFMR1 has been shown to play a role in the proper development of higher-order fine dendritic processes of MD sensory neurons in the PNS of Drosophila larvae. Most MD neurons elaborate extensive dendritic arbors just underneath the epidermis in each segment. To test whether MD neurons modulate crawling behavior, DFMR1 was expressed in MD neurons under the control of Gal4 109(2)80, which drives target gene expression in all MD neurons and less than 100 central neurons. This manipulation decreased the average direction change and increased the time percentage and duration of linear locomotion; together, these create a phenotype opposite that of dfmr1 mutant larvae. Furthermore, expression of normal DFMR1 protein by the same Gal4 driver in a dfmr1 mutant background rescued the defects in crawling behavior. These results are consistent with the notion that changes in DFMR1 activity in MD neurons affect the crawling behavior of Drosophila larvae (Xu, 2004).

To uncover the molecular mechanism underlying DFMR1 function, the association between DFMR1 and several mRNAs that encode known channel molecules was examined. A monoclonal antibody against DFMR1 was used to immunoprecipitate DFMR1-mRNP complexes and reverse transcription-polymerase chain reaction (RT-PCR) was used to demonstrate the presence or absence of a particular mRNA in the complexes. The mRNA encoding PPK1, an MD neuron subtype-specific member of the DEG/ENaC family, was associated with DFMR1 in vivo. Lysates from dfmr14 mutant larvae served as negative controls. No association was detected in the same immunoprecipitation experiment between DFMR1 and the mRNAs encoding Hyperkinetic (HK) and Shaker, both of which have been implicated in larval crawling behavior. To further investigate the association between DFMR1 and ppk1 mRNA, GST-DFMR1 fusion protein was purified and it was found that ppk1 mRNA could bind in vitro directly to GST-DFMR1 but not GST alone. To demonstrate the binding specificity, a competition binding experiment was performed. The affinity of DFMR1 for ppk1 mRNA was at least one order of magnitude higher than that for a control mRNA. These findings demonstrate that DFMR1 is associated with ppk1 mRNA in vivo and that they can directly bind to each other at least in vitro (Xu, 2004).

The level of ppk1 mRNA is regulated by DFMR1 in MD sensory neurons. dfmr1 mutant larvae have been shown to have slightly more higher-order dendritic branches of sensory neurons than wild-type larvae. It seems that ppk1 mRNA and dendrite development are independently regulated by DFMR1, because overexpression of Rac1 in MD neurons increased dendritic branching without affecting the level of ppk1 mRNA and locomotion behavior. Recent studies demonstrate that DFMR1 associates with microRNAs and proteins involved in the RNAi pathway. Although it was reported that DFMR1 could affect the efficiency of RNAi in vitro, it was found that DFMR1, unlike Ago2, is not required for efficient RNAi-mediated degradation of ppk1 mRNA in vivo. Deletion mutations were generated in ago2 and it was found that Ago2 also regulates the ppk1 mRNA level in a manner similar to DFMR1. Genetic-interaction studies further support the notion that the two molecules work in the same genetic pathway to control ppk1 mRNA level. A GST-Ago2 fusion protein was purified and it was found that Ago2 itself does not bind to ppk1 mRNA. These findings suggest a model in which DFMR1 binds to ppk1 mRNA and recruits Ago2 and presumably other components to regulate the level of ppk1 mRNA, which in turn modulates larval locomotion behavior. Interestingly, overexpression of PPK1 by itself does not affect locomotion behavior, possibly due to the fact that PPK1 is only a subunit of a functional channel. Therefore, other channel subunits or downstream components may be coordinately regulated by DFMR1. The association of DFMR1 and Ago1 has also been reported (Jin, 2004). Argonaute family proteins are involved not only in RNAi pathways but also in microRNA-mediated gene regulation. These studies provide strong evidence that sensory-channel molecules can be regulated by components associated with microRNA pathways (Xu, 2004).

Although DFMR1 is known to be an RNA binding protein, the RNAs that associate with it in vivo have not been systematically identified. Recent studies suggest that a small percentage of mRNAs are associated with FMR1-mRNA complexes in mouse brain and cell lines. Since DFMR1 and human FMR1 share a high degree of sequence homology and are likely to function similarly, DFMR1 might also regulate multiple mRNAs in Drosophila. It remains a major challenge to identify the key mRNA targets that mediate DFMR1's effect on a particular physiological pathway. In this study, by using both biochemical and genetic approaches, it has been shown that regulation of ppk1 mRNA by DFMR1 contributes to MD sensory neuron-modulated larval crawling behavior (Xu, 2004).

Because DEG/ENaC superfamily proteins are highly conserved through evolution, it would be of great interest to determine which DEG/ENaC channel molecules in mammals are also regulated by FMR1. Further understanding of the functional alterations in the sensory pathway caused by DFMR1/FMR1 mutations may provide deeper insights into the mechanisms underlying fragile X syndrome in humans (Xu, 2004).

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: PubMed).

Balboa binds to Pickpocket in vivo and is required for mechanical nociception in Drosophila larvae

The Drosophila gene pickpocket (ppk) encodes an ion channel subunit of the degenerin/epithelial sodium channel (DEG/ENaC) family. PPK is specifically expressed in nociceptive, class IV multidendritic (md) neurons and is functionally required for mechanical nociception responses. In a genome-wide genetic screen for other ion channel subunits required for mechanical nociception, this study identified a gene that was named balboa (also known as CG8546, ppk26). Interestingly, the balboa locus encodes a DEG/ENaC ion channel subunit highly similar in amino acid sequence to PPK. Moreover, laser-capture isolation of RNA from larval neurons and microarray analyses reveal that balboa is also highly enriched in nociceptive neurons. The requirement for Balboa and PPK in mechanical nociception behaviors and their specific expression in larval nociceptors led to an hypothesis that these DEG/ENaC subunits form an ion channel complex in vivo. In nociceptive neurons, Balboa::GFP proteins distribute uniformly throughout dendrites but remarkably localize to discrete foci when ectopically expressed in other neuron subtypes (where PPK is not expressed). Indeed, ectopically coexpressing ppk transforms this punctate Balboa::GFP expression pattern to the uniform distribution observed in its native cell type. Furthermore, ppk-RNAi in class IV neurons alters the broad Balboa::GFP pattern to a punctate distribution. Interestingly, this interaction is mutually codependent as balboa-RNAi eliminates Venus::PPK from the sensory dendrites of nociceptors. Finally, using a GFP-reconstitution approach in transgenic larvae, in vivo physical interactions among PPK and Balboa subunits were directly detected. Combined, these results indicate a critical mechanical nociception function for heteromeric PPK and Balboa channels in vivo (Mauthner, 2014).


DEVELOPMENTAL BIOLOGY
Embryonic

Drosophila has proven to be a good model for understanding the physiology of ion channels. Two novel Drosophila DEG/ENaC (degenerin/epithelial Na+ channel) proteins, Pickpocket (PPK) and Ripped Pocket (RPK) have been identified. Both appear to be ion channel subunits. Expression of RPK generates multimeric Na+ channels that are dominantly activated by a mutation associated with neurodegeneration. Amiloride and gadolinium, which block mechanosensation in vivo, inhibit RPK channels. Although PPK does not form channels on its own, it associates with and reduces the current generated by a related human brain Na+ channel. RPK transcripts are abundant in early stage embryos, suggesting a role in development. In contrast, PPK is found in sensory dendrites of a subset of peripheral neurons in late stage embryos and early larvae. In insects, such multiple dendritic neurons play key roles in touch sensation and proprioception and their morphology resembles human mechanosensory free nerve endings. These results suggest that PPK may be a channel subunit involved in mechanosensation (Adams, 1998).

To confirm that ppk cDNA is capable of encoding protein in vivo, COS-7 cells were transfected with an epitope-tagged ppk construct, and expressed protein was examined by Western blot. Expression of ppk cDNA yielded a 95-kD glycoprotein, that could be deglycosylated with endoglycosidase H or protein N-glycosidase. The deglycosylated form of Ppk protein migrated at 69 kD, consistent with its predicted molecular mass. Ppk can also be detected by Western blot using an antibody raised against a portion of its extracellular region (Adams, 1998).

To determine the tissue distribution of Ppk, Drosophila embryos and early first instar larvae were labeled with either an antisense ppk probe, or with anti-Ppk antibody. Using both methods, Ppk expression was detected exclusively in a subset of peripheral neurons. Expression became apparent in late stage embryos (stage 17) and was present in early larvae. To confirm that the cells expressing Ppk were peripheral neurons, embryos were double labeled with both anti-Ppk antibody and monoclonal antibody 22C10 that recognizes peripheral neurons. Cells expressing Ppk are also recognized by 22C10 (Adams, 1998).

The Drosophila peripheral nervous system (PNS) assumes a stereotypical pattern with three main types of neurons. External sensory (es) and chordotonal (ch) neurons innervate specific mechanosensory organs. These neurons each have a single uniterminal sensory dendrite. Multiple dendritic (md) neurons possess variable numbers of fine dendritic processes that lie beneath the epidermis. Higher magnification reveals Ppk staining on the surface of the cell bodies and in multiple fine processes that extend from the cell, indicating that Ppk is expressed in md neurons. On the basis of their dendritic morphology, md neurons are classified into three types; da (dendrites that arborize), td (tracheal-associated dendrites), and bd (bipolar dendrites). Moreover, like other PNS neurons, each md neuron occupies a well-defined and stereotypical position in the PNS. Based on their morphology, number and anatomical position, Ppk+ neurons represent a subset of da neurons. In the abdominal segments A1-A7, Ppk is expressed in one of the six dmd neurons, in the v'ada neuron, and in one of the five vmd neurons. In the thoracic segments T1-T3, Ppk is expressed in one of five dmd neurons and in one of five v'md neurons (Adams, 1998).

Since da neurons serve a variety of mechanosensory functions in insects, the neuronal expression pattern of Ppk suggested that it might play a role in mechanosensation. Along the dendrites of Ppk+ neurons, Ppk staining is observed at a number of discrete varicosities, giving the dendrites a beaded appearance. Interestingly, in the butterfly Pieris rapae crucivora, varicosities on the dendritic processes of da neurons are thought to be sites of mechanotransduction. Thus, the presence of Ppk in dendritic varicosities also suggest a mechanosensory function (Adams, 1998).

Amiloride sensitivity is a common characteristic of structurally related cationic channels that are associated with a wide range of physiological functions. In Caenorhabditis elegans, neuronal and muscular degenerins are involved in mechanoperception. In animal epithelia, a Na(+)-selective channel participates in vectorial Na+ transport. In the snail nervous system, an ionotropic receptor for the peptide FMRFamide forms a Na(+)-selective channel. In mammalian brain and/or in sensory neurons, ASIC channels form H(+)-activated cation channels involved in nociception linked to acidosis. A new member of this family has been cloned from Drosophila melanogaster. The corresponding protein displays low sequence identity with the previously cloned members of the super-family but it has the same structural organization. Its mRNA was detected from late embryogenesis (14-17 hours) and was present in the dendritic arbor subtype of the Drosophila peripheral nervous system multiple dendritic (md) sensory neurons. While the origin and specification of md neurons are well documented, their roles are still poorly understood. They could function as stretch or touch receptors, raising the possibility that this Drosophila gene product, called dmdNaC1 (Pickpocket), could also be involved in mechanotransduction (Darboux, 1998).

Sensory neurons in the Drosophila genital tract regulate female reproductive behavior

Females of many animal species behave very differently before and after mating. In Drosophila, changes in female behavior upon mating are triggered by the sex peptide (SP), a small peptide present in the male's seminal fluid. SP activates a specific receptor, the sex peptide receptor (SPR), which is broadly expressed in the female reproductive tract and nervous system. This study pinpoints the action of SPR to a small subset of internal sensory neurons that innervate the female uterus and oviduct. These neurons express both fruitless (fru), a marker for neurons likely to have sex-specific functions, and pickpocket (ppk), a marker for proprioceptive neurons. SPR expression in these fru+ ppk+ neurons is both necessary and sufficient for behavioral changes induced by mating. These neurons project to regions of the central nervous system that have been implicated in the control of reproductive behaviors in Drosophila and other insects (Häsemeyer, 2009).

SPR was initially identified in a genome-wide pan-neuronal RNAi screen. In this screen, the panneuronal elav-GAL4 driver was crossed to a genome-wide collection of RNAi transgenes, and female progeny were scored for egg-laying defects. Mated elav-GAL4 UAS-SPR-IR females lay very few eggs and remain sexually receptive, and thus, like SPR null mutants, behave as though they were still virgins. To define the cellular requirement for SPR function, the logic of this screen was inverted, crossing the UAS-SPR-IR transgene to a collection of 998 GAL4 lines and scoring the female progeny for egg-laying defects in the same fashion. In each of these lines, the GAL4 transcriptional activator is expressed in a random but stereotyped subset of cells, in which SPR function should now be inhibited by the UAS-SPR-IR transgene (Häsemeyer, 2009).

Fifty-nine lines were identified that resulted in a strong and reproducible egg-laying defect. Many of these lines were found to be broadly expressed, as revealed with a UAS-mCD8-GFP reporter. These lines were not examined further. More restricted neuronal expression was observed in seven lines, and for each of these a series of secondary assays was performed to confirm the egg-laying defect and to assess the receptivity of both virgin and mated females. For all seven GAL4 lines, SPR knockdown resulted in reduced egg laying and increased remating of mated females, but little if any change in the receptivity of virgin females. These defects were indistinguishable from those observed upon panneuronal SPR knockdown with the elav-GAL4 driver, or in SPR null mutant females. For the most restricted of the positive GAL4 lines, ppk-GAL4, it was confirmed that these defects can indeed be attributed to a diminished response to SP. It was then determined that SPR is required in ppk+ sensory neurons in the female reproductive tract (Häsemeyer, 2009).

This study describes a set of internal ppk+ fru+ sensory neurons in the female reproductive tract and provides evidence that SPR functions in these neurons to trigger the behavioral changes induced by SP upon mating. This conclusion rests on two complementary sets of observations. First, SPR is required in both ppk+ and fru+ cells, because postmating responses are eliminated upon knockdown of SPR in either cell population. Second, SPR is sufficient in either ppk+ or fru+ cells alone, as expression in either restores the postmating response in SPR null mutant females. This forces the conclusion that SPR acts exclusively in cells that are both ppk+ and fru+. The sensory neurons innervating the uterus are the only cells that were identified that express both of these markers. There are typically four to six such cells, and it is not yet known if they are functionally equivalent, or if egg laying and receptivity are regulated by two distinct cell subtypes (Häsemeyer, 2009).

Silencing synaptic transmission of ppk+ fru+ neurons mimics the activity of SP, in that they both cause virgin females to become unreceptive and initiate egg laying. Thus, an attractive hypothesis is that activation of SPR by SP reduces the synaptic output of these neurons. Like other ppk+ neurons, the ppk+ fru+ uterus neurons are probably mechanosensory. They may therefore have an important function as uterus stretch receptors in the coordination of sperm transfer, fertilization, and egg release. They may have two distinct functional states, depending on the presence or absence of SP. Because receptivity can be genetically uncoupled from egg production and egg laying, it is inferred that SP can also act independently of any stretch signal in the uterus. Modulation of receptivity and egg laying might be mediated through either distinct ppk+ fru+ subtypes or distinct central synapses (Häsemeyer, 2009).

How might SP regulate these sensory neurons? Two possibilities are envisioned. First, the ppk+ fru+ neurons may detect SP in the reproductive tract and alter their firing rate accordingly. In this model, passage of SP into the hemolymph would not be required to induce the postmating response. A second possibility is that SP enters the circulatory system and acts presynaptically to modulate the release of these neurons at their central targets. The fact that SP can indeed be detected in the hemolymph of mated females does not in itself exclude the former possibility. At least some effects of SP, such as stimulating juvenile hormone synthesis in the corpus allatum, probably do require SP to enter the hemolymph. Similarly, the fact that SP triggers a postmating response even when injected directly into the hemolymph is also consistent with either model. The somata and some processes of the ppk+ fru+ neurons lie outside the uterus and would be readily accessible to factors in the hemolymph. A neural rather than a circulatory route has been proposed to mediate postmating responses in several species of moths. However, this conclusion is based upon the loss of this response upon nerve cord transection, a result predicted by both of these models. Thus, both models are consistent with currently available evidence from studies in Drosophila and other species, and distinguishing between them will require detailed studies of the physiological properties of the ppk+ fru+ neurons in response to SP (Häsemeyer, 2009).

The central targets of the ppk+ fru+ sensory neurons include the abdominal and/or subesophageal ganglia -- regions of the CNS likely to contain circuits that mediate behavioral responses to mating. The abdominal ganglion houses the octopaminergic neurons that are believed to regulate the release and passage of mature eggs from the ovary to the uterus. It is suspected that these neurons are direct or indirect targets of the ppk+ fru+ sensory neurons and that these circuits serve to ensure that ovulation and oviposition are coordinated with the presence of sperm (Häsemeyer, 2009).

Some ppk+ fibers project from the abdominal trunk nerve right through to the SOG, potentially forming a direct neural connection from the reproductive tract to the brain. It is suspected that these projections may feed into circuits that regulate female receptivity and other postmating behaviors. Virgin females are enticed to mate by the male's courtship song. Most auditory sensory neurons project to the mechanosensory neuropil in the lateral SOG, close to the terminal arborizations of the ppk+ neurons. The proximity of the auditory processing centers and the ascending ppk+ projections raises the attractive possibility that mating modulates an early step in song processing. The SOG also contains processes of the Ilp7 neurons, which function in egg-laying site selection after mating. Direct evidence for mating-induced changes in SOG circuit function is lacking in flies but has been obtained in other insects. In some species of moth, mating induces a long-term inhibition of the SOG neurosecretory cells that regulate female pheromone biosynthesis, making mated females less attractive to other males (Häsemeyer, 2009).

Having identified sensory neurons that detect SP in the reproductive tract, it will now be important to characterize the central pathways that process these signals to regulate female behavior. In the olfactory system, sensory neurons that detect pheromones are fru+, as are their postsynaptic partners in the brain. Given that the sensory neurons that detect SP are also fru+, and many fru+ neurons are also located in both the abdominal and subesophageal ganglia, it is enticing to think that a similar logic may apply in these pathways too. Elucidating the operation of these circuits should reveal how the female CNS integrates both external and internal information to switch between two very different behavioral patterns (Häsemeyer, 2009).

Identification of Ppk26, a DEG/ENaC channel functioning with Ppk1 in a mutually dependent manner to guide locomotion behavior in Drosophila.

A major gap in understanding of sensation is how a single sensory neuron can differentially respond to a multitude of different stimuli (polymodality), such as propio- or nocisensation. The prevailing hypothesis is that different stimuli are transduced through ion channels with diverse properties and subunit composition. In a screen for ion channel genes expressed in polymodal nociceptive neurons, this study identified Ppk26, a member of the trimeric degenerin/epithelial sodium channel (DEG/ENaC) family, as being necessary for proper locomotion behavior in Drosophila larvae in a mutually dependent fashion with coexpressed Ppk1 (Pickpocket), another member of the same family. Mutants lacking Ppk1 and Ppk26 were defective in mechanical, but not thermal, nociception behavior. Mutants of Piezo, a channel involved in mechanical nociception in the same neurons, did not show a defect in locomotion, suggesting distinct molecular machinery for mediating locomotor feedback and mechanical nociception (Gorczyca, 2014).

This study has identified Ppk26, an DEG/ENaC channel subunit that is coexpressed with Ppk1 in class IV da neurons. Consistent with the model that Ppk26 and Ppk1 may be subunits of the same channel, it was found that Ppk1 and Ppk26 colocalize in class IV da neurons, they form a complex in heterologous expression systems, and they show nonadditive and nonredundant mutant phenotypes in vivo. Ppk26 protein was found in somatic, dendritic, and axonal compartments, and plasma membrane insertion was observed in terminal dendrites. Ppk1 and Ppk26 were reciprocally required for normal trafficking and/or insertion to the plasma membrane, further supporting the notion that these two channel subunits interact in vivo. This study shows that, as is the case for MEC Degenerin channels, mutations at the Degenerin position of Ppk26 lead to loss of class IV da neuron integrity. It was also found that Ppk26 function plays essential roles in normal larval locomotion, particularly in turning behavior (Gorczyca, 2014).

Overexpression of EGFP-tagged MEC channels in C. elegans has been reported to result in a punctate localization, leading to suggestions that each of these puncta represents a mechanosensory apparatus. In light of the speculation that contact of dendrites with subcuticular epidermis is part of the apparatus that senses mechanical stimuli, it is intriguing that both Ppk1 and Ppk26 were found on the surface of distal and higher-order dendrites, consistent with channel function in this compartment. Mutants in Ppk1 and Ppk26 showed defects in the frequency of turning of freely crawling larvae. Moreover, loss of function of either or both Ppk26 and Ppk1 had the same effect on larval locomotion. These findings support the notion that Ppk26 and Ppk1 may act in the same pathway—perhaps in the same channel complex—in mechanosensation that is important for proper locomotion (Gorczyca, 2014).

Class IV da neurons are known to express the DEG/ENaC channel subunit Ppk1 (Adams, 1998), an observation that was confirmed in this study. Given that DEG/ENaC channels are trimeric ion channels that typically require the assembly of different subunits for proper function, the specific coexpression of Ppk26 in class IV da neurons raises the possibility that Ppk1 and Ppk26 may correspond to different subunits of the same mechanosensory channel in vivo. This is supported by the findings of biochemical interactions between Ppk1 and Ppk26, their null mutant phenotypes that indicate nonredundancy, and by the reciprocal requirements of Ppk1 and Ppk26 for normal trafficking or insertion to the plasma membrane . The mechanism for this mutual dependence is currently unknown (Gorczyca, 2014).

Notably, inspection of the developmental expression profile of ppk1 RNA and ppk26 RNA revealed a similar time course, with the absolute levels of RNA for ppk26 twice as high as those of RNA for ppk1 throughout development. Although it is uncertain that this quantitative difference in RNA levels reflects a similar quantitative difference in the levels of Ppk1 and Ppk26 subunits, it is tempting to speculate that two Ppk26 subunits and one Ppk1 subunit may be assembled to form a surface-expressed trimeric channel (Gorczyca, 2014).

Studies in many systems have suggested that mutations in the Degenerin domain of DEG/ENaC lead to loss of cell integrity and perhaps degeneration; however, this behavior has not yet been observed in Drosophila Pickpocket family members. The Degenerin position is localized in the wrist region, close to the mouth of the pore. Thus, it has been suggested that Degenerin mutations change the properties of the channel, increasing open time probability and perhaps shifting its ion selectivity from Na+ to Ca2+. Given the tight regulation of Ca2+ within cells and its involvement in critical cellular processes, this increase in Ca2+ permeability may lead to loss of cellular homeostasis, in a process that has been dubbed excitotoxicity. Consistent with these findings, it was also observed that when a Degenerin mutation was introduced into Ppk26 that was overexpressed in class IV da neurons, it resulted in a marked reduction in dendritic arbor size. This suggests that the Ppk26-Deg mutation leads to toxicity in the sensory neurons, as has been observed in DEG/ENaC channels in C. elegans, but not in Ppk1. Whereas it is likely that the function of the pore structure of these channels is evolutionarily conserved, it is unclear why Ppk26-Deg has a more potent effect than Ppk1-Deg. One possibility is that the Ppk26-Deg residue makes a larger contribution to pore structure than Ppk1-Deg, because of its intrinsic structure or because of a potential 2:1 stoichiometry in vivo (Gorczyca, 2014).

Larval locomotion is likely regulated by sensory input provided by sensory neurons in the body wall, which may in turn modulate the motoneurons innervating the body wall. The C. elegans mechanosensitive TRPN channel TRP-4 acts in the DVA neuron to coordinate bending behavior and body posture through positive and negative modulation. It seems likely that class IV da neurons likewise provide some information for sensory modulation of locomotion through a mechanosensory mechanism, but future work will need to determine if this is indeed the case (Gorczyca, 2014).

Proprioception is a mechanosensory process involving sensory neurons that transduce the mechanical information related to body position or characteristics of the environment for the generation of appropriate behavioral output, such as the turning locomotor behavior that is essential for foraging larvae. This study has identified a member of the DEG/ENaC family of proteins, Ppk26, which acts together with Ppk1 likely as subunits of a channel important for mechanosensation. The results suggest that perhaps a major site of mechanosensory transduction is located in class IV da neuron dendritic processes. The behavioral phenotypes of larvae with Ppk1 and Ppk26 knockdown in class IV da neurons, as well as the respective null mutants or double mutants, suggest that a deficit in these channels interferes with the ability of the animal to execute proper turning behavior, raising the possibility that the two subunits could be involved in proprioceptively sensing the deformation of the cuticle. Whether class IV neurons function as proprioceptors still needs to be directly demonstrated, and future experiments will be needed to address the relationship between class IV neural activity and body position (Gorczyca, 2014).

Numerous studies have implicated the class IV da neurons in both thermal and mechanical nociception behavior. This study found that whereas Ppk1 and Ppk26 are important for mechanical nociception behavior, they are dispensable for thermal nociception behavior. While Ppk1 and Ppk26 channels indeed moonlight during two processes in the same neuron, namely, mechanical nociception and proprioception, these channels must be playing a specific role as they are not involved in thermal response by the same neuron (Gorczyca, 2014).

Whereas the class I da neurons and bd neurons are implicated in proprioception for the regulation of sequential contractions use the TRPN channel NompC as the sensor, class IV da neurons rely on the DEG/ENaC channel likely composed of Ppk26 and Ppk1 for the regulation of turning behavior as well as mechanical nociception, perhaps through sensing a mechanical signal at the cuticle. Interestingly, this study found that Piezo, a bona fide mechanotransducing ion channel involved in class IV da neuron mechanotransduction and required for mechanical nociception, does not appear to be involved in turning behavior, suggesting that different combinations of ion channels may serve different mechanosensory functions in the same neuron (Gorczyca, 2014).


EFFECTS OF MUTATION

Sensory mechanisms controlling the timing of larval developmental and behavioral transitions require the Drosophila DEG/ENaC subunit, Pickpocket1

Growth of multicellular organisms proceeds through a series of precisely timed developmental events requiring coordination between gene expression, behavioral changes, and environmental conditions. In Drosophila larvae, the essential midthird instar transition from foraging (feeding) to wandering (non-feeding) behavior occurs prior to pupariation and metamorphosis. The timing of this key transition is coordinated with larval growth and size, but physiological mechanisms regulating this process are poorly understood. Results presented in this study show that Drosophila larvae associate specific environmental conditions, such as temperature, with food in order to enact appropriate foraging strategies. The transition from foraging to wandering behavior is associated with a striking reversal in the behavioral responses to food-associated stimuli that begins early in the third instar, well before food exit. Genetic manipulations disrupting expression of the Degenerin/Epithelial Sodium Channel subunit, Pickpocket1(PPK1) or function of PPK1 peripheral sensory neurons caused defects in the timing of these behavioral transitions. Transient inactivation experiments demonstrated that sensory input from PPK1 neurons is required during a critical period early in the third instar to influence this developmental transition. Results demonstrate a key role for the PPK1 sensory neurons in regulation of important behavioral transitions associated with developmental progression of larvae from foraging to wandering stage (Ainsley, 2008; full text of article).

Developmental timing of a sensory-mediated larval surfacing behavior correlates with cessation of feeding and determination of final adult size

Controlled organismal growth to an appropriate adult size requires a regulated balance between nutrient resources, feeding behavior and growth rate. Defects can result in decreased survival and/or reproductive capability. Since Drosophila adults do not grow larger after eclosion, timing of feeding cessation during the third and final larval instar is critical to final size. This study demonstrates that larval food exit is preceded by a period of increased larval surfacing behavior termed the Intermediate Surfacing Transition (IST) that correlates with the end of larval feeding. This behavioral transition occurred during the larval Terminal Growth Period (TGP), a period of constant feeding and exponential growth of the animal. IST behavior is dependent upon function of a subset of peripheral sensory neurons expressing the Degenerin/Epithelial sodium channel (DEG/ENaC) subunit, Pickpocket1 (PPK1). PPK1 neuron inactivation or loss of PPK1 function caused an absence of IST behavior. Transgenic PPK1 neuron hyperactivation caused premature IST behavior with no significant change in timing of larval food exit resulting in decreased final adult size. These results suggest a peripheral sensory mechanism functioning to alter the relationship between the animal and its environment thereby contributing to the length of the larval TGP and determination of final adult size (Wegman, 2010).

The primary job description for insect larvae could be simplistically summarized as eating and growing. This sustained activity during their first week or so of life is necessary to fulfill their most critical function as a safe container nurturing the essential imaginal disks that will eventually be transformed into adult structures during morphogenesis. Since maintained association with an adequate food source is key to survival, larvae display numerous food-associated behaviors in response to a variety of environmental cues. Significant changes in larval responses to these cues during the third and final larval instar result in the major transition from foraging to wandering behavior. Results presented in this study and elsewhere (Ainsley, 2008), suggest that these two major larval stages, foraging (in the food) and wandering (out of the food) actually consist of a series of separable innate behaviors meant to alter the relationship between the animal and its environment (Wegman, 2010).

Midway through the third and final instar, larvae stop feeding and enter 'wandering stage' when they exit the food source completely in search of an appropriate pupation site. During this transition period, larvae display striking changes in body position and the spatial relationship between larvae and the food source. The constant feeding observed in foraging stage animals is associated with maintenance of a vertical anterior mouthhooks-down larval body position with only the posterior spiracles, the external openings to the larval tracheal system, protruding from the surface of the food. This body position allows constant contact with the food source for feeding and also maintains exposure of the posterior spiracle openings to the atmosphere to allow respiratory exchange. Full body immersion in the moist food source also prevents potential larval dessication resulting from excessive exposure to a drying external atmosphere. However, pupariation within the moist food source often results in lethality caused by suffocation due to occlusion of spiracular air exchange or by failure of adult eclosion due to inability to exit the pupal case. As a consequence, although this head-down full body immersion position is beneficial during larval foraging stages, it is essential for the animal to move to the surface of the food prior to pupariation. Results presented in this study show that changes in the spatial relationship between larvae and the food source during the final instar is modulated by activity of a subset of peripheral sensory neurons expressing the DEG/ENaC subunit PPK1 (Wegman, 2010).

Evolved differences between insect species also suggest that food surfacing behavior and complete food exit are genetically separable. Of the many Drosophila species that have been characterized, D. melanogaster is one of the few that actually fully exits the food source for pupariation. Entry into 'wandering' behavior in D. melanogaster is normally associated with complete larval exit from the food and movement up the sides of culture vials in search of a moderately dry pupation site. Many closely related Drosophila species choose to simply move to the food surface where they pupate. In these species, the change in body position from the vertical head-down foraging position to the food surface appears to be the key behavioral transition necessary for survival through metamorphosis. Although it is likely that this striking difference in larval behavior represents an adaptive response to environmental or food conditions, genetic and/or physiological explanations for this difference are not understood (Wegman, 2010).

The developmental transition in larval thermotactic preference reflected as dispersal vs. ARS behavior was initiated early in the third instar correlating with the previously characterized critical period for PPK1 neuron function from 80 to 90 h AEL (Ainsley, 2008). This timing coincides with the appearance of IST behavior suggesting that these two behaviors reflect the same developmental and behavioral transition preceding larval food exit. As further evidence of the functional correlation of these two intermediate behaviors, IST behavior was also dependent upon PPK1 function and activity of the PPK1 sensory neurons. IST behavior was absent in ppk1 null mutant larvae and sharply suppressed in transgenic animals with electrically silenced PPK1 neurons. In addition, hyperactivation of PPK1 neurons caused a premature and enhanced appearance of IST behavior paralleling the previously observed alterations in larval thermotactic behavior (Wegman, 2010).

The key role of PPK1 protein for normal IST behavior suggests that the temporal timing of PPK1 expression may function as a central regulatory switch for the developmental timing of this behavioral transition. However, previous studies have shown that PPK1 expression first appears in mdIV neurons in late embryos and is sustained into late third instar stages (Ainsley, 2008). These results indicate that developmentally regulated control of PPK1 expression is likely not a potential mechanism for control of IST behavior (Wegman, 2010).

Induction of premature and enhanced IST behavior did not lead to premature final food exit but caused a significant decrease in final adult size. This result is consistent with premature feeding termination caused by IST behavior. Therefore, these innate behaviors are genetically and functionally separable and the observed IST behavior is not simply the beginning step of final food exit and wandering behavior (Wegman, 2010).

Both the IST and ARS behavioral transitions correlate roughly with the proposed timing of larval critical size assessment early in the third instar. As discussed earlier, the developmental period between critical size assessment and larval food exit known as the TGP is essential for growth to final maximum size. If disrupted either behaviorally or metabolically, a decrease in TGP duration should result in a decrease in final adult size. This is consistent with the observed effect of PPK1 neuron hyperactivation on final adult size (Wegman, 2010).

Expression of the PPK1 DEG/ENaC subunit is tightly restricted to the mdIV neurons in the larval body wall and two bipolar neurons innervating the posterior spiracles (ps) (Ainsley, 2008). Although possible functional relationships between mdIV neurons and ps neurons are not clear, both morphological and anatomical differences suggest distinct physiological functions. The md/da sensory neurons within the larval body wall have been spatially and morphologically divided into four subclasses based upon complexity of their dendritic arbors and their relative location within the larval PNS. The mdIV neurons are just one subclass of the larger collection of md/da neurons present within the larval body wall. Although experimental characterization of the md/da neurons has been extremely useful in studies aimed at understanding the development of dendritic fields, the physiological functions and/or any interactions between the md/da subgroups remain poorly understood (Wegman, 2010).

The mdIV neurons have been implicated as nociceptive neurons involved in activating a writhing escape response when exposed to noxious heat. This escape response has been functionally attributed to the necessity for Drosophila larvae to protect themselves from parasitoid wasps.The mdIV neurons and PPK1 have been shown to be required for a larval nocifensive response to noxious mechanical stimuli (Zhong, 2010). The relevance of these responses to harsh thermal or mechanical stimulation within the normal endogenous larval environment remains uncertain. In experimental conditions reported in this study, hyperactivation of the mdIV neurons using either the constitutively-active PPK1[S511V] isoform or the low-threshold voltage-gated sodium channel, NaChBaceGFP, did not result in induction of the previously reported 'nocifensive' response (Wegman, 2010).

PPK1 neurons appear to play a much more prominent role in the normal larval response to its environment than simply a response to harsh stimuli. This supports the possibility of a polymodal role for this class of sensory neurons that may depend upon more than one source of activation signal. It must also be noted that the ppk1GAL4 driver transposon used in these studies and in previous studies demonstrating a nociceptive mdIV function does not distinguish between the mdIV neurons and the single PPK1-expressing bipolar neuron innervating each posterior spiracle (ps) (Ainsley, 2008). In addition, there are distinct morphological differences between the three mdIV neurons (ddaC, v'ada and vdaB) within each larval hemisegment. The mdIV ddaC neuron displays a more extensive and symmetrical dendritic arbor than the v'ada and vdaB neurons. Their relative dorsal/ventral locations within the body wall may also expose each mdIV subtype to a different range of stimuli whether mechanical or chemical. Therefore, results produced using the ppk1GAL4 transposon would not detect any differences in the relative contributions of mdIV subtypes or the ps neurons to any of the proposed functional roles (Wegman, 2010).

Feeding larvae detect and respond to multiple sensory inputs providing information about food resources and their immediate environment. Responses can be separated into distinct innate behaviors meant to coordinate internal growth signals with changing environmental conditions. The current findings demonstrate that the PPK1 neurons contribute to the regulation of larval developmental timing and feeding behavior transitions within normal environmental parameters. Results presented in this study have focused upon the larval PPK1-expressing neurons and their role in larval feeding behavior, however, other recent work has also described a role for PPK1-expressing neurons in regulation of adult feeding behavior (Ribeiro, 2010). Although a clear functional connection between the physiological roles of larval and adult PPK1 neurons is not yet apparent, both appear to contribute in some way to monitoring of external sensory information from food sources as input to help modulate internal nutrient homeostasis (Wegman, 2010).

Pickpocket is a DEG/ENaC protein required for mechanical nociception in Drosophila larvae

Highly branched Class IV multidendritic sensory neurons of the Drosophila larva function as polymodal nociceptors that are necessary for behavioral responses to noxious heat (>39°C) or noxious mechanical (>30 mN) stimuli. However, the molecular mechanisms that allow these cells to detect both heat and force are unknown. This study reports that the pickpocket(ppk) gene, which encodes a Degenerin/Epithelial Sodium Channel (DEG/ENaC) subunit, is required for mechanical nociception but not thermal nociception in these sensory cells. Larvae mutant for pickpocket show greatly reduced nociception behaviors in response to harsh mechanical stimuli. However, pickpocket mutants display normal behavioral responses to gentle touch. Tissue specific knockdown of pickpocket in nociceptors phenocopies the mechanical nociception impairment without causing defects in thermal nociception behavior. Finally, optogenetically-triggered nociception behavior is unaffected by pickpocket RNAi which indicates that ppk is not generally required for the excitability of the nociceptors. Interestingly, DEG/ENaCs are known to play a critical role in detecting gentle touch stimuli in C. elegans and have also been implicated in some aspects of harsh touch sensation in mammals. These results suggest that neurons which detect harsh touch in Drosophila utilize similar mechanosensory molecules (Zhong, 2010).

The situation for ppk is distinct from that of the painless gene which is required for both thermal and mechanical nociception responses. In heterologous cells, Painless currents can be activated by heat but not by osmotic pressure. This supports a direct role for Painless in the transduction of thermal nociception stimuli but a direct role as a mechanosensor remains unproven. Interestingly, heat activated Painless currents are strongly affected by intracellular calcium ions. This sensitivity to calcium suggests that the function of Painless in mechanical nociception pathways could feasibly be downstream of Pickpocket. For example, if Pickpocket functions as a direct mechanosensor, calcium influx through downstream voltage gated channels might indirectly sensitize Painless currents. In this model, the function of Painless would serve as an amplifier of mechanically gated Pickpocket currents (Zhong, 2010).

Although the results implicate a specific role for Pickpocket in mechanosensory function, a role for Pickpocket itself as a subunit of a mechanotransduction channel remains unproven. The ultimate proof of this hypothesis would require the detection of mechanically gated currents in a biochemically reconstituted system involving purified Pickpocket protein. However, expression of Pickpocket in heterologous cells has not been found to produce currents and the absence of currents by heterologously Pickpocket proteins likely indicates that additional factors present in vivo are needed for the function of this channel subunit. Indeed, co-expression of MEC-4 and MEC-10 is required for expression of these channels in heterologous expression systems; it is thus likely that another DEG/ENaC subunit in Drosophila is required for Pickpocket to form a functional channel. In future studies, using the mechanical nociception assay described in this study, it should be possible to identify additional Drosophila DEG/ENaC subunits that are specifically required for mechanical signaling in the multidendritic neurons. These would represent candidates for the additional subunits that may be required for the formation of a functional Pickpocket channel (Zhong, 2010).

Nevertheless, given the specificity of the phenotype, a model where Pickpocket functions as a component of a mechanotransduction complex seems likely especially given the well established functional role of DEG/ENaCs in the C. elegans mechanotransduction complex. The sub-cellular localization of the Pickpocket protein is found in discrete varicosities on the dendrites which is reminiscent of the punctate localization of the mechanotransduction complex of the touch receptor neuron processes in C. elegans (Zhong, 2010).

Although the mechanical nociception stimulus used in the assays used in this study seems qualitatively distinct from the gentle touch stimulus used in C. elegans studies the results suggest that the molecular machinery involved may be similar. This is somewhat surprising since the mec-4 and mec-10 genes of C. elegans are required for gentle touch responses in C. elegans, but mec-4 and mec-10 mutants show normal behavioral responses to harsh touch. This may suggest the existence of additional mechanotransduction pathways in the C. elegans touch neurons, or alternatively C. elegans may utilize distinct high threshold mechanosensory neurons. Consistent with the former possibility calcium responses to harsh touch in MEC-4 expressing neurons are still observed in mec-4 mutant ALM neurons. Consistent with the latter possibility worms with laser ablated gentle touch neurons still show behavioral responses to harsh touch. Indeed, although mec-10 itself is not required for harsh touch responses, it has been proposed that harsh touch detection in the worm may be mediated by high-threshold PVD neurons which express MEC-10. Interestingly, mice that are mutant for the ASIC3 DEG/ENaC channel show reduced sensitivity to noxious pinch. Thus, it is possible that the function of Pickpocket in Drosophila neurons could be similar to that of ASIC3 in mouse nociceptive neurons (Zhong, 2010).

It is clear that distinct mechanosensory pathways are likely to exist within organisms. In addition to the role of Pickpocket in harsh touch responses, gentle touch to the body wall of the Drosophila larva is thought to be detected by ciliated chordotonal neurons which utilize the TRP channels Nomp-C, Iav, and Nan for mechanotransduction. Similarly in C.elegans, distinct mechanosensory neurons rely on distinct signaling mechanisms (Zhong, 2010).

This identification of Pickpocket as a potential mechanotransducer in the Drosophila nociceptive neurons suggests a widespread and evolutionarily conserved role for DEG/ENaCs in neurosensory mechanotransduction. Furthermore, the pickpocket mutant phenotype genetically separates mechanical nociception from thermal nociception in Drosophila (Zhong, 2010).

Neural circuitry underlying Drosophila female postmating behavioral responses

After mating, Drosophila females undergo a remarkable phenotypic switch resulting in decreased sexual receptivity and increased egg laying. Transfer of male sex peptide (SP) during copulation mediates these postmating responses via sensory neurons that coexpress the sex-determination gene fruitless (fru) and the proprioceptive neuronal marker pickpocket (ppk) in the female reproductive system. Little is known about the neuronal pathways involved in relaying SP-sensory information to central circuits and how these inputs are processed to direct female-specific changes that occur in response to mating. This study demonstrates an essential role played by neurons expressing the sex-determination gene doublesex (dsx) in regulating the female postmating response. Shared circuitry was uncovered between dsx and a subset of the previously described SP-responsive fru+/ppk+-expressing neurons in the reproductive system. In addition, sexually dimorphic dsx circuitry was identified within the abdominal ganglion (Abg) that was critical for mediating postmating responses. Some of these dsx neurons target posterior regions of the brain while others project onto the uterus. It is proposed that dsx-specified circuitry is required to induce female postmating behavioral responses, from sensing SP to conveying this signal to higher-order circuits for processing and through to the generation of postmating behavioral and physiological outputs (Rezával, 2012).

These results show that in the female, dsx neurons associated with the internal genitalia not only form a component part of the previously described fru+/ppk+ network, but in fact define a more minimal SP-responsive neural circuit capable of inducing postmating changes, such as reduced receptivity, increased levels of rejection, and egg deposition (Rezával, 2012).

In addition to these 'classic' postmating behavioral responses, it was also noted that SP signaling to dsx neurons induces postmating changes in locomotor activity between unmated and mated females. Studies have shown that Drosophila males court immobilized females less than moving females; essentially, males react to changes in female locomotion, suggesting a causal link between female locomotion and increased courtship levels. It has been proposed that males are 'acoustically tuned' to signals generated by active females, stimulating increased courtship by changing the attention state of the male. Therefore, female mobility appears to contribute to her 'sex appeal' and decreased locomotion in mated females is likely to affect the male's willingness to copulate (Rezával, 2012).

The female's nervous system must have the capacity to receive, and interpret, postcopulatory signals derived from the male seminal package to direct physiological and behavioral responses required for successful deposition of fertilized eggs. It was demonstrated that two dsx clusters, composed of three bilateral neurons of the uterus, comprise a more defined component of the SP-responsive sensory circuit. In addition, the majority of other dsx neurons originating on the internal genitalia were shown to coexpress ppk. As ppk neurons are mechanosensory, these may be acting as uterine stretch receptors, facilitating sperm and egg transport, fertilization, and oviposition. Silencing neural function of ppk neurons appears to inhibit egg deposition, presumably by impeding egg transport along the oviducts. Similarly, in dsxGal4 females expressing TNT no egg deposition is ever observed, with unfertilized eggs atrophying in the lateral oviducts. In contrast, when fru+ neurons are silenced, deposition of successfully fertilized eggs is still observed, suggesting that different subsets of the dsx+/fru+/ppk+ SP-responsive sensory circuit may direct distinct postmating behavioral responses. As SP has been detected in the hemolymph of mated females, it has been suggested that this peptide could pass from the reproductive tract into the hemolymph to reach CNS targets. The fact that neither receptivity nor oviposition was restored to control levels when ppk-Gal80 (or Cha-Gal80) was expressed in dsxGal4/UAS-mSP flies opens the possibility that SP expression might affect additional dsx neurons in the CNS (Rezával, 2012).

Triggering of postmating responses via SP reception appears to occur via a small number of neurons expressing SPR on the female reproductive tract; however, SPR is also found on surface regions of the CNS as well as in endocrine glands and other reproductive tissues. Surprisingly, SPR may even be detected in the Drosophila male CNS, where no exposure to SP would be expected, and in insects that apparently lack SP-like. SPRs are therefore potentially responsive to other ligands, performing functions other than those associated with postmating responses in the diverse tissues in which SPRs are expressed (Rezával, 2012).

Extensive coexpression was found of dsx-expressing cells and SPR in the epithelium of the lower oviduct and spermathecae in females. However, mSP expression (or SPR downregulation) specifically in spermathecal secretory cells (SSC) or oviduct epithelium cells had no effect on receptivity or egg laying. In agreement with rescue experiments using neuronal Gal80 drivers to intersect Gal4-responsive UAS expression in dsx cells, this suggests that these cells are neither neuronal nor directly involved in SP-mediated postmating behaviors. SPR staining in the CNS was more difficult to determine given the limitations of the antibody; while no colocalization in the brain was observed, apparent coexpression was observed between SPR and a small subset of ventral (Rezával, 2012).

The results indicate that dsx-Abg neurons are required for the induction and regulation of specific components of the postmating response. It has been shown that inhibition of neurotransmission in apterous-expressing Abg neurons impairs SP-mediated postmating changes in receptivity and oviposition, emphasizing the importance of these neurons in the modulation of postmating responses (Rezával, 2012).

The level of dsx neuronal expression within the Abg and their associated fascicles projecting to the brain, where they form extensive presynaptic arborizations within the SOG, coupled with the effects that impairment of function in these neurons has on postmating responses, speaks to the involvement of these neurons in relaying information from the reproductive tract to the brain. That dsx-Abg neurons also project, and form presynaptic arborizations on the uterus, and that the effects on postmating responses when their function is impaired again argue that these neurons play a direct role in mediating processes such as egg fertilization and oviposition. Interestingly, most dsx intersecting neurons are specific to females. Sex-specific behaviors can arise from either shared circuits between males and females that operate differently and/or sex-specific circuits that result from the presence/absence of unique circuit components in one sex versus the other. The results support the latter (Rezával, 2012).

The VNC has been implicated in the modulation of postmating responses, with an identified focus specifically involved in ovulation and transfer of eggs into the uterus for fertilization. Octopaminergic modulatory neurons located at the distal tip of the VNC projecting to the reproductive tract are required for triggering ovulation, possibly by regulating muscle contractions in the ovaries and oviducts. Since earlier studies have shown that ablation of the pars intercerebralis revealed an additional focus for egg laying in the head, and the brain appears to be required for sexual behaviors, such that decapitated virgin females neither mate nor lay eggs, it seems likely that neurons in the Abg also require signals from the brain to regulate postmating responses such as egg transport, fertilization, and deposition (Rezával, 2012 and references therein).

Higher-order circuits in the female brain must be capable of integrating sensory inputs from the olfactory, auditory, and reproductive systems to decide between the alternative actions of acceptance or rejection of the male. Early gynandromorph studies mapped a region of the dorsal brain that must be female for an animal to be receptive; it has been recently shown that the majority of dsx neuronal clusters are located in this region. While neurons coexpressing dsx and fru in male brains define a more restricted circuitry for determining male mating decisions, in females no overlap between dsx+ and fru+ neurons is observable in the brain. It is also important to note that the sex-specific Fru isoform is absent in females; thus any circuits that are actively specified in the female are likely to depend on the female isoform DsxF. Most dsx neurons in the brain are found in the lateral protocerebrum, a region where multiple sensory inputs are thought to be integrated and discrete motor actions selected and coordinated. Further high-resolution functional and connectivity mapping will help to define which neurons participate in specific pre- and postmating behaviors in the female, allowing circuit architecture to be integrated with underlying cellular and synaptic properties. Future experiments will define what activity patterns trigger these behaviors and what activity patterns correlate with these behaviors (Rezával, 2012).

Drosophila nociceptors mediate larval aversion to dry surface environments utilizing both the painless TRP channel and the DEG/ENaC subunit, PPK1

A subset of sensory neurons embedded within the Drosophila larval body wall have been characterized as high-threshold polymodal nociceptors capable of responding to noxious heat and noxious mechanical stimulation. They are also sensitized by UV-induced tissue damage leading to both thermal hyperalgesia and allodynia very similar to that observed in vertebrate nociceptors. This study shows that the class IV multiple-dendritic (mdIV) nociceptors are also required for a normal larval aversion to locomotion on to a dry surface environment. Drosophila larvae are acutely susceptible to desiccation displaying a strong aversion to locomotion on dry surfaces severely limiting the distance of movement away from a moist food source. Transgenic inactivation of mdIV nociceptor neurons resulted in larvae moving inappropriately into regions of low humidity at the top of the vial reflected as an increased overall pupation height and larval desiccation. This larval lethal desiccation phenotype was not observed in wild-type controls and was completely suppressed by growth in conditions of high humidity. Transgenic hyperactivation of mdIV nociceptors caused a reciprocal hypersensitivity to dry surfaces resulting in drastically decreased pupation height but did not induce the writhing nocifensive response previously associated with mdIV nociceptor activation by noxious heat or harsh mechanical stimuli. Larvae carrying mutations in either the Drosophila TRP channel, Painless, or the degenerin/epithelial sodium channel subunit Pickpocket1 (PPK1), both expressed in mdIV nociceptors, showed the same inappropriate increased pupation height and lethal desiccation observed with mdIV nociceptor inactivation. Larval aversion to dry surfaces appears to utilize the same or overlapping sensory transduction pathways activated by noxious heat and harsh mechanical stimulation but with strikingly different sensitivities and disparate physiological responses (Johnson, 2012).

Regulation of food intake by mechanosensory ion channels in enteric neurons

Regulation of food intake is fundamental to energy homeostasis in animals. The contribution of non-nutritive and metabolic signals in regulating feeding is unclear. This study shows that enteric neurons play a major role in regulating feeding through specialized mechanosensory ion channels in Drosophila. Modulating activities of a specific subset of enteric neurons, the posterior enteric neurons (PENs), results in 6-fold changes in food intake. Deficiency of the mechanosensory ion channel PPK1 gene (pickpocket) or RNAi knockdown of its expression in the PENS result in a similar increase in food intake, which can be rescued by expression of wild-type PPK1 in the same neurons. Finally, pharmacological inhibition of the mechanosensory ion channel phenocopies the result of genetic interrogation. Together, this study provides the first molecular genetic evidence that mechanosensory ion channels in the enteric neurons are involved in regulating feeding, offering an enticing alternative to current therapeutic strategy for weight control (Olds, 2014: PubMed).


EVOLUTIONARY HOMOLOGS

Characterization of Mec-4, a C. elegans DEG/ENaC superfamily member involved in mechanosensory transduction

Three dominant mutations of mec-4, a gene needed for mechanosensation, cause the touch-receptor neurons of Caenorhabditis elegans to degenerate. With deg-1, another C. elegans gene that can mutate to induce neuronal degeneration and that is similar in sequence, mec-4 defines a new gene family. Cross-hybridizing sequences are detectable in other species, raising the possibility that degenerative conditions in other organisms may be caused by mutations in similar genes. All three dominant mec-4 mutations affect the same amino acid. Effects of amino-acid substitutions at this position suggest that steric hindrance may induce the degenerative state (Driscoll, 1991).

Aberrant ion channel activity plays a causative role in several human disorders. Inappropriately regulated channel activity also appears to be the basis for neurodegeneration induced by dominant mutations of Caenorhabditis elegans mec-4, a member of the degenerin gene family postulated to encode a subunit of a mechanosensory channel. The degenerin gene family has been defined by two C. elegans genes, mec-4 and deg-1, which can mutate to gain-of-function alleles that induce degeneration of specific groups of neurons. A related mammalian gene, rat alpha-rENaC, induces an amiloride-sensitive Na+ current when introduced to Xenopus oocytes, strongly suggesting that degenerin genes encode ion channel proteins. Deduced amino-acid sequences of the degenerins include two predicted membrane-spanning domains. Conserved amino acids within the second membrane-spanning domain (MSDII) are critical for MEC-4 activity and specific substitutions within MSDII, whether encoded in cis or in trans to a mec-4(d) mutation, block or delay the onset of degeneration. Remarkably, MSDII from two other family members, C. elegans deg-1 and rat alpha-rENaC, can functionally substitute for MEC-4 MSDII in chimaeric proteins. These results support a structural model for a mechanosensory channels in which multiple MEC-4 subunits are oriented such that MSDII lines the channel pore, and a neurodegeneration model, in which aberrant ion flow through this channel is a key event (Hong, 1994).

The process by which mechanical stimuli are converted into cellular responses is poorly understood, in part because key molecules in this mode of signal transduction, the mechanically gated ion channels, have eluded cloning efforts. The Caenorhabditis elegans mec-4 gene encodes a subunit of a candidate mechanosensitive ion channel that plays a critical role in touch reception. Comparative sequence analysis of C. elegans and Caenorhabditis briggsae mec-4 genes was used to initiate molecular studies that establish MEC-4 as a 768-amino acid protein that includes two hydrophobic domains theoretically capable of spanning a lipid bilayer. Immunoprecipitation of in vitro translated Mec-4 protein with domain-specific anti-MEC-4 antibodies and in vivo characterization of a series of mec-4lacZ fusion proteins both support the hypothesis that MEC-4 crosses the membrane twice. The MEC-4 amino- and carboxy-terminal domains are situated in the cytoplasm and a large domain, which includes three Cys-rich regions, is extracellular. Definition of transmembrane topology defines regions that might interact with the extracellular matrix or cytoskeleton to mediate mechanical signaling (Lai, 1996). Mechanosensory signaling mediated by mechanically gated ion channels constitutes the basis for the senses of touch and hearing and contributes fundamentally to the development and homeostasis of all organisms. Despite this profound importance in biology, little is known of the molecular identities or functional requirements of mechanically gated ion channels. This study reports a genetically based structure-function analysis of the candidate mechanotransducing channel subunit MEC-4, a core component of a touch-sensing complex in Caenorhabditis elegans and a member of the DEG/ENaC superfamily. Molecular lesions have been identified in 40 EMS-induced mec-4 alleles and residue and domain function have been further probed using site-directed approaches. This analysis highlights residues and subdomains critical for MEC-4 activity and suggests possible roles for these in channel assembly and/or function. A class of substitutions is described that disrupt normal channel activity in touch transduction but remain permissive for neurotoxic channel hyperactivation, and expression of an N-terminal MEC-4 fragment is shown to interfer with in vivo channel function. These data advance working models for the MEC-4 mechanotransducing channel and identify residues, unique to MEC-4 or the MEC-4 degenerin subfamily, that might be specifically required for mechanotransducing function. Because many other substitutions identified by this study affect residues conserved within the DEG/ENaC channel superfamily, this work also provides a broad view of structure-function relations in the superfamily as a whole. Because the C. elegans genome encodes representatives of a large number of eukaryotic channel classes, it is suggested that similar genetic-based structure-activity studies might be generally applied to generate insight into the in vivo function of diverse channel types (Hong, 2000).

Mechanosensory transduction in touch receptor neurons is believed to be mediated by DEG/ENaC (degenerin/epithelial Na+ channel) proteins in nematodes and mammals. In the nematode Caenorhabditis elegans, gain-of-function mutations in the degenerin genes mec-4 and mec-10 (denoted mec-4(d) and mec-10(d), respectively) cause degeneration of the touch cells. This phenotype is completely suppressed by mutation in a third gene, mec-6, that is needed for touch sensitivity. This last gene is also required for the function of other degenerins. mec-6 encodes a single-pass membrane-spanning protein with limited similarity to paraoxonases, which are implicated in human coronary heart disease. This gene is expressed in muscle cells and in many neurons, including the six touch receptor neurons. MEC-6 increases amiloride-sensitive Na+ currents produced by MEC-4(d)/MEC-10(d) by approximately 30-fold, and functions synergistically with MEC-2 (a stomatin-like protein that regulates MEC-4(d)/MEC-10(d) channel activity) to increase the currents by 200-fold. MEC-6 physically interacts with all three channel proteins. In vivo, MEC-6 co-localizes with MEC-4, and is required for punctate MEC-4 expression along touch-neuron processes. It is proposed that MEC-6 is a part of the degenerin channel complex that may mediate mechanotransduction in touch cells (Chelur, 2002).

In C. elegans, genes encoding components of a putative mechanotransducing channel complex have been identified in screens for light-touch-insensitive mutants. A long-standing question, however, is whether identified MEC proteins act directly in touch transduction or contribute indirectly by maintaining basic mechanoreceptor neuron physiology. In this study, the genetically encoded calcium indicator cameleon was used to record cellular responses of mechanosensory neurons to touch stimuli in intact, behaving nematodes. A gentle touch sensory modality is defined that adapts with a time course of approximately 500 ms and primarily senses motion rather than pressure. The DEG/ENaC channel subunit MEC-4 and channel-associated stomatin MEC-2 are specifically required for neural responses to gentle mechanical stimulation, but do not affect the basic physiology of touch neurons or their in vivo responses to harsh mechanical stimulation. These results distinguish a specific role for the MEC channel proteins in the process of gentle touch mechanosensation (Suzuki, 2003).

Characterization of Drosophila Ripped pocked, DEG/ENaC superfamily member

The molecular and functional characteristics of Ripped pocked (Rpk) were examined. Expression of an epitope-tagged Rpk construct in COS-7 cells generated a 73-kD glycoprotein that can be deglycosylated to its predicted molecular mass of 65 kD. On Northern analysis, an rpk probe detected transcripts in only embryonic and adult RNA, where a major 3.4-kb transcript was observed, as well as two smaller less-abundant messages (Adams, 1998).

To determine the embryonic expression pattern of rpk transcripts, in situ hybridization to whole mount embryos was performed using an antisense rpk probe. In contrast to ppk transcripts, rpk transcripts are detected in early stage (0-3 h) embryos, but are not present in later stages of embryogenesis. Furthermore, in early stage embryos, rpk transcripts are not localized to a specific embryonic region or cell type. In Drosophila embryos, zygotic transcription does not initiate until the third hour of development. Because rpk mRNA is detected in embryos before the initiation of zygotic transcription, this result suggests that embryonic rpk message is of maternal origin, and that Rpk may play a role in early development (Adams, 1998).

When expressed in Xenopus oocytes, Rpk generates small whole cell Na+ currents that are reversibly blocked by amiloride. Rpk is impermeable to K+, as shown by the elimination of inward current when external Na+ is replaced with K+. Thus, in contrast to Ppk, Rpk forms functional ion channels by itself (Adams, 1998).

In several C. elegans degenerins, mutation of a specific residue near M2, the 'Deg' mutation, causes a dominant form of neurodegeneration suggestive of constitutive ion channel activity. Similarly, BNC1 containing a Deg mutation (BNC1G430V) is activated, producing much larger currents in Xenopus oocytes. To learn whether Rpk could also be activated by the Deg mutation, a valine residue was incorporated at the appropriate position (residue 524). Like wild-type Rpk, RpkA524V generates Na+-selective currents that are reversibly inhibited by amiloride. However, RpkA524V currents are 20-50 times larger than wild-type Rpk currents. This indicates that the Deg mutation activates Rpk. RpkA524V is slightly more permeable to Li+ than Na+ but was impermeable to K+. RpkA524V is significantly more sensitive to amiloride than wild-type Rpk. Gadolinium, an inhibitor of mechanosensation and some stretch-activated channels, also reversibly inhibits RpkA524V current (Adams, 1998).

Individual DEG/ENaC proteins are subunits that form homo- or hetero-multimeric ion channels. Because DEG/ ENaC proteins with Deg mutations produce a genetically dominant phenotype in C. elegans, it is thought that the Deg mutation in one or a few subunits might activate the channel complex, producing larger currents. The hypothesis that the Deg mutation is dominant at the molecular level was tested by asking if channels composed of both wild-type Rpk and RpkA524V would generate small or large Na+ currents. Coexpression of Rpk and RpkA524V generate large Na+ currents that are similar in size to those generated by RpkA524V alone. However, the amiloride sensitivity of the current, was similar to that generated by wild-type Rpk alone. These observations indicate that the increase in current amplitude depends on RpkA524V, and the low amiloride sensitivity depends on wild-type Rpk. Thus, the data suggest that at least two subunits combine to produce multimeric channels, that the A524V mutation dominantly activates the channel, and that Ala524 dominantly determines amiloride sensitivity. Gadolinium also inhibits wild-type Rpk current, and gadolinium sensitivity is not significantly altered by the presence of wild-type Rpk in a complex with RpkA524V. Coexpression of Ppk with Rpk or RpkA524V does not significantly alter the amount, ionic selectivity, or amiloride sensitivity of Rpk or RpkA524V current. Thus, it appears that Ppk and Rpk are not subunits of the same ion channel but likely have distinct physiological roles (Adams, 1998).

Drosophila DEG/ENaC pickpocket genes are expressed in the tracheal system, where they may be involved in liquid clearance

The Drosophila tracheal system and mammalian airways are branching networks of tubular epithelia that deliver oxygen to the organism. In mammals, the epithelial Na+ channel (ENaC) helps clear liquid from airways at the time of birth and removes liquid from the airspaces in adults. The hypothesis was tested that related Drosophila degenerin (DEG)/ENaC family members might play a similar role in the fly. Among 16 Drosophila DEG/ENaC genes, called pickpocket (PPK) genes, 9 were found expressed in the tracheal system. By in situ hybridization, expression appeared in late-stage embryos after tracheal tube formation, with individual PPK genes showing distinct temporal and spatial expression patterns as development progressed. Promoters for several PPK genes drove reporter gene expression in the larval and adult tracheal systems. Adding the DEG/ENaC channel blocker amiloride to the medium inhibits liquid clearance from the trachea of first instar larvae. Moreover, when RNA interference is used to silence PPK4 and PPK11, larvae fail to clear tracheal liquid. These data suggest substantial molecular diversity of DEG/ENaC channel expression in the Drosophila tracheal system where the PPK proteins likely play a role in Na+ absorption. Extensive similarities between Drosophila and mammalian airways offer opportunities for genetic studies that may decipher further the structure and function of DEG/ENaC proteins and development of the airways (Liu, 2003a).

Contribution of Drosophila DEG/ENaC genes to salt taste

The ability to detect salt is critical for the survival of terrestrial animals. Based on amiloride-dependent inhibition, the receptors that detect salt have been postulated to be DEG/ENaC channels. Drosophila DEG/ENaC genes Pickpocket11 (ppk11) and Pickpocket19 (ppk19) are expressed in the larval taste-sensing terminal organ and in adults on the taste bristles of the labelum, the legs, and the wing margins. When Ppk11 or Ppk19 function is disrupted, larvae lose their ability to discriminate low concentrations of Na+ or K+ from water, and the electrophysiologic responses to low salt concentrations are attenuated. In both larvae and adults, disrupting Ppk11 or Ppk19 affects the behavioral response to high salt concentrations. In contrast, the response of larvae to sucrose, pH 3, and several odors remains intact. These results indicate that the DEG/ENaC channels Ppk11 and Ppk19 play a key role in detecting Na+ and K+ salts (Liu, 2003b).


REFERENCES

Search PubMed for articles about Drosophila pickpocket

Adams, C. M., Anderson, M. G., Motto, D. G., Price, M. P., Johnson, W. A. and Welsh, M. J. (1998). Ripped pocket and Pickpocket, novel Drosophila DEG/ENaC subunits expressed in early development and in mechanosensory neurons. J. Cell Biol. 140: 143-152. 9425162

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Biological Overview

date revised: 5 February 2015

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