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).
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 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).
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).
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).
Red author names indicate recommended papers.
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date revised: 10 August 2010
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