Centromere proteins CENP-C and CAL1 functionally interact in meiosis for centromere clustering,pairing, and chromosome segregation

Meiotic chromosome segregation involves pairing and segregation of homologous chromosomes in the first division and segregation of sister chromatids in the second division. Although it is known that the centromere and kinetochore are responsible for chromosome movement in meiosis as in mitosis, potential specialized meiotic functions are being uncovered. Centromere pairing early in meiosis I, even between nonhomologous chromosomes, and clustering of centromeres can promote proper homolog associations in meiosis I in yeast, plants, and Drosophila. It was not known, however, whether centromere proteins are required for this clustering. We exploited Drosophila mutants for the centromere proteins centromere protein-C (CENP-C) and chromosome alignment 1 (CAL1) to demonstrate that a functional centromere is needed for centromere clustering and pairing. The cenp-C and cal1 mutations result in C-terminal truncations, removing the domains through which these two proteins interact. The mutants show striking genetic interactions, failing to complement as double heterozygotes, resulting in disrupted centromere clustering and meiotic nondisjunction. The cluster of meiotic centromeres localizes to the nucleolus, and this association requires centromere function. In Drosophila, synaptonemal complex (SC) formation can initiate from the centromere, and the SC is retained at the centromere after it disassembles from the chromosome arms. Although functional CENP-C and CAL1 are dispensable for assembly of the SC, they are required for subsequent retention of the SC at the centromere. These results show that integral centromere proteins are required for nuclear position and intercentromere associations in meiosis.Centromeres are the control centers for chromosomes, and thus are essential for accurate segregation in cell division. Centromeres are the DNA regions with a specialized chromatin structure upon which the kinetochore is built. The kinetochore is a complex of at least 100 proteins that contains the proteins to bind microtubules, motors to move on or destabilize microtubules, as well as checkpoint proteins monitoring kinetochore–microtubule attachment (1). The ability of the kinetochore to control microtubule binding and chromosome movement is essential for proper segregation in both mitosis and meiosis. In meiosis, additional constraints are placed on kinetochore function to ensure that homologs segregate in the first division and that segregation of sister chromatids is deferred until the second division (2). Recent studies indicate that in addition, the centromere itself may influence homolog segregation by controlling homolog pairing and formation of the synaptonemal complex (SC) (3).In prophase of meiosis I, the homologs must pair and ultimately become attached, usually by recombination and crossing-over. By quantifying centromere number through prophase I, it has been observed that centromeres pair in yeast, plants, and Drosophila (3). Perhaps unexpectedly, this pairing can be between nonhomologous centromeres; in yeast, this has been proposed as a mechanism to prevent recombination around the centromere, as centromere pairing resolves from initially being nonhomologous to being homologous (4, 5). Homologous centromere pairing may play a critical role in ensuring segregation of chromosomes that do not undergo crossing-over, possibly by affecting orientation of the kinetochores (3, 6–8).The centromere also regulates synapsis via the formation of the SC. SC formation initiates at the centromere and sites of cross-over formation in yeast, and the centromere is the first site for SC formation in Drosophila prophase I (9, 10). In addition, the SC persists at the centromere in yeast and Drosophila after the SC present along the chromosome arms has disassembled late in prophase I (7, 9, 11). Although SC assembly does not begin at centromeres in mouse meiosis, it persists at the centromeres and appears to promote proper segregation (12, 13).Another centromere property has been observed in Drosophila oocytes. In most organisms, the centromeres are clustered together at one site at the onset of meiosis, likely a remnant of their configuration in mitosis, but this clustering breaks down as centromeres arrange in pairs (3, 4). In Drosophila, however, the centromeres remain clustered until exit from prophase I at oocyte maturation (9, 10, 14). Although an essential role for centromere clustering has not been demonstrated, it may facilitate homolog pairing, synapsis, or accurate segregation, particularly given that the homologous telomeres do not pair into a bouquet formation in Drosophila meiosis (15, 16). Components of the SC are necessary for centromere clustering, as is the cohesion protein ORD (9, 14).The studies on centromere pairing and clustering define centromere geography within the meiotic nucleus, but they did not test whether centromere structure or function was involved. Centromeres have specialized nucleosomes with a histone H3 variant, centromere protein-A (CENP-A) (17). Incorporation of CENP-A into centromere chromatin is regulated precisely, although it occurs at distinct cell cycle times in different cell types, varying between late mitosis and G1 (17). In vertebrates, a complex of 15 proteins, the constitutive centromere-associated network (CCAN), is present on the CENP-A chromatin throughout the cell cycle and is crucial for assembling kinetochore proteins (1). In Drosophila, the entire CCAN complex has not been identified, although the CENP-C protein is present (18). Another Drosophila protein, CAL1, binds to CENP-A (called CID in Drosophila) in a prenucleosomal complex, and CAL1 is required for loading CID (19–22). CAL1 interacts with both CID and CENP-C, and all three proteins show interdependency for centromere localization (21, 23).Little is known about the activities of these centromere proteins in meiosis. In fission yeast, CENP-C has been demonstrated to be critical for kinetochore–microtubule binding in meiosis and also to control kinetochore orientation in meiosis I (24). The timing of assembly of kinetochore and centromere proteins onto meiotic chromosomes has been examined in mouse spermatocytes (25) and in Drosophila spermatocytes and sperm (26, 27). RNAi studies have shown that CAL1 and CENP-C (the latter to a lesser extent) are needed for CID localization in Drosophila male meiosis, with reduction in the levels of any of these three proteins being associated with meiotic segregation errors (26). Drosophila males differ from most organisms in not undergoing recombination or forming an SC, and centromere clustering does not occur (28). A question of particular interest that has yet to be addressed is whether centromere architecture and function are required for centromere clustering and pairing in meiosis.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.     

Distinct signaling of Drosophila chemoreceptors in olfactory sensory neurons

In Drosophila, olfactory sensory neurons (OSNs) rely primarily on two types of chemoreceptors, odorant receptors (Ors) and ionotropic receptors (Irs), to convert odor stimuli into neural activity. The cellular signaling of these receptors in their native OSNs remains unclear because of the difficulty of obtaining intracellular recordings from Drosophila OSNs. Here, we developed an antennal preparation that enabled the first recordings (to our knowledge) from targeted Drosophila OSNs through a patch-clamp technique. We found that brief odor pulses triggered graded inward receptor currents with distinct response kinetics and current–voltage relationships between Or- and Ir-driven responses. When stimulated with long-step odors, the receptor current of Ir-expressing OSNs did not adapt. In contrast, Or-expressing OSNs showed a strong Ca²⁺-dependent adaptation. The adaptation-induced changes in odor sensitivity obeyed the Weber–Fechner relation; however, surprisingly, the incremental sensitivity was reduced at low odor backgrounds but increased at high odor backgrounds. Our model for odor adaptation revealed two opposing effects of adaptation, desensitization and prevention of saturation, in dynamically adjusting odor sensitivity and extending the sensory operating range.From insects to mammals, the sense of smell begins with odor detection by olfactory sensory neurons (OSNs) (1–6). Recently, rapid advances have been made in understanding chemoreceptors in Drosophila OSNs (7–9). To date, Drosophila is the only model organism for which odor selectivity is known for most of its odorant receptors (Ors) (10, 11), and an Or expression pattern has been mapped to OSNs (12, 13). In addition, another family of chemoreceptors called ionotropic receptors (Irs) has been identified and characterized (14–16). These two types of chemoreceptors respond to different odors, thus endowing Drosophila OSNs with unique and complementary properties for odor detection (17). In contrast to the advanced molecular understanding of these two types of chemoreceptors, the mechanisms of their cellular signaling in native OSNs remain unclear, particularly hampered by the technical difficulty of carrying out patch-clamp recordings of Drosophila OSNs.Drosophila OSNs are encased in hair-like sensilla in the antennae and maxillary palps, with each sensillum containing the dendrites of one to four OSNs that are wrapped by sheath cells (18). The responses of native Drosophila OSNs to odors have traditionally been measured by electroantennogram (EAG) (19), which extracellularly measures the potentials across the entire antenna. In addition, single-sensillum recording (SSR) was developed to provide a higher spatial resolution by measuring the local field potentials (LFPs) from a single sensillum (20–24). These methods, especially SSR, have greatly advanced understanding of the odor selectivity of both Ors and Irs (10, 11, 14). However, because sheath cells and other OSNs also contribute to EAG and SSR signals (25), the response characteristics obtained by such measurements are often contaminated. Patch-clamp recordings of single OSNs could ideally overcome this issue while facilitating the experimental manipulations of a cell’s membrane potential; however, this standard method has unfortunately not yet been routinely applied to Drosophila OSNs.Here, we developed a Drosophila antennal preparation and succeeded in performing patch-clamp recordings of single identified OSNs. By using a fast solution change system to deliver liquid-phase odor stimuli, we investigated the response properties of odor-induced receptor currents of Drosophila OSNs. We found that OSNs expressing Ors exhibited slow response kinetics, outward receptor current rectification, and strong adaptation to odors. We further demonstrated that this adaptation was produced by a Ca²⁺ influx into OSNs because it could be eliminated by voltage clamping at positive holding potentials, by removing extracellular Ca²⁺, or by removing internal free Ca²⁺ with a Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Importantly, in contrast to the long-held view that adaptation simply increases sensitivity, we found that Or-mediated adaptation selectively reduced odor-signaling gain at low odor backgrounds but increased the gain at high odor backgrounds, thereby extending the dynamic odor-operating range. In contrast, odor-induced receptor currents in Ir-expressing OSNs showed fast response kinetics and, surprisingly, did not adapt.     

Mouse model implicates GNB3 duplication in a childhood obesity syndrome

Obesity is a highly heritable condition and a risk factor for other diseases, including type 2 diabetes, cardiovascular disease, hypertension, and cancer. Recently, genomic copy number variation (CNV) has been implicated in cases of early onset obesity that may be comorbid with intellectual disability. Here, we describe a recurrent CNV that causes a syndrome associated with intellectual disability, seizures, macrocephaly, and obesity. This unbalanced chromosome translocation leads to duplication of over 100 genes on chromosome 12, including the obesity candidate gene G protein β3 (GNB3). We generated a transgenic mouse model that carries an extra copy of GNB3, weighs significantly more than its wild-type littermates, and has excess intraabdominal fat accumulation. GNB3 is highly expressed in the brain, consistent with G-protein signaling involved in satiety and/or metabolism. These functional data connect GNB3 duplication and overexpression to elevated body mass index and provide evidence for a genetic syndrome caused by a recurrent CNV.Body weight exhibits a heritability of ∼70% (1–3), with both rare and common alleles contributing to obesity. Few cases of obesity are explained by mutations in single genes, with most confined to the well-known leptin/melanocortin pathway (4, 5). Genome-wide association studies (GWASs) have identified alleles that confer a small risk for elevated body mass index (BMI) and other measures of obesity, and some common variants involve candidate genes that are highly expressed in the central nervous system (6–9). However, the functional mechanism by which most GWAS loci contribute to obesity is unknown.Recently, large deletions and duplications that represent copy number variation (CNV) have been linked to early onset obesity in children (10–14). Rare CNVs have proven to be highly penetrant in familial and spontaneous cases of obesity that are sometimes concomitant with intellectual disability (ID) and/or other neurodevelopmental disorders. Rare but recurrent CNVs are particularly useful to pinpoint critical regions or candidate genes, because their shared genotype can be correlated with a common phenotype. For example, recurrent deletions of 16p11.2 are enriched in multiple obesity cohorts vs. controls (10–12).Mouse models have been instrumental in unraveling the genetic mechanisms and signaling pathways involved in obesity, which are critical for the development of therapeutics. Notably, ob/ob mutant mice that lack leptin are morbidly obese and exhibit other features, including hyperphagia, infertility, decreased immune function, and energy and body temperature dysfunction (15, 16). In humans, congenital leptin deficiency causes early onset hyperphagia, hypogonadism, impaired immunity, and severe obesity (17). For both ob/ob mice and leptin-deficient humans, leptin replacement ameliorates many of these symptoms (18–20). Mouse models of other metabolic disorders have also proven insightful in teasing apart the physiological effects of genes involved in obesity (21–24).Here, we report a syndrome caused by a recurrent chromosomal translocation associated with obesity as well as ID, seizures, macrocephaly, and eczema. Moreover, we have recapitulated the obesity phenotype in a transgenic mouse model and identified the gene responsible for elevated weight gain. This CNV duplication causes a highly penetrant form of early onset obesity.     

Differential effect of aneuploidy on the X chromosome and genes with sex-biased expression in Drosophila

Global analysis of gene expression via RNA sequencing was conducted for trisomics for the left arm of chromosome 2 (2L) and compared with the normal genotype. The predominant response of genes on 2L was dosage compensation in that similar expression occurred in the trisomic compared with the diploid control. However, the male and female trisomic/normal expression ratio distributions for 2L genes differed in that females also showed a strong peak of genes with increased expression and males showed a peak of reduced expression relative to the opposite sex. For genes in other autosomal regions, the predominant response to trisomy was reduced expression to the inverse of the altered chromosomal dosage (2/3), but a minor peak of increased expression in females and further reduced expression in males were also found, illustrating a sexual dimorphism for the response to aneuploidy. Moreover, genes with sex-biased expression as revealed by comparing amounts in normal males and females showed responses of greater magnitude to trisomy 2L, suggesting that the genes involved in dosage-sensitive aneuploid effects also influence sex-biased expression. Each autosomal chromosome arm responded to 2L trisomy similarly, but the ratio distributions for X-linked genes were distinct in both sexes, illustrating an X chromosome-specific response to aneuploidy.Changes in chromosomal dosage have long been known to affect the phenotype or viability of an organism (1–4). Altering the dosage of individual chromosomes typically has a greater impact than varying the whole genome (5–7). This general rule led to the concept of “genomic balance” in that dosage changes of part of the genome produce a nonoptimal relationship of gene products. The interpretation afforded these observations was that genes on the aneuploid chromosome produce a dosage effect for the amount of gene product present in the cell (8).However, when gene expression studies were conducted on aneuploids, it became known that transacting modulations of gene product amounts were also more prevalent with aneuploidy than with whole-genome changes (9–14). Assays of enzyme activities, protein, and RNA levels revealed that any one chromosomal segment could modulate in trans the expression of genes throughout the genome (9–15). These modulations could be positively or negatively correlated with the changed chromosomal segment dosage, but inverse correlations were the most common (10–13). For genes on the varied segment, not only were dosage effects observed, but dosage compensation was also observed, which results from a cancelation of gene dosage effects by inverse effects operating simultaneously on the varied genes (9, 10, 14–18). This circumstance results in “autosomal” dosage compensation (14, 16–18). Studies of trisomic X chromosomes examining selected endogenous genes or global RNA sequencing (RNA-seq) studies illustrate that the inverse effect can also account for sex chromosome dosage compensation in Drosophila (15, 19–21). In concert, autosomal genes are largely inversely affected by trisomy of the X chromosome (15, 19, 21).The dosage effects of aneuploidy can be reduced to the action of single genes whose functions tend to be involved in heterogeneous aspects of gene regulation but which have in common membership in macromolecular complexes (8, 22–24). This fact led to the hypothesis that genomic imbalance effects result from the altered stoichiometry of subunits that affects the function of the whole and that occurs from partial but not whole-genome dosage change (8, 22–25). Genomic balance also affects the evolutionary trajectory of duplicate genes differently based on whether the mode of duplication is partial or whole-genome (22, 23).Here we used RNA-seq to examine global patterns of gene expression in male and female larvae trisomic for the left arm of chromosome 2 (2L). The results demonstrate the strong prevalence of aneuploidy dosage compensation and of transacting inverse effects. Furthermore, because both trisomic males and females could be examined, a sexual dimorphism of the aneuploid response was discovered. Also, the response of the X chromosome to trisomy 2L was found to be distinct from that of the autosomes, illustrating an X chromosome-specific effect. Genes with sex-biased expression, as determined by comparing normal males and females, responded more strongly to trisomy 2L. Collectively, the results illustrate the prevalence of the inverse dosage effect in trisomic Drosophila and suggest that the X chromosome has evolved a distinct response to genomic imbalance as would be expected under the hypothesis that X chromosome dosage compensation uses the inverse dosage effect as part of its mechanism (15).     

Phagocytosis genes nonautonomously promote developmental cell death in the Drosophila ovary

Programmed cell death (PCD) is usually considered a cell-autonomous suicide program, synonymous with apoptosis. Recent research has revealed that PCD is complex, with at least a dozen cell death modalities. Here, we demonstrate that the large-scale nonapoptotic developmental PCD in the Drosophila ovary occurs by an alternative cell death program where the surrounding follicle cells nonautonomously promote death of the germ line. The phagocytic machinery of the follicle cells, including Draper, cell death abnormality (Ced)-12, and c-Jun N-terminal kinase (JNK), is essential for the death and removal of germ-line–derived nurse cells during late oogenesis. Cell death events including acidification, nuclear envelope permeabilization, and DNA fragmentation of the nurse cells are impaired when phagocytosis is inhibited. Moreover, elimination of a small subset of follicle cells prevents nurse cell death and cytoplasmic dumping. Developmental PCD in the Drosophila ovary is an intriguing example of nonapoptotic, nonautonomous PCD, providing insight on the diversity of cell death mechanisms.Programmed cell death (PCD) is the genetically controlled elimination of cells that occurs during organismal development and homeostasis. Cells are considered dead when they have undergone irreversible plasma membrane permeabilization or have become completely fragmented (1). Apoptosis is the most well-characterized form of PCD, however there are at least a dozen cell death modalities that are morphologically, biochemically, and genetically distinct (2, 3). Two examples of nonapoptotic cell death are autophagic cell death and necrosis, but there are several alternative cell death mechanisms that are less well understood.Nonapoptotic PCD occurs on a large scale in the Drosophila ovary. Drosophila females can produce hundreds of eggs during their lifetime, and for every egg that is formed, developmental PCD of supporting nurse cells (NCs) occurs. However, the mechanisms of developmental PCD in the Drosophila ovary are poorly understood. Each egg forms from a 16-cell germ-line cyst, comprised of the single oocyte and 15 NCs that support the oocyte throughout 14 stages of oogenesis (4, 5). Hundreds of somatically derived follicle cells (FCs) surround the germ-line cyst, forming an egg chamber. At stage 11 of oogenesis, NCs rapidly transfer (“dump”) their cytoplasm into the oocyte. Concurrently, the NCs asynchronously undergo developmental PCD, resulting in mature stage 14 egg chambers that no longer contain any NCs (4–6). Interestingly, caspases, proteases associated with apoptosis, play only a minor role in the death of the NCs in late oogenesis (7–9). Furthermore, combined inhibition of caspases and autophagy does not significantly block NC death during late oogenesis (10). To date, defining the major mechanism of developmental PCD in the Drosophila ovary has remained elusive.An intriguing possibility is that the somatic FCs non–cell-autonomously promote developmental PCD of the NCs during late oogenesis. Non–cell-autonomous regulation of PCD occurs when a cell or group of cells extrinsically initiates or promotes the death of another cell. This concept challenges the idea that PCD is largely a self-regulated, autonomous suicide program in which a cell controls its own demise. One well-characterized example of non–cell-autonomous control of PCD is apoptosis induced by the death ligands Fas or TNF (11, 12).Another type of non–cell-autonomous PCD is phagoptosis (or primary phagocytosis), in which engulfing cells directly cause the death of other cells via “murder” or “assisted suicide.” Phagoptosis is distinct from the engulfment of cell corpses, as the engulfing cell plays an active role in the death of a cell, rather than simply degrading a cell that died via another mechanism. The defining characteristic of phagoptosis is that inhibition of phagocytosis leads to a failure in cell death (13, 14). Phagoptosis has been demonstrated in activated microglia that phagocytose viable neurons, resulting in their destruction (13–15). Entosis is another example of non–cell-autonomous PCD, often referred to as “cell cannibalism,” in which a viable cell invades another cell, where it is degraded by lysosomes. Entosis is distinct from phagoptosis, as the inhibition of phagocytosis genes does not prevent entosis (16). Phagocytosis has also been shown to promote PCD in Caenorhabditis elegans, although this is an example of assisted suicide, as dying cells also require apoptotic machinery (17, 18).Genetic studies in C. elegans have identified two partially redundant signaling pathways that control phagocytosis: the cell death abnormality (CED)-1, 6, 7 and CED-2, 5, 12 pathways (19–21). The CED-1, 6, 7 and CED-2, 5, 12 pathways act in parallel to promote the activation of CED-10, a Rac GTPase responsible for cytoskeletal rearrangements that allow for internalization of the cell corpse. In Drosophila, the roles of the Ced-1, 6, 7 and Ced-2, 5, 12 pathways appear to be conserved. The CED-1 ortholog, Draper, is a transmembrane protein that localizes to the surface of the engulfing cell and acts as a receptor to recognize dying cells. Draper was first shown to be required for engulfment of apoptotic neurons in the embryonic central nervous system with mutants displaying lingering cell corpses (22). Additionally, Draper has been shown to be important in several other contexts including the engulfment of severed axons, bacteria, imaginal disc cells, hemocytes, and apoptotic NCs in midoogenesis (23–27). In addition to Draper, other Drosophila engulfment receptors include Croquemort (28) and integrins (29–31). Croquemort is related to CD36, a scavenger receptor involved in engulfment in mammals (32), and integrins also act as engulfment receptors in C. elegans and mammals (33, 34). The upstream activators of the Ced-2, 5, 12 pathway are largely unknown, although integrins may activate the pathway (34). As in C. elegans, it appears that Ced-12 and draper function in separate pathways in Drosophila. Ced-12 and draper have been shown to function in distinct steps in axon clearance (35). In macrophages, Ced-12 has been shown to function in a separate pathway from simu, a bridging molecule that acts upstream of draper (36). A number of other engulfment genes have been identified in Drosophila, and their molecular interactions are under active investigation (36–39).Given the minor role for apoptosis and autophagic cell death during developmental PCD in the Drosophila ovary, we investigated the possibility that the FCs non–cell-autonomously promote NC death. Previously we showed that FCs of the Drosophila ovary are capable of phagoptosis in midoogenesis when phagocytosis genes are overexpressed (27), and we questioned whether phagocytosis genes might normally function to control cell death in late oogenesis. Indeed, we found that the phagocytosis genes draper and Ced-12/ELMO are required in the FCs for NC removal in late oogenesis and that they function partly in parallel. We also show that the FCs non–cell-autonomously control events associated with the death of the NCs, including nuclear envelope permeabilization, acidification, and DNA fragmentation. Furthermore, the genetic ablation of stretch FCs disrupted all cellular changes associated with developmental PCD of the NCs. Therefore, PCD of the NCs is a unique model of a naturally occurring developmental cell death program that is nonapoptotic and non–cell-autonomously controlled.     

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

Mechanical stimuli, including tactile and sound signals, convey a variety of information important for animals to navigate the environment and avoid predators. Recent studies have revealed that Drosophila larvae can sense harsh or gentle touch with dendritic arborization (da) neurons in the body wall and can detect vibration with chordotonal organs (Cho). Whether they can also detect and respond to vibration or sound from their predators remains an open question. Here we report that larvae respond to sound of wasps and yellow jackets, as well as to pure tones of frequencies that are represented in such natural sounds, with startle and burrowing behaviors. The larval response to sound/vibration requires Cho neurons and, to a lesser extent, class IV da neurons. Our calcium imaging and electrophysiological experiments reveal that Cho neurons, but not class IV da neurons, are excited by natural sounds or pure tones, with tuning curves and intensity dependence appropriate for the behavioral responses. Furthermore, our study implicates the transient receptor potential (TRP) channels NOMPC, NANCHUNG, and INACTIVE, but not the dmPIEZO channel, in the mechanotransduction and/or signal amplification for the detection of sound by the larval Cho neurons. These findings indicate that larval Cho, like their counterparts in the adult fly, use some of the same mechanotransduction channels to detect sound waves and mediate the sensation akin to hearing in Drosophila larvae, allowing them to respond to the appearance of predators or other environmental cues at a distance with behaviors crucial for survival.In the life of insects, sound signals mediate important information that is used in various contexts, ranging from courtship to detection of predators or prey (1, 2). Insects are equipped with sensitive receptor organs for detection of sounds and the underlying neural network enabling recognition and localization of the targets in a complex environment (3). Chordotonal organs (Cho), the main hearing organs for insects, are specialized mechanosensitive organs found in the Insecta and Crustacea (4–7) that may serve as proprioceptors or as cutaneous mechanoreceptor organs (8).In the adult fruit fly Drosophila melanogaster, Cho neurons of the Johnston organ in the antenna mediate both hearing and sensing of gravity and wind (9, 10). Larval Cho have similar developmental programs (11) and structures as their adult counterparts, suggesting that they might be capable of sensing sound/vibration. Previous studies have shown that larval Cho play a role in low-temperature sensation (12, 13). Moreover, mistargeting of Cho axons in the ventral nerve cord (VNC) impairs the larval response to tactile vibration (14), suggesting that Cho neurons might be involved in vibration sensing as well.The mechanotransduction channels in sound sensation have been studied extensively for decades. Several transient receptor potential (TRP) channels and proteins have been implicated in mechanotransduction in the Cho neurons of adult flies. TRP channels usually form nonselective cation channels, which have been found in many types of sensory neurons as well as other cell types. They function as sensors for both intrinsic signals and external stimuli (15, 16). The TRP channel NOMPC (TRPN1) is thought to be a component of the transduction complex (17). Loss of NOMPC eliminates mechanical response in touch-sensitive neurons, and amino acid substitutions in the putative pore domain of the channel can alter ion selectivity (18–21). The loss of NOMPC does not entirely eliminate sound-evoked response in the adult Drosophila auditory nerve, however. Two other Drosophila TRP channels, NANCHUNG (NAN) and INACTIVE (IAV), likely function as heteromers (22), and mutations of either channel render the fly deaf (22, 23). These TRPV family members also could be part of the transduction complex. The precise roles of NOMPC/NAN/IAV in the mechanotransduction of Cho neurons remain unclear. PAINLESS, a Drosophila TRPA channel (24), along with dmPIEZO, one of the first mechanotransduction channels identified in Drosophila, are involved in mechanical nociception (25); however, whether they have a role in sound sensing is unknown.In the present study, by combining in vivo recording and behavioral tests, we are able to study the capabilities of Drosophila larvae to detect and respond to sound and to evaluate the role of Cho neurons in this behavior. We also explored the potential roles of different TRP channels and dmPIEZO channels in Cho neurons for sound sensation.     

BMP signaling is required for the generation of primordial germ cells in an insect

Two modes of germ cell formation are known in animals. Specification through maternally inherited germ plasm occurs in many well-characterized model organisms, but most animals lack germ plasm by morphological and functional criteria. The only known alternative mechanism is induction, experimentally described only in mice, which specify germ cells through bone morphogenetic protein (BMP) signal-mediated induction of a subpopulation of mesodermal cells. Until this report, no experimental evidence of an inductive germ cell signal for specification has been available outside of vertebrates. Here we provide functional genetic experimental evidence consistent with a role for BMP signaling in germ cell formation in a basally branching insect. We show that primordial germ cells of the cricket Gryllus bimaculatus transduce BMP signals and require BMP pathway activity for their formation. Moreover, increased BMP activity leads to ectopic and supernumerary germ cells. Given the commonality of BMP signaling in mouse and cricket germ cell induction, we suggest that BMP-based germ cell formation may be a shared ancestral mechanism in animals.There are two well-characterized modes of animal germ cell specification. In the inheritance mode, observed in Drosophila melanogaster, Caenorhabditis elegans, and Xenopus laevis, maternally provided cytoplasmic determinants (germ plasm) specify a subset of early embryonic cells as germ cells. In contrast, mice specify their germ line through the induction mode, in which a zygotic cell–cell signaling mechanism specifies germ cells later in development. We previously hypothesized that the inductive mode was ancestral among metazoans and that the inheritance mode had evolved independently in multiple derived lineages (1, 2). Consistent with this hypothesis, multiple basally branching insects do not segregate maternally provided germ plasm, unlike the relatively derived Drosophila model (3, 4). However, experimental evidence for the inductive mode was available only for salamanders (5, 6) and mice (7–10), and to date, inferences of induction outside of vertebrates have been based on gene expression and cytological data (1, 11–16).Because Drosophila is highly derived with respect to many aspects of development (17), we examined germ cell development in the cricket Gryllus bimaculatus, a basally branching insect that may shed light on putative ancestral mechanisms of specifying germ cells. We previously showed that unlike Drosophila, Gryllus primordial germ cell (PGC) specification requires zygotic mechanisms rather than germ plasm or the oskar germ-line determinant (4, 18). However, the signals that might induce PGC formation in Gryllus remained unknown. Because mammals require the highly conserved bone morphogenetic protein (BMP) pathway to specify PGCs (8–10, 19, 20), we investigated BMP signaling as a candidate for regulating inductive germ cell specification in Gryllus.     

From the Cover: Integrated 3D view of postmating responses by the Drosophila melanogaster female reproductive tract,obtained by micro-computed tomography scanning

Physiological changes in females during and after mating are triggered by seminal fluid components in conjunction with female-derived molecules. In insects, these changes include increased egg production, storage of sperm, and changes in muscle contraction within the reproductive tract (RT). Such postmating changes have been studied in dissected RT tissues, but understanding their coordination in vivo requires a holistic view of the tissues and their interrelationships. Here, we used high-resolution, multiscale micro-computed tomography (CT) scans to visualize and measure postmating changes in situ in the Drosophila female RT before, during, and after mating. These studies reveal previously unidentified dynamic changes in the conformation of the female RT that occur after mating. Our results also reveal how the reproductive organs temporally shift in concert within the confines of the abdomen. For example, we observed chiral loops in the uterus and in the upper common oviduct that relax and constrict throughout sperm storage and egg movement. We found that specific seminal fluid proteins or female secretions mediate some of the postmating changes in morphology. The morphological movements, in turn, can cause further changes due to the connections among organs. In addition, we observed apparent copulatory damage to the female intima, suggesting a mechanism for entry of seminal proteins, or other exogenous components, into the female’s circulatory system. The 3D reconstructions provided by high-resolution micro-CT scans reveal how male and female molecules and anatomy interface to carry out and coordinate mating-dependent changes in the female’s reproductive physiology.Mating induces physiological changes in females that enhance fertility. Many of these changes result from the transfer of seminal fluid, a complex chemical mixture containing sperm, male-derived molecules, and membranous vesicles (1–6), acting in the context of, and in conjunction with, female molecules and physiology. In insects and mammals, examples include seminal components that induce contraction of the female reproductive tract (RT) musculature (7–10), stimulate ovulation (11–13), regulate semen coagulation (14, 15), aid sperm movement and storage in the female RT (16, 17), and modulate female immune responses postmating (18, 19).Drosophila melanogaster is a powerful system with which to dissect how male-derived molecules interact with female physiology to cause the cascade of postmating responses required for fertility. Drosophila males transfer >200 different seminal fluid proteins (SFPs) to females during mating (19–21). Within the female RT, SFPs induce a series of conformational changes of the uterus (16, 22). Specific SFPs increase ovulation and oviposition rates (12, 13, 23), promote sperm storage (16, 24, 25), increase feeding (26), decrease intestinal transit rates (27, 28), and increase synaptic development at female RT neuromuscular junctions (10). Although much is known about the molecules and interactions that mediate these female postmating responses, next to nothing is known about the physical mechanics of the postmating changes and their response to male and female molecules.The physical act of mating, SFPs, and sperm receipt each modulate the release of signaling molecules in the Drosophila female RT (29). The Drosophila female RT is a complex system whose subregions differ in function and in the release and reuptake of signaling molecules; each region has a characteristic temporal and spatial identity postmating. Thus, postmating physiological changes are not simply a sequence of independent events but rather a coordinated process required for optimal fertility. Previous studies carried out on dissected tissues or specimens did not allow observation of the interactions and coordination among RT organs in situ within mating, or mated, flies.To visualize spatially the coordinated and concurrent organ rearrangements within the confines of the female abdomen during insemination through to egg laying, we used high-resolution, multiscale micro-computed tomography (CT) scans. These scans give a holistic 3D view of the interrelationship among reproductive organs in situ, as females respond to mating and its chemical components. With views ranging from 400-nm to 4-μm voxel sizes of the interior of female flies before, during, and at several times after mating, we observed looping, opening/closing, and repositioning of RT organs within the female. These data allow us to revisit, and newly define, effects of male and female molecules on female postmating physiology.