Cyclic oscillations in the level of phosphatidylinositol 3-phosphate in phagosomes, regulated by two phosphoinositide kinases and one phosphatase, are critical for phagosome maturation and degradation of apoptotic cells. C. elegans, the Class II PI3K PIKI-1 plays a novel and crucial role in producing phagosomal PtdIns(3)P. PIKI-1 is recruited to extending pseudopods and nascent phagosomes prior to the appearance of PtdIns(3)P in a manner dependent on the large GTPase dynamin (DYN-1). PIKI-1 and VPS-34 act in sequence to provide overlapping pools of PtdIns(3)P on phagosomes. Inactivating both piki-1 and vps-34 completely abolishes the production of phagosomal PtdIns(3)P and disables phagosomes from recruiting multiple essential maturation factors, resulting in a complete arrest of apoptotic-cell degradation. We have further identified MTM-1, a PI 3-phosphatase that antagonizes the activities of PIKI-1 and VPS-34 by down-regulating PtdIns(3)P on phagosomes. Remarkably, persistent appearance of phagosomal PtdIns(3)P, as a result of inactivating mtm-1, blocks phagosome maturation. Our findings demonstrate that the proper oscillation pattern of PtdIns(3)P on phagosomes, programmed by the coordinated activities of two PI3Ks and one PI 3-phosphatase, is critical for phagosome maturation. They further shed light on how the temporally controlled reversible phosphorylation of phosphoinositides regulates the progression of multi-step cellular events.Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a signaling molecule important for many membrane trafficking events, including phagosome maturation. The level of PtdIns(3)P on phagosomes oscillates in two waves during phagosome maturation. However, the physiological significance of such oscillation remains unknown. Currently, the Class III PI 3-kinase (PI3K) Vps34 is regarded as the only kinase that produces PtdIns(3)P in phagosomal membranes. We report here that, in the nematode  During animal development and in adulthood many cells are programmed to die by an active process called apoptosis. These dead or dying apoptotic cells are swiftly taken up by scavenger cells into membrane-bound compartments—phagosomes—where they are subsequently degraded when other intracellular organelles containing digestive enzymes fuse with phagosomes—a process called phagosome maturation. Phagocytosis of apoptotic cells is important for tissue remodeling in development and to prevent harmful inflammatory and autoimmune responses. In nematode worms—a model organism in which to study apoptosis—phagosome maturation is accompanied by two waves of the signaling molecule phosphatidylinositol 3-phosphate (PtdIns(3)P) in this compartment: one that forms soon after the formation of the phagosome and lasts for 10–15 minutes, and a second, weaker one 10 minutes later that lasts until the apoptotic cell is fully digested. In this study, we investigated the mechanism that regulates the timing and length of these two waves. We found that they are established by the sequential and combined action of three enzymes: two phosphoinositide 3-kinases, which add a phosphate group to the 3′ site of PtdIns, and one phosphoinositide 3-phosphatase, which removes it. We showed that inactivation of both kinases depleted phagosomes of PtdIns(3)P and resulted in the arrest of phagosome maturation and degradation of apoptotic cells. In addition, the timely turnover of PtdIns(3)P catalyzed by the phosphatase was critical for the step-wise progress of phagosome maturation. Our findings suggest that reversible phosphorylation of phophoinositides, catalyzed by distinct sets of kinases and phosphatases, might be a general mechanism to drive multi-step intracellular membrane trafficking events. PtdIns(3)P is a phosphorylated phosphatidylinositol (PtdIns) species that is embedded in distinct membrane domains and plays important roles in many membrane trafficking events, including endocytic trafficking, retrograde trafficking, autophagy, and phagosome maturation P is dynamically enriched on phagosomes in two consecutive waves: the initial burst, which appears upon the closure of a phagocytic cup and dissipates after 10–15 min, and a subsequent reappearance of a relatively weaker PtdIns(3)P signal approximately 10 min later, which lasts until an apoptotic cell is fully degraded In metazoan, such as the nematode C. elegans, reduces the efficiency of phagosome maturation, indicating that PtdIns(3)P is important for promoting phagosome maturation PtdIns(3)P is a 3′-phosphorylated form of PtdIns. Among the three classes of known phosphoinositide 3-kinases (PI3Ks), Vps34, the sole Class III PI3K, is known to specifically produce PtdIns(3)P on intracellular membranes, such as endosomes, phagosomes, and autophagosomes, to regulate diverse membrane trafficking events  optics as highly refractive, button-like objects referred to as “cell corpses” C. elegans, after being rapidly engulfed and contained inside phagosomes, apoptotic cells are degraded via a pathway initiated by the phagocytic receptor CED-1, which is transiently clustered on the surface of extending pseudopods and nascent phagosomes C. elegans homolog of mammalian dynamins, to phagosomes C. elegans, we identified LST-4/SNX-9, SNX-1, and SNX-6, three PX and BAR domain-containing sorting nexins, as PtdIns(3)P effectors recruited to phagosomal surfaces by PtdIns(3)P and subsequently acting in two parallel pathways to drive the incorporation of endosomes and lysosomes into phagosomes During the development of C. elegans vps-34 mutants was much milder than that from the ced-1 or dyn-1 single mutants, or the lst-4; snx-1; snx-6 triple mutants Interestingly, the phagosome maturation-delay/arrest phenotype observed from 3C. elegans Class II PI3K, in phagosome maturation.Besides Vps34, Class II PI3Ks are also able to produce PtdIns(3)P from PtdIns, whereas Class I PI3Ks primarily produce PtdInsP2 into PtdIns or PtdIns(5)P, respectively C. elegans, rescued the endocytosis defect of vps-34 mutants, suggesting that MTM-1 antagonizes the PI 3-kinase activity of VPS-34 during endosomal trafficking In a closely related aspect, little is known about the factors that down-regulate the initial peak of phagosomal PtdIns(3)P, or the physiological significance of PtdIns(3)P oscillation during phagosome maturation. Promising candidates able to down-regulate PtdIns(3)P level include PtdIns(3)P phosphatases. The myotubularin phosphatases are a family of PI phosphatases that convert PtdIns(3)P or PtdInsPC. elegans Class II PI3K PIKI-1 in the degradation of apoptotic cells. We further revealed the differential and complementary roles of PIKI-1 and VPS-34 in the production and maintenance of phagosomal PtdIns(3)P. Moreover, we have uncovered a novel function of MTM-1 in modulating the dynamic pattern of PtdIns(3)P on phagosomes. We found that the prompt down-regulation of phagosomal PtdIns(3)P, like its robust production on nascent phagosomes, is pivotal for driving phagosome maturation. Our work revealed a regulatory system that issues a precise temporal control over the PtdIns(3)P cycling pattern on phagosomes and ensures the efficient degradation of apoptotic cells.In this report, we identified a novel role of the vps-34 would result in a complete depletion of phagosomal PtdIns(3)P, we monitored the level of PtdIns(3)P on the surface of phagosomes using a previously established PtdIns(3)P reporter, 2xFYVE::GFP, which was expressed in engulfing cells under the control of Pced-1 and specifically associated with PtdIns(3)P To evaluate whether inactivating vps-34 by RNA interference (RNAi) using two non-overlapping RNAi constructs reduced the percentage of gonadal phagosomes labeled with 2xFYVE::GFP to 54% or 58%, respectively (vps-34(RNAi) animals displayed a mild cell-corpse removal defective (Ced) phenotype resulting from un-removed cell corpses, evident by the modestly increased numbers of germ cell corpses in the gonad P on gonadal phagosomes.In the gonad of wild-type adult hermaphrodites, 86% of germ cell corpses, recognized under the DIC optics, were labeled with bright 2xFYVE::GFP on their surfaces , indicatectively . Furtherhe gonad  and S1A,vps-34(h510)(m−z−)  homozygous null mutant embryos that lost both the maternal and the zygotic expression of vps-34, which is likely to represent the vps-34 complete loss-of-function phenotypes P in a more strict manner, we created a strain that produced enotypes .vps-34(h510)(m−z−) mutant embryos, the number of PtdIns(3)P-labeled cytoplasmic puncta, which represented early endosomal particles, was nearly abolished, consistent with the known function of VPS-34 in the production of PtdIns(3)P on early endosomes (vps-34 reduces but does not completely abolish the production of PtdIns(3)P on phagosomes. Additional PtdIns(3)P-producing activity besides VPS-34 must exist on phagosomal membranes.In ndosomes  [35]. Mondosomes . Consistndosomes . Howeverndosomes , indicattm3171, a deletion mutation of piki-1, which encodes the only C. elegans Class II PI3K (piki-1(tm3171) mutation also resulted in moderate Ced phenotypes in embryos throughout mid- and late-embryonic stages  mutant hermaphrodites, the percentage of phagosomes labeled with 2xFYVE::GFP was reduced to 15% of the wild-type level (piki-1(tm3171) and vps-34(RNAi) animals indicates that in gonadal sheath cells, PIKI-1 plays a major role in producing PtdIns(3)P on phagosomes (In the gonad of pe level . The majpe level . These oagosomes .piki-1(tm3171) mutant embryos, the percentage of PtdIns(3)P-labeled phagosomes was reduced to 40% of the wild-type level P in somatic engulfing cells.In mid-stage (2-fold stage) pe level , indicatvps-34(h510)(m−z−) mutant animals, which are embryonic and larval lethal (piki-1(tm3171) mutant animals undergo normal development, and are viable and fertile. In addition, unlike vps-34(RNAi) animals, piki-1(tm3171) animals display normal endocytosis activities (piki-1(tm3171) mutant and wild-type embryos  mutant adult hermaphrodites, persistent germ cell corpses were all observed inside phagosomes: only 13% of these phagosomes were labeled with PtdIns(3)P on their surfaces that appeared like bright GFP circles, the rest of them lacked PtdIns(3)P on their surfaces and appeared as dark spheres inside the host cells , we analyzed the internalization status of germ cell corpses (piki-1(tm3171) mutants were fully engulfed and remained undegraded inside phagosomes ; vps-34(RNAi) hermaphrodites and observed a near complete depletion of phagosomal PtdIns(3)P (−z−)vps-34(h510) double mutant embryos, PtdIns(3)P was no longer detected on phagosomal surfaces or on any cytoplasmic puncta (vps-34(RNAi) is potent in inactivating VPS-34 in the gonadal sheath cells since only a residual PtdIns(3)P-production activity was left on phagosomes in piki-1(tm3171); vps-34(RNAi) double mutant animals.Since the inactivation of either VPS-34 or PIKI-1 only partially impaired the PtdIns(3)P production on phagosomes, we further monitored PtdIns(3)P in the gonad of dIns(3)P . Similarc puncta . These ovps-34; piki-1 double mutant, in which PtdIns(3)P is no longer detectable on phagosomal surfaces, provides a suitable tool for addressing this question.Previously, without an effective method to completely block the production of PtdIns(3)P on phagosomes, it was impossible to determine whether PtdIns(3)P was absolutely essential for triggering phagosome maturation or merely contributed to phagosome maturation as one of multiple signaling molecules. The piki-1(tm3171); vps-34(RNAi) double mutants, a larger number of persistent germ cell corpses were observed than in any PI3K single mutants (piki-1(tm3171) single mutants, all cell corpses were engulfed inside phagosomes in piki-1(tm3171); vps-34(RNAi) double mutants (−z−)vps-34(h510) double mutant embryos displayed a much stronger Ced phenotype than any single mutants (In the adult gonads of  mutants . As in p mutants . At 4-fo mutants . These o−z−)vps-34(h510) double mutant embryos. We used a previously established time-lapse recording technique to monitor the maturation process of three well-defined phagosomes, C1, C2, and C3, which were formed inside three adjacent hypodermal cells at ∼330 min post-first cleavage (vps-34(h510)(m−z−); piki-1(tm3171) double mutant embryos, SNX-1 and LST-4 failed to accumulate on phagosomes P effectors that drive the degradation of apoptotic cells cleavage  [8],[33]cleavage . In contagosomes , indicatagosomes . This obpiki-1 would affect the recruitment of three small GTPases, RAB-5, RAB-2 , and RAB-7, to phagosomal surfaces. During phagosome maturation, these membrane-tethering factors are sequentially recruited from the cytoplasm of the host cells to phagosomal surfaces, where they facilitate the fusion of intracellular organelles to phagosomes piki-1(tm3171) mutant hermaphrodites, however, the percentages of phagosomes labeled with GFP::RAB-5, GFP::RAB-2, or GFP::RAB-7 were strongly reduced to 9%, 16%, or 23%, respectively P in regulating phagosome maturation, we examined whether the inactivation of ectively , indicatd to 10% . These rrab-5, rab-2, and rab-7 loss-of-function background. Whereas rab-2 and rab-7 mutations did not affect the presentation of PtdIns(3)P on phagosomes, inactivation of rab-5 by RNAi greatly reduced the percentage of PtdIns(3)P-labeled phagosomes (To further determine the relationships between PtdIns(3)P and the RAB GTPases, we examined the production of PtdIns(3)P on phagosomes in each of the agosomes , Text S1To determine why the activities of two PI3Ks, which produce an identical signaling molecule PtdIns(3)P on phagosomes, are both needed for efficient phagosome maturation, we monitored the pattern of PtdIns(3)P appearance on phagosomes over time in wild-type, each of the single PI3K mutant, and double PI3K mutant embryos . As repo−z−)vps-34(h510) double mutant embryos, PtdIns(3)P was completely absent from phagosome surfaces throughout the entire recording period (90 min) (In (90 min) , causingvps-34(h510)(m−z−) single mutant embryos, PtdIns(3)P was robustly produced on nascent phagosomes immediately after engulfment, as in the wild-type background (piki-1(tm3171) single mutant embryos, an obvious delay of PtdIns(3)P production on nascent phagosomes was observed P, we characterized a PIKI-1::GFP reporter expressed in engulfing cells under the control of P mutants , indicate domain  or carrye domain  [39] lose domain , indicatIn both embryos and the gonads of adult hermaphrodites, PIKI-1::GFP was primarily localized to the cytoplasm and was enriched on phagosomal surfaces . During dyn-1(n4039) null mutant embryos. We failed to observe any significant enrichment of PIKI-1::GFP on the surfaces of either the extending pseudopods or nascent phagosomes P agosomes . This revps-34 mutants suggests the existence of a previously unidentified activity that antagonizes PI3Ks by removing PtdIns(3)P from phagosomes.In wild-type embryos, despite the sequential and combined activities of PIKI-1 and VPS-34, an obvious temporal gap, during which PtdIns(3)P is almost undetectable on phagosomes, was observed between the two PtdIns(3)P peaks  and S6F.C. elegans PI 3-phosphatase MTM-1 in regulating PtdIns(3)P dynamics during phagosome maturation. In embryos homozygous for mtm-1(op309), a partial loss-of-function mutation mtm-1(op309) mutant embryos than in wild-type embryos (mtm-1(op309) embryos, with 27% phagosomes lasting longer than 3.6 times of the average length in wild-type embryos P, we examined the function of  embryos  and S10. embryos  and S10.mtm-1(op309) embryos, aided by CED-1C (the intracellular domain of CED-1)::GFP expressed in the engulfing cells mtm-1(op309) mutant embryos, phagosome duration was mildly yet significantly  prolonged, with the average duration 38% longer than the wild-type control P has any functional significance has not been explored. To address this question, we measured the rate of phagosome maturation in  control . This remtm-1(op309) mutation is a missense mutation (G106E) that only partially inactivates MTM-1 mtm-1(RNAi)  gonads were engulfed inside gonadal sheath cells (ced-5(n1812) null mutants, which are primarily defective in the engulfment of germ cell corpses (mtm-1 is primarily required for phagosome maturation.The -1(RNAi) , which r-1(RNAi) . This stseverity . Aided bth cells . This ph corpses , and indmtm-1(RNAi) increased the percentage of PtdIns(3)P-labeled gonadal phagosomes from 86% to 100% , we observed the transient association of MTM-1 with the extending pseudopods throughout engulfment and with nascent phagosomes for approximate 13 min ; piki-1(tm3171) double mutant animals carrying the PtdIns(3)P reporter 2xFYVE::GFP. In both embryos and adult hermaphrodite gonads, mtm-1(op309); piki-1(tm3171) double mutants had significantly fewer numbers of persistent cell corpses than piki-1(tm3171) single mutants (mtm-1(op309); piki-1(tm3171) double mutants than in piki-1(tm3171) single mutants (mtm-1 function significantly rescued the defects caused by piki-1(tm3171) mutation in both PtdIns(3)P production and phagosome maturation. The suppression effect of the Ced phenotype of piki-1(tm3171) mutants by the mtm-1(op309) mutation became more evident when examined in strains not carrying Pced-12xFYVE::gfp, which might compete with endogenous PtdIns(3)P effectors for PtdIns(3)P (piki-1(tm3171); mtm-1(RNAi) animals, the number of persistent germ cell corpses was drastically reduced to 15% of that observed in mtm-1(RNAi) animals and 29% of that in piki-1(tm3171) single mutant animals (piki-1(tm3171) and mtm-1(RNAi), each of which caused a strong Ced phenotype, efficiently rescued each other's defect in phagosome maturation. These results clearly indicate that MTM-1 and PIKI-1 antagonize each other's activity and underscore the importance of a balanced PtdIns(3)P production on phagosomes.To determine the functional relationship between MTM-1 and the two PI3Ks, we first analyzed  mutants . Further mutants . These odIns(3)P . More st animals . Therefovps-34(RNAi) resulted in a relative weak Ced phenotype in the adult hermaphrodite gonads (vps-34(RNAi) modestly reduced the severity of the Ced phenotype of mtm-1(RNAi) animals (vps-34(RNAi) is much weaker than that of piki-1(tm3171) mutation, suggesting that PIKI-1 is the major PI3K that counteracts the activity of MTM-1.e gonads . vps-34P production, was no longer suppressible by mtm-1(RNAi)  , clearlyC. elegans provides a unique opportunity for studying the molecular mechanisms and the functional significance of the temporal changes of PtdIns(3)P on intracellular membranes because the dynamics of PtdIns(3)P can be readily monitored, using time-lapse microscopy, on a relatively large object (diameter >2 µm) throughout the entire maturation process (∼50 min or longer). In this report, using genetic and live-cell imaging approaches, we identified a temporal regulation mechanism that programs the two-wave cycling pattern of PtdIns(3)P on phagosomal surfaces P-mediated cellular events, phagosome maturation in surfaces . This meC. elegans Class II PI3K, in producing a dynamic pool of PtdIns(3)P on nascent phagosomes and in initiating the degradation of apoptotic cells inside phagosomes. The combined activities of PIKI-1 and VPS-34 account for most if not all PtdIns(3)P molecules on the surface of phagosomes. To our knowledge, this is the first example that two PI3Ks, which belong to different classes, contribute to overlapping pools of PtdIns(3)P on a particular organelle and coordinately regulate the same cellular event.The physiological functions of Class II PI3Ks are only starting to be understood. Mammalian Class II PI3Ks are implicated in the production of a dynamic PtdIns(3)P pool on the plasma membrane in response to external stimuli piki-1 and vps-34 single mutants and vps-34; piki-1 double mutants, defects in PtdIns(3)P-production correlate quantitatively with defects in cell-corpse removal, indicating that the lack of phagosomal PtdIns(3)P is the primary cause for the Ced phenotype. Our observations further indicate that the contribution of PIKI-1 and VPS-34 to phagosome maturation depends on the tissue context: in the adult hermaphrodite gonad, PIKI-1 apparently plays a major role in producing phagosomal PtdIns(3)P.In piki-1 mutants were specifically defective in the engulfment of apoptotic cells based on the observation of extra cell corpses in animals, which displayed the unique button-like morphology under DIC microscope piki-1 mutants that resulted in the accumulation of cell corpses was misinterpreted previously The accumulation of persistent cell corpses could result from defects in either the engulfment or the degradation of apoptotic cells, two consecutive cellular events executed through different mechanisms Mammals have three Class II PI3K isoforms, which display differential expression patterns P pattern on phagosomal surfaces: whereas PIKI-1 initiates the production of PtdIns(3)P on nascent phagosomes, VPS-34 acts to maintain the level of phagosomal PtdIns(3)P for the subsequent period . The funC. elegans, we and others have found that RAB-5 is required for the production of phagosomal PtdIns(3)P P, consistent with the role of PIKI-1 in initiating PtdIns(3)P production on nascent phagosomes. The phagosome association of PIKI-1 relies on DYN-1, the key organizer of phagosome maturation events, further suggesting that PIKI-1 might be recruited by DYN-1, directly or indirectly, to its site of action . This fidIns(3)P  [13]. WedIns(3)P  and S7. dIns(3)P .vps-34(m−z−) null mutants, piki-1 homozygous deletion mutants are viable and fertile, and do not display any obvious endocytosis defects ; piki-1 double mutation) that completely blocked phagosomal PtdIns(3)P production, which allowed us to determine quantitatively how fundamental the role of PtdIns(3)P is in initiating phagosome maturation. The vps-34(m−z−); piki-1 double mutations resulted in a Ced phenotype as severe as that caused by mutations in known key regulators and executors of cell-corpse degradation, such as mutations in DYN-1 and triple mutations that completely inactivate SNX-9, SNX-1, and SNX-6, three PtdIns(3)P effectors Previous studies of Vps34 established the principle that PtdIns(3)P is important for phagosome maturation VPS-34 has been implicated in the recruitment of RAB-5 and RAB-7 to phagosomes MTM1, a member of the myotubularin PI phosphatase family, helps maintain the optimal level of PtdIns(3)P on the surface of multiple kinds of intracellular membranes by converting PtdIns(3)P to PtdIns (reviewed in mtm-1(op309) mutation slows down phagosome maturation in embryos. Likewise, mtm-1(RNAi) results in a dramatic phagosome maturation arrest in adult gonads. These results demonstrate that despite being a negative regulator of PtdIns(3)P, MTM-1 is an essential positive regulator for phagosome maturation. Furthermore, the piki-1 deletion mutation and mtm-1(RNAi) mutually suppresses the phagosome maturation defects of each other. In addition, vps-34(RNAi) partially suppresses the phagosome maturation defects caused by mtm-1(RNAi). On the contrary, mtm-1(RNAi)'s suppression effect no longer exists in piki-1; vps-34(RNAi) double mutant backgrounds. Together, these observations demonstrate that MTM-1 directly antagonizes the function of the two PI3Ks on phagosomes by dephosphorylating their product, PtdIns(3)P. The association of MTM-1 with the extending pseudopods and nascent phagosomes correlates, completely and partially, with the PtdIns(3)P-producing activities of PIKI-1 and VPS-34 on phagosomes, respectively, further supporting the model that MTM-1 antagonizes the activities of PIKI-1 and VPS-34 and results in the gap period between the two PtdIns(3)P waves on phagosomes P might be critical for the timely dissociation of certain initial phagosome maturation factors from phagosomes as well as the subsequent association of certain other, perhaps yet-to-be identified maturation factors that act at later stages of phagosome maturation . The dynpiki-1 and vps-34 resulted in the accumulation of persistent cell corpses and that the ability of MTM-1 to suppress the engulfment defect of ced-1 and ced-6 mutants relied on the functions of PIKI-1 and VPS-34. Zou et al.'s work identified the antagonizing genetic interaction between MTM-1 and the two PI3Ks during cell-corpse removal C. elegans, the production of PtdIns(3)P is initiated on nascent phagosomes after the completion of engulfment, and PtdIns(3)P has not been detected on the extending pseudopods Recently, MTM-1 was reported to negatively regulate the engulfment of apoptotic cells, one step prior to the degradation of apoptotic cells 2C. elegans MTM-1 might play opposite roles in engulfment and degradation, two consecutive steps of apoptotic-cell removal, by down-regulating different phosphoinositide species.On the other hand, Neukomm et al. (2011), who independently identified the function of MTM-1 as a negative regulator of cell-corpse engulfment, proposed that during engulfment, MTM-1 performs its function through dephosphorylating another substrate, PtdInsPThis study, together with a few recent reports, have highlighted the physiological importance of the antagonizing activities of PI kinases and phosphatases in the regulation of PtdIns(3)P dynamics on various intracellular membranes C. elegans strains were grown at 20°C as previously described www.wormbase.org), and this work, except when noted otherwise: LGI, vps-34(h510)unc-108/rab-2(n3263)mtm-1(op309)rab-7(ok511) ced-5(n1812); LGV, unc-76(e911); LGX, piki-1(tm3171), piki-1(ok2346), dyn-1(n4039)piki-1(tm3171) and piki-1(ok2346) alleles were characterized after being out-crossed for four times. Transgenic worms were generated by microinjection as previously described unc-76(+)] unc-76(e911) mutant adult hermaphrodites and transgenic animals were identified as non-Unc animals. Transgenes are maintained in animals as extra-chromosomal arrays and, when necessary, introduced into different genetic backgrounds by crosses.piki-1 cDNA was amplified from a mixed-stage C. elegans cDNA library  using polymerase chain reaction (PCR). Pced-1piki-1::gfp was constructed by cloning the piki-1 cDNA into the multi-cloning sites of pZZ829, a plasmid carrying ced-1P, a 3′ gfp tag, and the unc-54 3′ UTR ced-1piki-1::gfp and generate Pced-1piki-1(Δkinase)::gfp. To generate Pced-1piki-1(K1059A)::gfp, the K1059A mutation was introduced into Pced-1piki-1::gfp using the QuickChange Site-directed Mutagenesis Kit (Stratagene). To make Pced-1gfp::mtm-1, mtm-1 cDNA was amplified from the mixed-stage C. elegans cDNA library using PCR and subsequently cloned into pZZ956, a plasmid carrying ced-1P, a 5′ gfp tag, and the unc-54 3′ UTR.The 3, mounted on 4% agarose pads, and observed under DIC microscopy. Somatic embryonic cell corpses were scored in the embryos at different developmental stages as described by DIC microscopy was performed with an Axionplan 2 compound microscope  equipped with Nomarski DIC optics, a digital camera  and imaging software . Worms were immobilized with 25 mM NaNAn Olympus IX70-Applied Precision DeltaVision microscope equipped with standard epifluorescent filter sets and Photometris Coolsnap digital camera was used to capture fluorescence images, which were deconvolved and processed by the Applied Precision SoftWoRx software. To score the numbers of somatic or germ phagosomes labeled by GFP or mRFP reporters on their surfaces, z-sections of DIC and fluorescent images spanning the entire depth of embryos or adult gonads, respectively, were captured and analyzed vps-34(RNAi) experiments in mtm-1(RNAi) and vps-34(RNAi) experiments in E. coli strain HT115 transformed with RNAi constructs. For RNAi experiments starting at L1 stage, mid-L4-stage animals were retransferred to fresh RNAi plates. 48 h after L4 stage, the number of germ cell corpses per gonad arm were scored under DIC and fluorescent microscopy. The RNAi-by-feeding vector pPD129.36 vps-34 were generated by cloning PCR-amplified cDNA fragments (vps-34(N) and vps-34(C)) corresponding to the N- and C-terminal halves of VPS-34 into vector pPD129.36 using the following primers: vps-34(N) (5′-ttgggaacacgaggatgatg-3′ and 5′-gttcaggatcagctacacag-3′), vps-34(C) (5′-taaaggagtccatc-3′ and 5′-ttgtcaagatgacgatcacc-3′). The RNAi feeding constructs for mtm-1 were generated by cloning PCR-amplified cDNA fragments corresponding to the N- and C-terminal halves of MTM-1 into vector pPD129.36 using the following primers: mtm-1(N) (5′-gcgccccgggatggattcacaatttattg-3′ and 5′-atatcccgggctcgccgagcttctttac-3′), mtm-1(C) (5′-aaaggaaattttcagccaatgtt-3′ and 5′-tgcacataaagaaagcaaaatga-3′).RNAi experiments were performed using feeding protocol as previously described Electron microscopy was performed as previously described Figure S1vps-34(RNAi) and vps-34(m−z−) animals (related to vps-34(RNAi)-treated worms could be further enhanced by overexpressing 2xFYVE::GFP reporter in engulfing cells. The numbers of germ cell corpses were scored in adult hermaphrodites treated with vps-34 RNAi for 48 h from L4 stage. Animals either carried or did not carry the Pced-12xFYVE::gfp transgenic array, as indicated. vps-34(N) and vps-34(C) are two independent RNAi feeding constructs targeting different regions of vps-34 coding sequence. Data are presented as mean ± standard deviation (SD). n, number of animals scored. (B) The depletion of both maternal and zygotic vps-34 products (indicated by m−z−) resulted in the arrest of animal development at earlier stages than solely depleting zygotic vps-34 products (indicated by m+z−). The percentages of animals arrested during embryogenesis or at each of the four larval stages were shown as bar graph.The phenotypes of (TIF)Click here for additional data file.Figure S2piki-1 and phenotype of two piki-1 deletion mutants  Domain structure of wild-type PIKI-1 and the predicted truncated forms of PIKI-1 encoded by two deletion alleles. Shaded domains indicate the domains, part of which were deleted by each deletion allele. UIM, Ubiquitin-interacting motif; RBD, Ras-binding domain; C2. Protein kinase C conserved region 2; PIK, Phosphoinositide 3-kinase, accessory domain; Kinase, Phosphoinositide 3-kinase, catalytic domain; PX, PhoX homologous domain. (C) DIC images of part of gonadal arms of adult hermaphrodites at 48 h after L4 stages. Arrows indicate germ cell corpses. Dorsal is to the top. Scale bars, 20 µm. (D) The numbers of germ cell corpses in adult hermaphrodites of different ages are displayed in a bar graph. Germ cell corpses were scored every 12 h after the L4 stage. Fifteen animals were scored for each data point. Data are presented as mean ± SD. (E) The numbers of somatic cell corpses at different embryonic stages are displayed in a bar graph. At least 15 embryos were scored for each data point. Data are presented as mean ± SD.Gene structure of (TIF)Click here for additional data file.Figure S3piki-1(tm3171) mutants are normal for endocytosis. (A–B) The endocytosis (A) and redistribution (B) of yolk is normal in piki-1(tm3171) mutant oocytes and embryos, respectively, monitored by the YP170::GFP reporter. (A) Epifluorescence (a–b) and DIC (c–d) images of adult hermaphrodite gonad in wild-type  and piki-1(tm3171)  adult hermaphrodites. Filled arrows indicate oocytes filled with YP170::GFP, filled arrowheads indicate embryos, and open arrows indicate spermathecae. Scale bars, 10 µm. (B) Epifluorescence and DIC images of wild-type (a–f) and piki-1(tm3171) (g–m) embryos at different stages (labeled as min post the first cleavage). Arrows indicate intestinal precursor cells, which are enriched with YP170::GFP. Solid lines indicate the head region in 450-min-stage embryos, from which the YP170::GFP is depleted. Scale bars, 10 µm. (C–D) The endocytosis of ssGFP (secreted GFP) by coelomocytes is normal in piki-1(tm3171) mutant adults, monitored with the Pmyo-3::ssGFP reporter. (C) Epifluorescence (a–b) and DIC (c–d) images of wild-type and piki-1(tm3171) mutant adults. Arrows indicate coelomocytes. Scale bars, 20 µm. (D) Efficiency of endocytosis of ssGFP by coelomocytes in wild-type and piki-1(tm3171) mutant adults. n, number of animals analyzed.(TIF)Click here for additional data file.Figure S4ced-12xFYVE::gfp in gonadal sheath cells. Animals were analyzed at 48 h after L4 stages. Arrows and arrowheads indicate 2xFYVE::GFP(+) and 2xFYVE::GFP(−) phagosomes, respectively. One open arrowhead in (b) indicates a blob of unengulfed yolk resulted from defects in endocytosis caused by rab-5(RNAi). Dorsal is to the top. Scale bars, 20 µm.RAB-5, but not RAB-2 or RAB-7, is required for the production of PtdIns(3)P on phagosomes (related to (TIF)Click here for additional data file.Figure S5p<0.05 by independent Student's t-test. “NS” indicates non-significant differences. (B) Histogram distribution of the phagosome duration in embryos expressing indicated transgenes. The duration of phagosomes also displayed as mean ± sd. n, the number of C1, C2, and C3 phagosomes measured.The overexpression of SNX-1 or LST-4 does not affect phagosome maturation (related to (TIF)Click here for additional data file.Figure S6p<0.05, independent Student's t-test. “NS” indicates non-significant differences. (B) A diagram showing that the over-expressed 2xFYVE::GFP molecules may compete with endogenous PtdIns(3)P effectors for the interaction with phagosomal PtdIns(3)P. (C–D) Time-lapse images of the degradation of C3 phagosomes in wild-type embryos expressing transgenes Pced-1 CED-1C::gfp(C) or Pced-1 2xFYVE::gfp at a relatively high level. (D) “0 min” represents the time point when a C3 cell corpse was just fully internalized by its engulfing cell, ABplaapppp, and the newly formed phagosome was recognizable as a dark sphere inside GFP(+) engulfing cell. Anterior is to the top. Ventral faces readers. Arrows indicate cell corpses C1, C2, or C3; arrowheads indicate their corresponding engulfing cells; open arrowheads in D(d) indicate nuclei. Scale bars, 5 µm. (E–F) Time-lapse images of the degradation of C3 phagosomes in wild-type embryos expressing transgenes Pced-1 CED-1C::gfp (E) or Pced-1 2xFYVE::gfp at low level (F). “0 min” is when engulfment just completed. Scale bars, 2 µm.The sequestration effect of 2xFYVE::GFP reporter on phagosomal PtdIns(3)P is dependent on the expression level of the transgene (related to (TIF)Click here for additional data file.Figure S7n, the number of C1, C2, and C3 phagosomes analyzed.The timing of the transient enrichment of RAB-5 on nascent phagosomes is not affected by the expression of the 2xFYVE::GFP reporter. (A–B) Time-lapse recording of the dynamic phagosomal localization of GFP::RAB-5 on C2 phagosomes in wild-type embryos that expressed GFP::RAB-5 alone (A) or that co-expressed GFP::RAB-5 and high level of 2xFYVE::mRFP (B). “0 min” represents the time point when engulfment is just complete. Scale bars, 2 µm. (C) The relative GFP::RAB-5 signal intensity, represented as the ratio of GFP::RAB-5 signal intensity on the surface of phagosomes to that in the nearby cytoplasm of the host cell, was measured from images in (A–B) and plotted over time. (D) Quantification of the timing of the initial appearance of RAB-5 on phagosomes and the duration of RAB-5 in association with phagosomes. Data are presented as mean ± SD. (TIF)Click here for additional data file.Figure S8Three additional examples of the recruitment of PIKI-1 to the extending pseudopods and nascent phagosomes prior to the production of phagosomal PtdIns(3)P (related to (TIF)Click here for additional data file.Figure S9C. elegans PIKI-1, H. sapiens PI3KC2α, and D. melanogaster PI3K68D (related to ced-1piki-1(K1059A)::gfp, is labeled by a red frame.Sequence alignment of the kinase domain of class II PI 3-kinases that include (TIF)Click here for additional data file.Figure S10mtm-1(op309) mutants (related to mtm-1(op309) mutant (B) embryo were monitored by 2xFYVE::GFP. “0 min” is the time point when engulfment is just completed. Arrows and arrowheads indicate the phagosome maturation stages covered by the first and the second waves of PtdIns(3)P on phagosomes, respectively. Scale bars, 2 µm. (C) The relative PtdIns(3)P signal intensity, represented as the ratio of 2xFYVE::GFP signal intensity on the surface of phagosomes to that in the nearby cytoplasm of the host cell, was measured from images in (A–B) and plotted over time.Additional examples of the enhanced and prolonged PtdIns(3)P signal on the phagosomes in (TIF)Click here for additional data file.Text S1vps-34 gene products; characterization of piki-1 deletion alleles; normal endocytosis in piki-1 mutants; the requirement of RAB-5 for phagosomal PtdIns(3)P production; and the characterization of the effect of SNX-1::GFP, LST-4::GFP and 2xFYVE::GFP overexpression on phagosome maturation.Supplemental text includes the following sections. Depletion of maternal and zygotic (DOCX)Click here for additional data file.


The diverse cell types and the precise synaptic connectivity between them are the cardinal features of the nervous system. Little is known about how cell fate diversification is linked to synaptic target choices. Here we investigate how presynaptic neurons select one type of muscles, vm2, as a synaptic target and form synapses on its dendritic spine-like muscle arms. We found that the Notch-Delta pathway was required to distinguish target from non-target muscles. APX-1/Delta acts in surrounding cells including the non-target vm1 to activate LIN-12/Notch in the target vm2. LIN-12 functions cell-autonomously to up-regulate the expression of UNC-40/DCC and MADD-2 in vm2, which in turn function together to promote muscle arm formation and guidance. Ectopic expression of UNC-40/DCC in non-target vm1 muscle is sufficient to induce muscle arm extension from these cells. Therefore, the LIN-12/Notch signaling specifies target selection by selectively up-regulating guidance molecules and forming muscle arms in target cells.DOI:http://dx.doi.org/10.7554/eLife.00378.001 The development of the nervous system involves the formation of complex networks of connections between diverse cell types, such as motor neurons, interneurons and pyramidal cells. However, the mechanisms by which individual cells are programmed to acquire particular identities, and how they are instructed to form connections with other specific cells, remain unclear.In many species, the Notch signaling pathway has a role in setting up these networks. Notch is a transmembrane protein, which means that it has one component inside the cell and another outside. When a ligand binds to the extracellular part of Notch, this causes the receptor to break in two. The intracellular domain then travels to the nucleus where it can influence gene expression.C. elegans), which has two Notch receptors, is often used to study the formation of neuronal networks because each worm has only around 300 neurons, and they are connected in roughly the same way in each worm. C. elegans relies on two types of cell that are very similar to each other—type-1 and type-2 vulval muscle cells—to lay eggs, and the neurons that trigger egg-laying form synaptic connections on specialized structures called muscle arms. However, these structures are found only in type-2 vulval muscle.The nematode worm (To investigate the mechanisms underlying the formation of the egg-laying circuit, Li et al. screened large numbers of mutant worms to find animals that lacked muscle arms. They identified a number of such mutants, which laid fewer eggs compared to wild-type worms, and found that they all had mutations in genes that encode for proteins or ligands that are involved in the LIN-12/Notch pathway. This pathway mediates cell–cell interactions that help to specify cell fates.Li et al. showed that type-2 vulval muscle cells develop muscle arms when their neighbors—type-1 vulval muscle cells and vulval epithelial cells—produce enough ligand to activate the LIN-12 Notch receptor on the type-2 vulval muscle cells. They also identified two of the downstream targets of LIN-12, and found that artificially expressing one of these in type-1 vulval muscle cells is sufficient to trigger the formation of muscle arms.The work of Li et al. provides further evidence that the Notch signalling pathway, which is well known for its role in early development, also acts at later developmental stages to determine cell fate and patterns of connectivity.DOI:http://dx.doi.org/10.7554/eLife.00378.002 Drosophila, many transmembrane molecules, including semaphorin, capricious and teneurins, have been suggested to guide synaptic target selection between olfactory receptor neurons (ORN) and projection neurons (PN)  , 2012. Ions (PN) . In vertons (PN) . This laons (PN) . Similar neurons . Anotheruron AIY . In addiuron AIY . IntriguWhile it is likely that transcription factors ultimately regulate the expression of cell surface molecules to determine the target specificity, few examples are well characterized. In one such example, the even-skipped transcription factor impacts long-range axon guidance choices through regulating a Netrin receptor, UNC-5 . HoweverC. elegans, lin-12 encodes one of the two homologs of Notch receptor  specification  but not type 1 vulval muscles (vm1)    . These ses (vm1) . The HSNes (vm1) . SYG-2 ies (vm1) . While tes (vm1) .10.7554/lin-12 functions cell-autonomously to up-regulate the activity of the guidance molecules UNC-40/DCC and MADD-2, which in turn generates and guides the development of vm2 postsynaptic muscle arms. The ectopic expression of UNC-40 in vm1 cells that normally do not form muscle arms is sufficient to induce the extension of muscle arm-like membrane structures from vm1, suggesting a deterministic role of LIN-12/Notch and UNC-40/DCC in postsynaptic target selection.To understand how cell specification was coordinated with synaptic target selection, we explored the cellular and molecular mechanisms underlying vm2 postsynaptic muscle arm development. We found that postsynaptic muscle arm development in egg-laying circuit is mediated by LIN-12/Notch-dependent cell specification. The canonical LIN-12/Notch signaling pathway is specifically activated in the target vm2 cells by the ligand APX-1/DSL expressed in two adjacent cell types, the secondary vulval epithelial cells and the non-target vm1 cells. C. elegans egg-laying circuit consists four type 1 vulval muscle cells (vm1) and four type 2 vulval muscle cells (vm2), which form two X-shaped structures near the vulval opening (mCherry::rab-3) to label presynaptic vesicles in HSN or VC neurons, receptively  mutant, in which the HSN neurons undergo programmed cell death in embryonic stage (egl-1(n986);lin-39(n709) double mutant animals which lacked both HSN and VC neurons (unc-104(e1265) mutant animals, in which the synaptic vesicles were absent from the presynaptic regions in both HSN and VC neurons . We therefore asked whether the HSN neurons are required for vm2 specification and muscle arm development by examining the vm2 morphology in the ic stage . Surprisic stage . Several neurons , the vm2 neurons . Second, neurons , vm2 mus neurons . These rlin-3(e1417) mutant animals, in which vulval epithelial cell fate is abnormal  mutant animals, while the muscle morphology of both vm1 and vm2 were largely normal, the SER-1B::GFP labeled vm2 muscle arm structures were completely absent (lin-12(wy750) mutant, indicating that the mutation specifically affected postsynaptic differentiation and development (lin-12(wy750) mutants either by differential interference contrast (DIC) microscopy or the localization pattern of the ajm-1::gfp apical junction marker at several development stages (apx-1(wy750) and sel-12(wy760), were very similar to the lin-12(wy750) mutant in their specific defective vm2 muscle arm development and normal vulval muscle development  mutants, all three markers exhibits wild type pattern, suggesting that many molecular signatures of the M lineage are intact in the lin-12(wy750) mutants, while the development of the postsynaptic specializations completely fails (lin-12(wy750) mutant, the position where vm1 is linked to the body wall is always on the dorsal side of the connection between vm2 and body wall. No significant difference was found on the intersection angle between the vulval muscle and the vulval slit, as well as the relative size of vm2 to vm1 (Svm2/Svm1) . We considered the possibility that the absence of muscle arms in vm2 could be due to the change of vm2 cell fate to a different cell. We therefore examined several cell-type-specific markers for each differentiation stage: the  lineage  and an eg system . We also muscles . In lin-ly fails . Furtherly fails . In bothm2/Svm1) . In summIf vm2 muscle arms play crucial functional roles in the synaptic connections of the egg-laying circuit, mutants with defective vm2 muscle arm development should show altered egg-laying behavior. As a measure of this behavior, we examined the eggs freshly-laid by the wild-type animals and the muscle arm defective mutants. First, the muscle arm defective mutants laid notably fewer eggs compared to the wild-type animals . A more lin-12(wy750) and apx-1(wy755) mutants, and the physiological basis for their reduced egg-laying. By co-expressing the Ca2+ reporter GCaMP3 and the calcium-insensitive fluorescent protein mCherry in the vulval muscles, we were able to perform ratiometric Ca2+ imaging in behaving wild-type, lin-12(wy750), and apx-1(wy755) mutant animals. In wild-type animals, simultaneous Ca2+ transients in the anterior and posterior vulval muscles, which we call ‘double’ transients, drive synchronous muscle contraction and release of eggs (2+ transients (magnitude < 150% ΔR/R) drive small vulval muscle twitching contractions that do not result in release of eggs. Such twitch transients are sometimes ‘single’ transients, limited to either the anterior or posterior muscles, or smaller magnitude double transients. Double transients result in larger peaks in our ratiometric trace recordings because the anterior and posterior signals are summed  and apx-1(wy755) vm2 muscle arm mutants were still able to execute single anterior and posterior vulval muscle Ca2+ transients, which was presumably due to the intact minor synaptic inputs from the ventral nerve cord alongside the vulval muscles  and apx-1(wy755) mutants had a twofold increase in the frequency of Ca2+ transient peaks (lin-12(wy750) and apx-1(wy755) mutants had significantly fewer synchronous double transients compared to the wild type (2+ transients of lin-12(wy750) and apx-1(wy755) mutants led to opening of the vulva, egg-laying would still occur, indicating that their egg-laying defect was not caused by structural problems in the vulva itself  and apx-1(wy755) mutants, the average anterior/posterior delay in double Ca2+ transients increased to ∼800 ms (lin-12(wy750) and apx-1(wy755) mutants are not necessary for excitation of the vulval muscles, but suggest they are required for the synchronous excitation of anterior and posterior vulval muscles needed to successfully execute egg-laying behavior.To quantify the anterior/posterior coordination in double Ca ∼100 ms , as near ∼100 ms . In both ∼800 ms  and ante ∼800 ms . Togethesel-12 early-stop allele wy756  contained a single missense mutation (G473R) in one of the conserved extracellular EGF domains of the LIN-12 protein (lin-12(wy750) was a loss-of-function allele. First, trans-heterozygotes between lin-12(wy750) and a canonical loss-of-function allele lin-12(n676n930) presented similar muscle arm defects (data not shown). Second, a lin-12 genomic fragment including 5 kb of upstream regulatory sequence and the entire coding region of lin-12 strongly rescued the muscle arm phenotype of lin-12(wy750) mutants  mutant animals, it is likely that the missense mutation we isolated represents a partial loss-of-function allele. Three apx-1 mutant alleles independently identified from the screen also contain missense mutations in the extracellular domain. One early-stop mutation in the sel-12 coding region leads to additional pleiotropic effects on both vulval muscle patterning and vulval morphogenesis, while the other missense mutation of sel-12 causes specific muscle arm defect , in which LIN-12 activity is normal at the permissive temperature (15°C) but disrupted at the restrictive temperature (25°C). Consistent with previous behavioral studies, lin-12(n676n930) mutant animals grown at the permissive temperature had no egg-laying defect and showed intact vulval muscle morphology and vm2 muscle arms, whereas animals grown at the restrictive temperature retained significantly more unlaid eggs inside their bodies and displayed vulval morphogenesis and vulval muscle arm defects. We then shifted lin-12(n676n930) mutant animals from the permissive temperature to the restrictive temperature at different larval stages and examined the vulval muscle morphogenesis and vm2 muscle arm development in the resulting adults. Most of the animals shifted from 15°C to 25°C prior to mid-L4 stage showed muscle arm defects similar to those of lin-12(wy750), whereas the wild-type muscle arm phenotype was observed in most of lin-12(n676n930) animals that were shifted after mid-L4 stage (Previous studies suggest a late requirement for behavior . The ‘labehavior . We hypoL4 stage .10.7554/lin-12(n676n930) mutant animals at the permissive temperature until the early-L3 stage, when the AC/VU decision and VPC specification processes were complete but vulval muscle cells had not yet been generated  was inserted into a fosmid containing the lin-12 genomic sequence at the 3′ end of the lin-12 coding region (lin-12::SL2::mCherry). This recombineered fosmid fully rescued the lin-12 muscle arm phenotype  mutant and analyzed mosaic animals in which expression of this transgene was lost in some of the tissues. Forty-five of 48 animals with lin-12 expressed in the vm2 cells (vm2+) showed a rescued, wild-type muscle arms (lin-12 expression in the vm2 cell (vm2-) . The mosaic rescue analyses argue that lin-12 functions cell-autonomously in vm2 to specify vm2 postsynaptic development through regulating muscle arm formation.We next investigated where cle arms , whereasl (vm2-) . On the lin-12 in vm2, we made two predictions. First, the ligand(s) of LIN-12 should be localized in the surrounding cells that directly contact vm2 to activate LIN-12/Notch pathway. Second, downstream components of LIN-12/Notch signaling pathway should also function in vm2 cells to regulate vm2 specification and muscle arm development. APX-1/Dsl is a good candidate as the relevant ligand because apx-1(wy755), but not other known LIN-12 ligand mutants including dsl-1 and lag-2, showed the muscle arm phenotype similar to that of lin-12(wy750) (lin-12(wy750); apx-1(wy755) double mutant did not show an enhanced muscle arm defect . We observed the expression of apx-1 in both secondary vulval epithelial cells and vm1 muscle cells during the L4 stage, when LIN-12/Notch signaling was activated to promote vm2 muscle arm development  mutants  of sel-12, the C. elegans homolog of presenilin (sel-12 encodes the γ-secretase that releases the LIN-12/NOTCH intracellular domain (NICD) by ligand-induced cleavage , consistent with the cell-autonomous requirement of LIN-12/Notch signaling in vm2 (lag-1 construct (lin-12(wy750), apx-1(wy750) and sel-12(wy760) mutants (hlh-29 transcription reporter (hlh-29::gfp), a known target gene of the canonical LIN-12/Notch pathway in vm2, was also dramatically down-regulated in lin-12(wy750), apx-1(wy755) and sel-12(wy760) mutants  mutant. Compared with wild-type animals, apx-1(wy755) mutant animals showed significantly reduced expression of lin-12 in vm2 cells, whereas the lin-12 expression in vulval epithelial cells was not affected (apx-1 expression in the lin-12(wy750) mutant  mutant . These rlin-12   , bottom,unc-40(n324) and madd-2(ok2226) null mutants showed specific vulval muscle arm defects with largely normal vulval muscle morphology  and neogenin in vertebrates serving as a receptor for the UNC-6/Netrin ligand , was reprphology . A signirphology . These dm growth . Consistm1 cells . Moreovem1 cells . These runc-40 and madd-2 functioned downstream of the LIN-12/Notch pathway, we compared the expression pattern of UNC-40 and MADD-2 in wild-type animals and lin-12(wy750) mutants. For these analyses, we used two types of expression constructs: translational reporters and transcriptional reporters. The translational reporters contain the promoter, coding and 3′UTR sequences in which the GFP is inserted in frame with the coding sequences. The fluorescence signal from this type of reporter reflects transcriptional activity, translational regulation and protein stability. The transcriptional reporters only contain the promoter regions and report the transcriptional activity. In wild-type animals, both UNC-40 and MADD-2 translational reporters were preferentially expressed in vm2 cells (lin-12(wy750) mutant (lin-12 was required for the expression of unc-40 and madd-2 in vm2.To investigate whether m2 cells . UNC-40:) mutant , supportunc-40 and madd-2 was regulated by lin-12, we examined the transcriptional reporters for unc-40 and madd-2 to measure the transcriptional activity of these genes. Similar transcription pattern was observed for unc-40 and madd-2. Both unc-40 and madd-2 were preferentially transcribed in vm2 cells, and the transcriptional activities of both promoters were dramatically suppressed in the lin-12(wy750) mutant  mutant . These dunc-40 or madd-2 expression was an output of LIN-12/Notch signaling sufficient for muscle arm formation, we asked whether the overexpression of unc-40 or madd-2 with an exogenous promoter in vulval muscles could bypass the requirement of lin-12. We found that the expression of unc-40, but not madd-2, in the lin-12(wy750) mutant was sufficient to induced muscle arm-like membrane extension in vm2 cells in ∼41% (36 of 88) of the animal , hinting that the activity of unc-40 in vm1 might be required. Since both unc-40 and madd-2 mutants showed muscle arm phenotypes, we investigated whether they functioned in the same or parallel pathways. We generated an unc-40(n324); madd-2(ok2226) double mutant and found slightly enhanced muscle arm defects in the double mutants  null mutant (unc-40 largely rescued the muscle arm defects of madd-2(ok2226) mutant, suggesting that madd-2 likely functioned synergistically with unc-40 to promote muscle arm formation  weak allele could be caused directly by the reduction of LIN-12/Notch activity, or indirectly by the disruption of other signaling(s) due to slight alternations of LIN-12/Notch-dependent vulval morphogenesis or other LIN-12/Notch-dependent events during early development. Using a temperature sensitive allele, we showed that disruption of lin-12 activity at a very late developmental stage caused vm2 muscle arm defects along with egg-laying defects. We never observed the loss or alternation of vulval muscles in these new weak alleles by examining several cell-type-specific markers  mutant is not likely the consequence of a complete cell-fate conversion. Even in the muscle lineage, several cell-type specific reporters displayed wild type patterns in lin-12(wy750) mutant (While ) mutant , supportlin-12 and apx-1 were found in the extracellular domains, but not in the signal transducing cytosolic domain of LIN-12. In addition, a nonsense mutation in sel-12 caused strong vulval phenotypes, while a missense mutation in sel-12 showed specific vm2 muscle arm phenotype, further arguing for the difference in the dosage requirement for these developmental events. Along the same line, a lag-1 dominant negative construct expressed in the vulval muscles led to muscle arm defects, suggesting that the same canonical LIN-12/Notch signaling pathways is required for the muscle arm development. Second, the lin-12(e2621) allele, showed muscle arm defects  (data not shown). Together, these data strongly argue that vulval morphogenesis requires a low level of LIN-12/Notch signaling activation, while vm2 postsynaptic specification requires a relatively high level of LIN-12/Notch activity. Consistent with this model, mir-61::gfp, another transcription reporter expressed in VPCs during its specification process, does not change in the lin-12(wy750) mutant (data not shown), suggesting that the alternation of LIN-12/Notch signaling activity in lin-12(wy750) has little or no effect in VPC specification.We favor the first dosage hypothesis for several reasons. First, the new mutations in  defects  with int defects . Sequenclin-12 expression is down-regulated in the apx-1(wy755) mutant, while the apx-1 expression does not change in the lin-12(wy750) mutant. These results indicate a different unilateral induction of LIN-12/Notch signaling activated by APX-1.The function of LIN-12 in the AC/VU decision is a case of classic lateral inhibition. A small initial difference in LIN-12/Notch activity between two adjacent cells is amplified by reciprocal feedback mechanisms. The enhanced difference of LIN-12/Notch activity eventually leads to alternative cell fates . Howeverlin-12 is expressed and cell-autonomously required in the postsynaptic target cell vm2, and that the activity of canonical LIN-12/Notch signaling pathway is involved, we speculate that the putative guidance receptors could be the downstream target genes of the canonical LIN-12/Notch pathway. While the canonical LIN-12/Notch signaling has been demonstrated to function in axon guidance and dendrite morphogenesis in fly and mammals  mutants. Both UNC-40 and MADD-2 are expressed in the postsynaptic vm2 cells but show little or no expression in the non-target vm1 cells. Cell-autonomous rescue of UNC-40 and MADD-2 in the vm2 cells further supports the notion that these two proteins function in vm2 to direct muscle arm development. Interestingly, the expression of the translational reporters for both genes in vm2 is dramatically down-regulated in lin-12(wy750) mutant, suggesting that both genes function downstream of LIN-12/Notch signaling pathway.UNC-40/DCC is a good candidate of the putative guidance receptor. receptor . It has receptor . More rereceptor . Indeed,C. elegans body wall muscle arms (unc-6 mutants (data not shown), suggesting the existence of a novel ligand of UNC-40 that functions in guiding the muscle arm extension. The putative ligand provided by vulval epithelial cells could be either a short-range cue that is associated on membrane or locally secreted, or a globally diffusive cue. We favor the former prediction based on the observation that the relative distance between the vulval muscles and the vulval opening is crucial. SM migration is affected in egl-15(n484) mutant, which causes posteriorly displaced vulval muscles descended from an SM that stops prematurely during the migration  plates. N2 Bristol strain worms were used as the wild-type reference. The following mutants were used in this study: unc-104(e1265) II, egl-1(n986) V, lin-3(e1417) IV, lin-39(n709) III, egl-15(n484) X, unc-40(e271) I, unc-40(n324) I. The mutants strains unc-32(e189); lin-12(n676n930) III, lin-12(n941)/eT1 (III); him-5(e1467)/eT1[him-5(e1467)] (V), apx-1(or3)/uT1 , sel-12(ty11) X, dsl-1(ok810) IV, glp-1(e2141) III and madd-2(ok2226) V were obtained from Caenorhabditis Genetics Center. lin-12(e2621) III was kindly provided by Iva Greenwald.Worms were maintained at 20°C on OP50 vsIs147 II (Punc-103e::mCD8::mCherry), vsIs149 V (Punc-103e::mCD8::mCherry), vsSi1 II (Punc-103e::unc-103e::gfp single copy), wyIs321 IV , wyIs333 I .ncIs13 II (Pajm-1::ajm-1::gfp), cyIs4 V (Pcat-1::cat-1::gfp), ayIs2 IV (Pegl-15::gfp), kyIs235 V (Punc-86::snb-1::yfp), leEx1444 (Phlh-29::gfp), from CGC, Phlh-29::gfp III, kindly provided by Casonya Johnson, arIs107 (Pmir-61::gfp), kindly provided by Iva Greenwald, ayIs4 I (Pegl-17::gfp), syIs60 II (P(F47B8.6)::gfp), ayIs6 X (Phlh-8::gfp), vsIs4 IV (Prgs-2::gfp), kindly provided by Jun Liu.wyEx5200 (Plin-12::lin-12::SL2::mCherry), arIs98 (Papx-1::2*nls::yfp), kindly provided by Iva Greenwald, wyEx604 (Punc-40+minigene::unc-40::gfp), wyEx5657 (Punc-40+minigene::gfp), trIs31 (Pmadd-2::madd-2::gfp), kindly provided by Peter Roy, wyEx5586 (Pmadd-2::mCD8::gfp).wyEx5402 (Punc-103e::unc-40::gfp), wyEx5658 (Pegl-15::unc-40::gfp), wyEx5281 (Psel-12::sel-12), wyEx5293 (Punc-103e::sel-12 #1), wyEx5294 (Punc-103e::sel-12 #2), wyEx5171 (Papx-1::apx-1), wyEx5197 (Pegl-17::apx-1 #1), wyEx5198 (Pegl-17::apx-1 #2), wyEx5451 (Pegl-15::apx-1 #1), wyEx5452 (Pegl-15::apx-1 #2), wyEx5269 (Punc-103e::dnLag-1 #1), wyEx5270 (Punc-103e::dnLag-1 #2), wyEx5506 (Pegl-15::mCD8::gfp), wyEx5697 (Pegl-17::mCD8::mCherry), wyEx5698 (Punc-103e::madd-2::gfp #1), wyEx5700 (Punc-103e::madd-2::gfp #2).lin-12(wy750) III; lite-1(ce314) X, LX1893 apx-1(wy755) V; lite-1(ce314) X, LX1848 vsIs153  IV; lite-1(ce314) X, LX1889 lin-12(wy750) II; vsIs153 IV; lite-1(ce314) X, LX1890 apx-1(wy755) V; vsIs153 IV; lite-1(ce314) XLX1892 wyIs333. Worms were mutagenized with 50 mM EMS. Complementation test, SNIP-SNP mapping, rescue, and sequencing were performed according to standard protocols.Alleles were isolated from an F2 semi-clonal screen of 3500 haploid genomes using the strain All plasmids for transgenic worm lines were made using pPD49.26 vector, ΔpSM or pSM vector, a derivative of pPD49.26 .unc-103e gene was amplified from genomic DNA by PCR using the following oligonucleotides: 5′-GGA ACT AGT TCA TGC CTA TTT TAT ATT TAC AAT ATT TTA G-3′, and 5′-TCA GCG CCC GGG ACC ACC ACC ACC ACA ACC-3′. This fragment was ligated into pPD49.26 to generate pKMC189. mCD8 coding sequence (lin-15 rescuing plasmid at 50 ng/μl (lin-15(n765ts) animals and then integrated into chromosomes using UV/Trimethylpsoralen. Four independent integrants including vsIs147 and vsIs149 were recovered and backcrossed to the wild type.Approximately 2.6-kb DNA fragment upstream of the start site of sequence  fused to50 ng/μl  into theunc-103 exons 5 to 11 were PCR-amplified from worm genomic DNA and inserted into the unc-103e cDNA at unique BstXI and AccI sites to generate pKMC100. Coding sequence of GFP was inserted near the 3′ end of unc-103e in a region encoding five consecutive glycine residues to make pKMC179. The unc-103e::gfp DNA fragment was then amplified from pKMC179 by PCR and inserted into the MoSCI vector that derived from pCFJ151 (ttTi5606 Mos1 transposon. vsSi1 was isolated and confirmed by DNA sequencing and expression of GFP. pCFJ151  to generPunc-4::mCherry::rab-3) at 1 ng/μl were injected with Podr-1::dsRed at 25 ng/μl into N2 worms and then integrated into chromosomes using UV/Trimethylpsoralen. wyIs321 was recovered and backcrossed to the wild type six times.pKMC180 at 5 ng/μl and pPL38 (ser-1b cDNA was PCR-amplified from worm cDNA library and subcloned into pKMC192 to replace the mCD8 sequence using the NheI and BamHI sites to generate pPL57. pPL57 at 5 ng/μl and pPL39 (Punc-86::mCherry::rab-3) at 1 ng/μl were injected with Podr-1::dsRed at 25 ng/μl into N2 worms and then integrated into chromosomes using UV/Trimethylpsoralen. wyIs333 was recovered and backcrossed to the wild type six times.SL2::mCherry(kanR+) DNA fragment from pOL007 (lin-12 genomic sequence using the fosmid recombineering approach (Plin-12::lin-12::SL2::mCherry). pPL102 was injected at 5 ng/μl with Podr-1::dsRed at 25 ng/μl into lin-12(wy750); wyIs333 worms.m pOL007  was amplapproach  to genersel-12 cDNA was PCR-amplified from worm cDNA library and ligated into ΔpSM using AscI and SalI sites to generate pPL154. Approximately 2.5-kb DNA fragment upstream of the start site of sel-12 gene was amplified from worm genomic DNA by PCR using the following oligonucleotides: 5′-ACG CGT CGA CGC ATA TAA TAA ACA CTT TTG AGA GAC TTG TG-3′, and 5′-AAG GAA AAA AGC GGC CGC GTA TTC ACA CGA GTA CAG TCT GA-3′. This DNA fragment was then inserted into pPL154 using NotI and AscI sites to generate pPL155 (Psel-12::sel-12). pPL155 at 15 ng/μl was injected with Podr-1::dsRed at 25 ng/μl into sel-12(wy760); wyIs333 worms.unc-103e promoter from pKMC192 was subcloned into pPL154 using NotI and AscI sites to generate pPL157 (Punc-103e::sel-12). pPL157 at 5 ng/μl was injected with Podr-1::gfp at 25 ng/μl into sel-12(wy760); wyIs333 worms. Two independent extrachromosomal arrays were recovered.apx-1 cDNA was PCR-amplified from worm cDNA library and ligated into ΔpSM using AscI and XmaI sites to generate pPL118. Approximately 4-kb DNA fragment upstream of the start site of apx-1 gene was amplified from worm genomic DNA by PCR using the following oligonucleotides: 5′- AAG GAA AAA AGC GGC CGC GAT TTG AAT ATT TAT TAG TTT GCC AGG T-3′, and 5′- TTG GCG CGC CAG TAC AGG ATC GTG TGC TAG A-3′. This DNA fragment was then inserted into pPL118 using NotI and AscI sites to generate pPL120 (Papx-1::apx-1). pPL120 at 10 ng/μl was injected with Podr-1::gfp at 25 ng/μl into apx-1(wy755); wyIs333 worms.egl-17 gene was amplified from worm genomic DNA by PCR using the following oligonucleotides: 5′-ACA TGC ATG CCT ATG CAG CAT TGG AGG ATG ATG-3′, and 5′-TTG GCG CGC CAG CTC ACA TTT CGG GCA CCT GAA-3′. This DNA fragment was then inserted into pPL118 using NotI and AscI sites to generate pPL135 (Pegl-17::apx-1). pPL135 at 15 ng/μl was injected with Podr-1::gfp at 25 ng/μl into apx-1(wy755); wyIs333 worms. Two independent extrachromosomal arrays were recovered.Approximately 4-kb DNA fragment upstream of the start site of egl-15 gene was amplified from worm genomic DNA by PCR using the following oligonucleotides: 5′-ACA TGC ATG CAA TCG CAC AGA TTA AAT TAC ACT TC-3′, and 5′-TTG GCG CGC CGA TGA TAT GTA TCA GGC AAA CAT TC-3′. This DNA fragment was then inserted into pPL118 using NotI and AscI sites to generate pPL194 (Pegl-15::apx-1). pPL194 at 15 ng/μl was injected with Podr-1::gfp at 25 ng/μl into apx-1(wy755); wyIs333 worms. Two independent extrachromosomal arrays were recovered.Approximately 1-kb DNA fragment upstream of the start site of lag-1 cDNA was PCR-amplified from worm cDNA library. A g to a single nucleotide mutation was made by overlapping PCR to generate R397H alternation. The product was confirmed by sequencing and then inserted into ΔpSM using AscI and SalI sites to generate pPL141. unc-103e promoter from pKMC192 was subcloned into pPL141 using SphI and AscI sites to generate pPL145 (Punc-103e::dnLag-1). pPL145 at 30 ng/μl was injected with Podr-1::gfp at 25 ng/μl into wyIs333 worms. Two independent extrachromosomal arrays were recovered.egl-15 promoter from pPL194 was subcloned into pKMC192 to replace the unc-103e promoter and generate pPL213 (Pegl-15::mCD8::gfp). pPL213 at 5 ng/μl was injected with Podr-1::DsRed at 25 ng/μl into vsIs147 worms.egl-17 promoter from pPL135 was subcloned into pKMC191 to replace the unc-103e promoter and generate pPL247 (Pegl-17::mCD8::mCherry). pPL247 at 10 ng/μl was injected with Podr-1::DsRed at 25 ng/μl into arIs98 worms.unc-103e promoter from pKMC192 was subcloned into pHMT15 (mig-13 promoter and generate pPL180 (Punc-103e::unc-40::gfp). pPL180 at 15 ng/μl was injected with Podr-1::DsRed at 25 ng/μl into vsIs147 worms.o pHMT15  using Spegl-15 promoter from pPL194 was subcloned into pHMT15 (mig-13 promoter and generate pPL185 (Pegl-15::unc-40::gfp). pPL185 at 10 ng/μl was injected with Podr-1::DsRed at 25 ng/μl into vsIs147 worms.o pHMT15  using Spunc-40 gene was amplified from worm genomic DNA by PCR using the following oligonucleotides: 5-AAG GAA AAA AGC GGC CGC GGG TTC TGA AAT AGA AAA AAG TTG TTT CG-3′, and 5′-TTG GCG CGC CTG CCT CTG GTC TAG CAG CCG CC-3′. This DNA fragment was then inserted into pSM using NotI and AscI sites to generate pPL309 (Punc-40+minigene::gfp). pPL309 at 5 ng/μl was injected with Podr-1::DsRed at 25 ng/μl into vsIs147 worms.The regulatory region containing ∼0.7 kb of sequence 5′ upstream to the initiator methionine in exon 1, with exons 1–5, introns 1–5  of madd-2 cDNA was PCR-amplified from worm cDNA library and inserted into pSM using AscI and XmaI sites to generate pPL197. unc-103e promoter from pKMC192 was subcloned into pPL197 using SphI and AscI sites to generate pPL201 (Punc-103e::madd-2::gfp). pPL201 at 50 ng/μl was injected with Podr-1::DsRed at 25 ng/μl into vsIs147 worms. Two independent extrachromosomal arrays were recovered.madd-2 gene was amplified from worm genomic DNA by PCR using the following oligonucleotides: 5′-ACA TGC ATG CTG CAA GTC TAA TAG GGA GGC-3′, and 5′-TTG GCG CGC CTT TTG AAG GGA CTG AAA TTT GAG T-3′. This DNA fragment was then inserted into pKMC192 using SphI and AscI sites to replace the unc-103e promoter and generate pPL219 (Pmadd-2::mCD8::gfp). pPL219 at 15 ng/μl was injected with Podr-1::DsRed at 25 ng/μl into vsIs147 worms.Approximately 2.5-kb DNA fragment upstream of the start site of C. elegans using a Plan-Apochromat 63 3 1.4 objective on a Zeiss LSM710 confocal microscope. Worms were immobilized using a mixture of 200 mM 2,3-butanedione monoxime  and 2.5 mM levamisole (Sigma-Aldrich) in 10 mM Na HEPES.Fluorescence images were captured in live For quantifying the vulval muscle sizes and the intersection angles between vulval slit and vulval muscles, 12–16 confocal images were used for each genotype. Each vulval muscle was lineated by polygonal lines in ImageJ to obtain the size. The middle points of the connection lines where vm1 was linked to the body wall, and the tips of vm2 cells were used as the landmarks to measure the intersection angles.Twenty L4-stage worms of each genotype were isolated and allowed to develop 30 hr at 20°C into gravid adults. The worms were transferred to a fresh plate and allowed to lay eggs for 2 hr at 20°C. The adult worms were removed before the total number of each plate and the stage of each egg were scored. The eggs were classified into one of three developmental stages: 1–8 cell, eight-cell to comma, or post-comma. Three independent assays were performed with at least 50 eggs quantified in total for each genotype. Fisher's exact test was performed to compare statistical significance between two groups.2+ imaging in the vulval muscles, we used as a control strain LX1890 vsIs153; lite-1(ce314), which expresses GCaMP3 and mCherry from the unc-103e promoter and is blue light insensitive, as previously described (lite-1(ce314) mutation (lin-12(wy750) or apx-1(wy755) animals to generate LX1892 lin-12(wy750); lite-1(ce314) and LX1893 apx-1(wy755); lite-1(ce314), respectively. LX1890 vsIs153; lite-1(ce314) animals were crossed with LX1892 lin-12(wy750); lite-1(ce314) and LX1893 apx-1(wy755); lite-1(ce314) to generate LX1889 lin-12(wy750); vsIs153; lite-1(ce314) and LX1890 vsIs153; apx-1(wy755); lite-1(ce314).To perform Caescribed . KG1180 mutation  were cro2+ imaging of behaving animals was performed as previously described . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent . However, the data do document an interesting muscle differentiation program that precedes postsynaptic differentiation and the participation of the muscle cell in synapse development through muscle arm formation. An appropriate interpretation should focus on the muscle arm phenotype, the developmental program for muscle arm formation controlled by Notch, and the involvement of these processes in synapse development. However, the concept of synapse specificity, highlighted in the title and throughout the text, is an interpretation that is not directly addressed by the data. The text and title should be substantially revised to address this issue.lin-12 mutants could clarify this issue. In any case, this needs to be more directly addressed in the Discussion.2. There was discussion among the reviewers regarding whether the absence of Notch signaling in vm2 partially switches fate or has a more selective disruption of the muscle arm growth program. The analysis of more fate markers and a more detailed description of morphological features of the vm2 muscle  in  1. The major concern of all three reviewers centers on the interpretation of the muscle arm phenotype as a defect in synapse specificity. The data do not address a “synaptic function” of Notch in this system . However, the data do document an interesting muscle differentiation program that precedes postsynaptic differentiation and the participation of the muscle cell in synapse development through muscle arm formation. An appropriate interpretation should focus on the muscle arm phenotype, the developmental program for muscle arm formation controlled by Notch, and the involvement of these processes in synapse development. However, the concept of synapse specificity, highlighted in the title and throughout the text, is an interpretation that is not directly addressed by the data. The text and title should be substantially revised to address this issue.We agree with the reviewers that our data do not address the role of the Notch in synaptic function, similar to the reports by Alberi et al. and de Bivort et al. In those two studies, the authors documented the function of the Notch pathway in synaptic plasticity in the mature nervous system. In our manuscript, we are studying the role of Notch during the development of the neural circuit.We also agree with the reviewers that we are studying a “muscle differentiation program that precedes postsynaptic differentiation and the participation of the muscle cell in synapse development through muscle arm formation”. We have made changes in the title and throughout the text to more precisely reflect the specific function in muscle arm development.However, we do feel that our data strongly indicate that the Notch-mediated muscle arm formation is a critical determinant of target specificity in the egg-laying circuit. We started the project with a forward genetic screen on a marker that labeled the postsynaptic specializations on the muscle arms. In the partial loss-of-function mutant alleles of the Notch pathway that we isolated in this synaptic morphology screen, many aspects of the muscle differentiation remain intact, suggesting that high level of the Notch signaling activity is specifically required for the postsynaptic development. In other words, the vm2 cells absolutely require high level of Notch signaling to become the postsynaptic target of the presynaptic HSN neurons.The next question is how LIN-12 affects muscle arm target selection. We found that LIN-12 activity is only found in vm2 but not vm1. Furthermore, LIN-12 acts cell-autonomously to activate the expression of UNC-40/DCC and MADD-2 in vm2. These two molecules in turn are required for vm2 muscle arm formation and targeting. Most importantly, to demonstrate that the LIN-12/Notch-controlled molecular program is directly involved in target selection during neural circuit assembly, we showed that UNC-40 is sufficient to induce muscle arm-like membrane protrusions when misexpressed in vm1. Taken together, these data suggest that the high-level activity of LIN-12 distinguishes vm2 from vm1 in the process of forming muscle arms and becoming the postsynaptic targets in the egg-laying circuit.2. There was discussion among the reviewers regarding whether the absence of Notch signaling in vm2 partially switches fate or has a more selective disruption of the muscle arm growth program. The analysis of more fate markers and a more detailed description of morphological features of the vm2 muscle  in lin-12 mutants could clarify this issue. In any case, this needs to be more directly addressed in the Discussion.egl-15::gfp , we have included analyses of two additional cell-type specific markers (hlh-8::gfp and rgs-2::gfp) . hlh-8::gfp labels the M lineage, which include vm1, vm2, and the uterine muscles. This marker is only present in differentiating muscles and is absent in mature muscles. rgs-2::gfp is a specific marker for the uterine muscle. In the lin-12(wy750) mutant animals, the expression of hlh-8::gfp is found in all three classes of muscles only during L3-L4, indistinguishable from the wild type controls. Similarly, in the lin-12(wy750) mutant animals, the expression of rgs-2::gfp is found only in the uterine muscles, indistinguishable from the wild type controls. Together, we have examined three differentiation markers for the vm2 and related muscle lineage. We found no obvious gross cell fate changes in the lin-12(wy750) mutant. We want to remind the reviewers that this lin-12 allele is a partial loss-of-function allele, which showed no vulval morphogenesis phenotypes (another cell fate phenotype). We think the lack of cell fate phenotype is likely due to the remaining LIN-12/Notch activity in this strain.We thank the reviewers for the suggestions. We have performed several additional experiments. In the revised manuscript, besides the vm1 specific marker lin-12(wy750) and unc-40(n324) mutants, further supporting the notion that the phenotype of lin-12(wy750) is restricted on the muscle arm structure.We have also followed the reviewers' suggestion and characterized the morphology of the vulval muscles quantitatively. The results are now presented in Figure 1–figure supplements 5 and 6. We show that the general vulval muscle morphology is not affected in


To reduce rates of VRE colonization/disease, we introduced a multimodal hospital-wide bleach-based cleaning program (BBCP) that included a new product (sodium hypochlorite 1000ppm + detergent), new standardised (routine and detailed) cleaning practices & modified glove/gown protocols to rely on alcohol-based handrub & sleeveless aprons. Rates of VRE pre- & post-BBCP were compared.vs post-BBCP (Period B1 & B2 – Feb-July & Aug-Jan 2010/11). Rates of VRE bacteremia (per 100 patients blood cultured [100PBC]) & of urinary tract infection [UTI] were compared - Period A vs B1 & B2.Patients in 4 high-risk wards  were screened on admission & weekly for rectal VRE colonization, & rates were compared pre-BBCP (Period A [6 mo] – Feb-July 2009) vs 181/2129 [Period B1] vs 143/2141 [Period B2], p<0.0001), despite an increase in screening compliance  and a stable rate of on-admission VRE colonization . VRE bacteremia declined from 0.48/100PBC (14/2935) pre-BBCP to 0.08/100PBC (5/6194) during the 12 mo post-BBCP (p=0.0002), with a reduction in UTI cases (24 [A] vs 19 [B1] vs 17 [B2]).A 37% reduction in newly recognised VRE colonizations was observed post-BBCP (208/1948 patients screened [Period A] The BBCP was associated with a significant reduction in rates of both new VRE colonizations (37% decrease) & VRE disease. This approach potentially represents a new paradigm in the management of VRE.None declared.


Superparamagnetic iron oxide nanoparticle (SPIO)-based molecular imaging agents targeting macrophages have been developed and successfully applied in animal models of myocardial infarction.The purpose of this clinical trial was to investigate whether molecular magnetic resonance imaging (MRI) of macrophages using ferucarbotran (Resovist®) allows improved visualization of the myocardial (peri-)infarct zone compared to conventional gadolinium-based necrosis/fibrosis imaging in patients with acute myocardial infarction.The clinical study NIMINI-1 was performed as a prospective, non-randomised, non-blinded, single agent phase III clinical trial. Twenty patients who had experienced either an acute ST-elevation or non-ST-elevation myocardial infarction (STEMI/NSTEMI) were included to this study. Following coronary angiography, a first baseline cardiovascular magnetic resonance (CMR) study (pre-SPIO) was performed within seven days after onset of cardiac symptoms. A second CMR study (post-SPIO) was performed either 10min, 4h, 24h or 48h after ferucarbotran administration. The CMR studies comprised cine-CMR, T2-weighted “edema” imaging, T2*-weighted cardiac imaging and T1-weighted late-gadolinium-enhancement (LGE) imaging.The median extent of short-axis in-plane LGE was 28% (IQR 19-31%). Following Resovist® administration the median extent of short-axis in-plane T2*-weighted hypoenhancement  was 0% . A significant in-slice increase (>3%) in the extent of T2*-weighted “hypoenhancement” (post-SPIO compared to pre-SPIO) was seen in 6/16 patients (38%). However, no patient demonstrated “hypoenhancement” in T2*-weighted images following Resovist® administration that exceeded the area of LGE.T2/T2*-weighted MRI aiming at non-invasive myocardial macrophage imaging using the approved dose of ferucarbotran does not allow improved visualization of the myocardial (peri-) infarct zone compared to conventional gadolinium-based necrosis/fibrosis imaging.


We previously validated a grass pollen nasal challenge model to record clinical outcomes and local biomarkers in nasal fluid . Here we18 grass pollen allergics underwent nasal allergen challenges; 15 grass allergics had matched diluent challenges. Symptom scores and peak nasal inspiratory flow were recorded; nasal fluid was collected using polyurethane sponges, then extracted by centrifugation across microfilters. Fluid was then tested for Th2 cytokines by Mesoscale 7-plex multi-spot plate assay. A further 6 allergics underwent nasal allergen challenges with collection of fluid using both polyurethane sponges and Leukosorb filter strips; nasal fluid was analysed by Milliplex cytokine/chemokine magnetic bead multiplex assay.Allergen vs diluent challenges; multi-spot plate assay: symptoms increased, and peak nasal flow decreased, following allergen but not diluent challenge . Levels of IL-4 (p<0.01), IL-5 and IL-13 (both P<0.001) were maximally increased at 5 hours compared to pre-challenge; no significant increases were seen following diluent challenge. Between group differences  for IL-4, -5 and -13 were seen at 4 and 6 hours .Allergen challenge; magnetic bead assay: IL-5 was increased at 6 hours , with IL-13 and IL-4 also showing a trend towards an increase . Eotaxin and MDC were increased at 6 hours ; IL-6 was elevated at 2 hours . Levels of IL-17A, IL-27, IL-23, IFN-gamma and IL-12p70 were low and did not change significantly after allergen challenge. High levels of IL-8 were detected, maximal at baseline, but did not change significantly after challenge. Polyurethane sponges proved superior to filters for all measurements.Grass pollen nasal challenge is associated with a strong local Th2, but not Th1, response detectable in nasal secretions. Despite elevation of IL-6, there is no significant local Th17 response, up to 6 hours. Responses are allergen-driven and independent of the intervention and any diurnal variation. Multiplex assays are capable of detecting cytokines and chemokines in low volumes (25mcl) of nasal fluid by both multi-spot and micro-bead techniques.