| Salivary gland is an important secretory gland in animals.Salivary glands from Drosophila and other insects generally consist of two symmetrical glands and a common duct that connects to the mouth,and each gland composes of columnar epithelial secretory cells surrounding a lumen.The well-documented functions of Drosophila salivary glands are to produce and secrete various products including some proteins into the lumen by exocrine way,and thsese products are then involved in multiple physiological processes,such as food lubrication,food digestion,immunity,and the interaction between insects and host plants.Interestingly,previous studies in mammals have reported that salivary glands may have endocrine roles as well.For example,Parotin hormone is produced from bovine salivary glands and may be secreted into the general circulation to promote cell growth.However,the endocrine function of animal salivary gland has not been rigorously established.During systemic growth in animals,all tissues and organs adapt to body growth through appropriate growth rate and finally reaches a suitable size compared with body size.Animal systemic growth is mainly regulated by two classic nutrient-sensing pathways,namely,insulin/insulin-like growth factors(IIS)and mechanistic target of rapamycin(m TOR)signaling.In drosophila,insulin-like peptide 2(Dilp2)is secreted from insulin-producing cells(IPCs)of larval brain and functions as a master regulator of systemic growth.Genetically ablating the IPCs causes systemic growth retardation and Dilp2 overexpression promotes systemic growth.In addition,m TOR signaling in the fat body as a nutrient sensing organ regulates larval growth by coupling with the IIS signaling.Although salivary gland growth is coordinated with organism growth in Drosophila,whether it plays endocrine roles in systemic growth remains unclear.In this study,we used Drosophila as a model to determine whether salivary glands have endocrine function by combining genetic,molecular,biochemistrical,and proteomic approaches at individual,tissue,and cell scales.We demonstrated that salivary glandsecreted peptide Sgsf promotes systemic growth by modulating Dilp2 secretion from brain IPCs.Major findings are summarized as following:1.Ablation of the Drosophila salivary glands retard systemic growthTo determine the role of salivary glands in the growth and development of Drosophila larvae,based on the GAL4 / UAS system,we genetically ablated them via ectopic expression of the apoptosis-inducing genes Grim or Rpr using the Sg-Gal4 driver,which is specifically expressed in salivary glands.The effect of salivary gland ablation on Drosophila growth and development were then investigated.Strikingly,ablation of salivary glands decreased pupal volume as well as the size of larval wing disc,fat body cells,and brain at 120 h after egg laying(AEL).In addition,ablation of salivary glands similarly decreased the body size,body weight,and wing size of adult flies.Collectively,our results indicate that Drosophila salivary glands are involved in the regulating systemic growth.We further detected whether the systemic growth arrest caused by larval food intake or digestion.Bromophenol blue sodium salt dye food feed larvae showed that salivary gland-ablated larvae could feed and digest normally.Besides,considering the food became somewhat dry and hard after salivary gland ablation,we detected whether the food condition had an effect on organism systemic growth.We pretreated food by using GFP-labeled larvae to keep the food moist and soft and made the larvae live in this food condition.The results showed that salivary gland-ablated larvae still developed into smaller pupae.Knockdown of exocrine related gene in salivary gland by using RNA interference(RNAi)did not affect pupal volume.Above results showed that salivary gland ablation did not affect larval food intake and digestion;dry and hard food condition as well as knockdown exocrine related gene did not affect systemic growth.We further explored whether salivary gland ablation-caused systemic growth arrest was due to a change in feeding and digestion abilities.Bromophenol blue sodium salt dye food experiments showed that larvae with salivary gland ablation could feed and digest food normally.In addition,the food where Drosophila larvae with ablated salivary glands were raised was somewhat dry and hard,we next asked whether this food condition influences systemic growth.We pretreated medium with green fluorescent protein labeled normal Drosophila to keep the medium moist and soft,and then placed salivary glandablated larvae in this conditioned medium and found that salivary gland-ablated larvae still developed into smaller pupae.Morover,we performed salivary gland-specific RNA interference(RNAi)of some secretory factors related to exocrine functions of salivary glands.The results revealed that this did not change pupal volume.Collectively,salivary gland ablation did not affect larval feeding and digestion;the food condition caused by salivary gland ablation had no obvious impact on systemic growth;salivary glandspecific RNAi of some exocrine factors did not affect systemic growth.2.Salivary gland-derived Sgsf peptide is secreted into the hemolymphIncreasing evidence in Drosophila reveals that several nonneuronal tissues,such as the fat body and wing discs,can secrete peptides into the hemolymph via endocrine way to regulate body growth and development.Because Drosophila salivary gland ablation induced a retardation of systemic growth,it raised the possibility that salivary glands might secrete a growth factor-like peptide into the hemolymph to regulate systemic growth in an endocrine manner.Therefore,we analyzed the proteomes of the hemolymph and salivary glands using LC-MS/MS approach to identify potential proteins secreted from salivary glands.As a result,we identified 544 proteins in the hemolymph of the control,294 proteins in Sg>Grim hemolymph,and 191 proteins in Sg>Rpr hemolymph.Further analaysis showed that a previously uncharacterized protein with a signal peptide sequence,CG44956,was lost in the hemolymph following salivary gland ablation.It was specifically expressed in larval salivary glands and we named it as a salivary glandderived secreted factor(Sgsf).Furhter Western blot results show that Sgsf protein levels in salivary gland and hemolymph exhibited a dynamic change during larval development.Sgsf protein level was lower in salivary gland and higher in the hemolymph at the early L3 stage(84 h AEL).But,Sgsf protein level was higher in salivary gland and lower in the hemolymph at the end L3 stage(120 h AEL).Immunofluorescence showed that Sgsf was not secreted into the lumen.Moreover,we found that Sgsf protein could be detected in both salivary glands and hemolymph following a transgenic overexpression of the complete Sgsf gene containing signal peptide sequence.However,following a transgenic overexpression of the Sgsf gene without signal peptide sequence,Sgsf could only be detected in salivary glands,but not in the hemolymph.Similarily,an investigation in Drosophila S2 cells revealed that Sgsf protein containing signal peptide could be detected in the cultured medium of S2 cells following transient overexpression.However,overexpression of a truncated form of Sgsf lacking the signal peptide was undetectable.Collectively,our results confirm that Sgsf protein specifically expressed in Drosophila salivary glands can be secreted into the hemolymph via endocrine way.3.Sgsf promotes systemic growthTo characterize the role for secrting salivary gland specific Sgsf protein into hemolymph,we used CRISPR/Cas9 method to performe site-specific knockout of the Drosophila Sgsf gene.A Sgsf mutant allele with two genomic deletions induced by different sg RNAs was generated,which caused sgsf frameshift mutation.Sgsf protein could not be detected in either salivary glands or the hemolymph of Sgsf knockout flies.Compared to controls,Sgsf knockout flies phenocopied salivary gland ablation and exhibited a decrease in pupal volume,the size of larval organs,the nuclear size of salivary gland and fat body cells,and the size and weight of the adult body.Moreover,we generated two RNAi lines targeting different regions of the Sgsf gene and observed that salivary gland-specific Sgsf RNAi decreased pupal volume.Conversely,salivary glandspecific Sgsf overexpression increased pupal volume and the size of larval organs.The decrease in pupal volume caused by Sgsf knockout could also be rescued by salivary gland-specific Sgsf overexpression.Altogether,these data suggest that salivary glandderived Sgsf is secreted into the hemolymph to promote systemic growth.4.Sgsf regulates systemic growth by modulating Dilp2 secretion from the IPCs and IIS/m TOR signaling in the fat bodySince Drosophila systemic growth is mainly regulated by the IIS/m TOR signalling pathway.Thus,we tested whether Sgsf modulates IIS/m TOR signaling.Interestingly,phosphorylation levels of Akt,S6 K and 4E-BP,key effectors of IIS/m TOR signaling,were decreased in the fat body of Sgsf knockout flies.Consistently,the levels of phosphorylated Akt,S6 K,and 4E-BP were also decreased in the fat body following salivary gland ablation.Furthermore,increasing IIS/m TOR signaling in the fat body of Sgsf knockout flies by using Cg-Gal4 to drive an overexpression of insulin receptor In R,m TOR or S6 K rescued the decreased pupal volume of Sgsf mutant flies.Finally,feeding salivary gland-ablated larvae with high-protein food could also rescued the abnormal size associated with salivary gland ablation.Taken together,the above results show that Sgsf protein secreted from Drosophila salivary glands promotes systemic growth by mediating IIS/m TOR signaling.Given that Dilp2,an insulin-like peptided secreted from the IPCs of the brain into hemolymph,is a key ligand of IIS/m TOR signaling and plays essential role in systemic growth,we further investigated the effect of Sgsf protein on dilp2 secretion.Compared to the control at 120 h AEL,Sgsf knockout caused Dilp2 accumulation in the IPCs but a decrease in Dilp2 levels in the hemolymph.Besides,either salivary gland ablation or salivary gland-specific Sgsf knockdown also led to Dilp2 accumulation in the IPCs,but salivary gland-specific Sgsf overexpression promoted Dilp2 secretion.Strikingly,both Dilp2 accumulation and reduced pupal volume could be restored by Dilp3-Gal4 drivermediated bacterial sodium channel(Na Ch Bac)overexpression in the IPCs of the Sgsf knockout flies.Finally,we performed an ex vivo assay and confirmed the effect of Sgsf on Dilp2 secretion from the IPCs.Altogether,our results indicate that Sgsf regulates systemic growth by promoting the secretion of Dilp2 from the IPCs and modulating IIS/m TOR signaling in the fat body.5.Ecdysone regulates the expression and secretion of sgsfThe steroid hormone ecdysone controls larval development and metamorphosis in Drosophila.At the early stage of the last instar larvae(approximately 84 h AEL),a lowlevel ecdysone pulse appears in the hemolymph and promotes larval growth,while at the end of the larval development(approximately 120 h AEL),a high-level ecdysone pulse appears in the hemolymph,and inhibits the feeding and stops the growth.We observed that the transcription level of Sgsf was gradually decreased from 84 to 120 h AEL,and in contrast to a gradual accumulation in salivary glands,Sgsf protein levels gradually decreased in cell-free hemolymph during the last larval instar.This trendency is likely associated with dynamic changes of ecdysone pulses.Therefore,we performed salivary gland-specific RNAi of ecdysone receptor gene Ec R at different stages during the last larval instar to check the effects of ecdysone on the expression and secretion of sgsf.Ec R RNAi in salivary gland at 84 h AEL decreased Sgsf transcription,indicating Sgsf expression in salivary glands is activated by low-level ecdysone pulse at the early stage of the last instar larvae.In addition,Ec R RNAi in salivary gland at 120 h AEL enhanced Sgsf secretion into the hemolymph,indicating Sgsf secretion is inhibited by high-level ecdysone pulse at the end of the larval development. |
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