The Mechanism Of Dally-like Regulate Morphogens Transportat In Posterior Compartment Of Drosophila Wing Discs

Heparin sulfate proteoglycan(HSPGs),as a macromolecular substance on the cell surface and extracellular matrix,regulates the formation of morphogens concentration gradients including the control of morphogens movement,signal transduction and intracellular transport.There are two corresponding homologous genes dally and dally-like(dlp)in Drosophila.It has been studied that Dally regulates the transport and concentration gradient formation of Wg,Hh and Dpp.Dlp also plays an important role in regulating the formation of Wg concentration gradient.In addition,dlp plays a regulatory role in the long-range transmission of Drosophila wing discs Hh to the forward compartment.However,in the area where Hh is produced in the posterior compartment,how does dlp regulate the release and long-range transport of Hh,have not seen systematic research yet.In addition,it has not been reported whether dlp has a regulatory relationship on the transportation of Dpp.This project uses the model organism Drosophila to conduct research,focusing on the mechanism of dlp regulating the development of the wing posterior compartment.The main research contents are as follows:1.Effects of HSPGs expression level on wing discs and adult wing development of Drosophila.The development of Drosophila wing discs and adult wing were observed by manipulating the expression levels of dlp and dally.It was found that overexpression of dlp and repression of dally resulted in smaller wing discs area of the expression region,while overexpression of dally and repression of dlp had no significant effect on the area change of the expression region.After eclosion,there was no significant change in the wing area of the adult female Drosophila en>dally,except for that,the wing area of the adult had shrunk.Through the detection of cell proliferation rate by Brd U and PH3 antibody staining,and it was found that the cell proliferation rate in the expression region remained unchanged,indicating that the reduction of cell proliferation rate caused by overexpression of dlp or repression of dally was not caused by the reduced cell proliferation rate.Detection of cell apoptosis by Caspase3found that overexpression of dlp driven by en-Gal4 would lead to cell apoptosis,expression of the factor p35 that inhibits cell apoptosis,and the reduction of the posterior compartment area was partially rescued.It shows that the reduction of wing discs area caused by overexpression of dlp is related to cell apoptosis.2.To rescue a phenotype that manipulates HSPGs levels in the posterior compartment of the wing discs result the region reduction.By detecting the downstream target Omb of the Dpp signaling pathway,it was found that when the expression of UAS-dlp and UAS-dally-RNAi was driven in the posterior compartment,and the expression of Omb was down-regulated,indicating that the Dpp signal pathway was inhibited.Co-expression of Tkv ^QD,the persistently active receptor of Dpp signal,could completely rescue en>dally-RNAi,suggesting that the inhibition of dally induced posterior compartment shrinkage is mainly due to the obstruction of Dpp signaling pathway.en>dlp with overexpression of dpp had excessive proliferation in the anterior compartment of wing discs,suggesting that the excess dpp in the posterior compartment could be transported in the posterior compartment,so excessive Dlp did not affect the transport of Dpp.Overexpression of dpp did not have a rescue effect on the phenotype of reduced of posterior compartment area caused by en>dlp.However,co-expression of Tkv ^QDhad a significant rescue effect,the continuously activated receptor of dpp,suggesting that excessive Dlp hindered the signal transduction of extracellular Dpp.In addition,by interfering with tumor suppressor Wts in the Hippo signaling pathway,it was found that wts-RNAi had a certain rescue effect on en>dlp and en>dally-RNAi.3.dlp control of Hh remote delivery in the posterior compartment.Overexpression of Hh in the wing posterior compartment can cause excessive proliferation of the wing anterior compartment and up-regulation of the expression level of the target gene ptc.Hh negatively regulates the expression of dlp,while dlp promotes the expression of Hh.When hh was overexpressed in the posterior compartment and dlp was overexpressed,the phenotype of the wing discs posterior compartment was not rescued,and the anterior compartment was not over-proliferated.The band of Hh's target ptc was narrowed,indicating that excessive dlp restriction excessive Hh in the posterior compartment was transported to the anterior compartment;after RNA repression of dlp,the area of the anterior compartment did not increase,and the expression band of Ptc narrowed.Further studies showed that when hh was overexpressed in the posterior compartment alone,GFP-labeled Hh could be seen to be evenly distributed in the posterior compartment.When hh and dlp were co-expressed,abnormal Hh aggregation occurred,thus restricting the long-distance transport of Hh.4.Effects of overexpressed dlp on the development of appendage organs in Drosophila.Overexpression of dlp not only affects wing size,but also affects the development of other organs in Drosophila.When the adults overexpressing dlp were observed,it was found that there were some phenotypes such as unilateral wing loss,unilateral wing wrinkle,bilateral wing loss and bilateral wing loss,etc.In addition,in the case of wing loss,tissues similar to legs would be formed in the hinged area.The adult haltere is either partially missing or transformed into a leg-like structure.The experimental results show that overexpression of dlp can lead to abnormal organ development,and the growth of haltere and some legs in the wrong position.Therefore,an appropriate amount of dlp plays an important role in the correct configuration and development of organs.In conclusion,HSPGs participates in the regulation of the release and transmission such as Dpp,Hh,and other morphogens,and play an important role in regulating the organ development in Drosophila.Excessive dlp can cause Hh to gather into clusters,which restricts the long-distance transportation of Hh.