Drosophila Melanogaster As A Model Organism To Study The Function Of The Pathogenic Gene SLC20A2 Of Idiopathic Basal Ganglia Calcification

SLC20A2 has been identified as the first pathogenic gene for the idiopathic basal ganglia calcification（IBGC）by our research group in 2012.According to the statistics,there are about 40%of IBGC cases caused by mutations in SLC20A2,therefore it has been deemed to the primary pathogenic gene for IBGC.SLC20A2 encodes a typeⅢsodium-dependent inorganic phosphate transporter 2（PiT2）,which is broadly expressed in different tissues with widely varied expression levels,this demonstrate that SLC20A2 may play a key role as a housekeeping gene.PiT2 as a cell membrane transporter protein involves in binding amphotropic murine leukemia virus（A-MuLV）and maintenance of inorganic phosphate homeostasis.A topological organization model of PiT2 has 12transmembrane domains with extracellular N-and C-terminal extremities,and a large central intracytoplasmic domain（loop7）.While the SLC20A2 truncation mutant lacking loop7 proving that this domain is dispensable for P_i transport and retroviral recognition function of PiT2.So far,exact functions of the loop7 domain in PiT2 remain poorly understood,the role it plays in the PiT2 functions is one of our primary object of research.In this dissertation,we investigated the function of the pathogenic gene SLC20A2 in vivo using Drosophila melanogaster as a model organism.At first,the protein sequences BLAST result showed that the homologs of human SLC20A1 and SLC20A2 in the Drosophila genome both were dPiT,which was the only typeⅢsodium-dependent inorganic phosphate transporter in Drosophila.The protein sequences of dPiT were 38%identical and 56%similar to human PiT2.Afterwards,we constructed dPiT loss of function mutants dPiT^21-4 and dPiT^15-1 through the CRISPR/Cas9 technology.The dPiT^21-4and the dPiT^15-1 mutants were frameshift in dPiT,respectively.They carried one base pair deletion at 62^th and 536^th nucleotides,and produced two truncated 43 and 190 amino acids peptides,as well as only 20 and 178 amino acids in the N-terminal of both peptides were in common with wild type dPiT protein,independently.To investigate the function of loop7 domain in dPiT during Drosophila development,we constructed two mutant flies using the UAS-Gal4 system for overexpression,dPiT-Δloop7 and dPiT-Δloop7-GFP,respectively.Ubiquitous or neuronal overexpression of dPiT-GFP in dPiT loss of function mutant dPiT^21-4/Df（3L）ED4470 background by actin-Gal4 or elav-Gal4,respectively,rescued the embryonic lethality of dPiT loss of function mutant.However,ubiquitous or neuronal overexpression of dPiT-Δloop7-GFP in dPiT loss of function background by actin-Gal4 or elav-Gal4 could not rescue the embryonic lethality.These results suggest that dPiT is an essential gene for Drosophila development,and the loop7 domain of dPiT is crucial for the function of dPiT.By means of dissecting the third instar larvae and immunostaining,both dPiT-GFP and dPiT-Δloop7-GFP,when driven by elav-Gal4 in the wild-type background,were abundantly expressed in the cell body of Drosophila brain and ventral ganglions.While dPiT-GFP could also be detected in the axon and the terminal of NMJ,there were little distribution of dPiT-Δloop7-GFP in the axon,and it was hardly detectable in the NMJ.Furthermore,dPiT-GFP was found to be located in all parts of the sensory neuron system,including the cell body and dendrite branches.Nonetheless,dPiT-Δloop7-GFP mainly existed in the cell body and hardly observed in the dendritic branches.These results indicate that loop7 domain affects subcellular localization of dPiT in neurons in vivo.To determine whether dPiT plays a role in Drosophila NMJ synaptic development,we observed and analyzed the morphology of NMJ 4 from the abdominal segment 3 of the third instar larvae.We found that knocking down dPiT or overexpressing dPiT-Δloop7-GFP in nervous system was associated with striking NMJ abnormalities as compared with controls or flies overexpressing dPiT-GFP.In addition,Western blotting of the immunoprecipitates exhibited an interaction between dPiT and Futsch in the Drosophila nervous system.Futsch is the only one representative of the whole MAP1 family,and it plays an important role in maintaining the stability of microtubule and neuronal development.Taking advantage of dPiT and futsch mutants,we made the genetic hybrid and immunostaining assays,found that dPiT and Futsch function in a common pathway to regulate NMJ synaptic growth.These results suggest that dPiT is crucial for normal development of Drosophila NMJ synapsesIn summary,these in vivo studies reveal that dPiT is an essential gene for the regulation of Drosophila development.Deletion of loop7 domain affects subcellular distribution of dPiT and normal morphology of NMJ synapses.These results indicate that the loop7 domain is required for the function of dPiT and normal development of Drosophila NMJ synapses.Meanwhile,we demonstrate that dPiT might regulates Drosophila NMJ development through interaction with Futsch.These discoveries provide a theoretical foundation for studying the function of human PiT2,encoded by the pathogenic gene SLC20A2 of IBGC,during the neuronal development.