Using Drosophila Wing Imaginal Disc To Study The Role Of Cizl In Organ Growth Control And The Underlying Mechanism

Background:Precise control of organ growth is essential for organ development and function.Organ size is determined by cell volume and cell number.And the latter is the consequence of cell proliferation and cell death.Drosophila wing imaginal disc is the precursor structure of the adult wing and a commonly-used model to study organ size control and pattern formation.The growth of wing disc is restrained to the larval stage.The final size of wing discs determines the size of adult wings.CIZ1 is a nuclear matrix protein.Mammalian CIZ1 has been reported to participate in DNA replication,G1/S transition and X chromosome inactivation.However,whether CIZ1 is involved in organ growth and size control is unknown.Previously through RNAi screen,our lab found Ciz1,the Drosophila homolog of CIZ1,is essential for wing disc cell survival.Objective:In this study,we aim to determine the role of Drosophila Ciz1 in wing growth and development and to analyze the underlying mechanism.Methods and results:1)To determine the role of Drosophila Ciz1 in wing growth and development,we ubiquitously knocked down Ciz1 under tub-Gal4.Ubiquitous knockdown of Ciz1 resulted in larval lethality.The average size of the wing discs with Ciz1 knocked down(Ciz1KD)was only 40%of that of the control wing discs at the same stage.We specifically knocked down Ciz1 in Drosophila wing discs using C765-Gal4 or nub-Gal4,and found that the adult wing size was reduced by half compared to the control group.These data suggest Ciz1 is essential for Drosophila wing growth.2)To clarify the mechanism by which Ciz1 regulates wing growth,we examined the cell size,cell proliferation and apoptosis in the Ciz1KD and control wing discs.We found that knocking down Ciz1 resulted in increased cell size,reduced proliferation and increased apoptosis.Inhibition of apoptosis in Ciz1KD wing discs did not rescue the reduced adult wing size.These data suggest the reduced size of CizKD wings was mainly due to inhibition of cell proliferation.3)To figure out how Ciz1 regulates proliferation,we adopted immunostaining against pH3 and EdU and the FUCCI cell cycle reporter.We found knocking down Ciz1 did not affect DNA replication but resulted in G2 arrest.Next,we measured the mRNA and protein level of Cyclin B(CycB),an important driver for G2/M transition,using qRT-PCR and immunostaining.We found knocking down Ciz1 led to decreased levels of CycB mRNA and protein.Ciz1 may promote G2/M transition by regulating CycB level.4)Human CIZ1 is a nuclear protein.We found Drosophila Ciz1 is also localized to the nucleus by staining the overexpressed Ciz1 protein.The full-length Drosophila Ciz1 contains 919 amino acids,with two C2H2 zinc finger motifs at 476-498 amino acid(aa)and 571-593aa,respectively.We constructed three plasmids expressing the N-terminal 475 amino acids of Ciz1(Ciz1-N),476-919aa of Ciz1(Ciz1-C)and a Ciz1 fragment with 476-593aa deleted(Ciz1ΔZF),which no longer contains the two zinc finger motifs,respectively.We generated transgenic flies using these plasmids.We found that both Ciz1-N and Ciz1ΔZF localize to the nucleus,and that Ciz1-C resides in the cytosol,suggesting the nuclear localization signal is in the N-terminal half.We further analyzed subcellular localization of different fragments of Ciz1,and concluded that the nuclear localization signal of Ciz1 resides in 73-131aa.5)To determine the regions that mediate Ciz1 regulation of proliferation and apoptosis,we overexpressed Ciz1-N,Ciz1-NLSC(73-131+476-919)and Ciz1ΔZF in Ciz1KD wing discs,respectively.We found Ciz1ΔZF could rescue the increased apoptosis,decreased proliferation and reduced wing size caused by knockdown of Ciz1,indicating that the zinc finger motifs are not required for regulation of proliferation and apoptosis by Ciz1.Expression of Ciz1-N but not Ciz1-NLSC rescued the reduced proliferation,decreased CycB level and reduced wing size caused by knocking down Ciz1,indicating Ciz1-N is responsible for regulating CycB level and cell proliferation.Neither Ciz-N nor Ciz1-NLSC alone could suppress apoptosis induced by Ciz1 knockdown,indicating that the apoptosis regulation by Ciz1 requires both parts.In this study,we for the first time demonstrated that Ciz1 is essential for wing disc growth and cell proliferation.We also analyzed the mechanism by which Ciz1 regulates proliferation and identified the regions mediating Ciz1 regulation of proliferation and apoptosis.Our study provides basis for the molecular function study of Ciz1.