Identification And Functional Study Of Novel Slmb And Rn Mutants In Drosophila

Objective:From the mutant strains with wing development defects screened in the previous work,two phenotypes were selected for the next stage study to explore the causes of wing development defects and identify the related genes.Methods:mf917 performed direct mosaic clone analysis and mosaic clone analysis to observe the phenotypes of adult wings and eyes and dissect wing discs.The target genes and reporter genes of Notch,Wnt,Hippo and Hh signaling pathways were detected by immunofluorescence staining to determine the correlation between mf917 and these signaling pathways.Reverse PCR was used to identify the insertion site of Minos transposon in mf917 and the affected genes.mf917 lethality and phenotype recovery were detected by precise excision of the transposon.Complementation experiments were performed with mf917 and another reported mutation to further verify the correlation between them.For another selected phenotype,the homozygous mutant adult with wing-related developmental defect was directly observed,and the insertion site inserted by the transposon were also identified by reverse PCR.Complementation test were performed by crossing three different mutations in pairs,and complementation experiments were also use by using the corresponding deficiency line to verify the correlation between the identified genes and this phenotype.Results:（1）mosaic clone analysis of the mutant strain mf917 showed that the adult phenotypes of the offspring were notched,ectopic bristle,vein disorder and eye hyperplasia.（2）By direct mosaic cloning and mosaic cloning analysis,the wing disc of the third instar larvae of mf917 was dissected,and the homozygous mutant clones were small and well-shaped,and the disc wild-type region was significantly enlarged.（3）There was no significant change in the expression of Notch signaling target genes cut and wingless in mf917 homozygous mutant clones.（4）Wnt signaling reporter genes Dll-lac Z and fz3-lac Z were upregulated in mf917 homozygous mutant clones.（5）The expression of Hippo signaling reporter genes Cyc E-lac Z and ex⁶⁹⁷-lac Z was decreased and the expression of bantam sensor-GFP was increased in the mf917homozygous mutant clone.（6）The expression of ptc-lac Z and Ci protein was increased in the mf917homozygous mutant clone in the anterior compartment of the wing disc,and the hyperplasia was more obvious in the anterior wild-type region than in the posterior region of the wing disc.（7）Reverse PCR sequencing results showed that mf917 was a new effective slmb mutant.（8）Complementation experiments using a slmb^{MI03650-TG4.0} of slmb showed that mutation in slmb were indeed responsible for the phenotype and lethality of mf917.（9）The mf1354,mf2843 and mf3565 homozygous flies all showed shortened proximal wings and abnormal veins.（10）mf1354,mf2843 and mf3565 were all inserted into different sites of rn gene,which were identified as three new effective mutants of rn.（11）The defective phenotypes of mf1354,mf2843 and mf3565 were indeed caused by the rn gene mutation by crossing the mutant lines with the deficiency line containing the rn gene deletion and the complementation experiments of the three mutant lines.Conclusions:One novel effective mutant of slmb and three novel effective mutants of rn were identified.slmb functions as a negative regulator of Wnt,Hippo and Hh signaling pathways.This study provides more efficient mutant resources for further study of gene functions of slmb and rn.