Identifications And Functional Studies Of Three Novel Alleles Of Serrate In Drosophila

Objective:To identify genes regulating the Notch signal pathway and further study their functions,we selected several mutant strains from the previous phenotypic screening for our research.Methods:Several mutations,which were identified to be associated with Notch wing phenotypes,were selected to repeat the direct mosaic cloning analysis at the first.Immunofluorescence staining using Cut and Wingless as primary antibodies respectively was performed to determine the correlation of the selected mutant line with Notch signaling pathway.Then,the immunofluorescence staining was also conducted to investigate whether the selected mutant strain affects the expressions of Notch and Delta at protein level.Subsequently,the MARCM system was used to explore the actuating range of the selected mutant strain in Notch pathway by showing the epistatic interaction between the selected strain and the components of Notch signaling.And the genomic DNA of flies’ mutants was extracted.Inverse PCR was performed and PCR products were sequenced.Then the sequnces were BLAST with Drosophila genome to find the insertion site of Minos element and identify the gene affected by Minos insertion.What’s more,the complementation experiments were conducted to determine the consistency between phenotype and the mutant gene based on the BLAST results.Ultimately,the transposon was removed from the genome of the chosen mutation to verify the screening results and exclude the influence of genetic background.Results:(1)The mutant strain mf157 was able to cause severe nicks and shrinkage of fly wings after induction of direct mosaic clone.(2)The expressions of Notch signal target gene cut and wingless were missing in mf157 homozygous mutant clone cell populations by mosaic analysis and immunofluorescence staining.(3)Homozygous mutant clones of mf157 displayed no significant change in expression of Notch and Delta at protein level.(4)mf157 functions in the signaling-sending cells of Notch pathway,its actuating range is at the same level as the ligands or the upstream of ligands.(5)Sequencing revealed that mf157 is a new allele of the serrate gene.In addition,two other lethal mutant lines,mf553 and mf167,were also identified as fresh effective mutations of serrate.(6)The results of complementary assay between mf157,mf553 and mf167 illustrated that the serrate mutations led to the lethality in the respective strains.(7)Transposon hop out experiments confirmed that the phenotype of mutant mf157 is caused by the serrate mutation,but there is an additional lethal mutational background in mf157,and the background does not affect the transduction process of Notch signaling.Conclusions:Here,we obtained three novel alleles of serrate,the ligand gene of Notch signaling pathway.Our works supply additional and effective genetic resources as well as new ideas for further study in the functions of serrate and regulation of Notch signaling.