The Molecular Mechanism Of Dilated Pupils By Mutation Of Itprl Gene In Mouse

Ethylnitrosourea(ENU)is recognized as one of the most effective mutagenic agents in mice.By using ENU,a large number of mouse mutants with a variety of phenotypes were recovered for the study of gene function and the generation of human disease models.Dp2(Dilated pupil mutation 2)was identified as a new ENU-induced mutant with a dilated pupil phenotype.Here,we report that the abnormal phenotype is due to a mutation in the Itprl gene,which causes unfolded protein response in the iris constrictor muscles.1 A Dilated pupils phenotype mouse(Dp2)obtained by ENU mutagenesisIn a dominant ENU mutagenesis screen for eye abnormal,we identified a mouse mutation Dp2 with bilaterally dilated pupils phenotype at 2 weeks.After mating the Dp2 with wild-type B6 mice,359 of 770 percentage of the progeny were recorded to have the dilated pupils phenotype.Interestingly,almost all pupils of Dp2 recovered normal size at 4 weeks.Hematoxylin and eosin staining showed that Dp2 have intact and similar iris to littermate control mice.The results indicating that the defect in Dp2 is in the pathway of the pupillary light response or in the muscles of the iris,hereby excluding iris hypoplasia(coloboma iridis).To further analyze the defect,a 1%pilocarpine solution(anonselective muscarinic cholinergic receptor agonist)was applied as drops in the eyes of Dp2 mice.The mutant pupils fail to constrict indicating that the defect in Dp2 is in the iris constrictor muscle.The above results showed that the large pupillary phenotype of Dp2 mice is mydriasis which caused by contraction defect of pupillary sphincter.2 Genetic mapping and mutation screening of the gene mutation that causes dilated pupils phenotype in Dp2 mouseFor initial mapping,we tested genomic DNA from 50 N2 samples with microsatellite markers across the whole genome.The mutation was mapped to chromosome 6 and had 1 exchange with marker D6Mit230,which was located at 45.74cM.In order to further refine the map position,genomic DNA from 400 N2 mutants was analyzed using microsatellite markers on chromosome 6.The mutation was mapped to a 4.78Mb region on chromosome 6,between microsatellite D6Mit149 and D6Mit148.The region contained 10 protein coding genes(Arl8b,Bhlhe40,Crbn,Edeml,Il5ra,Itprl,Lrrn1,Setmar,Sumf1 and Trnt1),2 unclassified non-coding RNA gene(0610040F04Rik and Gm17055).Sequence analysis of this genes using DNA or mRNA of tempret revealed no apparent nucleotide changes in Dp2 mice except for Itprl.In the Itprl gene,we identified a G5927A transition mutation in exon 46,which is predicted to result in a C1976Y amino acid change in the open reading frame.Sequence alignment across multiple species revealed that Cys-1976 is a highly evolutionarily conserved amino acid in IP3R1 suggesting conservation of function.3 Mechanism analysis of dilated pupils phenotype caused by Itprl mutation in Dp2 miceImmunohistochemistry results using Anti-IP3R1 antibody showed no obvious difference between wild-type and Dp2 mice.Myosin light chain plays an important role in regulation of both smooth muscle and nonmuscle cell contractile activity via its phosphorylation.To test whether Itprl mutation lead to alterations of phosphorylation level of myosin light chain in the iris constrictor muscles of Dp2 mice,we performed immunohistochemistry on eye sections at 2 and 4 weeks mice.The results showed that,compared with wild-type mice,the phosphorylation level of myosin light chain was significantly decreased in 2 weeks Dp2 mice.Immunohistochemistry results using anti-GRP78 BiP antibody showed that,compared with wild-type mice,BIP level was increased in 2 weeks Dp2 mice.There was no significant difference in recovered Dp2 mice and wild-type littermates(4W).Conclusion:In this study,a mouse model with dilated pupils was obtained by ENU mutagenesis.Gene localization and identification revealed that the phenotype was caused by the Itprl mutation.Further studies revealed that the Itprl mutation caused unfolded protein reaction in the endoplasmic reticulum,which caused temporary loss of part of IP3R1 function and lead a short-term dilated pupils phenotype in Dp2 mice.