The Molecular Mechanism And Genetic Characteristics Of An ENU-Induced Dilated Pupils Mouse Model

N-ethy1-N-nitrosourea (ENU) is a powerful point mutagen that can generate random mutations in the mouse genome. Following an ENU-mutagenesis screen for dominant and recessive mutations, 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.NrglmlYzcm (neuregulin1; mutation1, Yangzhou University Comparative Medicine Center, hereafter Dp1) 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 Nrgl gene, which causes a reduction in muscarinic receptors in the sphincter papillae. Interestingly, the Dp1dilated pupil phenotype is inherited with very low penetrance in heterozygous mice and with complete penetrance in homozygous mice.1A Dilated pupil phenotype obtained by ENU mutagenesisDp1is an ENU-induced mutation conveying either a unilateral or bilaterally dilated pupil phenotype that can range from partial to severe. When illuminated, the eyes of affected Dp1mice have no pupillary response to light, and the sphincter pupillae fail to act.The founder Dp1male mouse, which was the progeny of an ENU-treated B6male mouse and an untreated B6female mouse,had a unilateral dilated pupil phenotype. After mating the mutant with B6mice, a very low percentage of the progeny (3/115) were recorded to have the unilateral dilated pupil phenotype. Interestingly, crosses among the three heterozygous progeny resulted in five out of34abnormal progeny, of which four had bilaterally dilated pupils. All progeny generated by crossing the above four bilaterally dilated pupil individuals with each other had a dilated pupil phenotype.2Mapping of the mutation gene that causes dilated pupil phenotype of Dp1mouseFor initial mapping, we tested genomic DNA from25N2samples with microsatellite markers across the whole genome. We observed no exchange of the markers D8Mit171and D8Mit4with the dilated pupil phenotype and found no significant linkages with other chromosomal loci.In order to further refine the map position, we crossbred F1mice and reduced the critical interval to a1.52-Mb region between the single nucleotide polymorphism (SNP) rs32829041and D8Mit4using118F2, dilated pupil offspring. The region contained7protein coding genes (Dusp26, Rnf122, BC019943, Mak16, Fut10,7420700N18Rik. and Nrgl),1miRNA gene (Mir1186) and1snoRNA gene (Snord13).3Identification of the mutation gene that causes dilated pupil phenotype of Dp1mouseSequence analysis of the exons and flanking intronic sequences using DNA or mRNA of these genes revealed no apparent nucleotide changes in Dp1mice except for Neuregulin-1(Nrg1). In the Nrg1gene, we discovered a G to A transition mutation, which flanked exon E59, encoding for the EGFβ domain, in the5’splice donor site.In order to assess the effect of the G to A substitution on EGFβ-type Nrg1mRNA splicing, we amplified the sequences from RNA harvested from the brains of mice homozygous for the Nrgl mutation and wild-type B6mice using primers specific for CRD-Nrg1and Ig-Nrg1, respectively. For both CRD-Nrgl and Ig-Nrgl RT-PCR products, electrophoresis results revealed three bands from both Dp] and wild-type B6mice. The middle band (band b), corresponding to NRG Is with a β1-type EGF sequence (see below), was predominant in the brain of wild-type B6mice. Quantitative analysis by densitometry shows that, in homozygous mice, the relative yield of band b in CRD-Nrgl and Ig-Nrgl is only42.6%and32.8%, respectively, of that in wild-type B6mice.To determine the sequences amplified by RT-PCR analysis, individual bands generated from both wild-type and Dp1mice were purified and used directly as templates for nucleotide sequencing. Sequence analysis of the middle band of CRD-Nrgl and Ig-Nrgl from wild-type mice revealed wild-type sequences corresponding to CRD-β1and Ig-β1Nrg1. However, sequence analysis of the middle band of CRD-Nrgl and Ig-Nrgl in Dpl/Dpl mice revealed a mixed sequence. Sequence analysis of the lowest band (band c) from both wild-type and Dpl/Dpl mice revealed alternative splicing transcripts that lacked exons E59and E24when compared to the corresponding band b sequence from wildtype mice. To our knowledge, these are new transcripts that have not previously been reported. Sequence analysis of the top band (band a) of CRDNrg1and Ig-Nrgl from wild-type and Dpl/Dpl mice revealed a mixed sequence.We cloned the bands with mixed sequences into a T vector and then sequenced the plasmids. Sequencing of these bands revealed differences in the transcripts between Dpl/Dpl and wild-type mice. The majority of the transcripts found in Dpl/Dpl bypass the mutated splice donor site by splicing over exon E59, activating a cryptic splice site, or transcribing through exon E59into the adjacent sequence. These types of protein isoforms were expected, and the results show that, in the mutant, there is a decrease in, but not an elimination of, EGFβ-type Nrgl isoforms. This decrease is partially compensated for by increased expression of the alpha forms, inactive isoforms (without EGFβ,and EGFa domains), and truncated proteins (without the EGFc, EGFβ,and EGFa domains).4Mechanism analysis of dileatd pupil phenotype caused by Nrgl mutation in miceTo analyze the defect that caused the dilated pupil phenotype and cure the abnormal phenotype, we used drugs to clinically constrict or enlarge the pupils. A1%pilocarpine solution (a nonselective muscarinic cholinergic receptor agonist) was first applied as drops in the eyes of mice with severe and partially dilated pupil phenotypes. but it did not alter pupil size in mice with either phenotype (full or partial dilation of the pupil). In contrast, administration of a1%atropine solution, a muscarinic cholinergic receptor antagonist which can compete for AchR with Ach (we considered the drug effect to be a blocking of the AchR), resulted in the further loosening of the sphincter pupillae, which led to complete mydriasis in partially dilated pupil mice. Taken together, these results suggest that the number of muscarinic receptors might be decreased in the iris constrictor muscles of mutant mice, while the number of muscarinic receptors in partially dilated pupil mice is higher than in mice with a severely dilated pupil phenotype. The contractile extent of the sphincter corresponds to the amount of Ach and the number of AchR. Due to the decrease in AchR in partially dilated pupil mice, an excess of Ach with a limited number of AchR cannot lead to complete muscle contraction. In addition to pilocarpine, we used drops of a1%neostigmine solution (a cholinesterase inhibitor) or both solutions together in dilated pupil mice, but both drugs failed to constrict the mutant pupils (full or partial dilation of the pupil). After intraperitoneal administration of pilocarpine or neostigmine in Dp1mice, severe salivation and mild lachrimation were observed, suggesting that AchRs in the glands of Dp1mice were not (or only slightly) affected.M receptors comprise five distinct subtypes (M1-M5). M3is known to play a dominant role in eliciting smooth muscles contraction, and M3-/-mice showing a partially dilated pupil phenotype have been reported. To further validate the reduction of M receptors in the sphincter papillae of Dp1mice, we performed immunohistochemistry tests using an anti-M3antibody and found a significant reduction of M3receptors in the sphincter pupillae of Dp1/Dp1mice.5Genetic model testing of dilated pupil phenotypeTo determine the inheritance fashion of the dilated pupil phenotype, genetic model testing based on genotyping for a Tai I restriction site polymorphism was carried out by intercrossing unaffected Dpl/+mice, crossing Dp1/Dp1mice with unaffected Dp1/+mice, crossing Dp1/Dp1mice with wild-type mice, and intercrossing Dp1/Dp1mice, respectively. The results show that Dp1is a mutation with dilated pupil phenotype that is inherited with a very low penetrance (5/111) when heterozygous and with complete penetrance when homozygous.Additionally, in Dp1/+×Dp1/+offspring, the ratio of genotype of Dp1/+×Dp1/+offspring differed significantly from the expected1:2:1(homozygous mice: heterozygous mice:wild type) ratio (0.01<P<0.05, chi-square test) indicating viability effect.