Fine localization of a new cataract locus, Kec, on mouse chromosome 14 and exclusion of candidate genes as the gene that causes cataract in the Kec mouse

A mouse with cataract, Kec, was generated from N-ethyl-N-nitrosourea (ENU) mutagenesis. Cataract in the Kec mouse was observable at about 5 weeks after birth and this gradually progressed to become completely opaque by 12 weeks. Dissection microscopy revealed that vacuoles with a radial or irregular shape were located primarily in the cortex of the posterior and equatorial regions of the lens. At the late stage, the lens structure was distorted, but not ruptured. This cataract phenotype was inherited in an autosomal recessive manner. We performed a genetic linkage analysis using 133 mutant and 67 normal mice produced by mating Kec mutant (BALB/c) and F1 (C57BL/6 x Kec) mice. The Kec locus was mapped to the 3 cM region encompassed by D14Mit34 and D14Mit69. In addition we excluded coding sequences of 9 genes including Rcbtb2, P2ry5, Itm2b, Med4, Nudt15, Esd, Lcp1, Slc25a30, and 2810032E02Rik as the candidate gene that causes cataract in the Kec mouse.     

Regulation of lens fiber cell differentiation by transcription factor c-Maf.

To elucidate the regulatory mechanisms underlying lens development, we searched for members of the large Maf family, which are expressed in the mouse lens, and found three, c-Maf, MafB, and Nrl. Of these, the earliest factor expressed in the lens was c-Maf. The expression of c-Maf was most prominent in lens fiber cells and persisted throughout lens development. To examine the functional contribution of c-Maf to lens development, we isolated genomic clones encompassing the murine c-maf gene and carried out its targeted disruption. Insertion of the beta-galactosidase (lacZ) gene into the c-maf locus allowed visualization of c-Maf accumulation in heterozygous mutant mice by staining for LacZ activity. Homozygous mutant embryos and newborns lacked normal lenses. Histological examination of these mice revealed defective differentiation of lens fiber cells. The expression of crystallin genes was severely impaired in the c-maf-null mutant mouse lens. These results demonstrate that c-Maf is an indispensable regulator of lens differentiation during murine development.     

Targeted ablation of alpha-crystallin-synthesizing cells produces lens-deficient eyes in transgenic mice

Genetic ablation techniques were used to study the role of the lens in mammalian eye development. Ablation was accomplished by microinjecting murine eggs with chimeric DNA constructs in which the alpha A-crystallin gene regulatory sequence (-366 to +46) was fused to the highly cytotoxic diphtheria toxin gene coding sequence. For genetic ablation to be successful the promoter regulating expression should be specific and completely silent in cells necessary for normal mouse development. In this report, we describe the generation and analysis of transgenic mice with this readily discernible phenotype: aphakia or eyes without lens. Of the 109 live-born pups, eight carried the transgene and could be grouped according to the apparent severity of eye malformations. Lines 4, 5 and 6 founder (F0) mice had the most severe phenotype. Histological analysis revealed: marked reduction in eye size, total absence of lens, increased retinal cell density and extensive whorling of the retinal fibre layers. The line 1 F0 mouse displayed a distinct lens opacity and lines 2, 3 and 8 F0 mice were mosaics with a relatively mild, but most unusual phenotype. Their eyes contained a small, highly vacuolated lens. The progeny of these mosaics that inherited the transgene, however, again exhibited the severe phenotype. The aberrant structures of the eyes in which complete genetic ablation of the lens has been achieved suggest that the lens plays a pivotal role in the development of multiple components of the murine eye.     

Dense cataract and microphthalmia--new spontaneous mutation in BALB/c mice

We describe a new spontaneous mutation in BALB/c mice that causes abnormal phenotype, such as congenital cataract and microphthalmia. This abnormality was found to be inheritable because offspring with the same abnormality were produced by backcrossing the abnormal male to its normal female parent. Results of various crosses made to determine the mode of inheritance indicated that this abnormality is attributable to mutation of an autosomal recessive gene. Slit lamp examination of the mutant eyes revealed total lenticular opacity, disturbed typical iris pattern, and abnormal pupillary muscle development. Histologic changes in mutant eyes between gestation day 13 and postnatal day 1 indicated various eye and lens abnormalities, including microphthalmia; underdeveloped iris, optic stalk, cornea, and retina; degenerated lens fibers with lost fibrillar structure; and vacuoles of various sizes at the posterior border of the lens. Mild opacity of the lens was found to progress with age and became denser, resembling mature cataract, and occupying the lens completely at the age of six to eight weeks. We, therefore, temporarily designated this abnormality as dense cataract and microphthalmia, with the gene symbol dcm.     

A 1-bp deletion in the γC-crystallin leads to dominant cataracts in mice

To date around 140 genetic alleles have been identified as being responsible for mouse cataract pathology, including Crya, Cryb, Cryg, Maf, Pax6, Pitx3, Sox, Connexins, MIP, and Lim-2. We obtained a dominant cataract mouse model from a spontaneous mutation in the F1 hybrids of outbred strain ICR mice crossed to the inbred strain BALB/cJ mice. Heterozygous and homozygous mutants expressed a nuclear cataract in both eyes. In 8-day-old mice, histological analysis showed that polygon epithelial cells were in the equatorial region and cortex underneath, and vacuole and sponge-like degeneration were in the cortical area underneath the posterior lens capsule. The nucleus of the lens was a deeply stained pink, with the shorter fibers losing their normal arrangement. For the entire eye, there was a blank zone in the equatorial region in 8-day-old mice; however, there was a certain degree of atrophy in cornea tension and retina in the lens in 3-month-old mice. The lens had been serious damaged in the homozygous mutants. For mutation mapping, heterozygous carriers were mated to wild-type C3H/HeJ mice, and offspring (F1 generation) with cataracts were backcrossed to the wild-type C3H/HeJ mice again. N2 mice with cataracts were used for genotyping. Using genome-wide linkage analysis, the mutation was mapped to chromosome 1 and the Cryg gene cluster between two markers was confirmed as the candidate gene. After direct sequencing the cDNA of the Cryg gene cluster, a 1-bp deletion was found in exon 3 of the Crygc gene, leading to a stop codon at the 76th amino acid of exon 3 which results in production of a truncated protein in mutant mice (Leu160Stop). Bioinformatic analysis of the mutant γC-crystallin reveals that the COOH-terminal of the mutant protein deletes a β-sheet, which affects the function of the lens proteins and leads to the development of cataracts.     

Mechanism of insolubilization by a single-point mutation in alphaA-crystallin linked with hereditary human cataracts

AlphaA-crystallin is a small heat shock protein that functions as a molecular chaperone and a lens structural protein. The R49C single-point mutation in alphaA-crystallin causes hereditary human cataracts. We have previously investigated the in vivo properties of this mutant in a gene knock-in mouse model. Remarkably, homozygous mice carrying the alphaA-R49C mutant exhibit nearly complete lens opacity concurrent with small lenses and small eyes. Here we have investigated the 90 degrees light scattering, viscosity, refractive index, and bis-ANS fluorescence of lens proteins isolated from the alphaA-R49C mouse lenses and found that the concentration of total water-soluble proteins showed a pronounced decrease in alphaA-R49C homozygous lenses. Light scattering measurements on proteins separated by gel permeation chromatography showed a small amount of high-molecular mass aggregated material in the void volume which still remains soluble in alphaA-R49C homozygous lens homogenates. An increased level of binding of beta- and gamma-crystallin to the alpha-crystallin fraction was observed in alphaA-R49C heterozygous and homozygous lenses but not in wild-type lenses. Quantitative analysis with the hydrophobic fluorescence probe bis-ANS showed a pronounced increase in fluorescence yield upon binding to alpha-crystallin from mutant as compared with the wild-type lenses. These results suggest that the decrease in the solubility of the alphaA-R49C mutant protein was due to an increase in its hydrophobicity and supra-aggregation of alphaA-crystallin that leads to cataract formation. Our study further shows that analysis of mutant proteins from the mouse model is an effective way to understand the mechanism of protein insolubilization in hereditary cataracts.     

Establishment of a knock-in mouse model with the SLC26A4 c.919-2A>G mutation and characterization of its pathology

Recessive mutations in the SLC26A4 gene are a common cause of hereditary hearing impairment worldwide. Previous studies have demonstrated that different SLC26A4 mutations may have different pathogenetic mechanisms. In the present study, we established a knock-in mouse model (i.e., Slc26a4(tm1Dontuh/tm1Dontuh) mice) homozygous for the c.919-2A>G mutation, which is a common mutation in East Asians. Mice were then subjected to audiologic assessment, a battery of vestibular evaluations, and inner ear morphological studies. All Slc26a4(tm1Dontuh/tm1Dontuh) mice revealed profound hearing loss, whereas 46% mice demonstrated pronounced head tilting and circling behaviors. There was a significant difference in the vestibular performance between wild-type and Slc26a4(tm1Dontuh/tm1Dontuh) mice, especially those exhibiting circling behavior. Inner ear morphological examination of Slc26a4(tm1Dontuh/tm1Dontuh) mice revealed an enlarged endolymphatic duct, vestibular aqueduct and sac, atrophy of stria vascularis, deformity of otoconia in the vestibular organs, consistent degeneration of cochlear hair cells, and variable degeneration of vestibular hair cells. Audiologic and inner ear morphological features of Slc26a4(tm1Dontuh/tm1Dontuh) mice were reminiscent of those observed in humans. These features were also similar to those previously reported in both knock-out Slc26a4(-/-) mice and Slc26a4(loop/loop) mice with the Slc26a4 p.S408F mutation, albeit the severity of vestibular hair cell degeneration appeared different among the three mouse strains.     

Circling mouse, a spontaneous mutant in the inner ear.

A spontaneous mutant was established in the ICR mouse strain. The affected mice became hyperactive at about 7 days of age, and then showed circling behavior. The body weight decreased significantly 2 weeks after birth, and developmental defects were revealed in the middle ear, cochlea, cochlear nerve, and semicircular canal areas. The mutation was inherited by an autosomal single recessive gene and is referred to as cir.     

Comparison of the incidence of 5-azacytidine-induced exencephaly between MT/HokIdr and Slc:ICR mice.

The incidence of 5-azacytidine-induced exencephaly was compared between MT/HokIdr strain (MT) and Slc:ICR strain (ICR) mice. MT mice have a genetic predisposition for exencephaly, but ICR mice do not. Pregnant mice were given 5-azacytidine (1 mg/kg to 100 micrograms/kg) injected intraperitoneally on Day 7.5 of gestation (vaginal plug day = Day 0.5), and fetuses were observed for external malformations on Day 18.5 of gestation. One hundred micrograms/kg 5-azacytidine induced exencephaly in MT mice but not in ICR mice, and 1 mg/kg 5-azacytidine resulted in resorptions in MT mice but caused exencephaly in ICR mice. These results indicated that MT mice had 10-fold more sensitivity to 5-azacytidine than ICR mice. It seems likely that less than effective doses of teratogens for animals without genetic predispositions are still effective in inducing malformations in animals with a genetic predisposition for malformations. When 4-somite-stage embryos of both MT and ICR mice were cultured in rat serum supplemented with 5-azacytidine, 0.02 micrograms/ml 5-azacytidine induced the failure of closure of cephalic neural tube in MT embryos but not in ICR embryos, and 0.2 micrograms/ml 5-azacytidine induced severe growth retardation in MT embryos but in ICR embryos it only induced embryos with smaller heads and fewer somites than in control. These results indicated that MT mouse embryos in culture also had a 10-fold-increased sensitivity to 5-azacytidine compared with ICR mouse embryos, suggesting maternal effects play no significant role in their increased sensitivity to 5-azacytidine.     

AlphaA-crystallin expression prevents gamma-crystallin insolubility and cataract formation in the zebrafish cloche mutant lens

Cataracts, the loss of lens transparency, are the leading cause of human blindness. The zebrafish embryo, with its transparency and relatively large eyes, is an excellent model for studying ocular disease in vivo. We found that the zebrafish cloche mutant, both the cloche(m39) and cloche(S5) alleles, which have defects in hematopoiesis and blood vessel development, also have lens cataracts. Quantitative examination of the living zebrafish lens by confocal microscopy showed significant increases in lens reflectance. Histological analysis revealed retention of lens fiber cell nuclei owing to impeded terminal differentiation. Proteomics identified gamma-crystallin as a protein that was substantially diminished in cloche mutants. Crystallins are the major structural proteins in mouse, human and zebrafish lens. Defects in crystallins have previously been shown in mice and humans to contribute to cataracts. The loss of gamma-crystallin protein in cloche was not due to lowered mRNA levels but rather to gamma-crystallin protein insolubility. AlphaA-crystallin is a chaperone that protects proteins from misfolding and becoming insoluble. The cloche lens is deficient in both alphaA-crystallin mRNA and protein during development from 2-5 dpf. Overexpression of exogenous alphaA-crystallin rescued the cloche lens phenotype, including solubilization of gamma-crystallin, increased lens transparency and induction of lens fiber cell differentiation. Taken together, these results indicate that alphaA-crystallin expression is required for normal lens development and demonstrate that cataract formation can be prevented in vivo. In addition, these results show that proteomics is a valuable tool for detecting protein alterations in zebrafish.     

Targeted disruption of Slc2a8 (GLUT8) reduces motility and mitochondrial potential of spermatozoa

GLUT8 is a class 3 sugar transport facilitator which is predominantly expressed in testis and also detected in brain, heart, skeletal muscle, adipose tissue, adrenal gland, and liver. Since its physiological function in these tissues is unknown, we generated a Slc2a8 null mouse and characterized its phenotype. Slc2a8 knockout mice appeared healthy and exhibited normal growth, body weight development and glycemic control, indicating that GLUT8 does not play a significant role for maintenance of whole body glucose homeostasis. However, analysis of the offspring distribution of heterozygous mating indicated a lower number of Slc2a8 knockout offspring (30.5:47.3:22.1%, Slc2a8(+/+), Slc2a8(+/-), and Slc2a8(-/-) mice, respectively) resulting in a deviation (p=0.0024) from the expected Mendelian distribution. This difference was associated with lower ATP levels, a reduced mitochondrial membrane potential and a significant reduction of sperm motility of the Slc2a8 knockout in comparison to wild-type spermatozoa. In contrast, number and survival rate of spermatozoa were not altered. These data indicate that GLUT8 plays an important role in the energy metabolism of sperm cells.     

A deletion in a cis element of Foxe3 causes cataracts and microphthalmia in rct mice

The Rinshoken cataract (rct) mutation, which causes congenital cataracts, is a recessive mutation found in SJL/J mice. All mutants present with opacity in the lens by 2?months of age. The rct locus was mapped to a 1.6-Mb region in Chr 4 that contains the Foxe3 gene. This gene is responsible for cataracts in humans and mice, and it plays a crucial role in the development of the lens. Furthermore, mutation of Foxe3 causes various ocular defects. We sequenced the genomic region of Foxe3, including the coding exons and UTRs; however, no mutations were discovered in these regions. Because there were no differences in Foxe3 sequences between the rct/rct and wild-type mice, we inferred that a mutation was located in the regulatory regions of the Foxe3 gene. To test this possibility, we sequenced a 5' noncoding region that is highly conserved among vertebrates and is predicted to be the major enhancer of Foxe3. This analysis revealed a deletion of 22-bp located approximately 3.2-kb upstream of the start codon of Foxe3 in rct mice. Moreover, we demonstrated by RT-PCR and in situ hybridization that the rct mutant has reduced expression of Foxe3 in the lens during development. We therefore suggest that cataracts in rct mice are caused by reduced Foxe3 expression in the lens and that this decreased expression is a result of a deletion in a cis-acting regulatory element.     

Short-limbed dwarfism: slw is a new allele of Npr2 causing chondrodysplasia

Short-limbed dwarfism (SLW) is a new mutant mouse characterized by a dwarf phenotype with markedly short body, limbs, and tail. In the present study, we investigated the skeletal phenotypes of the SLW mouse and determined the chromosomal localization to identify the gene responsible for the phenotypes (slw). Skeletal preparations stained with alcian blue and alizarin red revealed that longitudinal growth of the extremities of the affected (slw/slw) mice was significantly reduced in comparison with that of normal mice, whereas the positions and numbers of skeletal elements were normal. Histological examination of tibial growth plates of the affected mice showed that the numbers of proliferating and hypertrophic chondrocytes were obviously diminished. These phenotypes resembled to those of human chondrodysplasias caused by defective chondrocyte proliferation and differentiation. We mapped the slw locus on an 11.7-cM interval of the proximal region of mouse chromosome 4 by linkage analysis. Furthermore, allelism test using Npr2(cn) locus, a mutant allele of Npr2 gene encoding a natriuretic peptide receptor B, revealed that slw locus is an allele of the Npr2 gene. These results suggest that the dwarf phenotype of the SLW mouse is caused by the disturbed endochondral ossification, and a mutation in the Npr2 gene is expected to be responsible for the phenotypes of the SLW mouse.     

Immunofluorescent studies on aphakia,a mutation of a gene involved in the control of lens differentiation in the mouse embryo

Aphakia, an autosomal recessive single gene mutation in the mouse, seriously affects the development of the ocular lens. Up to advanced stages of lens invagination morphogenesis proceeds normally. In the late lens cup and early lens vesicle stage, however, the epithelium of the lens rudiment becomes disorganized and the lumen of the vesicle fills up with rounded cells, apparently released from the epithelium. The lens stalk persists frequently. Probably as a consequence of the aphakic state other parts of the eye secondarily become abnormal.Immunofluorescent studies were done on embryonic normal and aphakia eyes with antisera against adult mouse crystallins. In the normal embryo the first positive reactions were found in the late lens cup stage ($\text{10}\text{3}\text{4}\text{–11}$ days of gestation). By Day 12 all cells of the lens vesicle were brightly fluorescent. A day later the cells of the posterior wall, now lens fibers, had elongated sufficiently to obliterate the lumen of the vesicle. The entire organ was highly fluorescent, indicating that all of its cells contained large amounts of crystallins. The mutant lens, studied over the same time span, showed no reaction at all. The most likely explanation is, that the multiple structural genes, which normally must be involved in the production of the crystallins, are not expressed up to this time in the mutant.The combination of morphological and biochemical defects suggests that the gene involved in the mutation somehow functions in the control of lens differentiation.     

Roles of the 15-kDa Selenoprotein (Sep15) in Redox Homeostasis and Cataract Development Revealed by the Analysis of Sep 15 Knockout Mice

The 15-kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase. Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In this study, we developed and characterized Sep15 KO mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteine-rich UDP-glucose:glycoprotein glucosyltransferase-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable and fertile, showed normal brain morphology, and did not activate endoplasmic reticulum stress pathways. However, parameters of oxidative stress were elevated in the livers of these mice. We found that Sep15 mRNA was enriched during lens development. Further phenotypic characterization of Sep15 KO mice revealed a prominent nuclear cataract that developed at an early age. These cataracts did not appear to be associated with severe oxidative stress or glucose dysregulation. We suggest that the cataracts resulted from an improper folding status of lens proteins caused by Sep15 deficiency.     

Development of a novel pink-eyed dilution mouse model showing progressive darkening of the eyes and coat hair with aging

Oca2^p-cas (oculocutaneous albinism II; pink-eyed dilution castaneus) is a coat color mutant gene on mouse chromosome 7 that arose spontaneously in wild Mus musculus castaneus mice. Mice homozygous for Oca2^p-cas usually exhibit pink eyes and gray coat hair on the non-agouti genetic background, and this ordinary phenotype remains unchanged throughout life. During breeding of a mixed strain carrying this gene on the C57BL/6J background, we discovered a novel spontaneous mutation that causes darkening of the eyes and coat hair with aging. In this study, we developed a novel mouse model showing this unique phenotype. Gross observations revealed that the pink eyes and gray coat hair of the novel mutant young mice became progressively darker in color by approximately 3 months after birth. Light and transmission-electron microscopic observations revealed a marked increase in melanin pigmentation of coat hair shafts and choroid of the eye in the novel mice compared to that in the ordinary mice. Sequence analysis of Oca2^p-cas revealed a 4.1-kb deletion involving exons 15 and 16 of its wild-type gene. However, there was no sequence difference between the two types of mutant mice. Mating experiments suggested that the novel mutant phenotype was not inherited in a simple fashion, due to incomplete penetrance. The novel spontaneous mutant mouse is the first example of progressive hair darkening animals and is an essential animal model for understanding of the regulation mechanisms of melanin biosynthesis with aging.     

Disrupted fat absorption attenuates obesity induced by a high-fat diet in Clock mutant mice

The Clock gene is a core component of the circadian clock in mammals. We show here that serum levels of triglyceride and free fatty acid were significantly lower in circadian Clock mutant ICR than in wild-type control mice, whereas total cholesterol and glucose levels did not differ. Moreover, an increase in body weight induced by a high-fat diet was attenuated in homozygous Clock mutant mice. We also found that dietary fat absorption was extremely impaired in Clock mutant mice. Circadian expressions of cholecystokinin-A (CCK-A) receptor and lipase mRNAs were damped in the pancreas of Clock mutant mice. We therefore showed that a Clock mutation attenuates obesity induced by a high-fat diet in mice with an ICR background through impaired dietary fat absorption. Our results suggest that circadian clock molecules play an important role in lipid homeostasis in mammals.     

Inactivation of the mitochondrial carrier SLC25A25 (ATP-Mg2+/Pi transporter) reduces physical endurance and metabolic efficiency in mice

An ATP-Mg(2+/)P(i) inner mitochondrial membrane solute transporter (SLC25A25), which is induced during adaptation to cold stress in the skeletal muscle of mice with defective UCP1/brown adipose tissue thermogenesis, has been evaluated for its role in metabolic efficiency. SLC25A25 is thought to control ATP homeostasis by functioning as a Ca(2+)-regulated shuttle of ATP-Mg(2+) and P(i) across the inner mitochondrial membrane. Mice with an inactivated Slc25a25 gene have reduced metabolic efficiency as evidenced by enhanced resistance to diet-induced obesity and impaired exercise performance on a treadmill. Mouse embryo fibroblasts from Slc25a25(-/-) mice have reduced Ca(2+) flux across the endoplasmic reticulum, basal mitochondrial respiration, and ATP content. Although Slc25a25(-/-) mice are metabolically inefficient, the source of the inefficiency is not from a primary function in thermogenesis, because Slc25a25(-/-) mice maintain body temperature upon acute exposure to the cold (4 °C). Rather, the role of SLC25A25 in metabolic efficiency is most likely linked to muscle function as evidenced from the physical endurance test of mutant mice on a treadmill. Consequently, in the absence of SLC25A25 the efficiency of ATP production required for skeletal muscle function is diminished with secondary effects on adiposity. However, in the absence of UCP1-based thermogenesis, induction of Slc25a25 in mice with an intact gene may contribute to an alternative thermogenic pathway for the maintenance of body temperature during cold stress.     

Alpha-actinin-4-mediated FSGS: an inherited kidney disease caused by an aggregated and rapidly degraded cytoskeletal protein

Focal segmental glomerulosclerosis (FSGS) is a common pattern of renal injury, seen as both a primary disorder and as a consequence of underlying insults such as diabetes, HIV infection, and hypertension. Point mutations in the alpha-actinin-4 gene ACTN4 cause an autosomal dominant form of human FSGS. We characterized the biological effect of these mutations by biochemical assays, cell-based studies, and the development of a new mouse model. We found that a fraction of the mutant protein forms large aggregates with a high sedimentation coefficient. Localization of mutant alpha-actinin-4 in transfected and injected cells, as well as in situ glomeruli, showed aggregates of the mutant protein. Video microscopy showed the mutant alpha-actinin-4 to be markedly less dynamic than the wild-type protein. We developed a "knockin" mouse model by replacing Actn4 with a copy of the gene bearing an FSGS-associated point mutation. We used cells from these mice to show increased degradation of mutant alpha-actinin-4, mediated, at least in part, by the ubiquitin-proteasome pathway. We correlate these findings with studies of alpha-actinin-4 expression in human samples. "Knockin" mice with a disease-associated Actn4 mutation develop a phenotype similar to that observed in humans. Comparison of the phenotype in wild-type, heterozygous, and homozygous Actn4 "knockin" and "knockout" mice, together with our in vitro data, suggests that the phenotypes in mice and humans involve both gain-of-function and loss-of-function mechanisms.