Interacting loci cause severe iris atrophy and glaucoma in DBA/2J mice.

Glaucomas are a major cause of blindness. Visual loss typically involves retinal ganglion cell death and optic nerve atrophy subsequent to a pathologic elevation of intraocular pressure (IOP). Some human glaucomas are associated with anterior segment abnormalities such as pigment dispersion syndrome (PDS) and iris atrophy with associated synechiae. The primary causes of these abnormalities are unknown, and their aetiology is poorly understood. We recently characterized a mouse strain (DBA/2J) that develops glaucoma subsequent to anterior segment changes including pigment dispersion and iris atrophy. Using crosses between mouse strains DBA/2J (D2) and C57BL/6J (B6), we now show there are two chromosomal regions that contribute to the anterior segment changes and glaucoma. Progeny homozygous for the D2 allele of one locus on chromosome 6 (called ipd) develop an iris pigment dispersion phenotype similar to human PDS. ipd resides on a region of mouse chromosome 6 with conserved synteny to a region of human chromosome 7q that is associated with human PDS. Progeny homozygous for the D2 allele of a different locus on chromosome 4 (called isa) develop an iris stromal atrophy phenotype (ISA). The Tyrpl gene is a candidate for isa and likely causes ISA via a mechanism involving pigment production. Progeny homozygous for the D2 alleles of both ipd and isa develop an earlier onset and more severe disease involving pigment dispersion and iris stromal atrophy.

Ocular retardation mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation.

Ocular retardation (or) is a murine eye mutation causing microphthalmia, a thin hypocellular retina and optic nerve aplasia. Here we show that mice carrying the OrJ allele have a premature stop codon in the homeobox of the Chx10 gene, a gene expressed at high levels in uncommitted retinal progenitor cells and mature bipolar cells. No CHX10 protein was detectable in the retinal neuroepithelium of orJ homozygotes. The loss of CHX10 leads both to reduced proliferation of retinal progenitors and to a specific absence of differentiated bipolar cells. Other major retinal cell types were present and correctly positioned in the mutant retina, although rod outer segments were short and retinal lamination was incomplete. These results indicate that Chx10 is an essential component in the network of genes required for the development of the mammalian eye, with profound effects on retinal progenitor proliferation and bipolar cell specification or differentiation. off

Nineteen paracentric chromosomal inversions in mice.

Using mutagens on sperm and spermatids we have produced nineteen chromosomal inversions in mice. The levels of radiation and chemical mutagen we used induced inversions in about one per cent of the animals screened. Nine inversions have been transmitted through successive generations, and the particular frequency of first meiotic anaphase bridges manifested by each inversion remained constant. The cytological properties of first meiotic anaphases varied considerably among the inversions. Chromosomal locations of five inversions are known. Four of the five are fully viable in homozygous condition.

The Hox-2 homeo box gene complex on mouse chromosome 11 is closely linked to Re.

Restriction fragment length polymorphisms have been identified between inbred strains of mice for the homeo box gene complex Hox-2. These genetic markers were used to follow the segregation of different Hox-2 alleles among recombinant inbred strains of mice and among the progeny of a three point genetic cross. The results place the Hoax-2 locus approximately 1 cM from the rex (Re) locus on mouse chromosome 11.

Autosomal dominant mouse cataract (Lop-10). Consistent differences of expression in heterozygotes.

The clinical and histologic features are reported of an autosomal dominant mouse cataract that was first observed as a new mutation in a cross between BALB/cJ and AKR/J. In the homozygous state, the eyes were microphthalmic, and a dense white cataract was present when the eyes opened at day 12. Histologic changes were apparent from birth and as early as 18 days' gestation. Liquefaction started by day 4, and herniation of lens contents posteriorly was seen at day 11. Heterozygous mice had variable expression depending both on their genetic background and age. When the single gene was expressed fully, the cataract appeared as a fetal nuclear white opacity; partial expression gave a nuclear haze to snowflake nuclear opacities. Lop-10 appeared to be an excellent model for studying variable expression of a dominant gene.

Retinal degeneration in motor neuron degeneration: a mouse model of ceroid lipofuscinosis.

PURPOSE: To evaluate the retinal degeneration of the motor neuron degeneration (mnd) mouse, and to confirm its inheritance pattern and gene location. METHODS: In screening the mnd/mnd mouse for ocular disease, a retinal degeneration was found that was evaluated by serial electroretinography, histology, electron microscopy, indirect ophthalmoscopy, and genetic and linkage analysis. RESULTS: In homozygous mnd mice, photoreceptor and outer nuclear layers show cell loss by 5 weeks after birth. By 2 months, the peripheral retina is preferentially thinner than central retina, and by 6 months the entire retina is reduced in thickness. The electroretinogram was extinguished by 6 months. Transmission electron microscopy at 3 and 6 months showed distinct cytoplasmic inclusions characteristic of the curvilinear profiles seen in human ceroid lipofuscinosis. Genetic analyses show that the retinal degeneration in mnd mice is inherited as a single autosomal gene with recessive expression, and a three-point cross placed the retinal degeneration at the mnd locus on the proximal end of mouse chromosome 8. Crosses with other known strains with retinal degeneration were normal. CONCLUSIONS. The mnd mouse model is similar to the juvenile onset Spielmeyer-Vogt form of ceroid lipofuscinosis (Batten disease), and provides a good model for the retinal degeneration found in these patients.

Corn1: a mouse model for corneal surface disease and neovascularization.

PURPOSE: To describe a new mouse model of corneal surface disease and neovascularization. METHODS: Anatomic changes were demonstrated in corn1 and control A.By/SnJ mice from day 10 of gestation of 8 months of age by routine techniques of light microscopic and scanning electron microscopy. Corneal epithelial cell kinetics were evaluated by labeling cells in the "S" phase of the cell cycle by intraperitoneal injection of tritiated thymidine. Labeled cells were counted under 250X magnification, and the length of the corneal epithelial chord was measured by morphometric techniques. Results were expressed as labeled cells per linear millimeter of corneal epithelium. The corn1 locus was mapped using selected back-crosses. RESULTS: Corn1 is characterized by early, irregular thickening of the corneal epithelium, development of stromal neovascularization by 20 days of age, and cataract by 48 days of age. Corneal epithelial cell kinetics demonstrated prominent labelling of corn1 mice at 30 days of age compared to the control mice. Corn1 behaves as an autosomal recessive gene and is located on mouse chromosome 2, approximately 5.2 cM from the agouti locus. Heterozygotes have no corneal disease. CONCLUSIONS: Corn1 mice, with genetically determined corneal epithelial hyperplasia and stromal neovascularization, may be particularly useful in studies of neovascularization and corneal surface proliferative disease.

Essential iris atrophy, pigment dispersion, and glaucoma in DBA/2J mice.

PURPOSE: To characterize ocular abnormalities associated with iris atrophy in DBA/2J mice and to determine whether mice of this strain develop elevated intraocular pressure (IOP) and glaucoma. METHODS: Different approaches, including slit-lamp biomicroscopy, ophthalmoscopic examination, ultrasound backscatter microscopy, and histology were used to examine the eyes of DBA/2J mice ranging from 2 to 30 months old. IOP was measured in DBA/2J mice of different ages. RESULTS: DBA/2J mice were found to develop pigment dispersion, iris transillumination, iris atrophy, anterior synechias, and elevated IOP. IOP was elevated in most mice by the age of 9 months. These changes were followed by the death of retinal ganglion cells, optic nerve atrophy, and optic nerve cupping. The prevalence and severity of these lesions increased with age. Optic nerve atrophy and optic nerve cupping was present in the majority of mice by the age of 22 months. CONCLUSIONS: DBA/2J mice develop a progressive form of secondary angle-closure glaucoma that appears to be initiated by iris atrophy and the associated formation of synechias. This mouse strain represents a useful model to evaluate mechanisms of pressure-related ganglion cell death and optic nerve atrophy, and to evaluate strategies for neuroprotection.

Retinal degeneration 6 (rd6): a new mouse model for human retinitis punctata albescens.

PURPOSE: To characterize the genetics and phenotype of a new mouse mutant with retinal degeneration, rd6, that is associated with extensive, scattered, small white retinal dots seen ophthalmoscopically. METHODS: The phenotype was characterized using ophthalmoscopy, fundus photography, electroretinography, light microscopy, immunocytochemistry, and electron microscopy. Genetic characterization and linkage analysis studies were performed using standard methods. RESULTS: The inheritance pattern of rd6 is autosomal recessive. Linkage analysis mapped rd6 to mouse Chromosome 9 approximately 24 cM from the centromere, suggesting that the human homolog may be on chromosome 11q23. Ophthalmoscopic examination of mice homozygous for rd6 revealed discrete subretinal spots oriented in a regular pattern across the retina. The retinal spots appeared by 8 to 10 weeks of age and persisted through advanced stages of retinal degeneration. Histologic examination revealed large cells in the subretinal space, typically juxtaposed to the retinal pigment epithelium. The white dots seen on fundus examination corresponded both in distribution and size to these large cells. By 3 months of age, the cells were filled with membranous profiles, lipofuscin-like material, and pigment. These cells reacted strongly with an antibody directed against a mouse macrophage-associated antigen. Photoreceptor cells progressively degenerated with age, and an abnormal electroretinogram was initially detected between 1 and 2 months of age. CONCLUSIONS: The fundi of mice homozygous for rd6 exhibit phenotypic similarities to the human flecked retinal disorder retinitis punctata albescens. Thus, rd6/rd6 mice may be a model for understanding the etiology of this or similar disorders. The relationship between the aberrant subretinal cells and the concomitant photoreceptor degeneration remains to be established.

Selection for maximum longevity in mice.

In both mice and men, during the adult life span, aging causes an exponential increase in vulnerability to almost all pathologies. Thus, aging is a serious public health problem. Altering the basic mechanisms that control normal aging would be a powerful approach to reduce damage from aging processes, so research identifying these mechanisms is of vital importance. Because life spans are determined by the first biological system to malfunction, it is likely that basic mechanisms are involved in life span extension of animals already having maximum normal life spans for the species. When life spans of a species are extended, all biological systems must function for unusually long times. If there are a limited number of genes for basic mechanisms that control aging rates in multiple biological systems, then life spans can be extended relatively easily. If not, extending maximum life spans would require changes in impractically large numbers of genes, all genes involved in functional life spans of every biological system. In fact, life spans appear to increase rapidly during evolution, suggesting that changes in only a few genes are required. These genes are likely to control underlying mechanisms timing aging in multiple biological systems. The purpose of selection for increased life span is to identify these genes. An important potential problem is that all species have many defective genetic alleles that can cause early disease and death. Selection studies must be designed to distinguish between altering basic mechanisms of aging, and simply avoiding early pathologies due to defective alleles. Animal models that are short lived for their species should be avoided, because their deaths almost always result from genetic defects unrelated to mechanisms of normal aging. During selection, alleles not causing early pathologies may appear to increase life spans by replacing defective alleles in genetic regions linked to early pathologies; however, these affect early disease, not basic mechanisms of aging. A more subtle potential problem is that caloric restriction increases life spans in mice. Selection for long lived mice should focus on more basic mechanisms than breeding mice that voluntarily consume fewer calories. The fact that aging rates in different biological systems are not necessarily coordinated in different individuals suggests that normal aging is timed by more than one mechanism. Thus, the objective in selection for maximum longevity is to capture the entire set of alleles that increase longevity in a species. Wild populations are not practical to use, despite some theoretical advantages, as genes retarding aging would be confounded with those reducing the stress of captivity. Currently we use four-way crosses of inbred strains that represent maximal genetic diversity. Genetic regions important in increasing longevity will be identified using microsatellite markers distinguishing each of the four starting strains over the entire genome. Other genetic techniques proven useful for studying characteristics that are quantitatively controlled by multiple genes may also be useful in studying mechanisms timing aging; these techniques include diallele crosses, recombinant inbred lines, bilineal congenic lines and correlated genetic markers.

Identification of a missense mutation in the alphaA-crystallin gene of the lop18 mouse.

PURPOSE: The mouse lop18 (lens opacity 18) mutation causes a white cataract obvious at weaning age. It soon progresses to a large white nuclear cataract with mild cortical changes. The mutation maps to mouse Chromosome 17 in close linkage to the alphaA-crystallin (Crya) gene, which encodes one of the major vertebrate eye lens proteins. Here we report the identification of a missense mutation in the alphaA-crystallin gene of lop18/lop18 mutant mice. METHODS: PCR primers were designed based on the alphaA-crystallin gene sequence from GenBank and PCR products were sequenced. RESULTS: We have analysed the sequence of the alphaA-crystallin gene from the lop18/lop18 mouse and identified a missense mutation. This mutation is tightly associated with the cataract phenotype, as no recombination was detected in 112 meioses. CONCLUSIONS: Our results suggest that a missense mutation in the alphaA-crystallin gene is responsible for the lop18/lop18 phenotype and Cryalop18 should be used as a gene symbol for the lop18 mutation.

Mouse chromosome fragility.

When cultures of fibroblast-like cells from inbred mouse strains RBC/Dn and AEJ/GnRk were exposed to 5-fluorodeoxyuridine (FUdR), non-random strain-specific distributions of chromosome gaps, breaks and exchanges were observed. Throughout the genomes there appeared to be specific sites at which lesions occurred preferentially. Two strain-specific fragile sites were identified in strain RBC/Dn at G-band 15A2, and at G-band 19B in strain AEJ/GnRk. Constitutive fragile sites at G-bands 12A2 and 18A2 were identified in both strains. A strain-specific marker at G-band 9B was found in strain AEJ/GnRk. The fragile sites reported here provide an animal model for the study of chromosome fragility as well as polymorphic markers for linkage studies.

Mapping X-linked ophthalmic diseases. III. Provisional assignment of the locus for blue cone monochromacy to Xq28.

Blue cone monochromacy (BCM) is an infrequent X-linked retinal disorder typified by poor central visual acuity and color discrimination, early onset of nystagmus, variable degrees of myopia and astigmatism, and a nearly normal retinal appearance. The physiologic functions of rods and blue cones are preserved. The regional location of the genetic mutation causing BCM has been unknown. We have applied the modern molecular techniques of analysis of restriction fragment length polymorphisms to three multigenerational kindreds in which BCM is segregating. Significant linkage is established to two DNA markers, DXS15 and DXS52, each of which maps to the vicinity of Xq28. Regional localization of the locus for BCM has the potential to improve carrier detection and to provide antenatal diagnosis in families at risk for the disease.

Electroretinograms in carriers of blue cone monochromatism.

We recorded full-field electroretinograms from seven female obligate carriers of X-linked blue cone monochromatism and eight daughters of obligate carriers. We observed that all obligate carriers had one or more of the following abnormalities: delayed cone b-wave implicit times to 30-Hz white flicker, loss of the a1 oscillation in responses to single flashes of white light under dark-adapted conditions, subnormal b-wave amplitudes to single flashes of white light under dark-adapted conditions, and subnormal cone responses to 30-Hz white flicker. All had normal rod responses to blue light. Three of eight daughters of obligate carriers had abnormal electroretinograms comparable to those recorded from obligate carriers. These obligate carriers have a partial but comparable deficiency of red and green cone function.

Prevalence of retinitis pigmentosa in Maine.

Between 1976 and 1980, medical and social service sources were used to ascertain cases of retinitis pigmentosa in Maine (1980 population, I, 124,660). As of July 1, 1980, 241 clinically prevalent cases of retinitis pigmentosa were ascertained. Extensive pedigrees were collected for 185 of the subjects and medical records were obtained. One hundred fourteen cases were further evaluated by clinical examination including electroretinography. Adjusting for incorrect diagnosis (eight of 114, 7%) and underascertainment (23 of 185, 12.5%), we estimated that prevalence of retinitis pigmentosa in Maine is 236 cases, 21 per 100,000 population or 1:4,756. Excluding Usher and Bardet-Biedl syndromes, the prevalence is 1:5,193. Estimated birth incidence of persons who will become affected with non-syndrome retinitis pigmentosa is 1:3,544. Incidence of newly diagnosed cases per year is about six per 1,000,000 population. Among kindreds, 16 of 85 (19%) were autosomal dominant, 55 of 85 (65%) autosomal recessive or isolated cases, seven of 85 (8%) X-linked recessive, and seven of 85 (8%) not classified by mode of transmission.

Maximum life spans in mice are extended by wild strain alleles.

The genes that control basic aging mechanisms in mammals are unknown. By using two four-way crosses, each including a strain derived from wild, undomesticated stocks, we identified two quantitative trait loci that extend murine life spans by approximately 10%. In one cross, the longest-lived 18% of carriers of the D8Mit171 marker allele from the MOLD/Rk strain, Mus m. molossinus, outlived the longest lived 18% of noncarriers by 129 days (P = 5.4 x 10(-5)); in a second cross, carriers of the D10Mit267 allele from the CAST/Ei strain, Mus m. castaneus, outlived noncarriers by 125 days ( P = 1.6 x 10(-6)). In both crosses, P < 1.0 x 10(-4 )is considered significant. Because these life span increases required that all essential biological systems function longer than normal, these alleles most likely retarded basic aging mechanisms in multiple biological systems simultaneously.

Ontogeny of susceptibility to the convulsant, Ro 5-4864, and its relationship to audiogenic seizure susceptibility in inbred mice.

The postnatal development of susceptibility to the convulsant effects of Ro5-4864 (4'-chlorodiazepam) was characterized in two inbred mouse strains (DBA/2J and BALB/c ByJ) which as adults differ markedly in their response to this convulsant. Onset of susceptibility to a dose of Ro5-4864 which caused a high frequency of clonic seizures in adults was observed at 10 days of age in DBA/2 mice, but not until 35 days in BALB/c By mice. At 14 days of age an abrupt increase in susceptibility to Ro5-4864-induced tonic seizures was found in DBA/2 but not BALB/c By mice. Both the peak of tonic seizure susceptibility (21 days) and the time course of its subsequent age-dependent decline closely paralleled the change in audiogenic seizure susceptibility in the DBA/2 strain. PK11195 (40 mg/kg) blocked Ro5-4864 (25 mg/kg)-induced, age-dependent tonic seizures but had no effect on clonic seizure induction in the same mice. These observations establish that both the susceptibility to Ro5-4864 in adult mice and the postnatal time course for development of susceptibility to this convulsant are inherently different in these two strains of mice. The lack of coincidence between the developmental onset of susceptibility to Ro5-4864-induced seizures and the onset of supersensitivity to Ro5-4864-induced tonic seizures during the period of peak audiogenic seizure susceptibility in DBA/2 mice implies that more than one neurochemical mechanism is involved in the ability of Ro5-4864 to induce seizures in this strain. However, the blockade of Ro5-4864-induced tonic seizures by PK11195 suggests that peripheral type benzodiazepine receptors may mediate this effect.

Complex genetic determinants of susceptibility to methylxanthine-induced locomotor activity changes.

The intent of this study was to investigate the role of inheritance in the determination of susceptibility to methylxanthine-induced behavioral changes. Two strains of inbred mice, SWR and CBA, which differ significantly in their response to caffeine- and theophylline-induced stimulation of locomotor activity, were used in classical genetic crosses to produce reciprocal F1 hybrids, reciprocal backcross progeny F2 progeny. Theophylline dose response curves in the reciprocal F1 hybrid strains were identical to each other and to their methylxanthine-responsive (CBA) parent. These results indicated that theophylline responsiveness behaved as a simple autosomal dominant trait. Behavioral responses of these F1 hybrid strains to caffeine showed the same maximal enhancement of locomotor activity as their CBA progenitor at a dose 10 mg/kg IP, but locomotor activity stimulation also occurred at 32 mg/kg IP, a dose which inhibited their CBA parent. These data suggest that the genes specifying caffeine responsiveness differ from those encoding theophylline responsiveness. For both caffeine and theophylline, behavioral phenotypes and their expected frequencies of occurrence among backcross and F2 progeny differed significantly from the segregation ratios expected for a trait determined by a single gene. These non-Mendelian segregation ratios suggest that locomotor activity stimulation by both of these methylxanthines is polygenically determined. It was anticipated that the same genetically encoded neurochemical mechanism would underlie the difference in behavioral response to the two methylxanthines. However, no significant correlation between caffeine-induced and theophylline-induced stimulation of locomotor activity was observed among progeny derived from backcrosses of F1 self-crosses.(ABSTRACT TRUNCATED AT 250 WORDS)

Different genes specify hyporesponsiveness to seizures induced by caffeine and the benzodiazepine inverse agonist, DMCM.

Two strains of inbred mice differed significantly in their susceptibility to tonic seizures induced by caffeine and the benzodiazepine inverse agonist, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The hyporesponsive strain, SWR, was not less susceptible to the convulsant action of other chemical convulsants, an observation which indicated that the response differences between the strains were pharmacologically specific. These observations and drug interaction studies suggested that caffeine-induced seizures might be mediated through an "inverse" agonist-like action of caffeine on benzodiazepine receptors associated with GABA receptor-benzodiazepine receptor-chloride ionophore complex. To determine whether the coincident alteration in susceptibility to DMCM and caffeine resulted from a single mutational change or was the result of two different genetic changes occurring coincidentally between these two strains of mice, progeny from conventional Mendelian crosses (F1, F2 and reciprocal backcrosses) were analyzed for the co-segregation of susceptibility to DMCM and caffeine. The inheritance of DMCM sensitivity was consistent with a single autosomal gene determinant in which the allele specifying increased responsiveness was dominant to the allele determining hyporesponsiveness. The frequent occurrence of recombinant phenotypes (e.g., caffeine hyporesponsive but DMCM sensitive mice) among F2 and backcross progeny established that different genetic determinants encode DMCM susceptibility and caffeine susceptibility in these two strains of mice. Thus, while these data establish a simply inherited difference in benzodiazepine responsiveness between the two mouse strains, they also indicate that this pair of strains is inappropriate for a genetic analysis aimed at probing the relationship between caffeine-induced seizures and the benzodiazepine receptor.