DBA/2J is an established mouse model for inherited glaucoma that shows iris pigment dispersion, IOP elevation and optic nerve degeneration. D2 mice are homozygous for both the GpnmbR150X
mutations. Both of these mutations are necessary for the severe iris pigment disease that leads to IOP elevation and glaucoma [16
]. Although DBA/2J is an experimentally tractable model of glaucoma, it is an inbred mouse strain and so no strain matched DBA/2J controls without glaucoma have been available. Here we describe the generation of two derivative strains of DBA/2J (D2.Tyrp1B6GpnmbB6
), where either the mutant alleles of both Gpnmb
or only Gpnmb
have been replaced by wild-type versions. D2.Tyrp1B6GpnmbB6
mice contain a maximum of 3.3% of B6-derived sequence on a D2 background. In the case of D2-Gpnmb+
, the only known difference compared to D2 is a single base change in the Gpnmb
gene. Neither D2.Tyrp1B6GpnmbB6
strains develop IOP elevation or glaucoma.
Since determining that the Gpnmb
mutation recently arose on the DBA/2J background [10
], we have produced a further control strain, D2.Tyrp1B6Gpnmb+
, that is currently being characterized. This strain contains a congenic interval flanking Tyrp1
but is improved over D2.Tyrp1B6GpnmbB6
in that it lacks a congenic interval around Gpnmb
(as the Gpnmb+
allele is the ancestral DBA/2J allele). This strain is wild-type for both glaucoma inducing genes and is not expected to develop any D2 glaucoma-associated phenotypes. Although characterization of large numbers of these mice is a lengthy undertaking, initial analyses agree with the findings reported here for D2.Tyrp1B6GpnmbB6
mice, with no evidence of iris disease, IOP elevation or glaucoma.
Regarding the control strains, the following points are pertinent. Although they lack all iris disease and glaucoma, the D2.Tyrp1B6GpnmbB6 strain does not typically reproduce as well as the D2-Gpnmb+ strain and appears to have an increased incidence of early deaths (based on retrospective observations, but no specific spontaneous death study has been conducted). Although not well characterized, the decreased fecundity appears to be related to the Tyrp1B6 allele on a D2 background, since other D2 strains that we have been producing with this allele have exhibited a similar phenotype. These issues do not prevent the use of this strain. However, since the D2-Gpnmb+ strain lacks these issues and has no B6 derived interval, we recommend it as a control when studying glaucomatous neurodegeneration. Although the D2-Gpnmb+ strain still develops a mild iris disease, glaucomatous nerve damage was clearly absent in the vast majority, if not all, of these mice. Thus, despite the spread in IOP, the highest IOPs were not significant enough to induce glaucomatous axon loss. When using these control strains, it is important to remember that they may still have some undefined but glaucoma relevant phenotype(s).
It is clear that glaucoma is a multifactorial disease and D2 mice are likely to have susceptibility factors in addition to those associated with the Gpnmb and Tyrp1 genes. Thus, although these strains do not develop glaucoma, as defined by the absence of nerve damage, they may still have other phenotypes that render the D2 background susceptible to glaucoma. Such phenotypes may or may not have contributed to nerve the damage detected in a small percentage of mice.
function in melanosomes, organelles that produce pigment. Manifestation of both IPD and ISA in D2 eyes is dependent upon active pigment production [16
]. The mechanisms involved in IPD and ISA and the subsequent IOP elevation are complex and in addition to pigment production appear to involve immunity [21
]. Ongoing functional mouse genetics experiments complemented with a variety of immunological, molecular and cellular techniques are vital to unravel these mechanisms. The control strains described here will be a valuable resource in elucidating the biological mechanisms involved in pigment-related iris disease. In addition to ocular-related diseases, these strains provide an important resource to test the roles of Tyrp1
in other diseases. Tyrp1
is the most common melanoma antigen and Gpnmb
is used as a marker for metastatic melanomas [23
] and has recently been identified as a potential molecular therapeutic target in patients with glioblastoma multiforme [24
may also be important in antigen presenting cells that control immune responses [21
To improve our understanding of glaucoma, it is essential that genes and pathways involved in IOP elevation and glaucomatous RGC and optic nerve degeneration are identified. By analyzing gene expression profiles in D2 mice at various ages [25
], and conducting subsequent functional tests, it is possible to identify differentially expressed genes important for the onset and/or progression of glaucoma. Individual mouse strains, such as D2, have a unique collection of alleles affecting many biological processes and that affect gene expression levels and protein activity [26
]. Therefore, many gene expression differences are observed between D2 and other naturally occurring strains that do not get glaucoma [27
]. This can complicate experiments and make it difficult to prioritize genes for subsequent functional testing. Additionally, it is possible that strain differences can mask some expression changes that occur in D2 mice and are relevant to the glaucoma. By enabling analysis of age- and strain-matched control mice, the control strains reported here alleviate the difficulties of controlling experiments using D2 mice.