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Papillorenal syndrome is an autosomal dominant disease caused by mutations in the PAX2 transcription factor gene. Patients often exhibit congenital excavation of the optic nerve and a spectrum of congenital kidney abnormalities. Using a novel mouse model of this syndrome (C57BL/6J Pax2A220G/+), we investigated the effect of Pax2 haploinsufficiency on optic nerve axon number. Because Pax2 expression and retinal pigment epithelium pigmentation have a mutually-exclusive relationship during development and because tyrosinase (Tyr) has been shown to modify the penetrance of other ocular development genes, we also investigated whether tyrosinase modified the mutant Pax2 phenotype.
C57BL/6J Pax2A220G/+Tyr+/+mice were crossed with mice of the same genetic background (C57BL/6J) that are homozygous for an effective null allele of tyrosinase (Tyrc-2J/c-2J) over two generations to create mice with four distinct genotypes: Pax2A220G/+Tyr+/c-2J, Pax2A220G/+Tyr c-2J/c-2J, Pax2+/+Tyrc-2J/+ and Pax2+/+Tyrc-2J/c-2J. Mouse optic nerves were examined clinically and histologically. Axon number was assessed in a masked fashion in optic nerves from mice of all four genotypes and compared to parental strains.
Mice heterozygous for a Pax2 mutation show reduced optic nerve axon number compared to age-matched controls. Tyrosinase does not appear to modify this phenotype.
Our results show that Pax2 is important in determining axon number in mouse optic nerve. The developmental effects of tyrosinase and Pax2 mutation appear to act via different pathways.
Papillorenal syndrome (also known as renal-coloboma syndrome, MIM#120330) is an autosomal dominant disorder characterized by congenital optic nerve and kidney abnormalities. Mutations in the paired-box transcription factor gene, PAX2, have been described in patients with papillorenal syndrome.1-4 Ocular features of papillorenal syndrome range from asymptomatic abnormalities in retinal blood vessel patterning and optic pits to large excavations of the optic nerve head associated with reduced best-corrected visual acuity.5 Although most mutations that lead to papillorenal syndrome are predicted to result in complete loss-of-function of PAX2,6 a few individuals with missense mutations have been reported.7, 8
During mouse development, Pax2 is expressed in the developing kidney, optic cup, otic vesicle and in other parts of the central nervous system,(e.g., the midbrain-hindbrain boundary.)9-12 Initially, Pax2 is expressed in the entire ventral optic vesicle without a defined proximal-distal gradient. As invagination of the optic cup and lens vesicle proceed, however, expression becomes more restricted to the proximal portions of the optic cup and stalk.9Pax2 expression follows the margins of the optic fissure and is down regulated as the fissure closes,13 after which time its expression is limited to the optic stalk.
The down-regulation of Pax2 in the optic cup also occurs as the presumptive retinal pigment epithelium (RPE) develops visible pigmentation. In fact, there is a mutually-exclusive relationship between pigmentation and Pax2 expression such that presumptive RPE cells that are pigmented do not express Pax2 and cells expressing Pax2 are unpigmented.12, 13 Torres et al. found that Pax2 mutant mice showed abnormal extension of the RPE into the optic stalk.12 These observations suggest some developmental relationship between Pax2 and pigmentation.
Ocular pigment production begins with the rate-limiting step of tyrosine hydroxylation by the enzyme tyrosinase (Tyr). Several lines of evidence suggest that Tyr may have a role in ocular development. It is well-recognized that defects in Tyr result in optic nerve axonal misdirection at the optic chiasm of mammals, such that there is an abnormal preponderance of crossed fibers.14 Furthermore, Libby et al. found that Tyr was a modifier of anterior segment development in mice homozygous for a mutation in Cyp1b1, a gene known to cause congenital glaucoma in humans.15 In this case, homozygosity for the mutant Tyrc-2J allele significantly worsened the morphologic and physiologic phenotype of homozygous Cyb1b1 knock-out mice.
Because patients with papillorenal syndrome have congenital optic nerve abnormalities often associated with decreased best-corrected visual acuity, we investigated the effect of Pax2 mutation on axon number in a papillorenal syndrome mouse model. Furthermore, we investigated whether Tyr modified the optic nerve phenotype and/or optic nerve axon number in our papillorenal syndrome model system. Our results indicate that mutation of Pax2 results in a reduced number of axonal fibers in mouse optic nerve and that Tyr does not significantly modify this phenotype.
(Further details given as online supplement.)
We have identified and characterized a novel mouse model of papillorenal syndrome through a collaborative ethylnitrosourea mutagenesis screen of C57BL/6J mice (unpublished data). In brief, these mice carry a c.A220G mutation in the Pax2 gene, which is predicted to change a well-conserved threonine residue to an alanine (T74A) in the critical paired domain of the protein. Mice heterozygous for the A220G mutation in Pax2 (C57BL/6J Pax2A220G/+) exhibit congenital optic nerve excavation (Figure 1A,B) and kidney abnormalities similar to that observed in patients with papillorenal syndrome. In fact, the position of this mutation is identical to one of the few missense mutations in Pax2 reported in humans.7 Heterozygous mice were used for comparison to wild-type for two reasons: 1) patients with PRS carry only one mutation in PAX2 and 2) homozygosity for this mutation is lethal in mice in the late embryonic or neonatal period, making their analysis difficult. C57BL/6J mice are phenotypically black and are considered wild-type at the Tyr locus.
C57BL/6JTyrc-2J/c-2J mice (MGI ID# 1855985, Jackson Laboratories, Bar Harbor, ME) are phenotypically albino due to a G291T (Arg77Leu) mutation in the Tyr gene that is a functional null at the protein level.16, 17 Because this albino strain is on the same genetic background as our Pax2 mutant, any changes observed should be due to the individual mutations in Pax2 and Tyr. Mice were bred over two generations as shown in Figure 2 to create progeny with four distinct genotypes: Pax2A220G/+Tyr+/c-2J, Pax2A220G/+Tyr c-2J/c-2J, Pax2+/+Tyrc-2J/+, and Pax2+/+Tyrc-2J/c-2J. These progeny were compared to the parental (C57BL/6JPax2A220G/+Tyr+/+) and wild-type (C57BL/6J Pax2+/+Tyr+/+) mice (Stock #000664, Jackson Labs, Bar Harbor, ME). All mice analyzed were age 3 months and strain-matched.
Optic nerves were dissected from mice according to published methods.18
Samples were imaged on a Carl-Zeiss Axiovert 200 microscope with Axiocam MRc5 camera (Carl Zeiss MicroImaging, Inc., Thornwood, NY). Axon number was determined using four fields within the periphery of each optic nerve (Figure 3A) at 40x magnification (Figure 3B). The observer was not aware of the sample's genotype during image acquisition or analysis. Images were analyzed using NIH ImageJ software, which has been validated for axon counting.19, 20 NIH ImageJ is an image processing and analysis program that was developed at the Research Services Branch of the National Institute of Mental Health. It allows for particle counting and analysis of an image in a Java-based platform. Initially all photographs are converted into an 8-bit gray scale image. By adjusting the contrast, brightness and threshold values, a true representative image of the picture was obtained (Figure 3C). Areas of clear miscounting or discrepancy were identified by visual inspection and eliminated from analysis. NIH ImageJ software then assessed total number of “particles” (i.e., axon number) in each high power field. Care was taken to minimize any overlap in the four high-power fields obtained for each nerve.
Data were analyzed using the Non-linear Mixed Effect library (version 3.1-78) in R (a programming language, version 2.4.1, http://www.r-project.org/). A linear mixed effect model was used, with axon count as the outcome variable. Fixed-effect predictor variables were Pax2 and tyrsosinase genotypes. Random effects for mouse and eye were included in the model; these random effects implicitly adjust for correlation between measurements obtained from individual eyes of individual mice.
Mice heterozygous for the Pax2 c.A220G mutation exhibit congenital excavation of the optic nerve, similar to patients with papillorenal syndrome (Figure 1B and Figure 4). The morphology of the retrobulbar optic nerve and the optic chiasm in Pax2A220G/+ mice is grossly normal (Figure 1C). However, upon microscopic examination, the number of axons per high-power field in C57BL/6J Pax2A220G/+ mice was significantly decreased (Figure 5). The statistical model predicts an average decrease of 1086 (34.7%, P < .0001) in axon count per high-powered field attributable to the Pax2A220G allele. For example, the model estimates an axon count of 3130 per high-powered field in Pax2+/+Tyr+/c-2J mice and that this count is reduced by 1086 axons per high-powered field (34.7%) in Pax2+/A220G Tyr+/c-2J mice.
Mice homozygous for the c-2J allele of tyrosinase exhibited lack of pigmentation, as anticipated, but showed no clinical modification of the optic nerve excavation seen in Pax2A220G/+ mice (Figure 4). Upon microscopic analysis, the number of axons per high-power field was not significant altered by the dosage of the Tyrc-2J allele (Figure 5). Therefore, although a relationship between pigment production in the RPE and Pax2 expression may exist, this relationship does not appear to involve determination of optic nerve axon number.
Our study examined two questions: 1) does mutation of Pax2 quantitatively affect optic nerve axon number in mice and 2) is the Pax2 mutant phenotype modified by tyrosinase? Although we found that mice heterozygous for the Pax2A220G allele had a quantitative reduction in optic nerve axon number, this phenotype was not significantly modified by tyrosinase.
Pax2 is a member of the paired-box family of transcription factors, which play critical roles in development.21 Previous studies of Pax2 in mouse ocular development have shown congenital optic nerve excavation similar to that seen in our model.22 The Krd (kidney and renal defect) mouse contains a 7cM transgene-induced deletion on chromosome 19 that includes the Pax2 locus.23 These mice exhibit retinal thinning, particularly in the inner nuclear and retinal ganglion cell layers. Although these results suggest that Pax2 regulates optic nerve axon number, the deletion in this mouse includes many other genes that could potentially contribute to its phenotype. Similarly, Favor et al. describe retinal thinning in the Pax1Neu/+ mouse, which carries a frameshift mutation early in the Pax2 sequence that is predicted to cause a complete loss of protein function.22 Homozygous, directed knock-out of the Pax2 gene results in smaller optic nerves.12 Torres et al. hypothesize that this is due to lack of optic nerve glial differentiation in Pax2-/- embryos, but also comment that a reduced number of axons could contribute to this phenotype. Our results therefore complement, augment and quantify these previous observations.
Why does haploinsufficiency for Pax2 result in a reduced number of optic nerve axons? Presuming that the number of optic nerve axons is related to the number of retinal ganglion cells, this decrease could be due to a reduced number of retinal progenitor cells differentiating into ganglion cells and/or an increase in the ganglion cell loss after differentiation. Retinal ganglion cells are the first cells to differentiate from retinal progenitor cells. They appear at the time of optic fissure closure, as Pax2 expression is diminishing at the edges of the fissure and is being limited to the optic stalk.9, 13 This pattern of expression would not easily explain a decrease in the number of retinal ganglion cells being formed across the retina. On the other hand, Pax2 is required for the proper differentiation of optic stalk neuroepithelium into glial cells.9, 12Pax2 expressing cells persist late into embryogenesis as a ring of cells at the optic disc separating the retinal ganglion cells axons from the retinal neuroepithelium of the optic stalk.13 We posit that a lack of structural and trophic support normally provided by glial cells and/or aberrant cell-cell interactions in Pax2-deficient mice leads to early death of a population of ganglion cells and a resultant decrease in optic nerve axons.
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