Experimental Eye Research 128 (2014) 83e91

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Heterozygote Wdr36-deficient mice do not develop glaucoma €rz a, Marcus Koch a, Martin Gallenberger a, Markus Kroeber a, Loreen Ma a a Rudolf Fuchshofer , Barbara M. Braunger , Takeshi Iwata b, Ernst R. Tamm a, * €tsstr. 31, D-93053 Regensburg, Germany Institute of Human Anatomy and Embryology, University of Regensburg, Universita Molecular & Cellular Biology Division, National Institute of Sensory Organs, National Hospital Organization Tokyo Medical Center, 2-5-1 Higashigaoka, Meguro-ku, Tokyo 152-8902, Japan a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 March 2014 Received in revised form 2 September 2014 Accepted in revised form 23 September 2014 Available online 26 September 2014

There is an ongoing controversy regarding the role of WDR36 sequence variants in the pathogenesis of primary open-angle glaucoma (POAG). WDR36 is a nucleolar protein involved in the maturation of 18S rRNA. The function of WDR36 is essential as homozygous Wdr36-deficient mouse embryos die before reaching the blastocyst stage. Here we provide a detailed analysis of the phenotype of heterozygous Wdr36-deficient mice. Loss of one Wdr36 allele causes a substantial reduction in the expression of Wdr36 mRNA. In the eyes of Wdr36þ/ animals, the structure of the tissues involved in aqueous humor circulation and of the optic nerve head are not different from that of control littermates. In addition, one-yearold Wdr36þ/ animals do not differ from wild-type animals with regards to intraocular pressure and number of optic nerve axons. The susceptibility of retinal ganglion cells to excitotoxic damage induced by NMDA is similar in Wdr36þ/ and wild-type animals. Moreover, the amount of optic nerve axonal damage induced by high IOP is not different between Wdr36þ/ and wild-type mice. Transgenic overexpression of mutated Del605-607 Wdr36 in Wdr36þ/ animals does not cause changes in the number of optic nerve axons or susceptibility to excitotoxic damage. In addition, analysis of 18S rRNA maturation in Del605-607 Wdr36þ/ or Wdr36þ/ mice does not show obvious differences in rRNA processing or in the amounts of precursor forms when compared to wild-type animals. Our data obtained in Wdr36þ/ mice do not support the assumption of a causative role for WDR36 in the pathogenesis of POAG. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Primary open-angle glaucoma Animal model Genetics

1. Introduction Glaucoma is defined as a chronic, degenerative optic neuropathy which is characterized by damage of optic nerve axons at the lamina cribrosa leading to typical visual field defects (Kwon et al., 2009; Quigley, 2011). In the most common form, primary openangle glaucoma (POAG), the resistance to outflow of aqueous humor is commonly elevated in the trabecular meshwork outflow pathways causing an increase in intraocular pressure (IOP) (Johnson, 2006; Tamm et al., 2007). Several prospective, randomized multicenter clinical studies have identified IOP as the most critical risk factor for the development of optic nerve axonal damage in POAG (Collaborative Normal-Tension Glaucoma Study Group, 1998a, b; Higginbotham et al., 2004; Kass et al., 2002; Leske et al., 2003; The AGIS Investigators, 2000). Susceptibility to POAG commonly depends on complex trait inheritance resulting from interactions of multiple genetic factors

* Corresponding author. E-mail address: [email protected] (E.R. Tamm). http://dx.doi.org/10.1016/j.exer.2014.09.008 0014-4835/© 2014 Elsevier Ltd. All rights reserved.

and the influence of environmental exposures (Fan and Wiggs, 2011; Fingert, 2011; Liu and Allingham, 2011). In rare cases, POAG may also be inherited as a Mendelian trait, and up to now at least 17 loci (GLC1A to GLC1Q) that contribute to the susceptibility of POAG have been identified (Fan and Wiggs, 2011; Fingert, 2011; Liu and Allingham, 2011). Among the genes that have been detected within these loci are myocilin (MYOC) (Stone et al., 1997), optineurin (OPTN) (Rezaie et al., 2002), TANK-binding kinase-1 (TBK1) (Fingert et al., 2011), neurotrophin-4 (NT4) (Pasutto et al., 2009), and the WD repeat domain 36 gene (WDR36) (Monemi et al., 2005). Sequence variants in WDR36 have originally been reported to be associated with GLC1G-linked glaucoma mapped to 5q22.1 (Monemi et al., 2005). Subsequent studies have confirmed the presence of rare WDR36 sequence variants that appear to be associated with POAG in some populations (Fan et al., 2009; Miyazawa et al., 2007; Pasutto et al., 2008; Weisschuh et al., 2007). Nonetheless, several studies have failed to confirm a causative role for WDR36 in POAG (Fingert et al., 2007; Hauser et al., 2006; Hewitt et al., 2006). In addition, two additional POAG pedigrees have been identified with 5q22.1-linked POAG that harbor no

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WDR36 mutations and indicate the presence of a different glaucoma gene in this region (Kramer et al., 2006; Pang et al., 2006). One study reported that patients with WDR36 sequence variants suffer from a more severe disease phenotype than those without indicating rather a modifying than a causative role of WDR36 in POAG (Hauser et al., 2006). WDR36 is expressed in multiple ocular and non-ocular tissues, and high amounts of its mRNA have been detected in heart, skeletal muscle, pancreas, liver, and placenta (Monemi et al., 2005). The structural hallmark of WDR36 is the presence of multiple WD40 repeats which are conserved structural motifs of approximately 40 amino acids that often terminate in a tryptophaneaspartic acid (WD) dipeptide. According to a computed protein structure, WDR36 contains 14 WD40 repeats which fold into two connected sevenbladed b-propellers (Footz et al., 2009). The primary structure of WDR36 shows similarity to that of Utp21 (Footz et al., 2009), an essential nucleolar ribonucleoprotein in the yeast Saccharomyces cerevisiae. Utp21 is part of the small subunit (SSU) processome (Bernstein et al., 2004), a large ribonucleoprotein complex containing more than 40 proteins, which is required for the maturation of the 18S rRNA of the SSU of the ribosome (Kressler et al., 2010). Recent data obtained in animal models strongly suggest a similar role for WDR36 in vertebrates. In cells from both zebrafish (Skarie and Link, 2008) and humans (Gallenberger et al., 2011), WDR36 specifically localizes to their nucleolus. Loss-of-function phenotypes in zebrafish result in reduced levels of 18S rRNA (Skarie and Link, 2008). Depletion of WDR36 in human cells causes a delay of 18S rRNA processing and augments apoptotic cell death (Gallenberger et al., 2011). Moreover, homozygous Wdr36-deficient mouse embryos die before implantation (Gallenberger et al., 2011), a finding commonly observed in mice with targeted null mutations in genes that encode for proteins involved in ribosomal RNA synthesis or processing (Chen et al., 2008; Lerch-Gaggl et al., 2002; Matsson et al., 2004; Newton et al., 2003; Romanova et al., 2006; Zhang et al., 2008). In light of the conflicting data regarding the role of WDR36 sequence variants in the pathogenesis of POAG, we investigated here in detail the phenotype of mutant heterozygous Wdr36-deficient mice. We provide evidence that loss of one Wdr36 allele neither causes POAG nor enhances the susceptibility of retinal ganglion cells and their axons to damage induced by excitotoxicity or an increase in IOP. Moreover, the transgenic overexpression of mutant Wdr36 did not cause obvious changes in the ocular phenotype of heterozygous Wdr36-deficient mice. Our results do not support a causative role for WDR36 in the pathogenesis of POAG. 2. Material and methods 2.1. Animals Wdr36-deficient mice in a C57BL/6J background (Gallenberger et al., 2011), bB1-CTGF mice in a CD1 background (Junglas et al., 2012) and Del605-607 mice (Chi et al., 2010), in a BDF1/C57BL6N background, were genotyped as described previously (Chi et al., 2010; Gallenberger et al., 2011; Junglas et al., 2012). Mice were housed at a 12 h light/dark cycle under standardized conditions of 62% air humidity and 21  C room temperature. Before enucleation of the eyes, mice were anaesthetized with CO2 and killed by atlanto-occipital dislocation. All animal procedures performed in this study complied with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and with institutional guidelines. IOP of transgenic mice and wild-type littermates was measured using a TonoLab tonometer (Tiolat OY, Helsinki, Finland) as described previously (Junglas et al., 2012; Kroeber et al., 2010).

Measurements were conducted non-invasively in eyes of sedated mice (90e120 mg/kg ketamine and 6e10 mg/kg xylazine hydrochloride injected i.p.) at the same time of day. Each reading was comprised of six measurements averaged automatically. Highly and moderately variable readings were excluded. An average of six readings was considered as a single result. 2.2. Light and electron microscopy Eyes were enucleated and fixed in Karnovsky's solution (2.5% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M cacodylate buffer) for 24 h (Karnovsky, 1965). After rinsing in 0.1 M cacodylate buffer, post fixation was accomplished in a mixture of 1% OsO4 and 0.8% potassium ferrocyanide in 0.1 M cacodylate buffer for 2 h at 48  C. Eyes were then dehydrated in a graded series of ethanol and embedded in Epon (Serva). Semithin sections (1 mm) were collected on uncoated glass slides and stained with methylene blue/azure II (Richardson et al., 1960). Ultrathin sections were mounted on uncoated copper grids, stained with uranyl acetate and lead citrate and examined on a Zeiss Libra transmission electron microscope (Carl Zeiss AG). Myelinated optic nerve axons were visualized by paraphenylendiamin (PPD, Roth) staining of Epon-embedded semithin sections (Schultze, 1972). In brief, 1% PPD in 98% ethanol was freshly prepared and stored at daylight for 3 days prior to use until the solution had darkened. The solution is stable for 1 week in the dark at 48  C. Optic nerve cross sections were stained for 2e3 min at room temperature and staining was differentiated with changes of 100% ethanol. To count the total number of optic nerve axons, PPD stained cross sections were visualized by bright field microscopy using a 100 oil immersion objective for highest resolution. Myelinated axons were identified and counted software assisted (Axiovision software 3.0, Carl Zeiss AG). The examiners were masked as to the nature of the samples. 2.3. Preimplantation stage embryos Since in Del605-607 animal, the expression of mutated WDR36 is driven by an ectopic transgene, while the Wdr36 wildtype locus is still intact, we were able to generate by crossbreeding Wdr36þ/  /Del605-607 mice. For timed pregnancies, Wdr36þ//Del605-607 females received an i.p. injection of serum gonadotropin from a pregnant mare (5 IU/animal; Sigma), followed 48 h later by human chorionic gonadotropin [hcG] (5 IU/animal; Sigma). Mice were then allowed to mate with Wdr36þ/ males. The males were removed the next morning and the females were examined for the presence of a vaginal plug. Preimplantation stage embryos (zygotes at 0.5 dpc) were collected by flushing the oviduct of plugged females with M2 medium (Sigma). Flushed embryos were cultured individually in KSOM (Millipore) microdrop cultures at 37  C in humified air containing 5% CO2 over several days. The growth patterns of embryos were microscopically examined and photographed. DNA was prepared by incubation of individual embryos with 20 ml of lysis buffer (50 mM TriseHCl, 0.5% Triton X-100, proteinase K [500 mg/ml], pH 8.0) for 4 h at 55  C, followed by incubation at 90  C for 10 min Wdr36 genotyping of preimplantation stage embryos was performed as described previously (32). For detection of the Del605-607 Wdr36 transgene in early stage embryos a nested PCR strategy was used. Primers were placed over exoneintron boundaries for only the transgene Del605-607 Wdr36 gene is amplified. In the first round 1 ml of embryonic DNA was used to amplify a 659 bp fragment in transgene embryos in a 17 ml reaction mixture containing further standard buffer, 0.1 mM of each primer (Nested forward1: 50 -GTGAGCAGGGAAGCCTACAG-30 ; Nested reverse1: 50 AGGTGGGAGCCTTACTGACA-30 ), 1 mM dNTPs, 2.5 mM MgCl2, 12 mM glycerol, 0.2 mM cresol red (Sigma) and 0.25 U Taq-

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Polymerase (New England Biolabs). Second round PCR was performed in a reaction mixture comparable to that described above containing 1 ml of first round reaction mixture and second round primers (Nested forward2: 50 -TGTTGCCATTGGTCTTGTGT-30 ; Nested reverse2: 50 -AAGCACTATGCCCCATTCTG-30 ) which resulted in the amplification of a 359 bp fragment in Del605-607 Wdr36 transgene embryos. The cycling conditions consisted of an initial 2 min denaturation step at 94  C, followed by 30 cycles (35 cycles in the second round PCR) for 20 s at 94  C, 20 s at 59  C and an elongation for 45 s at 72  C.

anesthetized with ketamine [120 mg/kg body weight, i.m.] and xylazine [8 mg/kg body weight] and the ocular surface was disinfected by an iodine tincture. To induce retinal damage, 3 ml of NMDA [10 mM dissolved in PBS] (Sigma) were injected into the vitreous body of one eye while the fellow was treated with PBS. Mice were sacrificed 3 weeks after injection. Eyes and optic nerves were isolated and fixed in 2% paraformaldehyde/2.5% glutaraldehyde over night.

2.4. RNA isolation and analysis

All results are expressed as mean ± SEM. Comparisons between the mean variables of 2 groups were made by a 2-tailed Student's ttest. Statistical significance was calculated using SPSS (IBM). To evaluate the significance of the intergroup difference, analysis of variance (ANOVA) with a Bonferroni post hoc test was used. P values less than 0.05 were considered to be statistically significant.

Total RNA was extracted with TRIzol (Invitrogen) according to manufacturer's recommendations. Structural integrity of RNA samples was confirmed by electrophoresis using 1% (w/v) agarose gels. The RNA concentration was determined by absorbance at 260 nm. First strand cDNA was prepared from total RNA using the iScript cDNA Synthesis Kit (BioRad) according to the manufacturer's instructions. Real-time RT-PCR was performed on a BioRad iQ5 Real-Time PCR Detection System (BioRad) with the temperature profile as follows: 40 cycles of 10 s melting at 95  C, 40 s of annealing and extension at 60  C. Sequences of primer pairs were 50 -AGCAAAGTGCGTGAGGAGTT-30 (forward) and 50 ACAAGTCCCCCTCCTTCTTG-30 (reverse) for LaminA which was used as housekeeping gene, and 50 -TGTCAGTAAGGCTCCCACCT-30 (forward) and 50 -CATCCCAGTCACTTTGACGA-30 (reverse) for Wdr36. Primers were purchased from Invitrogen and extended over exoneintron boundaries. RNA that was not reverse transcribed served as negative control for real-time PCR. For Northern blot analysis, RNA was size fractionated on a 1% agarose gel containing 3% formaldehyde and blotted onto a positively charged nylon membrane (Roche). After transfer, the blot was cross-linked using an UV Stratalinker 1800 (Stratagene) followed by methylene blue staining (0.03% methylene blue, 0.3 M sodium acetate, pH 5.2) to assess the amount and quality of the RNA. Prehybridization was performed for 45 min at 68  C in a Hybridizer HB-1000 (UVP Laboratory Products, Upland, CA) using the Dig EasyHyb-buffer (Roche). mmITS1-antisense rRNA probe was generated of genomic DNA using mmITS-1 fwd (50 CGAGGTGTCTGGAGTGAGGT-30 ) and mmITS-1-T7 rev (50 -TAATACGACTCACTATAGGGCATGGAGTCTGAGGGAGAGC-30 ) primers. Wdr36 mRNA probe was generated with primer pairs mmWdr36 fwd (50 -ATGTCAGTAAGGCTCCCACC-3) and mmWdr36-T7 rev (50 TAATACGACTCACTATAGGGAGAAGGAAGGTCCCAAGTCC-30 ) in a PCR by use of cDNA as template. DIG labeling was performed according to DIG RNA Labeling Kit (Roche) instructions. Hybridization was performed for 16e18 h at 68  C. Membranes were washed 2 times for 5 min with 20 SSC and 0.1% SDS at room temperature followed by two washes with 0.2% SSC and 0.1% SDS at 70  C for 15 min. After an additional washing step with maleic acid wash buffer (0.1 M maleic acid, 0.15 M sodium chloride, 0.3% Tween20, pH 7.5) the membrane was blocked for 30 min in 1 DIG-blocking reagent (Roche) at room temperature. Then blot was incubated for 30 min with Anti-DIG-antibody (1:10,000 in 1 DIG-blocking reagent; Roche), washed with maleic acid wash buffer (2  15 min, RT) and equilibrated in detection buffer (100 mM TriseHCl, 100 mM NaCl; pH 9.5) for 10 min. For detection, blots were incubated with chemiluminescence substrate CDP-STAR (Roche) and documented in an LAS 3000 Intelligent Dark box (Fujifilm). 2.5. NMDA-induced retinal damage NMDA-mediated retinal damage was induced as reported previously (Seitz and Tamm, 2013). Briefly, mice were deeply

2.6. Statistics

3. Results 3.1. Heterozygous deficiency for Wdr36 does not cause glaucoma Heterozygous Wdr36-deficent mice were viable and fertile. Northern blot analyses from organs with high Wdr36 expression showed a substantial reduction of specific mRNA in Wdr36þ/ animals (Fig. 1A) compared to control littermates. No structural differences were observed upon light microscopy of heart, kidney or liver in Wdr36þ/ animals when compared to wild-type littermates (not shown). When RNA from the eye was analyzed by quantitative real time RT-PCR, the amounts of mRNA for Wdr36 were only 51% ± 12% of that seen in wild-type littermates (p < 0.05), a finding that was entirely consistent with the disruption of one Wdr36 allele (Fig. 1B). We next investigated the phenotype of the tissues involved in aqueous humor turnover in the anterior eye. By light microscopy, the structure of the ciliary body did not differ between Wdr36þ/ animals and their wild-type littermates (Fig. 2A). In addition, the chamber angle was open both in Wdr36þ/ and wild-type mice (Fig. 2A, B). Transmission electron microscopy showed the typical lamellae of the trabecular meshwork next to Schlemm's canal, which both appeared to be normal in Wdr36þ/ animals (Fig. 2B). IOP did not differ between one-year-old Wdr36þ/ mice and wildtype littermates (Fig. 3A), a finding which was consistent with

Fig. 1. A. Northern blot analysis for Wdr36 mRNA (arrow) in RNA from heart and kidney of Wdr36/þ mice and wild-type (WT) littermates. Ribosomal RNA was stained with methylene blue. B. Quantitative real time RT-PCR for Wdr36 mRNA in RNA from whole eyes of adult Wdr36þ/ mice and wild-type littermates. LaminA was used as reference gene. The mean value obtained with wild-type RNA was set at 1 (mean ± SEM, n ¼ 6, *p  0.05).

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Fig. 2. A. Semithin sections (Richardson's stain) through the chamber angle region of an adult Wdr36þ/ mouse and its wild-type littermate. In both animals, the chamber angle is open (arrow). No structural abnormalities are seen in the ciliary body (CB). B. By transmission electron microscopy, no obvious structural differences are present in trabecular meshwork (TM) and Schlemm's canal (SC) between adult Wdr36þ/ mice and wild-type littermates. AC. Anterior chamber.

the normal structural phenotype of the aqueous humor circulation system. Now we turned our attention to the back of the eye and investigated the optic nerve head. The axon bundles entering the nerve head were of comparable thickness in both Wdr36þ/ and wild-type littermates (Fig. 3B). Moreover, when the total number of axons in cross sections through the optic nerve was counted in oneyear-old animals, no difference between both Wdr36þ/ and wildtype mice was observed (Wdr36þ/: 47,647 ± 5485, WT: 42,485 ± 6167, n  11, Fig. 3C). Finally, we investigated if the reduction in Wdr36 expression in Wdr36þ/ mice attenuates the processing of 18S ribosomal RNA comparable to findings in WDR36deficient human trabecular meshwork cells (Gallenberger et al., 2011). We generated a probe specific for the mouse internal transcribed spacer-1 (ITS-1) that allows the simultaneous detection of 47S/45S, 41S, 34S and 20S pre-rRNAs which are precursors of 18S rRNA (Gallenberger et al., 2011). As expected, the ITS-1 probe hybridized with 47/45S, 41S, 34S and 20S pre-rRNAs which all contain ITS-1, but not with mature 18S rRNA (Fig. 4A). Neither the amount of the various pre-rRNA species (Fig. 4A) nor that of mature 18S rRNA (Fig. 4B) showed any quantitative differences between RNA from Wdr36þ/ and wild-type mice strongly indicating that loss of one Wdr36 allele did not cause relevant functional deficits of the SSU processome. In summary, the phenotype analysis of Wdr36þ/ mice did not detect any functional or structural changes leading to a pathogenic process comparable to that seen in primary open-angle glaucoma.

Fig. 3. A. IOP measurements in one-year-old Wdr36þ/ mice and wild-type (WT) littermates (mean ± SEM, n  13). B. Semithin sections (Richardson's stain) through the optic nerve head of an adult Wdr36þ/ mouse and its wild-type littermate. The axon bundles entering the optic nerve (ON) head (arrows) are of comparable thickness in both animals. C. Cross sections through optic nerves (paraphenylenediamine stain) of a one-year-old Wdr36þ/ mouse and its wild-type littermate. D. Number of optic nerve axons in one-year-old Wdr36þ/ mice and wild-type littermates (mean ± SEM, n  11).

3.2. Heterozygous deficiency for Wdr36 does not increase the susceptibility to damage of retinal ganglion cells We now wondered if heterozygote deficiency for Wdr36 induces in retinal ganglion cells and/or their axons a higher vulnerability to injury. To this end we induced excitotoxic damage by injection of NMDA into the vitreous. Three weeks after injection of NMDA in one eye and of PBS into the contralateral eye, we observed a substantial and significant (p < 0.01) reduction in the number of optic nerve axons in both Wdr36þ/ (43,525 ± 1691 versus 15,544 ± 915, n ¼ 5) and wild-type (42,853 ± 1963 versus 15,320 ± 1134, n ¼ 6) animals (Fig. 5). There was no significant difference though, when PBS- or NMDA-injected eyes were compared between Wdr36þ/

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Fig. 4. A. Northern blot analysis of 18S rRNA maturation in RNA from the whole eye of a Wdr36þ/ mouse and its wild-type littermate. B. Methylene blue staining serves as a control for equal loading.

and wild-type animals (Fig. 5). Our next approach was to induce axonal damage of the optic nerve by elevated IOP. To this end, we crossed Wdr36þ/ mice with transgenic bB1-CTGF mice. In bB1CTGF mice, higher amounts of connective tissue growth factor (CTGF) in the aqueous humor cause an increase in IOP and subsequent glaucomatous damage of the optic nerve (Junglas et al., 2012). In three-month-old bB1-CTGF (18.4 ± 1.96; p ¼ 0.001) and Wdr36þ//bB1-CTGF mice (18.39 ± 1.45; p ¼ 0.0002), we observed a significant increase in IOP when compared to wild-type littermates (15.41 ± 1.74) (Fig. 6A). Moreover, there were significantly fewer axons in the optic nerves of three-month-old bB1-CTGF (37,764 ± 2864; p > 0.05) and Wdr36þ//bB1-CTGF mice (37,131 ± 2749; p > 0.05) than in wild-type animals (44,781 ± 3126) (Fig. 6B). We assumed that the CTGF-induced increase in IOP had caused glaucomatous optic nerve damage. Noteworthy, there were no significant differences between three-month-old bB1-CTGF and Wdr36þ//bB1-CTGF mice with regards to the levels of IOP or the

Fig. 6. Intraocular pressure (A, box and whisker blots showing median, upper and lower quartile, and smallest and greatest values in the distribution) and number of optic nerve axons (B) in three-month-old wild-type (WT), bB1-CTGF, and Wdr36þ/  /bB1-CTGF-mice. **p  0.01, *p  0.05, n ¼ 14 (A), n ¼ 6 (B).

amount of axonal loss in the optic nerve (Fig. 6A, B). We concluded that heterozygous deficiency for Wdr36 did not increase the susceptibility of retinal ganglion cells and their axons to injury induced by excitotoxicity or increase in IOP.

3.3. The Wdr36 Del605-607 transgene does not rescue homozygous Wdr36-deficient mice from early embryonic death

Fig. 5. Number of optic nerve axons in wild-type (WT) and Wdr36þ/-deficient littermates three weeks after injection of PBS into the vitreous of one eye and of NMDA into that of the contralateral eye. Mean ± SEM, n  5.

Transgenic Del605-607 mice overexpress a mutant form of Wdr36 under control of the chicken beta-actin promoter and CMV enhancer (pCAGGS) driving an ubiquitous strong expression (Chi et al., 2010). The transgene that was inserted into the genome of Del605-607 mice carries a 3 amino acid deletion (Del605-607) in the Wdr36 cDNA. The deletion includes the D606 position which is equivalent to the human D658G variant that was found to be associated with a severe phenotype in glaucoma patients (Monemi et al., 2005). Other studies found the D658G variant at a comparable frequency in both POAG patients and in control subjects (Fingert et al., 2007; Hauser et al., 2006), indicating that the variant is not sufficient to cause POAG in humans. Del605-607 mice suffer from a loss of peripheral retinal ganglion cells and connecting

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amacrine cell synapses at very high (16 months) age (Chi et al., 2010). To analyze the function of mutated Del605-607 Wdr36 in a Wdr36-deficient background, we crossed Wdr36þ/ mice with Del605-607 animals. Since the pCAGGs promoter is ubiquitously active from the 2-cell stage on (Okabe et al., 1997), we first analyzed if mutated Del605-607 WDR36 is able to prevent the preimplantation lethality of Wdr36/ embryos (Gallenberger et al., 2011). We took zygotes to culture and observed their development to blastocyst stage. Wild-type and Del605-607 transgenic embryos reached blastocyst stage (Fig. 7). In contrast, Wdr36/ and Wdr36/  /Del605-607 embryos ceased to develop and degenerated 3.5 days post coitum. In degenerating embryos, the inner cavity or blastocoel of blastocysts did not form and the embryonic cells became detached from the Zona pellucida to clump together to a cluster (Fig. 7). 3.4. The phenotype of 6-week-old heterozygous Wdr36-deficient mice is not changed by the expression of mutated Wdr36 We next analyzed if the expression of mutant Del605-607 Wdr36 mRNA causes changes in the phenotype of heterozygous Wdr36-deficient mice. We first analyzed the expression of wildtype and mutated Wdr36 by real time RT-PCR of RNA from the whole eye of 6-week-old animals to observe an almost 20-fold higher expression of Del605-607 Wdr36 than that of wild-type Wdr36 both in wild-type (p  0.05) and Wdr36þ/ mice (p  0.01) (Fig. 8A). Del605-607 mice were generated in a mixed BDF1/C57BL6N background (40) while Wdr36-deficient mice are bred in a CD1 background (32). When animals from both lines were crossed, wild-type mice that did not carry the transgene showed a smaller number of optic nerve axons (34,062 ± 3569, mean ± SEM, n ¼ 4) than previously observed in the CD1 background. Still, the number of axons in 6-week-old Wdr36þ//Del605-607 animals (35,185 ± 5517, mean ± SEM, n ¼ 4) did not significantly differ from that of their wild-type littermates (Fig. 8B). To investigate if the strong expression of mutated Del605-607 in a Wdr36þ/ background attenuates the processing of ribosomal RNA, we again performed Northern blot analysis using the mouse ITS-1 probe. No quantitative difference regarding the amounts of 47S/45S, 41S, 34S and 20S pre-rRNAs, or mature 18S rRNA was detected (Fig. 9).

Fig. 7. Phenotype of wild-type (WT) and of Wdr36-deficient embryos with and without Del605-607 Wdr36 transgene. Embryos were photographed at 4.5 dpc. Wildtype and heterozygous Wdr36-deficient embryos develop normally and reach blastocyst stage at 4.5 dpc. In contrast, homozygous Wdr36-deficient embryos do not reach blastocyst stage and degenerate at 4.5 dpc. Accordingly, the cells become detached from the Zona pellucida and clump together in a cluster. The presence of the Del605607 Wdr36 transgene has no influence on the fate of Wdr36-deficient embryos.

Fig. 8. A. Quantitative real-time RT-PCR for Wdr36 mRNA in RNA from the whole eye of wild-type (WT), Wdr36þ/, and Wdr36þ//Del605-607 mice. LaminA was used as reference gene. The mean value obtained with wild-type RNA was set at 1 (mean ± SEM, n ¼ 6, *p  0.05, **p  0.01). B. Number of optic nerve axons in 6-weekold Wdr36þ/ and Wdr36þ//Del605-607 littermates (mean ± SEM, n ¼ 4).

Fig. 9. A. Northern blot analysis of 18S rRNA maturation in RNA from the whole eye of a wild-type (WT) mouse and its Wdr36þ//Del605-607 littermate. B. Methylene blue staining serves as a control for equal loading.

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Finally we induced an excitotoxic damage by injection of NMDA into the vitreous of 6-week-old animals. Three weeks after injection of NMDA in one eye and of PBS into the contralateral eye, we observed a substantial and significant (p  0.01) reduction in the number of optic nerve axons in both wild-type (35,561 ± 2045 versus 15,256 ± 1810, n ¼ 7) and Wdr36þ//Del605-607 animals (34,555 ± 3922 versus 13,898 ± 2090, n ¼ 7) animals (Fig. 10). There was no significant difference though, when PBS- or NMDA-injected eyes were compared between wild-type and Wdr36þ//Del605-607 animals (Fig. 10).

4. Discussion We conclude that lack of one Wdr36 allele is not a predisposing factor for POAG. This conclusion rests upon (1) the observation that heterozygous Wdr36-deficient mice do not develop any detectable structural changes in the optic nerve or the tissues involved in aqueous humor circulation, (2) the lack of any detectable deficits in 18S rRNA processing in both Wdr36-deficient and mutated Del605607 Wdr36 mice, (3) the finding that IOP is not increased in heterozygous Wdr36-deficient mice, (4) the fact that heterozygous deficiency for Wdr36 does not increase the susceptibility of retinal ganglion cells and their axons to damage induced by high IOP or excitotoxicity, and (5) the observation that transgenic overexpression of mutated Del605-607 Wdr36 in Wdr36þ/ animals does not cause changes in the number of optic nerve axons or susceptibility to excitotoxic damage. In light of the conflicting data regarding the role of WDR36 sequence variants in the pathogenesis of POAG, our results appear to provide additional evidence against a causative role for WDR36 in human glaucoma. Data obtained in both zebrafish (Skarie and Link, 2008) and mouse (Gallenberger et al., 2011) strongly indicate that WDR36 is an essential nucleolar ribonucleoprotein which is part of the SSU processome required for the maturation of 18S rRNA. Clearly, ribosome production is a critical metabolic activity that is essential for life. Over the last few decades, several rare genetic diseases collectively known as ribosomopathies have been attributed to defects in ribosome function or ribosome biogenesis (Freed et al., 2010; Narla and Ebert, 2010). Well-studied examples are

Fig. 10. Number of optic nerve axons in wild-type (WT) and Wdr36þ//Del605-607 littermates three weeks after injection of PBS into the vitreous of one eye and of NMDA into that of the contralateral eye. Mean ± SEM, n ¼ 7.

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Diamond-Blackfan anemia (OMIM 105650), a rare congenital erythroblastopenia, the 5q- syndrome (OMIM 153550), a subtype of myelodysplastic syndrome, or Treacher Collins syndrome (OMIM 154500) an autosomal dominant craniofacial disorder. Ribosomopathies show quite distinct clinical phenotypes that are restricted to specific cell types and most often involve bone marrow failure and/or craniofacial or other skeletal defects. Loss-of-function mutations leading to haploinsufficiency are the common pathogenic mechanism leading to disruption or delay in ribosomal biogenesis. The resulting nucleolar stress in turn activates a p53 signaling pathway (Chakraborty et al., 2011; Fumagalli and Thomas, 2011) which is very likely part of a surveillance system that monitors ribosomal integrity. Major outcomes of the activated p53 response are cell cycle arrest and apoptosis which cause or contribute to the phenotype seen in ribosomopathies (Fumagalli and Thomas, 2011). Indeed, the phenotype of mutant mouse models suffering from ribosomopathies may be rescued upon cross-breeding with p53deficient mice (Barlow et al., 2010; Chakraborty et al., 2011; McGowan and Mason, 2011). P53 is upregulated in several neurodegenerative diseases (Morrison et al., 2003) and the partial loss of WDR36 function might hypothetically cause or contribute to apoptotic cell death of retinal ganglion cells, a hallmark of glaucoma (Quigley, 2011). Indeed, we recently showed that the knock-down of Wdr36 expression in cultured cells causes a delay in 18S rRNA processing, induces the expression of p53 and causes apoptotic cell death (Gallenberger et al., 2011). Nevertheless, the data of the present study strongly indicate that the loss of one Wdr36 allele in the living mouse does not cause obvious changes in 18S rRNA processing. In addition, there is no evidence that apoptotic cell death of retinal ganglion cells is enhanced, neither in the normal eye nor after injury. Our data clearly show that mice are capable to compensate for the loss of one Wdr36 allele. Similar compensatory mechanisms may well be present in humans and may account for the relative high number of WDR36 sequence variants in the normal population. We cannot exclude though that in the presence of additional genetic modifications compensatory mechanisms may fail in humans. In support of this are data from a recent study providing some evidence for a genetic interaction between WDR36 and P53 sequence variants in POAG susceptibility (Blanco-Marchite et al., 2011). Certainly, the possibility remains that WR36 sequence variants cause POAG by gain-of-function effects. To analyze if gain-offunction effects might cause a delay in rRNA processing or increase susceptibility to apoptotic cell death in heterozygous Wdr36deficient mice, we crossed the mice with transgenic Del605-607 mice that express a mutated form of Wdr36 with a 3 amino acid deletion (Chi et al., 2010). The deletion includes the D606 position which is equivalent to the human D658G variant that was reported to be associated with a severe phenotype in glaucoma patients (Monemi et al., 2005). Molecular modeling indicates that the deletion removes a hydrogen bond that is required to stabilize the anti-parallel beta-sheet of the 6th beta-propeller in the second domain (Chi et al., 2010). Indeed, in our study the transgenic expression could not rescue homozygous Wdr36-deficient mice from early embryonic death strongly indicating that the mutation prevented synthesis of a functional protein. The data are in agreement with those reported by Footz et al. (2009) who introduced the D658G sequence variant into UTP21 which is the homologous gene in yeast. Combined with disruption of STI1 (which synthetically interacts with UTP1), the variant caused a decrease in cell viability in a yeast model system and an accumulation of pre-rRNA strongly indicating diminished function of the mutated variant. It should be noted though that the human D658G variant was found in two studies at a comparable frequency in both POAG patients and in

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control subjects (Fingert et al., 2007; Hauser et al., 2006), findings that clearly indicate that the variant is not sufficient to cause POAG. Our data show that putative gain-of-function effects caused by mutated Del605-607 Wdr36 are unlikely to act on rRNA processing or the susceptibility to apoptotic cell death of retinal ganglion cells in young animals. 16-month-old Del605-607 mice have been reported to develop progressive retinal degeneration of retinal ganglion cells and amacrine synapses in the peripheral retina with normal IOP (Chi et al., 2010). The strong overexpression of the transgene apparently impairs biological properties of retinal ganglion cells as their axon outgrowth in culture conditions is reduced (Chi et al., 2010). A possible mechanism might be that the expression of transgenic Del605-607 Wdr36 results in the accumulation of misfolded protein and initiates an unfolded protein response that leads to cell stress and finally apoptotic cell death. At the moment, we do not have data to support this hypothesis. In addition, it remains to be shown if a similar effect causes or contributes to POAG in humans harboring WDR36 sequence variants.

Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (FOR 1075, TP5 to E.R.T.). We greatly appreciate the expert technical assistance of Silvia Babl, Angelika Pach and Margit Schimmel.

References Barlow, J.L., Drynan, L.F., Hewett, D.R., Holmes, L.R., Lorenzo-Abalde, S., Lane, A.L., Jolin, H.E., Pannell, R., Middleton, A.J., Wong, S.H., Warren, A.J., Wainscoat, J.S., Boultwood, J., McKenzie, A.N., 2010. A p53-dependent mechanism underlies macrocytic anemia in a mouse model of human 5q- syndrome. Nat. Med. 16, 59e66. Bernstein, K.A., Gallagher, J.E., Mitchell, B.M., Granneman, S., Baserga, S.J., 2004. The small-subunit processome is a ribosome assembly intermediate. Eukaryot. Cell. 3, 1619e1626. Blanco-Marchite, C., Sanchez-Sanchez, F., Lopez-Garrido, M.P., Inigez-deOnzono, M., Lopez-Martinez, F., Lopez-Sanchez, E., Alvarez, L., RodriguezCalvo, P.P., Mendez-Hernandez, C., Fernandez-Vega, L., Garcia-Sanchez, J., CocaPrados, M., Garcia-Feijoo, J., Escribano, J., 2011. WDR36 and P53 gene variants and susceptibility to primary open-angle glaucoma: analysis of gene-gene interactions. Invest. Ophthalmol. Vis. Sci. 52, 8467e8478. Chakraborty, A., Uechi, T., Kenmochi, N., 2011. Guarding the 'translation apparatus': defective ribosome biogenesis and the p53 signaling pathway. Wiley Interdiscip. Rev. RNA 2, 507e522. Chen, H., Li, Z., Haruna, K., Semba, K., Araki, M., Yamamura, K., Araki, K., 2008. Early pre-implantation lethality in mice carrying truncated mutation in the RNA polymerase 1-2 gene. Biochem.. Biophys. Res. Commun. 365, 636e642. Chi, Z.L., Yasumoto, F., Sergeev, Y., Minami, M., Obazawa, M., Kimura, I., Takada, Y., Iwata, T., 2010. Mutant WDR36 directly affects axon growth of retinal ganglion cells leading to progressive retinal degeneration in mice. Hum. Mol. Genet. 19, 3806e3815. Collaborative Normal-Tension Glaucoma Study Group, 1998a. Comparison of glaucomatous progression between untreated patients with normal-tension glaucoma and patients with therapeutically reduced intraocular pressures. Am. J. Ophthalmol. 126, 487e497. Collaborative Normal-Tension Glaucoma Study Group, 1998b. The effectiveness of intraocular pressure reduction in the treatment of normal-tension glaucoma. Collaborative Normal-Tension Glaucoma Study Group. Am. J. Ophthalmol. 126, 498e505. Fan, B.J., Wang, D.Y., Cheng, C.Y., Ko, W.C., Lam, S.C., Pang, C.P., 2009. Different WDR36 mutation pattern in Chinese patients with primary open-angle glaucoma. Mol. Vis. 15, 646e653. Fan, B.J., Wiggs, J.L., 2011. Glaucoma: genes, phenotypes, and new directions for therapy. J. Clin. Invest 120, 3064e3072. Fingert, J.H., 2011. Primary open-angle glaucoma genes. Eye (Lond.) 25, 587e595. Fingert, J.H., Alward, W.L., Kwon, Y.H., Shankar, S.P., Andorf, J.L., Mackey, D.A., Sheffield, V.C., Stone, E.M., 2007. No association between variations in the WDR36 gene and primary open-angle glaucoma. Arch. Ophthalmol. 125, 434e436. Fingert, J.H., Robin, A.L., Stone, J.L., Roos, B.R., Davis, L.K., Scheetz, T.E., Bennett, S.R., Wassink, T.H., Kwon, Y.H., Alward, W.L., Mullins, R.F., Sheffield, V.C., Stone, E.M., 2011. Copy number variations on chromosome 12q14 in patients with normal tension glaucoma. Hum. Mol. Genet. 20, 2482e2494.

Footz, T.K., Johnson, J.L., Dubois, S., Boivin, N., Raymond, V., Walter, M.A., 2009. Glaucoma-associated WDR36 variants encode functional defects in a yeast model system. Hum. Mol. Genet. 18, 1276e1287. Freed, E.F., Bleichert, F., Dutca, L.M., Baserga, S.J., 2010. When ribosomes go bad: diseases of ribosome biogenesis. Mol. Biosyst. 6, 481e493. Fumagalli, S., Thomas, G., 2011. The role of p53 in ribosomopathies. Semin. Hematol. 48, 97e105. €sl, M.R., Gallenberger, M., Meinel, D.M., Kroeber, M., Wegner, M., Milkereit, P., Bo Tamm, E.R., 2011. Lack of WDR36 leads to preimplantation embryonic lethality in mice and delays the formation of small subunit ribosomal RNA in human cells in vitro. Hum. Mol. Genet. 20, 422e435. Hauser, M.A., Allingham, R.R., Linkroum, K., Wang, J., LaRocque-Abramson, K., Figueiredo, D., Santiago-Turla, C., del Bono, E.A., Haines, J.L., Pericak-Vance, M.A., Wiggs, J.L., 2006. Distribution of WDR36 DNA sequence variants in patients with primary open-angle glaucoma. Invest. Ophthalmol. Vis. Sci. 47, 2542e2546. Hewitt, A.W., Dimasi, D.P., Mackey, D.A., Craig, J.E., 2006. A glaucoma case-control study of the WDR36 gene D658G sequence variant. Am. J. Ophthalmol. 142, 324e325. Higginbotham, E.J., Gordon, M.O., Beiser, J.A., Drake, M.V., Bennett, G.R., Wilson, M.R., Kass, M.A., 2004. The ocular hypertension treatment study: topical medication delays or prevents primary open-angle glaucoma in African American individuals. Arch. Ophthalmol. 122, 813e820. Johnson, M., 2006. What controls aqueous humour outflow resistance? Exp. Eye Res. 82, 545e557. Junglas, B., Kuespert, S., Seleem, A.A., Struller, T., Ullmann, S., Bosl, M., Bosserhoff, A., Kostler, J., Wagner, R., Tamm, E.R., Fuchshofer, R., 2012. Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork. Am. J. Pathol. 180, 2386e2403. Karnovsky, M.J., 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron-microscopy. J. Cell. Biol. 27, 137e138. Kass, M.A., Heuer, D.K., Higginbotham, E.J., Johnson, C.A., Keltner, J.L., Miller, J.P., Parrish 2nd, R.K., Wilson, M.R., Gordon, M.O., 2002. The ocular hypertension treatment study: a randomized trial determines that topical ocular hypotensive medication delays or prevents the onset of primary open-angle glaucoma. Arch. Ophthalmol. 120, 701e713 discussion 829-730. Kramer, P.L., Samples, J.R., Monemi, S., Sykes, R., Sarfarazi, M., Wirtz, M.K., 2006. The role of the WDR36 gene on chromosome 5q22.1 in a large family with primary open-angle glaucoma mapped to this region. Arch. Ophthalmol. 124, 1328e1331. Kressler, D., Hurt, E., Bassler, J., 2010. Driving ribosome assembly. Biochim. Biophys. Acta 1803, 673e683. Kroeber, M., Davis, N., Holzmann, S., Kritzenberger, M., Shelah-Goraly, M., Ofri, R., Ashery-Padan, R., Tamm, E.R., 2010. Reduced expression of Pax6 in lens and cornea of mutant mice leads to failure of chamber angle development and juvenile glaucoma. Hum. Mol. Genet. 19, 3332e3342. Kwon, Y.H., Fingert, J.H., Kuehn, M.H., Alward, W.L., 2009. Primary open-angle glaucoma. N. Engl. J. Med. 360, 1113e1124. Lerch-Gaggl, A., Haque, J., Li, J., Ning, G., Traktman, P., Duncan, S.A., 2002. Pescadillo is essential for nucleolar assembly, ribosome biogenesis, and mammalian cell proliferation. J. Biol. Chem. 277, 45347e45355. Leske, M.C., Heijl, A., Hussein, M., Bengtsson, B., Hyman, L., Komaroff, E., 2003. Factors for glaucoma progression and the effect of treatment: the early manifest glaucoma trial. Arch. Ophthalmol. 121, 48e56. Liu, Y., Allingham, R.R., 2011. Molecular genetics in glaucoma. Exp. Eye. Res. 93, 331e339. Matsson, H., Davey, E.J., Draptchinskaia, N., Hamaguchi, I., Ooka, A., Leveen, P., Forsberg, E., Karlsson, S., Dahl, N., 2004. Targeted disruption of the ribosomal protein S19 gene is lethal prior to implantation. Mol. Cell. Biol. 24, 4032e4037. McGowan, K.A., Mason, P.J., 2011. Animal models of Diamond Blackfan anemia. Semin. Hematol. 48, 106e116. Miyazawa, A., Fuse, N., Mengkegale, M., Ryu, M., Seimiya, M., Wada, Y., Nishida, K., 2007. Association between primary open-angle glaucoma and WDR36 DNA sequence variants in Japanese. Mol. Vis. 13, 1912e1919. Monemi, S., Spaeth, G., DaSilva, A., Popinchalk, S., Ilitchev, E., Liebmann, J., Ritch, R., Heon, E., Crick, R.P., Child, A., Sarfarazi, M., 2005. Identification of a novel adultonset primary open-angle glaucoma (POAG) gene on 5q22.1. Hum. Mol. Genet. 14, 725e733. Morrison, R.S., Kinoshita, Y., Johnson, M.D., Guo, W., Garden, G.A., 2003. p53dependent cell death signaling in neurons. Neurochem.. Res. 28, 15e27. Narla, A., Ebert, B.L., 2010. Ribosomopathies: human disorders of ribosome dysfunction. Blood 115, 3196e3205. Newton, K., Petfalski, E., Tollervey, D., Caceres, J.F., 2003. Fibrillarin is essential for early development and required for accumulation of an intron-encoded small nucleolar RNA in the mouse. Mol. Cell. Biol. 23, 8519e8527. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T., Nishimune, Y., 1997. 'Green mice' as a source of ubiquitous green cells. FEBS Lett. 407, 313e319. Pang, C.P., Fan, B.J., Canlas, O., Wang, D.Y., Dubois, S., Tam, P.O., Lam, D.S., Raymond, V., Ritch, R., 2006. A genome-wide scan maps a novel juvenile-onset primary open angle glaucoma locus to chromosome 5q. Mol. Vis. 12, 85e92. Pasutto, F., Mardin, C.Y., Michels-Rautenstrauss, K., Weber, B.H., Sticht, H., ChavarriaSoley, G., Rautenstrauss, B., Kruse, F., Reis, A., 2008. Profiling of WDR36 missense variants in German patients with glaucoma. Invest. Ophthalmol. Vis. Sci. 49, 270e274.

M. Gallenberger et al. / Experimental Eye Research 128 (2014) 83e91 Pasutto, F., Matsumoto, T., Mardin, C.Y., Sticht, H., Brandstatter, J.H., MichelsRautenstrauss, K., Weisschuh, N., Gramer, E., Ramdas, W.D., van Koolwijk, L.M., Klaver, C.C., Vingerling, J.R., Weber, B.H., Kruse, F.E., Rautenstrauss, B., Barde, Y.A., Reis, A., 2009. Heterozygous NTF4 mutations impairing neurotrophin-4 signaling in patients with primary open-angle glaucoma. Am. J. Hum. Genet. 85, 447e456. Quigley, H.A., 2011. Glaucoma. Lancet 377, 1367e1377. Rezaie, T., Child, A., Hitchings, R., Brice, G., Miller, L., Coca-Prados, M., Heon, E., Krupin, T., Ritch, R., Kreutzer, D., Crick, R.P., Sarfarazi, M., 2002. Adult-onset primary open-angle glaucoma caused by mutations in optineurin. Science 295, 1077e1079. Richardson, K.C., Jarret, L., Finke, H., 1960. Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol. 35, 313e323. Romanova, L.G., Anger, M., Zatsepina, O.V., Schultz, R.M., 2006. Implication of nucleolar protein SURF6 in ribosome biogenesis and preimplantation mouse development. Biol. Reprod. 75, 690e696. Schultze, W.H., 1972. Über das Paraphenylendiamin in der histologischen €rbetechnik und über eine neue Schnellf€ Fa arbemethode der Nervenmarkscheide am Gefrierschnitt. Zentralbl. Pathol 36, 639e640. Seitz, R., Tamm, E.R., 2013. N-methyl-D-aspartate (NMDA)-mediated excitotoxic damage: a mouse model of acute retinal ganglion cell damage. Methods Mol. Biol. 935, 99e109.

91

Skarie, J.M., Link, B.A., 2008. The primary open-angle glaucoma gene WDR36 functions in ribosomal RNA processing and interacts with the p53 stressresponse pathway. Hum. Mol. Genet. 17, 2474e2485. Stone, E.M., Fingert, J.H., Alward, W.L.M., Nguyen, T.D., Polansky, J.R., Sunden, S.L.F., Nishimura, D., Clark, A.F., Nystuen, A., Nichols, B.E., Mackey, D.A., Ritch, R., Kalenak, J.W., Craven, E.R., Sheffield, V.C., 1997. Identification of a gene that causes primary open angle glaucoma. Science 275, 668e670. Tamm, E.R., Toris, C.B., Crowston, J.G., Sit, A., Lim, S., Lambrou, G., Alm, A., 2007. Basic science of intraocular pressure. In: Weinreb, R.N., Brandt, J.D., GarwayHeath, D., Medeiros, F. (Eds.), Intraocular Pressure. Reports and Consensus Statements of the 4th Global AIGS Consensus Meeting on Intraocular Pressure. Kugler Publications, Amsterdam, pp. 1e14. The AGIS Investigators, 2000. The advanced glaucoma intervention study (AGIS): 7. The relationship between control of intraocular pressure and visual field deterioration. Am. J. Ophthalmol. 130, 429e440. Weisschuh, N., Wolf, C., Wissinger, B., Gramer, E., 2007. Variations in the WDR36 gene in German patients with normal tension glaucoma. Mol. Vis. 13, 724e729. Zhang, J., Tomasini, A.J., Mayer, A.N., 2008. RBM19 is essential for preimplantation development in the mouse. BMC Dev. Biol. 8, 115.