|Molecular Vision 2007;
Received 12 March 2007 | Accepted 17 August 2007 | Published 27 August 2007
Embryonic expression of the optineurin (glaucoma) gene in different stages of mouse development
1Molecular Ophthalmic Genetics Laboratory, Departments of Surgery and 2Pharmacology, University of Connecticut Health Center, Farmington, CT
Correspondence to: Mansoor Sarfarazi, University of Connecticut Health Center, Molecular Ophthalmic Genetics Laboratory, 263 Farmington Ave., Farmington, CT, 06030-1110; Phone: (860) 679-3629; FAX: (860) 679-7524; email: email@example.com
Purpose: To analyze optineurin (Optn) gene expression in various embryonic stages of mouse development by whole mount in situ hybridization.
Methods: FVB/NcrlBR mouse embryos (10.5 and 13.5 dpc) were collected by timed breeding experiments. A 712 bp Optn cDNA fragment was amplified by PCR and cloned into a transcription vector pCR®II-TOPO®. Digoxigenin labeled sense and antisense RNA probes were generated by in vitro transcription. The labeled RNA probe was localized using an anti-digoxigenin antibody conjugated with alkaline phosphatase. Colorimetric detection was performed with substrate solution containing, 4-nitro-blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP).
Results: This study revealed that the developing eye represents a major expression site for Optn. At both 10.5 and 13.5 dpc a strong specific expression was detected in the outer layer of the optic cup (future pigment layer of the retina). This is in contrast to the expression of another glaucoma gene, Cyp1b1, the expression of which at this state is only limited to the inner (neural) layer of the optic cup (future nervous layer of the retina). Inspection of sections from the cephalic region of whole mounts also revealed limited Optn staining in the lens as well as in the optic nerve. A second Optn expression domain was detected at the base of the developing forelimb. The biological significance of this observation is not clear and remains to be determined.
Conclusions: Eye and forelimb were identified as two major sites for expression of the Optn gene. These findings suggest that Optn expression is triggered during early stages of eye development. Expression of the Optn gene in ocular tissues during mouse embryogenesis correlates with the presence and distribution of the optineurin protein, as previously reported in adult ocular tissues. These findings are also in agreement with the predicted function of Optn protein in the eye and the role of its ortholog in human glaucoma. Further investigations are required to determine the molecular mechanisms of Optn in the developing murine forelimb.
Glaucoma is one of the leading causes of blindness worldwide [1,2]. The disease is caused by gradual degeneration of retinal ganglion cells and secondary loss of axons that carry visual information from eyes to the brain. Our previous studies revealed that human mutations in the optic neuropathy inducing protein (Optineurin; OPTN) gene are associated with the GLC1E-linked  primary open-angle glaucoma (POAG) phenotype . Our further studies on optineurin cloning, with genomic and protein characterization in both mouse and Macaca mulatta showed that both OPTN and its encoded protein are evolutionary conserved [5,6]. As the mouse is a well-established animal model for study of mammalian development, this study was designed to investigate the spatial and temporal expression of Optn mRNA during various stages of mouse embryogenesis by in situ hybridization.
Male and female FVB/NCrlBR mice were purchased from a commercial breeder (Charles River Laboratories, Wilmington, MA). For timed mating experiments, breeding units (one male and two females) were set at 3:30-4:00 PM. Inspection for a vaginal plug was performed before 9:00 AM on the following day. For staging purposes, the day that a vaginal plug was observed was designated as 0.5 days post-conception (dpc). Embryos were dissected from pregnant mice at eight stages of their development between 8.5 to 13.5 dpc. Embryos were freed from extra-embryonic membranes, rinsed immediately with phosphate-buffered saline (PBS), and fixed overnight in fresh 4% paraformaldehyde in PBS at 4 °C. The next day, they were dehydrated through a methanol series (25%, 50%, 75%, and 2X 100%) and stored in 100% methanol at -20 °C. All animal manipulations were conducted in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and were approved by The Animal Care Committee (ACC) of the University of Connecticut Health Center.
In situ hybridization
The in situ hybridization protocol used in this study has already been described in detail [7,8]. Briefly, a 712 bp Optn-specific cDNA fragment, corresponding to amino acid residues 83-320 (GenBank AY071834) was amplified by the PCR primers 5'-GCC TGT TGT TTG AGA TGC AA-3' and 3'-TGT GCC TCT TGA AGC TCC TT-5' and subcloned into the pCR®II-TOPO® transcription vector (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Antisense and sense RNA probes, labeled with digoxigenin-UPT, were generated by in vitro transcription of linearized plasmids with SP6 and T7 RNA polymerase (Roche Molecular Biochemicals, Mannheim, Germany) according to the protocol supplied by the manufacturer. The digoxigenin-labeled RNA was localized using a Fab fragment from a sheep anti-digoxigenin antibody, conjugated with alkaline phosphatase. Colorimetric detection of the digoxigenin-labeled probe was performed with substrate solution containing 350 mg/ml of nitroblue tetrazolium (NBT) and 175 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP). For sectioning, the whole mounts were fixed with 4% paraformaldehyde at 4 °C overnight, washed several times in 1X PBS and, equilibrated overnight in 30% sucrose in PBS. Next, the embryos were mounted on cryostat chuck with Tissue Tek® OCT (Sakura Finetek U.S.A., Inc., Torrance, CA). Sections of 20-50 μm were cut on cryostat and mounted under a coverslip with Gel/MountTM (Biomeda Corp., Foster City, CA).
In order to decipher the Optn gene function, we used nonisotopic whole-mount in situ hybridization of mRNA on a series of murine embryos and investigated Optn expression patterns during early embryogenesis. Inspection of the whole-mount embryos reveled strong and well-delineated Optn expression domain in the optic vesicle at age 10.5 dpc (Figure 1A-C). Optn transcripts were detectable at the forelimb buds as the second expression domain (Figure 1B,D). Examination of optic vesicle sections at age 10.5 dpc revealed a strong expression of Optn in the outer layer of the optic cup (future pigment layer of retina; Figure 2). At age 13.5 dpc, expression of Optn in the developing eye became even more prominent. The outer layer of the optic cup (future pigment layer of retina) showed a confined positive staining at a higher power view (Figure 3A,B). A slight expression signal was also present at the lens fibers and the anterior epithelium of the lens (Figure 3C). Similarly, the developing optic nerve and the axons of ganglion cells might show some expression of Optn mRNA (Figure 3D). However, difficulties of probe penetration into whole embryos, a common limiting factor for application of the whole-mount in situ hybridization assay, may have led to undetectable expression domains, particularly in tissues located more deeply and internally.
In our previous studies, we reported the association of mutations in human optineurin with adult-onset primary open-angle glaucoma . We further characterized the mouse ortholog of the human OPTN gene and showed a high degree of conservation between their gene and protein sequences . Expression analysis of optineurin gene in multiple adult mouse tissues by northern blotting revealed presence of Optn transcripts in various tissues . The optineurin protein in mouse is expressed in ocular tissues similar to its human and Macaca mulatta counterparts and it is associated intracellularly to vesicular structures near the nucleus [5,6]. The objective of this study was to further characterize ocular manifestation of Optn, and to profile its mRNA expression during eye development in the mouse embryo. Whole-mount in situ hybridization during days 10.5-13.5 of mouse embryos revealed very prominent expression in two specific domains, the eye and forelimb bud. By RT-PCR (reverse transcriptase PCR) the Optn transcript was detectable at 7.0 dpc in the mouse embryo , however, the small size and fragility of the embryos at ages of 8.5 and 9.0 dpc made it more difficult to observe the eye vesicle during our in situ hybridization evaluation. Similar to our findings, De Marco et al. , by in situ hybridization, also observed a prominent expression of Optn RNA in developing murine eyes at 10.5 dpc. Expression of Optn RNA in adult murine ocular tissues (i.e., cornea, lens, sclera, and remarkably in retina) have been observed by northern blot analysis [9,10]. Specific localization of Optn transcripts in ocular tissues of mouse embryos are in strong agreement with our previous findings and distribution of optineurin protein in lens fibers and the retinal layers of adult mouse eye . Our previous observation  has been further confirmed by other independent studies that showed expression of optineurin protein in the anterior segment, retina, and optic nerve by immunohistochemistry [9,10]. The restricted ocular expression throughout embryogenesis implies that Optn mRNA expression is developmentally regulated in a tissue-specific manner. Our findings further suggest that during embryogenesis, Optn may play a pivotal role in overall development of the eye. In adult murine eyes, the optineurin protein is highly expressed by retinal ganglion cells (RGCs) [5,9,10]. Furthermore, a transgenic mouse for the optineurin E50K mutation has recently been developed [11,12]. This study reported that overexpression of optineurin mutation promoted RGCs to go through apoptosis and thus leads to the cupping of the optic disc while the intraocular pressure remains within a normal range [11,12]. This observed phenotype is the most important resemblance to the reported clinical condition in glaucomatous neurodegeneration [13,14] and is compatible with the glaucomatous phenotype caused by the Optn-E50K mutation [4,15].
Future studies are needed to fully define the molecular mechanisms of Optn expression in the developing murine forelimb. Murine forelimb initiation precedes hindlimb development by roughly one gestational day at the age of 9.5 dpc. The presence of Optn transcripts only in the forelimb could be due to different timing of gene expression between the limbs or, alternatively, Optn could acts as a forelimb specific gene. A different transcriptome, as well as expression timing profiles, between the fore- and hindlimb has been reported . There is a remarkable conservation of gene transcripts between the two limb types, however, differential expression and utilization of commonly expressed genes, together with the small number of genes that are expressed in a specific limb type, may lead to limb-type-specific differentiation .
Nonisotopic whole-mount in situ hybridization of mRNA has greatly facilitated the precise three-dimensional localization of transcripts from genes whose expression is important during development. Due to the high sensitivity, expression profiling by PCR frequently delivers data that is difficult to prioritize. For example, PCR methods would detect expression of Optn and Cyp1b1 (Cytochrome P4501b1), a gene that is frequently mutated in patients with primary congenital glaucoma (PCG), in a wide range of tissues. Contrarily, as presented here and by our previous study  the whole mount in situ hybridization only identified the eye as a major expression domain in the developing embryo for both genes. This organ is significantly involved with the two different pathological phenotypes of glaucoma (PCG and POAG), both associated with the mutations in Cyp1b1 and Optn genes, respectively. Therefore, whole mount in situ hybridization delivers expression data that is biologically significant. The very restrictive and specific ocular expression of Optn as revealed in this study suggests that like the PCG-causative gene of Cyp1b1, the Optn may also play a significant role during the early stages of eye development. Interestingly the expression domains of Cyp1b1 and Optn do not overlap. Optn expression is limited to the outer layer of the optic cup while Cyp1b1 is expressed in the inner (neural) layer of the optic cup (future nervous layer of retina) . Specific expression profiles and involvements of both Cyp1b1 and Optn during early stages of eye development are compatible with their reported roles in two human phenotypes of primary congenital glaucoma and primary open angle glaucoma. Furthermore, comparable phenotypes as observed in the Cyp1b1-null mouse  and Optn-E50K transgenic animals [11,12] further corroborate the roles of these two genes in early stages of eye development and in human glaucoma.
This work was supported by NIH grant EY-014959.
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