Localization of MYOC Transcripts in Human Eye and Optic Nerve by In Situ Hybridization

Abstract

purpose. To evaluate MYOC (myocilin) gene expression at the RNA level in normal intact human eyes and optic nerve using in situ hybridization.

methods. Normal human eyes and optic nerves from donors 62 to 83 years of age with no history of glaucoma were fixed, embedded in paraffin, and sectioned. Sections were hybridized with ³⁵S-labeled sense and antisense riboprobes derived from a full-length MYOC cDNA.

results. High levels of MYOC expression were observed throughout the trabecular meshwork as well as in the most anterior nonfiltering meshwork (Schwalbe’s line), in the scleral spur, and in the endothelial lining of Schlemm’s canal. MYOC transcripts were also detected in the anterior corneal stroma, in the ciliary muscle, beneath the anterior border of the iris, in the iris stroma, and in the sclera. Expression in the retrolaminar region of the optic nerve was present in the pial septa that divide the nerve fiber bundles, in the perivascular connective tissue surrounding the central retinal vessels, and in the dura mater, arachnoid, and pia mater of the meningeal sheath surrounding the optic nerve.

conclusions. MYOC gene expression in the trabecular meshwork, Schlemm’s canal, scleral spur, and ciliary muscle indicates a structural or functional role for myocilin in the regulation of aqueous humor outflow that may influence intraocular pressure. MYOC expression in the optic nerve suggests that changes in the structural, metabolic, or neurotropic support of the optic nerve may influence its susceptibility to glaucomatous damage.

Primary open-angle glaucoma (POAG) is the most common form of glaucoma in the United States, affecting 1% to 2% of the population more than 40 years of age, and is the second leading cause of blindness. ¹ POAG is a slowly progressive optic neuropathy that results in irreversible damage to the ganglion cell layer and nerve fiber layer of the retina, death of optic nerve axons, and collapse of the lamina cribrosa, leading to excavation of the optic nerve head and visual field loss. Elevated intraocular pressure caused by an increase in aqueous humor outflow resistance through the trabecular meshwork is frequently associated with POAG. Ocular hypertension is a major risk factor for the disease, and modulation of intraocular pressure continues to be the mainstay of glaucoma therapy. 

After the identification of the myocilin gene (MYOC) and its association with juvenile-onset open-angle glaucoma (JOAG) and typical late-onset POAG, ^{2 3 4} much effort has been focused on understanding the normal role of myocilin in the eye, the effects of alterations in myocilin protein levels, and the contribution of dysfunctional forms of myocilin to the pathophysiology of POAG. Myocilin is a novel 57-kDa olfactomedin-related protein of yet undetermined function. Although the role of the olfactomedin-like domain in the pathophysiology of POAG is unknown, the evolutionary conservation of olfactomedin ⁵ and the frequency of pathogenic mutations observed in the related myocilin domain ^{6 7} and the influence of this domain on myocilin subunit interaction, ^{8 9} possible phosphorylation, ¹⁰ Triton solubility, ¹¹ and translational processing, ¹² imply that it plays an important role in the correct structure or function of the protein. Myocilin is found in multiple forms, both cellularly and extracellularly, ^{8 9 13 14 15 16 17 18 19} as well as in cultured cells derived from human trabecular meshwork and Schlemm’s canal. ^{8 13 15 16 18} It has been immunolocalized throughout the human eye, ¹⁹ in the trabecular meshwork of normal and glaucomatous human eyes, ^{13 14} and in the connecting cilium of mouse photoreceptor cells. ²⁰  

One hypothesis is that altered myocilin expression or an altered form of the polypeptide may obstruct aqueous humor outflow through the trabecular meshwork and into Schlemm’s canal, leading to ocular hypertension. ^{8 13} A recent report of myocilin immunolocalization in the optic nerve suggests that it also may be a target of glaucomatous damage in MYOC-linked POAG. ¹⁹  

The MYOC gene is widely expressed at the mRNA level, as assessed by Northern blot analysis and reverse transcription–polymerase chain reaction (RT-PCR) analysis of numerous adult human and mouse tissues. ^{8 20 21 22 23 24 25 26} In contrast, the level of expression in developing mouse embryos, embryonic mouse eyes, and human fetal and newborn tissue is relatively low. ^{21 25 27} Examination of dissected human ocular tissues or derived cell lines by Northern blot analysis and RT-PCR, as well as in situ hybridization analysis of mouse eyes and human trabecular meshwork, has demonstrated widespread MYOC expression in a number of structures, including the ciliary body, trabecular meshwork, iris, sclera, choroid, and retina. ^{13 20 21 22 26 27 28 29}  

To date, there has been no comprehensive analysis of MYOC gene expression at the mRNA level in the intact human eye. To carefully evaluate expression in normal human eyes, we used in situ hybridization to localize MYOC transcripts in ocular tissues and expanded the study to include the optic nerve, which is the primary site of glaucomatous optic neuropathy. The widespread MYOC gene expression observed in this study suggests an important role for myocilin in the structure and function of the eye. 

Methods

Collection of Human Eyes

Five human eyes from five donors 62 to 83 years of age were obtained within 6 hours after death from the Iowa Lions Eye Bank. Donors had no known history of glaucoma or other eye diseases. Anterior segments of the enucleated eyes were isolated by a circumferential cut made posterior to the iris. The optic nerve head together with the adjacent optic nerve was dissected from the enucleated globe. Tissue was fixed overnight at room temperature in Pen-Fix (Richard Allan Medical, Richland, MI), dehydrated in graded ethanols, and embedded in paraffin. 

In Situ Hybridization

Serial 7-μm sections were mounted onto slides (Superfrost Plus; Fisher Scientific, Fairlawn, NJ) and were hybridized with ³⁵S-labeled sense and antisense MYOC riboprobes generated from a full-length MYOC cDNA ²³ that was subcloned into pBluescript II SK (Stratagene, La Jolla, CA), linearized, and transcribed in vitro using T3 and T7 RNA polymerases. Hybridization with labeled sense RNA riboprobes served as controls for nonspecific hybridization, and in all cases, no specific hybridization was observed. In situ hybridization was performed as described previously. ²⁵ Briefly, tissue sections mounted on slides were hybridized overnight at 50°C in 50% formamide, 1× STE (0.3 M NaCl, 20 mM Tris [pH 8.0], and 1 mM EDTA), 80 μg/ml denatured salmon sperm DNA, 1× Denhardt’s solution, 10% dextran sulfate, 500 μg/ml yeast tRNA, and 0.1 M dithiothreitol (DTT). After hybridization, slides were washed twice in 5× SSC-0.01 M DTT at 50°C for 30 minutes each, and once in 2× SSC-50% formamide at 60°C for 30 minutes After treatment with RNAses A and T1, slides were further washed in 2× SSC at 37°C, 0.1× SSC at 50°C, and 0.1× SSC at room temperature for 15 minutes each. After dehydration, slides were dipped in photographic emulsion (NT2-B; Eastman Kodak; Rochester, NY) and exposed for 1 to 2 weeks at 4°C. 

Slides were developed and counterstained with hematoxylin and photographed with bright-field and dark-field microscopy. Images were collected digitally on a light microscope (Diaplan; Leitz, Rockleigh, NJ) with a cooled CCD camera (model DEI-750; Optronix, Goletta, GA). Images were converted to gray scale and sharpened, with brightness adjusted by computer (Photoshop; Adobe, San Jose, CA). The montages were laid out (IRIS Showcase; Silicon Graphics, Mountain View, CA) on a work station (Indy; Silicon Graphics). 

Northern Blot Analysis

Freshly dissected postmortem human neurosensory retina and retinal pigment epithelium (RPE)-choroid-sclera were frozen in liquid nitrogen and stored at −70°C until use. Total cellular RNA was prepared using RNA-STAT-60 (Tel-Test B, Friendswood, TX), and poly(A) mRNA was isolated (MessageMaker mRNA Isolation System; Gibco, Gaithersburg, MD). One microgram of poly(A) mRNA and RNA standards were electrophoresed through a denaturing 0.8% agarose-formaldehyde gel, and the RNA was transferred to a nylon membrane (Gene Screen Plus; NEN, Boston, MA) using a standard method. The blot was hybridized with a gel-purified insert of the MYOC cDNA plasmid described. ³²P-dCTP DNA labeling, hybridization, and autoradiography were performed as described previously. ²⁵ The blot was stripped of radioactivity and rehybridized with a cDNA probe for β-actin (Clontech, Palo Alto, CA) to assess equal loading of RNA. 

Results

Localization of MYOC Transcripts in the Anterior Segment of the Eye

To examine MYOC gene expression at the mRNA level in the intact normal human eye, transcripts were localized in the anterior segment by in situ hybridization. As shown in Figure 1A , MYOC was widely expressed in longitudinal sections of the eye anterior segment in a pattern that was consistently observed in all five donor eyes. High levels of expression were observed throughout the trabecular meshwork. MYOC transcripts were also present in the anterior corneal stroma, beneath the iris anterior surface, in the iris stroma, in the scleral spur, in the ciliary muscle, and in the sclera. No signal was detected using the control sense strand riboprobe (Fig. 1B) . The autofluorescence observed in the iris posterior layer, ciliary epithelium, and retinal pigment epithelium using both sense and antisense riboprobes is due to the presence of pigment when viewed using dark-field optics and is not a positive hybridization signal. 

As seen in more detail in Figures 2A 2B and 2C , elevated MYOC expression was readily detectable in the most anterior nonfiltering region of the trabecular meshwork in the zone of transition between the corneal and trabecular endothelium known as Schwalbe’s line. High levels of expression were also observed throughout the trabecular meshwork, in the contractile cells of the scleral spur (the region of the sclera between the ciliary body and Schlemm’s canal), and in the endothelial lining of Schlemm’s canal. 

In the ciliary body, a relatively high level of MYOC expression was observed in the ciliary muscle that regulates aqueous humor outflow through trabecular and uveoscleral pathways (Figs. 2D 2E 2F) . In other regions of the eye, MYOC expression was noted beneath the anterior border of the iris and in the iris stroma (Figs. 3A 3B 3C ), in a punctate pattern surrounding scleral fibroblasts (Figs. 3D 3E 3F) , and in the anterior corneal stroma (Fig. 4) . To discount the possibility that the MYOC antisense riboprobe corresponding to the full-length cDNA may have cross-hybridized with other olfactomedin-related transcripts, we used an MYOC 3′untranslated region (UTR)–specific antisense riboprobe for in situ hybridization with adjacent sections and observed the same signal localization as that seen with the cDNA antisense riboprobe (data not shown). 

MYOC Expression in the Neurosensory Retina

As shown in Figure 5 , MYOC expression in the human retina was undetectable by in situ hybridization analysis. Our results may be the consequence of postmortem retinal tissue fragility or may be due to a low level of MYOC expression that was undetectable as the result of the high-stringency hybridization conditions and posthybridization washes used in our study. 

To determine the abundance of MYOC mRNA in retinal tissue, poly(A) mRNA was isolated from freshly dissected human neurosensory retina and from the RPE-choroid-sclera and analyzed by Northern blot analysis. As shown in Figure 6 , MYOC expression was undetectable in the neurosensory retina after prolonged autoradiography, suggesting that MYOC mRNA abundance in this tissue is low or that the tissue had undergone partial degradation. The signal observed in the RPE-choroid-sclera layer can be attributed to MYOC transcripts observed in the sclera as seen by in situ hybridization (Figs. 1 3) . 

Localization of MYOC Transcripts in the Optic Nerve

MYOC transcripts were localized in the retrolaminar region of the optic nerve in sections cut approximately 500 μm distal to the optic nerve head, by using in situ hybridization (Figs. 7) . Adjacent sections stained with Luxol fast blue verified myelination of the optic nerve axons (data not shown). As seen in greater detail in Figures 8A 8B and 8C , MYOC expression was noted in the perivascular tissue surrounding the central retinal artery and vein. MYOC expression was also observed in the dura mater, the outer layer of the meningeal sheath composed of dense bundles of collagen and elastic tissue that surrounds and protects the intraorbital optic nerve (Figs. 8D 8E 8F) . MYOC transcripts were also detected in the intermediate meningeal layer, the arachnoid, which is made up of delicate connective tissue trabeculae lined by meningothelial cells. Consistent with our earlier observation of MYOC expression in the pia mater of the adult mouse brain, ²⁵ MYOC transcripts were also observed in the pia mater of the human optic nerve; the innermost meningeal layer consisting of fibrous tissue with multiple small blood vessels. MYOC expression was noted in the pial septa, a vascularized connective tissue derived from the pia mater and composed of collagen, elastic tissue, fibroblasts, nerves, and small arterioles and venules. The pial septa divide the optic nerve fibers into bundles and provide mechanical support for the nerve bundles as well as metabolic support to the axons and glial cells as they traverse the optic canal. 

Discussion

The progressive neuronal damage that is characteristic of POAG most likely represents the culmination of various types of injury, including chronic trauma from pressure on the retinal ganglion cell body or axon, ischemia caused by vascular compromise, neurochemical damage, or an accelerated activation of the retinal ganglion cell apoptotic pathway. ^{30 31} The elevated intraocular pressure frequently associated with POAG is correlated with compression, stretching, and remodeling of the extracellular matrix of the lamina cribrosa and astrocytes of the optic nerve head. ^{32 33} These structures normally provide mechanical and nutritive support to the retinal ganglion cells as they exit the eye. During the progression of POAG, changes in the connective tissue support of the optic nerve head may increase the susceptibility of axonal damage, resulting in interference with regional axoplasmic transport, compromised blood flow, or mechanical impingement of the nerve axons in the optic nerve head. In addition to studies of the optic nerve head, the trabecular meshwork and ciliary muscle also play important roles in regulating intraocular pressure in the anterior segment of the eye. Dysregulation of ciliary muscle function or damage to the trabecular meshwork result in elevated intraocular pressure that is frequently associated with POAG and may contribute to the optic neuropathy. 

After the identification of the MYOC gene and its association with POAG, ⁴ much effort has been focused on understanding the role of myocilin in the pathophysiology of POAG. In this report, we used in situ hybridization to localize MYOC transcripts in normal human ocular tissues and optic nerve. Expression throughout the anterior segment was widespread. High levels of MYOC expression were seen throughout the trabecular meshwork, in agreement with previously published results using Northern blot analysis. ^{21 27} Expression was also present in the scleral spur and in Schwalbe’s line, the nonfiltering region of the trabecular meshwork in the zone of transition between the corneal and trabecular endothelium. The elevated MYOC expression pattern seen in the trabecular meshwork is significant, because ocular hypertension is thought to result from increased aqueous humor outflow resistance through the trabecular meshwork, particularly the juxtacanalicular tissue and inner wall of Schlemm’s canal. ³⁴ Although the mechanism underlying outflow resistance in glaucoma is not yet clear, it is associated with ultrastructural and biochemical changes in the trabecular meshwork including deposition of extracellular material within the meshwork and beneath the endothelial lining of Schlemm’s canal. ³⁵  

A role for myocilin in the structure or function of the trabecular meshwork was first suggested by its synthesis and secretion into the culture media of human meshwork cells after long-term treatment with dexamethasone. ^{8 13} MYOC transcripts were subsequently localized in the trabecular meshwork of normal human eyes by in situ hybridization, ²⁹ and myocilin immunostaining in normal eyes has been reported throughout the trabecular meshwork and in the anterior nonfiltering region of the meshwork (Schwalbe’s line) and more abundantly in these regions of glaucomatous eyes. ^{14 19} MYOC gene expression in both the trabecular meshwork and ciliary muscle supports a role for altered MYOC expression or an altered form of the polypeptide that may be dysregulated in the diseased state and contribute to ocular hypertension. ^{8 13} It is noteworthy that not all patients with POAG who bear MYOC mutations have elevated intraocular pressure. A recent report of a relatively young patient with POAG with normal ocular tension, who had MYOC Gln368Stop mutation in exon 3, ³⁶ which is usually associated with moderately elevated intraocular pressure POAG, ^{6 7} suggests a more complex disease pathogenesis that may involve other proteins that interact with myocilin and merits further investigation. 

In addition to MYOC expression in the trabecular meshwork, transcripts were also detected in keratocytes of the anterior corneal stroma, beneath the anterior border of the iris, in the iris stroma, in scleral fibroblasts, and in the ciliary muscle. Expression was undetectable in the ciliary epithelium. This result differs somewhat from reports of Myoc expression in the mouse ciliary epithelium using in situ hybridization, ²⁸ myocilin immunolocalization in the ciliary epithelium and ciliary muscle of the human ciliary body, ¹⁹ and RT-PCR analysis that demonstrated a high level of gene expression in cultured human ciliary muscle cells compared with a relatively low level of expression in a human nonpigmented ciliary epithelial cell line. ²⁶ One explanation for our results is that a low level of MYOC expression in the nonpigmented ciliary epithelium together with our use of higher stringency in situ hybridization conditions and posthybridization washes compared with those of Takahashi et al. ²⁸ may have resulted in absence of detectable expression. 

In contrast to reports of Myoc expression in whole mouse retina, as assessed by Northern blot analysis ²⁴ ; in murine retinal photoreceptor cells and the ganglion cell layer, as assessed by in situ hybridization ²⁸ ; and in the human retinal nerve fiber layer and the inner and outer layers of photoreceptors, as assessed by myocilin immunostaining, ¹⁹ we were unable to detect MYOC expression in the human retina. MYOC transcripts in human retina have also been reported to be undetectable by Northern blot analysis ²⁶ or to be present in low abundance after prolonged autoradiography. ²¹ The integrity of the poly(A) mRNA used for our Northern blot analysis and the adjacent retinal tissue sections used for in situ hybridization analysis were verified independently and were shown to be reactive with a probe for NR2E3, a nuclear receptor gene associated with enhanced S cone syndrome, that is expressed in the neurosensory retina in greater abundance than MYOC. ³⁷ Although we cannot rule out the possibility of partial postmortem RNA degradation, our inability to detect MYOC transcripts in the neurosensory retina most likely resulted from a low level of gene expression coupled with the high-stringency hybridization conditions used in the in situ hybridization analysis. 

We have provided new data in the present study that MYOC transcripts are localized in the retrolaminar region of the optic nerve—notably, in the perivascular tissue surrounding the central retinal vessels, in the vascularized pial septa that divide and support the nerve fiber bundles, and in the dura mater, arachnoid, and pia mater of the meninges surrounding the optic nerve. These specialized connective tissues contribute structural support to the central retinal artery and vein, mechanical and nutritive support to the retinal ganglion axons as they traverse the optic nerve, and structural support to the optic nerve itself. We have also detected MYOC expression more anteriorly in glial cells of the optic nerve head using in situ hybridization and immunostaining of normal and glaucomatous eyes (unpublished results, 2000). According to recent reports, myocilin has been immunolocalized in human cultured optic nerve head astrocytes and lamina cribrosa cells derived from normal eyes, ³⁸ in cultured astrocytes derived from glaucomatous eyes, ³⁹ and in the optic nerve axons and lamina cribrosa astrocytes of the intact normal optic nerve head. ¹⁹ Although we did not observe MYOC expression in optic nerve axons, one hypothesis suggests that myocilin is translated in the perikarya of optic nerve ganglions cells in the retina and transported to the optic nerve by axoplasmic flow. ¹⁹  

MYOC expression in the optic nerve head is significant, because this is the site of glaucomatous optic neuropathy, whether associated with a normal or elevated intraocular pressure. It is commonly believed that the site of damage to retinal ganglion axons is at the level of the lamina cribrosa. ⁴⁰ The lamina cribrosa, a fibroelastic connective tissue composed of a specialized extracellular matrix organized into a sievelike meshwork lined by astrocytes, provides mechanical and nutritive support to the axons as they leave the eye. During the progression of glaucoma, changes in the structural support of axons in the lamina cribrosa appear to reflect an aberrant remodeling of the lamina cribrosa’s extracellular matrix, leading to collapse of the cribriform plates and misalignment of its channels that may lead to axoplasmic flow obstruction. ⁴⁰ Studies indicate that the astrocytes may play a major role in the remodeling process. ³⁵ It is not yet clear whether this remodeling is the primary cause of glaucomatous injury or whether other insults, such as elevated intraocular pressure, ischemia, or axonal loss trigger the remodeling of the lamina cribrosa. MYOC expression in the optic nerve adds to a growing body of evidence suggesting that changes in the structural, metabolic, or neurotropic support of the optic nerve may influence its susceptibility to glaucomatous damage. Further analysis of the role of normal myocilin, altered myocilin levels, and dysfunctional myocilin in the structure or function of the optic nerve will provide new insight into the pathophysiology of both normal tension and hypertension in POAG. 

 Supported by the National Institutes of Health Grant R01 EY-10564. VCS is an Associate Investigator for the Howard Hughes Medical Institute.

 Submitted for publication March 16, 2000; revised May 24, 2000; accepted May 31, 2000.

 Commercial relationships policy: E (AC); all others N.

 

Figure 1.

 

Detection of MYOC transcripts in longitudinal sections of normal human eye anterior segments by in situ hybridization. (A) Antisense MYOC riboprobe was localized in the anterior cornea, trabecular meshwork, scleral spur, ciliary muscle, iris, and sclera. This expression pattern was consistently observed in all five donor eyes. (B) Sense (control) MYOC riboprobe shows no hybridization. The autofluorescence observed in the iris posterior layer, ciliary epithelium, and RPE is due to the presence of pigment when viewed using dark-field optics and is not a positive hybridization signal. (C) Bright-field optics illustrate the morphology of the anterior segment. C, Cornea; TM, trabecular meshwork; SC, sclera; L, lens; CB, ciliary body. Original magnification, ×7.5.

Figure 2.

 

Detection of MYOC transcripts in the trabecular meshwork and ciliary muscle of the normal human eye by in situ hybridization. The iridocorneal angle and ciliary body shown in Figure 1 is presented here in more detail. (A) Elevated MYOC expression was noted in the most anterior nonfiltering region of the trabecular meshwork (TM; Schwalbe’s line, SL) and throughout the trabecular meshwork. Signal was also observed in the scleral spur and in the endothelial lining of Schlemm’s canal (SC). Iris (I). The autofluorescence observed in the iris posterior layer and in the ciliary epithelium is due to the presence of pigment when viewed with dark-field optics, using both the MYOC antisense (A) and sense (B; control) riboprobes and is not a positive hybridization signal. (C) Bright-field optics illustrate tissue morphology. (D) MYOC transcripts were present in the ciliary muscle (CM) of the ciliary body. Autofluorescence in the ciliary epithelium is as in (A) and (B), using antisense (D) and sense (E; control) riboprobes. (F) Bright-field optics illustrate tissue morphology. Original magnification, ×55.

Figure 3.

 

Detection of MYOC transcripts in the iris and sclera using in situ hybridization. (A, B, and C) MYOC expression was observed beneath the anterior border of the iris and in the iris stroma. Autofluorescence in the iris posterior layer is as in Figures 2A and 2B , using both MYOC antisense (A) and sense (B; control) riboprobes. (C) Bright-field optics illustrate tissue morphology. (D, E, and F) MYOC expression was observed in a punctate pattern surrounding scleral fibroblasts. Autofluorescence in the RPE is as in (A) and (B) when using the antisense (D) and sense (E; control) riboprobes. (F) Bright-field optics illustrate tissue morphology. Original magnification, ×27.

Figure 4.

 

Detection of MYOC transcripts in the cornea using in situ hybridization. (A) MYOC expression was noted in the keratocytes of the anterior corneal stroma. (B) No signal was detected using the MYOC sense (control) riboprobe. (C) Bright-field optics illustrate tissue morphology. Original magnification, ×27.

Figure 5.

 

MYOC expression in human neurosensory retina. Retinal MYOC expression using antisense (A) and sense (control) (B) riboprobes was undetectable using in situ hybridization. (C) Hematoxylin and eosin–stained section illustrates retinal morphology. The autofluorescence in the RPE (rpe) in (A) and (B) is as in Figures 2A and 2B . gcl, ganglion cell layer; ipl, inner plexiform layer; inl, inner nuclear layer; opl, outer plexiform layer; onl, outer nuclear layer; os, outer segment; rpe, retinal pigment epithelium; ch, chorioid; sc, sclera. Original magnification, ×100.

Figure 6.

 

Northern blot analysis of retinal mRNA. Northern blot analysis of 1μ g of poly(A) mRNA isolated from dissected normal human neurosensory retina and RPE-choroid-sclera and hybridized with a ³²P-labeled MYOC cDNA probe.

Figure 7.

 

Detection of MYOC transcripts in the retrolaminar region of the human optic nerve using in situ hybridization. (A) Antisense MYOC riboprobe was hybridized to optic nerve sections cut 500 μm distal to the optic nerve head and analyzed using dark-field microscopy. Expression was observed in the sclera (SC), dura mater (D), arachnoid (A), pia mater (P), the septa (S) that divide the nerve fibers into bundles, and the perivascular tissue surrounding the central retinal vessels (CRV). (B) MYOC sense (control) riboprobe showed no hybridization. The autofluorescence in the RPE and choroid in (A) and (B) is as in Figures 2A and 2B . (C) Bright-field optics illustrate tissue morphology. Original magnification, ×15.

Figure 8.

 

Detection of MYOC transcripts surrounding the central retinal vessels and in the meninges of the optic nerve using in situ hybridization. Regions of Figure 7 are shown in greater detail. (A) MYOC expression was noted in the perivascular connective tissue surrounding the central retinal vessels (CRV). (B) No signal was detected using the MYOC sense (control) riboprobe. (C) Bright-field optics illustrate tissue morphology. (D) MYOC expression was observed in the pial septa (S), the pia mater (P), the arachnoid (A), and the dura mater (D) of the meninges surrounding the retrolaminar region of the optic nerve. (E) No signal was seen using the MYOC sense (control) riboprobe. (F) Bright-field optics illustrate tissue morphology. Original magnification, ×75.

The authors thank Jim Jung-Ching Lin and Rebecca Reiter for the use of the in situ hybridization facility; the donors and their families, Gregory Hageman and the Lions Eye Bank for the kind gift of human tissue; Adam Kanis for human retinal tissue collection; the Blodi Ocular Pathology Laboratory for tissue processing and embedding, and Martin Cassell, Beata Rymgayllo–Jankowska, and Andrew Lotery for helpful discussions.