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Form-deprivation myopia in chick induces limited changes in retinal gene expression.

ABSTRACT: Evidence has implicated the retina as a principal controller of refractive development. In the present study, the retinal transcriptome was analyzed to identify alterations in gene expression and potential signaling pathways involved in form-deprivation myopia of the chick.One-week-old white Leghorn chicks wore a unilateral image-degrading goggle for 6 hours or 3 days (n = 6 at each time). Total RNA from the retina/(retinal pigment epithelium) was used for expression profiling with chicken gene microarrays (Chicken GeneChips; Affymetrix, Santa Clara, CA). To identify gene expression level differences between goggled and contralateral nongoggled eyes, normalized microarray signal intensities were analyzed by the significance analysis of microarrays (SAM) approach. Differentially expressed genes were validated by real-time quantitative reverse transcription-polymerase chain reaction (qPCR) in independent biological replicates.Small changes were detected in differentially expressed genes in form-deprived eyes. In chickens that had 6 hours of goggle wear, downregulation of bone morphogenetic protein 2 and connective tissue growth factor was validated. In those with 3 days of goggle wear, downregulation of bone morphogenetic protein 2, vasoactive intestinal peptide, preopro-urotensin II-related peptide and mitogen-activated protein kinase phosphatase 2 was validated, and upregulation of endothelin receptor type B and interleukin-18 was validated.Form-deprivation myopia, in its early stages, is associated with only minimal changes in retinal gene expression at the level of the transcriptome. While the list of validated genes is short, each merits further study for potential involvement in the signaling cascade mediating myopia development.

SUBMITTER: McGlinn AM

PROVIDER: S-EPMC1983368 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Form-deprivation myopia in chick induces limited changes in retinal gene expression.

McGlinn Alice M AM Baldwin Donald A DA Tobias John W JW Budak Murat T MT Khurana Tejvir S TS Stone Richard A RA

Investigative ophthalmology & visual science 20070801 8

<h4>Purpose</h4>Evidence has implicated the retina as a principal controller of refractive development. In the present study, the retinal transcriptome was analyzed to identify alterations in gene expression and potential signaling pathways involved in form-deprivation myopia of the chick.<h4>Methods</h4>One-week-old white Leghorn chicks wore a unilateral image-degrading goggle for 6 hours or 3 days (n = 6 at each time). Total RNA from the retina/(retinal pigment epithelium) was used for express ...[more]

PMID: 17652709

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Project description:BACKGROUND:The putative myopia-controlling receptor is thought to be muscarinic acetylcholine receptor subtype M4 , because mamba toxin-3 can inhibit form-deprivation myopia in chicks at a far lower concentration than atropine. However, mamba toxin-3 is equally potent at the human α1A -, α1D -, and α2A -adrenoceptors. To test the hypothesis that α-adrenoceptors might be involved in regulation of eye growth, the treatment effects of α2 -adrenoceptor agonists brimonidine, clonidine, and guanfacine, and antagonist yohimbine, on form-deprivation myopia in the chick were measured. METHODS:Right eyes of White Leghorn chicks were goggled with diffusers to induce form-deprivation myopia; left eyes were left open as controls. Goggled eyes were injected intravitreally with 20 μL of vehicle, or 2, 20, or 200 nmol of brimonidine, clonidine, guanfacine, or yohimbine, 24, 72, and 120 hours after goggle application. Alternatively, myopia was inhibited physiologically by goggle removal for two hours, and the α2 -adrenoceptor antagonist, yohimbine, was injected to test whether it could block this type of myopia inhibition. One day after the last injection, refractive error and axial length were measured. RESULTS:Brimonidine (20 and 200 nmol) and clonidine (200 nmol) effectively inhibited experimentally induced increases in negative refractive error and axial elongation. All doses of guanfacine significantly inhibited induced negative refractive error, but only 20 and 200 nmol significantly inhibited axial elongation. Yohimbine had no effect on form-deprivation myopia, but 200 nmol reduced the myopia-inhibiting effect of goggle removal. CONCLUSION:High concentrations of α2 -adrenoceptor agonists, similar to those required by atropine, inhibited chick form-deprivation myopia; antagonism by yohimbine had no effect. High-concentration yohimbine partially interfered with emmetropisation in form-deprived chicks experiencing normal vision for two hours per day. These data support the hypothesis that treatment with high concentrations of adrenergic drugs can affect experimentally induced myopia and normal visual processes.

Project description:Juvenile primates develop myopia when their visual experience is degraded by lid fusion. In response to the abnormal visual input, retinal neural networks cause an excessive growth of the postequatorial segment of the eye, but the mechanism underlying this axial elongation is unknown. By combining analysis of gene expression, injection of the thymidine analog 5-bromo-2'-deoxyuridine and immunocytochemistry, we show that the retinal periphery in both juvenile rhesus macaques and green monkeys harbors a population of mitotically active neuroprogenitor cells that proliferate when the visual experience is altered by lid fusion. Furthermore, the number of dividing cells is highly correlated with the axial elongation of the eye and the resulting myopic refractive error. Thus, the retina undergoes active growth during the postnatal development of the primate eye. This growth is modulated by the visual input and accelerates considerably when the eye develops axial myopia. cDNA subtractions, construction of cDNA libraries and differential screening were performed as previously described (Tkatchenko et al, 2000). Two subtractions were carried out resulting in two cDNA libraries. One library was enriched in clones potentially up-regulated in the retina of the closed eye and the other was enriched in cDNAs potentially down-regulated. One thousand clones from each library were analyzed, and 535 differentially-expressed cDNAs were amplified by PCR and spotted onto glass slides coated with poly-L-lysine (five duplicates for each cDNA). The slides were then processed, hybridized with Cy3- and Cy5-labeled complex cDNA probes and washed in 0.2xSSC as described at http://cmgm.stanford.edu/pbrown/protocols/. Two hybridizations with color swap were carried for each monkey. After hybridization, slides were scanned using a GenePix 4000B microarray scanner (Axon Instruments, Union City, CA). The images were acquired and processed using the GenePix Pro 5.1 software package (Axon Instruments). The data obtained were normalized against GAPDH expression level and mean values (the reported values) and P-values (probability associated with a Student's t-test) were calculated for duplicate experiments (n = 10) with color swap using Microsoft Excel. Genes were defined as differentially regulated if P< 0.05. Cluster analysis was performed using the Genesis software package (provided by Alexander Sturn, Graz University of Technology, Austria).

Project description:PURPOSE: Aquaporins (AQP) form a family of specialized water channels known to transport water across cell membranes and reduce osmotic gradients. The isoform AQP4 is highly expressed in the astroglia of the brain and Müller cells in the retina. In the brain, AQP4 play a role in the control of cerebral edema by shunting excess fluid into blood vessels and by upregulating during conditions of hyperosmolarity. Thus, on the basis of the hyperosmolarity seen across the retina and choroid of hatchling chickens made myopic by form deprivation (FD), we predicted an upregulation of retinal AQP4 expression during induction of myopia. METHODS: Two-day-old hatchling chicks were monocularly form-deprived for 48, 72, or 96 h, and then after biometric assessment, the eyes of these animals and the normal controls of the same age were enucleated. Retinal tissue was prepared either for western blot analysis to show the presence of the AQP4 protein in the chick retina or for immunolocalization using polyclonal AQP4 antibodies to determine regional distribution and intensity of labeling during the induction of form deprivation myopia (FDM). RESULTS: As expected, ultrasonography demonstrated that all post hatchling eyes showed rapid elongation with occluded eyes elongating faster than fellow eyes or normal controls and becoming progressively more myopic with the duration of visual deprivation. Western blot analyses revealed an approximately 30 kDa band immunoreactive for AQP4 protein and confirmed the presence of AQP4 in chicks. Immunohistochemical staining showed the greatest positive immunoreactivity for antibodies to AQP4 in the inner retina along the vitreoretinal interface, nerve fiber layer, ganglion cell layer, and inner plexiform layer in all animals. The control eyes showed relatively constant levels of AQP4 expression until day 5 after which the level appeared to decrease. By comparison, positive AQP4 immunoreactivity in the nerve fiber layer increased significantly over the first 48 h in form-deprived eyes and in fellow eyes and then decreased over the next 48 h but not to the level of expression in the normal untreated eyes. CONCLUSIONS: This is the first study to demonstrate the presence of AQP4 protein in the chick retina and to associate AQP4 expression in the inner retina with the initiation of form deprivation and the period of fastest axial elongation. This increased expression of AQP4 channels near the vitread border during the time of rapid growth suggests a role for AQP4 as a conduit for movement of retinal fluid into the vitreous in form-deprived chicks.

Dataset Information

Form-deprivation myopia in chick induces limited changes in retinal gene expression.

Publications

Form-deprivation myopia in chick induces limited changes in retinal gene expression.

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