Retinoic Acid in Ocular Growth Regulation

All-trans-retinoic acid (atRA) is a metabolite of vitamin A (retinol) and is required for growth and development of a variety of organ systems in all higher animals from fish to humans. Evidence is accumulating to suggest that atRA may also be an important molecular signal in the postnatal control of eye size. Choroidal synthesis of atRA is modulated during periods of visually-induced changes in ocular growth and has pronounced effects on eye growth and refraction in several animal models of myopia. Choroidal atRA synthesis is exclusively regulated by expression of the enzyme, retinaldehyde dehydrogenase 2 (RALDH2). In chicks and humans, RALDH2 is synthesized by a unique population of uncharacterized extravascular stromal cells concentrated in the proximal choroid. The identification of choroidal atRA and RALDH2 as visually induced ocular growth regulators provides the potential for new therapeutic targets for the treatment of childhood myopia. The objective of this chapter is to discuss what is presently known about atRA biosynthesis and transport in the eye during visually guided eye growth and how this research can contribute to a better understanding of the mechanisms underlying the development of myopia

Ocular expression of avian thymic hormone: changes during the recovery from induced myopia

Purpose: Several studies suggest that postnatal ocular growth is under the control of factors within the eye that regulate the rate of scleral extracellular matrix remodeling and the rate of ocular elongation. A microarray analysis was employed to identify some of the factors involved in the regulation of visually guided ocular growth. Gene expression was compared in the retina-retinal pigmented epithelium (RPE)-choroid of chick eyes that were decelerating in the rate of ocular growth (“recovering ” from myopia) as compared with contralateral control eyes. Methods: Form-deprivation myopia was induced in the right eyes of two-day-old chicks by the application of translucent occluders. Following 10 days of deprivation, occluders were removed and chicks were provided unrestricted vision for an additional 1–7 days (recovery). After one and four days of recovery, chicks were sacrificed, retina, RPE, and choroid were isolated, and mRNA was subjected to microarray analysis using a chicken immune system 4000 gene microarray. In addition, whole eyes and isolated ocular tissues (retina and RPE, choroid, sclera, and extraocular muscle) of treated and control eyes were subjected to real-time PCR, immunohistochemistry, and western blot analyses to verify gene expression results. Results: Following one day of recovery, only one gene, avian thymic hormone (ATH) was highly upregulated (+12.3 fold). ATH gene and protein expression were confirmed in the retina and choroid as well as in the sclera and extraocular muscle. A significant increase in ATH protein was detected in choroids from treated eyes following four days of recover

Single cell transcriptomics identifies distinct choroid cell populations involved in visually guided eye growth

IntroductionPostnatal ocular growth is regulated by a vision-dependent mechanism, termed emmetropization, which acts to minimize refractive error through coordinated growth of the ocular tissues. The choroid participates in the emmetropization process via the production of scleral growth regulators that control ocular elongation and refraction.MethodsTo elucidate the role of the choroid in emmetropization, we used single-cell RNA sequencing (scRNA-seq) to characterize the cell populations in the chick choroid and compare gene expression changes in these cell populations when the eye is undergoing emmetropization. Emmetropization was modeled by inducing form deprivation myopia in 3 -4 day old chicks via application of translucent occluders to one eye for a period of 10 days followed by 24 hrs of unrestricted vision (by removing the occluder). Numerous studies have previously shown that visual form deprivation stimulates axial elongation and myopia, and upon removal of the occluder, the myopic chick eye rapidly slows its rate of axial elongation to recover from the induced myopia and regain emmetropia. The contralateral, untreated eyes served as controls.ResultsUMAP clustering analysis identified 24 distinct cell clusters in all chick choroids. 7 clusters were identified as fibroblast subpopulations; 5 clusters represented different populations of endothelial cells; 4 clusters were CD45+ macrophages, T cells and B cells; 3 clusters were Schwann cell subpopulations; and 2 clusters were identified as melanocytes. Additionally, single populations of RBCs, plasma cells and neuronal cells were identified. Significant changes in gene expression between control and treated choroids were identified in 17 cell clusters, representing 95% of total choroidal cells. The majority of gene expression changes were relatively small (< 2 fold). The greatest changes in gene expression were identified in a rare cell population (0.11% - 0.49% of total choroidal cells). This population expressed neuron-specific genes as well as several opsin genes suggestive of a rare neuronal cell population that is potentially light sensitive.DiscussionOur results, for the first time, provide a comprehensive profile of the major choroidal cell types and their gene expression changes during the process of emmetropization and provide insights into the canonical pathways and upstream regulators that coordinate postnatal ocular growth

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Choroidal Regulation of Scleral Glycosaminoglycan Synthesis during Recovery from Induced Myopia

PURPOSE. The present study was undertaken to examine the relationship between choroidal permeability and scleral glycosaminoglycan synthesis rates during the development of and recovery from form deprivation myopia. METHODS. Form deprivation myopia was induced in chicks for 10 days and was followed by a period of unrestricted vision for 0 to 15 days (recovery). Choroidal permeability was quantified by measuring albumin leakage from choroidal blood vessels into suprachoroidal fluid using Evans blue. Scleral sulfated glycosaminoglycan synthesis was assessed on punches of sclera obtained immediately after extraction of suprachoroidal fluid for permeability measurements or after incubation with suprachoroidal fluid by measuring the amount of 35 SO 4 incorporated into glycosaminoglycans over a period of 4 hours at 37°C. Suprachoroidal fluid was subjected to size fractionation and proteinase digestion to characterize the bioactive fractions from recovering and control chick eyes. RESULTS. Recovery from prior form deprivation was associated with a significant increase in choroidal permeability, compared with that of myopic eyes and contralateral control eyes, and was coincident with a significant decrease in scleral sulfated glycosaminoglycan synthesis rates in treated eyes compared with contralateral control eyes. Suprachoroidal fluid isolated from recovering chick eyes significantly inhibited scleral glycosaminoglycan synthesis compared with suprachoroidal fluid from control eyes (-54%; P Ͻ 0.01; ANOVA). Preliminary characterization of suprachoroidal fluid suggested that all inhibitory activity in suprachoroidal fluid fractions specific to recovering eyes is present in molecular weight fractions of less than 10 kDa. CONCLUSIONS. The results of this study suggest that increased choroidal permeability coincides with a decrease in the rate of scleral glycosaminoglycan synthesis during recovery from myopia. The authors speculate that increased choroidal permeability may represent a mechanism for controlling the rate of delivery of bioactive factors to the sclera to regulate the rate of glycosaminoglycan synthesis in the posterior sclera. (Invest Ophthalmol Vis Sci

Melatonin receptors in chick ocular tissues: Implications for a role of melatonin in ocular growth regulation

PURPOSE. The influences of diurnal rhythms involving a variety of ocular parameters are implicated in the development of myopia. The purpose of this study was to determine the expression of the melatonin receptor subtype proteins in chick ocular tissues and to examine the role of the circadian signaling molecule melatonin in normal ocular growth and the exaggerated ocular growth associated with the development of myopia. METHODS. Expression of the Mel 1a , Mel 1b , and Mel 1c melatonin receptor proteins in ocular tissues was examined by Western blot analyses, slot blot analyses, and immunocytochemistry. For examining the effect of melatonin on ocular growth, chicks were maintained on a 12-hour light-dark cycle and were monocularly form-vision deprived in one eye with a translucent occluder for 5 days. During the 5-day treatment period, chicks were injected systemically during the early dark period with melatonin (0.6 mg) or 2% ethanol vehicle control. Ocular dimensions of normal and deprived eyes were examined by high frequency A-scan ultrasound. RESULTS. Immunocytochemical analysis of chick ocular tissues revealed the cellular distribution of the Mel 1a receptor subtype in the cornea, choroid, sclera, and retina. Western blot and slot blot analyses demonstrated that all three receptors were present in these tissues and they demonstrated distinct diurnal rhythms of protein expression in the retina-RPE-choroid, with peak levels of Mel 1a and Mel 1b occurring during the night and peak levels of Mel 1c during the day. Systemic administration of melatonin resulted in significant changes in anterior chamber depth, vitreous chamber depth, and choroidal thickness of form-deprived and/or control eyes. CONCLUSIONS. Results of this study show that all three melatonin receptor subtypes are expressed in retinal and extraretinal ocular tissues of the chick eye. The finding that administration of melatonin alters the growth of several ocular tissues in both control and form-deprived eyes suggests that melatonin, acting through specific melatonin receptors in ocular tissues, plays a role in ocular growth and development. This conclusion suggests that the action of melatonin, combined with expression of melatonin receptors, is involved in the regulation of the diurnal rhythm of ocular growth. (Invest Ophthalmol Vis Sci