Mapping the origin and fate of myeloid cells in distinct compartments of the eye by single-cell profiling.

Similar to the brain, the eye is considered an immune-privileged organ where tissue-resident macrophages provide the major immune cell constituents. However, little is known about spatially restricted macrophage subsets within different eye compartments with regard to their origin, function, and fate during health and disease. Here, we combined single-cell analysis, fate mapping, parabiosis, and computational modeling to comprehensively examine myeloid subsets in distinct parts of the eye during homeostasis. This approach allowed us to identify myeloid subsets displaying diverse transcriptional states. During choroidal neovascularization, a typical hallmark of neovascular age-related macular degeneration (AMD), we recognized disease-specific macrophage subpopulations with distinct molecular signatures. Our results highlight the heterogeneity of myeloid subsets and their dynamics in the eye that provide new insights into the innate immune system in this organ which may offer new therapeutic targets for ophthalmological diseases.

Melanoblasts Populate the Mouse Choroid Earlier in Development Than Previously Described.

Purpose: Human choroidal melanocytes become evident in the last trimester of development, but very little is known about them. To better understand normal and diseased choroidal melanocyte biology we examined their precursors, melanoblasts (MB), in mouse eyes during development, particularly their relation to the developing vasculature and immune cells. Methods: Naïve B6(Cg)-Tyrc-2J/J albino mice were used between embryonic (E) day 15.5 and postnatal (P) day 8, with adult controls. Whole eyes, posterior segments, or dissected choroidal wholemounts were stained with antibodies against tyrosinase-related protein 2, ionized calcium binding adaptor molecule-1 or isolectin B4, and examined by confocal microscopy. Immunoreactive cell numbers in the choroid were quantified with Imaris. One-way ANOVA with Tukey's post hoc test assessed statistical significance. Results: Small numbers of MB were present in the presumptive choroid at E15.5 and E18.5. The density significantly increased between E18.5 (381.4 ± 45.8 cells/mm2) and P0 (695.2 ± 87.1 cells/mm2; P = 0.032). In postnatal eyes MB increased in density and formed multiple layers beneath the choriocapillaris. MB in the periocular mesenchyme preceded the appearance of vascular structures at E15.5. Myeloid cells (Ionized calcium binding adaptor molecule-1-positive) were also present at high densities from this time, and attained adult-equivalent densities by P8 (556.4 ± 73.6 cells/mm2). Conclusions: We demonstrate that choroidal MB and myeloid cells are both present at very early stages of mouse eye development (E15.5). Although MB and vascularization seemed to be unlinked early in choroidal development, they were closely associated at later stages. MB did not migrate into the choroid in waves, nor did they have a consistent relationship with nerves.

Mitf-family transcription factor function is required within cranial neural crest cells to promote choroid fissure closure.

A crucial step in eye development is the closure of the choroid fissure (CF), a transient structure in the ventral optic cup through which vasculature enters the eye and ganglion cell axons exit. Although many factors have been identified that function during CF closure, the molecular and cellular mechanisms mediating this process remain poorly understood. Failure of CF closure results in colobomas. Recently, MITF was shown to be mutated in a subset of individuals with colobomas, but how MITF functions during CF closure is unknown. To address this issue, zebrafish with mutations in mitfa and tfec, two members of the Mitf family of transcription factors, were analyzed and their functions during CF closure determined. mitfa;tfec mutants possess severe colobomas and our data demonstrate that Mitf activity is required within cranial neural crest cells (cNCCs) during CF closure. In the absence of Mitf function, cNCC migration and localization in the optic cup are perturbed. These data shed light on the cellular mechanisms underlying colobomas in individuals with MITF mutations and identify a novel role for Mitf function in cNCCs during CF closure.

Synchronized tissue-scale vasculogenesis and ubiquitous lateral sprouting underlie the unique architecture of the choriocapillaris.

The choriocapillaris is an exceptionally high density, two-dimensional, sheet-like capillary network, characterized by the highest exchange rate of nutrients for waste products per area in the organism. These unique morphological and physiological features are critical for supporting the extreme metabolic requirements of the outer retina needed for vision. The developmental mechanisms and processes responsible for generating this unique vascular network remain, however, poorly understood. Here we take advantage of the zebrafish as a model organism for gaining novel insights into the cellular dynamics and molecular signaling mechanisms involved in the development of the choriocapillaris. We show for the first time that zebrafish have a choriocapillaris highly similar to that in mammals, and that it is initially formed by a novel process of synchronized vasculogenesis occurring simultaneously across the entire outer retina. This initial vascular network expands by un-inhibited sprouting angiogenesis whereby all endothelial cells adopt tip-cell characteristics, a process which is sustained throughout embryonic and early post-natal development, even after the choriocapillaris becomes perfused. Ubiquitous sprouting was maintained by continuous VEGF-VEGFR2 signaling in endothelial cells delaying maturation until immediately before stages where vision becomes important for survival, leading to the unparalleled high density and lobular structure of this vasculature. Sprouting was throughout development limited to two dimensions by Bruch's membrane and the sclera at the anterior and posterior surfaces respectively. These novel cellular and molecular mechanisms underlying choriocapillaris development were recapitulated in mice. In conclusion, our findings reveal novel mechanisms underlying the development of the choriocapillaris during zebrafish and mouse development. These results may explain the uniquely high density and sheet-like organization of this vasculature.

The cellular bases of choroid fissure formation and closure.

Defects in choroid fissure (CF) formation and closure lead to coloboma, a major cause of childhood blindness. Despite genetic advances, the cellular defects underlying coloboma remain poorly elucidated due to our limited understanding of normal CF morphogenesis. We address this deficit by conducting high-resolution spatio-temporal analyses of CF formation and closure in the chick, mouse and fish. We show that a small ventral midline invagination initiates CF formation in the medial-proximal optic cup, subsequently extending it dorsally toward the lens, and proximally into the optic stalk. Unlike previously supposed, the optic disc does not form solely as a result of this invagination. Morphogenetic events that alter the shape of the proximal optic cup also direct clusters of outer layer and optic stalk cells to form dorsal optic disc. A cross-species comparison suggests that CF closure can be accomplished by breaking down basement membranes (BM) along the CF margins, and by establishing BM continuity along the dorsal and ventral surfaces of the CF. CF closure is subsequently accomplished via two distinct mechanisms: tissue fusion or the intercalation of various tissues into the inter-CF space. We identify several novel cell behaviors that underlie CF fusion, many of which involve remodeling of the retinal epithelium. In addition to BM disruption, these include NCAD downregulation along the SOX2+ retinal CF margin, and the protrusion or movement of partially polarized retinal cells into the inter-CF space to mediate fusion. Proximally, the inter-CF space does not fuse or narrow and is instead loosely packed with migrating SOX2+/PAX2+/Vimentin+ astrocytes until it is closed by the outgoing optic nerve. Taken together, our results highlight distinct proximal-distal differences in CF morphogenesis and closure and establish detailed cellular models that can be utilized for understanding the genetic bases of coloboma.

Expression, regulation and function of miR-126 in the mouse choroid vasculature.

MicroRNA miR-126 has been shown to be required for proper angiogenesis in several models. However, its expression, regulation and function in the mouse choroid remain unclear. Our previous data has shown that miR-126 expression is enriched in the endothelial cells (ECs) of the mouse choroid. Here we report that a 5.5 kb Egfl7/miR-126 promoter drives the expression of miR-126 in the choroid ECs during choroidal vascular development. The expression of miR-126 in the ECs is regulated by flow stress likely through Krüppel-like transcriptional factors. miR-126-/- mice show mildly delayed choroidal vascular development, but adult knockout mice develop periphery choroidal vascular lesions. This study suggests that miR-126 is largely dispensable for mouse choroidal development but required for maintaining choroidal vasculature integrity.

Development of the hyaloid, choroidal and retinal vasculatures in the fetal human eye.

The development of the ocular vasculatures is perfectly synchronized to provide the nutritional and oxygen requirements of the forming human eye. The fetal vasculature of vitreous, which includes the hyaloid vasculature, vasa hyaloidea propria, and tunica vasculosa lentis, initially develops around 4-6 weeks gestation (WG) by hemo-vasculogenesis (development of blood and blood vessels from a common progenitor, the hemangioblast). This transient fetal vasculature expands around 12 WG by angiogenesis (budding from primordial vessels) and remains until a retinal vasculature begins to form. The fetal vasculature then regresses by apoptosis with the assistance of macrophages/hyalocytes. The human choroidal vasculature also forms by a similar process and will supply nutrients and oxygen to outer retina. This lobular vasculature develops in a dense collagenous tissue juxtaposed with a cell constitutively producing vascular endothelial growth factor (VEGF), the retinal pigment epithelium. This epithelial/endothelial relationship is critical in maintaining the function of this vasculature throughout life and maintaining it's fenestrated state. The lobular capillary system (choriocapillaris) develops first by hemo-vasculogenesis and then the intermediate choroidal blood vessels form by angiogenesis, budding from the choriocapillaris. The human retinal vasculature is the last to develop. It develops by vasculogenesis, assembly of CXCR4+/CD39+ angioblasts or vascular progenitors perhaps using Muller cell Notch1 or axonal neuropilinin-1 for guidance of semaphorin 3A-expressing angioblasts. The fovea never develops a retinal vasculature, which is probably due to the foveal avascular zone area of retina expressing high levels of antiangiogenic factors. From these studies, it is apparent that development of the mouse ocular vasculatures is not representative of the development of the human fetal, choroidal and retinal vasculatures.

Zebrafish zic2 controls formation of periocular neural crest and choroid fissure morphogenesis.

The vertebrate retina develops in close proximity to the forebrain and neural crest-derived cartilages of the face and jaw. Coloboma, a congenital eye malformation, is associated with aberrant forebrain development (holoprosencephaly) and with craniofacial defects (frontonasal dysplasia) in humans, suggesting a critical role for cross-lineage interactions during retinal morphogenesis. ZIC2, a zinc-finger transcription factor, is linked to human holoprosencephaly. We have previously used morpholino assays to show zebrafish zic2 functions in the developing forebrain, retina and craniofacial cartilage. We now report that zebrafish with genetic lesions in zebrafish zic2 orthologs, zic2a and zic2b, develop with retinal coloboma and craniofacial anomalies. We demonstrate a requirement for zic2 in restricting pax2a expression and show evidence that zic2 function limits Hh signaling. RNA-seq transcriptome analysis identified an early requirement for zic2 in periocular neural crest as an activator of alx1, a transcription factor with essential roles in craniofacial and ocular morphogenesis in human and zebrafish. Collectively, these data establish zic2 mutant zebrafish as a powerful new genetic model for in-depth dissection of cell interactions and genetic controls during craniofacial complex development.

Lutein and its oxidized forms in eye structures throughout prenatal human development.

The presence of carotenoids in the vitreous body, retina, lens, retinal pigment epithelium together with choroid (hereinafter RPE), and ciliary body and iris together with choroidal stroma (hereinafter CBI) was studied throughout the second trimester of prenatal development of the human eye. It has been found that the vitreous body, retina, and RPE contain lutein and its oxidized forms. Zeaxanthin was not found in the tissues studied. The presence of lutein in the vitreous body is transient and no longer detected after 28 weeks of gestation. Lutein was not detected in the lens and CBI, but its oxidized forms were found. The presence of carotenoids in different tissues of the eye in the course of normal eye development and the antioxidant role of carotenoids are discussed.

The hyaloid vasculature facilitates basement membrane breakdown during choroid fissure closure in the zebrafish eye.

A critical aspect of vertebrate eye development is closure of the choroid fissure (CF). Defects in CF closure result in colobomas, which are a significant cause of childhood blindness worldwide. Despite the growing number of mutated loci associated with colobomas, we have a limited understanding of the cell biological underpinnings of CF closure. Here, we utilize the zebrafish embryo to identify key phases of CF closure and regulators of the process. Utilizing Laminin-111 as a marker for the basement membrane (BM) lining the CF, we determine the spatial and temporal patterns of BM breakdown in the CF, a prerequisite for CF closure. Similarly, utilizing a combination of in vivo time-lapse imaging, ß-catenin immunohistochemistry and F-actin staining, we determine that tissue fusion, which serves to close the fissure, follows BM breakdown closely. Periocular mesenchyme (POM)-derived endothelial cells, which migrate through the CF to give rise to the hyaloid vasculature, possess distinct actin foci that correlate with regions of BM breakdown. Disruption of talin1, which encodes a regulator of the actin cytoskeleton, results in colobomas and these correlate with structural defects in the hyaloid vasculature and defects in BM breakdown. cloche mutants, which entirely lack a hyaloid vasculature, also possess defects in BM breakdown in the CF. Taken together, these data support a model in which the hyaloid vasculature and/or the POM-derived endothelial cells that give rise to the hyaloid vasculature contribute to BM breakdown during CF closure.

Developmentally Regulated Production of meso-Zeaxanthin in Chicken Retinal Pigment Epithelium/Choroid and Retina.

PURPOSE: meso-Zeaxanthin is a carotenoid that is rarely encountered in nature outside of the vertebrate eye. It is not a constituent of a normal human diet, yet this carotenoid comprises one-third of the primate macular pigment. In the current study, we undertook a systematic approach to biochemically characterize the production of meso-zeaxanthin in the vertebrate eye. METHODS: Fertilized White Leghorn chicken eggs were analyzed for the presence of carotenoids during development. Yolk, liver, brain, serum, retina, and RPE/choroid were isolated, and carotenoids were extracted. The samples were analyzed on C-30 or chiral HPLC columns to determine the carotenoid composition. RESULTS: Lutein and zeaxanthin were found in all studied nonocular tissues, but no meso-zeaxanthin was ever detected. Among the ocular tissues, the presence of meso-zeaxanthin was consistently observed starting at embryonic day 17 (E17) in the RPE/choroid, several days before its consistent detection in the retina. If RPE/choroid of an embryo was devoid of meso-zeaxanthin, the corresponding retina was always negative as well. CONCLUSIONS: This is the first report of developmentally regulated synthesis of meso-zeaxanthin in a vertebrate system. Our observations suggest that the RPE/choroid is the primary site of meso-zeaxanthin synthesis. Identification of meso-zeaxanthin isomerase enzyme in the developing chicken embryo will facilitate our ability to determine the biochemical mechanisms responsible for production of this unique carotenoid in other higher vertebrates, such as humans.

Sox4 regulates choroid fissure closure by limiting Hedgehog signaling during ocular morphogenesis.

SoxC transcription factors play critical roles in many developmental processes, including neurogenesis, cardiac formation, and skeletal differentiation. In vitro and in vivo loss-of-function studies have suggested that SoxC genes are required for oculogenesis; however the mechanism was poorly understood. Here, we have explored the function of the SoxC factor Sox4 during zebrafish eye development. We show that sox4a and sox4b are expressed in the forebrain and periocular mesenchyme adjacent to the optic stalk during early eye development. Knockdown of sox4 in zebrafish resulted in coloboma, a structural malformation of the eye that is a significant cause of pediatric visual impairment in humans, in which the choroid fissure fails to close. Sox4 morphants displayed altered proximo-distal patterning of the optic vesicle, including expanded pax2 expression in the optic stalk, as well as ectopic cell proliferation in the retina. We show that the abnormal ocular morphogenesis observed in Sox4-deficient zebrafish is caused by elevated Hedgehog (Hh) signaling, and this is due to increased expression of the Hh pathway ligand Indian Hedgehog b (ihhb). Consistent with these results, coloboma in sox4 morphants could be rescued by pharmacological treatment with the Hh inhibitor cyclopamine, or by co-knockdown of ihhb. Conversely, overexpression of sox4 reduced Hh signaling and ihhb expression, resulting in cyclopia. Finally, we demonstrate that sox4 and sox11 have overlapping, but not completely redundant, functions in regulating ocular morphogenesis. Taken together, our data demonstrate that Sox4 is required to limit the extent of Hh signaling during eye development, and suggest that mutations in SoxC factors could contribute to the development of coloboma.

Whole genome expression profiling of normal human fetal and adult ocular tissues.

To study growth and development of ocular tissues, gene expression patterns in normal human fetal versus adult eyes were compared. Human retina/retinal pigment epithelium, choroid, sclera, optic nerve* and cornea* tissues were dissected from fetal (24 week gestational age) (N = 9; *N = 6), and adult (N = 6) normal donor eyes. The Illumina(®) whole genome expression microarray platform was used to assess differential expression. Statistical significance for all comparisons was determined using the Benjamin and Hochberg False Discovery Rate (FDR, 5%). Significant gene expression fold changes > 1.5 were found in adult versus fetal retina/RPE (N = 1185), choroid (N = 6446), sclera (N = 1349), and cornea (N = 3872), but not optic nerve. Genes showing differential expression were assessed using Ingenuity Pathway Analysis (IPA) for enriched functions and canonical pathways. In all tissues, development, cell death/growth, cancer functions, and signaling canonical pathways were enriched. There was also a general trend of down-regulation of collagen genes in adult tissues.

Co-expression of endothelial and neuronal nitric oxide synthases in the developing vasculatures of the human fetal eye.

BACKGROUND: Nitric oxide (NO) is a multifunctional gaseous molecule that regulates various physiological functions in both neuronal and non-neuronal cells. NO is synthesized by nitric oxide synthases (NOSs), of which three isoforms have been identified. Neuronal NOS (nNOS) and endothelial NOS (eNOS) constitutively produce low levels of NO as a cell-signaling molecule in response to an increase in intracellular calcium concentration. Recent data have revealed a predominant role of eNOS in both angiogenesis and vasculogenesis. METHODS: The immunohistochemical localization of nNOS and eNOS was investigated during embryonic and fetal ocular vascular development from 7 to 21 weeks gestation (WG) on sections of cryopreserved tissue. RESULTS: eNOS was confined to endothelial cells of developing vessels at all ages studied. nNOS was prominent in nuclei of vascular endothelial and smooth muscle cells in the fetal vasculature of vitreous and choriocapillaris. nNOS was also prominent in the nuclei of CXCR4(+) progenitors in the inner retina and inner neuroblastic layer. CONCLUSIONS: These findings demonstrate co-expression of n- and eNOS isoforms in different compartments of vasoformative cells during development. Nuclear nNOS was present in vascular and nonvascular progenitors as well as endothelial cells and pericytes. This suggests that nNOS may play a role in the transcription regulatory systems in endothelial cells and pericytes during ocular hemo-vasculogenesis, vasculogenesis, and angiogenesis.

Organogenesis of mild ocular coloboma in FLS mice: failure of basement membrane disintegration at optic fissure margins.

Fatty Liver Shionogi (FLS) mice have been shown to develop a hereditary disorder characterized by localized retinochoroidal defects of the ventral fundus very similar to human typical ocular coloboma without microphthalmia. The objective of this study was to determine when and how the failure of the optic fissure closure occurs, and to clarify the disturbed mechanism of basement membrane disintegration during embryonal stage in FLS mice. Fetuses at day 11.5-15.5 of gestation were obtained from dams of FLS and BALB/c strain of mice. Coronal serial sections through the eye were examined by light and electron microscopy. The sections were followed by observation of the basement membrane using reaction with periodic acid-Schiff (PAS) reagent and immunohistochemical staining with anti-Laminin and anti-Type IV collagen antibodies. Both optic fissure margins closely approached each other up to GD 11.5 in all FLS and BALB/c embryos. The inner and outer layers of the optic cup did not normally fuse at midlenticular levels of the optic fissure in almost 70% of FLS fetuses by GD 15.5, whereas both margins were completely fused in all BALB/c fetuses of the same gestational day. In the FLS fetuses at GD 12.5, rolling on one side of fissure margins and consequent asymmetry were observed at the ventral optic fissure. The basement membrane persisted after the close contact of both sides of the fissure margins during GD 11.5 and 15.5. Ultrastructurally, the basal lamina was not disintegrated and mesenchymal cells intervened between the two neuroepithelial layers, resulting in complete separation of both fissure margins at GD 13.0. It is highly probable that the disturbed basement membrane disintegration right before optic fissure closure causes mild ocular coloboma without microphthalmia in FLS mice.

Evidence of hematopoietic differentiation, vasculogenesis and angiogenesis in the formation of human choroidal blood vessels.

Human fetal eyes 8-40 weeks gestation (WG) were examined using markers to hematopoietic stem cells (HSC), vascular precursor cells (VPC), monocytes/macrophages and endothelial cells (EC). Electron microscopy and bromo-deoxyuridene labeling were undertaken to confirm the existence of solid vascular cords and to demonstrate vasculogenesis and angiogenesis in developing choroidal tissue. Our results demonstrated that the earliest incipient choroid consisted of vimentin(+) mesenchymal precursor cells which downregulated vimentin expression with maturation. Our observations lead us to conclude that these vimentin(-)/CD34(+)/CD44(+)/CD133(+) HSCs then differentiated into three distinct lineages: single isolated CD34(-)/CD39(+) VPCs that formed solid vascular cords which lumenized and became lined with CD34(+) vascular ECs; CD34(--+)/CD14(+)/CD68(+) monocytes that differentiated into tissue macrophages; and CD133(+)/CD34(--+)/α-smooth muscle actin(+) mural precursor cells that matured into smooth muscle cells and pericytes. Blood vessel formation occurred throughout the whole choroid simultaneously, indicative of in situ differentiation. Vasculogenesis, as evidenced by lumenization of solid vascular cords, was responsible for the formation of the entire choroidal area with angiogenesis, in all three layers of the choroid, only adding to vascular density. These results suggest that formation of the human choroid involves three processes: HSC differentiation, vasculogenesis and angiogenesis. Since vasculogenesis takes place independently of VEGF(165), further insights regarding the molecular mechanisms of vasculogenesis are required to better inform future treatments of choroidal neovascularization.

Modulation of MCT3 expression during wound healing of the retinal pigment epithelium.

PURPOSE: MCT3 is a proton-coupled monocarboxylate transporter preferentially expressed in the basolateral membrane of the retinal pigment epithelium (RPE) and has been shown to play an important role in regulating pH and lactate concentrations in the outer retina. Decreased expression of MCT3 in response to trauma or disease could contribute to pathologic changes in the retina. The present study followed the expression of MCT3 after wounding and re-epithelialization of chick RPE explant and human fetal (hf) RPE cultures. METHODS: Immunofluorescence microscopy and immunoblotting were performed to determine changes in MCT expression after scratch wounding and re-epithelialization of chick RPE/choroid explant cultures and hfRPE cell monolayers. RESULTS: MCT3 expression and basolateral polarity were maintained in chick RPE/choroid explant cultures and hfRPE monolayers. Wounding resulted in loss of MCT3 and the upregulation of MCT4 expression in migrating cells at the edge of the wound. On re-epithelialization, MCT3 was detected in chick and hfRPE cells when cells became hexagonally packed and pigmented. However, in hfRPE cells, MCT4 was consistently expressed throughout the epithelial monolayer. RPE cells at the edges of chick explants and hfRPE cultures with a free edge expressed MCT4 but not MCT3. CONCLUSIONS: Wounding of RPE monolayers resulted in dedifferentiation of the cells at the edge of the wound, as evidenced by a loss of MCT3 and increased MCT4 expression. Collectively, these findings suggest that both cell-cell and cell-substrate interactions are essential in directing and maintaining differentiation of the RPE and expression of MCT3.

Development of the human choriocapillaris.

Vasculogenesis and/or angiogenesis are thought to be the major mechanisms for new vessel formation during development. A third mechanism, haemo-vasculogenesis, has been described in which blood vessel and blood cells (haematopoiesis (expression of CD34(+)) and erythropoiesis (presence of epsilon chain of haemoglobin or Hb-epsilon(+))) differentiate from a common precursor, the haemangioblast. This review describes the mechanism(s) for development of human choroidal vascular from 6 until 22 weeks gestation (WG). Endothelial cell or EC (CD31, CD34, CD39, VEGFR-2) and angioblast (CD39, VEGFR-2) markers were present in choriocapillaris (CC) by 7 WG through 22 WG. From 6 to 8 WG, many erythroblasts (nucleated Hb-epsilon(+) RBCs) were observed in the CC layer. Erythroblasts (Hb-epsilon(+)) were also positive for CD34, CD31, and/or VEGFR-2. Proliferation of vascular cells (Ki67+), suggesting angiogenesis, was not observed until 12 WG. TEM analysis demonstrated that CC was structurally immature even at 11 WG: no basement membrane, absence of pericytes, and poorly formed lumens that were filled with filopodia. Contiguous fenestrations and significant PV-1 (protein in diaphragms of fenestrations) were not observed until 21-22 WG. Smooth muscle actin was prominent at 20 WG and the maturation of pericytes was confirmed by TEM. Therefore, the embryonic CC appears to form initially by haemo-vasculogenesis (Hb-epsilon(+)/CD31(+) cells), whereas angiogenesis (CD34(+)/Ki67(+)) appears to be the mode of intermediate and large choroidal vessel development later in the foetus. Contiguous fenestrations, mature pericytes, and EC basal lamina occur late in development, around 22 WG, which coincides with photoreceptors developing inner segments.

IFN{gamma} regulates retinal pigment epithelial fluid transport.

The present experiments show that IFNgamma receptors are mainly localized to the basolateral membrane of human retinal pigment epithelium (RPE). Activation of these receptors in primary cultures of human fetal RPE inhibited cell proliferation and migration, decreased RPE mitochondrial membrane potential, altered transepithelial potential and resistance, and significantly increased transepithelial fluid absorption. These effects are mediated through JAK-STAT and p38 MAPK signaling pathways. Second messenger signaling through cAMP-PKA pathway- and interferon regulatory factor-1-dependent production of nitric oxide/cGMP stimulated the CFTR at the basolateral membrane and increased transepithelial fluid absorption. In vivo experiments using a rat model of retinal reattachment showed that IFNgamma applied to the anterior surface of the eye can remove extra fluid deposited in the extracellular or subretinal space between the retinal photoreceptors and RPE. Removal of this extra fluid was blocked by a combination of PKA and JAK-STAT pathway inhibitors injected into the subretinal space. These results demonstrate a protective role for IFNgamma in regulating retinal hydration across the outer blood-retinal barrier in inflammatory disease processes and provide the basis for possible therapeutic interventions.