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.

Optic cup morphogenesis requires neural crest-mediated basement membrane assembly.

Organogenesis requires precise interactions between a developing tissue and its environment. In vertebrates, the developing eye is surrounded by a complex extracellular matrix as well as multiple mesenchymal cell populations. Disruptions to either the matrix or periocular mesenchyme can cause defects in early eye development, yet in many cases the underlying mechanism is unknown. Here, using multidimensional imaging and computational analyses in zebrafish, we establish that cell movements in the developing optic cup require neural crest. Ultrastructural analysis reveals that basement membrane formation around the developing eye is also dependent on neural crest, but only specifically around the retinal pigment epithelium. Neural crest cells produce the extracellular matrix protein nidogen: impairing nidogen function disrupts eye development, and, strikingly, expression of nidogen in the absence of neural crest partially restores optic cup morphogenesis. These results demonstrate that eye formation is regulated in part by extrinsic control of extracellular matrix assembly.This article has an associated 'The people behind the papers' interview.

Dissecting the transcriptome landscape of the human fetal neural retina and retinal pigment epithelium by single-cell RNA-seq analysis.

The developmental pathway of the neural retina (NR) and retinal pigment epithelium (RPE) has been revealed by extensive research in mice. However, the molecular mechanisms underlying the development of the human NR and RPE, as well as the interactions between these two tissues, have not been well defined. Here, we analyzed 2,421 individual cells from human fetal NR and RPE using single-cell RNA sequencing (RNA-seq) technique and revealed the tightly regulated spatiotemporal gene expression network of human retinal cells. We identified major cell classes of human fetal retina and potential crucial transcription factors for each cell class. We dissected the dynamic expression patterns of visual cycle- and ligand-receptor interaction-related genes in the RPE and NR. Moreover, we provided a map of disease-related genes for human fetal retinal cells and highlighted the importance of retinal progenitor cells as potential targets of inherited retinal diseases. Our findings captured the key in vivo features of the development of the human NR and RPE and offered insightful clues for further functional studies.

Primary cilia in retinal pigment epithelium development and diseases.

Retinal pigment epithelium (RPE) is a highly polarized epithelial monolayer lying between the photoreceptor layer and the Bruch membrane. It is essential for vision through participating in many critical activities, including phagocytosis of photoreceptor outer segments, recycling the visual cycle-related compounds, forming a barrier to control the transport of nutrients, ions, and water, and the removal of waste. Primary cilia are conservatively present in almost all the vertebrate cells and acts as a sensory organelle to control tissue development and homeostasis maintenance. Numerous studies reveal that abnormalities in RPE lead to various retinal diseases, such as age-related macular degeneration and diabetic macular oedema, but the mechanism of primary cilia in these physiological and pathological activities remains to be elucidated. Herein, we summarize the functions of primary cilia in the RPE development and the mutations of ciliary genes identified in RPE-related diseases. By highlighting the significance of primary cilia in regulating the physiological and pathological processes of RPE, we aim to provide novel insights for the treatment of RPE-related retinal diseases.

Optic vesicle morphogenesis requires primary cilia.

Arl13b is a gene known to regulate ciliogenesis. Functional alterations in this gene's activity have been associated with Joubert syndrome. We found that in Arl13 null mouse embryos the orientation of the optic cup is inverted, such that the lens is abnormally surrounded by an inverted optic cup whose retina pigmented epithelium is oddly facing the surface ectoderm. Loss of Arl13b leads to the disruption of optic vesicle's patterning and expansion of ventral fates. We show that this phenotype is consequence of miss-regulation of Sonic hedgehog (Shh) signaling and demonstrate that the Arl13b-/- eye phenotype can be rescued by deletion of Gli2, a downstream effector of the Shh pathway. This work identified an unexpected role of primary cilia during the morphogenetic movements required for the formation of the eye.

Vitamin C- and Valproic Acid-Induced Fetal RPE Stem-like Cells Recover Retinal Degeneration via Regulating SOX2.

Retinal pigment epithelial (RPE) cell replacement therapy has provided promising outcomes in the treatment of retinal degenerative diseases (RDDs), but the resulting limited visual improvement has raised questions about graft survival and differentiation. Through combined treatment with vitamin C and valproic acid (together, VV), we activated human fetal RPE (fRPE) cells to become highly proliferative fetal RPE stem-like cells (fRPESCs). In this study, we report that SOX2 (SRY-box 2) activation contributed to mesenchymal-epithelial transition and elevated the retinal progenitor and mesenchymal stromal markers expressions of fRPESCs. These fRPESCs could differentiate into RPE cells, rod photoreceptors, and mesenchymal lineage progenies under defined conditions. Finally, fRPESCs were transplanted into the subretinal space of an RDD mouse model, and a photoreceptor rescue benefit was demonstrated. The RPE and rod photoreceptor differentiation of transplanted fRPESCs may account for the neural retinal recovery. This study establishes fRPESCs as a highly proliferative, multi-lineage differentiation potential (including RPE, rod photoreceptor, and mesenchymal lineage differentiation), mesenchymal-to-epithelial-transitioned retinal stem-like cell source for cell-based therapy of RDDs.

PRPS polymerization influences lens fiber organization in zebrafish.

BACKGROUND: The self-assembly of metabolic enzymes into filaments or foci highlights an intriguing mechanism for the regulation of metabolic activity. Recently, we identified the conserved polymerization of phosphoribosyl pyrophosphate synthetase (PRPS), which catalyzes the first step in purine nucleotide synthesis, in yeast and cultured mammalian cells. While previous work has revealed that loss of PRPS activity regulates retinal development in zebrafish, the extent to which PRPS filament formation affects tissue development remains unknown. RESULTS: By generating novel alleles in the zebrafish PRPS paralogs, prps1a and prps1b, we gained new insight into the role of PRPS filaments during eye development. We found that mutations in prps1a alone are sufficient to generate abnormally small eyes along with defects in head size, pigmentation, and swim bladder inflation. Furthermore, a loss-of-function mutation that truncates the Prps1a protein resulted in the failure of PRPS filament assembly. Lastly, in mutants that fail to assemble PRPS filaments, we observed disorganization of the actin network in the lens fibers. CONCLUSIONS: The truncation of Prps1a blocked PRPS filament formation and resulted in a disorganized lens fiber actin network. Altogether, these findings highlight a potential role for PRPS filaments during lens fiber organization in zebrafish.

Dual roles of the retinal pigment epithelium and lens in cavefish eye degeneration.

Astyanax mexicanus consists of two forms, a sighted surface dwelling form (surface fish) and a blind cave-dwelling form (cavefish). Embryonic eyes are initially formed in cavefish but they are subsequently arrested in growth and degenerate during larval development. Previous lens transplantation studies have shown that the lens plays a central role in cavefish eye loss. However, several lines of evidence suggest that additional factors, such as the retinal pigment epithelium (RPE), which is morphologically altered in cavefish, could also be involved in the eye regression process. To explore the role of the RPE in cavefish eye degeneration, we generated an albino eyed (AE) strain by artificial selection for hybrid individuals with large eyes and a depigmented RPE. The AE strain exhibited an RPE lacking pigment granules and showed reduced expression of the RPE specific enzyme retinol isomerase, allowing eye development to be studied by lens ablation in an RPE background resembling cavefish. We found that lens ablation in the AE strain had stronger negative effects on eye growth than in surface fish, suggesting that an intact RPE is required for normal eye development. We also found that the AE strain develops a cartilaginous sclera lacking boney ossicles, a trait similar to cavefish. Extrapolation of the results to cavefish suggests that the RPE and lens have dual roles in eye degeneration, and that deficiencies in the RPE may be associated with evolutionary changes in scleral ossification.

Bardet-Biedl Syndrome proteins regulate cilia disassembly during tissue maturation.

Primary cilia are conserved organelles that mediate cellular communication crucial for organogenesis and homeostasis in numerous tissues. The retinal pigment epithelium (RPE) is a ciliated monolayer in the eye that borders the retina and is vital for visual function. Maturation of the RPE is absolutely critical for visual function and the role of the primary cilium in this process has been largely ignored to date. We show that primary cilia are transiently present during RPE development and that as the RPE matures, primary cilia retract, and gene expression of ciliary disassembly components decline. We observe that ciliary-associated BBS proteins protect against HDAC6-mediated ciliary disassembly via their recruitment of Inversin to the base of the primary cilium. Inhibition of ciliary disassembly components was able to rescue ciliary length defects in BBS deficient cells. This consequently affects ciliary regulation of Wnt signaling. Our results shed light onto the mechanisms by which cilia-mediated signaling facilitates tissue maturation.

RPE65 has an additional function as the lutein to meso-zeaxanthin isomerase in the vertebrate eye.

Carotenoids are plant-derived pigment molecules that vertebrates cannot synthesize de novo that protect the fovea of the primate retina from oxidative stress and light damage. meso-Zeaxanthin is an ocular-specific carotenoid for which there are no common dietary sources. It is one of the three major carotenoids present at the foveal center, but the mechanism by which it is produced in the eye is unknown. An isomerase enzyme is thought to be responsible for the transformation of lutein to meso-zeaxanthin by a double-bond shift mechanism, but its identity has been elusive. We previously found that meso-zeaxanthin is produced in a developmentally regulated manner in chicken embryonic retinal pigment epithelium (RPE)/choroid in the absence of light. In the present study, we show that RPE65, the isomerohydrolase enzyme of the vertebrate visual cycle that catalyzes the isomerization of all-trans-retinyl esters to 11-cis-retinol, is also the isomerase enzyme responsible for the production of meso-zeaxanthin in vertebrates. Its RNA is up-regulated 23-fold at the time of meso-zeaxanthin production during chicken eye development, and we present evidence that overexpression of either chicken or human RPE65 in cell culture leads to the production of meso-zeaxanthin from lutein. Pharmacologic inhibition of RPE65 function resulted in significant inhibition of meso-zeaxanthin biosynthesis during chicken eye development. Structural docking experiments revealed that the epsilon ring of lutein fits into the active site of RPE65 close to the nonheme iron center. This report describes a previously unrecognized additional activity of RPE65 in ocular carotenoid metabolism.

Effect of arsenic exposure on early eye development in zebrafish (Danio rerio).

Arsenic is a metalloid that contaminates drinking water supplies worldwide. Owing to concerns for human health, the World Health Organization and the US Environmental Protection Agency have established a safe level in drinking water of ≤10 ppb. Recently, arsenic exposure has also been linked to lower IQ values in children. The effect of arsenic on neurogenesis, specifically eye development, has not been widely explored. This study aimed to examine the effect of environmentally relevant concentrations of arsenic on early eye development by morphological and molecular analysis. The zebrafish, Danio rerio, was chosen to model the impact of arsenic on retinogenesis because of similarities to human eye development. Arsenic exposure to zebrafish embryos resulted in a significant increase in eye diameter at 14 days postfertilization. This was coupled with a trend in thinning of the retinal pigmented epithelium (RPE) layer in embryos exposed to 500 ppb arsenic. Reverse transcription-quantitative polymerase chain reaction analysis of genes associated with eye development revealed differential expression of Pax6a, Pax2a, Ngn1, Sox2 and Shha relative to control. Pax6a, Pax2a and Sox2 are important in the formation of the RPE. Proper formation of the RPE is necessary for growth of the sclera, which, in turn, is responsible for maintaining the shape of the eye. This could potentially be explained by the disruption of gene expression under arsenic exposure during critical time points in early eye development. These results provide insight into the effects arsenic may be having on early eye development in children exposed to contaminated drinking water supplies.

Reductive carboxylation is a major metabolic pathway in the retinal pigment epithelium.

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells that requires an active metabolism to maintain outer retinal homeostasis and compensate for oxidative stress. Using 13C metabolic flux analysis in human RPE cells, we found that RPE has an exceptionally high capacity for reductive carboxylation, a metabolic pathway that has recently garnered significant interest because of its role in cancer cell survival. The capacity for reductive carboxylation in RPE exceeds that of all other cells tested, including retina, neural tissue, glial cells, and a cancer cell line. Loss of reductive carboxylation disrupts redox balance and increases RPE sensitivity to oxidative damage, suggesting that deficiencies of reductive carboxylation may contribute to RPE cell death. Supporting reductive carboxylation by supplementation with an NAD+ precursor or its substrate α-ketoglutarate or treatment with a poly(ADP ribose) polymerase inhibitor protects reductive carboxylation and RPE viability from excessive oxidative stress. The ability of these treatments to rescue RPE could be the basis for an effective strategy to treat blinding diseases caused by RPE dysfunction.

Six3 in a small population of progenitors at E8.5 is required for neuroretinal specification via regulating cell signaling and survival in mice.

Neuroretina and retinal pigment epithelium (RPE) are differentiated from the progenitors in optic vesicles, but it is unclear when and how the two lineages are segregated. Manipulation of chick embryos reveals that the early anteroventral optic vesicle is crucial for neuroretinal development, but the molecular mechanism is unclear. Homeodomain transcription factor Six3 is required for neuroretinal specification and is dispensable for RPE formation, but the cell fates of Six3-deficient progenitors and the origins of remnant RPE are unknown. Here, we performed lineage tracing of Six3-Cre positive cells in wild-type and Six3-deficient mouse embryos. Six3-Cre positive progenies were found in a population of progenitors in the anteroventral optic pits/vesicles starting at E8.5, and were found in neuroretina, optic stalk, ventral forebrain, but not RPE, at E10.5. Six3-deletion in the small population of progenitors at E8.5 was sufficient to cause rostral expansion of Wnt8b and drastic reduction of Fgf8/MAPK signaling, ablating neuroretinal specification without affecting RPE. Lineage tracing revealed Six3-deficient progenitors at E8.5 were eventually lost and the remnant RPE was derived from Six3-Cre negative cells. Thus, Six3 in a small population of progenitors expressing Six3-Cre at E8.5 is required for neuroretinal specification via regulating cell signaling and survival in mice.

Complement component C3aR constitutes a novel regulator for chick eye morphogenesis.

Complement components have been implicated in a wide variety of functions including neurogenesis, proliferation, cell migration, differentiation, cancer, and more recently early development and regeneration. Following our initial observations indicating that C3a/C3aR signaling induces chick retina regeneration, we analyzed its role in chick eye morphogenesis. During eye development, the optic vesicle (OV) invaginates to generate a bilayer optic cup (OC) that gives rise to the retinal pigmented epithelium (RPE) and neural retina. We show by immunofluorescence staining that C3 and the receptor for C3a (the cleaved and active form of C3), C3aR, are present in chick embryos during eye morphogenesis in the OV and OC. Interestingly, C3aR is mainly localized in the nuclear compartment at the OC stage. Loss of function studies at the OV stage using morpholinos or a blocking antibody targeting the C3aR (anti-C3aR Ab), causes eye defects such as microphthalmia and defects in the ventral portion of the eye that result in coloboma. Such defects were not observed when C3aR was disrupted at the OC stage. Histological analysis demonstrated that microphthalmic eyes were unable to generate a normal optic stalk or a closed OC. The dorsal/ventral patterning defects were accompanied by an expansion of the ventral markers Pax2, cVax and retinoic acid synthesizing enzyme raldh-3 (aldh1a3) domains, an absence of the dorsal expression of Tbx5 and raldh-1 (aldh1a1) and a re-specification of the ventral RPE to neuroepithelium. In addition, the eyes showed overall decreased expression of Gli1 and a change in distribution of nuclear ß-catenin, suggesting that Shh and Wnt pathways have been affected. Finally, we observed prominent cell death along with a decrease in proliferating cells, indicating that both processes contribute to the microphthalmic phenotype. Together our results show that C3aR is necessary for the proper morphogenesis of the OC. This is the first report implicating C3aR in eye development, revealing an unsuspected hitherto regulator for proper chick eye morphogenesis.

Short term optical defocus perturbs normal developmental shifts in retina/RPE protein abundance.

BACKGROUND: Myopia (short-sightedness) affects approximately 1.4 billion people worldwide, and prevalence is increasing. Animal models induced by defocusing lenses show striking similarity with human myopia in terms of morphology and the implicated genetic pathways. Less is known about proteome changes in animals. Thus, the present study aimed to improve understanding of protein pathway responses to lens defocus, with an emphasis on relating expression changes to no lens control development and identifying bidirectional and/or distinct pathways across myopia and hyperopia (long-sightedness) models. RESULTS: Quantitative label-free proteomics and gene set enrichment analysis (GSEA) were used to examine protein pathway expression in the retina/RPE of chicks following 6 h and 48 h of myopia induction with - 10 dioptre (D) lenses, hyperopia induction with +10D lenses, or normal no lens rearing. Seventy-one pathways linked to cell development and neuronal maturation were differentially enriched between 6 and 48 h in no lens chicks. The majority of these normal developmental changes were disrupted by lens-wear (47 of 71 pathways), however, only 11 pathways displayed distinct expression profiles across the lens conditions. Most notably, negative lens-wear induced up-regulation of proteins involved in ATP-driven ion transport, calcium homeostasis, and GABA signalling between 6 and 48 h, while the same proteins were down-regulated over time in normally developing chicks. Glutamate and bicarbonate/chloride transporters were also down-regulated over time in normally developing chicks, and positive lens-wear inhibited this down-regulation. CONCLUSIONS: The chick retina/RPE proteome undergoes extensive pathway expression shifts during normal development. Most of these pathways are further disrupted by lens-wear. The identified expression patterns suggest close interactions between neurotransmission (as exemplified by increased GABA receptor and synaptic protein expression), cellular ion homeostasis, and associated energy resources during myopia induction. We have also provided novel evidence for changes to SLC-mediated transmembrane transport during hyperopia induction, with potential implications for signalling at the photoreceptor-bipolar synapse. These findings reflect a key role for perturbed neurotransmission and ionic homeostasis in optically-induced refractive errors, and are predicted by our Retinal Ion Driven Efflux (RIDE) model.

Rapid reverse genetic screening using CRISPR in zebrafish.

Identifying genes involved in biological processes is critical for understanding the molecular building blocks of life. We used engineered CRISPR (clustered regularly interspaced short palindromic repeats) to efficiently mutate specific loci in zebrafish (Danio rerio) and screen for genes involved in vertebrate biological processes. We found that increasing CRISPR efficiency by injecting optimized amounts of Cas9-encoding mRNA and multiplexing single guide RNAs (sgRNAs) allowed for phenocopy of known mutants across many phenotypes in embryos. We performed a proof-of-concept screen in which we used intersecting, multiplexed pool injections to examine 48 loci and identified two new genes involved in electrical-synapse formation. By deep sequencing target loci, we found that 90% of the genes were effectively screened. We conclude that CRISPR can be used as a powerful reverse genetic screening strategy in vivo in a vertebrate system.

Loss of laminin alpha 1 results in multiple structural defects and divergent effects on adhesion during vertebrate optic cup morphogenesis.

The vertebrate eye forms via a complex set of morphogenetic events. The optic vesicle evaginates and undergoes transformative shape changes to form the optic cup, in which neural retina and retinal pigmented epithelium enwrap the lens. It has long been known that a complex, glycoprotein-rich extracellular matrix layer surrounds the developing optic cup throughout the process, yet the functions of the matrix and its specific molecular components have remained unclear. Previous work established a role for laminin extracellular matrix in particular steps of eye development, including optic vesicle evagination, lens differentiation, and retinal ganglion cell polarization, yet it is unknown what role laminin might play in the early process of optic cup formation subsequent to the initial step of optic vesicle evagination. Here, we use the zebrafish lama1 mutant (lama1(UW1)) to determine the function of laminin during optic cup morphogenesis. Using live imaging, we find, surprisingly, that loss of laminin leads to divergent effects on focal adhesion assembly in a spatiotemporally-specific manner, and that laminin is required for multiple steps of optic cup morphogenesis, including optic stalk constriction, invagination, and formation of a spherical lens. Laminin is not required for single cell behaviors and changes in cell shape. Rather, in lama1(UW1) mutants, loss of epithelial polarity and altered adhesion lead to defective tissue architecture and formation of a disorganized retina. These results demonstrate that the laminin extracellular matrix plays multiple critical roles regulating adhesion and polarity to establish and maintain tissue structure during optic cup morphogenesis.

Regulation of melanosome number, shape and movement in the zebrafish retinal pigment epithelium by OA1 and PMEL.

Analysis of melanosome biogenesis in the retinal pigment epithelium (RPE) is challenging because it occurs predominantly in a short embryonic time window. Here, we show that the zebrafish provides an ideal model system for studying this process because in the RPE the timing of melanosome biogenesis facilitates molecular manipulation using morpholinos. Morpholino-mediated knockdown of OA1 (also known as GPR143), mutations in the human homologue of which cause the most common form of human ocular albinism, induces a major reduction in melanosome number, recapitulating a key feature of the mammalian disease where reduced melanosome numbers precede macromelanosome formation. We further show that PMEL, a key component of mammalian melanosome biogenesis, is required for the generation of cylindrical melanosomes in zebrafish, which in turn is required for melanosome movement into the apical processes and maintenance of photoreceptor integrity. Spherical and cylindrical melanosomes containing similar melanin volumes co-exist in the cell body but only cylindrical melanosomes enter the apical processes. Taken together, our findings indicate that melanosome number and shape are independently regulated and that melanosome shape controls a function in the RPE that depends on localisation in the apical processes.

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.

Differential behavioral outcomes following neonatal versus fetal human retinal pigment epithelial cell striatal implants in parkinsonian rats.

Following the failure of a Phase II clinical study evaluating human retinal pigment epithelial (hRPE) cell implants as a potential treatment option for Parkinson's disease, speculation has centered on implant function and survival as possible contributors to the therapeutic outcomes. We recently reported that neonatal hRPE cells, similar to hRPE cells used in the Phase II clinical study, produced short-lived in vitro and limited in vivo trophic factors, which supports that assumption. We hypothesize that the switch from fetal to neonatal hRPE cells, between the Phase I and the Phase II clinical trial may be partly responsible for the later negative outcomes. To investigate this hypothesis, we used two neonatal hRPE cell lots, prepared in a similar manner to neonatal hRPE cells used in the Phase II clinical study, and compared them to previously evaluated fetal hRPE cells for behavioral changes following unilateral striatal implantation in 6-hydroxydopamine-lesioned rats. The results showed that only fetal, not neonatal, hRPE cell implants, were able to improve behavioral outcomes following striatal implantation in the lesioned rats. These data suggest that fetal hRPE cells may be preferential to neonatal hRPE cells in restoring behavioral deficits.