Mutations in SPATA13/ASEF2 cause primary angle closure glaucoma.

Current estimates suggest 50% of glaucoma blindness worldwide is caused by primary angle-closure glaucoma (PACG) but the causative gene is not known. We used genetic linkage and whole genome sequencing to identify Spermatogenesis Associated Protein 13, SPATA13 (NM_001166271; NP_001159743, SPATA13 isoform I), also known as ASEF2 (Adenomatous polyposis coli-stimulated guanine nucleotide exchange factor 2), as the causal gene for PACG in a large seven-generation white British family showing variable expression and incomplete penetrance. The 9 bp deletion, c.1432_1440del; p.478_480del was present in all affected individuals with angle-closure disease. We show ubiquitous expression of this transcript in cell lines derived from human tissues and in iris, retina, retinal pigment and ciliary epithelia, cornea and lens. We also identified eight additional mutations in SPATA13 in a cohort of 189 unrelated PACS/PAC/PACG samples. This gene encodes a 1277 residue protein which localises to the nucleus with partial co-localisation with nuclear speckles. In cells undergoing mitosis SPATA13 isoform I becomes part of the kinetochore complex co-localising with two kinetochore markers, polo like kinase 1 (PLK-1) and centrosome-associated protein E (CENP-E). The 9 bp deletion reported in this study increases the RAC1-dependent guanine nucleotide exchange factors (GEF) activity. The increase in GEF activity was also observed in three other variants identified in this study. Taken together, our data suggest that SPATA13 is involved in the regulation of mitosis and the mutations dysregulate GEF activity affecting homeostasis in tissues where it is highly expressed, influencing PACG pathogenesis.

WDR34, a candidate gene for non-syndromic rod-cone dystrophy.

Rod-cone dystrophy (RCD), also called retinitis pigmentosa, is characterized by rod followed by cone photoreceptor degeneration, leading to gradual visual loss. Mutations in over 65 genes have been associated with non-syndromic RCD explaining 60% to 70% of cases, with novel gene defects possibly accounting for the unsolved cases. Homozygosity mapping and whole-exome sequencing applied to a case of autosomal recessive non-syndromic RCD from a consanguineous union identified a homozygous variant in WDR34. Mutations in WDR34 have been previously associated with severe ciliopathy syndromes possibly associated with a retinal dystrophy. This is the first report of a homozygous mutation in WDR34 associated with non-syndromic RCD.

A missense mutation in ASRGL1 is involved in causing autosomal recessive retinal degeneration.

Inherited retinal dystrophies are a group of genetically heterogeneous conditions with broad phenotypic heterogeneity. We analyzed a large five-generation pedigree with early-onset recessive retinal degeneration to identify the causative mutation. Linkage analysis and homozygosity mapping combined with exome sequencing were carried out to map the disease locus and identify the p.G178R mutation in the asparaginase like-1 gene (ASRGL1), segregating with the retinal dystrophy phenotype in the study pedigree. ASRGL1 encodes an enzyme that catalyzes the hydrolysis of L-asparagine and isoaspartyl-peptides. Studies on the ASRGL1 expressed in Escherichia coli and transiently transfected mammalian cells indicated that the p.G178R mutation impairs the autocatalytic processing of this enzyme resulting in the loss of functional ASRGL1 and leaving the inactive precursor protein as a destabilized and aggregation-prone protein. A zebrafish model overexpressing the mutant hASRGL1 developed retinal abnormalities and loss of cone photoreceptors. Our studies suggest that the p.G178R mutation in ASRGL1 leads to photoreceptor degeneration resulting in progressive vision loss.

267 Spanish Exomes Reveal Population-Specific Differences in Disease-Related Genetic Variation.

Recent results from large-scale genomic projects suggest that allele frequencies, which are highly relevant for medical purposes, differ considerably across different populations. The need for a detailed catalog of local variability motivated the whole-exome sequencing of 267 unrelated individuals, representative of the healthy Spanish population. Like in other studies, a considerable number of rare variants were found (almost one-third of the described variants). There were also relevant differences in allelic frequencies in polymorphic variants, including ∼10,000 polymorphisms private to the Spanish population. The allelic frequencies of variants conferring susceptibility to complex diseases (including cancer, schizophrenia, Alzheimer disease, type 2 diabetes, and other pathologies) were overall similar to those of other populations. However, the trend is the opposite for variants linked to Mendelian and rare diseases (including several retinal degenerative dystrophies and cardiomyopathies) that show marked frequency differences between populations. Interestingly, a correspondence between differences in allelic frequencies and disease prevalence was found, highlighting the relevance of frequency differences in disease risk. These differences are also observed in variants that disrupt known drug binding sites, suggesting an important role for local variability in population-specific drug resistances or adverse effects. We have made the Spanish population variant server web page that contains population frequency information for the complete list of 170,888 variant positions we found publicly available (http://spv.babelomics.org/), We show that it if fundamental to determine population-specific variant frequencies to distinguish real disease associations from population-specific polymorphisms.

Biallelic variants in TTLL5, encoding a tubulin glutamylase, cause retinal dystrophy.

In a subset of inherited retinal degenerations (including cone, cone-rod, and macular dystrophies), cone photoreceptors are more severely affected than rods; ABCA4 mutations are the most common cause of this heterogeneous class of disorders. To identify retinal-disease-associated genes, we performed exome sequencing in 28 individuals with "cone-first" retinal disease and clinical features atypical for ABCA4 retinopathy. We then conducted a gene-based case-control association study with an internal exome data set as the control group. TTLL5, encoding a tubulin glutamylase, was highlighted as the most likely disease-associated gene; 2 of 28 affected subjects harbored presumed loss-of-function variants: c.[1586_1589delAGAG];[1586_1589delAGAG], p.[Glu529Valfs(∗)2];[Glu529Valfs(∗)2], and c.[401delT(;)3354G>A], p.[Leu134Argfs(∗)45(;)Trp1118(∗)]. We then inspected previously collected exome sequence data from individuals with related phenotypes and found two siblings with homozygous nonsense variant c.1627G>T (p.Glu543(∗)) in TTLL5. Subsequently, we tested a panel of 55 probands with retinal dystrophy for TTLL5 mutations; one proband had a homozygous missense change (c.1627G>A [p.Glu543Lys]). The retinal phenotype was highly similar in three of four families; the sibling pair had a more severe, early-onset disease. In human and murine retinae, TTLL5 localized to the centrioles at the base of the connecting cilium. TTLL5 has been previously reported to be essential for the correct function of sperm flagella in mice and play a role in polyglutamylation of primary cilia in vitro. Notably, genes involved in the polyglutamylation and deglutamylation of tubulin have been associated with photoreceptor degeneration in mice. The electrophysiological and fundus autofluorescence imaging presented here should facilitate the molecular diagnosis in further families.

Cleavage of Mer tyrosine kinase (MerTK) from the cell surface contributes to the regulation of retinal phagocytosis.

Phagocytosis of apoptotic cells by macrophages and spent photoreceptor outer segments (POS) by retinal pigment epithelial (RPE) cells requires several proteins, including MerTK receptors and associated Gas6 and protein S ligands. In the retina, POS phagocytosis is rhythmic, and MerTK is activated promptly after light onset via the αvß5 integrin receptor and its ligand MFG-E8, thus generating a phagocytic peak. The phagocytic burst is limited in time, suggesting a down-regulation mechanism that limits its duration. Our previous data showed that MerTK helps control POS binding of integrin receptors at the RPE cell surface as a negative feedback loop. Our present results show that a soluble form of MerTK (sMerTK) is released in the conditioned media of RPE-J cells during phagocytosis and in the interphotoreceptor matrix of the mouse retina during the morning phagocytic peak. In contrast to macrophages, the two cognate MerTK ligands have an opposite effect on phagocytosis and sMerTK release, whereas the integrin ligand MFG-E8 markedly increases both phagocytosis and sMerTK levels. sMerTK acts as a decoy receptor blocking the effect of both MerTK ligands. Interestingly, stimulation of sMerTK release decreases POS binding. Conversely, blocking MerTK cleavage increased mostly POS binding by RPE cells. Therefore, our data suggest that MerTK cleavage contributes to the acute regulation of RPE phagocytosis by limiting POS binding to the cell surface.

The familial dementia gene revisited: a missense mutation revealed by whole-exome sequencing identifies ITM2B as a candidate gene underlying a novel autosomal dominant retinal dystrophy in a large family.

Inherited retinal diseases are a group of clinically and genetically heterogeneous disorders for which a significant number of cases remain genetically unresolved. Increasing knowledge on underlying pathogenic mechanisms with precise phenotype-genotype correlation is, however, critical for establishing novel therapeutic interventions for these yet incurable neurodegenerative conditions. We report phenotypic and genetic characterization of a large family presenting an unusual autosomal dominant retinal dystrophy. Phenotypic characterization revealed a retinopathy dominated by inner retinal dysfunction and ganglion cell abnormalities. Whole-exome sequencing identified a missense variant (c.782A>C, p.Glu261Ala) in ITM2B coding for Integral Membrane Protein 2B, which co-segregates with the disease in this large family and lies within the 24.6 Mb interval identified by microsatellite haplotyping. The physiological role of ITM2B remains unclear and has never been investigated in the retina. RNA in situ hybridization reveals Itm2b mRNA in inner nuclear and ganglion cell layers within the retina, with immunostaining demonstrating the presence of the corresponding protein in the same layers. Furthermore, ITM2B in the retina co-localizes with its known interacting partner in cerebral tissue, the amyloid ß precursor protein, critical in Alzheimer disease physiopathology. Interestingly, two distinct ITM2B mutations, both resulting in a longer protein product, had already been reported in two large autosomal dominant families with Alzheimer-like dementia but never in subjects with isolated retinal diseases. These findings should better define pathogenic mechanism(s) associated with ITM2B mutations underlying dementia or retinal disease and add a new candidate to the list of genes involved in inherited retinal dystrophies.

Whole-exome sequencing identifies LRIT3 mutations as a cause of autosomal-recessive complete congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous retinal disorder. Two forms can be distinguished clinically: complete CSNB (cCSNB) and incomplete CSNB. Individuals with cCSNB have visual impairment under low-light conditions and show a characteristic electroretinogram (ERG). The b-wave amplitude is severely reduced in the dark-adapted state of the ERG, representing abnormal function of ON bipolar cells. Furthermore, individuals with cCSNB can show other ocular features such as nystagmus, myopia, and strabismus and can have reduced visual acuity and abnormalities of the cone ERG waveform. The mode of inheritance of this form can be X-linked or autosomal recessive, and the dysfunction of four genes (NYX, GRM6, TRPM1, and GPR179) has been described so far. Whole-exome sequencing in one simplex cCSNB case lacking mutations in the known genes led to the identification of a missense mutation (c.983G>A [p.Cys328Tyr]) and a nonsense mutation (c.1318C>T [p.Arg440(∗)]) in LRIT3, encoding leucine-rich-repeat (LRR), immunoglobulin-like, and transmembrane-domain 3 (LRIT3). Subsequent Sanger sequencing of 89 individuals with CSNB identified another cCSNB case harboring a nonsense mutation (c.1151C>G [p.Ser384(∗)]) and a deletion predicted to lead to a premature stop codon (c.1538_1539del [p.Ser513Cysfs(∗)59]) in the same gene. Human LRIT3 antibody staining revealed in the outer plexiform layer of the human retina a punctate-labeling pattern resembling the dendritic tips of bipolar cells; similar patterns have been observed for other proteins implicated in cCSNB. The exact role of this LRR protein in cCSNB remains to be elucidated.

Concise review: reactive astrocytes and stem cells in spinal cord injury: good guys or bad guys?

Spinal cord injury (SCI) usually results in long lasting locomotor and sensory neuron degeneration below the injury. Astrocytes normally play a decisive role in mechanical and metabolic support of neurons, but in the spinal cord they cause injury, exerting well-known detrimental effects that contribute to glial scar formation and inhibition of axon outgrowth. Cell transplantation is considered a promising approach for replacing damaged cells and promoting neuroprotective and neuroregenerative repair, but the effects of the grafted cells on local tissue and the regenerative properties of endogenous neural stem cells in the injured spinal cord are largely unknown. During the last 2 decades cumulative evidence from diverse animal models has indicated that reactive astrocytes in synergy with transplanted cells could be beneficial for injury in multiple ways, including neuroprotection and axonal growth. In this review, we specifically focus on the dual opposing roles of reactive astrocytes in SCI and how they contribute to the creation of a permissive environment when combined with transplanted cells as the influential components for a local regenerative niche. Modulation of reactive astrocyte function might represent an extremely attractive new therapy to enhance the functional outcomes in patients.

First insights into the expression of VAX2 in humans and its localization in the adult primate retina.

VAX2 is a transcription factor specifically expressed in the ventral region of the prospective neural retina in vertebrates and is required for ventral eye specification. Despite its extensive analysis in vertebrates, the biological role of VAX2 in the human is presently unclear. This study was undertaken to investigate VAX2 in humans aiming to gain new knowledge into its involvement in retinal function. Here, we report VAX2 gene expression and protein localization in cultured cells and adult retina. RT-PCR experiments indicated that VAX2 is enriched in neuronal tissues. Moreover, we identified a novel isoform most abundantly expressed in the retina. We termed the known transcript (NM_012476) isoform-1, and the newly identified transcript as isoform-2. Analysis of protein localization in cultured cells revealed that isoform-1 localizes to the nucleus and isoform-2 is widely expressed within the cell; partial co-localization of isoform-2 and actin filaments was also observed. In nonhuman primate retina VAX2 was seen either in the nuclear or in the cytoplasmic compartment depending on the retinal cell type. In addition, a noteworthy enrichment of the signal was observed in the outer segment of cone photoreceptors. Overall, this study provides the first insights into the expression of VAX2 in humans and its localization in the adult primate retina. Moreover, preliminary characterization of alternative variants suggests an involvement of VAX2 in multiple cellular pathways. Our findings raise the interesting possibility for further investigation of VAX2 in the retina in health and disease.

Photoreceptor degeneration: genetic and mechanistic dissection of a complex trait.

The retina provides exquisitely sensitive vision that relies on the integrity of a uniquely vulnerable cell, the photoreceptor (PR). The genetic and mechanistic causes of retinal degeneration due to PR cell death--which occurs in conditions such as retinitis pigmentosa and age-related macular degeneration--are being successfully dissected. Over one hundred loci, some containing common variants but most containing rare variants, are implicated in the genetic architecture of this complex trait. This genetic heterogeneity results in equally diverse disease mechanisms that affect almost every aspect of PR function but converge on a common cell death pathway. Although genetic and mechanistic diversity creates challenges for therapy, some approaches--particularly gene-replacement therapy--are showing considerable promise.

Span poly-L-arginine nanoparticles are efficient non-viral vectors for PRPF31 gene delivery: An approach of gene therapy to treat retinitis pigmentosa.

Retinitis pigmentosa (RP) is the most common cause of inherited blindness in adults. Mutations in the PRPF31 gene produce autosomal dominant RP (adRP). To date there are no effective treatments for this disease. The purpose of this study was to design an efficient non-viral vector for human PRPF31 gene delivery as an approach to treat this form of adRP. Span based nanoparticles were developed to mediate gene transfer in the subretinal space of a mouse model of adRP carrying a point mutation (A216P) in the Prpf31 gene. Funduscopic examination, electroretinogram, optomotor test and optical coherence tomography were conducted to further in vivo evaluate the safety and efficacy of the nanosystems developed. Span-polyarginine (SP-PA) nanoparticles were able to efficiently transfect the GFP and PRPF31 plasmid in mice retinas. Statistically significant improvement in visual acuity and retinal thickness were found in Prpf31A216P/+ mice treated with the SP-PA-PRPF31 nanomedicine.

ATR localizes to the photoreceptor connecting cilium and deficiency leads to severe photoreceptor degeneration in mice.

Ataxia-telangiectasia and Rad3 (ATR), a sensor of DNA damage, is associated with the regulation and control of cell division. ATR deficit is known to cause Seckel syndrome, characterized by severe proportionate short stature and microcephaly. We used a mouse model for Seckel disease to study the effect of ATR deficit on retinal development and function and we have found a new role for ATR, which is critical for the postnatal development of the photoreceptor (PR) layer in mouse retina. The structural and functional characterization of the ATR(+/s) mouse retinas displayed a specific, severe and early degeneration of rod and cone cells resembling some characteristics of human retinal degenerations. A new localization of ATR in the cilia of PRs and the fact that mutant mice have shorter cilia suggests that the PR degeneration here described results from a ciliary defect.

Mutations in pre-mRNA processing factors 3, 8, and 31 cause dysfunction of the retinal pigment epithelium.

Mutations in the ubiquitously expressed pre-mRNA processing factors 3, 8, and 31 (PRPF3, PRPF8, and PRPF31) cause nonsyndromic dominant retinitis pigmentosa in humans, an inherited retinal degeneration. It is unclear what mechanisms, or which cell types of the retina, are affected. Transgenic mice with the human mutations in these genes display late-onset morphological changes in the retinal pigment epithelium (RPE). To determine whether the observed morphological changes are preceded by abnormal RPE function, we investigated its phagocytic function in Prpf3(T494M/T494M), Prpf8(H2309P/H2309P), and Prpf31(+/-) mice. We observe decreased phagocytosis in primary RPE cultures from mutant mice, and this is replicated by shRNA-mediated knockdown of PRPF31 in human ARPE-19 cells. The diurnal rhythmicity of phagocytosis is almost lost, indicated by the marked attenuation of the phagocytic burst 2 hours after light onset. The strength of adhesion between RPE apical microvilli and photoreceptor outer segments also declined during peak adhesion in all mutants. In all models, at least one of the receptors involved in binding and internalization of shed photoreceptor outer segments was subjected to changes in localization. Although the mechanism underlying these changes in RPE function is yet to be elucidated, these data are consistent with the mouse RPE being the primary cell affected by mutations in the RNA splicing factors, and these changes occur at an early age.

Brief report: astrogliosis promotes functional recovery of completely transected spinal cord following transplantation of hESC-derived oligodendrocyte and motoneuron progenitors.

Spinal cord injury results in neural loss and consequently motor and sensory impairment below the injury. Reactive astrocytes contribute to formation of glial scar, thus impeding axonal regeneration, through secretion of extracellular matrix molecules, chondroitin sulfate proteoglycans (CSPGs). In this study, we analyze lesion site tissue to reveal the possible mechanism underlying the functional recovery after cell transplantation of human embryonic stem cell (hESC)-derived oligodendrocyte progenitor cell (OPC) and motoneuron progenitors (MP) and propose that transplanted cells increase astrogliosis through the regenerative signaling pathways activated in the host tissue that may crucial for restoring locomotor ability. We show that the transplantation of hESC-derived OPC and MP promotes astrogliosis, through activation of Jagged1-dependent Notch and Jak/STAT signaling that support axonal survival. The transplanted cells in synergism with reactive astrocytes create permissive environment in which the expression of detrimental genes (Cspg, Tenascins, and genes involved in SLIT/ROBO signaling) was significantly decreased while expression of beneficial ones (Laminins and Fibronectin) was increased. According to our data, this mechanism is activated in all transplantation groups independently of the level of locomotor recovery. These results indicate that modifying the beneficial function of reactive astrocytes could be a feasible therapeutic strategy for spinal cord injury in future.

CNOT3 is a modifier of PRPF31 mutations in retinitis pigmentosa with incomplete penetrance.

Heterozygous mutations in the PRPF31 gene cause autosomal dominant retinitis pigmentosa (adRP), a hereditary disorder leading to progressive blindness. In some cases, such mutations display incomplete penetrance, implying that certain carriers develop retinal degeneration while others have no symptoms at all. Asymptomatic carriers are protected from the disease by a higher than average expression of the PRPF31 allele that is not mutated, mainly through the action of an unknown modifier gene mapping to chromosome 19q13.4. We investigated a large family with adRP segregating an 11-bp deletion in PRPF31. The analysis of cell lines derived from asymptomatic and affected individuals revealed that the expression of only one gene among a number of candidates within the 19q13.4 interval significantly correlated with that of PRPF31, both at the mRNA and protein levels, and according to an inverse relationship. This gene was CNOT3, encoding a subunit of the Ccr4-not transcription complex. In cultured cells, siRNA-mediated silencing of CNOT3 provoked an increase in PRPF31 expression, confirming a repressive nature of CNOT3 on PRPF31. Furthermore, chromatin immunoprecipitation revealed that CNOT3 directly binds to a specific PRPF31 promoter sequence, while next-generation sequencing of the CNOT3 genomic region indicated that its variable expression is associated with a common intronic SNP. In conclusion, we identify CNOT3 as the main modifier gene determining penetrance of PRPF31 mutations, via a mechanism of transcriptional repression. In asymptomatic carriers CNOT3 is expressed at low levels, allowing higher amounts of wild-type PRPF31 transcripts to be produced and preventing manifestation of retinal degeneration.

Expression of PRPF31 and TFPT: regulation in health and retinal disease.

PRPF31, a gene located at chromosome 19q13.4, encodes the ubiquitous splicing factor PRPF31. The gene lies in a head-to-head arrangement with TFPT, a poorly characterized gene with a role in cellular apoptosis. Mutations in PRPF31 have been implicated in autosomal dominant retinitis pigmentosa (adRP), a frequent and important cause of blindness worldwide. Disease associated with PRPF31 mutations is unusual, in that there is often non-penetrance of the disease phenotype in affected families, caused by differential expression of PRPF31. This study aimed to characterize the basic promoter elements of PRPF31 and TFPT. Luciferase reporter constructs were made, using genomic DNA from an asymptomatic individual with a heterozygous deletion of the entire putative promoter region. Fragments were tested by the dual-luciferase reporter assay in HeLa and RPE-1 cell lines. A comparison was made between the promoter regions of symptomatic and asymptomatic mutation-carrying individuals. A patient (CAN493) with adRP was identified, harbouring a regulatory region mutation; both alleles were assayed by the dual-luciferase reporter assay. Luciferase assays led to the identification of core promoters for both PRPF31 and TFPT; despite their shared gene architecture, the two genes appear to be controlled by slightly different regulatory regions. One functional polymorphism was identified in the PRPF31 promoter that increased transcriptional activation. The change was not, however, consistent with the observed symptomatic-asymptomatic phenotypes in a family affected by PRPF31-adRP. Analysis of the mutant promoter fragment from CAN493 showed a >50% reduction in promoter activity, suggesting a disease mechanism of functional haploinsufficiency-the first report of this disease mechanism in adRP.

Dominant PRPF31 mutations are hypostatic to a recessive CNOT3 polymorphism in retinitis pigmentosa: a novel phenomenon of "linked trans-acting epistasis".

Mutations in PRPF31 are responsible for autosomal dominant retinitis pigmentosa (adRP, RP11 form) and affected families show nonpenetrance. Differential expression of the wildtype PRPF31 allele is responsible for this phenomenon: coinheritance of a mutation and a higher expressing wildtype allele provide protection against development of disease. It has been suggested that a major modulating factor lies in close proximity to the wildtype PRPF31 gene on Chromosome 19, implying that a cis-acting factor directly alters PRPF31 expression. Variable expression of CNOT3 is one determinant of PRPF31 expression. This study explored the relationship between CNOT3 (a trans-acting factor) and its paradoxical cis-acting nature in relation to RP11. Linkage analysis on Chromosome 19 was performed in mutation-carrying families, and the inheritance of the wildtype PRPF31 allele in symptomatic-asymptomatic sibships was assessed-confirming that differential inheritance of wildtype chromosome 19q13 determines the clinical phenotype (P < 2.6 × 10(-7) ). A theoretical model was constructed that explains the apparent conflict between the linkage data and the recent demonstration that a trans-acting factor (CNOT3) is a major nonpenetrance factor: we propose that this apparently cis-acting effect arises due to the intimate linkage of CNOT3 and PRPF31 on Chromosome 19q13-a novel mechanism that we have termed "linked trans-acting epistasis."

Hypoxia increases the yield of photoreceptors differentiating from mouse embryonic stem cells and improves the modeling of retinogenesis in vitro.

Retinitis pigmentosa (RP), a genetically heterogeneous group of diseases together with age-related macular degeneration (AMD), are the leading causes of permanent blindness and are characterized by the progressive dysfunction and death of the light sensing photoreceptors of the retina. Due to the limited regeneration capacity of the mammalian retina, the scientific community has invested significantly in trying to obtain retinal progenitor cells from embryonic stem cells (ESC). These represent an unlimited source of retinal cells, but it has not yet been possible to achieve specific populations, such as photoreceptors, efficiently enough to allow them to be used safely in the future as cell therapy of RP or AMD. In this study, we generated a high yield of photoreceptors from directed differentiation of mouse ESC (mESC) by recapitulating crucial phases of retinal development. We present a new protocol of differentiation, involving hypoxia and taking into account extrinsic and intrinsic cues. These include niche-specific conditions as well as the manipulation of the signaling pathways involved in retinal development. Our results show that hypoxia promotes and improves the differentiation of mESC toward photoreceptors. Different populations of retinal cells are increased in number under the hypoxic conditions applied, such as Crx-positive cells, S-Opsin-positive cells, and double positive cells for Rhodopsin and Recoverin, as shown by immunofluorescence analysis. For the first time, this manuscript reports the high efficiency of differentiation in vivo and the expression of mature rod photoreceptor markers in a large number of differentiated cells, transplanted in the subretinal space of wild-type mice.

Cross species analysis of Prominin reveals a conserved cellular role in invertebrate and vertebrate photoreceptor cells.

The two fundamental types of photoreceptor cells have evolved unique structures to expand the apical membrane to accommodate the phototransduction machinery, exemplified by the cilia-based outer segment of the vertebrate photoreceptor cell and the microvilli-based rhabdomere of the invertebrate photoreceptor. The morphogenesis of these compartments is integral for photoreceptor cell integrity and function. However, little is known about the elementary cellular and molecular mechanisms required to generate these compartments. Here we investigate whether a conserved cellular mechanism exists to create the phototransduction compartments by examining the functional role of a photoreceptor protein common to both rhabdomeric and ciliated photoreceptor cells, Prominin. First and foremost we demonstrate that the physiological role of Prominin is conserved between rhabdomeric and ciliated photoreceptor cells. Human Prominin1 is not only capable of rescuing the corresponding rhabdomeric Drosophila prominin mutation but also demonstrates a conserved genetic interaction with a second photoreceptor protein Eyes Shut. Furthermore, we demonstrate the Prominin homologs in vertebrate and invertebrate photoreceptors require the same structural features and post-translational modifications for function. Moreover, expression of mutant human Prominin1, associated with autosomal dominant retinal degeneration, in rhabdomeric photoreceptor cells disrupts morphogenesis in ways paralleling retinal degeneration seen in ciliated photoreceptors. Taken together, our results suggest the existence of an ancestral Prominin-directed cellular mechanism to create and model the apical membranes of the two fundamental types of photoreceptor cells into their respective phototransduction compartments.