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As signalling organelles, primary cilia regulate their membrane G protein-coupled receptor (GPCR) content by ectocytosis, a process requiring localised actin dynamics at their tip to alter membrane shape.(1, 2) Mammalian photoreceptor outer segments comprise an expanse of folded membranes (discs) at the tip of highly-specialised connecting cilia (CC), in which photosensitive GPCRs like rhodopsin are concentrated. In an extraordinary feat of biology, outer segment discs are shed and remade daily.(3) Defects in this process, due to genetic mutations, cause retinitis pigmentosa (RP), an untreatable, blinding disease. The mechanism by which photoreceptor cilia generate outer segments is therefore fundamental for vision yet poorly understood. Here, we show the membrane deformation required for outer segment disc genesis is driven by dynamic changes in the actin cytoskeleton in a process akin to ectocytosis. Further, we showRPGR, a leading causal RP gene, regulates activity of actin binding proteins crucial to this process. Disc genesis is compromised inRpgrmouse models, slowing the actin dynamics required for timely disc formation, leading to aborted membrane shedding as ectosome-like vesicles, photoreceptor death and visual loss. Manipulation of actin dynamics partially rescues the phenotype, suggesting this pathway could be targeted therapeutically. These findings help define how actin-mediated dynamics control outer segment turnover.

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Fam151b is a mammalian homologue of the C. elegans menorin gene, which is involved in neuronal branching. The International Mouse Phenotyping Consortium (IMPC) aims to knock out every gene in the mouse and comprehensively phenotype the mutant animals. This project identified Fam151b homozygous knock-out mice as having retinal degeneration. We show they have no photoreceptor function from eye opening, as demonstrated by a lack of electroretinograph (ERG) response. Histological analysis shows that during development of the eye the correct number of cells are produced and that the layers of the retina differentiate normally. However, after eye opening at P14, Fam151b mutant eyes exhibit signs of retinal stress and rapidly lose photoreceptor cells. We have mutated the second mammalian menorin homologue, Fam151a, and homozygous mutant mice have no discernible phenotype. Sequence analysis indicates that the FAM151 proteins are members of the PLC-like phosphodiesterase superfamily. However, the substrates and function of the proteins remains unknown.

PurposeAlbinism is a clinically and genetically heterogeneous condition. Despite analysis of the nineteen known genes, ∼30% patients remain unsolved. We aimed to identify new genes involved in albinism.MethodsWe sequenced a panel of genes with known or predicted involvement in melanogenesis in 230 unsolved albinism patients.ResultsWe identified variants in theDopachrome tautomerase(DCT) gene in two patients. One was compound heterozygous for a 14 bp deletion in exon 9 and c.118T>A p.(Cys40Ser). The second was homozygous for c.183C>G p.(Cys61Trp). Both patients had mild hair and skin hypopigmentation, and classical ocular features. CRISPR/Cas9 was used in C57BL/6J mice to create mutations identical to the missense mutations carried by the patients, along with one loss-of-function indel mutation. When bred to homozygosity the three mutations revealed hypopigmentation of the coat, milder for Cys40Ser compared to Cys61Trp or the frameshift mutation. Histological analysis identified significant hypopigmentation of the retinal pigmented epithelium (RPE) indicating that defective RPE melanogenesis could be associated with eye and vision defects.DCTloss of function in zebrafish embryos elicited hypopigmentation both in melanocytes and RPE cells.ConclusionsDCTis the gene for a new type of oculocutaneous albinism that we propose to name OCA8.

The precise control of eye size is essential for normal vision. TMEM98 is a highly conserved and widely expressed gene which appears to be involved in eye size regulation. Mutations in human TMEM98 are found in patients with nanophthalmos (very small eyes) and variants near the gene are associated in population studies with myopia and increased eye size. As complete loss of function mutations in mouse Tmem98 result in perinatal lethality, we produced mice deficient for Tmem98 in the retinal pigment epithelium (RPE), where Tmem98 is highly expressed. These mice have greatly enlarged eyes that are very fragile with very thin retinas, compressed choroid and thin sclera. To gain insight into the mechanism of action we used a proximity labelling approach to discover interacting proteins and identified MYRF as an interacting partner. Mutations of MYRF are also associated with nanophthalmos. The protein is an endoplasmic reticulum-tethered transcription factor which undergoes autoproteolytic cleavage to liberate the N-terminal part which then translocates to the nucleus where it acts as a transcription factor. We find that TMEM98 inhibits the self-cleavage of MYRF, in a novel regulatory mechanism. In RPE lacking TMEM98, MYRF is ectopically activated and abnormally localised to the nuclei. Our findings highlight the importance of the interplay between TMEM98 and MYRF in determining the size of the eye., Author summary Having the correct eye size is important, too large and you will be short-sighted and too small and you will be far-sighted. Nanophthalmos, literally very small eye from the Greek, is a condition where the eye is very small but structurally normal. In addition to being farsighted such eyes are prone to glaucoma which can lead to loss of vision. Here we studied a protein called TMEM98 which is found in the membranes of the cells which form a layer at the back of eye called the retinal pigment epithelium (RPE). Mutations in TMEM98 have been found in nanophthalmos patients. Patients have one normal copy of the gene and one carrying a mutation. We removed Tmem98 from the RPE of mice in order to ascertain its function. We found, surprisingly, that rather than having small eyes this led to the development of very large eyes that were structurally fragile. We went on to identify protein partners of TMEM98 and found that it interacts with a protein called MYRF, mutations in which also cause nanophthalmos. This work demonstrates the importance of TMEM98 in eye size specification.

PURPOSEWe previously found a dominant mutation,Rwhs, causing white spots on the retina accompanied by retinal folds. Here we identify the mutant gene to beTmem98.In humans, mutations in the orthologous gene cause nanophthalmos. We modelled these mutations in mice and characterised the mutant eye phenotypes of these andRwhs.METHODSTheRwhsmutation was identified to be a missense mutation inTmem98by genetic mapping and sequencing. The humanTMEM98nanophthalmos missense mutations were made in the mouse gene by CRISPR-Cas9. Eyes were examined by indirect ophthalmoscopy and the retinas imaged using a retinal camera. Electroretinography was used to study retinal function. Histology, immunohistochemistry and electron microscopy techniques were used to study adult eyes.RESULTSAn I135T mutation ofTmem98causes the dominantRwhsphenotype and is perinatally lethal when homozygous. Two dominant missense mutations ofTMEM98, A193P and H196P are associated with human nanophthalmos. In the mouse these mutations cause recessive retinal defects similar to theRwhsphenotype, either alone or in combination with each other, but do not cause nanophthalmos. The retinal folds did not affect retinal function as assessed by electroretinography. Within the folds there was accumulation of disorganised outer segment material as demonstrated by immunohistochemistry and electron microscopy, and macrophages had infiltrated into these regions.CONCLUSIONSMutations in the mouse orthologue of the human nanophthalmos geneTMEM98do not result in small eyes. Rather, there is localised disruption of the laminar structure of the photoreceptors.

Purpose: We previously found a dominant mutation, Rwhs, causing white spots on the retina accompanied by retinal folds. Here we identify the mutant gene to be Tmem98. In humans, mutations in the orthologous gene cause nanophthalmos. We modeled these mutations in mice and characterized the mutant eye phenotypes of these and Rwhs. // Methods: The Rwhs mutation was identified to be a missense mutation in Tmem98 by genetic mapping and sequencing. The human TMEM98 nanophthalmos missense mutations were made in the mouse gene by CRISPR-Cas9. Eyes were examined by indirect ophthalmoscopy and the retinas imaged using a retinal camera. Electroretinography was used to study retinal function. Histology, immunohistochemistry, and electron microscopy techniques were used to study adult eyes. // Results: An I135T mutation of Tmem98 causes the dominant Rwhs phenotype and is perinatally lethal when homozygous. Two dominant missense mutations of TMEM98, A193P and H196P, are associated with human nanophthalmos. In the mouse these mutations cause recessive retinal defects similar to the Rwhs phenotype, either alone or in combination with each other, but do not cause nanophthalmos. The retinal folds did not affect retinal function as assessed by electroretinography. Within the folds there was accumulation of disorganized outer segment material as demonstrated by immunohistochemistry and electron microscopy, and macrophages had infiltrated into these regions. // Conclusions: Mutations in the mouse orthologue of the human nanophthalmos gene TMEM98 do not result in small eyes. Rather, there is localized disruption of the laminar structure of the photoreceptors.

In an international effort to assign function to all genes, and to identify new models of human disease, cohorts of mice produced from mutant embryonic stem cells are being screened in large-scale phenotyping pipelines. Hundreds of mouse lines, each mutant for a different gene, have been analyzed for developmental, hematological, immunological, metabolic, neurological, reproductive, and sensory parameters, which, thus, provide a comprehensive assessment of the consequences of gene ablation.1 Included in the sensory analysis is slit-lamp examination of the anterior chamber of the eye, and indirect ophthalmoscopy to view the retina and retinal vessels. The phenotype data can be viewed online and the mice are freely available. We describe here the detailed pathological and functional analysis of a mouse line mutant in the sodium/hydrogen ion exchange protein gene, Slc9a8, which was identified as having a retinal defect and no other abnormality other than male sterility. Sodium/hydrogen ion exchange proteins (NHEs) or cotransporter solute channels are encoded by the Slc9 gene family, comprising nine members: Slc9a1-9, encoding NHE1-9. Proteins NHE1 to 5 are located in the plasma membrane while NHE6 to 9 are found in membranes of cell organelles (reviewed previously2). These proteins contain 12 membrane spanning domains and catalyze the electroneutral transport of cations down a concentration gradient. Typically sodium ions are transported in and hydrogen ions out of the cell or organelle. Simultaneous transport of anions creates osmotic pressure and results in influx of water, regulating the pH and volume of cells and organelles. Sodium/hydrogen ion exchange is involved in aqueous humor dynamics and maintenance of intraocular pressure (IOP). Inhibition of sodium/hydrogen ion exchange in mice3 reduces IOP and at least one cotransporter, NHE1, is found expressed in the ciliary body, where aqueous dynamics are regulated. The single yeast NHE is necessary for correct sorting of proteins to the vacuole4 and, indeed, depletion of NHE8 in mammalian cells disrupts endosome trafficking.5 Six of the Slc9 family members have been mutated in mice and give rise to a range of phenotypes, from hyperactivity or ataxia and seizures (Slc9a1 and 6)6–8 to relatively mild gastric secretion and absorption defects (Slc9a2, 3, and 4).9–11 The gene Slc9a8 is widely expressed in adult mouse tissues, but is enriched in kidney, intestine, muscle, liver, and testes.12,13 The gene is expressed in the kidney proximal tubules, where reabsorption of secreted bicarbonate requires NHE function, although this is mostly via Slc9a3 (NHE3). However, Slc9a8 is expressed at higher levels in the neonatal kidney, and may partially compensate for NHE3 in Slc9a3 knockout animals, which have only a mild absorption defect.14 The Slc9a8 gene previously has been knocked out and the authors report that the mice have normal levels of serum sodium ions. They also report an increase in the length of the intestine, but a reduction in the number of goblet cells in the mutant mice with a concomitant reduction in mucin secretion and pH.13,15 Furthermore, it has been reported recently that the gene is expressed in the epithelial cells of the conjunctiva, cornea, and lacrimal glands and may have a role in the protection of these ocular epithelia.16 The male mutants also are sterile. Here, we identified and characterized a previously undescribed retinal defect of Slc9a8 knockout mice and located the origin of the phenotype to be abnormalities in the retinal pigmented epithelium.