OBJECTIVE: To investigate the long-term effects of high-dose recombinant human erythropoietin (rhEPO) administered during the perinatal period on retinal and visual function in children born extremely or very preterm.
METHODS: Randomized, double-blind clinical trial follow-up plus cohort study.
METHODS:  Setting: Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland.
METHODS: Extremely or very preterm-born children aged 7 to 15 years, previously randomized to receive either high-dose rhEPO or placebo in the perinatal period.
METHODS: participation in an ongoing neuropediatric study (EpoKids), written informed consent.
METHODS: previous ocular trauma or surgery; retinal or developmental disease unrelated to prematurity. Healthy control (HC) children of comparable age were recruited.
METHODS: term birth, informed consent.
METHODS: any ocular/visual abnormality, high refractive error. Intervention status (rhEPO/placebo) was unknown to examiners and subjects at examination, with examiners unblinded only after completion of all analyses.
METHODS: The electroretinogram (ERG) was performed with the RETeval device (LKC Technologies, Inc). Ophthalmological and orthoptic examinations excluded comorbidity in the prematurely born cohort and ocular diseases in the HC group.
METHODS: Scotopic and photopic ERG response amplitudes and peak times (6 amplitudes; 6 peak times). Secondary outcomes were habitual visual acuity and color discrimination performance (for descriptive summary only).
RESULTS: No differences in ERG parameters between EPO (n = 52; 104 eyes) and placebo (n = 35; 70 eyes) subgroups were observed (all corrected P > .05). Two cone system-mediated peak times were slightly slower in the placebo than HC (n = 52; 104 eyes) subgroup (coefficient/95% confidence interval = 0.53/0.21-0.85 and 0.36/0.13-0.60; P = .012 and .022); a predominantly rod system-mediated peak time was slightly faster in the EPO than the HC subgroup (coefficient/95% confidence interval = -4.33/-6.88 to -1.78; P = .011). Secondary outcomes were comparable across subgroups.
CONCLUSIONS: Administration of high-dose rhEPO to infants born extremely or very preterm during the perinatal period has no measurable effects on retinal function in childhood compared to placebo. Premature birth may cause small, likely clinically insignificant effects on retinal function in childhood, which may be partially mitigated by administration of rhEPO during the perinatal period.

The proneural transcription factor atonal basic helix-loop-helix transcription factor 7 (ATOH7) is expressed in early progenitors in the developing neuroretina. In vertebrates, this is crucial for the development of retinal ganglion cells (RGCs), as mutant animals show an almost complete absence of RGCs, underdeveloped optic nerves, and aberrations in retinal vessel development. Human mutations are rare and result in autosomal recessive optic nerve hypoplasia (ONH) or severe vascular changes, diagnosed as autosomal recessive persistent hyperplasia of the primary vitreous (PHPVAR). To better understand the role of ATOH7 in neuroretinal development, we created ATOH7 knockout and eGFP-expressing ATOH7 reporter human induced pluripotent stem cells (hiPSCs), which were differentiated into early-stage retinal organoids. Target loci regulated by ATOH7 were identified by Cleavage Under Targets and Release Using Nuclease with sequencing (CUT&RUN-seq) and differential expression by RNA sequencing (RNA-seq) of wildtype and mutant organoid-derived reporter cells. Additionally, single-cell RNA sequencing (scRNA-seq) was performed on whole organoids to identify cell type-specific genes. Mutant organoids displayed substantial deficiency in axon sprouting, reduction in RGCs, and an increase in other cell types. We identified 469 differentially expressed target genes, with an overrepresentation of genes belonging to axon development/guidance and Notch signaling. Taken together, we consolidate the function of human ATOH7 in guiding progenitor competence by inducing RGC-specific genes while inhibiting other cell fates. Furthermore, we highlight candidate genes responsible for ATOH7-associated optic nerve and retinovascular anomalies, which sheds light to potential future therapy targets for related disorders.

Super-enhancers (SEs) are expansive regions of genomic DNA that regulate the expression of genes involved in cell identity and cell fate. We recently identified developmental stage- and cell type-specific modules within the murine Vsx2 SE. Here, we show that the human VSX2 SE modules have similar developmental stage- and cell type-specific activity in reporter gene assays. By inserting the human sequence of one VSX2 SE module into a mouse with microphthalmia, eye size was rescued. To understand the function of these SE modules during human retinal development, we deleted individual modules in human embryonic stem cells and generated retinal organoids. Deleting one module results in small organoids, recapitulating the small-eyed phenotype of mice with microphthalmia, while deletion of the other module led to disruptions in bipolar neuron development. This prototypical SE serves as a model for understanding developmental stage- and cell type-specific effects of neurogenic transcription factors with complex expression patterns. Moreover, by elucidating the gene regulatory mechanisms, we can begin to examine how dysregulation of these mechanisms contributes to phenotypic diversity and disease.

OBJECTIVE: To study the long-term effects of perinatal high-dose recombinant human erythropoietin (rhEPO) on macular structural and vascular development in preterm children.
METHODS: Randomized, double-blind clinical trial follow-up plus cohort study.
METHODS: Setting: Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland.
METHODS: extremely or very preterm born children aged 7-15 years from an ongoing neuropediatric study (EpoKids). These had been previously randomized to receive either high-dose rhEPO or placebo perinatally.
METHODS: participation in the EpoKids Study, written informed consent (IC).
METHODS: previous ocular trauma or surgery; retinal or developmental disease unrelated to prematurity. Term-born children of comparable age were enrolled as a healthy control (HC) group.
METHODS: term birth, IC.
METHODS: any ocular or visual abnormality, high refractive error. Examiners were blinded regarding intervention status until completion of all analyses. (Participants/guardians remain blinded).
METHODS: Spectral-domain OCT scans (Heidelberg Spectralis system) and OCTA imaging (Zeiss PlexElite 9000) were obtained. Ophthalmological and orthoptic examinations excluded ocular comorbidities.
METHODS: OCT (central retinal thickness, CRT; total macular volume, TMV), superficial plexus OCTA (foveal avascular zone, FAZ; vessel density, VD; vessel length density, VLD) parameters and foveal hypoplasia grade according to published criteria.
RESULTS: Macular vessel density parameters (VD and VLD) were significantly lower (p =0.015, CI-95: 0.01 to 0.06 and p=0.015, CI-95: 0.74 to 3.64) in the EPO group (n= 52) when compared to placebo (n=35). No other significant differences were observed between the EPO and placebo group. When comparing the intervention subgroups to HC we found six significant differences in OCT and OCTA parameters (FAZ, VD, VLD and CRT comparing HC and EPO group; FAZ and CRT when comparing HC and placebo group).
CONCLUSIONS: Early high-dose rhEPO in infants born extremely or very preterm affects macular vessel density parameters compared to placebo. Premature birth (regardless of intervention status) affects retinal structure and vascular development. Our findings on macular vascular development do not contraindicate the administration of early high-dose EPO in preterm infants. For further understanding of the role of EPO on macular development and its clinical significance, future studies are needed.

The functionality of photoreceptors, rods, and cones is highly dependent on their outer segments (POS), a cellular compartment containing highly organized membranous structures that generate biochemical signals from incident light. While POS formation and degeneration are qualitatively assessed on microscopy images, reliable methodology for quantitative analyses is still limited. Here, we developed methods to quantify POS (QuaPOS) maturation and quality on retinal sections using automated image analyses. POS formation was examined during the development and in adulthood of wild-type mice via light microscopy (LM) and transmission electron microscopy (TEM). To quantify the number, size, shape, and fluorescence intensity of POS, retinal cryosections were immunostained for the cone POS marker S-opsin. Fluorescence images were used to train the robust classifier QuaPOS-LM based on supervised machine learning for automated image segmentation. Characteristic features of segmentation results were extracted to quantify the maturation of cone POS. Subsequently, this quantification method was applied to characterize POS degeneration in \"cone photoreceptor function loss 1\" mice. TEM images were used to establish the ultrastructural quantification method QuaPOS-TEM for the alignment of POS membranes. Images were analyzed using a custom-written MATLAB code to extract the orientation of membranes from the image gradient and their alignment (coherency). This analysis was used to quantify the POS morphology of wild-type and two inherited retinal degeneration (\"retinal degeneration 19\" and \"rhodopsin knock-out\") mouse lines. Both automated analysis technologies provided robust characterization and quantification of POS based on LM or TEM images. Automated image segmentation by the classifier QuaPOS-LM and analysis of the orientation of membrane stacks by QuaPOS-TEM using fluorescent or TEM images allowed quantitative evaluation of POS formation and quality. The assessments showed an increase in POS number, volume, and membrane coherency during wild-type postnatal development, while a decrease in all three observables was detected in different retinal degeneration mouse models. All the code used for the presented analysis is open source, including example datasets to reproduce the findings. Hence, the QuaPOS quantification methods are useful for in-depth characterization of POS on retinal sections in developmental studies, for disease modeling, or after therapeutic interventions affecting photoreceptors.

Atoh7 is transiently expressed in retinal progenitor cells (RPCs) and is required for retinal ganglion cell (RGC) differentiation. In humans, a deletion in a distal non-coding regulatory region upstream of ATOH7 is associated with optic nerve atrophy and blindness. Here, we functionally interrogate the significance of the Atoh7 regulatory landscape to retinogenesis in mice. Deletion of the Atoh7 enhancer structure leads to RGC deficiency, optic nerve hypoplasia, and retinal blood vascular abnormalities, phenocopying inactivation of Atoh7. Further, loss of the Atoh7 remote enhancer impacts ipsilaterally projecting RGCs and disrupts proper axonal projections to the visual thalamus. Deletion of the Atoh7 remote enhancer is also associated with the dysregulation of axonogenesis genes, including the derepression of the axon repulsive cue Robo3. Our data provide insights into how Atoh7 enhancer elements function to promote RGC development and optic nerve formation and highlight a key role of Atoh7 in the transcriptional control of axon guidance molecules.

UNASSIGNED: Platelet-activating factor (PAF), PAF receptor (PAFR), and PAF- synthesis/degradation systems are involved in essential CNS processes such as neuroblast proliferation, differentiation, migration, and synaptic modulation. The retina is an important central nervous system (CNS) tissue for visual information processing. During retinal development, the balance between Retinal Progenitor Cell (RPC) proliferation and differentiation is crucial for proper cell determination and retinogenesis. Despite its importance in retinal development, the effects of PAFR deletion on RPC dynamics are still unknown.
UNASSIGNED: We compared PAFR knockout mice (PAFR-/-) retinal postnatal development proliferation and differentiation aspects with control animals. Electrophysiological responses were analyzed by electroretinography (ERG).
UNASSIGNED: In this study, we demonstrate that PAFR-/- mice increased proliferation during postnatal retinogenesis and altered the expression of specific differentiation markers. The retinas of postnatal PAFR-/- animals decreased neuronal differentiation and synaptic transmission markers, leading to differential responses to light stimuli measured by ERG. Our findings suggest that PAFR signaling plays a critical role in regulating postnatal RPC cell differentiation dynamics during retinal development, cell organization, and neuronal circuitry formation.

X-linked retinoschisis (XLRS) is an early onset degenerative retinal disease characterized by cystic lesions in the middle layers of the retina. These structural changes are accompanied by a loss of visual acuity and decreased contrast sensitivity. XLRS is caused by mutations in the gene Rs1 which encodes the secreted protein Retinoschisin 1. Young Rs1-mutant mouse models develop key hallmarks of XLRS including intraretinal schisis and abnormal electroretinograms. The electroretinogram (ERG) comprises activity of multiple cellular generators, and it is not known how and when each of these is impacted in Rs1 mutant mice. Here we use an ex vivo ERG system and pharmacological blockade to determine how ERG components generated by photoreceptors, ON-bipolar, and Müller glial cells are impacted in Rs1 mutants and to determine the time course of these changes. We report that ERG abnormalities begin near eye-opening and that all ERG components are involved.

Mutations in NR2E3, a gene encoding an orphan nuclear transcription factor, cause two retinal dystrophies with a distinct phenotype, but the precise role of NR2E3 in rod and cone transcriptional networks remains unclear. To dissect NR2E3 function, we performed scRNA-seq in the retinas of wildtype and two different Nr2e3 mouse models that show phenotypes similar to patients carrying NR2E3 mutations. Our results reveal that rod and cone populations are not homogeneous and can be separated into different sub-classes. We identify a previously unreported cone pathway that generates hybrid cones co-expressing both cone- and rod-related genes. In mutant retinas, this hybrid cone subpopulation is more abundant and includes a subpopulation of rods transitioning towards a cone cell fate. Hybrid photoreceptors with high misexpression of cone- and rod-related genes are prone to regulated necrosis. Overall, our results shed light on the role of NR2E3 in modulating photoreceptor differentiation towards cone and rod fates and explain how different mutations in NR2E3 lead to distinct visual disorders in humans.

We recently identified PKN1 as a developmentally active gatekeeper of the transcription factor neuronal differentiation-2 (NeuroD2) in several brain areas. Since NeuroD2 plays an important role in amacrine cell (AC) and retinal ganglion cell (RGC) type formation, we aimed to study the expression of NeuroD2 in the postnatal retina of WT and Pkn1-/- animals, with a particular focus on these two cell types. We show that PKN1 is broadly expressed in the retina and that the gross retinal structure is not different between both genotypes. Postnatal retinal NeuroD2 levels were elevated upon Pkn1 knockout, with Pkn1-/- retinae showing more NeuroD2+ cells in the lower portion of the inner nuclear layer. Accordingly, immunohistochemical analysis revealed an increased amount of AC in postnatal and adult Pkn1-/- retinae. There were no differences in horizontal cell, bipolar cell, glial cell and RGC numbers, nor defective axon guidance to the optic chiasm or tract upon Pkn1 knockout. Interestingly, we did, however, see a specific reduction in SMI-32+ α-RGC in Pkn1-/- retinae. These results suggest that PKN1 is important for retinal cell type formation and validate PKN1 for future studies focusing on AC and α-RGC specification and development.